EPA
        United States
        Environmental Protection
        Agency
            Research And Development
            (MD-591)
EPA-600-4-91-003
July 1994
Shprt-Term Methods For
Estimating The Chronic Toxicity
Of Effluents And Receiving Water
To Marine And Estuaririe
Organisms

Second Edition

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                                                        EPA/600/4-91 /003
                                                        July 1994
9CRT-TBMIVE1KC6 FCR ESTIMtTIISG Tl-E OflCNIC TOXICITY OF EFFLJUBMT5

          RECEIVIN3 WATERS TO IvPRIIVE tbD ESTU4RINE CR3NI5M6
                          (Second Edition)
                             Edited by
             Donald J. Klenm1 and George E. IVbrrison2
           Teresa J. Nor berg-King3, Wi I I ian H. Peltier4
                      and IVbrgarete A. Heber5
  2Envirormsntal MbhStoring Systems Laboratory, Cincinnati, Ohio
  EnvirorrnentaI  Research  Laboratory, IMarragansett, Rhode  Island
       Environmental Research Laboratory, Duluth, Minnesota
         Environmental Services Division, Athens, Georgia
                ^Office of \A6ter, Washington,  D.C.
      EH/IR3WBMTAL M3MITORIN3 SvSTHvB LftBCRATCRY-CIISCINWI
                CFFICE OF RESEARCH A\D DEVELORVENT
               U.S.  BWIR3WENTAL  PRDTECTICM
                     ClrCihhATI, OHIO  45268

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                                  DISCLAIMER


   This document has been reviewed by the Environmental Monitoring Systems
Laboratory-Cincinnati (EMSL-Cincinnati), U. S. Environmental Protection Agency
(USEPA), and approved for publication.  The mention of trade names or
commercial products does not constitute endorsement or recommendation for use.
The results of data analyses by computer programs described in the section on
data analysis were verified using data commonly obtained from effluent
toxicity tests.  However, these computer programs may not be applicable to all
data, and the USEPA assumes no responsibility for their use.

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                                    FOREWORD


   Environmental measurements are  required to determine  the  quality  of  ambient
waters and the character of waste  effluent.  The  Environmental Monitoring
Systems Laboratory-Cincinnati (EMSL-Cincinnati) conducts research  to:
                                                          i
   •  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.

   •  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.
                                                          i
   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 second
edition of the marine and estuarine chronic toxicity test manual  for
effluents, first published (EPA/600/4-87/028)  by EMSL-Cincinnati  in May 1988.
It provides updated and standardized methods for estimating the chronic
toxicity of effluents and receiving waters to estuarine arid marine organisms
for use by the U. S.  Environmental  Protection Agency (USEPA)  regional
programs,  the state programs,  and the National  Pollutant Discharge Elimination
System (NPDES) permittees.

                                          Thomas A. Clark, Director
                                          Environmental  Monitoring Systems
                                          Laboratory-Cincinnati
                                     m

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                                                                                         1
                                    PREFACE
   This manual represents the second edition of the Agency's methods manual
for estimating the chronic toxicity of effluents and receiving waters to
marine and estuarine organisms initially published by USEPA's Office of
Research and Development, Environmental Monitoring and Support Laboratory
(EMSL-Cincinnati) in May 1988.  This edition reflects changes recommended by ,
the Toxicity Assessment Subcommittee of the Biological Advisory Committee,
USEPA headquarters, program offices, and regional staff, other Federal
agencies, state and interstate water pollution control programs, environmental
protection groups, trade associations, major industries, consulting firms,
academic institutions engaged in aquatic toxicology research, and other
interested parties in the private sector.

   The membership of the Toxicity Assessment Subcommittee, USEPA's Biological
Advisory Committee is as follows:

   William Peltier, Subcommittee Chairman,
      Environmental Services Division, Region 4
   Peter Nolan, Environmental Services Division, Region  1
   Jim Ferretti, Environmental Services Division, Region 2
   Ronald Preston, Environmental Services Division, Region 3
   Charles Steiner, Environmental Services Division, Region 5
   Evan Hornig, Environmental Services Division, Region  6
   Terry Hollister, Environmental Services Division, Region 6
   Michael Tucker, Environmental Services Division, Region 7
   Loys Parrish, Environmental Services Division, Region 8
   Peter Husby, Environmental Services Division, Region  9
   Joseph Cummins, Environmental Services Division, Region 10
   Gretchen Hayslip, Water Monitoring and Analysis Section, Region 10
   Bruce Binkley, National Enforcement Investigations Center, Denver
   Wesley Kinney, Environmental Monitoring Systems Laboratory-Las Vegas
   James Lazorchak, Environmental Monitoring Systems Laboratory-Cincinnati
   Douglas Middaugh, Environmental  Research Laboratory-Gulf Breeze
   George Morrison, Environmental Research Laboratory-Narragansett
   Teresa Norberg-King, Environmental Research Laboratory-Duluth
   Donald Klemm, Environmental Monitoring Systems Laboratory-Cincinnati
   Philip Lewis, Environmental Monitoring Systems Laboratory-Cincinnati
   Cornelius  I. Weber, Environmental Monitoring Systems  Laboratory-
     Cincinnati
   Richard Swartz, Environmental Research Laboratory-Newport
   Margarete  Heber, Human Health and Ecological Criteria Division, Office
     of Science & Technology  (OST), Office of Water (OW)
   Bruce Newton, Assessment and Watershed Protection Division, Office
     of Wetlands, Oceans, and Watersheds, OW
   Christopher Zarba, Health  and Ecological Criteria Division, Office
     of Science & Technology, OW
   Daniel Rieder, Hazard Evaluation Division, Office of  Pesticide
     Programs
   Jerry Smrchek, Health and  Environmental Review Division, Office
     of Toxic Substances

                                      iv

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Gail Hansen, Office of Solid Waste
Royal Nadeau, Emergency Response Team, Edison
                        Teresa J. Norberg-King
                        Chairman, Biological Advisory Committee
                        Regulatory Ecotoxicology Branch, ERL-Duluth

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                                   ABSTRACT
   This manual describes six short-term (one hour to nine days) estuarine and
marine methods for measuring the chronic toxicity of effluents and receiving
waters to five species: the sheepshead minnow, Cypn'nodon van'egatus; the
inland silverside, Menidia beryllina-, the mysid, Mysidopsis bahia; the sea
urchin, Arbacia punctulata; and the red macroalga, Champia parvula.  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 Spearman-Karber Method, and the Linear Interpolation
Method are provided in the Appendices.
                                       VI

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Foreword
Preface
Abstract
                                   CONTENTS              i

                                                                      Page
Tables    .  .  .
Acknowledgments
                                                                        vi
                                                                      'xii
                                                                     xviii
                                                                      xxix
Section Number                                                        Page

  1.  Introduction	1
  2.  Short-Term Methods for Estimating Chronic Toxicity ! .......  4
         Introduction   	 	  4
         Types of Tests   .  .  .  .	i	7
         Static Tests   	 	  8
         Advantages and Disadvantages of Toxicity Test Types  .....  8
  3.  Health and Safety   .  .  .  .	10
         General Precautions	10
         Safety Equipment   	 10
         General Laboratory and  Field Operations  .  .  .  .	10
         Disease Prevention	11
         Safety Manuals	11
         Waste Disposal	'•	11
  4.  Quality Assurance	12
         Introduction
                                                                        12
         Facilities,  Equipment,  and Test Chambers   .  .  .	12
         Test Organisms   	13
         Laboratory Water Used for Culturing and
           and Test Dilution Water  .	13
         Effluent and Receiving  Water Sampling and
           Handling   	13
         Test Conditions	13
         Quality of Test Organisms	14
         Food Quality   	14
         Acceptability of Chronic Toxicity Tests	 15
         Analytical Methods    	  ....... 16
         Calibration  and Standardization	16
         Replication  and Test Sensitivity	16
         Variability  in Toxicity Test  Results   	i.	16
         Test Precision	17
         Demonstrating Acceptable Laboratory Performance !.  	 18
         Documenting  Ongoing Laboratory Performance   ...  	 18
         Reference Toxicants	21
         Record Keeping	  „	21
         Video Tapes  of USEPA Culture  and Toxicity
           Test Methods
                                                                        21
         Supplemental  Reports  for Training Video Tapes   .........  22
  5.   Facilities,  Equipment,  and Supplies    	 .....  23
         General  Requirements    .	23
         Test  Chambers  ....  	 i.......  24
                                     vn

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                             CONTENTS (CONTINUED)

Section Number                                                        Page


         Cleaning Test Chambers and Laboratory Apparatus  ....... 24
         Apparatus and Equipment for Culturing and
           Toxicity Tests   	 25
         Reagents and Consumable Materials  	 25
         Test Organisms   	25
         Supplies	25
  6.  Test Organisms	27
         Test Species   	27
         Sources of Test Organisms  	 28
         Life Stage   	29
         Laboratory Culturing   	 29
         Holding and Handling of Test Organisms   	29
         Transportation to the Test Site	.30
         Test Organism Disposal	31
  7.  Dilution Water	32
         Types of Dilution Water	32
      ,   Standard, Synthetic Dilution Water   	 32
         Use of Receiving Water as Dilution Water	 . 34
         Use of Tap Water as Dilution Water   	 36
         Dilution Water Holding   	 36
  8.  Effluent and Receiving Water Sampling, Sample Handling,
           and Sample Preparation for Toxicity Tests  	 37
         Effluent Sampling	37
         Effluent Sample Types  	 37
         Effluent Sampling Recommendations  	 38
         Receiving Water Sampling   	 39
         Effluent and Receiving Water Sample Handling,
           Preservation, and Shipping   	 40
         Sample Receiving   	 41
         Persistence of Effluent Toxicity During Sample
           Shipment and Holding   	41
         Preparation of Effluent and Receiving Water Samples
           for Toxicity Tests	41
         Preliminary Toxicity Range-finding Tests   .  	 45
         Multiconcentration (Definitive) Effluent
           Toxicity Tests   	 45
         Receiving Water Tests  	 46
  9.  Chronic Toxicity Test Endpoints and Data Analysis   .  . .  .  .  . .47
         Endpoints	47
         Relationship between Endpoints Determined
           by Hypothesis Testing and Point Estimation  Techniques  ... 48
         Precision	50
         Data Analysis	50
         Choice of Analysis   	 52
         Hypothesis Tests   	 54
         Point Estimation Techniques  .  .	56


                                     viii

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                             CONTENTS (CONTINUED)
                                                     •
Section Number                                                 '       Page


 10.  Report Preparation	j	58
         Introduction   	 ....... 	 58
         Plant Operations	58
         Source of Effluent, .Receiving Water, and Dilution Water  ... 58
         Test Methods	59
         Test Organisms	 59
         Quality Assurance  	  	 59
         Results	59
         Conclusions and Recommendations   	 	 60
 11.  Test Method:  Sheepshead Minnow, Cyprinodon van'eg&tus, Larval
       Survival and Growth Test Method 1004.0 ..*... 	 61
           Scope and Application   	  ............ 61
           Summary of Method	  . 61
           Interferences	61
           Safety	62
           Apparatus and Equipment   	 62
           Reagents and Consumable Materials	[	64
           Effluent and Receiving Water Collection,  Preservation, and
             Storage	\	  . 74
           Calibration and Standardization	!	74
           Quality Control	,	74
           Test Procedures	; .'	74
           Summary of Test Conditions and  Test Acceptability Criteria  . 84
           Acceptability of Test Results	j ....... 84
           Data Analysis	J .	84
           Precision and Accuracy	« '	116
 12.  Test Method:  Sheepshead Minnow, Cyprinodon variegntus, Embryo-
        larval Survival and Teratogenicity Test  Method  1005.0 ....   124
           Scope and Application	1	124
           Summary of Method	1	124
           Interferences	'.	»	124
           Safety	*	125
           Apparatus and Equipment	i	125
           Reagents and Consumable Materials   	  . 	   127
           Effluent and Receiving  Water Collection,  Preservation, and
             Storage	I	137
           Calibration  and  Standardization	I	137
           Quality Control   .......  	  L 	   138
           Test Procedures	L	138
           Acceptability of Test Results	L	144
           Summary of  Test  Conditions and  Test Acceptability Criteria   144
           Data Analysis	L ......   144
           Precision and Accuracy	  .  L	160
 13.   Test Method:   Inland  Silverside, Menidia beryTlina, Larval
        Survival  and Growth Method 1006.0  	  I 	   163
           Scope  and Application	L	163
           Summary of  Method	L	163

                                      ix

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                             CONTENTS (CONTINUED)

Section Number                                                        Page


           Interferences  	  163
           Safety	164
           Apparatus and Equipment  	  164
           Reagents and Consumable Materials  	  167
           Effluent and Receiving Water Collection, Preservation, and
             Storage	177
           Calibration and Standardization  	  177
           Quality Control  	  177
           Test Procedures	177
           Summary of Test Conditions and Test Acceptability Criteria  186
           Acceptability of Test Results	186
           Data Analysis	186
           Precision and Accuracy 	  216
 14.   Test Method:  Mysid, Mysidopsis bahia, Survival,  Growth,  and
         Fecundity Test Method 1007.0	  222
           Scope and Application	222
           Summary of Method  	  222
           Interferences  	  222
           Safety	[  [  223
           Apparatus and Equipment  	  223
           Reagents  and Consumable Materials  	  225
           Effluent  and Receiving Water  Collection, Preservation, and
             Storage	 .  .  235
           Calibration and Standardization  .....  	  ...  235
           Quality Control   ......  	  235
           Test Procedures	  235
           Summary of Test Conditions  and Test Acceptability Criteria  243
           Acceptability of Test Results	243
           Data Analysis  	  243
           Precision and Accuracy	'  294
 15.   Test Method:   Sea Urchin,  Arbacia  punctulata, Fertilization Test
         Method 1008.0	   300
           Scope and Application	  .  300
           Summary of Method  	  300
           Interferences	  300
           Safety	'  [  301
           Apparatus and Equipment  	  301
           Reagents  and  Consumable Materials  	  302
           Effluent  and  Receiving Water  Collection,  Preservation,  and
             Storage	311
           Calibration  and  Standardization   	   311
           Quality Control   	   311
           Test  Procedures	;	311
           Summary of Test  Conditions  and  Test Acceptability Criteria  316
           Acceptability of  Test Results	316
           Data  Analysis	319
           Precision  and Accuracy	'.'.'.   333

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                             CONTENTS (CONTINUED)
                                                         I
Section Number                                                        Page


 16.  Test Method:  Red Macroalga, Champia parvuTa, Reproduction Test
         Method 1009.0	.  341
           Scope and Application	341
           Summary of Method	341
           Interferences	341
           Safety	i	342
           Apparatus and Equipment	  342
           Reagents and Consumable Materials  	 	  343
           Effluent and Receiving Water Collection, Preservation, and
             Storage	350
           Calibration and Standardization  	 	  351
           Quality Control	351
           Test Procedures	351
           Summary of Test Conditions and Test Acceptability Criteria  356
           Acceptability of Test Results	360
           Data Analysis	360
           Precision and Accuracy	373
Cited References	380
Bibliography	  391
Appendices	401
  A.  Independence,  Randomization, and Outliers ..... 	  403
  B.  Validating Normality and Homogeneity of Variance
       Assumptions	409
  C.  Dunnett's Procedure	419
  D.  T test with Bonferroni's Adjustment	432
  E.  Steel's Many-one Rank Test	'	438
  F.  Wilcoxon Rank Sum Test	443
  G.  Single Concentration Toxicity Test - Comparison
        of Control with 100% Effluent or Receiving Water	450
  H.  Probit Analysis	454
  I.  Spearman-Karber Method  	  457
  J.  Trimmed Spearman-Karber Method  	 	  462
  K.  Graphical Method	467
  L.  Linear Interpolation Method	471
  Cited References	482

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                                    FIGURES


SECTION 1-10

Number                                                                Page

  1.  Control (cusum) charts  	  20

  2.  Flowchart for statistical  analysis of test data	  53


SECTION 11

Number                                                                Page

  1.  Embryonic development of sheepshead minnow, Cypn'nodon
      variegatus	72

  2.  Data form for the sheepshead minnow,  Cypn'nodon variegatus,
      larval survival and growth test.  Daily record of
      larval survival and test conditions   	  80

  3.  Data form for the sheepshead minnow,  Cypn'nodon variegatus,
      larval survival and growth test.  Dry weights of
      larvae	83

  4.  Data form for the sheepshead minnow,  Cypn'nodon variegatus,
      larval survival and growth test.  Summary of test
      results   	85

  5.  Flowchart for statistical  analysis of the sheepshead minnow,
      Cypn'nodon variegatus,  larval  survival  data by hypothesis
      testing	89

  6.  Flowchart for statistical  analysis of the sheepshead minnow,
      Cypn'nodon variegatus,  larval  survival  data by
      point estimation	90

  7.  Plot of mean survival  proportion data in Table 5	93

  8.  Flowchart for statistical  analysis of the sheepshead
      minnow,  Cypn'nodon variegatus,  larval  growth data   	  101

  9.  Plot of weight data from sheepshead minnow,  Cypn'nodon
      variegatus,  larval  survival  and growth  test   	  103

 10.  Plot of raw  data,  observed means and  smoothed means
      for  the sheepshead minnow,  Cypn'nodon variegatus,
      growth data  from Tables  4  and  21	112
                                     xn

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                              FIGURES  (CONTINUED)


SECTION 11 (Continued)

Number                                                   j             Page

 11.  ICPIN program output for the IC25	  114

 12.  ICPIN program output for the IC50   	i	115


SECTION 12

Number                                                                Page
                                                         !
  1.  Data form for sheepshead-minnow, Cyprinodon variegatus,
      embryo-larval survival and teratogenicity test.  Daily record
      of embryo-larval survival/terata and test conditions	128
                                                         i
  2.  Embryonic development of sheepshead minnow, Cyprinodon
      variegatus	142
  3.  Flowchart for statistical analysis of sheepshead minnow,
      Cyprinodon variegatus, embryo-larval survival and
      teratogenicity test.  Survival and terata data                   148

  4.  Plot of sheepshead minnow, Cyprinodon variegatus, total
      mortality data from the embryo-larval test	150

  5.  Output for USEPA Probit Analysis program, Version 1.5  	  159


SECTION 13
                                                         i
Number                          •                         '             Page
  1.  Glass chamber with sump area
166
  2.   Inland silverside, Menidia beryllina   	  	   176
                                                         i
  3.   Data form for the  inland silverside, Menidia beryllina,
       larval survival and growth test.  Daily record of larval
       survival and test  conditions	j	182

  4.   Data form for the  inland silverside, Menidia beryllina,
       larval survival and growth test.  Dry  weights of
       larvae	i	185
                                                         i

  5.   Data form for the  inland silverside, Menidia beryllina, larval
       survival and growth test.  Summary of  test results   	   187
                                     xm

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                              FIGURES (CONTINUED)
SECTION 13 (Continued)
  Number                                                              Page
  6.  Flowchart for statistical analysis of the inland silverside,
      Henida beryl Una, survival data by hypothesis testing	191
  7.  Flowchart for statistical analysis of the inland silverside,
      Nenida beryllina, survival data by point estimation 	  192
  8.  Plot of mean survival proportion of the inland silverside,
      Menidia beryllina, larvae	  193
  9.  Output for USEPA Probit Analysis program, Version 1.5 	  203
 10.  Flowchart for statistical analysis of the inland silverside,
      Menidia beryllina, growth data  	  204
 11.  Plot of mean weights of inland silverside, Menidia beryllina,
      larval  survival and growth test   	206
 12.  Plot of the raw data, observed means, and smoothed
      means from Tables 13 and 19    	215
 13.  ICPIN program output for the IC25	217
 14.  ICPIN program output for the IC50   	218

SECTION 14
Number                                                                Page
  1.  Apparatus (brood chamber) for collection of juvenile mysids,
       Mysidopsis bahia   	  234
  2.  Data form for the mysid, Mysidopsis bahia, water quality
       measurements	240
  3.  Mature  female mysid, Mysidopsis bahia,  with eggs in oviducts  .  241
  4.  Mature  female mysid, Mysidopsis bahia,  with eggs in oviducts
      and developing embryos in the brood sac   	242
  5.  Mature  male mysid, Mysidopsis bahia   .  .	243
  6.  Immature mysid, Mysidopsis bahia,  (A) lateral  view,
      (B)  dorsal  view   	244
                                     xiv

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                              FIGURES  (CONTINUED)


SECTION 14 (Continued)

Number                                                                Page

  7.  Data form for the mysid, Mysidopsis bahia, survival and
       fecundity data	245

  8.  Data form for the mysid, Mysidopsis bahia, dry weight
       measurements	247

  9.  Flowchart for statistical analysis of mysid, Mysidopsis bahia,
      survival data by hypothesis testing	j .......  253

 10.  Flowchart for statistical analysis of mysid, Mysidopsis bahia,
      survival data by point  estimation  	i	  254

 11.  Plot of survival proportions of mysids, Mysidopsis hahia,
      at each treatment level   .	255
 12.  Output for USEPA Probit Analyis program, Version 1.5'  	  262

 13.  Flowchart for statistical analysis of mysid, Mysidopsis bahia,
      growth data	264

 14.  Plot of mean growth data for mysid, Mysidopsis bahia,  test   .  .  265
                                                          i
 15.  Plot of raw data, observed means, and smoothed means
      for the mysid, Mysidopsis bahia, growth data from
      Tables 13 and 20	274
  16.   ICPIN program  output  for the  IC25    	j	276

  17.   ICPIN program  output  for the  IC50	277
                                                          i
  18.   Flowchart  for  statistical  analysis  of mysid, Mysidopsis
       bahia,  fecundity  data   	j ......   278

  19.   Proportion of  female  mysids,  Mysidopsis  bahia,  with eggs   ...   280
                                                          i
  20.   Plot of the mean  proportion of females mysids,
       Mysidopsis bahia,  with  eggs    .	290
                                                          i

  21.   ICPIN program  output  for the  IC25	292

  22.   ICPIN program  output  for the  IC50	   293
                                       xv

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                              FIGURES (CONTINUED)


SECTION 15

Number                                                                Page

  1.  Data form for (1) fertilization test using sea urchin,
      Arbacia punctulata  	  304

  2.  Data form (2) for fertilization test using sea urchin,
      Arbacia punctulata  	  305

  3.  Data form (3) for fertilization test using sea urchin,
      Arbacia punctulata	306

  4.  Flowchart for statistical analysis of sea urchin,
      Arbacia punctulata, by point estimation   .... 	  321

  5.  Plot of mean percent of fertilized sea urchin,
      Arbacia punctulata, eggs  .  .	322

  6.  ICPIN program output for the IC25   '.	332

  7.  ICPIN program output for the IC50   	334


SECTION 16

Number                                                                Page

  1.  Life history of the red macroalga, Champia parvula  	  347

  2.  Apex of branch of female plant, showing sterile
      hairs and reproductive hairs (trichogynes)  	  349

  3.  A portion of the male thai 1 us  showing  spermatial  sori    ....  349

  4.  A magnified portion of a spermatial  sorus   	  350

  5.  Apex of a branch on a mature female  plant that was
      exposed to spermatia from a  male plant	350

  6.  Data form for the red macroalga, Champia parvula, sexual
      reproduction test.   Receiving  water  summary sheet   	  352

  7.  A mature cystocarp   	  355

  8.  Comparison of a  very young branch and  an immature
      cystocarp   	355
                                     xvi

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                             FIGURES  (CONTINUED)


SECTION 16 (Continued)

Number                                                                Page

  9.  An aborted cystocarp	356
                                                          i
 10.  Data form for the red macroalga, Champia parvula, sexual
      reproduction test..  Cystocarp data sheet  	 	  357

 11.  Flowchart for statistical analysis of the red macroalga,
      Champia parvula, data	362

 12.  Plot of the number of cystocarps per plant  . .  .	364

 13.  ICPIN program output for the IC25	374
 "                                                         !

 14.  ICPIN program output for the IC50	375
                                     xvii

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                                    TABLES


SECTION 1-10

Number                                                                Page

  1.  Rational Inter!aboratory study of chronic toxicity test
      precision, 1991:  Summary of responses using two reference
      toxicants   	18

  2.  Commercial suppliers of brine shrimp (Artemia) cysts  	  26

  3.  Preparation of 6P2 artificial seawater using reagent
      grade chemicals   	34

  4.  Oxygen solubility (mg/L) in water at equilibrium with
      air at 760 mm Hg	43

  5.  Percent unionized NH3 in aqueous  ammonia solutions:
      temperature 15-26°C and pH 6.0-8.9	  44


SECTION 11

Number                                                                Page

  1.  Reagent grade chemicals used in the preparation of GP2
      artificial seawater for the sheepshead minnow, Cypn'nodon
      van'egatus, toxicity test   	67

  2.  Preparation of test solutions at  a salinity of 20%o, using
      20%o salinity dilution water prepared from natural seawater,
      hypersaline brine, or artificial  sea salts  	  76

  3.  Summary of test conditions and test acceptability criteria
      for sheepshead minnow, Cypn'nodon van'egatus,  larval survival
      and growth test with effluents and receiving waters   	  86

  4.  Summary of survival  and growth data for sheepshead minnow,
      Cypn'nodon van'egatus, larvae exposed to an effluent for
      seven days	88

  5.  Sheepshead minnow, Cypn'nodon van'egatus, survival data   ....  92

  6.  Centered observations for Shapiro-Wilk's example  	  92

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

  8.  Coefficients and differences for  Shapiro-Wilk's example   ....  95
                                    xvm

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                              TABLES (CONTINUED)

SECTION 11 (Continued)

Number                                                                Page

  9.  Assigning ranks to the control and 6.25% effluent
      concentration for Steel's Many-one Rank Test  	 96

 10.  Table of ranks	.....'	96

 11.  Rank sums   	;	96

 12.  Data for example of Spearman-Karber Analysis  . .	98
                                                          i
 13.  Sheepshead minnow, Cyprinodon variegatus, growth data 	  102

 14.  Centered observations for Shapiro-Milk's example   . i	  102

 15.  Ordered centered observations for Shapiro-Wilk's example  '. .  .  104

 16.  Coefficients and differences for Shapiro-Wilk's example   . .  .  105

 17.  ANOVA table   . .	107

 18.  ANOVA table for Dunnett's Procedure example   . .	  109

 19.  Calculated t values	110

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

 21.  Single-laboratory precision of the sheepshead minnow,
      Cyprinodon variegatus, larval survival and growth  test
      performed in FORTY FATHOMS® artificial seawater, using
      larvae from fish maintained and spawned  in FORTY FATHOMS®
      artificial seawater, using copper (Cu) sulfate as  a reference
      toxicant  	 ......  117
                                                          i
 22.  Single-laboratory precision of the sheepshead minnow,
      Cyprinodon variegatus, larval survival and growth  test
      performed in FORTY FATHOMS® artificial seawater, using
      larvae from fish maintained and spawned  in FORTY FATHOMS®
      artificial seawater, using sodium dodecyl sulfate  (SDS) as
      a reference toxicant	  118
                                      xix

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                              TABLES (CONTINUED)

SECTION 11 (Continued)

Number                                                                Page

 23.  Single-laboratory precision of the sheepshead minnow,
      Cypn'nodon variegatus, larval survival and growth test
      performed in natural seawater, using larvae from fish
      maintained and spawned in natural seawater, using copper
      (Cu) sulfate as a reference toxicant	119

 24.  Single-laboratory precision of the sheepshead minnow,
      Cypn'nodon variegatus, larval survival and growth test
      performed in natural seawater, using larvae from fish
      maintained and spawned in natural seawater, using sodium
      dodecyl sulfate (SDS) as a reference toxicant   	  120

 25.  Single-laboratory precision of the sheepshead minnow,
      Cypn'nodon variegatus, larval survival and growth test
      performed in FORTY FATHOMS® artificial seawater, using
      larvae from fish maintained and spawned in FORTY FATHOMS®
      artificial seawater, and hexavalent chromium as a reference
      toxicant	121

 26.  Comparison of larval survival (LC50) and growth (IC50)
      values for the sheepshead minnow, Cypn'nodon variegatus,
      exposed to sodium dodecyl sulfate (SDS) and copper (Cu)
      sulfate in 6P2 artificial seawater medium or natural
      seawater	". . . .	122

 27.  Data from interlaboratory study of the sheepshead minnow,
      Cypn'nodon variegatus, larval survival and growth test,
      using an industrial effluent as a reference toxicant   	  123


SECTION 12

Number                                                                Page

  1.  Preparation of test solutions at a salinity of 20%o,
      using 20%o natural or artificial  seawater, hypersaline
      brine,  or artificial sea salts  	  132

  2.  Summary of test conditions and test acceptability criteria
      for sheepshead minnow, Cypn'nodon variegatus, embryo-larval
      survival and teratogenicity test with effluents and receiving
      waters	145

  3.  Sheepshead minnow, Cypn'nodon variegatus,  embryo-larval
      total mortality data	  149
                                      xx

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                              TABLES (CONTINUED)

SECTION 12 (Continued)

Number                                                                Page

  4.  Centered observation for Shapiro-Wilk's example . .	  149

  5.  Ordered centered observations for Shapiro-Wilk's example  . .  . t 151

  6.  Coefficients and differences for Shapiro-Wilk's example ....  152
                                                          i
  7.  ANOVA table   	!......  154

  8.  ANOVA table for Dunnett's Procedure example   . .	  156

  9.  Calculated t values	157
                                                                           .
  10. Data for Probit Analysis	  158

  11. Single-laboratory precision of the sheepshead minnow,
      Cyprinodon variegatus, embryo-larval survival and
      teratogenicity test performed in HW MARINEMIX® artificial
      seawater, using embryos from fish maintained and spawned
      in HW MARINEMIX® artificial seawater using copper (Cu)
      sulfate as a reference toxicant	161

  12. Single-laboratory precision of the sheepshead minnow',
      Cyprinodon variegatus, embryo-larval survival and
      teratogenicity test performed in HW MARINEMIX® artificial
      seawater, using embryos from fish maintained and spawned
      in HW MARINEMIX® artificial seawater using sodium dodecyl
      sulfate (SDS) as a reference toxicant   	 	  162
                                                          i


SECTION 13

Number                                                    i            Page

  1.  Preparation of 3 L saline water from deionized water and a
      hypersaline brine of 100%o needed for test solutions at
      20%o salinity   	|	170

  2.  Reagent grade chemicals used in the preparation of GP2
      artificial seawater for the inland silverside, Menidia
      beryTlina, toxicity test  	 ......  171
  3.  Summary of test conditions and test acceptability criteria
      for inland silverside, Menidia beryTTina, larval survival
      and growth test with effluents and receiving waters
                                      xxi
188

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                              TABLES (CONTINUED)
SECTION 13 (Continued)
Number                                                                Pa9e
  4.  Inland silverside, Mem'dia beryllina, larval survival data  .  .   194
  5.  Centered observations for Shapiro-WiIk's example  	   194
  6.  Ordered centered observations for Shapiro-WiIk's example  ...   195
  7.  Coefficients and differences for Shapiro-WiIk's example   ...   196
  8.  ANOVA table   	198
  9.  ANOVA table for Dunnett's Procedure example   	   199
 10.  Calculated t values	   200
 11.  Data for Probit Analysis	202
 12.  Inland silverside, Mem'dia beryllina, growth data    	   205
 13.  Centered observations for Shapiro-WiIk's example  	   205
 14.  Ordered centered observations for Shapiro-WiIk's example  . .  .   207
 15.  Coefficients and differences for Shapiro-WiIk's example   ...   208
 16.  ANOVA table	210
 17.  ANOVA table for Dunnett's Procedure example   	   211
 18.  Calculated t values   	212
 19.  Inland silverside mean growth response  after smoothing   ....   214
 20.  Single-laboratory precision of the  inland  silverside,
      Nenidia beryllina, survival and growth  test performed
      in  natural seawater, using larvae from  fish maintained
      and spawned in  natural seawater, and copper (Cu) as  a
      reference toxicant   	  	   219
 21.  Single-laboratory precision of the  inland  silverside,
      Mem'dia beryllina, survival and growth  test performed
      in  natural seawater, using larvae from  fish maintained
      and spawned in  natural seawater, and sodium dodecyl
      sulfate  (SDS) as  a reference toxicant  	  	  220
                                      xxn

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                              TABLES (CONTINUED)          i

                                                          i
SECTION 13 (Continued)                                    !

Number                                                                Page


 22.  Comparison of the single-laboratory precision of the inland
      silverside, Mem'dia beryllina, larval survival (LC50) and
      growth (IC50) values exposed to sodium dodecyl sulfate
      (SDS) or copper (Cu) sulfate, in GP2 artificial seawater
      medium or natural seawater   	 	  221


SECTION 14  -                                              i

Number                                                                Page

  1.  Reagent grade chemicals used in the preparation of GP2
      artificial seawater for the mysid, Mysidopsis bahia, toxicity
      test	228

  2.  Quantities of effluent, deionized water, and hypersaline
      brine (100%o) needed to prepare 1800 ml volumes of test
      solution with a salinity of 20%o	  229

  3.  Summary of test conditions and test acceptability criteria
      for the mysid, Mysidopsis bahia, seven day survival, growth,
      and fecundity test with effluents and receiving waters  ....  249

  4.  Data for Mysidopsis bahia 7-day survival, growth, and
      fecundity test	251
                                                          i
  5.  Mysid, Mysidopsis bahia, survival data  	 	  256

  6.  Centered observations for Shapiro-Wilk's example	  257

  7.  Ordered centered observations for Shapiro-Wilk's example  . .  .  258

  8.  Coefficients and differences for Shapiro-Wilk's example   . .  .  259

  9.  Assigning ranks to the control and 50 ppb concentration level
      for Steel's Many-one Rank Test	260

 10.  Table of ranks	i. ......  261
                                                          I
 11.  Rank sums	,  261
                                                     .
 12.  Data for Probit Analysis	  263
                                     xxm

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                              TABLES (CONTINUED)
SECTION 14 (Continued)
Number                                                                Page
 13.  Hysid, Mysidopsis bahia, growth data  	  263
 14.  Centered observations for Shapiro-Wilk's example  	  266
 15.  Ordered centered observations for Shapiro-Wilk's example  .  .  .  267
 16.  Coefficients and differences for Shapiro-Wilk's example   .  .  .  268
 17.  ANOVA table   .	270
 18.  ANOVA table for Dunnett's Procedure example   	  271
 19.  Calculated t values	272
 20.  Mysid, Mysidopsis bahia, mean growth response after
      smoothing   	273
 21.  Hysid, Mysidopsis bahia, fecundity data:  Percent females
      with eggs   	281
 22.  Centered observations for Shapiro-Wilk's example  	  282
 23.  Ordered centered observations for Shapiro-Wilk's example  .  .  .  283
 24.  Coefficients and differences for Shapiro-Wilk's example   .  .  .  283
 25.  ANOVA table   	285
 26.  ANOVA table for the t test with Bonferroni's
      Adjustment example  	  287
 27.  Calculated t values	 .  .  .  288
 28.  Mysid, Mysidopsis bahia, mean proportion of females
      with eggs   	289
 29.  Single-laboratory precision of the mysid, Mysidopsis bahia,
      survival, growth, and fecundity test performed in natural
      seawater, using juveniles from mysids cultured and spawned
      in natural seawater and copper (Cu) sulfate as a reference
      toxicant	295
                                     xxiv

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                              TABLES (CONTINUED)
                                                                         .
SECTION 14 (Continued)                                     j

Number                                                                Page
                       ,
 30.  Single-laboratory precision of the mysid, Mysidopsis bahia,
      survival, growth, and fecundity test performed in natural
      seawater, using juveniles from mysids cultured and spawned
      in natural seawater and sodium dodecyl sulfate (SDS) as a
      reference toxicant	i,	296

 31.  Comparison of survival (LC50), growth and fecundity  (IC50)
      results from 7-day tests with the mysid, Mysidopsis bah fa,
      using natural seawater (NSW) and artificial seawater (GP2) as
      dilution water and sodium dodecyl sulfate (SDS) as a
      reference toxicant	i	297

 32.  Comparison of survival (LC50), growth and fecundity  (IC50)
      results from 7-day tests with the mysid, Mysidopsis bahia,
      using natural seawater (NSW) and artificial seawater (GP2)
      as dilution water and copper (Cu) sulfate as a reference
      toxicant	;	298
                                                           I

 33.  Control results from 7-day survival, growth, and fecundity
      tests with the mysid, Mysidopsis bahia, using natural
      seawater and artificial seawater (GP2) as a dilution
      water	,,	299


SECTION 15

Number                                                                Page

  1.  Preparation of test solutions at a salinity of 30%o using
      natural seawater, hypersaline brine, or artificial sea salts   .  309

  2.  Reagent grade chemicals used in the preparation of GP2
      artificial seawater for the sea urchin, Arbacia punctulata
      toxicity test   	,;	310
                                                                  •
  3.  Summary of test conditions and test acceptability criteria
      for sea urchin, Arbacia punctulata, fertilization test with
      effluent and receiving waters   	,;	317

  4.  Data from sea urchin, Arbacia punctulata, fertilization test   .  319
                                                           i
  5.  Sea urchin, Arbacia punctulata, fertilization data   i 	  323

  6.  Centered observations for Shapiro-Wilk's example  .1 	  323

  7.  Ordered centered observations for Shapiro-Wilk's example  . .  .  324

                                      xxv

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                              TABLES (CONTINUED)

SECTION 15 (Continued)

Number                                                                Page

  8.  Coefficients and differences for Shapiro-Wilk's example   .  .  .  325

  9.  ANOVA table	326

 10.  ANOVA table for Dunnett's Procedure example   	  328

 11..  Calculated t values   	329

 12.  Sea urchin, Arbacia punctulata, mean proportion of
      fertilized eggs   	331

 13.  Single-laboratory precision of the sea urchin, Arbacia
      punctulata, fertilization test performed in FORTY FATHOMS®
      artificial seawater, using gametes from adults maintained
      in FORTY FATHOMS® artificial seawater, or obtained directly
      from natural sources, and copper (Cu) sulfate as reference
      toxicant	335

 14.  Single-laboratory precision of the sea urchin,
      Arbacia punctulata, fertilization test performed
      in FORTY FATHOMS® artificial seawater, using gametes
      from adults maintained in FORTY FATHOMS® artificial
      seawater, or obtained directly from natural sources,
      and sodium dodecyl sulfate (SDS) as a reference toxicant  ...  336

 15.  Single-laboratory precision of the sea urchin,
      Arbacia punctulata, fertilization test performed
      in natural seawater, using gametes from adults
      maintained in natural seawater and copper (Cu)
      sulfate as a reference toxicant	337

 16.  Single-laboratory precision of the sea urchin,
      Arbacia punctulata, fertilization test performed
      in natural seawater, using gametes from adults
      maintained in natural seawater and sodium dodecyl
      sulfate (SDS) as a reference toxicant   	  338

 17.  Single-laboratory precision of the sea urchin,
      Arbacia punctulata, fertilization test performed
      in 6P2, using gametes from adults maintained in
      6P2 artificial seawater and copper (Cu) sulfate and
      sodium dodecyl sulfate (SDS) as reference toxicants    ......  339
                                     xxvi

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                              TABLES (CONTINUED)

SECTION 15 (Continued)

Number                                                                Page
                                                              .
 18.  Single-laboratory precision of the sea urchin, Arbacia
      punctulata, fertilization test performed in natural sea.water,
      using gametes from adults maintained in natural seawater
      and copper (Cu) sulfate and sodium dodecyl sulfate (SDS)
      as reference toxicants	  340
SECTION 16

Number
Page
  1.  Nutrients to be added to natural seawater and to
      artificial seawater (GP2) described in Table 2  	  345
  2.  Reagent grade chemicals used in the preparation of GP2
      artificial seawater for use in conjunction with natural
      seawater for the red macroalga, Champia parvula, culturing
      and toxicity testing		346

  3.  Summary of test conditions and test acceptability criteria
      for red macroalga, Champia parvula, sexual reproduction
      test with effluents and receiving waters	'	358
                                                          i
  4.  Data from the red macroalga, Champia parvula, effluent
      toxicity test.  Cystocarp counts for individual plants and
      mean count per test chamber for each effluent concentration   .  361

  5.  The red macroalga, Champia parvula, sexual reproduction data  .  363

  6.  Centered observations for Shapiro-Wilk's example	  365

  7.  Ordered centered observations for Shapiro-Wilk's example  . .  .  366
                                                          I
  8.  Coefficients and differences for Shapiro-Wilk's example   . .  .  366

  9.  ANOVA table   	 ......  368

 10.  ANOVA table for Dunnett's Procedure example   	  370

 11.  Calculated t values   	 ......  371

 12.  Red macroalga, Champia parvula, mean number of cystocarps   .  .  372
                                     xxv

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                              TABLES (CONTINUED)

SECTION 16 (Continued)

Number                                                                Page

 13.  Single-laboratory precision of the red macroalga, Champia
      parvula, reproduction test performed in a 50/50 mixture
      of natural seawater and GP2 artificial seawater, using
      gametes from adults cultured in natural seawater.  The
      reference toxicant used was copper (Cu) sulfate   	  376

 14.  Single-laboratory precision of the red macroalga, Champia
      parvula, reproduction test performed in a 50/50 mixture of
      natural seawater and GP2 artificial seawater, using gametes
      from adults cultured in natural seawater.  The reference
      toxicant used was sodium dodecyl  sulfate (SDS)  	  377

 15.  Single-laboratory precision of the red macroalga,
      Champia parvula, reproduction test in natural seawater
      (30%o salinity).  The reference toxicant used was copper
      (Cu) sulfate	378

 16.  Single-laboratory precision of the red macroalga,
      Champia parvula, reproduction test in natural seawater
      (30%o salinity).  The reference toxicant used was sodium
      dodecyl sulfate (SDS)   	  379
                                    xxv m

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                                ACKNOWLEDGMENTS


   The principal authors of this document are Donald Klemm, Cornelius Weber,
Philip Lewis, James Lazorchak, Florence Fulk, and Timothy Neiheisel,
Environmental Monitoring Systems Laboratory-Cincinnati, Ohio; George Morrison,
Environmental Research Laboratory, Narragansett, Rhode Island; Dennis M.
McMullen, Technology Applications Incorporated, Cincinnati, Ohio and Cathy
Poore, Computer Sciences Corporation, Cincinnati, Ohio.  Contributors to
specific sections of this manual are listed below.

1. Sections 1-10; General Guidelines                      i

   Margarete Heber, OST, Office of Water
   Donald Klemm, EMSL-Cincinnati
   James Lazorchak, EMSL-Cincinnati
   Philip Lewis, EMSL-Cincinnati
   George Morrison, ERL-Narragansett
   Teresa Norberg-King, ERL - Duluth
   William Peltier, ESD, Region 4
-   Cornelius Weber, EMSL - Cincinnati

2. Sections 11-16; Toxicity Test Methods

   Margarete Heber, OST, Office of Water
   Donald Klemm, EMSL-Cincinnati
   George Morrison, ERL-Narragansett
   William Peltier, ESD, Region 4
   Quentin Pickering, EMSL-Cincinnati

3. Data Analysis (Section 9, 11-16, and Appendices)

   Florence Fulk, EMSL-Cincinnati
   Laura Gast, Technology Applications, Inc. (TAI)
   Cathy Poore, Computer Sciences Corporation (CSC)

     Review comments from the following persons are gratefully acknowledged:
                                                          i
Pam Comeleo, Science Application International Corporation, Environmental
  Research Laboratory, U.S. Environmental Protection Agency, Narragansett, RI
Randy Comeleo, Science Application International Corporation, Environmental
  Research Laboratory, U.S. Environmental Protection Agency, Narragansett, RI
Philip Crocker, Water Quality Management Branch, U.S. Environmental
  Protection Agency, Region 6, Dallas, TX
Dan Fisher, John Hopkins University, Queenstown, MD
Terry Hollister, Environmental Services Division, Houston Branch, U.S.
  Environmental Protection Agency, Region 6, Houston, TX
Glen Modica, Science Application International Corporation, Environmental
  Research Laboratory, U.S. Environmental Protection Agency, Narragansett, RI
Michael Morton, Permits Branch, U.S. Environmental Protection Agency,
  Region 6, Dallas, TX                                    I
Peter Nolan, Environmental  Services Division, U.S. Environmental  Protection

                                     xxix

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                          ACKNOWLEDGMENTS (CONTINUED)

  Agency, Region 1, Lexington, MA
Mark Tagliabue, Science Application International Corporation, Environmental
  Research Laboratory, U.S. Environmental Protection Agency, Narragansett, RI
Glenn Thursby, Environmental Research Laboratory, U.S. Environmental
  Protection Agency, Narragansett, Rhode Island
Jerry Smrchek, Office of  Pesticides and Toxic Substances, Environmental
Effects Branch, Health and. Environmental Review Division, U.S. Environmental\
  Protection Agency, Washington, DC
Amy Wagner, Laboratory Support Section, U.S. Environmental Protection
  Agency, San Francisco,  CA
Audrey Weber, Virginia Water Control Board, Glen Allen, VA

    Many useful public comments on the first edition of the marine and
estuarine toxicity test methods "Short-term Methods for Estimating the Chronic
Toxicity of Effluents and  Receiving Waters to Freshwater Organisms, EPA/600/4-
87/028 (USEPA, 1988a) were received in response to the proposed rule,
published in the Federal  Register, December 4, 1989 [FR 54(231):50216-§0224],
regarding the Agency's intent to include short-term chronic toxicity tests in
Table IA, 40 CFR Part 136. These comments were carefully considered in
preparation of the second  edition of the manual, and are included  in the
Public Docket for the rulemaking, located at room 2904, USEPA Headquarters,
Washington, D.C.

   Materials in this manual were taken in part from the following  sources:
Methods for Acute Toxicity Tests with Fish, Macroinvertebrates, and
Amphibians, Environmental  Research Laboratory, U.S. Environmental  Protection
Agency, Duluth, Minnesota, EPA-660/3-75/009 (USEPA, 1975); Handbook for
Analytical Quality Control in Water and Wastewater Laboratories, Environmental
Monitoring and Support Laboratory - Cincinnati, U.S. Environmental Protection
Agency, Cincinnati, Ohio,  EPA-600/4-79/019 (USEPA, 1979a); Methods for
chemical analysis of water and wastes, Environmental Monitoring and Support
Laboratory-Cincinnati, U.  S. Environmental Protection Agency, Cincinnati, OH,
EPA-600/4-79-020 (USEPA,  1979b); Interim NPDES Compliance Biomonitoring
Inspection Manual, Enforcement Division, Office of Water Enforcement, U.S.
Environmental Protection  Agency, Washington, D.C. (USEPA, 1979c);  Methods for
Measuring the Acute Toxicity of Effluents to  Freshwater and Marine Organisms,
Environmental Monitoring  and Support Laboratory - Cincinnati, U.S.
Environmental Protection  Agency, Cincinnati, Ohio, EPA-600/4-85/013 (USEPA,
1985a); Short-term Methods for Estimating the Chronic Toxicity of  Effluents
and Receiving Waters to Freshwater Organisms, Environmental Monitoring and
Support Laboratory - Cincinnati, U.S. Environmental Protection Agency,
Cincinnati, Ohio, EPA-600/4-85/014 (USEPA, 1985b); Users Guide to  the Conduct
and Interpretation of Complex Effluent Toxicity Tests at Estuarine/Marine
Sites, Environmental Research Laboratory-Narragansett (ERL-N), U.S.
Environmental Protection  Agency, Narragansett, Rhode Island (USEPA, 1987a);
NPDES Compliance Inspection Manual, Office of Water Enforcement and Permits
(EN-338), U.S. Environmental Protection Agency, Washington, D.C. (USEPA,
1988b); Short-term Methods for Estimating the Chronic Toxicity of  Effluents
and Receiving Waters to Marine and Estuarine Organisms, Environmental
Monitoring and Support Laboratory-Cincinnati, U.S. Environmental Protection

                                      xxx

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                          ACKNOWLEDGMENTS (CONTINUED)

Agency, Cincinnati, Ohio, EPA-600/4-87/028 (USEPA, 1988a); Short-term Methods
for Estimating the Chronic Toxicity of Effluents and Receiving Waters to
Freshwater Organisms, Environmental Monitoring Systems'Laboratory-Cincinnati,
U.S. Environmental Protection Agency, Cincinnati, Ohio, EPA/600/4-89/001
(USEPA, 1989a); Methods for Measuring the Acute Toxicity of Effluents to
Freshwater and Marine Organisms, Environmental Monitoring Systems Laboratory-
Cincinnati, U.S. Environmental Protection Agency, Cincinnati, Ohio, EPA/600/4-
90/027F (USEPA, 1993a); Short-term Methods for Estimating the Chronic Toxicity
of Effluents and Receiving Waters to Freshwater Organisms (Third Edition),
Environmental Monitoring Systems Laboratory-Cincinnati, U.S. Environmental
Protection Agency, Cincinnati, Ohio, EPA/600/4-91/002  (USEPA, 1993b); and the
Technical Support Document for Water Quality-based Toxic Control, Office of
Water Enforcement and Permits and Office of Water Regulations and Standards,
U.S. Environmental Protection Agency, Washington, D.C., EPA/505/2-90-001
(USEPA, 1991a).

   Five of the six methods in the manual were adapted  from methods developed
at the Environmental Research Laboratory-Narragansett.  Individuals
responsible for specific methods are as follows:  Melissa Hughes, Margarete
Heber, and Walter Berry, Science Applications International Corporation
(SAIC), and Steven Schimmel, USEPA, developed the sheepshead minnow larval
survival and growth test; Margarete Heber and Melissa  Hughes, SAIC, Steven
Schimmel, USEPA, and David Bengtson, University of Rhode Island, developed the
inland silverside, Mem'dia beryllina, larval survival  and growth test; Suzanne
Lussier, USEPA, Anne Kuhn and John Sewall, SAIC, developed the mysid,
Mysidopsia bahia, survival, growth, and fecundity test; Diane Nacci and
Raymond Walsh, SAIC, and Eugene Jackim, USEPA, developed the sea urchin,
Arbacia punctulata, fertilization test; and Glen Thursby, University of Rhode
Island, and Richard Steele, USEPA, developed the red macroalga, Champia
parvula, reproduction test.

   Terry Hollister, U.S. Environmental Protection Agency, Region 6, Houston,
Texas, adapted the sheepshead minnow, Cyprinodon van'egatus, embryo-larval
survival and teratogenicity test from the fathead minnow, ipimephales promelas,
embryo-larval test in EPA/660/4-85/014 (USEPA, 1985b).

   Debbie Hall, Bioassessment and Ecotoxicology Branch, and Mary Sullivan,
Quality Assurance Research Division, provided valuable secretarial assistance,
and Betty Thomas, Technical Information Manager, EMSL-Cincinnati, provided an
editorial review.
                                     xxxi

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                                    SECTION 1

                                  INTRODUCTION

                                                          j
 1.1  This manual  describes chronic toxicity tests for use in the National
 Pollutant Discharge Elimination System (NPDES) Permits Program to identify
 effluents and receiving waters containing toxic materials in chronically toxic
 concentrations.   The methods included in this manual are referenced in Table
 IA, 40 CFR Part  136 regulations and, therefore, constitute approved methods
 for chronic toxicity tests.   They are also suitable for determining the
 toxicity of specific compounds contained in discharges.  The tests may be
 conducted in a central  laboratory or on-site, by the regulatory agency or the
 permittee.
                                                          i

 1.2  The data are used  for NPDES permits development and to determine
 compliance  with  permit  toxicity limits.   Data can also be used to predict
 potential  acute  and chronic  toxicity in  the receiving water,  based on the
 LC50,  NOEC,  IC25,  or IC50 (see Section 9,  Chronic Toxicity Test Endpoints and
 Data Analysis) and appropriate dilution,  application,  and persistence factors.
 The tests  are performed as a part of self-monitoring permit requirements,
 compliance  biomonitoring inspections,  toxics  sampling inspections,  and special
 investigations.   Data from chronic  toxicity tests performied as part  of permit
 requirements are  evaluated during compliance  evaluation inspections  and
 performance  audit  inspections.

 1.3  Modifications of these  tests are  also used in toxicity reduction
 evaluations  and toxicity identification  evaluations  to identify the  toxic
 components of an  effluent, to  aid in the development and  implementation of
 toxicity reduction plans,  and  to  compare and  control  the  effectiveness of
 various  treatment  technologies  for  a given type of industry,  irrespective of
 the receiving water (USEPA,  1988c;  USEPA,  1989b;  USEPA,  1989c;  USEPA  1989d*
 USEPA, 1989e; USEPA,  1991a;  USEPA,  1991b;  and  USEPA,  1992).

 1.4 This methods  manual  serves as  a companion  to  the  acute toxicity test
 methods  for  freshwater  and marine organisms  (USEPA,  1993a), the  short-term
 chronic  toxicity test methods for freshwater  organisms  (USEPA,  1993b),  and the
 manual for evaluation of laboratories  performing  aquatic  toxicity tests
 (USEPA,  1991c).

 1.5  Guidance for  the implementation of toxicity tests  in the  NPDES  program  is
 provided in  the Technical Support Document for  Water Quality-Based Toxics
 Control  (USEPA, 1991a).

 1.6  These marine  and estuarine short-term toxicity tests are  similar  to  those
 developed for the  freshwater organisms to  evaluate the toxicity of effluents
 discharged to estuarine  and coastal marine waters  under the NPDES permit
 program.  Methods  are presented in this manual  for five species from four
 phylogenetic groups.  Five of the six methods were developed and extensively
 field tested by Environmental Research Laboratory-Narragansett (ERL-N).  The
methods vary  in duration from one hour and 20 minutes to nine days.

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1.7  The five species for which toxicity test methods are provided are:  the
sheepshead minnow, Cyprinodon variegatus; the inland silverside, Mem'dia
beryllina-, the mysid, Mysidopsis bahia; the sea urchin, Arbacia punctulata;
and the red macroalga, Champia parvula.

1.7.1  The tests included in this document are based on the following methods:

   1. "Guidance manual for conducting complex effluent and receiving water
      larval fish growth/survival studies with the sheepshead minnow,
      Cyprinodon variegatus," by Melissa M. Hughes, Margarete A. Heber, Steven
      C. Schimmel and Walter J. Berry,  1987, Contribution No. X104,
      Environmental  Research Laboratory, U.S. Environmental Protection Agency,
      Narragansett,  RI (USEPA, 1987b).

   2. "Guidance manual for rapid chronic toxicity test on effluents and
      receiving waters with larval  inland silversides, Mem'dia  beryllina,1  by
      Margarete A. Heber, Melissa M. Hughes, Steven C. Schimmel, and David
      Bengtson, 1987, Contribution  No.  792, Environmental Research Laboratory,
      U.S.  Environmental  Protection Agency, Narragansett, RI  (USEPA, 1987c).

   3. "Guidance manual for conducting  seven-day, mysid survival/growth/
      reproduction study  using the  estuarine mysid, Mysidopsis  bahia," by
      Suzanne M.  Lussier, Anne Kuhn, and John Sewall,  1987, Contribution  No.
      X106,  Environmental Research  Laboratory, U.S. Environmental  Protection
      Agency, Narragansett, RI  (USEPA,  1987d).

   4. "Guidance manual for conducting  sperm cell tests with the sea urchin,
      Arbacia punctulata, for  use  in testing complex  effluents," by Diane E.
      Nacci,  Raymond Walsh, and  Eugene Jackim,  1987,  Contribution  No.  X105,
      Environmental  Research  Laboratory, U.S.  Environmental Protection Agency,
      Narragansett,  RI  (USEPA,  1987e).

   5.  "Guidance manual for conducting  sexual reproduction tests with the
      marine macroalga,  Champia  parvula,  for use in  testing complex
       effluents," by Glenn B.  Thursby  and  Richard  L.  Steele,  1987,
       Contribution No. X103,  Environmental  Research  Laboratory, U.S.
       Environmental  Protection Agency, Narragansett,  RI  (USEPA, 1987f).

    6.  A nine-day, sheepshead  minnow,  Cyprinodon variegatus,  static-renewal,
       embryo-larval  survival  and teratogenicity test, developed by Terry
       Hollister,  USEPA,  Region 6,  Houston,  TX.

 1.7.2  Four of the methods  incorporate the chronic endpoints  of growth or
 reproduction (or both)  in addition to lethality.  The sheepshead minnow 9-day
 embryo-larval  survival  and teratogenicity test incorporates teratogenic
 effects in addition to lethality.   The sea urchin sperm cell  test uses
 fertilization as an endpoint and has the advantage of an extremely short
 exposure period (1 h and 20 min).

 1.8  The validity of the marine/estuarine methods in predicting adverse
 ecological impacts of toxic discharges was demonstrated in field studies
 (USEPA, 1986d).

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1.9  The use of any test species or test conditions other than those described
in the methods summary tables in this manual shall be subject to application
and approval of alternate test procedures under 40 CFR 136.4 and 40 CFR 136.5.

1.10  These methods are restricted to use by or under the supervision of
analysts experienced in the use or conduct of aquatic toxicity testing and the
interpretation of data from aquatic toxicity testing.  Each analyst must
demonstrate the ability to generate acceptable test results with these methods
using the procedures described in this methods manual.

/MCCD The manual  was PrePared in the established EMSL-Cincinnati format
(UotrA,  1983).                                            i

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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
endpoints that have been considered in tests to determine the adverse effects
of toxicants include death and survival, decreased reproduction and growth,
locomotor activity, gill ventilation rate, heart rate, blood chemistry,
histopathology, enzyme activity, olfactory function, and terata.  Since it is
not feasible to detect and/or measure all of these (and other possible)
effects of toxic substances on a routine basis, observations in toxicity tests
generally have been limited to only a few effects, such as mortality, growth,
and reproduction.

2.1.2  Acute lethality is an obvious and easily observed effect which accounts
for its wide use in the early period of evaluation of the toxicity of pure
compounds and complex effluents.  The results of these tests were usually
expressed as the concentration lethal to 50% of the test organisms (LC50) over
relatively short exposure periods  (one-to-four days).

2.1.3  As exposure periods of acute tests were lengthened, the LC50 and lethal
threshold concentration were observed to decline for many compounds.  By
lengthening the tests to include one or more complete life cycles and
observing the more subtle effects  of the toxicants, such as a reduction in
growth and reproduction, more accurate, direct, estimates of the threshold or
safe concentration of the toxicant could be obtained.  However, laboratory
life cycle tests may not accurately estimate the "safe" concentration of
toxicants because they are conducted with a limited number of species under
highly controlled, steady state  conditions, and the results do not include the
effects of the stresses to which the organisms would ordinarily be exposed in
the natural environment.

2.1.4  An early published account  of a  full life cycle, fish toxicity test was
that of Mount  and Stephan (1967).   In this  study,  fathead minnows, Pimephales
promelas, were exposed to a graded series of pesticide concentrations
throughout their life cycle, and the effects of the toxicant on survival,
growth, and reproduction were measured  and  evaluated.  This work was soon
followed  by full life cycle tests  using other  toxicants and fish species.

2.1.5  McKim  (1977)  evaluated the  data  from 56 full life cycle  tests,  32  of
which  used the fathead minnow,  Pimephales promelas, and concluded  that  the
embryo-larval  and  early juvenile life stages were  the most  sensitive stages.
He proposed the  use  of  partial  life  cycle  toxicity tests with the  early life
stages  (ELS)  of  fish to establish  water quality criteria.

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2.1.6  Macek and Sleight  (1977) found that exposure of critical life stages of
fish to toxicants provides estimates of chronically safe concentrations
remarkably similar to those derived from full life cycle toxicity tests.  They
reported that "for a great majority of toxicants, the concentration which will
not be acutely toxic to the most sensitive life stages is the chronically safe
concentration for fish, and that the most sensitive life stages are the
embryos and fry."  Critical life stage exposure was considered to be exposure
of the embryos during most, preferably all, of the embryogenic (incubation)
period, and exposure of the fry for 30 days post-hatch for warm water 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).
                                                          i
2.1.8  Published reports  of the results of ELS tests indicate that the
relative sensitivity of growth and survival as endpoints may be species
dependent, toxicant dependent, or both.  Ward and Parrish (1980)  examined the
literature on ELS tests that used embryos and juveniles of the sheepshead
minnow, Cyprinodon variegatus, and found that growth was not a statistically
sensitive indicator of toxicity in 16 of 18 tests.  They suggested that the
ELS tests be shortened to. 14 days posthatch and that growth be eliminated as
an indicator of toxic effects.
                        .
2.1.9  In a review of the literature on 173 fish full  life-cycle and ELS tests
performed to determine the chronically safe concentrations of a wide variety
of toxicants, such as metals,  pesticides,  organics,  inorganics,  detergents,
and complex effluents, 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.
                                                          j
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.,

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

2.1.11  The similarity of estimates of chronically safe concentrations of
toxicants derived from short-term, embryo-larval survival and teratogenicity
tests to those derived from full life-cycle tests has been demonstrated by
Birge et al. (1981), Birge and Cassidy (1983), and Birge et al. (1985).

2.1.12  Use of a seven-day, fathead minnow, Pimephales promelas, larval
survival and growth test was first proposed by Norberg and Mount at the 1983
annual meeting of the Society for Environmental Toxicology and Chemistry
(Norberg and Mount, 1983).  This test was subsequently used by Mount and
associates in field demonstrations at Lima, Ohio (USEPA, 1984), and at many
other locations (USEPA, 1985c, USEPA, 1985d; USEPA, 1985e; USEPA, 1986a;
USEPA, 1986b; USEPA, 1986c; USEPA, 1986d).  Growth was frequently found to be
more sensitive than survival in determining the effects of complex effluents.

2.1.13  Norberg and Mount  (1985) performed three single toxicant fathead
minnow larval growth tests with zinc, copper, and DURSBAN®, using dilution
water from Lake Superior.  The results were comparable to, and had confidence
intervals that overlapped with, chronic values reported  in the literature for
both ELS and full life-cycle tests.

2.1.14  USEPA (1987b) and USEPA (1987c) adapted the fathead minnow larval
growth and survival test for use with the sheepshead  minnow and the inland
silverside, respectively.  When daily renewal 7-day sheepshead minnow  larval
growth and survival tests  and 28-day  ELS tests were performed with industrial
and municipal effluents, growth was more sensitive than  survival in seven out
of 12 larval growth and survival tests, equally sensitive  in  four tests,  and
less  sensitive in only one test.   In  four cases, the  ELS test may have been
three to  10 times more sensitive to effluents than the larval growth and
survival  test.  In  tests using  copper, the No Observable Effect Concentrations
(NOECs) were the same for  both  types  of test, and growth was  the most
sensitive  endpoint  for both.   In  a  four laboratory comparison,  six of  seven
tests produced identical NOECs  for  survival  and growth  (USEPA,  1987a). Data
indicate  that the  inland silverside  is at least equally  sensitive or more
sensitive  to effluents and single  compounds  than the  sheepshead minnow, and
can  be  tested over  a wider salinity range, 5-30%>  (USEPA,  1987a).

2.1.15   Lussier et  al.  (1985)  and  USEPA  (1987e) determined that  survival  and
growth  are often as sensitive  as  reproduction  in 28-day  life-cycle tests  with
the  mysid, Mysidopsis bahia.

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  (r  = 0.85).   However,  the results of the  fertilization  test with the
five metals  did not correlate  well  with  the  results  from the  acute tests.

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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 parvuTa, sexual reproduction test.  Nine single compounds were used to
compare the effects on sexual reproduction using a two-week exposure and a
two-day exposure.  For six of the nine compounds tested, the chronic values
were the same for both tests.

2.1.18  The use of short-term toxicity tests in the NPDES Program is
especially attractive because they provide a more direct estimate of the safe
concentrations of effluents in receiving waters than was provided by acute
toxicity tests, at an only slightly increased level of effort, compared to the
fish full life-cycle chronic and 28-day ELS tests and the 28-day mysid life-
cycle test.                                              ;

2.2  TYPES OF TESTS

2.2.1  The selection of the test type will depend on the NPDES permit
requirements, the objectives of the test, the available resources, the
requirements of the test organisms, and effluent characteristics such as
fluctuations in effluent toxicity.

2.2.2  Effluent chronic toxicity is generally measured using a multi-
concentration, or definitive test, consisting of a control  and a minimum of
five effluent concentrations.  The tests are designed to provide dose-response
information, expressed as the percent effluent concentration that affects the
hatchability, gross morphological abnormalities, survival,  growth, and/or
reproduction within the prescribed period of time  (one hour and 20 minutes to
nine days).  The results of the tests are expressed in terms of either the
highest concentration that has no statistically significant observed effect on
those responses when compared to the controls or the estimated concentration
that causes a specified percent reduction in responses versus the controls.

2.2.3  Use of pass/fail tests consisting of a single effluent concentration
(e.g., the receiving water concentration or RWC) and a control is not
recommended.  If the NPDES permit has a whole effluent toxicity limit for
acute toxicity at the RWC, it is prudent to use that permit limit as the
midpoint of a series of five effluent concentrations.  This will ensure that
there  is sufficient  information on the dose-response relationship.  For
example, the effluent concentrations utilized in a test may be:
(1)  100% effluent,  (2) (RWC + 100)/2, (3) RWC,  (4) RWC/2, and (5) RWC/4.  More
specifically, if the RWC = 50%, the effluent concentrations used in the
toxicity test would  be 100%, 75%, 50%, 25%, and 12.5%.

2.2.4  Receiving (ambient) water toxicity tests commonly employ two
treatments, a control  and the undiluted receiving water, but may also consist
of  a series of receiving water dilutions.

2.2.5  A negative result from a chronic toxicity test does not preclude the
presence of toxicity.  Also, because of the potential temporal variability in
the toxicity of  effluents, a negative test result with a particular sample

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does not preclude the possibility that samples collected at some other time
might exhibit chronic toxicity.

2.2.6  The frequency with which chronic toxicity tests are conducted under a
given NPDES permit is determined by the regulatory agency on the basis of
factors such as the variability and degree of toxicity of the'waste,
production schedules, and process changes.

2.2.7  Tests recommended for use in this methods manual may be static non-
renewal or static renewal.  Individual methods specify which static type of
test is to be conducted.

2.3  STATIC TESTS

2.3.1  Static non-renewal tests - The test organisms are exposed to the same
test solution for the duration of the test.

2.3.2  Static-renewal tests - The test organisms are exposed to a fresh
solution of the same concentration of sample every 24 h or other prescribed
interval, either by transferring the test organisms from one test chamber to
another, or by replacing all or a portion of solution in the test chambers.

2.4  ADVANTAGES AND DISADVANTAGES OF TOXICITY TEST TYPES

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

   Advantages:

   1. Simple and inexpensive
   2. Very cost effective in determining compliance with permit conditions.
   3. Limited resources (space, manpower, equipment) required; would permit
      staff to perform many more tests in the same amount of time.
   4. Smaller volume of effluent required than for static renewal or flow-
      through tests.

   Disadvantages:

   1. Dissolved oxygen (DO) depletion may result from high chemical oxygen
      demand (COD), biological oxygen demand (BOD), or metabolic wastes.
   2. Possible loss of toxicants through volatilization and/or adsorption to
      the exposure vessels.
   3. Generally less sensitive than static renewal because the toxic
      substances may degrade or be adsorbed, thereby reducing the apparent
      toxicity.  Also, there is less chance of detecting slugs of toxic
      wastes, or other temporal variations in waste properties.

2.4.2  STATIC RENEWAL, SHORT-TERM TOXICITY TESTS:

   Advantages:

   1. Reduced possibility of DO depletion from high COD and/or BOD, or ill
      effects from metabolic wastes from organisms in the test solutions.

                                      8

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2. Reduced possibility of loss of toxicants through volatilization and/or
   adsorption to the exposure vessels.
3. Test organisms that rapidly deplete energy reserve:; 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

3.2.2.1  Each laboratory (including mobile laboratories) should be provided
with safety equipment such as first aid kits, fire extinguishers, fire
blankets, emergency showers, chemical spill clean-up kits, and eye fountains.

3.2.2.2  Mobile laboratories should be equipped with a telephone to enable
personnel to summon help in case of emergency.

3.3  GENERAL LABORATORY AND FIELD OPERATIONS

3.3.1  Work with effluents should be performed in compliance with accepted
rules pertaining to the handling of hazardous materials (see safety manuals

                                      10

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listed in Section 3, Health and Safety, Subsection 3.5).  It is recommended
that personnel collecting samples and performing toxicity tests 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.

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                                      j

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.


                                       11

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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 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).
                                      12

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 4.3  TEST ORGANISMS

 4.3.1  The test organisms used in the procedures described in this manual  are
 the sheepshead minnow,  Cypn'nodon van'egatus; the inland silverside, Menidia
 beryTlina; the mysid,  Mysidopsis bahia; the sea urchin,  Arbacia punctulata-,
•and the red macroalga,  Champia parvula.  The organisms used should be disease-
 free and appear healthy,  behave normally, feed well,  and Have low mortality in
 cultures, during holding, and in test control.  Test  organisms should be
 positively identified  to  species (see Section 6, Test Organisms).

 4.4  LABORATORY WATER  USED FOR CULTURING AND TEST DILUTION WATER

 4.4.1  The quality of  water used for test organism culturing and for dilution
 water used in toxicity tests is extremely important.   Water for these two  uses
 should come from the same source.  The dilution water used in effluent
 toxicity 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%o)
 prepared from natural  seawater, or artificial seawater prepared from
 commercial sea salts,  such as FORTY FATHOMS® or HW MARINEMIX®, if recommended
 in the method.  GP2  synthetic seawater, made from reagent grade chemical salts
 (30%o) in conjunction  with natural  seawater, may also be used if recommended.
 Hypersaline brine and  artificial  seawater can be used with Champia parvula
 only if they are accompanied by at least 50% natural  seawater.  Types of water
 are discussed in Section  5,  Facilities, Equipment,  and Supplies.  Water used
 for culturing and test  dilution water should be analyzed for toxic metals  and
 organics at least annually or whenever difficulty is  encountered in meeting
 minimum acceptability  criteria for control  survival and  reproduction or
 growth.  The concentration of the metals, Al, As,  Cr,  Co,  Cu,  Fe, Pb,  Ni,  Zn,
 expressed as total  metal, should not exceed 1 /jg/L each,  and Cd, Hg, and Ag,
 expressed as total  metal, should not exceed 100 ng/L  each.   Total
 organochlorine pesticides plus PCBs should be less than  50 ng/L (APHA,  1992).
 Pesticide concentrations  should not exceed USEPA's National  Ambient Water
 Quality chronic criteria  values where available.         ,

 4.5  EFFLUENT AND RECEIVING  WATER SAMPLING AND HANDLING

 4.5.1  Sample holding  times  and temperatures of effluent samples collected for
 on-site and off-site testing must conform to conditions  described in
 Section 8, Effluent  and Receiving Water Sampling,  Sample Handling,  and  Sample
 Preparation for Toxicity  Tests.

 4.6  TEST CONDITIONS
                                                          i
                                                          I
 4.6.1  Water temperature  and salinity must  be maintained within the limits
 specified for each test.   The temperature ofttest  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

                                                          j
                                       13

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specified for each test.  DO concentrations and pH should be checked at the
beginning of the test and daily throughout the test period.

4.7  QUALITY OF TEST ORGANISMS

4.7.1  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.

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.

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 quality of the toxicity test
data.  This is especially true for the unsaturated fatty acid content of brine
shrimp nauplii, Artemia.  Problems with the nutritional suitability of the
food will be reflected in the survival, growth, and reproduction of the test
organisms in cultures and toxicity tests.  Artemia cysts and other foods must
be obtained as described in Section 5, Facilities, Equipment, and Supplies.

4.8.2  Problems with the nutritional suitability of food will be reflected  in
the survival, growth, and reproduction of the test organisms in cultures and
toxicity tests.  If a batch of food is suspected to be defective, the
performance of organisms fed with the new food can be compared with the

                                      14

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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.
                                                         i
4.8.3  New batches of food used in culturing and testing should be analyzed
for toxic organics and metals or whenever difficulty is encountered in meeting
minimum acceptability criteria for control survival and reproduction or
growth.  If the concentration of total organochlorine pesticides exceeds
0.15 fj,g/g wet weight, or the concentration of total organochlorine pesticides
plus PCBs exceeds 0.30 ^g/g wet weight, or toxic metals (Al, As, Cr, Cd, Cu,
Pb, Ni, Zn, expressed as total metal) exceed 20 /j.g/g wet weight, the food
should not be used (for analytical methods, see AOAC, 1990; and USDA, 1989).

For foods (e.g., YCT) which are used to culture and test organisms, the
quality of the food should meet the requirements for the laboratory water used
for culturing and test dilution water as described in Section 4.4 above.

4.9  ACCEPTABILITY OF CHRONIC TOXICITY TESTS
                                                         i
4.9.1  The results of the sheepshead minnow, Cyprinodon variegatus, inland
silverside, Mem'dia beryl Una, or mysid, Mysidopsis bahia,,  tests are
acceptable if survival in the controls is 80% or greater.  The sea urchin,
Arbacia punctulata, test requires control egg fertilization equal to or
exceeding 50%.  However, greater than 90% fertilization may result in masking
toxic responses.  The red macroalga, Champia parvula, test is acceptable if
survival is 100%, and the mean number of cystocarps per plant should equal or
exceed 10.  If the sheepshead minnow, Cypn'ndon van'egatus, larval survival
and growth test is begun with less-than-24-h old larvae, the mean dry weight
of the surviving larvae in the control chambers at the end of the test should
equal or exceed 0.60 mg, if the weights are determined immediately, or 0.50 mg
if the larvae are preserved in a 4% formalin or 70% ethanol solution.  If the
inland silverside, Mem'dia beryllina, larval survival and growth test is begun
with larvae seven days old, the mean dry weight of the surviving larvae in the
control chambers at the end of the test should equal  or exceed 0.50 mg, if the
weights are determined immediately,  or 0.43 mg if the larvae are preserved in
a 4% formalin or 70% ethanol  solution.  The mean mysid dry weight of survivors
should be at least 0.20 mg.  Automatic or hourly feeding will  generally
provide control mysids with a dry weight.of 0.30 mg.   At least 50% of the
females should bear eggs at the end of the test, but  mysid fecundity is not a
factor in test acceptability.  However, fecundity must equal  or exceed 50% to
be used as an endpoint in the test.   If these criteria are not met, the test
must be repeated.
          .
4.9.2  An individual  test may be conditionally acceptable if temperature,  DO,
and other specified conditions fall  outside specification.';, depending on the
                                                         I
                                      15

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degree of the departure and the objectives of the tests (see test conditions
and test acceptability criteria summaries).  The acceptability of the test
will depend on the experience and professional judgment of the laboratory
investigator and the reviewing staff of the regulatory authority.  Any
deviation from test specifications must be noted when reporting data from a
test.

4.10  ANALYTICAL METHODS

4.10.1  Routine chemical and physical analyses for culture and dilution water,
food, and test solutions must include established quality assurance practices
outlined in USEPA methods manuals (USEPA, 1979a and USEPA, 1979b).

4.10.2  Reagent containers should be dated and catalogued when received from
the supplier, and the shelf life should not be exceeded.  Also, working
solutions should be dated when prepared, and the recommended shelf life should
be observed.

4.11  CALIBRATION AND STANDARDIZATION

4.11.1  Instruments used for routine measurements of chemical and physical
parameters, such as pH, DO, temperature, conductivity, and salinity, must be
calibrated and standardized according to instrument manufacturers procedures
as indicated in the general section on quality assurance  (see USEPA Methods
150.1, 360.1, 170.1, and 120.1 in USEPA, 1979b).  Calibration data are
recorded in a permanent log book.

4.11.2  Wet chemical methods used to measure hardness, alkalinity, and total
residual chlorine, must be standardized prior to use each day according to the
procedures for those specific USEPA methods (see USEPA Methods 130.2 and 310.1
in USEPA, 1979b).

4.12  REPLICATION AND TEST SENSITIVITY

4.12.1  The sensitivity of the tests will depend in part  on the number of
replicates per concentration, the significance level selected, and the type of
statistical analysis.   If the variability remains constant, the sensitivity of
the test will increase  as the number of replicates is increased.  The minimum
recommended number of replicates varies with the objectives of the test and
the statistical method  used for analysis of the data.

4.13  VARIABILITY IN TOXICITY TEST RESULTS

4.13.1  Factors which can affect test success and precision include:  (1) the
experience and skill of the laboratory, analyst; (2) test  organism age,
condition, and sensitivity; (3) dilution water quality; (4) temperature
control; (5) and the quality and quantity of food provided.  The results will
depend upon the species used and the strain or source of  the test organisms,
and test conditions, such as temperature, DO, food, and water quality.  The
repeatability or precision of toxicity tests is also a function of the number
of test organisms used  at each toxicant concentration.  Jensen (1972)
discussed the relationship between sample size (number of fish) and the

                                      16

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standard error of the test, and considered 20 fish per concentration as
optimum for Probit Analysis.'                             :

4.14  TEST PRECISION

4.14.1  The ability of the laboratory .personnel to obtain consistent, precise
results must be demonstrated with reference toxicants before they attempt to
measure effluent toxicity.  The single-laboratory precision of each type of
test to be used in a laboratory should be determined by performing at least
five or more tests with a reference toxicant.
        .

4.14.2  Test precision can be estimated by using the same strain of organisms
under the same test conditions, and employing a known toxicant, such as a
reference toxicant.

4.14.3  Interlaboratory precision of chronic toxicity tests using two
reference toxicants with the mysid, Mysidopsis bahia, and the inland
silverside, Mem'dia beryllina, is listed in Table 1.  Additional precision
data for each of the tests described in this manual are presented in the
sections describing the individual test methods.

4.14.4  Additional information on toxicity test precision is provided in the
Technical Support Document for Water Quality-based Toxic Control (see pp. 2-4,
and 11-15 in USEPA, 1991a).

4.14.5  In cases where the test data are used in Probit Analysis or other
point estimation techniques (see Section 9, Chronic Toxicity Test Endpoints
and Data Analysis), precision can be described by the mean, standard
deviation, and relative standard deviation (percent coefficient of variation,
or CV) of the calculated .endpoints from the replicated tests.  In cases where
the test data are used in the Linear Interpolation Method, precision can be
estimated by empirical confidence intervals derived by using the ICPIN Method
(see Section 9, Chronic Toxicity Test Endpoints and Data Analysis).  However,
in cases where the results are reported in terms of the Mo-Observed-Effect-
Concentration (NOEC) and Lowest-Observed-Effect-Concentration (LOEC) (see
Section 9, Chronic Toxicity Test Endpoints and Data Analysis), precision can
only be described by listing the NOEC-LOEC interval for each test.  It is not
possible to express precision in terms of a commonly used statistic.  However,
when all tests of the same toxicant yield the same NOEC-LOEC interval, maximum
precision has been attained.  The "true" no effect concentration could fall
anywhere within the interval, NOEC ± (LOEC minus NOEC).
                                                         I
4.14.6  It should be noted here that the dilution factor selected for a test
determines the width of the NOEC-LOEC interval and the inherent maximum
precision of the test.  As the absolute value of the dilution factor
decreases, the width of the NOEC-LOEC interval increases, and the inherent
maximum precision of the test decreases.  When a dilution factor of 0.3 is
used, the NOEC could be considered to have a relative uncertainty as high as ±
300%.  With a dilution factor of 0.5, the NOEC could be considered to have a
relative variability of ± 100%.  As a result of the variability of different
dilution factors, USEPA recommends the use of a > 0.5 dilution factor.  Other
factors which can affect test precision include:  test organism age,
condition, and sensitivity; temperature control; and feeding.
                                                         i

                                      17

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  TABLE 1.    NATIONAL INTERLABORATORY STUDY OF CHRONIC TOXICITY TEST
              PRECISION, 1991: SUMMARY OF RESPONSES USING TWO REFERENCE
              TOXICANTS1'2
      Organism
      Organism
Endpoint
No. Labs   KCl(mg/L)
SD
Endpoint
No. Labs   Cu(mg/L)
SD
CV(%)3
Hysidopsis
bahia



Survival, NOEC
Growth, IC25
Growth, IC50
Growth, NOEC
Fecundity, NOEC
34
26
22
32
25
NA
480
656
NA
NA
NA
3.47
3.17
NA
NA
NA
28.9
19.3
NA
NA
CV(%)
Nenidia
beryl 1 ina


Survival, NOEC
Growth, IC25
Growth, IC50
Growth, NOEC
19
13
12
17
NA
0.144
0.180
NA
NA
1.56
1.87
NA
NA
43.5
41.6
NA
     From a national study of inter!aboratory precision of toxicity test
     data performed in 1991 by the Environmental Monitoring Systems
     Laboratory-Cincinnati, U.S. Environmental Protection Agency,
     Cincinnati, OH 45268.  Participants included federal, state, and
     private laboratories engaged in NPDES permit compliance monitoring.
  *  Static renewal test, using 25%o modified GP2 artificial seawater.
     Percent coefficient of variation = (standard deviation X 100)/mean.
     Expressed as mean.
4.15  DEMONSTRATING ACCEPTABLE LABORATORY PERFORMANCE

4.15.1  It is a laboratory's responsibility to demonstrate its ability to
obtain consistent, precise results with reference toxicants before it performs
toxicity tests with effluents for permit compliance purposes.  To meet this
requirement, the intralaboratory precision, expressed as percent coefficient
of variation (CV%), of each type of test to be used in a laboratory should be
determined by performing five or more tests with different batches of test
organisms, using the same reference toxicant, at the same concentrations, with
the same test conditions (i.e., the same test duration, type of dilution
water, age of test organisms, feeding, etc.), and same data analysis methods.
A reference toxicant concentration series (0.5 or higher) should be selected
that will consistently provide partial mortalities at two or more
concentrations.

4.16  DOCUMENTING ONGOING LABORATORY PERFORMANCE

4.16.1  Satisfactory laboratory performance is demonstrated by performing at
least one acceptable test per month with a reference toxicant for each


                                      18

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toxicity test method commonly used in the laboratory.  For a given test
method, successive tests must be performed with the same reference toxicant,
at the same concentrations, in the same dilution water, using the same data
analysis methods,.  Precision may vary with the test species, reference
toxicant, and type of test.
                                                  *
4.16.2  A control chart should be prepared for each combination of reference
toxicant, test species, test conditions, and endpoints.  Toxicity endpoints
from five or six tests are adequate for establishing the control charts.
Successive toxicity endpoints (NOECs, IC25s, LCSOs, etc.) should be plotted
and examined to determine if the results (X,) are within prescribed limits
(Figure 1).  The types of control charts illustrated (see USEPA, 1979a) are
used to evaluate the cumulative trend of results from a series of samples.
For endpoints that are point estimates  (LCSOs and IC25s), the cumulative
mean (X) and upper and lower control limits  (± 2S) are recalculated with each
successive test result.  Endpoints from hypothesis tests (NOEC, NOAEC) from
each test are plotted directly on the control chart.  The control limits would
consist of one concentration interval above  and below the concentration
representing the central tendency.  After two years of data collection, or a
minimum of 20 data points, the control  (cusum) chart should be maintained
using only the 20 most recent data points.
                                                         I
4.16.3  The outliers, which are values  falling outside the upper and lower
control limits, and trends of increasing or  decreasing sensitivity, are
readily identified.  In the case of endpoints that are point estimates (LCBOs
and IC25s), at the Pp 05 probability level, one in 20 tests would be expected
to fall outside of the control limits by chance alone.  If more than one out
of 20 reference toxicant tests fall outside  the control limits, the effluent
toxicity tests conducted during the month in which the second reference
toxicant test failed are suspect, and should be considered as provisional and
subject to careful review.  Control limits for the NOECs will also be exceeded
occasionally, regardless of how well a  laboratory performs.

4.16.4   If the toxicity value from a given test with a reference toxicant fall
well outside the expected  range for the test organisms when using the standard
dilution water and other test conditions, the sensitivity of the organisms and
the overall credibility of the test system are suspect.  In this case, the
test procedure should  be examined for defects and should be repeated with a
different  batch  of test organisms.

4.16.5   Performance should improve with experience,  and the control limits for
endpoints  that are point estimates should gradually  narrow.  However, control
limits  of  ± 2S will be exceeded 5% of the time by chance alone, regardless of
how well a laboratory  performs.  Highly proficient laboratories which develop
very narrow control limits may be unfairly penalized if a test  result which
falls  just outside the control limits is rejected de facto.  For this reason,
the width  of the control limits should  be considered by the permitting
authority  in determining whether the outliers should be rejected.
                                       19

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                    o
                    UJ
                    O
                               UPPER CONTROL LIMIT
CENTRAL TENDENCY
                               LOWER CONTROL LIMIT
                               I ,  ,
                                   I  I  I I  I  I 1  I 1  III
                                      10
              15      20
                    O
                    O
                    HI


                    O
                            UPPER CONTROL LIMIT (X •*• 2 S)
                                CENTRAL TENDENCY
                           LOWER CONTROL LIMIT (X - 2 S)
                      0       5       10      15      20

                    TOXICITY TESTS WITH REFERENCE TOXICANTS
                                 _
                                 y =  j=i
                                        n
                          S =
                              \
                                             n
       n-1
          Where:    X± = Successive toxicity values  from  toxicity tests.



                   n  = Number of tests.



                   X  = Mean toxicity value.



                   S  = Standard deviation.


Figure 1.  Control  (cusum)  charts.  (A)  hypothesis  testing results;   (B) point

          estimates (LC, EC, or 1C).
                                     20

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4.17  REFERENCE TOXICANTS
                                                          i
                                                          I
4.17.1  Reference toxicants such as sodium chloride (NaClj, potassium chloride
(KC1), cadmium chloride (CdClJ,  copper sulfate (CuS04),  sodium dodecyl
sulfate (SDS), and potassium dichromate (K2Cr207), are suitable  for use in the
NPDES Program and other Agency programs requiring aquatic toxicity tests.
EMSL-Cincinnati 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 EMSL-Cincinnati electronic bulletin board, using a modem to access the
following telephone number: 513-569-7610.  Standard reference materials also
can be obtained from commercial supply houses, or can be prepared inhouse
using reagent grade chemicals.  The regulatory agency should be consulted
before reference toxicant(s) are selected and used.

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.
                                                          I
4.18.2  The regulatory authority should retain records pertaining to discharge
permits.  Permittees are required to retain records pertaining to permit
applications and compliance for a minimum of 3 years [40 CFR 122.41(j)(2)].

4.19  VIDEO TAPES OF USEPA CULTURE AND TOXICITY TEST METHODS

Three video-based training packages are available from National Technical
Information Service (NTIS), Department of Commerce, 5285 Port. Royal  Road,
Springfield, VA  22161.  Credit card orders can be placed by calling toll-free
(800) 788-6282, or by FAX at 703-321-8547, or by mail  at the above address.
For other information call 703-487-4650.

   1. Order # A18545:  Toxicity Test Methods for the Red nriacroalga,
      Champia parvula; the Sheepshead Minnow, Cypn'nodon van'egatus; the
      inland silverside, Mem'dia beryllina; and the Sea Urchin, Arbacia
      punctuTata.  Price $85.00.
                                      21

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   2. Order # A18657:  Mysids, Mysidopsis bahia, Culture and Toxicity Test.
      Trice $75.00.

4.20  SUPPLEMENTAL REPORTS FOR TRAINING VIDEO TAPES

4.20.1  Ordering information:  USEPA, Center for Environmental Research
Information, Cincinnati, OH 45268.

4.20.1.1  Sheepshead minnow, Cyprinodon van'egatus, and inland silverside,
Henidia beryllina, larval survival and growth toxicity tests (EPA/600/3-
90/075), 1990.

4.20.1.2  Red algae, Champia parvula, sexual reproduction (EPA/600/3-90/076),
1990.

4.20.1.3  Sperm cell test using the sea urchin, Arbacia punctulata,
(EPA/600-3-90/077), 1990.

4.20.2  Ordering information:  USEPA, Office of Water (EN-336), Washington,
D.C. 20460.

4.20.2.1  Mysids, Mysidopsis bahia, survival, growth, and fecundity test
(EPA/505/8-90-006a), 1990.
                                      22

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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 hypersaline brine derived from
natural seawater, or water made up from reagent grade chemicals (GP2) or
commercial (FORTY FATHOMS® or HW MARINEMIX®) artificial sea salts when
specifically recommended in the method.  Air used for aeration must be free of
oil and toxic vapors.  Oil-free air pumps should be used where possible.
Particulates can be removed from the air using BALSTON® Grade BX or equivalent
filters (Balston, Inc., Lexington, Massachusetts), and oil and other organic
vapors can be removed using activated carbon filters (BALSTOM®, 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.
                                                          i
5.1.3  Materials used for exposure chambers, tubing, etc., which come in
contact with the effluent and dilution water, should be carefully chosen.
Tempered glass and perfluorocarbon plastics  (TEFLON®) should be used whenever
possible to minimize sorption and leaching of toxic substances.  These
materials may be reused following decontamination.  Containers made of
plastics, such as polyethylene, polypropylene, polyvinyl chloride, TYGON®,
etc., may be used as test chambers or to ship, store, and transfer effluents
and receiving waters, but they should not be reused unless absolutely
necessary, because they might carry over adsorbed toxicants from one test to
another, if reused.  However, these containers may be repeatedly reused for
storing uncontaminated waters such as deionized or laboratory-prepared
dilution waters and receiving waters.  Glass or disposable polystyrene
containers can be used as test chambers.  The use of large (> 20 L) glass
carboys is discouraged for safety reasons.                j

5.1.4  New plastic products of a type not previously used should be tested for
toxicity before initial use by exposing the test organisms in the test system
where the material is used.  Equipment (pumps, valves, etc.) which cannot be

                                      23

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discarded after each use because of cost, must be decontaminated according to
the cleaning procedures listed below (see Section 5, Facilities, Equipment,
and Supplies, Subsection 5.3.2).  Fiberglass, in addition to the previously
mentioned materials, can be used for holding, acclimating, and dilution water
storage tanks, and in the water delivery system, but once contaminated with
pollutants the fiberglass should not be reused.  All material should be
flushed or rinsed thoroughly with the test media before using in the test.

5.1.5  Copper, galvanized material, rubber, brass, and lead must not come in
contact with culturing, holding, acclimation, or dilution water, or with
effluent samples and test solutions.  Some materials, such as several types of
neoprene rubber (commonly used for stoppers) may be toxic and should be tested
before use.

5.1.6  Silicone adhesive used to construct glass test chambers absorbs some
organochlorine and organophosphorus pesticides, which are difficult to remove.
Therefore, as little of the adhesive as possible should be in contact with
water.  Extra beads of adhesive inside the containers should be removed.

5.2  TEST CHAMBERS

5.2.1  Test chamber size and shape are varied according to size of the test
organism.  Requirements are specified in each toxicity test method.

5.3  CLEANING TEST CHAMBERS AND LABORATORY APPARATUS

5.3.1  New plasticware used for sample collection or organism exposure vessels
generally does not require thorough cleaning before use.  It is sufficient to
rinse new sample containers once with dilution water before use.  New,
disposable, plastic test chambers may have to be rinsed with dilution water
before use.   New glassware must be soaked overnight in 10% acid (see below)
and also should be rinsed well in deionized water and seawater.

5.3.2  All non-disposable sample containers, test vessels, pumps, tanks, and
other equipment that has come in contact with effluent must be washed after
use to remove surface contaminants, as described below.

   1. Soak 15 minutes in tap water and scrub with detergent, or clean in an
      automatic dishwasher.
   2. Rinse twice with tap water.
   3. Carefully rinse once with fresh dilute (10% V:V) hydrochloric acid or
      nitric acid to remove scale, metals and bases.  To prepare a 10%
      solution of acid, add 10 ml of concentrated acid to 90 ml of
      deionized water.
   4. Rinse twice with deionized water.
   5. Rinse once with full-strength, pesticide-grade acetone to remove
      organic compounds (use a fume hood or canopy).
   6. Rinse three times with deionized water.

5.3.3  All test chambers and equipment must be thoroughly rinsed with the
dilution water immediately prior to use in each test.


                                      24

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5.4  APPARATUS AND EQUIPMENT FOR CULTURIN6 AND TOXICITY TESTS

5.4.1  Apparatus and equipment requirements for culturing and toxicity tests
are specified in each toxicity test method.  Also, see USIEPA, 1993a.

5.4.2  WATER PURIFICATION SYSTEM

5.4.2.1   A good quality deionized water, providing 18 mega-ohm, laboratory
grade water, should be available in the laboratory and with sufficient
capacity for laboratory needs.  Deionized water may be obtained from
MILLIPORE® MILLI-Q®, MILLIPORE® QPAK™2 or equivalent system.  If large
quantities of high quality deionized water are needed, it may be advisable to
supply the laboratory grade water deionizer with preconditioned water from a
Culligen®,. Continental®, or equivalent.
                                                 •        ;
5.5  REAGENTS AND CONSUMABLE MATERIALS
                                                         i

5.5.1  SOURCE'S OF FOOD FOR CULTURE AND TOXICITY TESTS

   1. Brine Shrimp, Artemia sp. cysts -- A list of commercial sources is
      provided in Table 2.
   2. Frozen Adult Brine Shrimp, Artemia -- Available from most pet supply
      shops-or from San Francisco Bay Brand, 8239 Enterprise Dr., Newark, CA
      94560 (415-792-7200).
   3. Flake Food -- TETRAMIN® and BIORIL® or equivalent are available at most
      pet supply shops.
   4. Feeding requirements and other specific foods are indicated; in the
      specific toxicity test method.                             I

5.5.1.1   All food should be tested for nutritional suitability ahd chemically
analyzed for organochlorine pesticides, PCBs, and toxic metals (see Section 4,
Quality Assurance).                                              <
                                                         i
5.5.2  Reagents and consumable materials are specified in each toxicity test
method.  Also, see Section 4, Quality Assurance.

5.6  TEST ORGANISMS

5.6.1  Test organisms are obtained from inhouse cultures or commercial
suppliers (see specific toxicity test method; Sections 4, Quality Assurance
and 6, Test Organisms).

5.7  SUPPLIES
                                                         i
5.7.1  See toxicity test methods (see Sections 11-16) for specific supplies.
                                      25

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     TABLE 2.  COMMERCIAL SUPPLIERS OF BRINE SHRIMP (ARTEMIA) CYSTS1'2
Aquafauna Biomarine
P.O. Box 5
Hawthorne  CA 90250
Tel.
Fax.
(310
 310
(Great Sa
San Francisco Bay)

Argent Chemical
8702 152nd Ave. NE
Redmond, WA 98052
 973-5275
 676-9387
t Lake North Arm,
Tel.
Tel.
Fax.
 800)
 206,
 206
 426-6258
 855-3777
 885-2112
(Plat num Label - San Francisco Bay;
Gold Label - San Francisco Bay,
Brazil; Silver Label - Great
Salt Lake, Australia; Bronze
Label - China, Canada, other)

Bonneville Artemia  International, Inc.
P.O. Box 511113
Salt Lake City, UT  84151-1113
Tel. (801) 972-4704
Fax. (801) 972-4795

Ocean Star International
P.O. Box 643
Snowville, UT 84336
Tel. (801) 872-8217
Fax  (801) 872-8272
(Great Salt Lake)
Sanders Brine Shrimp Co.
3850 South 540 West
Ogden, UT 84405
Tel. (801) 393-5027
(Great Salt Lake)
Sea Critters Inc.
P.O. Box 1508
Tavernier, FL 33070
Tel. (305) 367-2672
                                       Aquarium Products
                                       180L Penrod Court
                                       Glen Burnie, MD 21061
Tel.
Fax.
Tel.
800
410
301
                                       (Columbia
368-2507
761-6458
761-2100
I.NVE Artemia Systems
Oeverstraat 7
B-9200 Baasrode, Belgium
Tel. 011-32-52-331320
Fax. 011-32-52-341205
(For marine species  - AF grade)
[smal 1 naupl i i ], UL grade [1 arge
nauplii], for freshwater species
-HI grade  [small  nauplii],  EG
[large nauplii]
                                       Golden West Artemia .
                                       411 East 100 South
                                       Salt Lake City, UT 84111
                                       Tel. (801) 975-1222
                                       Fax. (801) 975-1444

                                       San Francisco Bay Brand
                                       8239 Enterprise Drive
                                       Newark, CA 94560
                                       Tel. (510) 792-7200
                                       Fax. (510) 792-5360
                                       (Great Salt Lake,
                                        San Francisco Bay)

                                       Western Brine Shrimp
                                       957 West South Temple
                                       Salt Lake City, UT 84104
                                       Tel. (801) 364-3642
                                       Fax. (801) 534-0211
                                       (Great Salt Lake)
   List from David  A.  Bengtson,  University of Rhode Island, Narragansett,
   RI.
   The geographic  sources  from which the  vendors  obtain  the brine shrimp
   cysts are shown  in parentheses.
                                    26

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                                  SECTION  6

                                TEST ORGANISMS
6.1  TEST SPECIES
                                                         I
6.1.1 .The species used in characterizing the chronic toxicity of effluents
and/or receiving waters will depend on the requirements of the regulatory
authority and the objectives of the test.  It is essential that good quality
test organisms be readily available throughout the year from inhouse or
commercial sources to meet NPDES monitoring requirements.  The organisms used
in toxicity tests must be identified to species.  If there is any doubt as to
the identity of the test organisms, representative specimens should be sent to
a taxonomic expert to confirm the identification.

6.1.2  Toxicity test conditions and culture methods for the species listed in
Subsection 6.1.3 are provided in this manual  (also, see USEPA, 1993a).

6.1.3  The organisms used in the short-term tests described in this manual are
the sheepshead minnow, Cyprinodon variegatus; the inland silverside, Menidia
berynina; the mysid, Mysidopsis bahia; the sea urchin, Arbacia punctulata;
and the red macroalga, Champia parvuTa.
                                                         i
6.1.4  Some states have developed culturing and testing methods for indigenous
species that may be as sensitive or more sensitive, than the species
recommended in Subsection 6.1.3.  However, USEPA allows the use of indigenous
species only where state regulations require their use or prohibit importation
of the species in Section 6, Facilities, Equipment, and Supplies, Subsection
6.1.3.  Where state regulations prohibit importation of non-native fishes or
use of the recommended test species, permission must be requested from the
appropriate state agency prior to their use.

6.1.5  Where states have developed culturing and testing methods for
indigenous species other than those recommended in this manual, data comparing
the sensitivity of the substitute species and one or more of the recommended
species must be obtained in side-by-side toxicity tests with reference
toxicants and/or effluents, to ensure that the species selected are at least
as sensitive as the recommended species.  These data must be submitted to the
permitting authority (State or Region) if required.  USEPA acknowledges that
reference toxicants prepared from pure chemicals may not always be
representative of effluents.  However, because of the observed and/or
potential variability in the quality and toxicity of effluents, it is not
possible to specify a representative effluent.

6.1.6  Guidance for the selection of test organisms where the salinity of the
effluent and/or receiving water requires special consideration is provided in
the Technical Support Document for Water Quality-based Toxics Control (USEPA,
1991a).
                                            ,
   1. Where the salinity of the receiving water is < l%o, freshwater organisms
      are used regardless of the salinity of the effluent.
                                                         i
                                      27

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   2. Where the salinity of the receiving water is > l%o, the choice of
      organisms depends on state water quality standards and/or permit
      requirements.

6.2   SOURCES OF TEST ORGANISMS

6.2.1  The test organisms recommended in this manual can be cultured in the
laboratory using culturing and handling methods for each organism described in
the respective test method sections.  Also, see USEPA (1993a).

6.2.2  Inhouse cultures should be established wherever it is cost effective.
If inhouse cultures cannot be maintained or it is not cost effective, test
organisms should be purchased from experienced commercial suppliers (see
USEPA, 1993c).

6.2.3  Sheepshead minnows, inland silversides, mysids, and sea urchins may be
purchased from commercial suppliers.  However, some of these organisms (e.g.,
adult sheepshead minnows or adult inland silversides) may not always be
available from commercial suppliers and may have to be collected in the field
and brought back to the laboratory for spawning to obtain eggs and larvae.

6.2.4  If, because of their source, there is any uncertainty concerning the
identity of the organisms, it is advisable to have them examined by a
taxonomic specialist to confirm their identification.  For detailed guidance
on identification, see the individual toxicity test methods.

6.2.5  FERAL (NATURAL OCCURRING, WILD CAUGHT) ORGANISMS

6.2.5.1  The use of test organisms taken from the receiving water has strong
appeal, and would seem to be the logical approach.  However, it is generally
impractical and not recommended for the following reasons:

   1. Sensitive organisms may not be present in the receiving water because of
      previous exposure to the effluent or other pollutants.
   2. It is often difficult to collect organisms of the required age and
      quality from the receiving water.
   3. Most states require collection permits, which may be difficult to
      obtain.  Therefore, it is usually more cost effective to culture the
      organisms in the laboratory or obtain them from private, state, or
      Federal sources.  Fish such as sheepshead minnows and silversides, and
      invertebrates such as mysids, are easily reared in the laboratory or
      purchased.
   4. The required QA/QC records, such as the single-laboratory precision
      data, would not be available.
   5. Since it is mandatory that the identity of test organisms is known to
      the species level, it would be necessary to examine each organism caught
      in the wild to confirm its identity, which would usually be impractical
      or, at the least, very stressful to the organisms.
   6. Test organisms obtained from the wild must be observed in the
      laboratory for a minimum of one week prior to use, to ensure that they
      are free of signs of parasitic or bacterial infections and other adverse


                                      28

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

6.2.5.2  Guidelines for collection of natural occurring organisms are provided
in USEPA (1973); USEPA (1990a); and USEPA (1993c).

6.2.6  Regardless of their source, test organisms should be carefully observed
to ensure that they are free of, signs of stress and disease, and in good
physical condition.  Some species of test organisms, such as trout, can be
obtained from stocks certified as "disease-free."         |

6.3  LIFE STAGE                                           |

6.3.1  Young organisms are often more sensitive to toxicants than are adults.
For this reason, the use of early life stages, such as juvenile mysids and
larval fish, is required for all tests.  There may be special cases, however,
where the limited availability of organisms will require some deviation from
the recommended life stage.  In a given test, all organisms should be
approximately the same age and should be taken from the same source.  Since
age may affect the results of the tests, it would enhance the value and
comparability of the data if the same species in the same^life stages were
used throughout a monitoring program at a given facility.!

6.4  LABORATORY CULTURING

6.4.1   Instructions for culturing and/or holding the recommended test
organisms are included in specified test methods  (also, see USEPA, 1993a).

6.5  HOLDING AND HANDLING TEST ORGANISMS

6.5.1   Test organisms should not be subjected to changes of more than 3°C in
water temperature or 3%o in salinity in any  12 h period.

6.5.2   Organisms should be handled as little  as possible.  When handling is
necessary,  it should be done as gently, carefully,  and quickly as .possible to
minimize stress.  Organisms that  are dropped  or touch dry surfaces or are
injured during  handling must be discarded.   Dipnets are best for handling
larger  organisms.  These nets  are commercially available or can be made from
small-mesh  nylon netting, silk bolting  cloth, plankton netting, or similar
material. Wide-bore, smooth glass tubes  (4 to 8 mm  ID) with rubber bulbs or
pipettors (such  as  a PROPIPETTE®  or other pipettor) should  be used for
transferring  smaller organisms such as  mysids, and  larval fish.

6.5.3   Holding  tanks for fish  are supplied with a good quality water  (see
Section 5,  Facilities, Equipment, and Supplies) with a flow-through rate of  at
least  two tank-volumes per day.   Otherwise,  use a recirculation system where
the  water flows  through  an activated carbon  or undergravel  filter  to  remove
dissolved metabolites.   Culture water can also be piped through high  intensity
ultraviolet light  sources for  disinfection,  and to  photo-degrade dissolved
organics.
                                       29

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 6.5.4   Crowding  should  be  avoided  because  it  will  stress  the  organisms  and
 lower  the  DO  concentrations  to  unacceptable levels.   The  DO must  be  maintained
 at  a minimum  of  4.0  mg/L.  The  solubility  of  oxygen  depends on  temperature,
 salinity,  and altitude.  Aerate gently  if  necessary.

 6.5.5   The organisms should  be  observed carefully  each day for  signs of
 disease, stress,  physical  damage,  or  mortality.  Dead and abnormal organisms
 should be  removed as soon  as observed.   It is not  uncommon for  some  fish
 mortality  (5-10%)  to occur during  the first 48 h in  a holding tank because of
 individuals that  refuse  to feed on artificial  food and die of starvation.
 Organisms  in  the  holding tanks  should generally be fed as in the  cultures  (see
'culturing  methods in the respective methods).

 6.5.6   Fish should be fed  as much  as  they  will eat at least once  a day  with
 live brine shrimp nauplii, Artemia, or  frozen adult  brine shrimp  or  dry food
 (frozen food  should  be completely  thawed before use).  Adult brine shrimp can
 be  supplemented with commercially  prepared food such  as TETRAMIN® or BIORIL®
 flake  food, or equivalent.   Excess food and fecal material should be removed
 from the bottom of the tanks at least twice a week by siphoning.

 6.5.7   Fish should be observed  carefully each day for signs of  disease,
 stress, physical  damage, and mortality.  Dead and abnormal specimens  should be
 removed as soon as observed.  It is not uncommon to  have  some fish (5-10%)
 mortality  during  the first 48 h in a  holding  tank because of individuals that
 refuse to  feed on  artificial  food  and die  of  starvation.  Fish  in the holding
 tanks  should  generally be  fed as in the cultures (see culturing methods in the
 respective methods).

 6.5.8   A daily record of feeding,  behavioral  observations, and mortality
 should be  maintained.

 6.6  TRANSPORTATION  TO THE TEST SITE

 6.6.1   Organisms  are transported from the  base or supply  laboratory  to  a
 remote test site  in  culture  water  or  standard  dilution water in plastic bags
 or large-mouth screw-cap (500 ml)  plastic  bottles in  styrofoam coolers.
Adequate DO is maintained by replacing  the air above  the  water  in the bags
with oxygen from  a compressed gas  cylinder, and sealing 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.

                                      30

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 A group of organisms must not be used for a test if they appear to be
thy,  discolored,  or otherwise stressed,  or if mortality appears to
6.6.3
unhealthy,
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 can be used at all concentrations of effluent by
adjusting the salinity of the effluent to salinities specified for the
appropriate species test condition or to the salinity approximating that of
the receiving water, by adding sufficient dry ocean salts, such as FORTY
FATHOMS®, or equivalent, GP2, or hypersaline brine.

6.6.5  Saline dilution water can be prepared with deionized water or a
freshwater such as well water or a suitable surface water.  If dry ocean salts
are used, care must be taken to ensure that the added salts are completely
dissolved and the solution is aerated 24 h before the test organisms are
placed in the solutions.  The test organisms should be acclimated in synthetic
saline water prepared with the dry salts.  Caution: addition of dry ocean
salts to dilution water may result in an increase in pH.  (The pH of estuarine
and coastal saline waters is normally 7.5-8.3).

6.6.6  All effluent concentrations and the control(s) used in a test should
have the  same salinity.  The change in salinity upon acclimation at the
desired test dilution should not exceed 6%o.  The required salinities for
culturing and toxicity tests with estuarine and marine species are listed in
the test method sections.

6.7  TEST ORGANISM DISPOSAL

6.7.1  When the toxicity test(s) is concluded, all test organisms (including
controls) should  be humanely destroyed and disposed of in an  appropriate
manner.
                                31

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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 synthetic  (standard) dilution water is used.  If the test organisms
have been cultured in water which is different from the test dilution water, a
second set of controls, using culture water, should be included in the test.

7.1.1.2  If the objective of the test is to estimate the chronic toxicity of
the effluent in uncontaminated receiving water, the test may be conducted
using dilution water consisting of a single grab sample of receiving water (if
non-toxic), collected outside the influence of the outfall, or with other
uncontaminated natural water (surface water) or standard dilution water having
approximately the same salinity as the receiving water.  Seasonal variations
in the quality of receiving waters may affect effluent toxicity.  Therefore,
the salinity of saline receiving water samples should be determined before
each use.  If the test organisms have been cultured in water which is
different from the test dilution water, a second set of controls, using
culture water, should be included in the test.

7.1.1.3  If the objective of the test is to determine the additive or
mitigating effects of the discharge on already contaminated receiving water,
the test is performed using dilution water consisting of receiving water
collected outside the influence of the outfall.  A second set of controls,
using culture water, should be included in the test.

7.2  STANDARD, SYNTHETIC DILUTION WATER

7.2.1  Standard, synthetic, dilution water is prepared with deionized water
and reagent grade chemicals (GP2) or commercial sea salts (FORTY FATHOMS®, HW
HARINEMIX®) (Table 3).  The source water for the deionizer can be ground water
or tap water.

7.2.2  DEIONIZED WATER USED TO PREPARE STANDARD,  SYNTHETIC, DILUTION WATER

7.2.2.1  Deionized water is obtained from a MILLIPORE MILLI-Q®,  MILLIPORE®
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),

                                      32

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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 .

7.2.3.1  To prepare 20 L of a standard, synthetic, reconstituted seawater
(modified GP2), using reagent grade chemicals (Table 3), with a salinity of
31%o, 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 deion.ized water in a properly
      cleaned plastic carboy.
   2. Weigh reagent grade salts listed in Table 3 and  add, one  at a time, to
      the deionized water.  Stir well after adding each salt..
   3. Aerate the final solution at  a rate of 1 L/h for 24 h.
   4. Check the pH and salinity.

7.2.3.2  Synthetic seawater can also be prepared by adding commercial sea
salts, such as  FORTY  FATHOMS®, HW MARINEMIX®, or equivalent, to deionized
water.   For example,  thirty-one parts per thousand (31%o) FORTY FATHOMS® can
be prepared by  dissolving 31 g of sea salts per liter  of deionized water.   The
salinity of the resulting solutions should  be checked  with a refractometer.

7.2.4  Artificial  seawater  is to be used only if specified in the method.
EMSL-Cincinnati has  found FORTY  FATHOMS® artificial sea salts  (Marine
Enterprises,  Inc., 8755  Mylander Lane, Baltimore, MD  21204, 301-321-1189)
suitable for maintaining and spawning the  sheepshead  minnow, Cyprinodon
variegatus,  and for  its  use  in the  sheepshead minnow  larval survival and
growth  test,  suitable for maintaining  and  spawning the inland silverside,
Menidia  beryllina, and  for  its use  in  the  inland silverside larval  survival
and  growth  test,  suitable for culturing  and maintaining mysid shrimp,
Mysidopsis  bahia,  and its use  in the mysid  shrimp survival, growth,  and
fecundity  test, and  suitable for maintaining  sea urchins, Arbacia punctulata,
and  for  its  use in the  sea  urchin  fertilization test.   The USEPA  Region  6
Houston  Laboratory has  successfully used HW MARINEMIX® (Hawaiian  Marine
 Imports  Inc.,  P.O. Box  218687, Houston,  TX 77218, 713-492^7864) sea salts  to
maintain and  spawn sheepshead minnows,  and perform the larval  survival  and
growth test and the  embryo-larval  survival  and  teratogenicity test.  Also,  HW
MARINEMIX® sea salts has been  used  successfully to culture  and  maintain  the
mysid brood stock and perform  the  mysid  survival, growth,  fecundity test.   An
 artificial  seawater  formulation,  GP2  (Spotte  et al.,  1984), Table 3,  has been
 used by the Environmental  Research  Laboratory-Narragansett,  RI  for  all  but the
 embryo-larval  survival  and  teratogenicity  test.   The  suitability  of GP2  as a
medium for culturing organisms  has  not been determined.


                                       33

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  TABLE  3.     PREPARATION  OF GP2  ARTIFICIAL  SEAWATER  USING  REAGENT  GRADE

               CHEMICALS1'2'3
Compound
NaCl
Na2S04
KC1
KBr
Na2B407 • 10 H20
MgCl2 • 6 H20
CaCl2 • 2 H20
SrCl2 • 6 H20
NaHC03
Concentration
(g/L)
21.03
3.52
0.61
0.088
0.034
9.50
1 .32
0.02
0.17
Amount (g)
Required for
20 L
420.6
70.4
12.2
1.76
0.68
190.0
26.4
0.400
3.40
  J  Modified GP2 from Spotte et al.  (1984).
     The constituent salts and concentrations were taken from USEPA (1993a).
     The salinity is 30.89 g/L.
     6P2  can be  diluted  with  deionized  (DI)  water to  the  desired  test
     salinity.
7.3  USE OF RECEIVING WATER AS DILUTHON WATER

7.3.1  If the objectives of the test require the use of uncontaminated
receiving water as dilution water, and the receiving water is uncontaminated,
it may be possible to collect a sample of the receiving water close to the
outfall, but should be away from or beyond the influence of the effluent.
However, if the receiving water is contaminated, it may be necessary to
collect the sample in an area "remote" from the discharge site, matching as
closely as possible the physical and chemical characteristics of the receiving
water near the outfall.
                                      34

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 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.
                                   1
 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 ^m mesh openings prior to use.

 7.3.5  HYPERSALINE BRINE
                                                           I
 7.3.5.1  Hypersaline brine (HSB) has several advantages that make it  desirable
 for use in toxicity testing.  It can be made from any high quality, filtered
 seawater by evaporation, and can be added to deionized water to prepare
 dilution water, or to effluents or surface waters to increase their salinity.

 7.3.5.2  The ideal container for making HSB from natural sieawater is  one that
 (1) has a high surface to volume ratio, (2) is made of a rioncorrosive
 material, and (3) is easily cleaned (fiberglass containers are ideal).
 Special care should be used to prevent any toxic materials from coming in
 contact with the seawater being used to generate the brine.  If a heater is
 immersed directly into the seawater, ensure that the heater materials do not
 corrode or leach any substances that would contaminate the brine.  One
 successful method used is a thermostatically controlled heat exchanger made
 from fiberglass.  If aeration is used,  use only oil-free air compressors to
 prevent contamination.

 7.3.5.3  Before adding seawater to the brine generator, thoroughly clean the
 generator, aeration supply tube, heater, and any other materials that will be
 in direct.contact with the brine.  A good quality biodegradable detergent
 should be used, followed by several thorough deionized water rinses.   High
 quality  (and preferably high salinity)  seawater should be filtered to at least
 10 /um before placing into the brine generator.  Water should be collected on
 an incoming tide to minimize the possibility of contamination.

 7.3.5.4  The temperature of the seawater is increased slowly to 40°C.  The
 water should be aerated to prevent temperature stratification and to  increase

                                       35

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water evaporation.  The brine should be checked daily (depending on the volume
being generated) to ensure that the salinity does not exceed 100%o and that
the temperature does not exceed 40°C.  Additional seawater may be added to the
brine to obtain the volume of brine required.

7.3.5.5  After the required salinity is attained, the MSB should be filtered a
second time through a l-/zm filter and poured directly into portable containers
(20-L CUBITAINERS® or polycarbonate water cooler jugs are suitable).  The
containers should be -capped and labelled with the date the brine was generated
and its salinity.  Containers of HSB should be stored in the dark and
maintained under room temperature until used.

7.3.5.6  If a source of HSB is available, test solutions can be made by
following the directions below.  Thoroughly mix together the deionized water
and brine before mixing in the effluent.

7.3.5.7  Divide the salinity of the HSB by the expected test salinity to
determine the proportion of deionized water to brine.  For example, if the
salinity of the brine is 100%o and the test is to be conducted at 25%o, 100%o
divided, by 25%o = 4.0.  The proportion of brine is 1 part in 4 (one part brine
to three parts deionized water).

7.3.5.8  To make 1 L of seawater at 25%o salinity from a hypersaline brine of
100%o, 250 ml of brine and 750 ml of deionized water are required.

7.4  USE OF TAP WATER AS DILUTION WATER

7.4.1  The use of tap water in the reconstituting of synthetic (artificial)
seawater as dilution water is discouraged unless it is dechlorinated and fully
treated.   Tap water can be dechlorinated by deionization, carbon filtration,
or the use of sodium thiosulfate.  Use of 3.6 mg/L (anhydrous) sodium
thiosulfate will reduce 1.0 mg chlorine/L (APHA, 1992).   Following
dechlorination, total residual chlorine should not exceed 0.01 mg/L.  Because
of the possible 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.
                                      36

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                                  SECTION 8

           EFFLUENT AND RECEIVING MATER SAMPLING, SAMPLE HANDLING,
                  AND SAMPLE PREPARATION FOR TOXICITY TESTS
8.1  EFFLUENT SAMPLING                                    j
                                                          i
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).
                                                          j
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:

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.
                                      37

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   Disadvantages:

   1. Samples are collected over a very short period of time and on a
      relatively infrequent basis.  The chances of detecting a spike 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:

   1. Sampling equipment is more sophisticated and expensive, and must be
      placed on-site for at least 24 h.
   2. Toxicity spikes may not be detected because they are masked by dilution
      with less toxic wastes.

8.3  EFFLUENT SAMPLING RECOMMENDATIONS

8.3.1  When tests are conducted on-site, test solutions can be renewed daily
with freshly collected samples.

8.3.2  When tests are conducted off-site, a minimum of three samples are
collected.  If these samples are collected on Test Days 1,3, and 5, the first
sample would be used for test initiation, and for test solution renewal on Day
2.  The second sample would be used for test solution renewal on Days 3 and 4.
The third sample would be used for test solution renewal on Days 5, 6, and 7.

8.3.3  Sufficient sample must be collected to perform the required toxicity
and chemical tests.  A 4-L (1-gal) CUBITAINER® will provide sufficient sample
volume for most tests.

8.3.4  THE FOLLOWING EFFLUENT SAMPLING METHODS ARE RECOMMENDED:

8.3.4.1  Continuous Discharges

   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

                                      38

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      wastewater does not vary more than 10% in toxicity over a 24-h period,
      regardless of retention time, a single grab sample is collected for a
      single toxicity test.
   3. The retention time of the effluent in the wastewater treatment facility
      may be estimated from calculations based on the volume of the retention
      basin and rate of wastewater inflow.   However, the calculated retention
      time may be much greater than the actual time because of
      short-circuiting in the holding basin.  Where short-circuiting is
      suspected, or sedimentation may have reduced holding basin capacity, a
      more accurate estimate of the retention time can be obtained by carrying
      out a dye study.

8.3.4.2  Intermittent Discharges                          I

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).

8.4  RECEIVING WATER SAMPLING

8.4.1  Logistical problems and difficulty in securing sampling equipment
generally preclude the collection of composite receiving water samples for
toxicity tests.  Therefore, based on the requirements of the test, a single
grab sample or daily grab samples of receiving water is collected for use in
the test.
                                                          I
                                                          I
8.4.2  The sampling point is determined by the objectives of the test.  At
estuarine and marine sites, samples should be collected at mid-depth.

8.4.3  To determine the extent of the zone of toxicity in the receiving water
at estuarine and marine effluent sites, receiving water samples are collected
at several distances away from the discharge.  The time required for the
effluent-receiving-water mixture to travel to sampling points away from the
effluent, and the rate and degree .of mixing, may be difficult to ascertain.
Therefore, it may not be possible to correlate receiving water toxicity with
effluent toxicity at the discharge point unless a dye study is performed.  The
toxicity of receiving water samples from five stations in the discharge plume
can be evaluated using the same number of test vessels and test organisms as
used in one effluent toxicity test with five effluent dilutions.


                                      39

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8.5   EFFLUENT AND RECEIVING WATER SAMPLE HANDLING, PRESERVATION, AND SHIPPING

8.5.1  Unless the samples are used in an on-site toxicity test the day of
collection, it is recommended that they be held at 4°C until used to inhibit
microbial degradation, chemical transformations, and loss of highly volatile
toxic substances.

8.5.2  Composite samples should be chilled as they are collected.  Grab
samples should be chilled immediately following collection.

8.5.3  If the effluent has been chlorinated, total residual chlorine must be
measured immediately following sample collection.

8.5.4  Sample holding time begins when the last grab sample in a series is
taken (i.e., when a series of four grab samples are taken over a 24-h period),
or when a 24-h composite sampling period is completed.  If the data from the
samples are to be acceptable for use in the NPDES Program, the lapsed time
(holding time) from sample collection to first use of the sample in test
initiation must not exceed 36 h.  EPA believes that 36 h is adequate time to
deliver the sample to the laboratories performing the test in most cases.  In
the isolated cases, where the permittee can document that this delivery time
cannot be met, the permitting authority can allow an option for on-site
testing or a variance for an extension of shipped sample holding time.  The
.request for a variance in sample holding time, directed to the USEPA Regional
Administrator under 40 CFR 136.3(e), must include supportive data which show
that the toxicity of the effluent sample is not reduced (e.g., because of
volatilization and/or sorption of toxics on the sample container  surfaces) by
extending the  holding time beyond more than 36 h.  However,  in no case should
more than 72 h elapse between collection and first use of the sample.  In
static-renewal tests, the original sample may also be used to prepare test
solutions for renewal at 24 h and 48 h after test initiation, if stored at
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

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.
                                      40

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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  source water
before being filled with sample.  After use with receiving water or  effluents,
CUBITAINERS® and plastic jugs are punctured to prevent reuse.

8.5.7.3  Several sample shipping options are available,  including  Express
Mail, air express, bus, and courier service.  Express Mai] is delivered  seven
days a week.  Saturday and Sunday shipping and receiving  schedules of  private
carriers vary with the carrier.

8.6  SAMPLE RECEIVING

8.6.1  Upon arrival at the laboratory, samples are  logged in and the
temperature is measured and recorded.  If the samples are not
immediately prepared for testing, they are stored at 4°C  until  used.

8.6.2  Every effort must be made to initiate the test with an effluent sample
on the day of arrival in the laboratory, and the sample  holding time should
not exceed 36 h 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 samp-le salinity to the level appropriate for  objectives of
the study using hypersaline brine or artificial sea salts,

8.8.2  When aliquots are removed from the sample container, the head space
above the remaining sample should be held to a minimum.  Air which enters a
container upon removal of sample should be expelled by compressing the
container before reelosing, 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 urn mesh  openings.  Since filtering may increase the dissolved oxygen (DO)
in an effluent,  the DO should be determined prior to  filtering.   Low  dissolved

                                      41

-------
oxygen concentrations will indicate a potential problem in performing the test.
Caution:  filtration may remove some toxicity.

8.8.4  If the samples must be warmed to bring them to the prescribed test
temperature, supersaturation of the dissolved oxygen and nitrogen may become a
problem.  To avoid this problem, 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 4) or until
the DO is within the prescribed range (> 4.0 mg/L).  Caution:  avoid excessive
aeration.

8.8.4.1  Aeration during the test may alter the results and should be used only
as a last resort to maintain the required DO.  Aeration can reduce the apparent
toxicity of the test solutions by stripping them of highly volatile toxic
substances, or increase their toxicity by altering the pH.  However, the DO in
the test solution 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, conductivity or salinity, and total residual chlorine
are measured in the undiluted effluent or receiving water, and pH and
conductivity are measured in the dilution water.

8.8.5.1  It is recommended that total alkalinity and total hardness also be
measured in the undiluted effluent test water and the dilution water.

8.8.6  Total ammonia is measured in effluent and receiving water samples where
toxicity may be contributed by unionized ammonia (i.e., where total ammonia
•&. 5 mg/L).  The concentration (mg/L) of unionized (free) ammonia in a sample is a
function of temperature and pH, and is calculated using the percentage value
obtained from Table 5, under the appropriate pH and temperature, and multiplying
it by the concentration (mg/L) of total ammonia in the sample.

8.8.7  Effluents and receiving waters can be dechlorinated using 6.7 mg/L
anhydrous sodium thiosulfate to reduce 1 mg/L chlorine (APHA, 1992).  Note that
the amount of thiosulfate required to dechlorinate effluents is greater than the
amount needed to dechlorinate tap water, (see Section 7, Dilution Water).  Since
thiosulfate may contribute to sample toxicity, a thiosulfate control should be
used in the test in addition to the normal dilution water control.

8.8.8  The DO concentration in the samples should be near saturation prior to
use.  Aeration will bring the DO and other gases into equilibrium with air,
minimize oxygen demand, and stabilize the pH.  However, aeration during

                                        42

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TABLE 4. OXYGEN SOLUBILITY (MG/L)  IN WATER AT EQUILIBRIUM WITH AIR AT 760 MM
         HG (AFTER Richards and Corwin, 1956)

TEMP
(C°)
0
1
2
3
4
5
6
8
10
12
14
16
18
20
22
24
26
28
30
32

SALINITY (%o)
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
                                    43

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TABLE 5. PERCENT  UNIONIZED NH, IN AQUEOUS  AMMONIA SOLUTIONS:
         15-26°C  AND  pH 6.0-8.91
                                TEMPERATURE
pH
TEMPERATURE <°C)

6.0
6.1
6.2
6.3
6.4
6.5
6.6
6.7
6.8
6.9
7.0
7.1
7.2
7.3
7.4
7.5
7.6
7.7
7.8
7.9
8.0
8.1
8.2
8.3
8.4
8.5
8.6
8.7
8.8
8.9
15
0.0274
0.0345
0.0434
0.0546
0.0687
0.0865
0.109
0.137
0.172
0.217
0.273
0.343
0.432
0.543
0.683
0.858
1.08
1.35
1.70
2.13
2.66
3.33
4.16
5.18
6.43
7.97
9.83
12.07
14.7
17.9
16
0.0295
0.0372
0.0468
0.0589
0.0741
0.0933
0.117
0.148
0.186
0.234
0.294
0.370
0.466
0.586
0.736
0.925
1.16
1.46
1.83
2.29
2.87
3.58
4.47
5.56
6.90
8.54
10.5
12.9
15.7
19.0
17
0.0318
0.0400
0.0504
0.0634
0.0799
0.1005
0.127
0.159
0.200
0.252
0.317
0.399
0.502
0.631
0.793
0.996
1.25
1.57
1.97
2.46
3.08
3.85
4.80
5.97
7.40
9.14
11.2
13.8
16.7
20.2
18
0.0343
0.0431
0.0543
0.0683
0.0860
0.1083
0.136
0.171
0.216
0.271
0.342
0.430
0.540
0.679
0.854
1.07
1.35
1.69
2.12
2.65
3.31
4.14
5.15
6.40
7.93
9.78
12.0
14.7
17.8
21.4
•19
0.0369
0.0464
0.0584
0.0736
0.0926
0.1166
0.147
0.185
0.232
0.292
0.368
0.462
0.581
0.731
0.918
1.15
1.45
1.82
2.28
2.85
3.56
4.44
5.52
6.86
8.48
10.45
12.8
15.6
18.9
22.7
20
0.0397
0.0500
0.0629
0.0792
0.0996
0.1254
0.158
0.199
0.250
0.314
0.396
0.497
0.625
0.786
0.988
1.24
1.56
1.95
2.44
3.06
3.82
4.76
5.92
7.34
9.07
11.16
13.6
16.6
20.0
24.0
21
0.0427
0.0537
0.0676
0.0851
0.107
0.135
0.170
0.214
0.269
0.338
0.425
0.535
0.672
0.845
1.061
1.33
1.67
2.10
2.62
3.28
4.10
5.10
6.34
7.85
9.69
11.90
14.5
17.6
21.2
25.3
22
0.0459
0.0578
0.0727
0.0915
0.115
0.145
0.182
0.230
0.289
0.363
0.457
0.575
0.722
0.908
1.140
1.43
1.80
2.25
2.82
3.52
4.39
5.46
6.78
8.39
10.3
12.7
15.5
18.7
22.5
26.7
23
0.0493
0.0621
0.0781
0.0983
0.124
0.156
0.196
0.247
0.310
0.390
0.491
0.617
0.776
0.975
1.224
1.54
1.93
2.41
3.02
3.77
4.70
5.85
7.25
8.96
11.0
13.5
16.4
19.8
23.7
28.2
24 25
0.0530 0.0568
0.0667 0.0716
0.0901 0.0901
0.1134 0.1134
0.133 0.143
0.167 0.180
0.210 0.226
0.265 0.284
0.333 0.358
0.419 0.450
0.527 0.566
0.663 0.711
0.833 0.893
1.05 1.12
1.31 1.41
1.65 1.77
2.07 2.21
2.59 2.77
3.24 3.46
4.04 4.32
5.03 5.38
6.25 6.68
7.75 8.27
9.56 10.2
11.7 12.5
14.4 15.2
17.4 18.5
21.0 22.2
25.1 26,4
29.6 31.1
26
0.0610
0.0768
0.0966
0.1216
0.153
0.193
0.242
0.305
0.384
0.482
0.607
0.762
0.958
1.20
1.51
1.89
2.37
2.97
3.71
4.62
5.75
7.14
8.82
10.9
13.3
16.2
19.5
23.4
27.8
32.6
   Table  provided by  Teresa Norberg-King,  Duluth,  Minnesota.   Also
   Emerson et al.  (1975),  Thurston et al.  (1974), and USEPA  (1985a).
                                        see
                                     44

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collection, transfer, and preparation of samples should be minimized to reduce
the loss of volatile chemicals.

8.8.9  Mortality or impairment of growth or reproduction due to pH alone may
occur if the pH of the sample falls outside the range of 6.0 - 9.0.  Thus, the
presence of other forms of toxicity (metals and organics) in the sample may be
masked by the toxic effects of low or high pH.  The question about the presence
of other toxicants can be answered only by performing two parallel tests, one
with an adjusted pH, and one without an adjusted pH.  Freshwater samples are
adjusted to pH 7.0, and marine samples are adjusted to pH 8.0, by adding 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.  Cautiom:  if the sample
must also be used for the full-scale definitive test, the 36-h limit on holding
time (see Subsection 8.5.4) must not be exceeded before the definitive test is
initiated.  •

8.9.3  It should be noted that the toxicity (LC50) of a sample observed in a
range-finding test may be significantly different from the toxicity observed in
the follow-up, chronic, definitive test because: (1) the definitive test is
longer; and (2) the test may be performed with a sample collected at a different
time, and possibly differing significantly in the level of toxicity.

8.10  MULTICONCENTRATION (DEFINITIVE)  EFFLUENT TOXICITY TESTS

8.10.1  The tests recommended for use in determining discharge permit compliance
in the NPDES program are multiconcentration, or definitive,  tests which provide
(1) a point estimate of effluent toxicity in terms of an IC25,  IC50, or LC50, or
(2) a no-observed-effect-concentration (NOEC) defined in terms of mortality,
growth, reproduction, and/or teratogenicity and obtained by hypothesis testing.
The tests may be static renewal or static non-renewal.       i

8.10.2  The tests consist of a control  and a minimum of five;effluent
concentrations commonly selected to approximate a geometric series,  such as 100%,
50%,  25%, 12.5%, and 6.25%, using a > 0.5 dilution series.

8.10.3  These tests are also to be used in determining  compliance with permit
limits on the mortality of the receiving water concentration (RWC)  of effluents
by bracketing the RWC with effluent concentrations in the following manner:   (1)
100% effluent, (2)  [RWC + 100J/2, (3)  RWC,  (4) RWC/2, and (5)  RWC/4.   For

                                        45

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example, where the RWC = 50%, the effluent concentrations used in the test would
be 100%, 75%, 50%, 25%, and 12.5%.

8.10.4  If acute/chronic ratios are to be determined by simultaneous acute and
short-term chronic tests with a single species, using the same sample, both types
of tests must use the same test conditions, i.e., pH, temperature, water
hardness, salinity, etc.

8.11  RECEIVING WATER TESTS

8.11.1  Receiving water toxicity tests generally consist of 100% receiving water
and a control.  The total salinity of the control should be comparable to the
receiving water.

8.11.2  The data from the two treatments are analyzed by hypothesis testing to
determine if test organism survival in the receiving water differs significantly
from the control.  Four replicates and 10 organisms per replicate are required
for each treatment (see Summary of Test Conditions and Test Acceptability
Criteria in the specific test method).

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.
                                        46

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                                    SECTION 9

                CHRONIC TOXICITY TEST ENDPOINTS AND DATA ANALYSIS
9.1  ENDPOINTS
9 1.1  The objective of chronic aquatic toxicity tests with effluents and pure
compounds is to estimate the highest "safe" or "no-effect concentration" of these
substances.  For practical reasons, the responses observed in these tests are
usually limited to hatchability, gross morphological abnormalities, survival,
growth, and reproduction, and the results of the tests are usually expressed in
terms of the highest toxicant concentration that has no statistically significant
observed effect on these responses, when compared to the controls.  The terms
currently used to define the endpoints employed in the rapid, chronic and
sub-chronic toxicity tests have been derived from the terms previously used for
full life-cycle tests.  As shorter chronic tests were developed, it became common
practice to apply the same terminology to the endpoints.  The terms used in this
manual are as follows:

9.1.1.1  Safe Concentration - The highest concentration of toxicant that will
permit normal propagation of fish and other aquatic life in receiving waters.
The concept of a "safe concentration" is a biological concept, whereas the
"no-observed-effect concentration" (below) is a statistically defined
concentration.

9.1.1.2  No-Observed-Effect-Concentration  (NOEC) - The highest concentration of
toxicant to which organisms are exposed  in a full life-cycle or partial life-
cycle  (short-term) test,  that causes no  observable adverse effects on the test
organisms  (i.e., the  highest concentration of toxicant in which the values for
the observed responses are not  statistically significantly different from the
controls).  This value is used, along with other factors, to determine toxicity
limits  in  permits.

9.1.1.3   Lowest-Observed-Effect-Concentration  (LOEC)  - The "lowest  concentration
of toxicant to which  organisms  are exposed in  a life-cycle or partial life-cycle
 (short-term) test, which causes adverse  effects on  the test  organisms (i.e.,
where  the  values for  the observed  responses  are statistically significantly
different  from the controls).

9.1.1.4  Effective Concentration  (EC)  -  A  point estimate of  the toxicant
concentration that would cause  an  observable adverse  affect  on  a  quanta!,  "all or
nothing,"  response  (such as death,  immobilization,  or serious  incapacitation)  in
 a given percent  of the test organisms,  calculated  by  point estimation techniques.
 If the observable effect is death  or immobility, the  term, Lethal  Concentration
 (LC),  should  be  used  (see Subsection 9.1.1.5).  A  certain  EC or LC value might be
 judged from a  biological standpoint  to  represent  a threshold concentration,  or
 lowest concentration  that would cause an adverse effect  on |:he  observed  response.

 9.1.1.5  Lethal  Concentration  (LC)  - The toxicant  concentration that  would  cause
 death in a given percent of the test population.   Identical  to  EC when  the


                                         47                  !

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 observable adverse effect is death.   For example,  the  LC50  is  the  concentration
 of toxicant that would cause death in 50% of the test  population.

 9.1.1.6   Inhibition Concentration  (1C)  - The toxicant  concentration  that would
 cause  a  given  percent reduction  in a  nonquantal  biological  measurement  for  the
 test population.   For example, the IC25 is the  concentration of toxicant that
 would  cause a  25% reduction  in mean young per female or  in  growth  for the test
 population,  and  the IC50  is  the  concentration of toxicant that would cause  a 50%
 reduction  in the mean population responses.

 9.2   RELATIONSHIP BETWEEN ENDPOINTS  DETERMINED BY HYPOTHESIS  TESTING AND POINT
       ESTIMATION TECHNIQUES

 9.2.1  If  the  objective of chronic aquatic toxicity tests with effluents and pure
 compounds  is to  estimate  the highest  "safe or no-effect  concentration"  of these
 substances,  it is imperative to  understand how  the  statistical  endpoints of these
 tests  are  related to  the  "safe"  or "no-effect"  concentration.   NOECs and LOECs
 are determined by hypothesis testing  (Dunnett's  Test,  a  t test with  the
 Bonferroni  adjustment,  Steel's Many-One Rank Test,  or  the Wilcoxon Rank Sum Test
 with Bonferroni  adjustment),  whereas  LCs,  ICs,  and  ECs are  determined by point
 estimation  techniques (Probit Analysis,  the  Spearman-Karber Method,  the Trimmed
 Spearman-Karber Method, the  Graphical Method or  Linear Interpolation Method).
 There  are  inherent differences between  the use of a NOEC or LOEC derived from
 hypothesis  testing to estimate a "safe"  concentration, and  the use of a LC,  1C,
 EC, or other point estimates  derived  from curve  fitting, interpolation,  etc.

 9.2.2  Most  point estimates,  such  as  the LC,  1C, or EC are  derived from a
 mathematical model  that assumes  a  continuous  dose-response  relationship.  By
 definition,  any LC, 1C, or EC value is  an  estimate  of  some  amount of adverse
 effect.  Thus  the assessment  of  a  "safe"  concentration must be made from a
 biological  standpoint rather  than  with  a statistical test.  In this instance, the
 biologist must determine  some amount  of adverse  effect that is deemed to be
 "safe,"  in the sense  that from a practical biological viewpoint it will  not
 affect the normal  propagation of fish and  other  aquatic life in receiving waters.

 9.2.3  The use of NOECs and  LOECs, on the  other  hand, assumes  either (1) a
 continuous dose-response  relationship,  or  (2) a  non-continuous  (threshold)  model
 of the dose-response  relationship.

 9.2.3.1  In the case  of a continuous dose-response  relationship, it is also
 assumed that adverse  effects  that  are not  "statistically observable" are also not
 important from a  biological standpoint,  since they  are not pronounced enough to
test as statistically significant  against  some measure of the natural variability
of the responses.

9.2.3.2  In the case of non-continuous dose-response relationships, it is assumed
that there exists  a true threshold, or concentration below which there is no
adverse effect on  aquatic life,   and above which there is an adverse effect.   The
purpose of the statistical analysis in this case is to estimate as closely as
possible where that threshold lies.
                                        48

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9.2.3.3  In either case, it is important to realize that the amount of adverse
effect that is statistically observable (LOEC) or not observable (NOEC) is highly
dependent on all aspects of the experimental design, such as the number of
concentrations of toxicant, number of replicates per concentration, number of
organisms per replicate, and use of randomization.  Other factors that affect the
sensitivity of the test include the choice of statistical analysis, the choice of
an alpha level, and the amount of variability between responses at a given
concentration.

9.2.3.4  Where the assumption of a continuous dose-response relationship is made,
by definition some amount of adverse effect might be present! at the NOEC, but is
not great enough to be detected by hypothesis testing.

9.2.3.5  Where the assumption of a noncontinuous dose-response relationship is
made, the NOEC would indeed be an estimate of a "safe" or "rio-effect"
concentration if the amount of adverse effect that  appears at the threshold is
great enough to test as statistically significantly different from the controls
in the face of all aspects of the experimental design mentioned above.  If,
however, the amount of adverse effect at the threshold were not great enough to
test as statistically different, some amount of adverse  effect might be present
at the NOEC.  In any case, the estimate of the NOEC with hypothesis testing is
always dependent on the aspects of the experimental design mentioned above.  For
this reason, the reporting and examination of some  measure of the sensitivity of
the test (either the minimum significant difference -or the percent change from
the control that this minimum difference represents)  is  extremely important.

9.2.4   In  summary, the  assessment of a "safe" or  "no-effect" concentration cannot
be made from the results of statistical analysis  alone,  unless  (1) the
assumptions of  a strict threshold model are accepted, and  (!>) 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 van'egatus,  and the  red  macroalga, Champia parvula.   Birge  et  al.
 (1985)  reported that  LCls  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,  Cen'odaphm'a  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

                                        49

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 relationship between these two types of endpoints, especially when derived from
 effluent toxicity test data.

 9.3  PRECISION

 9.3.1  HYPOTHESIS TESTS

 9.3.1.1  When hypothesis tests are used to analyze toxicity test data,  it is not
 possible to express precision in terms of a commonly used statistic.   The results
 of the test are given in terms of two endpoints,  the No-Observed-Effect
 Concentration (NOEC) and the Lowest-Observed-Effect Concentration (LOEC).   The
 NOEC and LOEC are limited to the concentrations selected for the test.   The width
 of the NOEC-LOEC interval is a function of the dilution series,  and differs
 greatly depending on whether a dilution factor of 0.3 or 0.5 is  used  in the test
 design.  Therefore,  USEPA recommends the use of the > 0.5 dilution factor  (see
 Section 4,  Quality Assurance).  It is not possible to place confidence  limits  on
 the NOEC and LOEC derived from a given test,  and  it is difficult to quantify the
 precision of the NOEC-LOEC endpoints between  tests.   If the data from a series of
 tests performed with the same toxicant,  toxicant  concentrations,  and  test
 species,  were analyzed with hypothesis tests,  precision could only be assessed by
 a qualitative comparison of the NOEC-LOEC intervals,  with the understanding that
 maximum precision would be attained if all  tests  yielded the same NOEC-LOEC
 interval.   In practice,  the precision of results  of repetitive chronic  tests is
 considered  acceptable if the NOECs vary by no  more than one concentration
 interval  above or below a central  tendency.   Using these guidelines,  the  "normal"
 range of NOECs from  toxicity tests using a 0.5 dilution factor (two-fold
 difference  between adjacent concentrations), would be four-fold.

 9.3.2  POINT ESTIMATION  TECHNIQUES

 9.3.2.1   Point estimation techniques  have the  advantage of  providing  a  point
 estimate  of the toxicant concentration  causing a  given  amount  of  adverse
 (inhibiting)  effect,  the precision of which can be quantitatively assessed  (1)
 within  tests  by calculation of 95% confidence  limits,  and  (2)  across  tests  by
 calculating a standard deviation  and  coefficient  of variation.

 9.4   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

                                        50

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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 estimations approaches most probably would require the services
of a statistician to determine the appropriateness of the model (goodness of
fit), higher order linear or nonlinear models, confidence intervals for estimates
generated by inverse regression, etc.  In addition, point estimation or
regression approaches would require the specification by biologists or
toxicologists of some low level of adverse effect that would be deemed acceptable
or safe.  The statistical methods contained in this manual have been chosen
because they are (1) applicable to most of the different toxicity test data sets
for which they are recommended, (2) powerful statistical tests, (3) hopefully
"easily" understood by nonstatisticians, and (4) amenable to use without a
computer,  if necessary.                                      ;

9.4.2  PLOTTING THE DATA

9.4.2.1  The data should be plotted, both  as a preliminary step to help detect
problems and unsuspected trends or patterns in the  responses,  and as an aid in
interpretation of the results.  Further discussion  and plotted sets of data are
included in the methods  and the Appendices.

9.4.3  DATA TRANSFORMATIONS

9.4.3.1  Transformations of the data,  (e.g.,  arc  sine square root and logs),  are
used where necessary  to  meet  assumptions of the proposed  analyses, such as  the
requirement for normally distributed data.

9.4.4   INDEPENDENCE,  RANDOMIZATION,  AND OUTLIERS
                                                             i
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, arid should be based on
 the objectives for obtaining the toxicity data.
                                         51

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 9.4.5.2  In a statistical  analysis of toxicity data,  the choice of a particular
 analysis and the ability to detect departures from the assumptions of the
 analysis, such as the normal distribution of the data and homogeneity of
 variance, is also dependent on the number of replicates.  More than the minimum
 number of replicates may be required in situations where it is imperative to
 obtain optimal statistical  results, such as with tests used in enforcement cases
 or when it is not possible  to repeat the tests.   For  example,  when the data are
 analyzed by hypothesis testing,  the nonparametric alternatives cannot be used
 unless there are at least four replicates at each toxicant concentration.

 9.4.6  RECOMMENDED ALPHA LEVELS

 9.4.6.1  The data analysis  examples included in  the manual  specify an alpha level
 of 0.01 for testing the assumptions of hypothesis tests and an alpha level  of
 0.05  for the hypothesis tests themselves.   These levels are common and well
 accepted levels for this type of analysis and are presented as a recommended
 minimum significance level  for toxicity data analysis.

 9.5  CHOICE OF ANALYSIS

 9.5.1  The recommended statistical  analysis of most data from  chronic toxicity
 tests with aquatic organisms follows a decision  process illustrated in the
 flowchart in Figure 2.   An  initial  decision is made to  use  point estimation
 techniques (the Probit Analysis,  the Spearman-Karber  Method, the Trimmed
 Spearman-Karber Method,  the Graphical  Method,  or Linear Interpolation Method)
 and/or to use hypothesis testing  (Dunnett's Test,  the t test with  the Bonferroni
 adjustment,  Steel's Many-one Rank Test,  or Wilcoxon Rank Sum Test  with the
 Bonferroni  adjustment).  NOTE:  For the NPDES Permit Program, the point estimation
 techniques are the preferred statistical methods in calculating  end points for
 effluent toxicity tests.  If hypothesis testing  is  chosen,  subsequent decisions
 are made on  the appropriate procedure  for  a given  set of data, depending on  the
 results of tests  of assumptions,  as illustrated  in  the  flowchart.   A specific
 flow  chart is included  in the analysis section for  each test.

 9.5.2  Since a single  chronic toxicity test might yield information  on more  than
 one parameter (such as  survival,  growth, and reproduction), the  lowest estimate
 of a  "no-observed-effect concentration"  for any  of  the  responses.would be used as
 the "no-observed-effect  concentration"  for each  test.   It follows  logically  that
 in  the  statistical  analysis  of the  data, concentrations  that had a  significant
 toxic effect  on one of the  observed  responses would not  be  subsequently tested
 for an  effect on  some other  response.   This  is one  reason for excluding
 concentrations  that have shown a  statistically significant  reduction  in survival
 from  a  subsequent  hypothesis  test  for  effects on another parameter  such as
 reproduction.  A  second  reason is that  the  exclusion  of  such concentrations
 usually  results in  a more powerful  and  appropriate  statistical  analysis.  In
 performing the point estimation techniques  recommended  in this manual, an all-
data  approach  is  used.   For  example, data  from concentrations above the NOEC for
survival are  included in determining ICp estimates using the Linear  Interpolation
Method.
                                        52

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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 OF! MORE
                      REPLICATES?
                                                   YESJ
          EQUAL NUMBER OF
            REPLICATES?
T-TESTWITH
BONFERRONI
ADJUSTMENT
               EQUAL NUMBER OF
                 REPLICATES?
                                     YES
         T
                               NO
STEEL'S MANY-ONE
   RANK TEST
  WILCOXON RANK SUM
      TEST WITH
BONF:ERRONI ADJUSTMENT
                         ENDPOINT ESTIMATES
                             NOEC, LOEC
     Figure 2.  Flowchart for statistical  analysis of test data.

                                 53

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 9.5.3  ANALYSIS OF GROWTH AND REPRODUCTION DATA

 9.5.3.1  Growth data from the sheepshead minnow, Cyprinodon van'egatus,  and
 inland silverside, Menidia beryTlina, larval survival  and growth tests,  and the
 mysid, Mysidopsis bahia, survival, growth, and fecundity test, are analyzed using
 hypothesis testing according to the flowchart in Figure 2.  The above mentioned
 growth data may also be analyzed by generating a point estimate with the Linear
 Interpolation Method.  Data from effluent concentrations that have tested
 significantly different from the control for survival  are excluded from  further
 hypothesis tests concerning growth effects.   Growth is defined as the change in
 dry weight of the orginal  number of test organisms when group weights are
 obtained.   When analyzing the data using point estimating techniques, data from
 all  concentrations are included in the analysis.

 9.5.3.2  Fecundity data from the mysid,  Mysidopsis bahia,  test may be analyzed
 using hypothesis testing after an arc sine transformation according to the
 flowchart  in Figure 2.   The fecundity data from the mysid test may also  be
 analyzed by generating a point estimate  with the Linear Interpolation Method.

 9.5.3.3  Reproduction data from the red  macroalga,  Champia parvula,  test are
 analyzed using hypothesis  testing as illustrated in Figure 2.   The reproduction
 data from  the red macroalga test may also be analyzed  by generating a point
 estimate with the Linear Interpolation Method.

 9.5.4  ANALYSIS OF THE SEA URCHIN,  ARBACIA PUNCTULATA,  FERTILIZATION DATA

 9.5.4.1 Data from the  sea urchin,  Arbacia punctulata,  fertilization test may  be
 analyzed by hypothesis  testing after an  arc  sine transformation  according to the
 flowchart  in Figure 2.   The fertilization data  from the sea urchin test  may also
 be analyzed by generating  a point estimate with  the Linear Interpolation  Method.

 9.5.5  ANALYSIS OF MORTALITY DATA

 9.5.5.1 Mortality data are analyzed by  Probit Analysis,  if appropriate,  or other
 point estimation techniques,  (i.e.,  the  Spearman-Karber Method,  the  Trimmed
 Spearman-Karber Method,  or the Graphical  Method)  (see Appendices  G-I)  (see
 discussion  below).   The mortality data can also  be  analyzed  by hypothesis
 testing, after an  arc sine square root transformation  (see Appendices  B-F),
 according to  the flowchart in  Figure 2.

 9.6   HYPOTHESIS TESTS

 9.6.1   DUNNETT'S PROCEDURE

 9.6.1.1  Dunnett's  Procedure  is used  to determine the NOEC.  The  procedure
 consists of an  analysis  of variance  (ANOVA) to determine the error term,  which  is
then  used in  a multiple  comparison procedure for comparing each of the treatment
means with the control mean,  in a series of paired tests (see Appendix C)   Use
of Dunnett's  Procedure requires at least three replicates per treatment to  check
the assumptions  of the test.   In cases where the numbers of data points
 (replicates) for each concentration  are not equal, a t test may be performed with


                                       54

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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.                j

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.

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).
                                         55

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 9.6.3.2  It is necessary to have at least four replicates per toxicant
 concentration to use Steel's test.  Unlike Dunnett's procedure, the sensitivity
 of this test cannot be stated in terms of the minimum difference between
 treatment means and the control mean that can be detected as statistically
 significant.

 9.6.3.3  The estimate of the safe concentration is reported as the NOEC   A
 step-by-step example of the use of Steel's Many-One Rank Test is provided in
 Appendix E.

 9.6.4  WILCOXON RANK SUM TEST WITH THE BONFERRONI ADJUSTMENT

 9.6.4.1  The Wilcoxon Rank Sum Test is a nonparametric test for comparing a
 treatment with a control.   The data are ranked and the analysis proceeds exactly
 as in Steel's Test except that Bonferroni's adjustment for multiple comparisons
 is used instead of Steel's tables.   When Steel's  test can be used  (i.e., when
 there are equal  numbers of data points per toxicant concentration), it will  be
 more powerful  (able to detect smaller differences as statistically significant)
 than the Wilcoxon  Rank Sum Test with Bonferroni's-adjustment.

 9.6.4.2  The estimate of the safe concentration  is reported as  the NOEC.  A
 step-by-step example of the use of the Wilcoxon Rank Sum Test with Bonferroni
 adjustment  is  provided in  Appendix F.

 9.6.5  A CAUTION IN THE USE OF  HYPOTHESIS TESTING

 9.6.5.1  If in the  calculation  of an NOEC by  hypothesis  testing, two tested
 concentrations cause statistically  significant adverse effects,  but an
 intermediate concentration  did  not  cause  statistically significant  effects, the
 results should be used  with extreme caution.

 9.7   POINT  ESTIMATION TECHNIQUES

 9.7.1   PROBIT ANALYSIS

 9.7.1.1   Probit Analysis is used  to  estimate  an LCI,  LC50, EC1, or  EC50  and the
 associated  95% confidence interval.  The  analysis consists of adjusting  the data
 for mortality in the control, and then using  a maximum likelihood technique to
 estimate the parameters of  the underlying log tolerance distribution, which is
 assumed to  have a particular shape.

 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.
                                       56

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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, growth, fertilization, or fecundity of the
test organisms.  The procedure was  designed for general applicability in the
analysis of data from short-term chronic  toxicity tests.
                                                                 .
9.7.2.2  Use of the  Linear Interpolation  Method is based on the assumptions that
the responses  (1)  are monotonically non-increasing (the mean response for each
higher concentration is less than or equal to the mean response for the previous
concentration),  (2)  follow a piece-wise  linear response function,  and (3) are
from a random, independent, and representative sample  of test data.  The
assumption for piece-wise linear response cannot be tested statistically, and  no
defined  statistical  procedure  is provided to test the  assumption for
monotonicity.  Where the observed means  are not strictly monotonic by
examination, they  are adjusted  by smoothing.  In cases where the responses at  the
low toxicant concentrations are much higher than in the controls,  the smoothing
process  may result in a large  upward adjustment in the control mean.
                                                             j
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.                       j
                                         57

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                                    SECTION 10

                                REPORT PREPARATION
   The toxicity  data  are  reported,  together with  other  appropriate data.
following general format  and content are recommended for the report:
The
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. Eff1uent Samples
      a. Sampling point
         Collection dates and times
         Sample collection method
         Physical and chemical data
         Mean daily discharge on sample collection date
         Lapsed time from sample collection to delivery
         Sample temperature when received at the laboratory
      Receiving Water Samples
      a. Sampling point
      b. Collection dates and times
      c. Sample collection method
      d. Physical and chemical data
      e. Tide stages
      f. Sample temperature when received at the laboratory
      g. Lapsed time from sample collection to delivery
                                       58

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   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
   2.  Age
   3.  Life stage
   4.  Mean length  and weight  (where applicable)
   5.  Source
   6.  Diseases  and treatment  (where applicable)
    7.  Taxonomic key used  for  species  identification

 10.6   QUALITY ASSURANCE

    1.  Reference toxicant  used routinely; source
    2.  Date and  time of most recent reference toxicant test; test results and
       current control  (cusum) chart
    3.  Dilution  water used  in  reference toxicant test
    4.  Results (NOEC or,  where applicable, LOEC,  LC50, EC50, IC2:5 and/or IC50)
    5.  Physical  and chemical methods used

 10.7   RESULTS

    1.  Provide raw toxicity data in tabular form,  including daily records of
       affected organisms in each concentration (including controls), and plots of
       toxicity data
    2. Provide table of LC50s, NOECs,  IC25, IC50,  etc.
    3.  Indicate statistical methods to  calculate endpoints
    4. Provide summary table of physical and  chemical data
    5. Tabulate QA data

                                         59

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10.8  CONCLUSIONS AND RECOMMENDATIONS

   1. Relationship between test endpoints and permit limits.
   2. Action to be taken.
                                       60

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                                  SECTION 11

                                 TEST METHOD

                   SHEEPSHEAD MINNOW,  CYPRINODON VARIEGATUS
                       LARVAL SURVIVAL AND GROWTH TEST
                                METHOD 1004.0
11.1  SCOPE AND APPLICATION

11.1.1  This method,  adapted in part from USEPA (1987b), estimates the chronic
toxicity of effluents and receiving waters to the sheepshead minnow,
Cypn'nodon van'egatus, using newly hatched larvae in a seven-day,
static-renewal test.   The effects include the synergistic, antagonistic, and
additive effects of all the chemical, physical, and biological components
which adversely affect the physiological and biochemical functions of the test
species.                                                  i

11.1.2  Daily observations on mortality make it possible to also calculate
acute toxicity for desired exposure periods (i.e., 24-h, 48-h, 96-h LC50s).

11.1.3  Detection limits of the toxicity of an effluent or chemical are
organism dependent.

11.1.4  Brief excursions in toxicity may not be detected using 24-h composite
samples.  Also, because of the long sample collection period  involved in
composite sampling, and because the test chambers are not sealed, highly
volatile and  highly degradable toxicants present  in the source may  not  be
detected in the test.
                                                          i
11.1.5  This  method is commonly used in one of two forms:   (1) a definitive
test, consisting of a minimum of  five effluent concentration:; and a control,
and (2) a receiving water test(s), consisting of  one or more  receiving  water
concentrations  and a  control.

11.2  SUMMARY OF METHOD

11.2.1  Sheepshead minnow,  Cypn'nodon van'egatus, larvae  (preferably  less  than
24-h  old)  are exposed in  a  static renewal  system  for  seven  days  to  different
concentrations  of  effluent  or  to  receiving water.  Test results  are based  on
the survival  and weight  of  the  larvae.
                                                          i
11.3   INTERFERENCES

11.3.1  Toxic substances may  be introduced by  contaminants  in dilution  water,
glassware,  sample  hardware,  and testing equipment (see  Section  5,  Facilities,
Equipment,  and Supplies).

 11.3.2  Adverse effects  of low dissolved oxygen  concentrations  (DO),  high
concentrations of  suspended and/or dissolved solids,  and  extremes of  pH, may
mask the  effects  of toxic substances.

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 11.3.3   Improper effluent  sampling  and  handling  may  adversely  affect test
 results  (see Section  8,  Effluent  and  Receiving Water Sampling,  Sample
 Handling,  and Sample  Preparation  for  Toxicity Tests).

 11.3.4   Pathogenic  and/or  predatory organisms in the dilution  water and
 effluent may affect test organism survival,  and  confound  test  results.

 11.3.5   Food added  during  the  test  may  sequester metals and  other toxic
 substances  and reduce the  apparent  toxicity  of the test substance.  However,
 in  a growth test the  nutritional  needs  of  the organisms must be  satisfied,
 even if  feeding has the  potential to  confound test results.

 11.4  SAFETY

 11.4.1   See Section 3, Health  and Safety.

 11.5  APPARATUS AND EQUIPMENT

 11.5.1   Facilities  for holding and  acclimating test  organisms.

 11.5.2   Brine  shrimp, Artemia, culture  unit  -- see Subsection  11.6.14 below
 and Section 4,  Quality Assurance.

 11.5.3   Sheepshead  minnow  culture unit  --  see Subsection  11.6.15 below.  The
 maximum  number of larvae required per test will  range from a maximum of 360,
 if  15 larvae  are  used in each of four replicates, to a minimum of 180 per
 test, if 10 larvae  are used in each of  three replicates.  It is preferable to
 obtain the  test  organisms  from an in-house culture unit.  If it is not
 feasible to culture fish in-house,  embryos or newly  hatched larvae can be
 obtained from  other sources if shipped  in well oxygenated saline water in
 insulated containers.

 11.5.4   Samplers  -- automatic sampler,  preferably with sample cooling
 capability, that  can collect a 24-h composite sample of 5 L.

 11.5.5   Environmental chamber or equivalent facility with temperature control
 (25 ± 1°C).

 11.5.6   Water  purification system -- Millipore Milli-Q®, deionized water (DI)
 or equivalent.

 11.5.7   Balance -- Analytical, capable of accurately weighing to 0.00001 g.

 11.5.8   Reference weights,  Class S --  for checking performance of balance.
Weights  should bracket the expected weights of the weighing pans and the
expected weights of the pans plus fish.

11.5.9  Drying oven -- 50-105°C range, for drying larvae.

11.5.10  Air pump -- for oil-free air  supply.
                                      62

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11.5.11  Air lines,  and air stones -- for aerating water containing embryos or
larvae, or for supplying air to test solutions with low DO.

11.5.12  Meters, pH and DO -- for routine physical and chemical measurements.

11.5.13  Standard or micro-Winkler apparatus -- for determining DO (optional).

11.5.14  Dissecting microscope -- for checking embryo viability.

11.5.15  Desiccator -- for holding dried larvae.

11.5.16  Light box -- for counting and observing larvae.

11.5.17  Refractometer -- for determining salinity.

11.5.18  Thermometers, glass or electronic, laboratory grade -- for measuring
water temperatures.

11.5.19  Thermometers, bulb-thermograph or electronic-chart type'-- for
continuously recording temperature.

11.5.20  Thermometer, National Bureau of Standards Certified (see USEPA Method
170.1, USEPA, 1979b) -- to calibrate laboratory thermometers..
                                                          I
11.5.21  Test chambers --  four  (minimum of three) for each concentration and
control.  Borosilicate glass 1000 ml beakers or modified Norberg and Mount
(1985) glass chambers used in the short-term inland silverside  test may be
used.  It is recommended that each chamber contain a minimum of 50 ml/larvae
and allow adequate depth of test solution  (5.0 cm).  To avoid potential
contamination from the air and excessive evaporation of test solutions during
the test, the chambers should be covered with  safety glass plates or sheet
plastic  (6 mm thick).

11.5.22  Beakers  --  six Class A, borosilicate  glass or non-toxic plasticware,
1000 ml  for making test solutions.

11.5.23  Wash bottles  -- for deionized water,  for washing  embryos from
substrates and  containers, and for rinsing small glassware and  instrument
electrodes and  probes.                                    i
                                                          I
11.5.24  Crystallization dishes, beakers,  culture dishes  (1 L), or equivalent
-- for incubating embryos.

11.5.25  Volumetric  flasks and graduated cylinders -- Class A,  borosilicate
glass  or non-toxic plastic labware,  10-1000 ml for making  test  solutions.

11.5.26  Separatory  funnels,  2-L --  two  to four for cultuiring Artemia nauplii.

11.5.27   Pipets,  volumetric  — Class A,  1-100  ml.         j

11.5.28   Pipets,  automatic  --  adjustable,   1-100  ml.


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11.5.29   Pipets,  serological  --  1-10  ml,  graduated.
11.5.30   Pipet  bulbs  and  fillers  -- PROPIPET®,  or  equivalent.
11.5.31   Droppers,  and  glass  tubing with  fire polished  edges,  4 mm  ID  --  for
transferring  larvae.
11.5.32   Siphon with  bulb and clamp --  for  cleaning  test  chambers.
11.5.33   Forceps  -- for transferring  dead larvae to  weighing boats.
11.5.34   NITEX® or  stainless  steel mesh sieves  (<  150 /zm,  500  A*ITI, 3 to 5  mm)
-- for collecting Artemia nauplii and fish  embryos,  and for spawning baskets,
respectively.   (Nitex®  is available from  Sterling  Marine  Products,  18  Label
Street, Montclair,  NJ 07042;  201-783-9800).
11.6  REAGENTS  AND  CONSUMABLE MATERIALS
11.6.1  Sample  containers --  for  sample shipment and storage (see Section 8,
Effluent  and  Receiving  Water  Sampling, Sample Handling, and Sample  Preparation
for Toxicity  Tests).
11.6.2  Data  sheets (one  set  per  test)  -- for data recording.
11.6.3  Vials,  marked --  18-24 per test,  containing  4%  formalin or  70%
ethanol,  to preserve larvae (optional).
11.6.4  Weighing pans,  aluminum -- 18-24  per test.
11.6.5  Tape, colored --  for  labelling test chambers.
11.6.6  Markers, waterproof -- for marking  containers,  etc.
11.6.7  Buffers, pH 4,  pH  7,  and  pH 10  (or  as per  instructions of instrument
manufacturer) -- for standards and calibration check (see USEPA Method 150.1,
USEPA, 1979b).
11.6.8  Membranes and filling  solutions for dissolved oxygen probe  (see USEPA
Method 360.1, USEPA, 1979b),  or reagents  -- for modified Winkler analysis.
11.6.9  Laboratory quality control samples  and standards -- for calibration of
the above methods.
11.6.10   Reference toxicant solutions --  see Section 4, Quality Assurance.
11.6.11   Ethanol (70%) or formalin (4%) -- for use as a preservative for the
fish larvae.
11.6.12  Reagent water -- defined as distilled or deionized water that does
not contain substances which are toxic to the test organisms.
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11.6.13  Effluent, receiving water, and dilution water -- see Section 7,
Dilution Water, and Section 8, Effluent and Receiving Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests.

11.6.13.1  Saline test and dilution water -- The salinity of the test water
must be in the range of 20 to 32%o.  The salinity should vary by no more than
± 2%o among the chambers on a given day.  If effluent and receiving water
tests are conducted concurrently, the salinities of these tests should be
similar.  This test is not recommended for salinities less than 20%o.
                                                          I
11.6.13.2  The overwhelming majority of industrial and sewage treatment
effluents entering marine and estuarine systems contain little or no
measurable salts.  Exposure of sheepshead minnow larvae to these effluents
will require adjustments in the salinity of the test solutions.  It is
important to maintain a constant salinity across all treatments.  In addition,
it may be desirable to match the test salinity with that of the receiving
water.  Two methods are available to adjust salinities -- a hypersaline brine
derived from natural seawater or artificial sea salts.

11.6.13.3  Hypersaline brine  (HSB):  (HSB) has several advantages that make it
desirable for use in toxicity testing. It can be made from any high quality,
filtered seawater by evaporation, and can be added to the effluent or to
deionized water to increase the salinity.  HSB derived from natural seawater
contains the necessary trace metals, biogenic colloids, and some of the
microbial components necessary for adequate growth, survival, and/or
reproduction of marine and estuarine organisms, and may be stored for
prolonged periods without any apparent degradation.  However, if 100%o HSB is
used as a diluent, the maximum concentration of effluent that can be tested
will be 80% at 20%o salinity  and 70% at 30%o salinity.

11.6.13.3.1  The  ideal container for making brine from natural seawater is one
that (1) has a high surface to volume ratio, (2) is made of a non-corrosive
material, and  (3) is easily cleaned (fiberglass containers are ideal).
Special care should be used to prevent any toxic materials from coming in
contact with the  seawater being used to generate the brine.  If a heater is
immersed directly into the seawater, ensure that the heater materials do not
corrode or leach  any substances that would contaminate the brine.  One
successful method used is a thermostatically controlled heat exchanger made
from fiberglass.  If aeration is used, use only oil-free air compressors to
prevent contamination.                                    |
                                                          i
11.6.13.3.2  Before adding seawater to the brine generator, thoroughly clean
the generator, aeration supply tube, heater, and any other materials that will
be  in  direct contact with the brine.  A good quality biodegradable detergent
should be used,  followed by several (at least three) thorough deionized water
rinses.

11.6.13.3.3  High quality  (and preferably high  salinity) seawater should  be
filtered to at least 10 urn before placing into the brine generator.   Water should
be collected on an incoming tide to minimize the  possibility of contamination.
                                       65

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11.6.13.3.4  The temperature of the seawater is increased slowly to 40°C.  The
water should be aerated to prevent temperature stratification and to increase
water evaporation.  The brine should be checked daily (depending on volume being
generated) to ensure that the salinity does not exceed 100%o and that the
temperature does not exceed 40°C.  Additional seawater may be added to the brine
to obtain the volume of brine required.

11.6.13.3.5  After the required salinity is attained, the HSB should be filtered
a second time through a 1 //m filter and poured directly into portable containers
(20-L cubitainers or polycarbonate water cooler jugs are suitable).  The
containers should be capped and labelled with the date the HSB was generated and
its salinity.  Containers of HSB should be stored in the dark and maintained at
room temperature until used.

11.6.13.3.6  If a source of HSB is available, test solutions can be made by
following the directions below.  Thoroughly mix together the deionized water and
HSB before adding the effluent.

11.6.13.3.7  Divide the salinity of the HSB by the expected test salinity to
determine the proportion of deionized water to brine.  For example, if the
salinity of the brine is 100%o and the test is to be conducted at 20%o, 100%o
divided by 20%o = 5.0.  The proportion of brine is 1 part in 5 (one part brine to
four parts deionized water).  To make 1 L of seawater at 20%o salinity from a HSB
of 100%o, divide 1 L (1000 mL) by 5.0.  The result, 200 ml, is the quantity of
brine needed to make 1 L of seawater.  The difference, 800 ml, is the quantity of
deionized water required.

11.6.13.4  Artificial sea salts:  FORTY FATHOMS® brand sea salts (Marine
Enterprises, Inc., 8755 Mylander Lane, Baltimore, MD 21204; 301-321-1189) have
been used successfully at the EMSL-Cincinnati to maintain and spawn sheephead
minnows and perform the larval survival and growth test (see Section 7, Dilution
Water).  HW MARINEMIX® (Hawaiian Marine Imports, Inc., P.O. Box 218687, Houston,
TX 77218; 713-492-7864) sea salts have been used successfully at the USEPA Region
6 Houston Laboratory to maintain and spawn sheephead minnows and perform the
larval growth and survival test and the embryo-larval survival and teratogenicity
test.  In addition, a slightly modified version of the GP2 medium (Spotte et a!.,
1984) has been successfully used to perform the sheepshead minnow survival and
growth test (Table 1).  Artifical sea salts may be used for culturing sheepshead
minnows and for the larval survival and growth test if the criteria for
acceptability of test data are satisfied (see Subsection 11.12).

11.6.13.4.1  Synthetic sea salts are packaged in plastic bags and mixed with
deionized water or equivalent.  The instructions on the package of sea salts
should be followed carefully, and the salts should be mixed in a separate
container -- not in the culture tank.  The deionized water used, in hydration
should be in the temperature range of 21-26°C.  Seawater made from artificial sea
salts is conditioned (Spotte, 1973; Spotte et a!., 1984; Bower, 1983) before it
is used for culturing or testing.  After adding the water, place an air stone in
the container, cover, and aerate the solution mildly for 24 h before use.
                                        66

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11.6.13.4.2  The GP2 reagent grade chemicals (Table 1) should be mixed with
deionized (DI) water or its equivalent in a container other than the culture
or testing tanks.  The deionized water used for hydration should be 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 NaHC03 in 500 ml of
deionized water.  Add 2.5 ml of this stock solution for each liter of the GP2
artificial seawater.

  TABLE 1.      REAGENT GRADE CHEMICALS USED IN THE PREPARATION OF GP2
                ARTIFICIAL SEAWATER FOR THE SHEEPSHEAD MINNOW, CYPRINODON
                VARIEGATUS, TOXICITY TEST1'2'3
Compound Concentration
NaCl 21.03
Na2S04 3.52
KC1 0.61
KBr 0.088
Na2B407 • 10 H20 0.034
MgCl2 • 6 H20 9.50
CaCl2 • 2 H20 1.32
SrCl2 • 6 H20 0.02
NaHC03 0.17
Amount (g)
Required for
20 L
420.6
70.4 .
12.2
1.76
0.68
190.0
26.4
0.400
3.40
   1 Modified GP2 from Spotte et al.  (1984).              ,
   2 The constituent salts and concentrations were taken  from USEPA (1990b).
     The salinity is 30.89 g/L.                           !
     GP2 can be diluted with deionized  (DI) water to the  desired test
     salinity.                                            ;
11.6.14  BRINE SHRIMP, ARTEMIA, NAUPLII -- for feeding cultures and test
organisms

11.6.14.1  Newly-hatched Artemia nauplii  (see USEPA, 1993a) are used as food
for sheepshead minnow larvae  in toxicity  tests and in the maintenance of
continuous stock cultures.  Although there are many commercial sources of
brine shrimp cysts, the Brazilian or Colombian strains are currently preferred
because the supplies examined have had low concentrations of chemical residues
and produce nauplii of suitably small size.  For commercial sources of brine
shrimp, Artemia, cysts, see Table 2 of Section 5, Facilities, Equipment, and
Supplies and Section 4, Quality Assurance, Subsection 4.8.

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11.6.14.2  Each new batch of Artemia cysts must be evaluated for size
(Vanhaecke and Sorgeloos, 1980, and Vanhaecke et al.,  1980) and nutritional
suitability  (Leger et al.,  1985, and Leger et al., 1986) against known
suitable reference cysts by performing a side-by-side  larval growth test using
the "new" and "reference" cysts.  The "reference" cysts used in the
suitability  test may be a previously tested and acceptable batch of cysts, or
may be obtained from the Quality Assurance Research  Division, Environmental
Monitoring Systems Laboratory, Cincinnati, OH 45268; 513-569-7325.  A sample
of newly-hatched Artemia nauplii from each new batch of cysts should be
chemically analyzed.  The Artemia cysts should not be  used if the
concentration of total organochlorine pesticides exceeds 0.15 /zg/g wet weight
or the total concentration  of organochlorine pesticides plus PCBs exceeds
0.30 ng/g wet weight.  (For analytical methods see USEPA, 1982.)

11.6.14.3  Artemia nauplii  are obtained as follows:

  1.  Add 1 L of seawater,  or a solution prepared by adding 35.0 g uniodized
     salt (NaCl)  or artificial  sea salts to 1 L of deionized water,  to a 2-L
     separatory funnel,  or equivalent.
  2.  Add 10 ml Artemia cysts to the separatory funnel and aerate for 24 h at
     27°C.   (Hatching time varies with incubation  temperature and the
     geographic strain of Artemia used (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.   To prevent mortality,  do not leave .
     the concentrated nauplii  at the bottom of the funnel  more than  10 min
     without aeration.
  4.  Drain the nauplii  into a beaker or funnel  fitted with a < 150 ^m NITEX®
     or stainless steel  screen,  and rinse with seawater or equivalent before
     use.

11.6.14.4  Testing Artemia  nauplii as food for toxicity test organisms.

11.6.14.4.1  The primary criterion for acceptability of each new supply of
brine shrimp cysts is the ability of the.nauplii to support good survival and
growth of the sheepshead minnow larvae (see Subsection 11.12).  The larvae
used to evaluate the suitability of the brine shrimp nauplii must be of the
same geographical origin,  species, and stage of development as those used
routinely in the toxicity tests.  Sufficient data to detect differences in
survival and growth should  be obtained by using three replicate test vessels,
each containing a minimum of 15 larvae, for each type of food.

11.6.14.4.2  The feeding rate and frequency, test vessels, volume of control
water, duration of the test, and age of the nauplii at the start of the test,
should be the same as used  for the routine toxicity tests.
                                                                        "new"
11.6.14.4.3  Results of the brine shrimp nutrition assay, where there are only
two treatments, can be evaluated statistically by use of a t test.  The
food is acceptable if there are no statistically significant differences in
the survival and growth of the larvae fed the two sources of nauplii.
                                      68

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11.6.15  TEST ORGANISMS, SHEEPSHEAD MINNOWS, CYPRINODON VARIEGATUS

11.6.15.1  Brood Stock                                    i

11.6.15.1.1  Adult sheepshead minnows for use as brood stock may be obtained
by seine in Gulf of Mexico and Atlantic coast estuaries, from commercial
sources, or from young fish raised to maturity in the laboratory.  Feral brood
stocks and first generation laboratory fish are preferred, to minimize
inbreeding.

11.6.15.1.2  To detect disease and to allow time for acute mortality due to
the stress of capture, field-caught adults are observed in the laboratory a
minimum of two weeks before using as a source of gametes.  Injured or diseased
fish are discarded.
                                                          i
11.6.15.1.3  Sheepshead minnows can be continuously cultured in the laboratory
from eggs to adults.  The larvae, juvenile, and adult fish should be kept in
appropriate size rearing tanks, maintained at ambient laboratory temperature.
The larvae should be fed sufficient newly-hatched Artemia naitplii daily to
assure that live nauplii are always present.  Juveniles are fed frozen adult
brine shrimp and a commercial flake food, such as TETRA SM-80®, available from
Tetra Sales (U.S.A.), 201 Tabor Rd, Morris Plains, NJ 07950;  800-526-0650, or
MARDEL AQUARIAN® Tropical Fish Flakes, available from Mardel Laboratories,
Inc., 1958 Brandon Court, Glendale Heights, IL 60139; 312-351-0606, or
equivalent.  Adult fish  (age one month) are fed flake food three or four times
daily, supplemented with frozen adult brine shrimp.

11.6.15.1.3.1  Sheepshead minnows reach sexual maturity in three-to-five
months after hatch, and  have an average standard length of approximately 27 mm
for females and 34 mm for males.  At this time, the males begin to exhibit
sexual dimorphism and initiate territorial behavior.  When the fish reach
sexual maturity and are  to be used for natural spawning, the temperature
should be controlled at  18-20°C.
                                                       .
11.6.15.1.4  Adults can  be maintained in natural or artificial seawater in a
flow-through or recirculating, aerated system consisting of an all-glass
aquarium, or a "Living Stream" (Figid Unit, Inc., 3214 Sylvania Ave, Toledo,
OH 43613; 419-474-6971), or equivalent.
               •
11.6.15.1.5  The system  is equipped with an undergravel or outside biological
filter of shells Spotte  (1973) or Bower (1983) for conditioning the biological
filter), or a cartridge  filter, such as a MAGNUM® Filter, available from
Carolina Biological Supply Co., Burlington, NC 27215; 800-334-5551, or an
EHEIM® Filter, available from Hawaiian Marine Imports Inc., P.O. Box 218687,
Houston, TX 77218; 713-492-7864, or equivalent, at a salinity of 20-30%o and a
photoperiod of 16 h light/8 h dark.

11.6.15.2  Obtaining Embryos for Toxicity Tests (See USEPA, 1978)
                                                          i
11.6.15.2.1  Embryos can be shipped to the laboratory from an outside source
or obtained from adults  held in the laboratory.  Ripe eggs can be obtained
either by natural spawning or by intraperitoneal. injection of the females with

                                      69

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human chorionic gonadotrophin (HCG) hormone, available from United States
Biochemical Corporation, Cleveland, OH 44128; 216-765-5000.  If the culturing
system for adults is temperature controlled, natural spawning can be induced.
Natural spawning is preferred because repeated spawnings can be obtained from
the same brood stock, whereas with hormone injection, the brood stock is
sacrificed in obtaining gametes.

11.6.15.2.2  It should be emphasized that the injection and hatching schedules
given below are to be used only as guidelines.  Response to the hormone varies
from stock to stock and with temperature.  Time to hatch and percent viable
hatch also vary among stocks and among batches of embryos obtained from the
same stock, and are dependent on temperature, DO, and salinity.  The
coordination of spawning and hatching is further complicated by the fact that,
even under the most ideal conditions, embryos spawned over a 24-h period may
hatch over a 72-h period.  Therefore, it is advisable (especially if natural
spawning is used) to obtain fertilized eggs over several days to ensure that a
sufficient number of newly hatched larvae (less than 24 h old) will be
available to initiate a test.

11.6.15.2.3  Forced Spawning

11.6.15.2.3.1  HCG is reconstituted with sterile saline or Ringer's solution
immediately before use.  The standard HCG vial contains 1,000 IU to be
reconstituted in 10 mL of saline.  Freeze-dried HCG which comes with
premeasured and sterilized saline is the easiest to use.  Use of a 50 IU dose
requires injection of 0.05 ml of reconstituted hormone solution.
Reconstituted HCG may be used for several weeks if kept in the refrigerator.

11.6.15.2.3.2  Each female is injected intraperitoneally with 50 IU HCG on two
consecutive days, starting at least 10 days prior to the beginning of a test.
Two days following the second injection, eggs are stripped from the females
and mixed with sperm derived from excised macerated testes.  At least ten
females and five males are used per test to ensure that there is a sufficient
number (400) of viable embryos.

11.6.15.2.3.3  HCG is injected into the peritoneal cavity, just below the
skin, using as small a needle as possible.  A 50 IU dose is recommended for
females approximately 27 mm in standard length.  A larger or smaller dose may
be used for fish which are significantly larger or smaller than 27 mm.  With
injections made on days one and two, females which are held at 25°C should be
ready for stripping on days 4, 5, and 6.  Ripe females should show pronounced
abdominal swelling, and release at least a few eggs in response to a gentle
squeeze.  Injected females should be isolated from males.  It may be helpful
if fish that are to be injected are maintained at 20°C before injection, and
the temperature raised to 25°C on the day of the first injection.

11.6.15.2.3.4  Prepare the testes immediately before stripping the eggs from
the females.  Remove the testes from three-to-five males.  The testes are
paired, dark grey organs along the dorsal midline of the abdominal cavity.  If
the head of the male is cut off and pulled away from the rest of the fish,
most of the internal organs can be pulled out of the body cavity, leaving the
testes behind.  The testes are placed in a few mL of seawater until the eggs
are ready.

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11.6.15.2.3.5  Strip the eggs from the females, into a dish containing 50-100
ml of seawater, by firmly squeezing the abdomen.  Sacrifice the females and
remove the ovaries if all the ripe eggs do not flow out freely.  Break up any
clumps of ripe eggs and remove clumps of ovarian tissue and underripe eggs.
Ripe eggs are spherical, approximately 1 mm in diameter, and almost clear.

11.6.15.2.3.6  While being held over the dish containing the eggs, the testes
are macerated in a fold of NITEX® screen (250-500 n\n mesh) dampened with
seawater.  The testes are then rinsed with seawater to remove the sperm from
tissue, and the remaining sperm and testes are washed into the dish.  Let the
eggs and milt stand together for 10-15 min, swirling occasionally.

11.6.15.2.3.7  Pour the contents of the dish into a beaker, and insert an
airstone.  Aerate gently, such that the water moves slowly over the eggs, and
incubate at 25°C for 60-90 min.  After incubation, wash the eggs on a NITEX®
screen and resuspend them in clean seawater.  Examine the eggs period.ically
under a dissecting microscope until they are in the 2-8 cell stage.  (The
stage at which it is easiest to tell the developing embryos from the abnormal
embryos and unfertilized eggs; see Figure 1).  The eggs cam then be gently
rolled on a NITEX® screen and culled (see Section 6, Test Organisms).
                                                          I

11.6.15.2.4  Natural Spawning

11.6.15.2.4.1  Cultures of adult fish to be used for spawning are maintained
at 18-20°C until embryos are required.  When embryos are required, raise the
temperature to 25°C in the morning, seven or eight days before the beginning
of a test.  That afternoon, transfer the adult fish (generally, at least five
females and three males) to a spawning chamber (approximately, 20 x 35 x 22 cm
high; USEPA, 1978), which is a basket constructed of 3-5 mm NITEX® mesh, made
to fit a 57-L (15 gal) aquarium.  Spawning generally will begin within 24 h or
less.  Embryos will fall through the bottom of the basket and onto a
collecting screen (250-500 urn mesh) or tray below the basket.  Allow the
embryos to collect for 24 h.  Embryos are washed from the screen, checked for
viability, and placed in incubation dishes.  Replace the screens until a
sufficient number of embryos have been collected.  One-to-three spawning
aquaria can be used to collect the required number of embryos to run a
toxicity test.  To help keep the embryos clean, the adults are fed while the
screens are removed.

11.6.15.2.5  Incubation                                   \

11.6..15.2.5.1  Four hours post-fertilization, the embryos obtained by natural
or forced spawning are rolled gently with a finger on a 250-500 /m Nitex®
screen to remove excess fibers and tissue.  The embryos have adhesive threads
and tend to adhere to each other.  Gentle rolling on the screen facilitates
the culling process described below.  To reduce fungal conrtamination of the
newly spawned embryos after they have been manipulated, they should be placed
in a 250 fan sieve and briskly sprayed with seawater from a squeeze bottle.
                                      71

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Figure 1.     Embryonic development of sheepshead minnow,  Cypn'nodon
              variegatus:  A. Mature unfertilized egg,  snowing attachment
              filaments and micropyle, X33;  B.  Blastodisc  fully developed;
              C,D. Blastodisc, 8 cells; E. Blastoderm,  16  cells;
              F. Blastoderm, late cleavage stage; G.  Blastoderm with germ ring
              formed,  embryonic shield developing; H.  Blastoderm covers over
              3/4 of yolk, yolk noticeably constricted;  I.  Early embryo.   From
              Kuntz (1916).

                                      72

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Figure 1.     Embryonic development of sheepshead minnow, Cypn'nodon
              variegatus:  J. Embryo 48 h after fertilization, now segmented
              throughout, pigment on yolk sac and body, otoliths formed;
              K. Posterior portion of embryo free from yolk and moves freely
              within egg membrane, 72 h after fertilization; L. Newly hatched
              fish, actual length 4 mm; M. Larval fish 5 days after hatching,
              actual length 5 mm; N. Young fish 9 mm in length; 0. Young fish
              12 mm in length (CONTINUED).  From Kuntz (1916).

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11.6.15.2.5.2  Under a dissecting microscope, separate and discard abnormal
embryos and unfertilized eggs.  While they are checked, the embryos are
maintained in seawater at 25°C.  The embryos should be in Stages C-6,
Figure 1.

11.6.15.2.5.3  If the test is prepared with four replicates of 15 larvae at
each of six treatments (five effluent concentrations and a control), and the
combined mortality of eggs and larvae prior to the start of the test is less
than 20%, approximately 400 viable embryos are required at this stage.

11.6.15.2.5.4  Embryos are demersal.  They should be aerated and incubated at
25"C, at a salinity of 20-30%o and a 16-h photoperiod.  The embryos can be
cultured in either a flow-through or static system, using aquaria or
crystallization dishes.  However, if the embryos are cultured in dishes, it is
essential that aeration and daily water changes be provided, and the dishes be
covered to reduce evaporation that may cause increased salinity.  One-half to
three-quarters of the seawater from the culture vessels can be poured off and
the incubating embryos retained.  Embryos cultured in this manner should hatch
in six or seven days.

11.6.15.2.5.5  At 48 h post-fertilization, embryos are examined under a
microscope to determine development and survival.  Embryos should be in Stages
I and J, Figure 1.  Discard dead embryos.  Approximately 360 viable embryos
are required at this stage.

11.7  EFFLUENT AND RECEIVING WATER COLLECTION, PRESERVATION, AND STORAGE

11.7.1  See Section 8, Effluent and Receiving Water Sampling, Sample Handling,
and Sample Preparation for Toxicity Tests.

11.8  CALIBRATION AND STANDARDIZATION

11.8.1  See Section 4, Quality Assurance.

11.9  QUALITY CONTROL

11.9.1  See Section 4, Quality Assurance.

11.10  TEST PROCEDURES

11.10.1  TEST SOLUTIONS

11.10.1.1  Receiving Waters

11.10.1.1.1  The sampling point is determined by the objectives of the test.
At estuarine and marine sites, samples are usually collected at mid-depth.
Receiving water toxicity is determined with samples used directly as collected
or with samples passed through a 60 urn NITEX® filter and compared without
dilution, against a control.   Using four replicate chambers per test, each
containing 500-750 ml, and 400 ml for chemical analysis, would require
approximately 2.4-3.4 L or more of sample per test per day.


                                      74

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11.10.1.2  Effluents

11.10.1.2.1  The selection of the effluent test concentrations should be based
on the objectives of the study.  A dilution factor of 0.5 is commonly used.  A
dilution factor of 0.5 provides precision of ± 100%, and allows for testing of
concentrations between 6.25% and 100% effluent using only five effluent
concentrations (6.25%, 12.5%, 25.0%, 50.0%, and 100%).  Test precision shows
little improvement as dilution factors are increased beyond 0.5 and declines
rapidly if smaller dilution factors are used.  Therefore, USEPA recommends the
use of the > 0.5 dilution factor.  If 100%o HSB is used as a diluent, the
maximum concentration of effluent that can be tested will,be 80% at 20%o
salinity and 70% at 30%o salinity.

11.10.1.2.2  If the effluent is known or suspected to be highly toxic, a lower
range of effluent concentrations should be used (such as 25%, 12.5%, 6.25%,
3.12%, and 1.56%).  If a high rate of mortality is observed during the first
l-to-2 h of the test, additional dilutions at the lower range of effluent
concentrations should be added.

11.10.1.2.3  The volume of effluent required to initiate the test and for
daily renewal of four replicates (minimum of three) per concentration for five
concentrations of effluent and a control, each containing 750 ml of test
solution, is approximately 5 L.  Prepare enough test solution (approximately
3400 ml) at each effluent concentration to provide 400 ml additional volume
for chemical analyses (Table 2).

11.10.1.2.4  The salinity of effluent and receiving water tests for sheepshead
minnows should be between 20%o and 30%o.  If concurrent effluent and receiving
water testing occurs, the effluent test salinity should closely approximate
that of the receiving water test.  If an effluent is tested alone, select a
salinity between 20%o and 30%o, whichever comes closest to the salinity of the
receiving waters.  Table 2 illustrates the quantities of effluent, artificial
sea salts, hypersaline brine, or seawater needed to prepare 3 L of test
solution at each treatment level for tests performed at 20%o salinity.
                                                         i
11.10.1.2.5  Just prior to test initiation (approximately 1 h), the
temperature of sufficient quantity of the sample to make the test solutions
should be adjusted to the test temperature (25 ± 1°C) and maintained at that
temperature during the addition of dilution water.
                                                         i
11.10.1.2.6  Higher effluent concentrations (i.e., 25%, 50%, and 100%) may
require aeration to maintain adequate dissolved oxygen concentrations.
However, if one solution is aerated, all concentrations must be aerated.
Aerate effluent as it warms and continue to gently aerate test solutions in
the test chambers for the duration of the test.

11.10.1.2.7  Effluent dilutions should be prepared for all replicates in each
treatment in one beaker to minimize variability among the replicates.  The
test chambers are labelled with the test concentration and replicate number.
Dispense into the appropriate effluent dilution chamber.
                                      75

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TABLE 2.  PREPARATION OF TEST SOLUTIONS AT A SALINITY OF 20%o, USING 20%o
          SALINITY DILUTION WATER PREPARED FROM NATURAL SEAWATER,
          HYPERSALINE BRINE, OR ARTIFICIAL SEA SALTS
Effluent
Solution
1
2
3
4
5
Control
Total
Effluent
Cone. (%)
1001'2
50
25
12.5
6.25
0.0

Solutions To
Volume of
Effluent Solution
6800 mL
3400 mL Solution 1
3400 mL Solution 2
3400 mL Solution 3
3400 mL Solution 4


Be Combined
Volume of Diluent
Seawater (20%o)
	
+ 3400 mL
+ 3400 mL
+ 3400 mL
+ 3400 mL
3400 mL
17000 mL
  This  illustration  assumes:  (1)  the  use  of  750  mL  of  test  solution  in
  each  of four replicates  and 400 mL  for  chemical analysis  (total  of
  3,400 mL)  for the  control  and each  of five concentrations of  effluent
   (2) an effluent  dilution factor of  0.5,  and (3) the  effluent  lacks
  appreciable  salinity.  A sufficient initial  volume  (6,800 mL)  of
  effluent is  prepared  by  adjusting the salinity to the  desired  level.
  In this example, the  salinity is adjusted  by adding  artificial  sea
  salts to the 100%  effluent,  and preparing  a serial dilution using  20%o
  seawater (natural  seawater,  hypersaline brine, or artificial  seawater).
  Following addition of salts, the effluent  is stirred for  1 h  to  ensure
  that  the salts have dissolved.   The salinity of the  initial 6,800  mL of
  100%  effluent is adjusted  to 20%o by adding 136 g of dry  artificial sea
  salts (FORTY FATHOMS®).   Test concentrations are  then  made by  mixing
  appropriate  volumes of salinity-adjusted effluent and  20%o salinity
  dilution water to  provide  6,800 mL  of solution for each concentration.
  If hypersaline brine  alone (100%o)  is used to  adjust the  salinity  of
  the effluent,  the  highest  concentration of effluent  that  could be
  achieved would be  80% at 20%o salinity.  When  dry sea  salts are  used to
  adjust the salinity of the effluent,  it may be desirable  to use  a
  salinity control prepared  under the same conditions  and used  to
  determine survival  and growth.
  The same procedures would  be followed in preparing test concentrations
  at other salinities between 20%o and 30%o:  (1) the salinity of the bulk
  (initial)  effluent sample  would be  adjusted to the appropriate salinity
  using artificial sea  salts or hypersaline  brine,  and (2)  the  remaining
  effluent concentrations  would be prepared  by serial  dilution,  using a
  large batch  (17,000 mL)  of seawater for dilution  water, which  had  been
  prepared at  the  same  salinity as the effluent, using natural  seawater,
  or hypersaline or  artificial sea salts  and deionized water.
                                    76

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11.10.1.3  Dilution Water

11/10.1.3.1  Dilution water may be uncontaminated natural seawater (receiving
water), MSB prepared from natural seawater, or artificial seawater prepared
from FORTY FATHOMS® or 6P2 sea salts (see Table 1 and Section 7, Dilution
Water).  Other artificial sea salts may be used for culturing sheepshead
minnows and for the larval survival and growth test if the control criteria
for acceptability of test data are satisfied.

11.10.2  START OF THE TEST
                                                         I
11.10.2.1  Tests should begin as soon as possible, preferably within 24 h
after sample collection.  The maximum holding time following retrieval of the
sample from the sampling device should not exceed 36 h for off-site toxicity
tests unless permission is granted by the permitting authority.  In no case
should the sample be used in a test more than 72 h after sample collection
(see Section 8, Effluent and Receiving Water Sampling, Sample Handling, and
Sample Preparation for Toxicity Tests).

11.10.2.2  If the embryos have been incubating at 25°C, 30%o salinity, and a
16-h photoperiod, for 5 to 6 days with aeration and daily water renewals,
approximately 24 h prior to hatching, the salinity of the seawater in the
incubation chamber may be reduced from 30%> to the test salinity, if lower
than 30%o.  In addition to maintaining good water quality, reducing the
salinity and/or changing the water may also help to initiate hatching over the
next 24 h.  A few larvae may hatch 24 h ahead of the majority.  Remove these
larvae and reserve them in a separate dish, maintaining the same culture
conditions.  It is preferable to use only the larvae that hatch in the 24 h
prior to starting the test.  However, if sufficient numbers of larvae do not
hatch within the 24-h period, the larvae that hatch prior to 24 h are added to
the test organisms.  The test organisms are then randomly selected for the
test.  When eggs or larvae must  be shipped to the test site from a remote
location,  it may be necessary to use larvae older than 24-h because of the
difficulty in coordinating test  organism shipments with field operations.
However, in the latter case, the larvae should not be more than 48-h old at
the start  of the test and should all be within 24-h of the same age.

11.10.2.3  Label the test chambers with a marking pen.  Use of color coded
tape to  identify each treatment  and replicate.  A minimum of five effluent
concentrations and a control are used for each test.  Each treatment
(including controls) should have four  (minimum of three) replicates.  For
exposure chambers, use  1000 ml beakers, non-toxic disposable plasticware, or
glass  chambers with a sump area  as illustrated in the inland silverside test
method  (see Section 13).
                                                         i
11.10.2.4  Prepare the  test solutions and  add to the test chambers.

11.10.2.5  The test is  started by  randomly placing larvae from the common pool
into each  test chamber  until each  chamber  contains 15  (minimum of 10) larvae,
for a  total of 60  larvae  (minimum  of 30) for each concentration  (see
Appendix A).  The  amount  of water  added to the chambers when transferring the
larvae  should  be kept to  a minimum to avoid  unnecessary dilution  of the test
concentrations.

                                      77

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 11.10.2.6   The chambers  may be  placed  on  a  light  table  to  facilitate  counting
 the  larvae.

 11.10.2.7   Randomize  the position  of the  test  chambers  at  the  beginning  of  the
 test (see  Appendix A).   Maintain the chambers  in  this configuration throughout
 the  test.   Preparation of a position chart  may be helpful.

 11.10.3  LIGHT,  PHOTOPERIOD,  SALINITY,  AND  TEMPERATURE

 11.10.3.1   The light  quality and intensity  should be at ambient  laboratory
 levels, which  is approximately  10-20 ME/m/s,  or  50 to  100 foot candles
 (ft-c), with a photoperiod  of 16 h light  and 8 h  darkness.  The  water
 temperature in the test  chambers should be  maintained at 25 ±  1°C.  The  test
 salinity should  be in the range of 20  to  30%o  to  accommodate receiving waters
 that may fall  within  this range.  Conduct of this test  at  salinities  less than
 20%o may cause an  unacceptably  low growth response and  thereby invalidate the
 test.  The salinity should  vary by no  more  than ± 2%o among the  chambers on a
 given day.  If effluent  and receiving  water tests are conducted  concurrently,
 the  salinities of  these  tests should be similar.

 11.10.4  DISSOLVED OXYGEN (DO)  CONCENTRATION

 11.10.4.1  Aeration may  affect  the toxicity of effluents and should be used
 only as a  last resort to  maintain a  satisfactory  DO.  The DO should be
 measured on new  solutions at  the start  of the  test (Day 0) and before daily
 renewal of test  solutions on  subsequent days.  The DO should not fall below
 4.0  mg/L (see  Section 8,  Effluent and  Receiving Water Sampling, Sample
 Handling,  and  Sample  Preparation for Toxicity  Tests).   If  it is necessary to
 aerate, all treatments and  the control  should  be  aerated.  The aeration rate
 should not exceed  100 bubbles/min, using  a  pipet  with a 1-2 mm orifice, such
 as a 1-mL  KIMAX® serological  pipet No.  37033,  or  equivalent.  Care should be
 taken to ensure  that  turbulence resulting from aeration does not cause undue
 stress on  the  fish.

 11.10.5  FEEDING

 11.10.5.1  Artemia  nauplii  are prepared as  described above.

 11.10.5.2  Sheepshead minnow  larvae  are fed newly-hatched (less than 24-h old)
Artemia nauplii  once  a day  from hatch day 0 through day 6; larvae are not fed
on day 7.  Feed  0.10  g nauplii per test chamber on days 0-2, and 0.15 g
nauplii per test chamber  on days 3-6.   Equal amounts of Artemia nauplii must
be added to each replicate  test chamber to minimize the variability of larval
weight.  Sufficient numbers of nauplii   should  be  fed to ensure that some
remain alive overnight in the test chambers.  An  adequate but not excessive
amount should  be provided to  each replicate on a daily  basis.   Feeding
excessive  amounts of  nauplii will  result  in a depletion in DO to a lower than
acceptable level (below 4.0 mg/L).   Siphon as much of the uneaten Artemia
nauplii as possible from  each chamber daily to ensure that the larvae
principally eat newly hatched nauplii.
                                      78

-------
11.10.5.3  On days 0-2, weigh 4 g wet weiight or pipette 4 ml of concentrated,
rinsed Artemia nauplii for a test with five treatments and a control.
Resuspend the 4 g Artemia in 80 mL of natural or artificial seawater .in a 100
ml beaker.  Aerate or swirl Artemia to maintain a thoroughly mixed suspension
of nauplii.  Dispense 2 ml Artemia suspension by pipette or adjustable syringe
to each test chamber.  Collect only enough Artemia in the pipette or syringe
for one test chamber or settling of Artemia may occur, resulting in unequal
amounts of Artemia being distributed to the replicate test chambers.

11.10.5.4  On days 3-6, weigh 6 g wet weight or pipette 6 ml Artemia
suspension for a test with five treatments and a control.  Resuspend the 6 g
Artemia in 80 ml of natural or artificial seawater in a 100 ml beaker.  Aerate
or swirl as 2 ml is dispensed to each test chamber.
                                                         i
11.10.5.5  If the survival rate in any test replicate on any day falls below
50%, reduce the volume of Artemia added to that test chamber by one-half
(i.e., from 2 ml to  1 ml)  and continue feeding one-half the volume through
day 6.  Record the time of feeding on data sheets  (Figure  2).

11.10.6  DAILY CLEANING OF TEST CHAMBERS
                                                         i

11.10.6.1  Before the  daily  renewal  of test  solutions, uneaten and dead
Artemia, dead fish larvae, and other debris  are removed from the bottom of the
test chambers with a siphon  hose.  As much of  the  uneaten  Artemia as possible
should be  siphoned from each  chamber to  ensure that the larvae principally eat
newly hatched nauplii.  Alternately, a large pipet (50 mL), fitted with a
safety pipet  filler  or rubber bulb,  can  be used.   Because  of their  small size
during the first  few days  of the  tests,  larvae are easily  drawn  into the
siphon tube when  cleaning  the test chambers.   By placing the test chambers on
a light  box,  inadvertent  removal  of  live larvae can be greatly reduced because
they  can  be more  easily  seen.   If the water  siphoned  from  the test  chambers  is
collected in  a white plastic tray, the live  larvae caught  in the  siphon can  be
retrieved and returned to  the appropriate test chamber.  Any incidence of
removal  of live  larvae from  the  test chambers  by the  siphon during  cleaning,
 and subsequent  return to  the chambers, should  be noted  in  the test  records.

 11.10.7   OBSERVATIONS DURING THE TEST

 11.10.7.1  Routine  Chemical  and  Physical  Determinations
                                                         I
 11.10.7.1.1   DO is  measured at the beginning and  end  of each  24-h exposure
 period in one test  chamber at each test  concentration and in  the control.

 11.10.7.1.2  Temperature,  pH, and salinity are measured at the  end of each
 24-h exposure period in one test chamber at each  test concentration and  in the
 control.  Temperature should also be monitored continuously,  observed and
 recorded daily for at least two locations in the environmental  control  system
 or the samples.   Temperature should be measured in a sufficient number of test
 vessels at least at the end of the test to determine the temperature variation
 in the environmental chamber.

 11.10.7.1.3  The pH is measured in the effluent sample each day.
                                                         I
                                       79

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-------
 11.10.7.1.4  Record all  the measurements on the data sheet  (Figure  2).

 11.10.7.2  Routine Biological  Observations

 11.10.7.2.1  The number  of live larvae in each  test  chamber are  recorded  daily
 (Figure 7), and the dead larvae are discarded.

 11.10.7.2.2  Protect the larvae from unnecessary disturbance during the test
 by  carrying out the daily test observations,  solution  renewals,  and removal of
 dead  larvae,  carefully.   Make  sure  the larvae remain immersed during the
 performance of the above operations.

 11.10.8  TEST SOLUTION RENEWAL

 11.10.8.1  The test solutions  are renewed daily using  freshly prepared
 solution,  immediately after cleaning  the test chambers.   For on-site toxicity
 studies,  fresh effluent  and receiving water samples  used  in  toxicity tests
 should  be collected daily,  and no more than 24  h should elapse between
 collection of the sample and use in the test  (see Section 8,  Effluent and
 Receiving Water Sampling,  Sample Handling,  and  Sample  Preparation for Toxicity
 Tests).   For  off-site tests, a minimum of three samples must  be  collected,
 preferably on days one,  three,  and  five.  Maintain the samples at 4°C until
 used.

 11.10.8.2  For test solution renewal,  the water level  in each chamber is
 lowered to a  depth of 7  to  10  mm, which leaves  15 to 20% of  the  test solution.
 New test  solution (750 mL)  should be  added  slowly by pouring  down the side of
 the test  chamber to avoid  excessive turbulence  and possible  injury  to the
 larvae.

 11.10.9   TERMINATION  OF  THE TEST

 11.10.9.1   The test is terminated after 7-d of  exposure.  At  test termination,
 dead larvae are removed  and discarded.   The surviving larvae  in  each test
 chamber (replicate)  are  counted and immediately  prepared as a group  for dry
 weight  determination, or are preserved  as a group in 4% formalin or  70%
 ethanol.   Preserved organisms  are dried and weighed within 7 days.   For
 safety, formalin  should  be  used under  a hood.

 11.10.9.2   For immediate drying and weighing, siphon or pour live larvae onto
 a 500 urn mesh  screen  in  a large beaker  to retain  the larvae and  allow Artemia
 and debris  to  be  rinsed  away.  Rinse the  larvae with deionized water to wash
 away salts  that might contribute to the dry weight.   Sacrifice the larvae in
 an  ice bath of  deionized water.

 11.10.9.3   Small  aluminum weighing pans can be used to dry and weigh the
 larvae.  Mark  for  identification an appropriate number of small aluminum
weighing pans  (one  per replicate).   Weigh to the nearest 0.01 rag, and record
 the weights (Figure 3).
                                      82

-------
Test Dates:
                        Species:.
     Pan
     No.
Cone.
  &
 Reo.
Initial
  Wt.
  (mq)
Final
 Wt.
 (nig)
Diff.
 (mg)
 No.
.arvae
Av. Wt./
 Larvae
  (mg)
 Figure 3.     Data  form  for the  sheepshead minnow,  Cyprinodon van'egatus,
               larval  survival  and growth test.   Dry weights of larvae (from
               USEPA 1987b).
                                       83

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 11.10.9.4  Immediately prior to drying,  rinse the preserved larvae in
 distilled water.   The rinsed larvae from each test chamber is  transferred  to  a
 tared weighing pan and dried at 60°C for 24 h or at 105°C for  a minimum of 6
 h.   Immediately upon removal from the drying oven,  the weighing pans are
 placed in a desiccator until weighed, to prevent the absorption of moisture
 from the air.   Weigh to the nearest 0.01 mg all  weighing  pans  containing dried
 larvae'and subtract the tare weight to determine the dry  weight of larvae  in
 each replicate.   Record the weights (Figure 3).   For each test chamber,  divide
 the  final  dry  weight by the number of original  larvae in  the test  chamber  to
 determine the  average individual  dry weight,  and record (Figure 3).  For the
 controls,  also calculate the mean weight' per surviving fish in the test
 chamber to evaluate if weights  met test  acceptable  criteria (see Section 12).
 Complete the summary data sheet (Figure  4)  after calculating the average
 measurements and  statistically  analyzing the dry weights  and percent survival.
 Average dry weights should be expressed  to  the  nearest 0.001 mg.

 11.11   SUMMARY OF TEST CONDITIONS AND TEST  ACCEPTABILITY  CRITERIA

 11.11.1   A summary of test conditions and test  acceptability criteria  is
 listed in  Table 3.

 11.12   ACCEPTABILITY OF TEST RESULTS

 11.12.1   The tests are acceptable if (1)  the  average survival  of control
 larvae equals  or  exceeds 80%, and (2)  the average dry weight per surviving
 unpreserved  control  larvae is equal  to or greater than  0.60 mg,  or (3) the
 average  dry  weight per surviving  preserved  control  larvae  is equal to or
 greater  than 0.50  mg.   The above  minimum weights  presume  that  the  age of the
 larvae at  the  start  of the test  is  less  than  or  equal  to  24 h.

 11.13  DATA  ANALYSIS

 11.13.1  GENERAL

 11.13.1.1  Tabulate  and  summarize  the data.   A sample  set  of survival and
 growth response data  is  listed  in  Table  4.

 11.13.1.2  The endpoints  of  toxicity  tests  using the  sheepshead minnow larvae
 are  based  on the adverse  effects  on  survival  and growth.   The  LC50, the  IC25,
 and  the  IC50 are calculated  using  point  estimation techniques  (see Section 9,
 Chronic  Toxicity Test  Endpoints and  Data  Analysis).   LOEC  and NOEC values,  for
 survival and growth,  are  obtained  using  a hypothesis  testing approach such f,as
 Dunnett's  Procedure  (Dunnett, 1955)  or Steel's Many-one Rank Test  (Steel,
 1959; Miller, 1981)(see  Section 9).  Separate analyses are  performed for the
 estimation of the  LOEC and NOEC endpoints and for the estimation of the  LC50,
 IC25 and IC50.   Concentrations at which there is no survival in any of the
test chambers are excluded from the statistical analysis of the NOEC and LOEC
for survival and growth,  but included in the estimation of the LC50, IC25 and
 IC50.  See the Appendices  for examples of the manual computations,  program
listings, and examples of data input and program output.
                                      84

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Test Dates:
Species:
Effluent Tested:
TREATMENT
NO. LIVE
LARVAE
SURVIVAL
(0/\
(/<,)
MEAN DRY VIT/
LARVAE (MG)'
± SD
SI6NIF. DIFF.
FROM CONTROL
(o)
MEAN
TEMPERATURE
(°C)
± SD
MEAN SALINITY
%0
± SD
AVE DISSOLVED
OXYGEN
(MG/L) ± SD
















































COMMENTS:
Figure 4.     Data form for the sheepshead minnow, Cypn'nodon variegatus,
              larval   survival  and growth test.  Summary of test results (from
              USEPA,  1987b).
                                      85

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TABLE 3.      SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA
              FOR THE SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, LARVAL
              SURVIVAL AND GROWTH TEST WITH EFFLUENTS AND RECEIVING WATERS
1.  Test type:
2.  Salinity:

3.  Temperature:
4.  Light quality:
5.  Light intensity:
6.  Photoperiod:
7.  Test chamber size:
8.  Test solution volume:

9.  Renewal of test solutions:
10. Age of test organisms:.

11. No. larvae per test chamber:
12. No. replicate chambers per
    concentration:
13. No. larvae per concentration:
14. Source of food:

15. Feeding regime:
 16.  Cleaning:

 17.  Aeration:
Static renewal
20%o to 32%o (± 2%o of the selected
test salinity)
25 ± 1'C
Ambient laboratory illumination
10-20 ME/m2/s (50-100 ft-c)  (ambient
laboratory levels)
16 h light, 8 h darkness
600 mL - 1 L beakers or equivalent
500-750 mL/replicate (loading and DO
restrictions must be met)
Daily
Newly hatched larvae (less than 24 h
old; 24-h range in age)
15 (minimum of 10)
4 (minimum of 3)

60 (minimum of 30)
Newly hatched Artemia nauplii, (less
than 24-h old)
Feed once a day 0.10 g wet weight
Artemia nauplii per replicate on Days
0-2; Feed 0.15 g wet weight Artemia
nauplii per replicate on Days 3-6
Siphon daily, immediately before test
solution renewal and feeding
None, unless DO falls below 4.0 mg/L,
then aerate all chambers.  Rate should
be less than 100 bubbles/min
                                     86

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TABLE 3.      SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA
              FOR THE SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, LARVAL
              SURVIVAL AND GROWTH TEST WITH EFFLUENTS AND RECEIVING WATERS
              (CONTINUED)
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:
Uncontaminated source of natural
seawater; deionized water mixed with
hypersaline brine or artificial sea
salts (HW Marinemix®, FORTY FATHOMS®,
GP2 or equivalent)
Effluent:  Minimum of 5 and a control
Effluents:  > 0.5
Receiving waters:

7 days
None, or > 0.5
Survival and growth (weight)
80% or greater survival in controls;
average dry weight per surviving
organism in control chambers should be
0.60 mg or greater, if unpreserved, or
0.50 mg or greater after no more than
7 days in 4% formalin or 70% ethanol

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 for Toxicity Tests,
Subsection 8.5.4)
6  L per day
                                     87

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  TABLE 4.       SUMMARY OF  SURVIVAL AND  GROWTH  DATA  FOR SHEEPSHEAD MINNOW,
                 CYPRINODON,VARIEGATUS, LARVAE EXPOSED TO AN  EFFLUENT FOR
                 SEVEN  DAYS1
Effl.
Cone.
(%)
0.0
6.25
12.5
25.0
50.0
100.0
Proportion of
Survival in Replicate
Chambers
A
1.0
1.0
1.0
1.0
0.8
0.0
B
1.0
1.0
1.0
1.0
0.8
0.0
C
1.0
0.9
1.0
1.0
0.7
0.0
D
1.0
1.0
1.0
0.8
0.6
0.0
Mean
Prop.
Surv
1.00
0.98
1.00
0.95
0.73
0.00
Avg Dry Wgt (mg)


1
1
1
1
0

Repl
A
.29
.27
.32
.29
.62
--
icate
B
1.32
1.00
1.37
1.33
0.56
--
In
Chambers
C
1.59
0.97
1.35
1.20
0.46
--
D
1.27
0.97
1.34
0.94
0.46
--
Mean
Dry Wgt
(mg)
1.368
1.053
1.345
1.190
0.525
--
  1  Four replicates of 10 larvae each.
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 SHEEPHEAD MINNOW, CYPRINODON VARIEGATUS
         SURVIVAL DATA

11.13.2.1  Formal statistical analysis of the survival data is outlined in
Figures 5 and 6.  The response used in the analysis is the proportion of
animals surviving in each test or control chamber.  Separate analyses are
performed for the estimation of the NOEC and LOEC endpoints and for the
estimation of the LC50 endpoint.  Concentrations at which there is no survival
in any of the test chambers are excluded from statistical analysis of the NOEC
and LOEC, but included in the estimation of the 1C, EC, and LC endpoint.

11.13.2.2  For the case of equal numbers of replicates across all
concentrations and the control, the evaluation of the NOEC and LOEC endpoints
is made via a parametric test, Dunnett's Procedure, or a nonparametric test,
Steel's Many-one Rank Test, on the arc sine square root transformed data.
                                      88

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         STATISTICAL ANALYSIS OF SHEEPSHEAD MINNOW LARVAL
                      SURVIVAL AND GROWTH TEST

                     SURVIVAL HYPOTHESIS TESTING
                               SURVIVAL DATA
                           PROPORTION SURVIVING
                                 ARC SINE
                             TRANSFORMATION
                            SHAPIRO-WILICS TEST
                  NORMAL DISTRIBUTION
                                                NON-NORMAL DISTRIBUTION
       HOMOGENEOUS
          VARIANCE
                              BARTLETTSTEST
                              HETEROGENEOUS
                                 VARIANCE
              EQUAL NUMBER OF
                REPLICATES?
          NO
YES
    T-TESTWITH
    BONFERRONI
    ADJUSTMENT
I YES
                      EQUAL NUMBER OF
                        REPLICATES?
                                                             NO
        STEEL'S MANY-ONE
           RANK TEST
             WILCOXON RANK SUM
                  TEST WITH
            BONFERRONI ADJUSTMENT
                             ENDPOINT ESTIMATES
                                NOEC, LOEC
Figure  5.     Flowchart for  statistical analysis  of the sheepshead  minnow,
             Cyprinodon variegatus, larval  survival data by hypothesis
             testing.

                                    89

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       STATISTICAL ANALYSIS OF SHEEPSHEAD MINNOW LARVAL
                     SURVIVAL AND GROWTH TEST

                     SURVIVAL POINT ESTIMATION
MORTALITY DATA
#DEAD
\
i
       TWO OR MORE
    PARTIAL MORTALITIES?
 NO
             YES
      IS PROBIT MODEL
       APPROPRIATE?
     (SIGNIFICANT X2 TEST)
NO
             YES
ONE OR MORE
PARTIAL MORTALITIES?
i
YES
r
NO

                              GRAPHICAL METHOD
                                   LC50
       PROBIT METHOD
     ZERO MORTALITY IN THE
     LOWEST EFFLUENT CONC.
    AND 100% MORTALITY IN THE
    HIGHEST EFFLUENT CONC.?
NO
                                      YES
SPEARMAN-KARBER
METHOD
i
r
                                                    TRIMMED SPEARMAN-
                                                      KARBER METHOD
                                LC50AND95%
                                CONFIDENCE
                                  INTERVAL
Figure 6,     Flowchart for statistical analysis  of the sheepshead minnow,
             Cyprinodon van'egatus,  larval survival data by  point estimation,

                                   90

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Underlying assumptions of Dunnett's Procedure, normality and homogeneity of
variance, are formally tested.  The test for normality is the Shapiro-Milk's
Test, and Bartlett's Test is used to test for homogeneity of variance.  If
either of these tests fails, the nonparametric test, Steel's Many-one Rank
Test, is used to determine the NOEC and LOEC endpoints.  If the assumptions of
Dunnett's Procedure are met, the endpoints are estimated by the parametric
procedure.                                                j

11.13.2.3  If unequal numbers of replicates occur among the concentration
levels tested, there are parametric and nonparametric alternative analyses.
The parametric analysis is a t-test with the Bonferroni adjustment (see
Appendix D).  The Wilcoxon Rank Sum Test with the Bonferroni adjustment is the
nonparametric alternative.

11.13.2.4  Probit Analysis (Finney, 1971; see Appendix H) is used to estimate
the concentration that causes a specified percent decrease in survival from
the control.  In this analysis, the total mortality data from all test
replicates at a given concentration are combined.  If the data do not fit the
Probit Analysis, the Spearman-Karber Method, the Trimmed Spearman-Karber
Method, or the Graphical Method may be used (see Appendices H-K).
                                                          i
11.13.2.5  Example of Analysis of Survival Data

11.13.2.5.1   This example uses the survival data from the Sheepshead Minnow
Larval Survival and Growth Test.  The proportion surviving in each replicate
must first be transformed by the arc sine square root transformation procedure
described in Appendix B.  The raw and transformed data, means and variances of
the transformed observations at each effluent concentration arid control are
listed in Table 5.  A plot of the survival proportions is provided in
Figure 7.  Since there was 100% mortality in all four replicates for the 100%
concentration, it was not included in the statistical analysis and was
considered a qualitative mortality effect.

11.13.2.6  Test for Normality

11.13.2.6.1  The first step of the test for normality is to center the
observations by subtracting the mean of all observations within a
concentration from each observation in that concentration.  The centered
observations are summarized in Table 6.

11.13.2.6.2  Calculate the denominator, D, of the statistic:


                                D = £ (X.-X)2
                                    i=l

     Where:   X1- =  the ith centered observation

              X  =  the overall mean of the centered observations

              n  =   the total  number of  centered observations

                      •
                                      91

-------
TABLE 5.  SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, SURVIVAL DATA


Repncate

RAW



ARC SINE
TRANSFORMED


Mean (Y,)
s*
i1

A
B
C
D
A
B
C
D




Control

1.0
1.0
1.0
1.0
1.412
1.412
1.412
1.412
1.412
0.0
1
Effluent Concentration (%)

6.25
1.0
1.0
0.9
1.0
1.412
1.412
1.249
1.412
1.371
0.007
2

12.5
1.0
1.0
1.0
1.0
1.412
1.412
1.412
1.412
1.412
0.0
3

25.0
1.0
1.0
1.0
0.8
• 1.412
1.412
1.412
1.107
1.336
0.023
4

50.0
0.8
0.8
0.7
0.6
1.107
1.107
0.991
0.886
1.023
0.011
5
     TABLE  6.   CENTERED  OBSERVATIONS  FOR  SHAPIRO-WILK'S  EXAMPLE
Effluent Concentration (%)
Replicate Control
6.25
A 0.0 0.041
B 0.0 0.041
C 0.0 -0.122
D 0.0 0.041
11.13.2.6.3 For this set of data,
n = 20
X = 1 (-0
20
D = 0.1236
12.5 25.0
0.0 0.076
0.0 0.076
0.0 0.076
0.0 -0.229


.001) = 0.000

50.0
0.084
0.084
-0.032
-0.137




                                 92

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11.13.2.6.4   Order the centered observations from smallest to largest

               X<1)sX(2)<  ... 

where XO) denotes the ith ordered observation.  The ordered observations for
this example are listed in Table 7.

11.13.2.6.5   From Table 4,  Appendix B, for the number of observations,  n,
obtain the coefficients a1?  a2,  ...  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 8.

11.13.2.6.6  Compute the test statistic, W, as follows:


                         W = -[fa, (x(n~i+u -X(i})}2
                             D ±=i


The differences x                   i         X
-------
       TABLE 8.  COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-MILK'S EXAMPLE
i
1
2
3
4
5
6
7
8
9
10
ai
0.4734
0.3211
0.2565
0.2085
0.1686
0.1334
0.1013
0.0711
0.0422
0.0140
X(n-,>1) _ X(i)
0.313
0.221
0.198
0.108
0.076
0.041
0.041
0.041
0.0
0.0

|X(20>
X!S

X07>
X(16)
x;«;-

fe

xai)

X(D
: X(2>
- x<3)
- x(4)
- x<5)
- x(6)
- x(7)
- x<8>
- x<9)
- x<10)
11.13.2.6.8  Since the data do not meet the assumption of normality, Steel's
Many-one Rank Test will be used to analyze the survival data.

11.13.2.7  Steel's Many-one Rank Test

11.13.2.7.1  For each control and concentration combination, combine the data
and arrange the observations in order of size from smallest to largest.
Assign the ranks (1, 2, ..., 8} to the ordered observations with a rank of 1
assigned to the smallest observation, rank of 2 assigned to the next larger
observation, etc.  If ties occur when ranking, assign the average rank to each
tied observation.

11.13.2.7.2  An example of assigning ranks to the combined data for the
control and 6.25% effluent concentration is given in Table 9.  This ranking
procedure is repeated for each control/concentration combination.  The
complete set of rankings is summarized in Table 10.   The ranks are next summed
for each effluent concentration,  as shown in Table 11.
                                      95

-------
TABLE 9.      ASSIGNING RANKS TO THE CONTROL AND 6.25% EFFLUENT
              CONCENTRATION FOR STEEL'S MANY-ONE RANK TEST
               Rank
       Transformed
       Proportion
       Surviving
                   Effluent
                   Concentration
1
5
5
5
5
5
5
5
1.249
1.412
1.412
1.412
1.412
1.412
1.412
1.412
6.25
6.25
6.25
6.25
Control
Control
Control
Control
 Repli-   Control
                         TABLE  10.  TABLE  OF  RANKS
                                    Effluent  Concentration  (%)
6.25
12.5
25.0
50.0
*>"•»
A
B
C
D
2Us 	
1.412
1.412
1.412
1.412
(5,4.5,5,6
(5,4.5,5,6
(5,4.5,5,6
(5,4.5,5,6
.5)
.5)
.5)
.5)
1.412
. 1.412
1.249
1.412
(5)
(5)
(D
(5)
1.412
1.412
1.412
1.412
(4.5)
(4.5)
(4.5)
(4.5)
1.412
1.412
1.412
1.107
(5)
(5)
(5)
(1)
1.107
1.107
0.991
0.886
(3.5)
(3.5)
(2)
(1)
                           TABLE 11.  RANK SUMS
         Effluent Concentration
                     Rank Sum
                    6.25
                    12.5
                    25.0
                    50.0
                        16
                        18
                        16
                        10
                                     96

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11.13.2.7.3  For this example,  determine if the survival  in any of the
effluent concentrations is significantly lower than the survival  in the
control.  If this occurs,  the rank sum at that concentration would be
significantly lower than the rank sum of the control.   Thus, compare the rank
sums for the survival at each of the various effluent  concentrations with some
"minimum" or critical rank sum, at or below which the  survival  would be
considered significantly lower than the control.  At a significance level of
0.05, the minimum rank sum in a test with four concentrations (excluding the
control) and four replicates is 10 (see Table 5, Appendix E).

11.13.2.7.4  Since the rank sum for the 50% effluent concentration is equal  to
the critical value, the proportion surviving in the 50% concentration is
considered significantly less than that in the control.  Since  no other rank
sums are less than or equal to the critical value, no  other concentrations
have a significantly lower proportion surviving than the control.  Hence, the
NOEC and the LOEC are the 25% and 50% concentrations,  respectively.

11.13.2.8  Calculation of the LC50

11.13.2.8.1  The data used for the calculation of the  LC50 is summarized in
Table 12.  For estimating the LC50, the data for the 100% effluent
concentration with 100% mortality is included.

11.13.2.8.2  Because there are at least two partial mortalities in this set  of
test data, calculation of the LC50 using Probit Analysis i<; recommended.  For
this set of data, however, the test for heterogeneity of variance was
significant.  Probit Analysis is not appropriate in this case.   Inspection of
the data reveals that, once the data is smoothed and adjusted,  the proportion
mortality in the lowest effluent concentration will not be zero although the
proportion mortality in the highest effluent concentration will be one.
Therefore, the Spearman-Karber Method is appropriate for this data.

11.13.2.8.3  Before the LC50 can be calculated the data must be smoothed and
adjusted.  For the data in this example, because the observed proportion
mortality for the 12.5% effluent concentration is less than the observed
response proportion for the 6.25% effluent concentration, the observed
responses for the control and these two groups must be averaged:


            Pcf =PiS =P/ a. 0-00+0-025+0.00 =  °-025  =f 0.0083
Where:  p^ = 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

                                      97

-------
be adjusted using Abbott's formula (Finney, 1971).   The  adjustment takes the
form: *
Where:  p^ - the  smoothed observed proportion  mortality  for the control

        P? • (Pi  - Po)  / (1 -  Po)
        p? = 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:
         Po
a _ _a _ _a _  Pi ~Po  _  0 . 0083-Q . 0083      0.00     n  n
)  ~ Pi - Pa -  	— -	—r—		  = ——	=0.0
                            1-0.0083
                                                      0.9917
                a _ P/-Po  _  0.05-0.0083  _  0.0417
              P3  =
                                1-0.0083
                                    0.9917
                                            = 0.042
              _ a    P/-PoS
              P4  =
                    i-PcT
               a =
                 ~
                   0.275-0.0083
                     1-0.0083
0.2667
0.9917
= 0.269
                _  1.000-0.0083  _ 0.9917  _
                     1-0.0083      0.9917  ~
                                                       _ ±  ...
                                                       ~   '
The smoothed,  adjusted response proportions for the  effluent concentrations
are shown in Table  12.

        TABLE  12.   DATA FOR EXAMPLE OF SPEARMAN-KARBER ANALYSIS
Effluent
Concentration
%
Control
6.25
12.5
25.0
50.0
100.0
Number
of Deaths
0
1
0
2
11
40
Number of
Organisms
Exposed
40
40
40
40
40
40
Mortality
Proportion
0.000
0.025
0.000
0.050
0.275
1.000
Smoothed
Mortality
Proportion
0.0083
0.0083
0.0083
0.0500
0.2750
1.0000
Smoothed,
Adjusted
Mortality
Proportion
0.000
0.000
0.000
0.042
0.269
1.000
                                     98

-------
11.13.2.8.5  Calculate the Iog10 of the  estimated  LC50, m,  as  follows:
                            m =
Where:  p* = the smoothed adjusted proportion mortality at concentration i

        Xf = the Iog10 of concentration i

        k  = the number of effluent concentrations tested, not including the
             control


11.13.2.8.5.1  For this example, the Iog10 of the  estimated LC50,  m,  is
calculated as follows:

      m = [(0.000 - 0.000) (0.7959 + 1.0969)]/2 +
          [(0.042 - 0.000) (1.0969 + 1.3979)]/2 +
          [(0.269 - 0.042) (1.3979 + 1.6990)]/2 +
          [(1.000 - 0.269) (1.6990 + 2.0000)]/2
                                                          I
        = 1.755873
                                                          i
11.13.2.8.6  Calculate the estimated variance of m as follows:


                         (  }
Where:  X,- =  the Iog10  of  concentration  i

        n,- =  the number of organisms tested at effluent concentration  i

        p? =  the smoothed adjusted observed proportion mortality  at effluent
              concentration i

        k  =  the number of effluent concentrations tested, riot  including the
              control
                                                          i
11.13.2.8.6.1   For this example, the estimated variance of m, V(m), is
calculated as follows:
                  ...
      V(m) =  (0.000)(1.000)(1.3979 - 0.7959)^/4(39) +
              (0.042)(0.958)(1.6990 - 1.0969)74(39) +
              (0.269)(0.731)(2.0000 - 1.3979)74(39)

           =  0.0005505

11.13.2.8.7   Calculate  the 95%  confidence  interval for m:  m ± 2.0 7 V(m)
                                       99

-------
 11.13.2.8.7.1   For  this  example,  the  95%  confidence  interval for m is
 calculated  as  follows:
             1.755873 ± 2 ^0.0005505 =  (1.754772, 1.756974)

 11.13.2.8.8  The  estimated LC50  and  a  95%  confidence  interval for the
 estimated  LC50  can  be found  by taking  base10 antilogs  of the above  values.

 11.13.2.8.8.1   For  this example, the estimated LC50 is calculated as follows:

             LC50 = antilog(m) - antilog(1.755873) =  57.0%.

 11.13.2.8.8.2   The  limits of the 95% confidence interval for the estimated
 LC50 are calculated by taking the antilogs of the upper and lower limits of
 the 95% confidence  interval  for m as follows:

            lower limit:   antilog(l.754772) = 56.9%

            upper limit:   antilog(1.756974) = 57.1%

 11.13.3   EXAMPLE OF ANALYSIS OF SHEEPSHEAD MINNOW,  CYPRINODON VARIEGATUS,
          GROWTH DATA

 11.13.3.1  Formal statistical analysis of the growth  data is outlined in
 Figure 8.  The  response used in the  statistical analysis is mean weight per
 original organism for each replicate.  The IC25 and IC50 can be calculated for
 the growth data via a point  estimation technique (see Section 9, Chronic
 Toxicity Test Endpoints and  Data Analysis).  Hypothesis testing can be used to
 obtain an NOEC  and  LOEC for  growth.  Concentrations above the NOEC for
 survival are excluded from the hypothesis test for growth effects.

 11.13.3.2  The  statistical analysis  using hypothesis  testing consists of a
 parametric test,  Dunnett's Procedure, and a nonparametric test,  Steel's
 Many-one Rank Test.  The underlying  assumptions of the Dunnett's Procedure,
 normality and homogeneity of variance, are formally tested.  The test for
 normality is the  Shapiro-Wilk's Test and Bartlett's Test is used to test for
 homogeneity of variance.  If either  of these tests fails, the nonparametric
 test, Steels' Many-one Rank Test, is used to determine the NOEC and LOEC
 endpoints.  If the  assumptions of Dunnett's Procedure are met, the endpoints
 are determined by the parametric test.

 11.13.3.3  Additionally, if unequal   numbers of replicates occur among the
 concentration levels tested there are parametric and nonparametric alternative
 analyses.  The parametric analysis is a t test with the Bonferroni  adjustment.
 The Wilcoxon Rank Sum Test with the  Bonferroni adjustment is the nonparametric
 alternative.  For detailed information on the Bonferroni adjustment,  see
 Appendix D.

 11.13.3.4  The data, mean and variance of the observations at each
 concentration including the control   are listed in Table 13.   A plot of the
mean weights for each treatment is provided in Figure 9.  Since  there is no

                                      100

-------
                                                                                        1
            STATISTICAL ANALYSIS OF SHEEPSHEAD MINNOW LARVAL
                         SURVIVAL AND GROWTH TEST
                                  GROWTH
                                 GROWTH DATA
                                MEAN DRY WEIGHT
     POINT ESTIMATION
    HYPOTHESIS TESTING
(EXCLUDING CONCENTRATIONS
 ABOVE NOEC FOR SURVIVAL)
    ENDPOINT ESTIMATE
        IC25, IC50
   SHAPIRO-WILK-S TEST
                       NON-NORMAL DISTRIBUTION
                    NORMAL DISTRIBUTION
         HOMOGENEOUS
           VARIANCE
                                BARTLETTSTEST
                             HETEROGENEOUS
                                VARIANCE
1
EQUAL NUM!
REPLJCAT
NO
r 1
3EROF
ES?
YES
T-TESTWITH niiMMPTPQ
BONFERRONI DUNNETTS
ADJUSTMENT TEST






i
EQU
R
YES
r
STEEL'S MANY-ONE
RANK TEST


t
ENDPOINT ESTIMATES
NOEC, LOEC
AL NUMBER OF
EPUCATIES?
1 r

NO
WILCOXON RANK SUM
TEST WITH
BONFERRONI ADJUSTMENT
1



Figure 8.      Flowchart for statistical  analysis of the sheepshead minnow,
              Cypn'nodon van'egatus,  larval growth data.

                                    101

-------
survival in the 100% concentration, it is not considered in the growth
analysis.  Additionally, since there is significant mortality in the 50%
effluent concentration, its effect on growth is not considered.
       TABLE 13.  SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, GROWTH DATA
  Replicate    Control
                                     Effluent Concentration (%)
6.25    12.5    25.0   50.0   100.0
A
B
C
D
MfanCY,.)
sf
1!
1.29
1.32
1.59
1.27
1.368
0.0224
1
1.27
1.00
0.97
0.97
1.053
0.0212
2
1.32
1.37
1.35
1.34
1.345
0.0004
3
1.29
1.33
1.20
0.94
1.190
0.0307 -
4 5
_
-
-
"
-
6
 11.13.3.5  Test  for  Normality

 11.13.3.5.1  The first  step  of  the  test  for  normality  is to center the
 observations by  subtracting  the mean  of  all  the  observations  ithin a
 concentration  from each observation in that  concentration.  The centered
 observations are summarized  in  Table  14.
       TABLE 14.   CENTERED OBSERVATIONS  FOR SHAPIRO-MILK'S  EXAMPLE
                                     Effluent Concentration  (%)
        Replicate     Control
 6.25
12.5
25.0
A
B
C
D
-0.078
-0.048
0.222
-0.098
0.217
-0.053
-0.083
0.083
-0.025
0.025
0.005
-0.005
0.100
0.140
0.010
-0.250
                                       102

-------
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                                 103

-------
11.13.3.5.2  Calculate the denominator, D, of the test statistic:
                               D =  £ (Xt-
                                   1=1
Where:     X,-  = the  ith  centered observation
           X  - the  overall  mean of the centered observations
           n  = the  total  number of centered observations.
For this set of data,  n  =  16
                      X  =  _L_(-0.004)  = -0.00025 = 0.00
                           16
                      D  =  0.2245
11.13.3.5.3  Order the centered observations from smallest to largest:
          x<1) is the ith ordered observation.   These ordered observations are
listed in Table 15.
       TABLE 15.  ORDERED  CENTERED OBSERVATIONS FOR SHAPIRO-MILK'S  EXAMPLE
i
1
2
3
4
5
6
7
8
xci>
-0.250
-0.098
-0.083
-0.083
-0.078
-0.053
-0.048
-0.025
i
9
10
11
12
13
14
15
16
xci>
-0.005
0.005
0.010
0.025
O.'lOO
0.140
0.217
0.222
                                     104

-------

 11.13.3.5.4   From Table 4,  Appendix B,  for the number of observations,  n,
 obtain  the  coefficients  a., a
                          .,  2,
 (n-l)/2  if n  is  odd.   For'the'data in "this example,'n
 values are listed  in  Table 16.
                                     ak where k is n/2 if n is even and
                                                         16  and  k =  8.   The
      TABLE 16.  COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE
                                       - X
                                          (i)
1
2
3
4
5
6
7
8
0.5056
0.3290
0.2521
0.1939
0.1447
0.1005
0.0593
0.0196
0.472
0.315
0.223
0.183
0.103
0.063
0.053
0.020
X(16)
X(15)
X(14>
X(13)
X(12)
xdD
X(10)
x<9)
I
- x<1)
- XC2)
- X<3)
J(4)
- x<5)
- X(6>
- X(7>
v<8)
~ A
11.13.3.5.5  Compute the test statistic, W, as follows:

                                   ± (X <*-
The differences x

For this set of data:

                    W =
                          (i)
                       - X   are  listed  in Table  16.
                            1    (0.4588)2 = 0.938
                         0.2245
11.13.3.5.6  The decision rule for this test is to compare W with the critical
value found in Table 6, Appendix B.  If the computed W is less than the
critical value, conclude that the data are not normally distributed.  For this
example, the critical value at a significance level of 0.01 and 16
observations (n) is 0.876.  Since W = 0.938 is greater than the critical
value, the conclusion of the test is that the data are normally distributed.
                                                          i
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
                                      105

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Bartlett's Test (Snedecor and Cochran, 1980).   The test statistic  is  as
f ol1ows:
V,)  in
                                                 in
                      B =
    Where:  V-   =   degrees of freedom for each effluent concentration and
             1       control, V, = (n,-  -  1)

            n,-   =   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
                    -52 _
                     C = 1 + [3 (p-1) ] -1 [ f 1/V, - ( f V,) -
 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,  -  4  for all  i).   Thus, V,- = 3 for all i.

 11.13.3.6.3  Bartlett's statistic  is  therefore:

                  B = [(12)ln(0.0137)  -  3 £ In(Si)]/1.139
                       [12(-4.290) - 3(-18.960)]/1.139

                       5.396/1.139

                       4.737
                                       106

-------
11.13.3.6.4  B is approximately distributed as chi-square with  p -  1  degrees
of freedom, when the variances are in fact the same.   Therefore,  the
appropriate critical value for this test,  at a significance level of  0.01  with
three degrees of freedom,  is 11.345.  Since B = 4.737 is  'less than  the
critical value of 11.345,  conclude that the variances are not different.
                                                         j
11.13.3.7  Dunnett's Procedure
                                                         I

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
Between p - 1
Within N - p
Total N - 1
Sum of Squares
(SS)
SSB
SSW
SST
1
Mean Square(MS)
(SS/df)
i
S* = SSB/(p-l)
s* = SSW/(N-P)
-
   Where: p  = number of concentration levels including the control

          N  = total number of observations n, + n,  ... + n
                                                  *        P
          n,- = number of observations in concentration i
                                                         !

         SSB = f,Tl/n±-G2/N      Between Sum of Squares:
        SST = £ £ YJ.j-G2/N      Total Sum of Squares
               2=1:7=1
        SSW = SST-SSB
Within Sum of Squares
                                    107

-------
6  - the grand total of all sample observations
                                                          ,  G = E z^

           I,-  - the total of the replicate measurements for concentration  i


          Y-5  = the jth observation for concentration  i (represents the mean
                dry weight of the mysids for concentration i  in test
                 chamber j)

11.13.3.7.2  For the data in this example:
          n
     N  = 16
                   n  - 4
     Ti = Y., + Y12 + Y13 + Y14 = 5.47
     Tj - Y2 + Y22 + Y23 + Y2t = 4.21
     T, - Y,] + Y^ + Y,, + Y,4 = 5.38
     T~4 = Y^J + Y42 + Y^ + Y44 = 4.76

     G  - T, + T2 + T3 + T4 =  19.82
   SSB = *tTl/n±-G*/N
         _1_(99.247)  -  (19.82)2  = 0.260
          4                16
   SST
          25.036  -  (19. 82)2  = 0.484
                       16
   SSW = SST-SSB

     s?  = SSB/CP-D
           SSW/(N-p)
            = 0.484 - 0.260 = 0.224


            0.260/(4-l)  = 0.087

            0.224/(16-4) = 0.019
                                       108

-------
11.13.3.7.3  Summarize these calculations in the ANOVA table (Table 18)
           TABLE  18.  ANOVA TABLE  FOR  DUNNETT'S  PROCEDURE  EXAMPLE
      Source        df        Sum of Squares        Mean Siquare(MS)
                                    (SS)                 (SS/df)
Between
Within
3
12
0.260
0.224
0.087
0.019
      Total         15            0.484
11.13.3.7.4  To perform the individual comparisons, calculate the t statistic
for each concentration, and control combination as follows:
                           ti =
Where:  Yf  = mean dry weight for effluent concentration i
        Y1  = mean dry weight for the control
        Sw  = square root of the within mean square
        n,  = number of replicates for the control
        n,-  = number of replicates for concentration i.
                                      109

-------
11.13.3.7.5  Table 19 includes the calculated t values for each concentration
and control combination.  In this example, comparing the 6.25% concentration
with the control, the calculation is as follows:

                        TABLE 19.  CALCULATED T VALUES
             Effluent Concentration (%)          i          t,-
6.25
12.5
25.0
2
3
4
3.228
0.236
1.824
                     .  _      (1.368-1.053)
                     Co •"*
                          [0.138^(1/4) + (1/4)]


11.13.3.7.6  Since the purpose of this test is to detect a significant
reduction in mean weight, a one-sided test is appropriate.  The critical value
for this one-sided test is found in Table 5, Appendix C. For an overall alpha
level of 0.05, 12 degrees of freedom for error and three concentrations
(excluding the control) the critical value is 2.29. The mean weight for
concentration i is considered significantly less than the mean weight for the
control if tt is greater than the critical  value.   Since t2  is  greater  than
2.29, the 6.25% concentration has significantly lower growth than the control.
However, the 12.5% and 25% concentrations do not exhibit this effect.  Hence
the NOEC and the LOEC for growth cannot be calculated.

11.13.3.7.7  To quantify the sensitivity of the test, the minimum significant
difference (MSD) that can be statistically detected may be. calculated:
                          MSD = d S^CL/nJ + (1/n)
Where:  d  = the critical value for Dunnett's Procedure

        Sw » the square root of the within mean square

        n  = the common number of replicates at each concentration
             (this  assumes equal replication at each concentration)

        n, » the number of replicates in the control.
                                      110

-------
11.13.3.7.8  In this example:
                      MSD = 2.29 (0.10)^(1/4) + (1/4)


                           = 2.29 (0.138)(0.707)

                           = 0.223

11.13.3.7.9  Therefore,  for this set of data, the minimum
difference that can
be detected as statistically significant is 0.223 mg.

11.13.3.7.10  This represents a 16% reduction in mean weight from the control.

11.13.3.8  Calculation of the ICp

11.13.3.8.1  The growth data from Table 4 are utilized in this example.  As
seen from Table 4 and Figure 7, the observed means are not monotonically non-
increasing with respect to concentration (mean response for each higher
concentration is not less than or equal to the mean response for the previous
concentration and the responses between concentrations do not follow a linear
trends).  Therefore, the means are smoothed prior to calculating the 1C.  In
the following discussion, the observed means are represented by Y,- and the
smoothed means by M,-.

11.13.3.8.2  Starting with the control mean, Y.,  = 1.368 and Y2 =  1.053,  we  see
that Y, > Y2.   Set M, = Y,.  Comparing Y2 to Y3,  Y2 < Y3.   \  -

11.13.3.8.3  Calculate the smoothed means:

                       M2 = M3  =  (Y2 + Y3)/2 = 1.199

11.13.3.8.4  Since Y, = 0 < Y5  =  0.525  < Y, = 1.190 < Y. - 1.345,  set M, =
1.199, M4 = 1.190,  M5  = 0.525,  and  set  M6 - 0.

11.13.3.8.5  Table 20 contains the response means and smoothed means and
Figure 10 gives a plot of the smoothed response curve.

     TABLE 20.  SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, MEAN GROWTH
                RESPONSE AFTER SMOOTHING
Effluent
Cone. (%)
Control
6.25
12.50
25.00
50.00
100.00
i
1
2
3
4
5
6
Response Means Smoothed Means
(mg) Y, (mg) M;
1.
1.
1.
1.
0.
0.
368
053
345
189
525
0
1.368
1.199
1.199
1.189
0.525
0.0
                                      111

-------
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                                                                                    T3 O)

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-------
11.13.3.8.6  An IC25 and IC50 can be estimated using the Linear  Interpolation
Method.  A 25% reduction in weight> compared to the controls, would  result  in
a mean dry weight of 1.026 mg, where M^l-p/lOO) = 1.368(1-25/100).  A 50%
reduction in mean dry weight, compared to the controls, would result in  a mean
dry weight of 0.684 mg.  Examining the smoothed means and their  associated
concentrations (Table 4), the response, 1.026 mg,  is bracketed by  C4 = 25.0%
effluent and C5 = 50.0% effluent.  The response (0.684 mg) is bracketed  by
C4 = 25.0% effluent and C5  =  50% effluent.
11.13.3.8.7  Using the equation from Section 4.2 of Appendix  M,  the  estimate
of the IC25 is calculated as follows:
                    ICp =
                    IC25 = 25.0 +  [1.368(1  -  25/100)  -  1.189]   (50.00 -  25.00)
                                                               (0.525 -  1.189)
                          31.2%.
11.13.3.8.8  Using the equation  frontSection  4.2  of Appendix L,  the estimate
of the  IC50  is calculated  as  follows:
                     ICp=Cj+ [ML (l-p/100) -AT,]
                                                (C
                    IC50  -  50.0  +  [1.368(1-50/100)  -  0.525]   (100.00-50.00)
                                                              (0.0 - 0.525)
                         =  44.0%.
 11.13.3.8.9  When  the  ICPIN  program was  used to analyze this set of data,
 requesting 80  resamples,  the estimate of the IC25 was 31.1512%.   The empirical
 95%  confidence interval  for  the true mean was 22.0420% and 36.3613%.  The
 computer  program output  for  the IC25 for this data set is shown  in Figure 11.

 11.13.3.8.10   When the ICPIN program was  used to  analyze this set  of data  for
 the  IC50, requesting  80 resamples, the estimate of the IC50 was 44.0230%.  The
 empirical 95% confidence  interval for the true mean was 39.1011% and 49.0679%.
 The  computer program  output is  shown  in Figure 12.
                                      113

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Cone. ID

Cone. Tested
Response
Response
Response
Response
1
2
3
4
1
0
1.29
1.32
1.59
1.27
2
6.25
1.27
1
.972
.97
3
12.5
1.32
1.37
1.35
1.34
4
25
. 1.29
1.33
1.2
.936
5
50
.62
.560
.46
.46
6
100
0
0
0
0
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent: Effluent
Test Start Date:    Test Ending Date:
Test Species: Cyprinodon variegatus
Test Duration:             7-d
DATA FILE: sheep.icp
OUTPUT FILE: sheep.i25
Cone.
ID
1
2
3
4
5
6
Number
Replicates
4
4
4
4
4
4
Concentration
%
0.000
6.250
12.500
25.000
50.000
100.000
Response
Means
1.368
1.053
1.345
1.189
0.525
0.000
Std. Pooled
Dev. Response Means
0.150
0.145
0.021
0.177
0.079
0.000
1.368
1.199
1.199
1.189
0.525
0.000
The Linear Interpolation Estimate:    31.1512   Entered P Value: 25

Number of Resamplings:   80
The Bootstrap Estimates Mean:  30.6175 Standard Deviation:     2.9490
Original Confidence Limits:   Lower:    25.4579 Upper:    34.4075
Expanded Confidence Limits:   Lower:    22.0420 Upper:    36.3613
Resampling time in Seconds:     1.70  Random Seed: -2137496326
                Figure 11.  ICPIN program output for the IC25.

                                      114

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Cone. ID
Cone. Tested 0 6.25
Response 1 1.29 1.27
Response 2 1.32 1
Response 3 1.59 .972
Response 4 1.27 .97
12.5 25
1.32 1.29
1.37 1.33
1.35 1.2
1.34 .936
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent: Effluent
Test Start Date: Test Ending Date:
Test Species: Cyprinodon variegatus
Test Duration: 7-d
DATA FILE: sheep. icp
OUTPUT FILE: sheep. i 50
50 100
.62 0
. 560 0
.46 0
.46 0

Cone
ID
1
2
3
4
5
6
The
Number Concentration
Replicates
4
4
4
4
4
4
Linear Interpolation
%
0.000
6.250
12.500
25.000
50.000
100.000
Estimate:
Response Std.
Means Dev.
Pooled
Response Means
1.368 0.150 1.368
1.053 0.145 1.199
1.345 0.021
1.189 0.177
1.199
1.189
0.525 0.079 0.525
0.000 0.000 0.000
44.0230 Entered P
Value: 50
Number of Resamplings:   80
The Bootstrap Estimates Mean:
Original Confidence Limits:
Expanded Confidence Limits:
Resampling time in Seconds:
 44.3444 Standard Deviation:     1.7372
Lower:    40.9468 Upper:    47.1760
Lower:    39.1011 Upper:    49.0679
  1.70  Random Seed: -156164614
                Figure 12.  ICPIN program output for the IC50.

                                      115                  !

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11.14  PRECISION AND ACCURACY

11.14.1  PRECISION

11.14.1.1  Single-Laboratory Precision

11.14.1.1.1  Data on the single-laboratory precision of the sheepshead minnow
larval survival and growth test using FORTY FATHOMS® artificial seawater,
natural seawater, and 6P2 with copper sulfate, sodium dodecyl sulfate, and
hexavalent chromium, as reference toxicants, are given in Tables 21-27.  The
IC25, IC50, or LC50 data (coefficient of variation), indicating acceptable
precision for'the reference toxicants (copper, sodium dodecyl sulfate, and
hexavalent chromium), are also listed in these Tables.

11.14.1.2  Multilaboratory Precision

11.14.1.2.1  Data from a study of multilaboratory test precision, involving a
total of seven tests by four participating laboratories, are listed in
Table 27. The laboratories reported very similar results, indicating good
inter!aboratory precision.  The coefficient of variation (IC25) was 44.2% and
(IC50) was 56.9%, indicating acceptable precision.

11.14.2  ACCURACY

11.14.2.1  The accuracy of toxicity tests cannot be determined.
                                      116

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TABLE 21.  SINGLE-LABORATORY PRECISION OF THE SHEEPSHEAD MINNOW,
           CYPRINODON  VARIEGATUS, LARVAL SURVIVAL AND GROWTH TEST
           PERFORMED IN FORTY FATHOMS® ARTIFICIAL SEAWATER, USING LARVAE
           FROM FISH MAINTAINED AND SPAWNED IN FORTY FATHOMS® ARTIFICIAL
           SEAWATER, JJSING COPPER (CU) SULFATE AS A REFIEREINCE
           TOXICANT1'2'3'*'5
Most
Test
Number
1
2
3
4
5
6
7
8
n:
Mean:
CV(%):
NOEC
(M9/L)
507
< 507
< 507
50r
< 507
50
50
50
5
NA
NA
IC25
IC50 Sensitive
(Mg/L) Og/L) Endpoint6
113.3
54.3
41.8
63.2
57.7
48.3
79.6
123.5
p
72.7
41.82
152.3 S
97.5 G
71.4 G
90.8 S
99.8 S
132.5 G
159.7 G
236.4 G
8
130.0
40.87











    Data from USEPA (1988a) and USEPA (1991a).
    Tests performed by Donald J. Klemm, Bioassessment and Ecotoxicology
    Branch, EMSL, Cincinnati, OH.
    All tests were performed using Forty Fathoms® synthetic seawater.
    Three replicate exposure chambers, each with 15 larvae, were used for
    the control and each copper concentration.  Copper concentrations
    used in Tests 1-6 were: 50, 100, 200, 400, and 800 ng/l.  Copper
    concentrations in Tests 7-8 were: 25, 50, 100, 200 and 400 A*g/L.
    Adults collected in the field.
    For a discussion of the precision of data from chronic toxicity test
    see Section 4, Quality Assurance.
    Endpoints:   G=growth;  S=survival.                   i
    Lowest concentration tested was 50 /zg/L (NOEC Range;; > 50* - 50
                                    117

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TABLE 22.   SINGLE-LABORATORY PRECISION OF THE SHEEPSHEAD MINNOW,
            CYPRINIDON VARIEGATUS, LARVAL SURVIVAL AND GROWTH TEST
            PERFORMED IN FORTY FATHOMS® ARTIFICIAL SEAWATER, USING LARVAE
            FROM FISH MAINTAINED AND SPAWNED  IN FORTY FATHOMS® ARTIFICIAL
            SEAWATER, USING SODIUM DODECYL SULFATE (SDS) AS A REFERENCE
            TOXICANT1'2'3'4'5'6

Test
Number
1
2
3
4
5
6
n:
Mean:
CV(%):
! Data from USEPA

NOEC
(mg/L)
1.0
1.0
1.0
0.5
1.0
0.5
6
NA
NA
(1988a) and USEPA

IC25
(mg/L)
1.2799
1.4087
2.3051
1.9855
1.1901
1.1041
6
1.5456
31.44
(1991a).

Most
IC50 Sensitive
(mg/L) Endpoint
1.5598
1.8835
2.8367
2.6237
1.4267
1.4264
6
1.9595
31.82
+ anrl I7^*n+ftVT/*r
S
S
S
G
S
G



\1 nn\/
     Branch,  EMSL,   Cincinnati,  OH.
    All  tests were  performed using Forty Fathoms® synthetic seawater.
    Three replicate exposure chambers,  each with 15 larvae, were used for
    the  control  and each SDS concentration.  SDS concentrations in Tests
    1-2  were: 1.0,  1.9,  3.9, 7.7, and 15.5 mg/L. SDS concentrations in
    Tests 3-6 were: 0.2, 0.5, 1.0, 1.9, and 3.9 mg/L.
    Adults collected in  the field.
    For  a discussion of  the precision of data from chronic toxicity tests
    see  Section  4,  Quality Assurance.
    NOEC Range:  0.5 -1.0 mg/L (this represents a difference of one
    exposure   concentration).
   Endpoints: G=growth;  S=survival
                                    118

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TABLE 23.  SINGLE-LABORATORY PRECISION OF THE  SHEEPSHEAD MINNOW,
           CYPRINIDON  VARIEGATUS,  LARVAL SURVIVAL  AND  GROWTH TEST
           PERFORMED IN NATURAL SEAWATER, USING  LARVAE  FROM FISH
           MAINTAINED AND SPAWNED  IN NATURAL SEAWATER,  USING COPPER (CU)
           SULFATE AS A REFERENCE  TOXICANT1-2'J'*'5'6      ;
       Test
       Number
                     NOEC
                    (M9/L)
IC25
IC50
    Most
Sensitive
Endpoint
1
2
3
4
5
n:
Mean:
CV(%):
1 Data from US EPA
125
31
125
125
125
5
NA
NA
(1988a) and USEPA
320.3
182.3
333.4
228.4'
437.5
5
300.4
33.0
(1991a).
437.5
323.0
484.4
343.8
NC8
4
396.9
19.2
1 r-m hi i ir»i
S
G
S
S
S


5
6


7
8
i v-j 1.0 f/ci i ui llltvj i/jr ucui yc riui IIOUII  aiiu  L.IIOC  IUl C I I U;  UIM.ll)  Uotrn,
Narragansett, RI.
Three replicate exposure chambers,  each with  10-15  larvae,  were  used
for the control and each copper concentration.   Copper concentrations
were: 31, 63, 125, 250, and  500 //g/L.
NOEC Range: 31 - 125 ng/L (this represents  a  difference of  two exposure
concentrations).
Adults collected in the field.
For a discussion of the precision of data from chronic toxicity  tests
see Section 4, Quality Assurance.
Endpoints:  G=growth; S=survival.
NC = No linear interpolation estimate could be calculated from the
     data,  since none  of the group  response means were less than 50
     percent of the control  response mean.
                                    119

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TABLE 24.  SINGLE-LABORATORY PRECISION OF THE SHEEPSHEAD MINNOW,
           CYPRINIDON  VARIEGATUS, LARVAL SURVIVAL AND GROWTH TEST
           PERFORMED IN NATURAL SEAWATER, USING LARVAE FROM FISH
           MAINTAINED AND SPAWNED IN NATURAL SEAWATER, USING SODIUM
           DODECYL SULFATE (SDS) AS A REFERENCE TOXICANT1 f2'3'4'5'6

Test
Number
1
2
3
4
5
n:
Mean:
CV(%) :
\ Data from USEPA
** Tfte*^ o nov»f nv^maH

NOEC
(mg/L)
2.5
1.3
1.3
1.3
1.3
5
NA
NA
(1988a) and USEPA
K\/ £anwia Mnvvi cnr

IC25
(mg/L)
2.9
NCI8
1.9
2.4
1.5
4
2.2
27.6
(1991a).
i anH Fl i CP Tr»V*i

Most
IC50 Sensitive
(mg/L) Endpoint
3.60
NC29
2.4
NC2
1.8
3
2.6
35.3
olln. FRI -N. U!
S
G
S
G
S



SFPA.
5
6


7
8
Narragansett, RI.                                                 •
Three replicate exposure chambers, each with 10-15 larvae, were used
for the control and each SDS concentration.  SDS concentrations were:
0.3, 0.6, 1.3, 2.5, and 5.0 mg/L.
NOEC Range: 1.3 - 2.5 mg/L (this represents a difference of one
exposure concentration).
Adults collected in the field.
For a discussion of the precision of data from chronic toxicity tests
see Section 4, Quality Assurance.
Endpoints: G=growth; S=survival.
NCI -   No linear interpolation estimate could be calculated from the
        data, since none of the group response means were less than 75
        percent of the control response mean.
NC2 =   No linear interpolation estimate could be calculated from the
        data, since none of the group response means were less than 50
        percent of the control response mean.
                                    120

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TABLE 25.    SINGLE-LABORATORY PRECISION OF THE SHEEPSHEAD MINNOW,
             CYPRINODON VARIEGATUS, LARVAL SURVIVAL AND  RO TH TEST
             PERFORMED IN FORTY FATHOMS® ARTIFICIAL SEAWATER, USING LARVAE
             FROM FISH MAINTAINED AND SPAWNED IN FORTY FATHOMS® ARTIFICIAL
             SEAWATER, AND HEXAVALENT CHROMIUM AS THE REFERENCE
             TOXICANT1'2'3-*'5
       Test
      Number
          1
          2
          3
          4
          5
 NOEC
(mg/L)
  2.0
  1.0
  4.0
  2.0
  1.0
 IC25
(mg/L)
   5.8
   2.9
   6.9
   2.4
   3.1
 IC50
(mg/L)
  11.4
   9.9
  11.5
   9.2
  10.8
  Most
Sensitive
Endpoint
     G
     G
     G
     G
     G
            n:
         Mean:
   5
  NA
  NA
   5
   4.2
  47.6
    5
   10.6
    9.7
    Tests performed by Donald Klemm, Bioassessment and Ecotoxicology
    Branch,  EMSL, Cincinnati, OH.                        .
    All  tests were performed using Forty Fathoms® synthetic seawater.
    Three replicate exposure chambers, each with 15 larvae, were used for
    the control and each hexavalent chromium concentration.  Hexavalent
    chromium concentrations used in Tests 1-5 were: 1.0, 2.0, 4.0, 8.0,
    16.0, and 32.0 mg/L.
    NOEC Range: 1.0 - 4.0 mg/L (this represents a difference of four
    exposure concentrations)
    Adults collected in the field.                       ;      .
    For a discussion of the precision of data from chronic toxicity tests
    see Section 4, Quality Assurance.
    Endpoints:  G=growth; S=survival.
                                     121

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 TABLE 26.   COMPARISON OF LARVAL SURVIVAL (LC50) AND GROWTH (IC50) VALUES
             FOR THE SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS,  EXPOSED TO
             SODIUM DODECYL SULFATE (SDS) AND COPPER (CU) SULFATE IN GP2
             ARTIFICIAL SEAWATER MEDIUM OR NATURAL SEAWATER172'*'4
 SDS  (mg/L)
COPPER
                                Survival
                         GP2
NSW
                         GP2
NSW
                           455
                           467
                           390
                                   412
                                   485
                                   528
                                                   Growth
GP2



Mean
CV (%)
7.49
8.70
8.38
8.19
7.7
8.13
8.87
8.85
8.62
4.9
7.39
8.63
8.48
8.17
8.3
8.41
8.51
9.33
8.75
5.8
                                                   GP2
             341
             496
             467
           NSW
           333
           529
           776
Mean
CV (%)
437
9.4
475
12.3
435
18.9
546
40.7
3
4
Tests performed by George Morrison and Glen Modica, ERL-N, USEPA
Narragansett, RI.
Three replicate exposure chambers, each with 10-15 larvae, were used
for the control and each SDS concentration.  SDS concentrations were-
0.3, 0.6, 1.3, 2.5, and 5.0 mg/L.
Adults collected in the field.
For a discussion of the precision of data from chronic toxicity tests
see Section 4, Quality Assurance.
                                   122

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TABLE 27.  DATA FROM INTERLABORATORY STUDY OF THE SHEEPSHEAD MINNOW,
           CYPRINODON VARIEGATUS, LARVAL SURVIVAL AND GROWTH TEST USING AN
           INDUSTRIAL EFFLUENT AS A REFERENCE TOXICANT1'2'3
Laboratory A


Laboratory B


Laboratory C


Laboratory D
       n:
    Mean:
                  Test
                 Number
1
2

1
2
1
2
                                          Most  Sensitive  Endooint
              NOEC
3.2 (S,G)
3.2 (S,G)

3.2 (S,6)
3.2 (S,G)

1.0 (S)
3.2  (S,G)
1.0  (G)
              7
             NA
             NA
                                                   IC25
7.4 (S)
7.6 (S)

5.7 (G)
5.7 (G)

4.7 (S)
7.4 (G)
5.2 (S)
                  7
                  5.5
                 44.2
                                                IC50
 7.4 (G)
14.3 (G)

 9.7 (G)
 8.8 (G)

 7.2 (S)
24.7 (G)
 7.2 (S)
                    7
                   11.3
                   56.9
 1   Data from USEPA (1987b), USEPA (1988a), and USEPA  (1991a).
 2   Effluent concentrations were: 0.32, 1.0, 3.2, 10.0, and 32.0/».
 3   NOEC Range: 1.0 - 3.2%  (this represents a difference of one exposure
    concentration).
 4   Endpoints: G=growth; S=survival.
                                      123

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                                   SECTION 12

                                   TEST METHOD

                    SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS
                 EMBRYO-LARVAL SURVIVAL AND TERATOGENICITY TEST
                                  METHOD 1005.0

 12.1  SCOPE AND APPLICATION

 12.1.1  This method, adapted in part from USEPA (1981) and USEPA (19875)
 estimates the chronic toxicity of effluents and receiving waters to the
 sneepsnead minnow, Cyprinodon van'egatus, using embryos and larvae in a
 nine-day, static renewal test.  The effects include the synergistic,
 antagonistic, and additive effects of all the chemical, physical, and
 biological components which adversely affect the physiological  and biochemical
 functions of the test organisms.  The test is useful in screening for
 teratogens because organisms are exposed during embryonic development.

 12.1.2  Daily observations on mortality make it possible to also calculate
 acute toxicity for desired exposure periods (i.e.,  24-h,  48-h,  96-h LC50s).

 12.1.3  Detection limits of the toxicity of an effluent or chemical  substance
 are organism dependent,

 12.1.4  Brief excursions in toxicity may not be detected  using  24-h composite
 samples.   Also,  because  of the long sample collection  period  involved in
 composite sampling,  and  because the test chambers are  not sealed,  highly
 volatile  and highly  degradable toxicants present in  the source  may  not  be
 detected  in  the  test.

 12.1.5 This test  is  commonly used  in  one of two forms:  (1) a definitive test
 consisting of a  minimum  of five  effluent concentrations and a control,  and (2)
 a receiving  water  test(s),  consisting  of one  or more receiving  water
 concentrations and a  control.

 12.2   SUMMARY OF METHOD

 12.2.1  Sheepshead minnow,  Cyprinodon  van'egatus, embryos and larvae are
 exposed in a static renewal system to different  concentrations  of effluent or
 to receiving water starting shortly after  fertilization of the  eggs through
 four days  posthatch.  Test  results are based on  the  total frequency of both
mortality  and gross morphological deformities  (terata).

 12.3   INTERFERENCES

12.3.1  Toxic substances may be introduced by contaminants in dilution water
glassware, sample hardware, and testing equipment (see Section 5, Facilities
Equipment, and Supplies).                                                   '
                                     124

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12 3 2  Adverse effects of low dissolved oxygen concentrations (DO), high
concentrations of suspended and/or dissolved solids, and extremes of pH may
mask the effect of toxic substances.

12 3 3  Improper effluent sampling and handling may adversely affect test
results (see Section 8, Effluent and Receiving Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests).

12 3 4  Pathogenic and/or predatory organisms in the dilution water and
effluent may affect test organism survival, and confound test results.

12.4  SAFETY

12.4.1  See Section 3, Health  and Safety.

12.5  APPARATUS AND EQUIPMENT

12.5.1   Facilities for holding and  acclimating test organisms.
                                                           i
12.5.2   Sheepshead minnow  culture unit --  see  Subsection 6.13  below.   To
perform toxicity  tests on-site or in  the laboratory,  sufficient  numbers  of
newly fertilized  eggs  must be  available, preferably from an in-house
sheepshead  minnow culture  unit.  If necessary, embryos can be  obtained from
outside sources  if shipped in  well  oxygenated  water in insulated containers.

 12 5 2 1 A test  using 15  embryos per test vessel  and four replicates per
 concentration, will  require 360 newly-fertilized embryos  at the  start of the
 test   A test with a minimum of 10  embryos per test vessel and three
 replicates  per concentration,  and with five effluent concentrations and a
 control, will require a minimum of  180 embryos at the start cif the test.

 12 5 3  Brine shrimp,  Artemia, culture unit -- for feeding sheepshead minnow
 larvae in the continuous culture unit, (see Subsection 6.12! below).

 12 5.4  Samplers -- automatic  sampler, preferably with sample cooling
 capability, that can collect  a 24-h composite sample of 5 L, and maintain
 sample temperature at 4°C.

 12.5.5  Environmental chamber or equivalent facility with!temperature control
 (25 ±  1°C).

 12.5.6  Water purification  system  --  Millipore Milli-Q®,  deionized water  (DI)
 or equivalent.                                     .

 12  5 7  Balance  -- analytical, capable  of accurately  weighing to  0.00001  g.
 Note-   An  analytical  balance  is not  needed for this test  but  is needed  for
 other  specified  toxicity  test methods with growth  endpoints.

  12  5 8 Reference weights,  Class S -- for checking the performance of the
  balance.   The reference weights  should  bracket  the expected weights  of
  reagents,  and the expected weights of the weighing pans and the weights of the
  weighing pans plus  larvae.                                i

                                       125

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 12.5.9  Air pump -- for oil free air supply.
.12.5.10  Air lines, and air stones -- for aerating water containing embryos,
 larvae, or supplying air to test solution with low DO.
 12.5.11  Meters, pH and DO -- for routine physical and  chemical  measurements.
 12.5.12  Standard or micro-Winkler apparatus -- for determining  DO (optional).
 12.5.13  Dissecting microscope -- for examining embryos and larvae.
 12.5.14  Light box -- for counting and observing embryos and larvae.
 12.5.15  Refractometer -- for determining salinity.
 12.5.16  Thermometers,  glass or electronic,  laboratory  grade --  for measuring
 water temperatures.
 12.5.17  Thermometers,  bulb-thermograph or electronic-chart type -- for
 continuously recording temperature.
 12.5.18  Thermometer,  National  Bureau of Standards Certified (see USEPA  Method
 170.1,  USEPA,  1979b)  -- to calibrate  laboratory thermometers.
 12.5.19  Test  chambers  --  four (minimum of  three),  borosilicate glass or
 non-toxic plastic labware per test concentration.   Care must be  taken to avoid
 inadvertently  removing  embryos  or larvae when  test solutions are decanted  from
 the  chambers.   To avoid potential  contanimation from the air and excessive
 evaporation  of test solutions during  the test,  the chambers should be covered
 with safety  glass plates or sheet plastic (6 mm thick).   The covers are
 removed only for observation and  removal  of  dead organisms.
 12.5.20  Beakers -- six Class A,  borosilicate  glass  or  non-toxic plasticware,
 1000 mL for  making  test solutions.
 12.5.21   Wash  bottles  --  for deionized  water,  for  washing embryos from
 substrates and  containers,  and  for rinsing small glassware  and instrument
 electrodes and  probes.
 12.5.22   Volumetric flasks  and  graduated  cylinders  -- Class  A, borosilicate
 glass or  non-toxic  plastic  labware, 10-1000  ml  for making test solutions.
 12.5.23   Pi pets,  volumetric  --  Class A,  1-100 ml.
 12.5.24   Pipets,  automatic  -- adjustable, 1-100  ml.
 12.5.25   Pipets,  serological  -- 1-10 ml,  graduated.
 12.5.26   Pipet bulbs and  fillers  -- PROPIPET®,- or equivalent.
 12.5.27  Droppers and glass tubing with fire polished aperatures, 4 mm ID --
for transferring embryos  and larvae.
                                      126

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12.5.28  Siphon with bulb and clamp -- for cleaning test chambers.
                                                          i
                                                          r
12.5.29  NITEX® or stainless steel mesh sieves, (< 150 im> 500 /un, and 3 to 5
mm) -- for collecting Artemia nauplii and fish embryos, and for spawning
baskets, respectively (NITEX® is available from Sterling Marine Products, 18
Label Street, Montclair, NJ 07042;  201-783-9800).
                                                         • i
12.6  REAGENTS AND CONSUMABLE MATERIALS

12.6.1  Sample containers -- for sample shipment and storage (see Section 8,
Effluent and Receiving Water Sampling, Sample Handling, and Sample Preparation
for Toxicity Tests).
                                                          i
12.6.2  Data sheets (one set per test) -- for data recording (see Figure 1).
                                                          i
12.6.3  Tape, colored -- for labelling test chambers.

12.6.4  Markers, waterproof -- for marking containers, etc.

12.6.5  Buffers, pH 4, pH 7, and pH  10 (or as per instructions of instrument
manufacturer) -- for standards and calibration check (see USEPA Method 150.1,
USEPA,  1979b).

12.6.6  Membranes and filling solutions for dissolved  oxygen probe (see USEPA
Method  360.1, USEPA, 1979b), or reagents  -- for modified Vfinkier  analysis.

12.6.7  Laboratory quality assurance  samples and standards --for calibration
of the  above methods.

12.6.8  Reference toxicant solutions  -- see Section 4, Quality Assurance.

12.6.9  Reagent water -- defined  as  distilled or deionized water  that does  not
contain substances which are toxic to the test organisms  (see Section 5,
Facilities,  Equipment,  and Supplies).
                                                     '
12.6.10  Effluent, receiving water,  and dilution water -- see Section 7,
Dilution  Water, and Section 8,  Effluent and Receiving  Water Sampling, Sample
Handling,  and Sample Preparation  for Toxicity Tests.

12.6.10.1   Saline test  and dilution  water -- The  salinity of the  test water
must be in the  range of 5 to 32%o.   The salinity  should vary no more than ± 2
%ol  among chambers on a given day.   If effluent and receiving water tests are
conducted concurrently,  the  salinities of the water should be similar.

12.6.10.2  The  overwhelming majority of  industrial and sewage treatment
effluents entering marine  and estuarine systems contain little  or no
measurable salts.   Exposure  of  sheepshead minnow  embryos  to these effluents
will  require adjustments in  the salinity  of the test  solutions.   It  is
 important to maintain a constant  salinity across  all  treatments.   If  In
 addition,  it may  be  desirable to  match the test salinity  with that of the
receiving water.  Two methods are available to  adjust  salinities  --a
 hypersaline brine derived  from  natural seawater or artificial sea salts.

                                       127

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Test Dates:
Type Effluent:
Effluent Tested:

Original pH: 	
       Species:
           Field:
Lab:
Test:
Salinity:
  D.O.:
 CONCENTRATION:
    Replicate  I
DAY
^Live/Dead
Embrvo-Larvae
Terata
Terno. (°C)
Salinity foot)
D.O. (m/L)
DH
0






1






2






3






4






5






6






7






8






9






 CONCENTRATION:
  Replicate II;
Comments:
DAY
^Live/Dead
Embrvo-Larvae
Terata
Temo. CO
Salinity (ppt)
D.O. (ma/L)
DH
0






1






2






3






4






5






6






7






8






9






Note:  Final endpoint for this test is total mortality (combined total  number
of dead embryos, dead larvae, and deformed larvae) (see Subsection 12.10.8 and
12.13).

Figure 1.   Data form for sheepshead minnow, Cypn'nodon variegatus,
            embryo-larval survival/teratogenicity test.  Daily record of
            embryo-larval survival/terata and test conditions.
                                      128

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CONCENTRATION:
Reolicate III:
DAY
#Live/Dead
Embrvo- Larvae
Terata
Temo. (°)
Salinitv foot)
D.O. fma/L)
PH
n






i






2






3






4






5






6






7






8






9






 CONCENTRATION:
   REPLICATE IV
DAY
#Live/Dead
Embrvo -Larvae
Terata
Temo. (°C)
Salinitv foot)
D.O. (mq/L)
pH
0





1





2





3





4





5





6





7





8





9





Comments:
Note:  Final endpoint for this test is total mortality (combined total number
of dead embryos, dead larvae, and deformed larvae) (see Subsection 12.10.8 and
12.13).
Figure 1.   Data form for sheepshead minnow, Cyprinodon vsn'egatus,
            embryo-larval survival/teratogenicity test.  Daily record of
            embryo-larval survival/terata and test conditions; (CONTINUED).
                                      129

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        1.3   Hypersaline  brine  (HSB):  HSB has several advantages that make it
 fm^}6 f°r +SQ tn tox1city testing.  It can be made from any high Sity
 filtered seawater by evaporation, and can be added to the effluent or to
 deionized water to increase the salinity.  HSB derived from natural seawater
           rLnnne%Sary  trace "^als' biogenic ™"°™s, and some of the
           components necessary for adequate growth, survival,  and/or
          ion or marine  and estuarine organisms, and may be stored for •
 iicprt ac a rf-i   f w?Jnout any apparent degradation.  However if 100% HSB is
 ,  •    uon dll"ent' tne maximum concentration of effluent that  can be tpstpri
 using HSB is limited to 80% at 20%. salinity,  and 70% at 30%0  salinity

 that'(l)3has Ihhiahesu f°ntainer (or makil?9 HSB from natural seawater is one
 SKI                                                                 ,.
 immersed directly into the seawater,  ensure  that  the  heater mateHals do not
            le        ^ubstances  that  would contaminate  the br?ne   Jne
                             thermostatica1^ controlled heat exchanger made
                               " US6d  ^ °n       -         Compressors to
     ;ln^:?   Bef°re  addin9  seawater to the brine generator, thoroughly clean
     generator,  aeration  supply tube, heater, and any other materials that w?ll
         e^^
contaminaton
                                          -°
                          incoming tide to minimize the possibility of

Jater^houlri hSeaoSe^Ure °f tje^wwater is increased  slowly to 40°C.   The
water eSratf™   T£  Jo prevent temperature stratification  and to increase
                                salinity  is  attained, the HSB should be
                                     130

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12.6.10.3.7  Divide the salinity of the HSB by the expected test salinity to
determine the proportion of deionized water to brine.  For example, if the
salinity of the HSB is 100%o and the test is to be conducted at 20%o, 100%o
divided by 20%o = 5.0.  The proportion of brine is 1 part in 5 (one part brine
to four parts deionized water).  To make 1 L of seawater at 20%o salinity from
a HSB of 100%o, divide 1 L (1000 ml) by 5.0.  The result, 200 ml, is the
quantity of HSB needed to make 1 L of sea water.  The difference, 800 ml, is
the quantity of deionized water required.                 j
                                                          i
12.6.10.3.8  Table 1 illustrates the composition of test solutions at 20%o if
they are prepared by serial dilution of effluent with 20%o salinity seawater.

12.6.10.4  Artificial sea salts:  HW MARINEMIX® brand sea salts  (Hawaiian
Marine Imports Inc., 10801 Kempwood, Suite 2, Houston, TX 77043) have been
used successfully at the USEPA, Region 6, Houston laboratory to  culture
sheepshead minnows and perform the embryo-larval survival and teratogenicity
test.  EMSL-Cincinnati has found FORTY FATHOMS® artificial sea salts (Marine
Enterprises, Inc., 8755 Mylander Lane, Baltimore, MD 21204;  301-321-1189), to
be suitable for culturing sheepshead minnows and for performing'the larval
survival and growth test and embryo-larval test.  Artificial sea salts may be
used for culturing sheepshead minnows and for the embryo larval  test if  the
criteria for acceptability of test data  are satisfied (see Subsection 12.11).

12.6.10.4.1  Synthetic sea salts are packaged in plastic bags and mixed  with
deionized water or equivalent.  The  instructions on  the package  of sea salts
should be followed carefully,  and salts  should  be mixed in a separate
container -- not the  culture tank.   The  deionized water used in  hydration
should be in the temperature range  of 21-26°C.  Seawater made from artificial
sea  salts is conditioned  (Spotte, 1973;  Spotte  et al.,  1984; Bower,  1983)
before it  is used  for culturing or  testing.  After  adding the water, place an
airstone  in the container, cover, and aerate the solution mildly for at  least
24 h before use.                                          '•

12.6.11   BRINE SHRIMP, ARTEMIA, CULTURE  --  for  feeding  cultures.

 12.6.11.1   Newly-hatched  Artemia nauplii  are used as food  in the sheepshead
minnow culture,  and  a brine  shrimp  culture  unit should  be prepared  (USEPA,
 1993a).   Although  there  are  many commercial  sources of  brine shrimp  cysts, the
Brazilian or Colombian strains are  currently preferred  because  the  supplies
examined have  had  low concentrations of  chemical  residues  and  produce nauplii
 of suitably small  size.   For commercial  sources of  brine  shrimp, Artemia,
 cysts,  see  Table  2 of Section  5,  Facilities,  Equipment,  and  Supplies;  and
 Section  4,  Quality Assurance.

 12.6.11.2  Each  new batch of Artemia cysts  must be  evaluated for size
 (Vanhaecke and Sorgeloos,  1980,  and Vanhaecke  et al.,  1980)  and nutritional
 suitability (Leger,  et al.,  1985;  Leger, et al.,  1986)  against  known suitable
 reference cysts  by performing a side by  side larval growth  test using  the
 "new"  and "reference" cysts.   The  "reference"  cysts used in  the suitability
 test may be a  previously tested and acceptable batch of cysts,  or  may  be
 obtained from  the Quality Assurance Research Division,  Environmental
                                                          j

                                      131

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TABLE  1.     PREPARATION  OF  TEST  SOLUTIONS  AT A  SALINITY OF  20%o,  USING
             20%o NATURAL OR ARTIFICIAL  SEAWATER,  HYPERSALINE  BRINE, OR
             ARTIFICIAL SEA  SALTS
                                           Solutions To  Be Combined
Effluent
Solution
1
2
3
4
5
Control
Total
Ef f 1 uent
Cone.
fo/\
(/o)
1001'2
50
25
12.5
6.25
0.0

Volume of Volume of Diluent
Effluent Seawater (20%o)
Solution
4000 mL
2000 mL Solution 1 + 2000 mL
2000 mL Solution 2 + 2000 mL
2000 mL Solution 3 + 2000 mL
2000 mL Solution 4 + 2000 mL
2000 mL
10000 mL
  This illustration assumes: (1) the use of 400 mL of test solution in
  each of four replicates and 400 mL for chemical analysis (total of
  2000 mL) for the control and five concentrations of effluent (2) an
  effluent dilution factor of 0.5, and (3) the effluent lacks appreciable
  salinity.  A sufficient initial volume (4000 mL) of effluent is prepared
  by adjusting the salinity to the desired level.  In this example, the
  salinity is adjusted by adding artificial sea salts to the 100%
  effluent, and preparing a serial dilution using 20%o seawater (natural
  seawater, hypersaline brine, or artificial seawater).  The salinity of
  the initial 4000 mL of 100% effluent is adjusted to 20%o by adding 80 g
  of dry artificial sea salts (HW MARINEMIX or FORTY FATHOMS®), and mixing
  for 1 h.  Test concentrations are then made by mixing appropriate
  volumes of salinity-adjusted effluent and 20%> salinity dilution water
  to provide 4000 mL of solution for each concentration.  If hypersaline
  brine alone <100%o) is used to adjust the salinity of the effluent,  the
  highest concentration of effluent that could be achieved would be 80% at
2 20%o salinity, and 70% at 30%o salinity.
  The same procedures would be followed in preparing test concentrations
  at other salinities between 20%o and 30%o:  (1) The salinity of the bulk
  (initial) effluent sample would be adjusted to the appropriate salinity
  using artificial sea salts or hypersaline brine, and (2) the remaining
  effluent concentrations would be prepared by serial dilution, using a
  large batch (10 L) of seawater for dilution water,  which had been
  prepared at the same salinity as the effluent,  using natural  seawater,
  hypersaline and deionized water.
                                    132

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Monitoring Systems Laboratory, Cincinnati, OH 45268; 513-569-7325.  A sample
of newly hatched Artemia nauplii from each new batch of cysts should be
chemically analyzed.  The Artemia cysts should not be used if the
concentration of total organic chlorine pesticides exceeds 0.15 ng/g wet
weight or the total concentration of organochlorine pesticides plus PCBs
exceeds 0.30 pg/g wet weight. (For analytical methods see USEPA, 1982).
12.6.11.3  Artemia nauplii are obtained as follows:

      1.    Add 1 L of seawater, or a solution prepared by adding 35.0 g
            uniodized salt (NaCl) or artificial sea salts to 1 L of deionized
            water, to a 2-L separatory funnel, or equivalent.
      2.    Add 10 ml Artemia cysts to the separatory funnel and aerate for
            24 h at 27°C.  (Hatching time varies with incubation temperature
            and the geographic strain of Artemia used (see USEPA, 1985d,
            USEPA,  1993a; and ASTM, 1993).
      3.    'After 24 h, cut off the air supply in the separatory funnel.
            Artemia nauplii are phototactic,  and will concentrate at the
            bottom of the funnel if it is covered for 5-10 minutes.  To
            prevent mortality, do not leave the concentrated nauplii at the
            bottom of the funnel more than 10 min without aeration.
      4.    Drain the nauplii into a beaker or funnel fitted with a <  150 /M
            NITEX® or stainless steel screen, and rinse with seawater  or
            equivalent before use.

12.6.11.4  Testing Artemia nauplii as food for toxicity te;st organisms.

12.6.11.4.1   The primary  criterion for acceptability of each new supply of
brine shrimp  cysts is the ability of the nauplii to support good survival and
growth  of  the sheepshead  minnow larvae.  The  larvae used to evaluate the
suitability of the brine  shrimp nauplii must  be of the same geographical
origin,  species, and  stage of development as  those used routinely in the
toxicity tests.  Sufficient data to detect differences in survival  and growth
should  be  obtained by using three replicate test vessels, each containing a
'minimum of 15 larvae, for each  type of food.
                                                          i
                                                          i
12.6.11.4.2   The feeding  rate and frequency,  test vessels, volume of control
water,  duration of the test,  and age of the nauplii at the start of the test,
should  be  the same as used for  the routine toxicity tests.
                                                          i

12.6.11.4.3   Results  of  the brine shrimp nutrition assay, 'where there  are only
two treatments, can  be evaluated statistically  by  use of  ai t test.  The  "new"
food is acceptable  if there are no  statistically  significant differences  in
the survival  and growth  of the  larvae  fed the two  sources of nauplii.

 12.6.11.4.4  The  average seven-day  survival of  larvae should be 80% or
greater, and  (2)  the  average  dry weight of  larvae  should  be  0.60 mg or
greater, if dried  and weighed  immediately after the test, or  (3) the  average
dry weight of larvae  should be  0.50 mg  or greater,  if the larvae are preserved
 in 4% formalin before drying  and weighing.  The above minimum  weights  presume
that the age  of the  larvae  at  the  start of  the  test  is not; greater  than  24  h.


                                      133

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 12.6.12   TEST ORGANISMS,  SHEEPSHEAD  MINNOWS,  CYPRINODON VARIEGATUS

 12.6.12.1  Brood  Stock

 12.6.12.1.1  Adult  sheepshead minnows  for  use as  brood  stock may  be  obtained
 by  seine  in Gulf  of Mexico  and Atlantic  coast estuaries,  from commercial
 sources,  or from  young fish raised to  maturity in the laboratory.  Feral  brood
 stocks and  first  generation laboratory fish are preferred,  to minimize
 inbreeding.

 12.6.12.1.2  To detect disease and to  allow time  for acute  mortality due  to
 the stress  of capture,  field-caught  adults are observed in  the  laboratory a
 minimum of  two weeks before using as a source of  gametes.   Injured or diseased
 fish are  discarded.

 12.6.12.1.3  Sheepshead minnows can  be continuously cultured in the  laboratory
 from eggs to  adults.   The larvae, juvenile, and adult fish  should be kept in
 appropriate size  rearing tanks, maintained at ambient laboratory  temperature.
 The larvae  should be fed sufficient  newly  hatched Artemia nauplii daily to
 assure that live  nauplii are always  present.   Juveniles are fed frozen adult
 brine shrimp  and  a  commercial flake  food,  such as TETRA SM-80®, available from
 Tetra Sales (U.S.A),  201 Tabor Road, Morris Plains, NJ  07950; 800-526-0650, or
 MARDEL AQUARIAN®  Tropical Fish Flakes, available  from Mardel Laboratories,
 Inc., 1958  Brandon  Court, Glendale Heights, IL 60139; 312-351-0606,  or
 equivalent.   Adult  fish are fed flake  food three  or four times daily,
 supplemented  with frozen adult brine shrimp.

 12.6.12.1.3.1  Sheepshead minnows reach  sexual  maturity in  three-to-five
 months after  hatch,  and have an average  standard  length of  approximately  27 mm
 for females  and 34 mm for males.  At this time, the males begin to exhibit
 sexual dimorphism and initiate territorial behavior.  When  the fish  reach
 sexual maturity and  are to  be used for natural  spawning, the temperature
 should be controlled  at 18-20°C.

 12.6.12.1.4   Adults  can be  maintained  in natural  or artificial seawater in a
 flow-through  or recirculating, aerated system consisting of an all-glass
 aquarium, or  a "Living  Stream" (Figid  Unit, Inc.,  3214  Sylvania Avenue,
 Toledo, OH  43613; 419-474-6971), or equivalent.

 12.6.12.1.5   The  system is  equipped with an undergravel or  outside biological
 filter-of shells  (see  Spotte, 1973 or  Bower,  1983  for conditioning the
 biological  filter),  or  a cartridge filter, such as a MAGNUM® Filter, available
 from Carolina  Biological Supply Co., Burlington,  NC 27215;  800-334-5551,  or an
 EHEIM® Filter, available from Hawaiian Marine  Imports Inc., P.O. Box 218687,
 Houston,   TX 77218; 713-492-7864, or equivalent, at a salinity of 20-30%o  and a
 photoperiod of 16 h  light/8  h dark.

 12.6.12.2  Obtaining  Embryos for Toxicity Tests

 12.6.12.2.1   Embryos  can be  shipped to the laboratory from  an outside source
or obtained from adults held in the laboratory.   Ripe eggs  can be obtained
either by natural  spawning or by intraperitoneal  injection  of the females with

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human chorionic gonadotrophin  (HCG) hormone, available from United States
Biochemical Corporation, Cleveland, OH 44128; 216-765-5000.  If the culturing
system for adults is temperature controlled, natural spawning can be induced.
Natural spawning is preferred  because repeated spawnings can be obtained from
the same brood stock, whereas  with hormone injection, the brood stock is
sacrificed in obtaining gametes.

12.6.12.2.2  It should be emphasized that the injection and hatching schedules
given below are to be used only as guidelines.  Response to the hormone varies
from stock to stock and with temperature.  Time to hatch and percent hatch
also vary among stocks and among batches of embryos obtained from the same
stock, and are dependent on temperature, DO, and salinity.
                                                               .
12.6.12.2.3  Forced Spawning
                             .
12.6.12.2.3.1  HCG is reconstituted with sterile saline or Ringer's solution
immediately before use.  The standard HCG vial contains 1,000 IU to be
reconstituted in 10 ml of saline.  Freeze-dried HCG which comes with
premeasured and sterilized saline is the easiest to use.  Use of a 50 IU dose
requires injection of 0.05 ml  of reconstituted hormone solution.
Reconstituted HCG may be used  for several weeks if kept in the refrigerator.

12.6.12.2.3.2  Each female is  injected intraperitoneally with 50 IU HCG on two
consecutive days, starting at  least 4 days prior to the beginning of a test.
Two days following the second  injection, eggs are stripped from the females
and mixed with sperm derived from excised macerated testes.  At least ten
females and five males are used per test to ensure that there is a sufficient
number of viable embryos.

12.6.12.2.3.3  HCG is injected into the peritoneal cavity, just below the
skin, using as small a needle  as possible.  A 50 IU dose is recommended for
females approximately 27 mm in standard length.  A larger or smaller dose may
be used for fish which are significantly larger or smaller than 27 mm.   With
injections made on days one and two, females which are held at 25°C should be
ready for stripping on Day 4.  Ripe females should show pronounced abdominal
swelling, and release at least a few eggs in response to a gentle squeeze.
Injected females should be isolated from males.  It may be helpful if fish
that are to be injected are maintained at 20°C before injection, and the
temperature raised to 25°C on  the day of the first injection.

12.6.12.2.3.4  Prepare the testes immediately before stripping the eggs from
the females.   Remove the testes from three-to-five males.   The testes are
paired, dark grey organs along the dorsal mid!ine of the abdominal cavity. . If
the head of the male is cut off and pulled away from the rest of the fish,
most of the internal organs can be pulled out of the body cavity,  leaving the
testes behind.  The testes are placed in a few ml of seawater until  the eggs
are ready.
                            •
12.6.12.2.3.5  Strip the eggs  from the females, into a dish containing  50-100
ml of seawater, by firmly squeezing the abdomen.   Sacrifice the females and
remove the ovaries if all the ripe eggs do not flow out freely.   Break  up any
                                                          i

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clumps of ripe eggs and remove clumps of ovarian tissue and underripe eggs.
Ripe eggs are spherical, approximately 1 mm in diameter, and almost clear.

12.6.12.2.3.6  While being held over the dish containing the eggs, the testes
are macerated in a fold of NITEX® screen (250-500 urn mesh) dampened with
seawater.  The testes are then rinsed with seawater to remove the sperm from
tissue, and the remaining sperm and testes are washed into the dish.  Let the
eggs and milt stand together for 10-15 min, swirling occasionally.

12.6.12.2.3.7  Pour the contents of the dish into a beaker, and insert an
airstone.  Aerate gently, such that the water moves slowly over the eggs, and
incubate at 25°C for 60-90 min.  After incubation, wash the eggs on a NITEX®
screen and resuspend them in clean seawater.

12.6.12.2.4  Natural Spawning

12.6.12.2.4.1  Short-term (Demand) Embryo Production

12.6.12.2.4.1.1  Adult fish should be maintained at 18-20°C in a temperature
controlled system.  To obtain embryos for a test, adult fish (generally, at
least eight-to-ten females and three males) are transferred to a spawning
chamber, with a photoperiod of 16 h light/8 h dark and a temperature of 25°C,
two days before the beginning of the test.  The spawning chambers are
approximately 20 X 35 X 22 cm high (USEPA, 1978), and consist of a basket of
3-5 mm NITEX® mesh, made to fit into a 57-L (15 gal) aquarium.  Spawning
generally will begin within 24 h or less.  The embryos will fall through the
bottom of the basket and onto a collecting screen (250-500 /jm mesh) or tray
below the basket.  The collecting tray should be checked for embryos the next
morning.  The number of eggs produced is highly variable.  The number of
spawning units required to provide the embryos needed to perform a toxicity
test is determined by experience.  If the trays do not contain sufficient
embryos after the first 24 h, discard the embryos, replace the trays, and
collect the embryos for another 24 h or less.  To help keep the embryos clean,
the adults are fed while the screens are removed.

12.6.12.2.4.1.2  The embryos are collected in a tray placed on the bottom of
the tank.  The collecting tray consists of ± 150 /^m NITEX® screen attached to
a rigid plastic frame.  The collecting trays with newly-spawned, embryos are
removed from the spawning tank, and the embryos are collected from the screens
by washing them with -a wash bottle or removing them with a fine brush.  The
embryos from several spawning units may be pooled in a single container to
provide a sufficient number to conduct the test(s).  The embryos are
transferred into a petri dish or equivalent, filled with fresh culture water,
and are examined using a dissecting microscope or other suitable magnifying
device.  Damaged and infertile eggs are discarded (see Figure 2).  It is
strongly recommended that the embryos be obtained from fish cultured in-house,
rather than from outside sources, to eliminate the uncertainty of damage
caused by shipping and handling that may not be observable, but which might
affect the results of the test.

12.6.12.2.4.1.3  After sufficient embryos are collected for the test, the
adult fish are returned to the (18-20°C) culture tanks.

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12.6.12.2.4.2  Sustained Natural Embryo Production

12.6.12.2.4.2.1  Sustained (long-term), daily, embryo production can be
achieved by maintaining mature fish in tanks, such as a (2I85--L or 75-gal)
LIVING STREAM® tank, at a temperature of 23-25°C.  Embryos are produced daily,
and when needed, embryo "collectors" are placed on the bottom of the tank on
the afternoon preceding the start of the test.  The next morning, the embryo
collectors are removed and the embryos are washed into a shallow glass culture
dish using artificial seawater.

12.6.12.2.4.2.2  Four embryo collectors, approximately 20 cm X 45 cm, will
approximately cover the bottom of the 285-L tank.  The collectors are
fabricated from plastic fluorescent light fixture diffusors (grids), with
cells approximately 14 mm deep X 14 mm square.  A screen consisting of 500 /*m
mesh is attached to one side (bottom) of the grid with silicone adhesive.  The
depth and small size of the grid protects the embryos from predation by the
adult fish.

12.6.12.2.4.2.3  The brood stock is replaced annually with feral stock.

12.6.12.2.5  Test Organisms                               I

12.6.12.2.5.1  Embryos spawned over a less than 24-h period, are used for the
test.  These embryos may be used immediately to start a test or may be placed
in a suitable container and transported for use at a remote location.  When
overnight transportation is required, embryos should be obtained when they are
no more than 8-h old.  This permits the tests at the remote site to be started
with less than 24-h old embryos.  Embryos should be transported or shipped in
clean, insulated containers, in well aerated or oxygenated fresh seawater or
aged artificial sea water of correct salinity, and should be protected from
extremes of temperature and any other stressful conditions during transport.
Instantaneous changes of water temperature when embryos are transferred from
culture unit water to test dilution water, or from transport container water
to on-site test dilution, should be less than 2°C.  Instantaneous changes of
pH, dissolved ions, osmotic strength, and DO should also be kept to a minimum.

12.6.12.2.5.2  The number of embryos needed to start the test will depend on
the number of tests to be conducted and the objectives.  If the test is
conducted with four replicate test chambers (minimum of three) at each
toxicant concentration and in the control, with 15 embryos; (minimum of 10) in
each test chamber, and the combined mortality of embryos prior to the start of
the test is less than 20%, 400 viable embryos are required for the test.

12.7  EFFLUENT AND RECEIVING WATER COLLECTION, PRESERVATION, AND STORAGE

12.7.1  See Section 8, Effluent and Receiving Water Sampling, Sample Handling,
and Sample Preparation for Toxicity Tests.

12.8  CALIBRATION AND STANDARDIZATION

12.8.1  See Section 4, Quality Assurance.


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12.9  QUALITY CONTROL

12.9.1  See Section 4, Quality Assurance.

12.10  TEST PROCEDURES

12.10.1  TEST SOLUTIONS

12.10.1.1  Receiving Waters

12.10.1.1.1  The sampling point is determined by the objectives of the test.
At estuarine and marine sites, samples are usually collected at mid-depth.
Receiving water toxicity is determined with samples used directly as collected
or with samples passed through a 60 urn NIT.EX® filter and compared without
dilution, against a control.  Using four replicate chambers per test, each
containing 400-500 mL, and 400 mL for chemical analysis, would require
approximately 2.0-2.5 L or more of sample per test per day.

12.10.1.2  Effluents

12.10.1.2.1  The selection of the effluent test concentration should be based
on the objectives of the study.  A dilution factor of 0.5 is commonly used.  A
dilution factor of 0.5 provides precision of ± 100%, and allows for testing of
concentrations between 6.25% and 100% effluent using only five effluent
concentrations (6.25%, 12.5%, 25%, 50%, and 100%).  Test precision shows
little improvement as dilution factors are increased beyond 0.5 and declines
rapidly if smaller dilution factors are used.  Therefore, USEPA recommends the
use of the z. 0.5 dilution factor.  If 100%o salinity MSB is used as a diluent,
the maximum concentration of effluent that can be tested will be 80% at 20%o
and 70% at 30%o salinity.

12.10.1.2.2  If the effluent is known or suspected to be highly toxic, a lower
range of effluent concentrations should be used (such as 25%, 12.5%, 6.25%,
3.12%, and 1.56%).  If a high rate of mortality is observed during the first
l-to-2 h of the test, additional dilutions at the lower range of effluent
concentrations should be added.

12.10.1.2.3  The volume of effluent required to initiate the test and for
daily renewal of four replicates (minimum of three) per concentration for five
concentrations of effluent and a control, each containing 400 mL of test
solution, is approximately 4 L.  Prepare enough test solution (approximately
3000 mL) at each effluent concentration to refill the test chambers and
provide at least 400 mL additional volume for chemical analyses.

12.10.1.2.4  Maintain the effluent at 4°C.  Plastic containers such as 8-20 L
cubitainers have proven successful for effluent collection and storage.

12.10.1.2.5  Just prior to test initiation (approximately 1 h), the
temperature of a sufficient quantity of the sample(s) to make the test
solutions should be adjusted to the test temperature (25 ± 1°C) and maintained
at that temperature during the addition of dilution water.


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 12.10.1.2.6  Higher  effluent  concentrations  (i.e.,  25%,  50%,  and  100%)  may
 require  aeration to  maintain  adequate  dissolved  oxygen  concentrations.
 However,  if one solution  is aerated, all  concentrations  must  be.aerated.
 Aerate effluent as it warms and  continue  to  gently  aerate  test  solutions  in
 the test  chambers for the duration  of  the test.

 12.10.1.2.7  Effluent dilutions  should be prepared  for  all  replicates  in  each
 treatment  in one beaker to minimize variability  among the  replicates.   The
 test chambers are labelled with  the test  concentration  and  replicate number.
 Dispense  into the appropriate effluent dilution  chamber.

 12.10.1-.3  Dilution  Water

 12.10.1.3.1  Dilution water may  be  uncontaminated natural  seawater  (receiving
 water), HSB prepared from natural seawater,  or artifical seawater prepared
 from FORTY FATHOMS®  or GP2 sea salts (see Table  3 and Section 7, Dilution
 Water).  Other artifical  sea  salts  may be used for  culturing  sheepshead
 minnows  if the control criteria  for acceptability of test data  are  satisfied.

 12.10.2  START OF THE TEST
                                                          I

 12.10.2.1  Tests should begin as soon  as  possible,  preferably within 24 h
 after sample collection.  For on-site  toxicity studies,  no  more than 24 h
 should elapse between collection of the effluent and use in an  embryo-larval
 study.  The maximum  holding time following retrieval of  the sample  from the
 sampling device should not exceed 36 h  for off-site toxicity  studies unless
 permission is granted by  the  permitting authority.  In no case  should the
 sample be used in a  test  more than  72  h after sample collection (see Section
 8, Effluent and Receiving Water  Sampling, Sample Handling,  and  Sample
 Preparation for Toxicity  Tests).

 12.10.2.2  Label the test chambers  with a marking pen.  Use color-coded tape
 to identify each treatment and replicate.  A minimum of  five effluent
 concentrations and a control  are used  for each effluent test.   Each
 concentration (including  controls)  is  to  have four replicates (minimum of
 three).  Use 500 ml beakers, crystallization dishes, nontoxic disposable
 plastic labware, or equivalent for  test chambers.

 12.10.2.3  Prepare the test solutions  (see Table 1) and add to the test
 chambers.

 12.10.2.4  Gently agitate and mix the  embryos to be used in the test in a
 large container so that eggs from different spawns are evenly dispersed.

 12.10.2.5  The test is started by randomly placing embryos from the common
 pool,  using a small  bore  (2 mm),  fire polished,  glass tube calibrated to
 contain approximately the desired, number of embryos, into each of four
 replicate test chamber,  until  each chamber contains 15 embryos  (minimum of
 10),  for a total of 60 embryos (minimum of 30) for each concentration (four
 replicates recommended,  three minimum)  (see Appendix A).  The amount of water
 added to the chambers when transferring the embryos should be kept to a
minimum to avoid unnecessary dilution of the test concentrations.

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12.10.2.6  After the embryos have been distributed to each test chamber,
examine and count them.  Remove and discard damaged or infertile eggs and
replace with undamaged embryos.  It may be more convenient and efficient to
transfer embryos to intermediate containers of dilution water for examination
and counting.  After the embryos have been examined and counted in the
intermediate container, assign them to the appropriate test chamber and
transfer them with a minimum of dilution water.

12.10.2.7  Randomize the position of the test chambers at the beginning of the
test (see Appendix A).  Maintain the chambers in this configuration throughout
the test.  Preparation of a position chart may be helpful.

12.10.3  LIGHT, PHOTOPERIOD, SALINITY, AND TEMPERATURE

12.10.3.1  The light quality and intensity should be at ambient laboratory
levels, approximately 10-20 ^E/m/s, or 50 to 100 foot candles (ft-c),  with a
photoperiod of 16 h of light and 8 h of darkness.  The test water temperature
should be maintained at 25 ± 1°C.  The salinity should be 5%o to 32%o ± 2%o to
accommodate receiving waters that may fall within this range.  The salinity
should vary no more than ± 2%o  among the chambers on a given day.  If
effluent and receiving water tests are conducted concurrently, the salinities
of these tests should be similar.

12.10.4  DISSOLVED OXYGEN (DO) CONCENTRATION

12.10.4.1  Aeration may affect the toxicity of effluents and should be used
only as a last resort to maintain satisfactory DO.  The DO should not fall
below 4.0 mg/L (see Section 8, Effluent and Receiving Water Sampling, Sample
Holding, and Sample Preparation for Toxicity Tests).  If it is necessary to
aerate, all treatments and the control should be aerated.  The aeration rate
should not exceed 100 bubbles/min, using a pipet with a 1-2 mm orifice, such
as a 1-mL KIMAX® Serological Pipet No. 37033, or equivalent.  Care should be
taken to ensure that turbulence resulting from the aeration does not cause
undue physical stress to the fish.

12.10.5  FEEDING

12.10.5.1  Feeding is not required.

12.10.6  OBSERVATIONS DURING THE TEST

12.10.6.1  Routine Chemical and Physical Determinations

12.10.6.1.1  DO is measured at the  beginning and end of each 24-h exposure
period at each test concentration and  in the control.

12.10.6.1.2  Temperature, pH,  and salinity are measured at the end of each
24-h exposure period  in one test chamber at each test concentration and in the
control.  Temperature should also be monitored continuously or observed and
recorded daily for at least two locations in the environmental control  system
or the samples.  Temperature should be measured  in a sufficient number  of test
                                      140

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chambers at least at the end of. the test to determine the temperature
variation in the environmental chambers.

12.10.6.1.3  The pH is measured in the effluent sample each day before new
test solutions are made.

12.10.6.1.4  Record all measurements on the data sheet (Figure 1).

12.10.6.2  Routine Biological Observations

12.10.6.2.1  At the end of the first 24 h of exposure, before renewing the
test solutions, examine and  count the embryos.  Remove the dead embryos  (milky
colored  and opaque) and record the number.  If the rate of mortality or  fungal
infection exceeds 20%  in the control chambers, or if  excessive
nonconcentration related mortality occurs,  terminate  the test and  start  a new
test with new  embryos.  If the above mortality conditions  do not occur,
continue the test for  the full nine days.

12  10  6  2.2  At  25°C,  hatching begins on  about the sixth day,.  After hatching
begins,  count  the number of  dead  and live embryos and the .number  of hatched,
dead,  live,  and  deformed and/or debilitated larvae,  dally  (see Figure  2  for
illustrations  of morphological development of  embryo and larva).   Deformed
larvae are  those with  gross  morphological  abnormalities  such  as  curved spines,
lack  of appendages,  lack of fusiform  shape (non-distinct mass),  a colored
beating heart  in an opaque  mass,  lack  of mobility,  abnormal  swimming,  or other
characteristics  that preclude survival.  Remove  dead embryos  and dead  and
deformed larvae  as  previously discussed and record  the numbers  for all test
 observations (see  Figure  2).

 12 10 6 2 3  Protect the  embryos  and  larvae from unnecessary disturbance
 during the test by carefully carrying out the daily test observations,
 solution renewals,  and removal  of dead organisms.   Make sure the test
 organisms remain immersed during the performance of the above operations.

 12.10.7  DAILY CLEANING OF TEST CHAMBERS                 j

 12 10 7 1  Since feeding is not required, test chambers are not cleaned daily
 unless  accumulation of particulate matter at the bottm of the tank causes  a
 problem.

 12.10.8 TEST SOLUTION RENEWAL

 12 10 8 1  The test solutions are renewed daily using freshly prepared
 solution, immediately  after cleaning the test chambers.   For on-site  toxicity
 studies, fresh  effluent and receiving  water samples  used  in toxicity  tests
 should  be collected daily,  and no more than 24 h should elapse between
 collection of the  sample and use  in the  test  (see Section 8,. Effluent and
 Receiving Water Sampling, Sample  Handling, and Sample Preparation for Toxicity
 Tests).  For  off-site  tests, a minimum of  three samples must be collected,
 preferably  on days  one, three, and five.   Maintain  the samples  at 4°C until
 used.
                                                          i

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Figure 2.   Embryonic development of sheepshead minnow,  Cypn'nodon variegatus:
            A. Mature unfertilized egg,  showing attachment filaments and
            micropyle, X33; B.  Blastodisc fully developed;  C,D.  Blastodisc,  8
            cells; E. Blastoderm, 16 cells;  F.  Blastoderm, late cleavage
            stage; 6. Blastoderm with germ ring formed,  embryonic shield
            developing; H.  Blastoderm covers over 3/4 of yolk,  yolk noticeably
            constricted; I. Early embryo.  From Kuntz (1916).

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Figure 2.   Embryonic development of sheepshead minnow,  Cypnnodonvanegatus:
            J  Embryo 48 h after fertilization, now segmented    throughout,
            pigment on yolk sac and body,  otoliths formed;  K. Posterior
            portion of embryo free from yolk and moves freely within egg
            membrane, 72 h after fertilization; L. Newly hatched fish,  actual
            length 4 mm; M. Larval fish 5 days after hatching, actual length  5
            mm; N. Young fish 9 mm in length; 0. Young fish 12 mm in length
            (CONTINUED).  From Kuntz (1916).            |

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  12.10.8.2  The test solutions  are  adjusted to the correct salinity and renewed

  mof waie? £eiS£# -0lJhCte?  SfPlf'  DuHng the dail* renewal process? 7^0
  UL»f«   £     1  r  •"  th^  chamber to ensure that the embryos and larvae
  SXSJ KUbm^d  dm;in9 uthe ^newal process.  New test solution (400 ml)
  pynnldnn%h   dKSl°Wly  ty Pour1n9  down the side of the test chamber to avoid
  exposing  the embryos  and larvae to excessive turbulence.

  12.10.8.3  Prepare test solutions daily, making a minimum of five
  concentrates  and a  control.  If concurrent effluent and receiving water
  testing occurs, the effluent test salinity should closely approximate that  of
  the receiving water test.   If an effluent is tested alone,  selec^a salinity
  which  approximately matches the salinity of the receiving waters   Table 1
  !U?S?  ?S     quantities of effluent,  seawater,  deionized  water,  and
  artificial  sea  salts needed to prepare 3 L of test solution  at each effluent
  concentration for  tests conducted at 20%o salinity.'                 ernuent

  12.10.9   TERMINATION OF THE TEST


  JnJ^A ThS-*uSt iS terminated after  nine  days  of  exposure,  or four days
  post-hatch, whichever comes  first.   Count  the number  of  surviving   dead   and
  deformed  and/or debilitated  larvae, and  record  the  numbers of  ewh. The
 nJSlTVJT6 ale treated  as  dead-   Keep  a  separate record of  the total
 solution       med larvae  for use  in ^porting  the  teratogenicity of the  test


 12.11   ACCEPTABILITY OF  TEST RESULTS
                                  ^ aCC6ptable' SUrvival in the controls must

 12.12   SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA


                   °f teSt C0nd1tions and test acceptability criteria is
 12.13  DATA ANALYSIS

 12.13.1  GENERAL

 12.13.1.1  Tabulate and summarize the data.

 JH?'1;.2  Tue endP°ints of this toxicity test are based  on  total mortality
 combined number of dead embryos, dead larvae,  and deformed larvae.  The EC
 endpoints are calculated using Probit Analysis (Finney, 1971)    LOEC and NOFr
 values, for total  mortality,  are obtained Jslng a hypothes s test approach

 (Steel'5 ?9^6tS lle^lpsn  (°?M^  J955)  or'steel's MarJ-owRanTSs*?
 ^teei, 1959,  Miller,  1981).   See the Appendices  for examples of the manual
computations,  program listings,  and  examples of data input and program Su?put.
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TABLE 2     SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
            THE SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, EMBRYO-LARVAL
            SURVIVAL AND TERATOGENICITY TEST WITH EFFLUENTS AND RECEIVING
            WATERS
1.  Test type:
2.  Salinity:

3.  Temperature:
4.  Light quality:
5.  Light intensity:

6.  Photpperiod:
7.  Test chamber  size:
8.  Test solution volume:

9.  Renewal  of test solutions:
10. Age of  test organisms:
 11. No. of  embryos per chamber:
 12. No. replicate test chambers
      per concentration:
 13. No. embryos per concentration:
 14.  Feeding regime:
 15. Aeration:
 16.  Dilution water:
 17. Test concentrations:
Static renewal
5%o to 32%o (± 2%o of the selected
test salinity)
25 ± 1°C
Ambient laboratory "light
                   i
10-20 AiE/m2/s,  or 50-100 ft-c (ambient
laboratory levels)
16 h light, 8 h darkness
400-500 mL
250-400 mL per replicate  (loading and
DO restrictions must be met)
Daily
less than  24 h old
15  (minimum of 10)
4 (minimum of 3)
60  (minimum of 30)
Feeding  not required
None  unless DO falls  below 4.0  mg/L
Uncontaminated source of natural
 seawater;  deionized water mixed with
 hypersaline  brine or  artificial sea
 salts (HW  Marinemix®,  FORTY FATHOMS®,
 GP2,  or equivalent)
 Effluents:  Minimum  of 5 and a  control
 Receiving  waters: 100% receiving water
 or minimum of 5  and  a control
                                     145

-------
 TABLE 2.
SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
THE SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, EMBRYO-LARVAL

™^NN?lS{°GENICITV TEST WITH EFFLUENTS AN° ««*'«*
 18. Dilution factor:


 19. Test duration:

 20. Endpoints:
21. Test  acceptability  criteria:

22. Sampling  requirements:
23. Sample volume required:
                        Effluent:   > 0.5
                        Receiving  waters:

                        9 days
None, or > 0.5
                        Percent hatch;  percent larvae dead or
                        with  debilitating morphological  and/or
                        behavior abnormalities such  as:  gross
                        deformities;  curved  spine;
                        disoriented,  abnormal  swimming
                        behavior;  surviving  normal larvae  from
                        original  embryos

                        80% or  greater  survival  in controls

                        For on-site tests, samples collected
                        daily and  used  within  24 h of  the  time
                        they are removed  from  the sampling
                        device.  For off-site  tests, a minimum
                        of three samples  are   collected on
                        days one, three,  and five with a
                       maximum holding time of 36 h before
                        first use  (see  Section 8, Effluent and
                       Receiving Water Sampling, Sample
                       Handling, and Sample Preparation for
                       Toxicity Tests,  Subsection 8.5.4)

                       5 L per day
                                   146

-------
12 13 1 3  The statistical tests described here must be used with a knowledge
of'the assumptions upon which the tests are contingent.  The assistance of a
statistician is recommended for analysts who are not proficient in statistics.

12 13 2  EXAMPLE OF ANALYSIS OF SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS,
EMBRYO-LARVAL SURVIVAL AND TERATOGENICITY DATA

12 13 2.1  Formal statistical analysis of the total mortality data is outlined
in Fiqure 3.  The response used in the analysis is the total mortality
proportion in each test or control chamber.  Separate analyses are performed
for the estimation of the NOEC and LOEC endpoints and for the estimation of
the EC endpoint.  Concentrations at.which there ^ 100% mortality in all of
the test chambers are excluded from the statistical analysis of the NOEC and
LOEC, but included in the estimation of the EC endpoints.

12.13.2.2  For the case of equal numbers  of replicates across; all
concentrations and the control, the evaluation of the NOEC  arid 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 ot
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 unequal  numbers of replicates occur among  the concentration
levels tested,  there are  parametric and nonparametric alternative analyses.
The parametric  analysis  is a t  test with the  Bonferroni  adjustment (see
Appendix D).  The Wilcoxon Rank Sum Test with  the Bonferroni  adjustment is the
 nonparametric alternative.

 12  13 2 4  Probit Analysis  (Finney, 1971; see Appendix H):is  used to estimate
 the concentration that causes a specified percent decrease in  survival from
 the control.  In this analysis, the total mortality data from all test
 replicates at a given concentration are combined.  If the data do not tit the
 Probit Analysis, the Spearman-Karber Method,  the Trimmed Spearman-Karber
 Method or the Graphical  Method may be used (see Appendice H-K).
                                                          i
 12.13.2.5  Example of Analysis of Survival Data -

 12 13.2.5.1  The data for this example are listed in Table 3.   Total
 mortality, expressed as  a proportion (combined total number of dead embryos,
 dead larvae and deformed larvae divided  by the number of embryos at start of
 test)  is the response of interest.  The total mortality proportion in each
 replicate must first be  transformed by the arc sine square root transformation
 procedure described in Appendix B.  The  raw and transformed data, means and
 variences of the transformed observations  at each SDS concentration and
 control are listed  in Table 3.  A plot of  the data is provided  in Figure  4.
 Since  there  is  100% total mortality in all replicates for  the 8.0 mg/L
 concentration,  it is not included  in this  statistical analysis  and is
 considered  a qualitative mortality effect.

                                       147

-------
        STATISTICAL ANALYSIS OF SHEEPSHEAD MINNOW EMBRYO-LARVAL
                     SURVIVAL AND TERATOGENICITY TEST
                                TOTAL MORTALITY
                         TOTAL NUMBER OF DEAD EMBRYOS,
                        DEAD LARVAE, AND DEFORMED LARVAE
        PROBIT ANALYSIS
     ARC SINE
 TRANSFORMATION
      ENDPOINT ESTIMATE
            EC1
SHAPIRO-WILJCSTEST
                    NORMAL DISTRIBUTION
                    NON-NORMAL DISTRIBUTION
          HOMOGENEOUS
            VARIANCE
                                BARTLETTSTEST
               EQUAL NUMBER OF
                 REPLICATES?
           NO
                         HETEROGENEOUS
                            VARIANCE
                         YES
                 EQUAL NUMBER OF
                   REPLICATES?
                                        YES
                                                              NO
TWITH
ERRONI
TMENT


DUNNETTS
TEST



STEEL'S MANY-ONE
RANK TEST
	 r-J


WILCOXON RANK SUM
TEST WITH
BONFERRONI ADJUSTMENT


                             ENDPOINT ESTIMATES
                                 NOEC, LOEC
Figure 3.   Flowchart  for  statistical analysis of sheepshead minnow,
           Cyprinodon varlegatus, embryo-larval survival and teratoqenicity
           test.   Survival  and terata data.
                                   148

-------
   TABLE 3.  SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, EMBRYO-LARVAL
             TOTAL MORTALITY DATA
                                               Concentration  (mq/L)
          Replicate     Control     0.5     1.0
                                2.0
                            4.0
                            8.0
RAW



A
B
c
D
0.
0.
0.
0.
1
0
1
0
0
0
0
0
.0
.2
.2
.1
0
0
0
0
.0
.1
.1
.2
0.3
0.1
0.2
0.4
0
0
0
0
.9
.7
.8
.8
1.0
1.0
1.0
1.0
ARC SINE
TRANS-
FORMED

Mean (Y,-)
i
1
A
B
C
D


0.322
0.159
0.322
0.159
0.241
0.009
1
0.159
0.464
0.464
0.322
0.352
0.021
2
0.159
0.322
0.322
0.464
0.317
0.016
3
0.580
0.322
0.464
0.685
0.513
0.024
4
1.249
0.991
1.107
1.107
1.114
0.011
5
12.13.2.6  Test for Normality

12.13.2.6.1  The first step of the test for normality is to center the
observations by subtracting the mean of all observations within a
concentration from,each observation in that concentration,!  The centered
observations are summarized in Table 4.
         TABLE 4.  CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE
     Replicate
Control
0.5
                                        SDS Concentration (mg/L)
1.0
2.0
                                                             4.0    8.0
A
B
C
D
0.081
-0.082
0.081
-0.082
-0.193
0.112
0.112
-0.030
-0.158
0.005
0.005
0.147
0.067
-0.191
0.135
-0.123
-0.049 -0.007
0.172
-0.007
.
-
-
-
                                      149

-------
                                                     OJ
                                                     as
                              *     *
                              *     *
                                    *
p    o>
*-o
oq
o
' i '  ' ' ' i ' '  ' ' i ' ' '  ' i '.' ' '  i ' ' ' '  i ' ' * ' i
dooo
                                       8
                                       g
                                       s
                                                      UJ
                                                      o

                                                      o
                                                      o

                                                  w  z
                                                  CD  HI
                                                  Pi

                                                  m
                                          u_
                                          111
                                                  I
                                                     .0

                                                     ev

                                                     CD
                                                     o
                                                     as
                                                     -a
                                                     i-
                                                     o
                                                     OS
                                                     -4->
                                                     o
                                                     o
                                                     •o
                                                     o
                                                     I
                                                     o
                                                     T3
                                                     OJ
                                                     0>
                                                                            Q.
                                                                            O)
                                                                            O)
 1V1O1 dO NOIlUOdOdd
                                                     o 
                                                    i— Q)
                                                    Q- +->
                                                     O)
                                                    • s-
       150

-------
12.13.2.6.2  Calculate the denominator,  D,  of the statistic:
                               D= £ (X± - X)
                                   1=1
          Where:   Xj = the ith centered observation


                   X  = the overall mean of the centered observations


                   n  - the total number of centered observations


12.13.2.6.3  For this set of data,    n = 20


                                            1
                                      X =
                                               (-0.005)  =0.000
                                           20



                                      D = 0.2428
                                                          I
                                                          I

 12.13.2.6.4  Order  the  centered  observations from  smallest  to  largest
                                    (n)
                X   < X   <  ... < X


where XC1) denotes the ith ordered observation.

this example are listed in Table 5.
                                                 The ordered observations for
       TABLE 5.   ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE
i
1
2
3
4
5
6
7
8
9
10
X(D
-0.193
-0.191
-0.158
-0.123
-0.082
-0.082
-0.049
-0.030
-0.007
-0.007
•?
X(i>
11 0.005
12 0.005
13 0.067
14
15
16
0.081
0.081
0.112
17 0.112
18 0.135
19 0.147
20

0.172

                                       151

-------
 12.13.2.6.5  From Table 4, Appendix B, for the number of observations, n,
 Ar^'f******'* 4* I* M A«HM ^.£>S A J _ ._. J. ... _   _        _-_!___   I  •    I J*  • f+    m
 obtain the coefficients a,, a2,  ...,  ak where  k  is  n/2  if  n  is  even  and
 (n-l)/2 if n is odd.  For the data in this example, n  = 20  and k =  10.
 values are listed in Table 6.
                                                The a;
       TABLE 6.  COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-MILK'S EXAMPLE
                           ai
1
2
3
4
5
6
7
8
9
10
0.4734
0.3211
0.2565
0.2085
0.1686
0.1334
0.1013
0.0711
0.0422
0.0140
0.365
0.338
0.293
0.235
0.194
0.163
0.130
0.097
0.012
0.012
X(20)
x<19)
x<18)
X(17)
X(16)
•X(15>
XCU)
x<13)
x<12>
XC11)
- X(1)
- x(2)
- x<3)
- x<4)
- x(5)
- x(6)
- x(7)
- x(8>
- x(9)
- x(10>
 12.13.2.6.6   Compute the test statistic,  W,  as follows:

                          w=±  ~
                              D
The differences x(n"i+1)  -  Xci) are listed in Table 6.   For the data in this
example,
                   W
1	 (0.4807)2 = 0.952
                       0.2428
12.13.2.6.7  The decision rule  for  this  test  is to  compare  W  as  calculated  in
Section 13.2.6.6 to a critical  value  found  in Table 6, Appendix  B.   If  the
computed W is less than the critical  value, conclude that the data  are  not
normally distributed.  For the  data in this example, the critical value at  a
significance level of 0.01 and  n =  20 observations  is 0.868.  Since W = 0.952
is greater than the critical value, conclude that the data  are normally
distributed.
12.13.2.7  Test for Homogeneity of Variance

12.13.2.7.1  The test used to examine whether the variation in mean proportion
mortality is the same across all toxicant concentrations including the


                                      152

-------
control, is Bartlett's Test (Snedecor and  Cochran,  1980).  The test statistic
is as follows:
                        in
                                                 In
                      B =
    Where:  V,-  =
       degrees of freedom for each  copper  concentration and
       control,  V,-  =  (nf - 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

n-  =  the number of replicates for concentration  i.
                               s2  =
                  c =
                                       1=1
                                       1=1
                                                  1=1
12.13.2.7.2  Since B is approximately  distributed as chi-square with p - 1
degrees of freedom when the variances  are  equal, the appropriate critical
value is obtained from a table of the  chi-square distribution for p - 1
degrees of freedom and a significance  level  of 0.01.   If B is less than the
critical value then the variances are  assumed to be equal.i

12.13.2.7.3  For the data in this example, M- = 3,  p = 5, F2   =  0.0162,  and
C = 1.133.  The calculated B value is:
                     B =
               (15) [ln(0.0162)]-3Eln<

                            1.33
                          15(-4.1227)  -  3(-20.9485)
                                       1.33
                       =  0.886
                                     153

-------
12.13.2.7.4  Since B is approximately distributed  as chi-square with p - 1
degrees of freedom when the variances are  equal, the appropriate critical
value for the test is 13.277 for a significance  level of 0.01.  Since B =
0.886 is less than the critical  value of 13.277, conclude that the variances
are not different.

12.13.2.8  Dunnett's Procedure

12.13.2.8.1  To obtain an estimate of the  pooled variance for the Dunnett's
Procedure,  construct an ANOVA table as described in Table 7.
                            TABLE 7.  ANOVA TABLE
        Source
df
Sum of Squares
     (SS)
Mean Square(MS)
    (SS/df)
Between
Within
Total
P
N
N
- 1
- P
- 1
SSB
SSW
SST
SB = SSB/(p-l)
S* = SSW/(N-p)

   Where:    p   -=  number of SDS concentration levels including the control

            N   -  total number of observations n1 + n2  ... + n

            n{ = number of observations in concentration  i


        SSB =  f,Tl/n±-G2/N       Between Sum of Squares
              1=1
        SST =  £ ^Ylj-Gz/N       Total Sum of Squares
        SSW = SST-SSB
             Within Sum of Squares
                                     154

-------
               the grand total of all sample observations,   G
                                                = £**
       Y,
        ij
the total of the replicate measurements  for  concentration  i

the jth observation for concentration  i  (represents  the
proportion surviving for toxicant concentration  i  in test
chamber j)
12.13.2.8.2  For the data in this example:

                   n1 = n2 =  n3 = n4 = n5 = 4
                   N  = 20
                        '11
                                        Y,/ = 0.962
                        '21 + Y22 + Y23 + Y24 = 1.409
                   T3 = Y31 + Y32 + Y33 + Y34 = 1.267
                   '    "  + Y,, + Y43 + Y44 = 2.051
                                 + Y53 + Y54 = 4-454
    i:: % +
    6=1,
                                  T3 + T4 = 10.143
                 SSB = f^Tl/ni-G2/N
                        1  (28.561)  -  (10.143)2  = 1.996
                        4               20
   SST =
                             YJ.,-G2/N
         7.383  -  (10.143)
                     20
                                             2.239
                  SSW = SST-SSB    =  2.239 - 1.996 = 0.243


                  S*  =  SSB/(p-l) =  1.996/(5-l) = 0.499
                                                          i
                                                          I
                  SJ;  =  SSW/(N-p) =  0.243/(20-5) = 0.016


12.13.2.8.3  Summarize these calculations  in the ANOVA table (Table 8)
                                      155

-------
              TABLE 8.  ANOVA TABLE FOR DUNNETT'S PROCEDURE EXAMPLE
        Source
df
Sum of Squares
     (SS)
Mean Square(MS)
    (SS/df)
Between
Within
Total
4
15
19
1.996
0.243
2.239
0.499
0.016

12.13.2.8.4  To perform the individual comparisons, calculate the t statistic
for each concentration, and control combination as follows:
                           fci =
Where:  Yf  = mean proportion surviving for concentration i

        Y!  * mean proportion surviving for the control

        Sw  - square root of the within mean square

        n1  = number of replicates for the control

        n-  = number of replicates for concentration  i.
Since we are looking for an increased response in percent of total  mortality
over control, the control mean is subtracted from the mean at a concentration.

12.13.2.8.5  Table 9 includes the calculated t values for each concentration
and control combination.  In this example, comparing the 0.5 mg/L
concentration with the control the calculation is as follows:
                             (0.352  -  0.241)

                          [0.1265^(1/4)+(1/4)]
                            = 1.241
12.13.2.8.6  Since the purpose of this test is to detect a significant
increase in total mortality, a one-sided test is appropriate.   The critical
value for this one-sided test is found in Table 5,  Appendix C.   For an  overall
alpha level of 0.05, 15 degrees of freedom for error and four  concentrations
(excluding the control) the critical  value is 2.36.  The mean  proportion  of

                                      156

-------
                         TABLE 9.  CALCULATED T VALUES
SDS Concentration (mg/L)
0.5
1.0
2.0
4.0
i
2
3
4
5
*'
1.241
0.850
3.041
9.760
total mortality for concentration "i" is considered significantly less than
the mean proportion of total mortality for the control  if t,-  is  greater  than
the critical value.  Therefore, the 2.0 mg/L and the 4.0 mg/L concentrations
have significantly higher mean proportions of total mortality than the
control.  Hence the NOEC is 1.0 mg/L and the LOEC is 2.0 mg/L.

12.13.2.8.7  To quantify the sensitivity of the test,  the minimum significant
difference (MSD) that can be detected statistically may be calculated.
                          MSD = d SCL/nJ + (l/n)
   Where: d = the critical value for Dunnett's procedure
'
         Sw =  the square root of the within mean square  [

         n, =  the number of replicates in the control.    \

          n =  The common number of replicates at each concentration  (this
               assumes equal replication at each concentration)


12.13.2.8.8  In this example:
                    MSD = 2.36  (0.1265)  v/d/4) + (1/4)


                         = 2.36 (0.1265)(0.7071)

                         = 0.211

12.13.2.8.9  The MSD (0.450)  is in transformed  units.   To determine the MSD  in
terms of percent survival, carry  out the following conversion.


                                     157                  !

-------
   1.   Add the MSD to the transformed  control  mean.
                          0.241 + 0.211 = 0.452
   2.   Obtain the untransformed values for the control  mean  and  the  sum
       calculated in 1.          -
                       [ Sine (0.241)  ]2 = 0.057
                       [ Sine (0.452)  ]2 = 0.191
   3.   The untransformed MSD (MSDU)  is determined by  subtracting the
       untransformed values from step  2.
                       MSDU = 0.191  -  0.057 =  0.134
12.13.2.8.10  Therefore, for this set of data, the minimum difference in mean
proportion of total mortality between the control  and any SDS concentration
that can be detected as statistically significant is 0.134.
12.13.2.8.11  This represents a 268%  increase in mortality from the control.
12.13.2.9  Calculation of the LC50
12.13.2.9.1  The data used for the Probit Analysis is summarized in Table 10.
To perform the Probit Analysis, run the USEPA Probit Analysis Program.
An example of the program input and output is supplied in Appendix H.
                      TABLE 10.   DATA FOR PROBIT ANALYSIS
SDS Concentration (mq/L)

Number Dead
Number Exposed
Control 0.5
4 4
40 40
1.0 2.0 4.0
28 32
40 40 40
8.0
40
40
12.13.2.9.2   For this example, the chi-square test for heterogeneity was not
significant.  Thus  Probit Analysis appears  appropriate for this set of data.
12.13.2.9.3   Figure 5 shows  the  output data for the  Probit Analysis of the
data from Table 10  using the USEPA Probit Program.
                                      158

-------
                        USEPA PROBIT ANALYSIS  PROGRAM
                      USED  FOR CALCULATING LC/EC VALUES
                                 Version  1.5
Probit Analysis of Sheepshead Minnow Embryo-Larval Survival and
Teratogenicity Data
        Cone.

     Control
      0.5000
      1.0000
      2.0000
      4.0000
      8.0000
 Number
Exposed

  40
  40
  40
  40
  40
  40
Number
Resp.

  4
  4
  2
  8
 32
 40
 Observed
Proportion
Responding

  0.1000
  0.1000
  0.0500
  0.2000
  0.8000
  1.0000
 Proportion
 Responding
Adjusted for
 Controls
  0.
  0,

  o!
  0,
0000
0174
0372
1265
7816
  1.0000
Chi - Square for Heterogeneity  (calculated)    =    0.883
Chi - Square for Heterogeneity  (tabular value) =    7.815
Probit Analysis of Sheepshead Minnow Embryo-Larval Survival  and
Teratogenicity Data


      Estimated LC/EC Values and Confidence  Limits
Point

LC/EC   1.00
LC/EC  50.00
    Exposure
      Cone.

      1.346
      3.018
              Lower           Upper
              95% Confidence Limits
              0.751
              2.539
                     1.776
                     3.455
           Figure  5.   Output  for USEPA Probit  Program,  Version  1.5.
                                    159

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12.14  PRECISION AND ACCURACY

12.14.1 PRECISION

12.14.1.1  Single-Laboratory Precision

12.14.1.1.1  Data on the single-laboratory precision of the sheepshead minnow
embryo-larval survival and teratogenicity test are available for eight tests
with copper sulfate and five tests with sodium dodecyl sulfate (USEPA, 1989a).
The data for the first five tests show that the same NOEC and LOEC,
240 ^g Cu/L and 270 /*g Cu/L, respectively, were obtained in all five tests,
which is the maximum level of precision that can be attained.  Three
additional tests (6-8) were performed with narrower (20 /KJ) concentration
intervals, to more precisely identify the threshold concentration.  The NOEC
and LOEC for these tests are 200 ^g and 220 /*g Cu/L, respectively.  For sodium
dodecyl sulfate, the NOEC's and LOEC's for all tests are 2.0 and 4.0 mg/L,
respectively.  The precision, expressed as the coefficient of variation (CV%),
is indicated in Tables 11-12.  For copper (Cu), the coefficient of variation,
depending on the endpoint used, ranges from 2.5% to 6.1% which indicates
excellent precision.  For sodium dodecyl sulfate (SDS), the coefficient of
variation, depending on the endpoint used, ranges from 11.7% to 51.2%,
indicating acceptable precision.

12.14.1.2  Multilaboratory Precision
12.14.1.2.1
available.
Data on the multilaboratory precision of this test are not yet
12.14.2 ACCURACY

12.14.2.1  The accuracy of toxicity tests cannot be determined.
                                      160

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TABLE 11.  SINGLE-LABORATORY PRECISION OF THE SHEEPSHEAD MINNOW,
           CYPRINODON VARIEGATUS, EMBRYO-LARVAL SURVIVAL AND
           TERATOGENICITY TEST PERFORMED IN HW MARINEMIX® ARTIFICIAL
           SEAWATER, USING EMBRYOS FROM FISH MAINTAINED AND SPAWNED IN HW
           MARINEMIX® ARTIFICIAL SEAWATER USING COPPER (CU) SULFATE AS
           REFERENCE TOXICANT1'2'3'4'5'6'7
Test
Number (ju<
1
2
3
4
5
6
7
8
n:
Mean:
CV(%):
\ Data from USEPA
^ T*-* <"• 4- t+ r\s\\*£f*u*mr*if\
EC1
EC5
EC10 EC50 NOEC
3/L) UgA) (/*g/L) Ug/L) (/*g/L)
173
*
*
182
171
*
*
195
4
180
6.1
(1988a)
r^\/ T«»rtirti
189
*
*
197
187
*
*
203
4
194
3.8
and USEPA
* Us\1 T 4 t*4"r\\f*
198 234 240
*
*
* 240
* 240
206 240 240
197 234 240
*
*
* < 200
* 220
208 226 220
4
4 7
202 233 NA
2.8
(1991a).
A/iiir»4--ir« D-i/\T/\ri-ioH
2.5 NA
LJ/M i o4- f\n
* -
Facility,  Environmental Services Division, Region 6, USEPA, Houston,
Texas.                                               i
Cyprinodon variegatus embryos used in the tests were less than 20 h
old when the tests began.  Two replicate test chambers were used for
the control and each toxicant concentration.  Ten embryos were
randomly added to each test chamber containing 250 ml. of test or
control water.  Solutions were renewed daily.  The temperature and
salinity of the test solutions were 24 + 1°C and 20%o, respectively.
Copper test concentrations were prepared using copper sulfate.
Copper concentrations for Tests 1-5 were: 180, 210, 240, 270, and 300
Mg/L-  Copper concentrations for Test 6 were: 220, 240, 260, 280, and
300 ^g/L. Copper concentrations for Tests 7-8 were: 200., 220, 240,
260, and 280 ^g/L. Tests were conducted over a two-week period.
Adults collected in the field.
NOEC Range: 200 - 240 ng/L (this represents a difference of two
exposure concentrations).
For a discussion of the precision of data from chronic toxicity tests
see Section 4, Quality Assurance.
Data did not fit the Probit model.
                                    161

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TABLE 12.  SINGLE-LABORATORY PRECISION OF THE SHEEPSHEAD MINNOW,
           CYPRINODON VARIEGATUS, EMBRYO-LARVAL SURVIVAL AND
           TERATOGENICITY TEST PERFORMED IN HW MARINEMIX® ARTIFICIAL
           SEAWATER, USING EMBRYOS FROM FISH MAINTAINED AND SPAWNED IN HW
           MARINEMIX® ARTIFICIAL SEAWATER USING SODIUM DODECYL SULFATE
           / c*r\c* \ A r* nrrmrfci^r" Trt v T /"* A HIT ' /1— *•* i^ » ^ *& t •
('
Test
Number
1
2
3
4
5
n:
Mean:
CV(%):
SDS) AS REFERENC
EC1
(mg/L)
1.7
*
0.4
1.9
1.3
4
1.3
51.2
E TOXICANT'
EC5
(mg/L)
2.0
*
0.7
2.2
1.7
4
1.6
41.6

EC10
(mg/L)
2.2
*
0.9
2.4
1.9
4
1.9
35.0

EC50
(mg/L)
3.1
*
2.5
3.3
3.0
4
2.9
11.7

NOEC
(mg/L)
2.0
4.0
2.0
2.0
2.0
5
NA
NA
  Data from USEPA (1988a) and USEPA (1991a).
  Tests performed by Terry Hollister, Aquatic Biologist, Houston Facility,
  Environmental Services Division, Region 6, USEPA, Houston, Texas.
  Cyprinodon van'egatus embryos used in the tests were less than 20 h old
  when the tests began.  Two replicate test chambers were used for the
  control and each toxicant concentration.  Ten embryos were randomly
  added to each test chamber containing 250 mL of test or control water.
  Solutions  were renewed daily.  The temperature and salinity of the test
  solutions were 24 + 1°C and 20%o, respectively.
  SDS concentrations for all tests were: 0.5, 1.0, 2.0, 4.0, and 8.0 mg/L.
  Tests were conducted over a three-week period.
  Adults collected in the field.
  NOEC Range: 2.0 - 4.0 mg/L (this represents a difference of two exposure
  concentrations).
  For a discussion of the precision of data from chronic toxicity tests
  see Section 4, Quality Assurance.
  - Data did not fit the Probit model.
                                    162

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                                  SECTION 13

                                 TEST  METHOD
                                                          I
       INLAND SILVERSIDE, HENIDIA BERYLLINA, LARVAL SURVIVAL AND GROWTH
                                METHOD 1006.0


13.1  SCOPE AND APPLICATION

13.1.1  This method estimates the chronic toxicity of effluents and receiving
waters to the inland silverside, Mem'dia beryllina, using seven-to-eleven day
old larvae in a seven day, static renewal test.  The effects include the
synergistic, antagonistic, and additive effects of all the chemical, physical,
and biological components which adversely affect the physiological and
biochemical functions of the test species.

13.1.2  Daily observations on mortality make it possible to also calculate
acute toxicity for desired exposure periods (i.e., 24-h, 48-h, 96-h LCBOs).

13.1.3  Detection limits of the toxicity of an effluent or chemical substance
are organism dependent.

13.1.4  Brief excursions in toxicity may not be detected using 24-h composite
samples.  Also, because of the long sample collection period involved in
composite sampling, and because the test chambers are not sealed, highly
volatile and highly degradable toxicants present in the source may not be
detected in the test.
                                                          I
13.1.5  This test is commonly used in one of two forms: (1) a definitive test,
consisting of a minimum of five effluent concentrations and a control, and (2)
a receiving water test(s), consisting of one or more receiving water
concentrations and a control.

13.2  SUMMARY OF METHOD

13.2.1  Inland silverside, Mem'dia berylTina,  7 to 11-d old larvae are exposed
in a static renewal system for seven days to different concentrations of
effluent or to receiving water.  Test results are based on the survival and
growth of the larvae.

13.3  INTERFERENCES

13.3.1  Toxic substances may be introduced by contaminants in dilution water,
glassware, sample hardware, and testing equipment (see Section 5, Facilities,
Equipment, and Supplies).

13.3.2  Adverse effects of low dissolved oxygen (DO) concentrations, high
concentrations of suspended and/or dissolved solids, and extremes of pH, may
mask or confound the effects of toxic substances.
                                      163

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13.3.3  Improper effluent sampling and handling may adversely affect test
results (see Section 8, Effluent and Receiving Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests).

13.3.4  Pathogenic and/or predatory organisms in the dilution water and
effluent may affect test organism survival, and confound test results.

13.3.5  Food added during the test may sequester metals and other toxic
substances and confound test results.

13.4  SAFETY

13.4.1  See Section 3, Health and Safety.

13.5  APPARATUS AND EQUIPMENT

13.5.1  Facilities for holding and acclimating test organisms.

13.5.2  Brine shrimp, Artemia, culture unit -- see Subsection 13.6.16 below
and Section 4, Quality Assurance.

13.5.3  Menidia beryllina culture unit -- see Subsection 13.6.17 below,
Middaugh and Hemmer (1984), Middaugh et al. (1986), USEPA (1987g) and USEPA
(1993a) for detailed culture methods.  This test requires from 180 to 360 7 to
11 day-old larvae.  It is preferable to obtain the test organisms from an in-
house culture unit.  If it is not feasible to culture fish in-house, embryos
or larvae can be obtained from other sources by shipping them in well
oxygenated saline water in insulated containers.

13.5.4  Samplers -- automatic sampler, preferably with sample cooling
capability, that can collect a 24-h composite sample of 5 L.

13.5.5  Environmental chamber or equivalent facility with temperature control
(25 ± 1-C).

13.5.6  Water purification system -- Mi Hi pore Milli-Q®, deionized water (DI)
or equivalent.

13.5.7  Balance, analytical -- capable of accurately weighing to 0.00001 g.

13.5.8  Reference weights, Class S -- for checking performance of balance.
Weights should bracket the expected weights of the weighing pans and the
expected weights of the weighing pans plus fish.

13.5.9  Drying oven -- 50-105°C range, for drying larvae.

13.5.10  Air pump -- for oil-free air supply.

13.5.11  Air lines, plastic or pasteur pipettes, or air stones -- for gently
aerating water containing the fragile larvae or for supplying air to test
solution with low DO.
                                      164

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 13.5.12  Meters, pH and DO -- for routine physical and chemical measurements.

 13.5.13  Standard or micro-Winkler apparatus -- for calibrating DO (optional).

 13.5.14  Desiccator -- for holding dried larvae.

 13.5.15  Light box -- for counting and observing larvae.

 13.5.16  Refractometer -- for determining salinity.

 13.5.17  Thermometers, glass or electronic,  laboratory grade -- for measuring
 water temperatures.

 13.5.18  Thermometers, bulb-thermograph or electronic chart type -- for
 continuously recording temperature.
                                                                            .
 13.5.19  Thermometer,  National  Bureau of Standards Certified (see USEPA Method
 170.1,  USEPA,  1979b)  -- to calibrate laboratory thermometers.

 13.5.20  Test  chambers -- four  (minimum of three)  chambers  per concentration
 The  chambers should  be borosilicate  glass or nontoxic disposable plastic
 labware.   To avoid  potential  contamination from the air and excessive
 evaporation  of test  solutions during the test,  the chambers should be  covered
 during  the test with  safety glass  plates or  sheet  plastic  (6 mm thick).

 13.5.20.1  Each test  chamber for the inland  silverside should  contain  a
 minimum of 750 ml of  test solution.   A modified Norberg and Mount  (1985)
 chamber (Figure 1), constructed of glass and silicone cement,  has  been  used
 successfully for this  test.   This  type of chamber  holds an  adequate column of
 test  solution  and incorporates a sump area from which  test  solutions can be
 siphoned  and renewed without  disturbing  the  fragile  inland  silverside  larvae
 Modifications  for the  chamber are  as  follows:   1)  200  ^m mesh  NITEX® screen
 instead of stainless steel  screen; and 2)  thin  pieces  of glass  rods cemented
 with  silicone  to the NITEX® screen to reinforce  the  bottom  and  sides to
 produce a  sump area in  one  end of  the chamber.   Avoid  excessive  use of
 silicone,  while  still  ensuring that  the  chambers do  not leak and the larvae
 cannot  get trapped or  escape  into  the sump area.  Once  constructed, check the
 chambers for leaks and  repair if necessary.  Soak the  chambers overnight in
 seawater  (preferably in flowing water) to  cure the silicone cement before use
 Other types  of glass test chambers,  such  as  the  1000 mL beakers used in the
 short-term sheepshead minnow  larval  survival  and growth test, may be used   It
 is recommended that each  chamber contain  a minimum of 50 ml per larvae and
 allow adequate depth of test  solution  (5.0 cm).

 Jn™'2,1 ,Beaker? " six class A> borosilicate glass or non-toxic plasticware,
 1000 ml for making test solutions.

 13.5.22  Mini-Winkler bottles -- for dissolved oxygen calibrations.

 13.5.23  Wash bottles -- for deionized water, for washing  embryos from
substrates and containers, and for rinsing 'small glassware and  instrument
electrodes and probes.

                                      165

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                                                   GLASS
                                           REINFORCEMENTS
                                                                SUMP
Figure 1.   Glass chamber with sump  area.  Modified from  Norberg and Mount
           (1985).  From USEPA (1987c).
13.5.24  Crystallization dishes,  beakers, culture dishes,  or equivalent -- for
incubating  embryos.
13.5.25  Volumetric flasks and graduated cylinders -- Class A, borosilicate
glass or non-toxic plastic labware,  10-1000 ml for making test solutions.
13.5.26  Separatory funnels, 2-L --  Two-four for culturing Artemia.
13.5.27  Pipets, volumetric -- Class A,  1-100 ml.
13.5.28  Pipets, automatic -- adjustable, 1-100 ml.
13.5.29  Pipets, serological -- 1-10 ml, graduated.
13.5.30  Pipet bulbs and fillers --  PROPIPET®, or equivalent.
13.5.31  Droppers,  and glass tubing  with fire polished edges,  4  mm ID  --  for
transferring  larvae.
13.5.32  Siphon with bulb and clamp  -- for  cleaning test chambers.
13.5.33  Forceps -- for  transferring dead larvae to weighing pans.
                                     166

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13.5.34  NITEX® mesh sieves (< 150 A*m, 500 //m, 3 to 5 mm) -- for collecting
Artemia nauplii and fish larvae.  (NITEX® is available from Sterling Marine
Products, 18 Label Street, Montclair, NJ 07042; 201-783-9800.)

13.6  REAGENTS AND CONSUMABLE MATERIALS

13.6.1  Sample containers -- for sample shipment and storage (see Section 8,
Effluent and Receiving Water Sampling, Sample Handling, and Sample Preparation
for Toxicity Tests).

13.6.2  Data sheets (one set per test) -- for data recording.

13.6.3  Tape, colored -- for labelling test chambers.

13.6.4  Markers, waterproof -- for marking containers, etc.

13.6.5  Vials, marked -- 24/test, containing 4% formalin or 70% ethanol, to
preserve larvae (optional).
                                                          i
13.6.6  Weighing pans, aluminum  -- 26/test (2 extra).

13.6.7  Buffers, pH 4, pH 7, and pH 10 (or as per instructions of instrument
manufacturer) for standards and calibration check (see USEPA Method 150.1,
USEPA, 1979b).                                            |
                                                           '
13.6.8  Membranes and filling solutions for dissolved oxygen probe (see USEPA
Method 360.1, USEPA, 1979b), or reagents -- for modified Winkler analysis.

13.6.9  Laboratory quality assurance samples and standards -- for the above
methods.

13.6.10  Reference toxicant solutions -- see Section 4, Quality Assurance.

13.6.11  Ethanol (70%) or formalin (4%) -- for use as a preservative for the
fish larvae.

13.6.12  Reagent water -- defined as distilled or deionized water that does
not contain substances which are toxic to the test organisms (see Section 5,
Facilities, Equipment, and Supplies).

13.6.13  Effluent, receiving water, and dilution water -- see Section 7,
Dilution Water; and Section 8, Effluent and Surface Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests.
                                                          I
13.6.13.1  Saline test and dilution water -- the salinity 'of the test water
must be in the range of 5 to 32%o.  The salinity should vary by no more than
± 2%o among the chambers on a given day.  If effluent and receiving water
tests are conducted concurrently, the salinities of these tests should be
similar.
                                                          i

13.6.13.2  The overwhelming majority of industrial and sewage treatment
effluents entering marine and estuarine systems contain little or no

                                      167

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measurable salts.  Exposure of Menidia beryl Una larvae to these effluents
will require adjustments in the salinity of the test solutions.  It is
important to maintain a constant salinity across all treatments.  In addition,
it may be desirable to match the test salinity with that of the receiving
water.  Artificial sea salts or hypersaline brine (100%o) derived from natural
seawater may be used to adjust the salinities.

13.6.13,3  Hypersaline brine (MSB):  MSB has several advantages that make it
desirable for use in toxicity testing.  It can be made from any high quality,
filtered seawater by evaporation, and can be added to the effluent or to
deionized water to increase the salinity.  MSB derived from natural seawater
contains the necessary trace metals, biogenic colloids, and some of the
microbial components necessary for adequate growth, survival, and/or
reproduction of marine and estuarine organisms, and may be stored for
prolonged periods without any apparent degradation.  However, if 100% HSB is
used as a diluent, the maximum concentration of effluent that can be tested
will be 70% at 30%o salinity and 80% at 20%o salinity.

13.6.13.3.1  The ideal container for making HSB from natural seawater is one
that (1) has a high surface to volume ratio, (2) is made of a noncorrosive
material, and (3) is easily cleaned (fiberglass containers are ideal).
Special care should be used to prevent any toxic materials from coming in
contact with the seawater being used to generate the brine.  If a heater is
immersed directly into the seawater, ensure that the heater materials do not
corrode or leach any substances that would contaminate the brine.  One
successful method used is a thermostatically controlled heat exchanger made
from fiberglass.  If aeration is used, use only oil free air compressors to
prevent contamination.

13.6.13.3.2  Before adding seawater to the brine generator, thoroughly clean
the generator, aeration supply tube, heater, and any other materials that will
be in direct contact with the brine.  A good quality biodegradable detergent
should be used, followed by several (at least three) thorough deionized water
rinses.

13.6.13.3.3  High quality (and preferably high salinity) seawater should be
filtered to at least 10 /^m before placing into the brine generator.  Water
should be collected on an incoming tide to minimize the possibility of
contamination.

13.6.13.3.4  The temperature of the seawater is increased slowly to 40°C.
The water should be aerated to prevent temperature stratification and to
increase water evaporation.  The brine should be checked daily (depending on
volume being generated) to ensure that salinity does not exceed 100%o and that
the temperature does not exceed 40°C.   Additional seawater may be added to the
brine to obtain the volume of brine required.

13.6.13.3.5  After the required salinity is attained,  the HSB should be
filtered a second time through a 1 urn filter and poured directly into portable
containers (20 L cubitainers or polycarbonate water cooler jugs are suitable).
The containers should be capped and labelled with the date the brine was
                                      168

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generated and its salinity.  Containers of HSB should be stored in the dark
and maintained at room temperature until used.
                                       .
13.6.13.3.6  If a source of HSB is available, test solutions can be made by
following the directions below.  Thoroughly mix together the deionized water
and brine before mixing in the effluent.

13.6.13.3.7  Divide the salinity of the HSB by the expected test salinity to
determine the proportion of deionized water to brine.  For example, if the
salinity of the HSB is 100%o and the test is to be conducted at 20%o, 100%o
divided by 20%o = 5.0.  The proportion of brine is one part in five (one part
brine to four parts deionized water).  To make 1 L of seawater at 20%o
salinity from a HSB of 100%o, divide 1 L (1000 ml) by 5.0.  The.result, 200
ml, is the quantity of HSB needed to make 1 L of seawater.  The difference,
800 ml, is the quantity of deionized water required.

13.6.13.3.8  Table 1 illustrates the composition of test solutions at 20%o if
they are made by combining effluent (0%o), deionized water arid HSB at 100%o
salinity.  The volume (ml) of brine required is determined by using the amount
calculated above.  In this case, 200 ml of brine is required for 1 L;
therefore, 600 ml would be required for 3 L of solution.  The volumes of HSB
required are constant.  The volumes of deionized water are determined by
subtracting the volumes of effluent and brine from the total volume of
solution:  3,000 ml - mL effluent - ml HSB = ml deionized water.

13.6.13.4  Artificial sea salts:  A modified GP2 artificial seawater
formulation (Table 2) has been successfully used to perform the inland
silverside survival and growth test.  The use of GP2 for holding and culturing
of adults is not recommended at this time.

13.6.13.4.1  The GP2 artificial sea salts (Table 2) should be mixed with
deionized (DI) water or its equivalent  in a container other than the culture
or testing tanks.  The deionized water  used for hydration should be between
21-26°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 gm NaHC03, in 500 ml
deionized water.  Add 2.5 ml of this stock solution for each liter of the GP2
artificial seawater.                                      j

13.6.14  ROTIFER CULTURE --for feeding  cultures and test organisms

13.6.14.1  At hatching Mem'dia beryl!ina larvae are too small to  ingest
Artemia nauplii  and must be  fed rotifers, Brachionus pliaitilis.  The rotifers
can be maintained  in continuous culture when  fed algae  (see Section 6 and
USEPA, 1987g).   Rotifers are cultured  in 10-15  L Pyrex® carboys  (with a drain
spigot near the  bottom) at 25-28°C and  25-35%o  salinity.  Four  12-L culture
carboys should be maintained simultaneously  to  optimize production.  Clean
carboys should be  filled with  autoclaved seawater.  Alternatively, an
immersion heater may be used to heat saline  water  in the carboy to 70-80°C for
1  h.


                                      169

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TABLE 1.  PREPARATION OF 3 L SALINE WATER FROM DEIONIZED WATER AND A
          HYPERSALINE BRINE OF 100%o NEEDED FOR TEST SOLUTIONS AT
          20%o SALINITY
Effluent
Concentration
(%)

80
40
20
10
5
Control
Volume of
Ef f 1 uent
(0%o)
(ml)
2400
1200
600
300
150
0
Volume of
Deionized
Water
(ml)
0
1200
1800
2100
2250
2400
Volume of
Hypersaline
Brine
(ml)
600
600
600
600
600
600
Total
Vol ume
(ml)

3000
3000
3000
3000
3000
3000
     Total
4,650
9,750
3,600
18,000
                                   170

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TABLE 2.   REAGENT GRADE CHEMICALS USED IN THE PREPARATION OF
           GP2 ARTIFICIAL SEAWATER FOR THE INLAND SILVERSIDE,
           MENIDIA BERYLLINA,  TOXICITY TESTU'S
Compound
NaCl
Na2S04
KC1
KBr
Na2B407 • 10 H20
MgCl2 • 6 H20
CaCl2 • 2 H20
SrCl2 • 6 H20
NaHC03
\ Modified GP2
Concentration
(9/L)
21.03
3.52
0.61
0.088
0.034
9.50
1.32
0.02
0.17
from Spotte et al . (1984)
Amount; (g)
Required for
20 L
420.6
70.4
12.2
1.76
0.68
190.0
26.4
0.400
3.40
1
  (19905). The salinity is 30.89 g/L.
  GP2 can be diluted with deionized (DI) water to the desired test
  salinity.
                            171

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13.6.14.2  When the water has cooled to 25-28°C, aerate and add a start-up
sample of rotifers (50 rotifers/mL) and food (about 1 L of a dense algal
culture).  The carboys should be checked daily to ensure that adequate food is
available and that the rotifer density is adequate.  If the water appears
clear, drain 1 L of culture water and replace it with algae.  Excess water can
be removed through the spigot drain and filtered through a < 60 /j,m mesh
screen.  Rotifers collected on the screen should be returned to the culture.
If a more precise measure of the rotifer population is needed, rotifers
collected from a known volume of water can be resuspended in a smaller volume,
killed with formalin and counted in a Sedgwick-Rafter cell.  If the density
exceeds 50 rotifers/mL, the amount of food per day should be increased to 2 L
of algae suspension.  The optimum density of approximately 300-400 rotifers/mL
may be reached in 7-10 days and is sustainable for 2-3 weeks.  At these
densities, the rotifers should be cropped daily.  Keeping the carboys away
from light will reduce the amount of algae attached to the carboy walls.  When
detritus accumulates, populations of ciliates, nematodes, or harpacticoid
copepods that may have been inadvertently introduced can rapidly take over the
culture.  If this occurs, discard the cultures.

13.6.15  ALGAL CULTURES -- for feeding rotifer cultures

13.6.15.1  Tetraselmus suecica or Chlorella sp.  (see USEPA, 1987a) can be
cultured in 20-L polycarbonate carboys that are  normally used for bottled
drinking water.  Filtered seawater is added to the carboys and then autoclaved
(110°C for 30 min).  After-cooling to room temperature, the carboys are placed
in a temperature chamber controlled at 18-20°C.  One liter of 7. suecica or
Chlorella sp. starter culture and 100 mL of nutrients are added to each
carboy.

13.6.15.2  Formula for algal culture nutrients.
13.6.15.2.1  Add 180 g NaNO
                           3>
12 g NaH2P04,
and 6.16 g EDTA to 12 L of
deionized water.  Mix with a magnetic  stirrer until all salts are dissolved
(at least 1 h).

13.6.15.2.2  Add 3.78 g  FeCl3 • 6 H20 and stir again.   The solution  should be
bright yellow.

13.6.15.2.3  The algal culture  is vigorously  aerated  via  a pipette  inserted
through  a foam  stopper at the top of the  carboy.  A dense algal culture  should
develop  in 7-10 days and should be  used  by day  14.  Thus, start-up  of cultures
should be made  on a daily or every  second day basis.   Approximately 6-8
continuous cultures will meet the feeding requirements of four  12-L rotifer
cultures.  When emptied, carboys are washed with  soap and water and rinsed
thoroughly with deionized water before reuse.

13.6.16  BRINE  SHRIMP, ARTEMIA, NAUPLII  -- for  feeding cultures and test
organisms

13.6.16.1  Newly hatched Artemia nauplii  are  used as  food for inland
silverside larvae in toxicity tests.  Although  there  are  many commercial
sources  of brine shrimp  cysts,  the  Brazilian  or Colombian strains are being
                                      172

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 used because the  supplies  examined  have  had  low  concentrations  of chemical
 residues  and produce  nauplii  of  suitably small size.   For  commercial  sources
 of brine  shrimp,  Artemia,  cysts,  see  Table 2 of  Section  5.,  Facilities,
 Equipment,  and Supplies  and Section 4, Quality Assurance.

 13.6.16.2   Each new batch  of  Artemia  cysts must  be  evaluated  for  size
 (Vanhaecke  and Sorgeloos,  1980,  and Vanhaecke et al.,  1980) and nutritional
 suitability (see  Leger et.  al., 1985;  Leger et al.,  1986) against  known
 suitable  reference cysts by performing a side by side  larval  growth test  using
 the "new" and "reference"  cysts.  The "reference" cysts  used  in the
 suitability test  may  be  a  previously  tested  and  acceptable  batch  of cysts,  or
 may be obtained from  the Quality  Assurance Research Division, Environmental
 Monitoring  Systems Laboratory, Cincinnati, OH 45268; 513-569-7325.  A sample
 of newly-hatched  Artemia nauplii  from each new batch of  cysts should  be
 chemically  analyzed.  The  Artemia cysts  should not  be  used  if the
 concentration of  total organochlorine pesticides  exceeds 0.15 /^g/g wet weight
 or that the total concentration of organochlorine pesticides plus PCBs does
 not exceed  0.30 »g/g  wet weight.  (For analytical methods, see USEPA 1982).

 13.6.16.2.1  Artemia  nauplii  are  obtained as  follows:

   1.   Add  1 L of seawater, or a  solution prepared  by  adding 35.0 g uniodized
       salt (NaCl) or artificial  sea  salts to 1  L of deioriized  water, to  a
       2-L  separatory funnel  or equivalent.
   2.   Add  10 ml  Artemia cysts to the separatory  funnel  arid aerate for 24 h
       at 27°C.   (Hatching time varies with  incubation temperature and the
       geographic strain of Artemia used  (see USEPA, 1985d; USEPA, 1993a; and
       ASTM, 1993.)
   3.   After 24 h, cut off the air supply in  the separatory funnel.   Artemia
       nauplii are phototactic and will  concentrate at the bottom of  the
       funnel  if  it is covered for 10-15 minutes to prevent mortality, do not
       leave the  concentrated nauplii at the  bottom of the funnel more than
       10 minutes without  aeration.
   4.   Drain the  nauplii into a beaker or funnel  fitted with <  150 ^m NITEX®
       or stainless steel  screen, and rinse with seawater or equivalent
       before use.

 13.6.16.3   Testing Artemia nauplii as food for toxicity test organisms.

 13.6.16.3.1  The primary criterion for acceptability of each new  supply of
 brine shrimp cysts is the ability of  the nauplii  to support good  survival  and
growth of the inland  silverside larvae (see Subsection 13.11).  The larvae
used to evaluate the  suitability  of the brine shrimp nauplii must be  of the
same geographical  origin, species, and stage  of development as those  used
routinely in the toxicity tests.    Sufficient data to detect differences in
survival  and growth should be obtained by using three replicate test chambers
each containing a minimum of 15 larvae,  for each  type of food.

13.6.16.3.2  The feeding rate and frequency,  test vessels and volume of
control water,  duration of the test, and age of the nauplii at the start of
the test,  should be the same as used for the routine toxicity tests.
                                                          I
                                                          i
                                      173

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13.6.16.3.3  Results of the brine shrimp nutrition assay, where there are only
two treatments, can be evaluated statistically by use of a t test.  The "new"
food is acceptable if there are no statistically significant differences in
the survival and growth of the larvae fed the two sources of nauplii.

13.6.16.4  Use of Artemia nauplii as food for inland silverside, Menidia
beryllina, larvae.

13.6.16.4.1  Menidia beryllina larvae begin feeding on newly hatched Artemia
nauplii about five days after hatching, and are fed Artemia nauplii daily
throughout the 7-day larval survival and growth test.  Survival of Menidia
beryllina larvae 7-9 days old is improved by feeding newly hatched (< 24 h
old) Artemia nauplii.  Equal amounts of Artemia nauplii must be fed to each
replicate test chamber to minimize the variability of larval weight.
Sufficient numbers of nauplii should be fed to ensure that some remain alive
overnight in the test chambers.  An adequate but not excessive amount should
be provided to each replicate on a daily basis.  Feeding excessive amounts of
nauplii will result in a depletion in DO to below an acceptable level (below
4.0 mg/L).  As much of the uneaten Artemia nauplii as possible should be
siphoned from each chamber prior to test solution renewal to ensure that the
larvae principally eat newly hatched nauplii.

13.6.17  TEST ORGANISMS, INLAND SILVERSIDE, MENIDIA BERYLLINA

13.6.17.1  The inland silverside, Menidia beryllina, is one of three species
in the atherinid family that are amenable to laboratory culture;  and one of
four atherinid species used for chronic toxicity testing.  Several atherinid
species have been utilized successfully for early life stage toxicity tests
using field collected (Goodman et al., 1985) and laboratory reared adults
(Middaugh and Takita, 1983; Middaugh and Hemmer, 1984; and USEPA,  1987g).  The
inland silverside, Menidia beryllina, populates a variety of habitats from
Cape Cod, Massachusetts, to Florida and west to Vera Cruz, Mexico  (Johnson,
1975).  It can tolerate a wide range of temperature, 2.9 - 32.5°C  (Tagatz and
Dudley,  1961; Smith, 1971) and salinity, of 0%o - 58%.  (Simmons,  1957;
Renfro, 1960), having been reported from the freshwaters of the Mississippi
River drainage basin  (Chernoff et al., 1981) to hypersaline lagoons  (Simmons,
1957).  Ecologically, Menidia spp.  are important as major prey  for many
prominent commercial  species  (e.g., bluefish (Pomatomus  saltatrix), mackerel
(Scomber scombrus),  and striped bass (Morone saxatilis)  (Bigelow  and
Schroeder,  1953).  The  inland silverside, Menidia beryllina, is a serial
spawner, and will spawn under controlled laboratory conditions.   Spawning can
be  induced  by  diurnal interruption  in the circulation  of water  in the culture
tanks  (Middaugh et al., 1986; USEPA, 1987a).   The eggs are demersal,
approximately  0.75 mm in diameter  (Hildebrand  and Schroeder, 1928),  and  adhere
to  vegetation  in  the  wild,  or to filter floss  in laboratory culture  tanks.
The larvae  hatch  in  6-7 days when  incubated  at 25°C and  maintained in seawater
ranging from 5%o  to  30%  (USEPA,  1987a).  Newly hatched  larvae  are 3.5-4.0 mm
in  total length  (Hildebrand,  1922).

13.6.17.2   Inland silverside, Menidia beryllina,  adults  (see USEPA,  1987g  and
USEPA,  1993a for  detailed  culture methods) may be cultured  in  the laboratory
or  obtained from  the Gulf  of  Mexico or Atlantic coast  estuaries throughout the

                                      174

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year  (Figure  2).   Gravid  females  can  be  collected  from low salinity waters
along  the Atlantic coast  during April  to July,  depending  on the  latitude.
The most productive and protracted  spawning  stock  can  be  obtained  from adults
brought into  the  laboratory.   Broodstocks, collected from local  estuaries
twice  each year  (in April  and  October),  will  become sexually active after
1-2 months and will  generally  spawn for  4-6  months.       j

13.6.17.3  The fish can be collected  easily  with a beach  seine  (3-6 mm mesh),
but the seine should not  be completely landed onto the beach.  Silversides  are
very sensitive to  handling and should  never  be  removed from the  water  by net
-- only by beaker  or bucket.
                                                          i
13.6.17.4  Samples may contain a  mixture of  inland silverside, Mem'dia
beryllina, and Atlantic silverside, Mem'dia  mem'dia, on the Atlantic coast  or
inland silverside  and tidewater silverside,  Mem'dia peninsulae,  on  the Gulf
Coast  (see USEPA,  1987g for additional information on  morphological
differences for identification).  Johnson  (1975) and Chernoff et al. (1981)
have attempted to  differentiate these  species.  In the northeastern  United
States, M. beryllina juveniles and adults  are usually  considerably  smaller
than M. mem'dia juveniles  and  adults  (Bengtson, 1984), and  can be separated
easily in the field  on that basis.

13.6.17.5  Record  the water temperature  and  salinity at each  collection site.
Aerate (portable air pump,  battery operated)  the fish  and transport  to the
laboratory as quickly as possible after  collection.  Upon 'arrival at the
laboratory, the fish and the water in  which  they were  collected  are
transferred to a tank at least 0.9 m in  diameter.  A filter  system  should be
employed to maintain water  quality (see  USEPA,  1987g).  Laboratory water is
added to the  tank  slowly,  and  the fish are acclimated  at the  rate of 2°C per
day, to a final  temperature of 25°C, and about  5%o salinity  per  day, to a
final salinity in  the range of 20%o -  32%o.   The seawater in  each tank should
be brought to a minimum volume of 150  L.   A density of about  50  fish/tank is
appropriate.   Maintain a photoperiod of  16 h  light/8 h dark.  Feed the  adult
fish flake food or  frozen brine shrimp twice daily and Artemia nauplii  once
daily.  Siphon the detritus from the bottom of  the tanks weekly.

13.6.17.6  Larvae  for a toxicity test can be  obtained  from the broodstock by
spawning onto polyester aquarium filter-fiber substrates,  15  cm  long X 10 cm
wide X 10 cm  thick, which are  suspended with  a  string 8-10 cm below the
surface of the water and in contact with  the  side of the holding tanks for
24-48 h,  14 days prior to the  beginning of a  test.   The floss should be gently
aerated by placing  it above an airstone,  and weighted down with  a heavy
non-toxic object.  The embryos, which are light yellow in  color,  can be seen
on the floss,  and are round and hard to the touch compared to the soft floss.

13.6.17.7  Remove as much floss as possible from the embryos.  The floss
should be stretched and teased to prevent the embryos  from clumping.  The
embryos should be incubated at the test salinity and lightly aerated.  At
25°C,  the embryos will  hatch in about 6-8 days.   Larvae arta fed about 500
rotifer larvae/day from hatch through four days post-hatch.  On Days 5  and  6,
newly hatched (less than 12-h old) Artemia nauplii  are mixed with the

                                                          i
                                      175

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                            A. Adult, ca. 64 mm SL
                          fcAji?-=*w :-f •*-'-!•-< jiff.'-'.  .'.. .11.1.-
                          r^SS^S^^AvTvwv^^vJSSscsw
                             F.  Larva, 6.7 mm TL
                             G.  Larva, 8.9 mm TL
Figure 2.   Inland  silverside,  Menidia beryllina:  A.   Adult,  ca. 64 mm SL; B.
            Egg  (diagrammatic), only bases of filaments shown; C. Egg, 2-cell
            stage;  D.  Egg,  morula stage; E. Advanced  embryo,  2 1/2 days after
            fertilization.   From Martin and Drewry  (1978).
                                       176

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rotifers, to provide a transition period.  After Day 7, only nauplii are fed,
and the age range for the nauplii can be increased from U!-h old to 24-h old.

13.6.17.8  Silverside larvae are very sensitive to handling and shipping
during the first week after hatching.  For this reason, if organisms must be
shipped to the test laboratory, it may be impractical to use larvae less than
11 days old because the sensitivity of younger organisms may result in
excessive mortality during shipment.  If organisms are to be shipped to a test
site, they should be shipped only as (1) early embryos, so that they hatch
after arrival, or (2) after they are known to be feeding viiell on Artemia
nauplii (8-10 days of age).  Larvae shipped at 8-10 days of age would be 9 to
11 days old when the test is started.  Larvae that are hatched and reared in
the test laboratory can be used at 7 days of age.

13.6.17.9  If four replicates of 15 larvae are used at each effluent
concentration and in the control, 360 larvae will be needed for each test.

13.7   EFFLUENT AND RECEIVING WATER COLLECTION, PRESERVATION, AND STORAGE

13.7.1  See Section 8, Effluent and Receiving Water Sampling, Sample Handling,
and Sample Preparation for Toxicity Tests.

13.8  CALIBRATION AND STANDARDIZATION

13.8.1  See Section 4, Quality Assurance.                 !

13.9  QUALITY CONTROL
                                            •
13.9.1  See Section 4, Quality Assurance.
                                                          I
13.10  TEST PROCEDURES

13.10.1  TEST SOLUTIONS
                                                          i
13.10.1.1  Receiving Waters

13.10.1.1.1  The sampling point is determined by the objectives of the test.
At estuarine and marine sites, samples are usually collected at mid-depth.
Receiving water toxicity is determined with samples used directly as collected
or with samples passed through a 60 ^m NITEX® filter and compared without
dilution, against a control.  Using four replicate chambers per test, each
containing 500-750 mL, and 400 mL for chemical analysis, would require
approximately 2.4-3.4 L or more of sample per day.

13.10.1.2  Effluents

13,10.1.2.1  The selection of the effluent test concentrations should be based
on the objectives of the study.  A dilution factor of 0.5 is commonly used.
A dilution factor of 0.5 provides precision of ± 100%, and allows for testing
of concentrations between 6.25% and 100% effluent using only five effluent
concentrations  (6.25%, 12.5%, 25%, 50%, and 100%).  Test precision shows
                                      177

-------
little improvement as dilution factors are increased beyond 0.5 and declines
rapidly if smaller dilution factors are used.  Therefore, USEPA
recommends the use of the > 0.5 dilution factor.  If 100% salinity MSB is used
as a diluent, the maximum concentration of effluent that can be tested will be
80% at 20%o salinity, and 70% at 30%o salinity.

13.10.1.2.2  If the effluent is known or suspected to be highly toxic, a lower
range of effluent concentrations should be used (such as 25%, 12.5%, 6.25%,
3.12%, and 1.56%).  If a high rate of mortality is observed during the first 1
to 2 h of the test, additional dilutions at the lower range of effluent
concentrations should be added.

13.10.1.2.3  The volume of effluent required to initiate the test and for
daily renewal of four replicates per treatment for five concentrations of
effluent and a control, each containing 750 ml of test solution, is
approximately 5 L.  Prepare enough test solution at each effluent
concentration to provide 400 ml additional volume for chemical analyses.

13.10.1.2.4  Tests should begin as soon as possible after sample collection,
preferably within 24 h.  The maximum holding time following retrieval of the
sample from the sampling device should not exceed 36 h for off-site toxicity
studies unless permission is granted by the permitting authority.  In no case
should the test be started more than 72 h after sample collection (see Section
8, Effluent and Receiving Water Sampling, Sample Handling, and Sample
Preparation for Toxicity Tests, subsection 8.5.4).

13.10.1.2.5  Just prior to test initiation (approximately 1 h), the
temperature of a sufficient quantity of the sample to make the test solution
should be adjusted to the test temperature (25 ± 1°C) and maintained at that
temperature during the addition of dilution waters.

13.10.1.2.6  Effluent dilutions should be prepared for all replicates in each
treatment in one beaker to minimize variability among the replicates.  The
test chambers are labelled with the test concentration and replicate number.
Dispense into the appropriate effluent dilution chamber.

13.10.1.3  Dilution Water

13.10.1.3.1  Dilution water may be uncontaminated natural seawater (receiving
water), HSB prepared from natural seawater, or artificial seawater prepared
from FORTY FATHOMS® or GP2 sea salts (see Table 2 and Section 7, Dilution
Water).  Other artificial sea salts may be used for culturing sheepshead
minnows and for the larval survival and growth test if the control criteria
for acceptability of test data are satisfied.

13.10.2  START OF THE TEST

13.10.2.1  Inland silverside larvae 7 to 11 days old can be used to start the
survival and growth test.  At this age, the inland silverside feed on
newly-hatched Artemia nauplii.  At 25°C, tests with inland silverside larvae
can be performed at salinities ranging from 5%o to 32%o.  If the test salinity
ranges from 16%o to 32%o, the salinity for spawning, incubation, and culture

                                      178

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of the embryos and larvae should be maintained within this salinity range.  If
the test salinity is in the range of 5%o to 15%o, the embryos may be spawned
at 30%o, but egg incubation and larval rearing should be at the test salinity.
If the specific salinity required for the test differs from the rearing
salinity, adjustments of 5%o daily should be made over the three days prior to
start of test.

13.10.2.2  One Day Prior to Beginning of Test

13.10.2.2.1  Set up the Artemia culture so that newly hatched nauplii will be
available on the day the test begins,  (see Section 7).

13.10.2.2.2  Increase the temperature of water bath, room.i or incubator to the
required test temperature (25 ± 1°C).

13.10.2.2.3  Label the test chambers with a marking pen.  Use of color coded
tape to identify each concentration and replicate is helpful.,  A minimum of
five effluent concentrations and a control should be selected for each test.
Glass test chambers, such as crystallization dishes, beakers, or chambers with
a sump area (Figure 1), with a capacity for 500-750 ml of test solution,
should be used.

13.10.2.2.4  Randomize the position of test chambers in the temperature-
controlled water bath, room, or incubator at the beginning of the test, using
a position chart.  Assign numbers for the position of each test chamber using
a table of random numbers or similar process (see Appendix A for an example of
randomization).  Maintain the chambers in this configuration throughout the
test, using a position chart.

13.10.2.2.5  Because inland silverside larvae are very sensitive to handling,
it is advisable to distribute them to their respective test chambers which
contain control water on the day before the test is to begin„  Each test
chamber should contain 15 larvae (minimum 10) and it is recommended that there
be four replicates (minimum of three) for each concentration and control.

13.10.2.2.6  Seven to 11 day old larvae are active and difficult to capture
and are subject to handling mortality.  Carefully remove larvae (2-3-at a
time) by concentrating them in a corner of the aquarium or; culture vessel, and
capture them with a wide-bore pipette, small petri dish, crystallization dish,
3-4 cm in diameter, or small pipette.  They are active and will  readily escape
from a pipette.  Randomly transfer the larvae (2-3 at a time) into each test
chamber until  the desired number (15) is attained.  See Appendix A for an
example of randomization.  After the larvae are dispensed, use a light table
to verify the number in each chamber.
                                      .

13.10.2.3  Before beginning the test remove and replace any dead larvae from
each test chamber.  The test is started by removing approximately 90% of the
clean seawater from each test chamber and replacing with the appropriate test
solution.
                                      179

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13.10.3  LIGHT, PHOTOPERIOD, SALINITY, AND TEMPERATURE

13.10.3.1  The light quality and intensity should be at ambient laboratory
levels, which is approximately 10-20 pE/m/s,  or 50 to 100 foot candles
(ft-c), with a photoperiod of 16 h of light and 8 h of darkness. The water
temperature in the test chambers should be maintained at 25 ± 1°C.  The test
salinity should be in the range of 5%o to 32%o, and the salinity should not
vary by more than ± 2%o among the chambers on a given day.  If effluent and
receiving water tests are conducted concurrently, the salinities of these
tests should be similar.

13.10.4  DISSOLVED OXYGEN (DO) CONCENTRATION

13.10.4.1  Aeration may affect the toxicity of effluents and should be used
only as a last resort to maintain satisfactory DO.  The DO should be measured
on new solutions at the start of the test (Day 0) and before daily renewal of
test solutions on subsequent days.  The DO should not fall below 4.0 mg/L (see
Section 8, Effluent and Receiving Water Sampling, Sample Handling and Sample
Preparation for Toxicity Tests).  If it is necessary to aerate, all
concentrations and the control should be aerated.  The aeration rate should
not exceed 100 bubbles/min, using a pi pet with a 1-2 mm orifice such as a 1-mL
KIMAX® serological pi pet No. 37033, or equivalent.  Care should be taken to
ensure that turbulence resulting from aeration does not cause undue stress to
the fish.

13.10.5  FEEDING

13.10.5.1  Artemia nauplii are prepared as described above.

13.10.5.2  The test larvae are fed newly-hatched (less than 24-h-old) Artemia
nauplii once a day from Day 0 through Day 6; larvae are not fed on Day 7.
Equal amounts of Artemia nauplii must be fed to each replicate test chamber to
minimize the variability of larval weight..  Sufficient numbers of nauplii
should be fed to ensure that some remain alive overnight in the test chambers.
An adequate, but not excessive amount of Artemia nauplii, should be provided
to each replicate on a daily basis.  Feeding excessive amounts of Artemia
nauplii will result in a depletion in DO to below an acceptable level.  Siphon
as much of the uneaten Artemia nauplii as possible from each chamber daily to
ensure that the larvae principally eat newly hatched nauplii.

13.10.5.3  On days 0-2, transfer 4 g wet weight or pipette 4 mL of
concentrated, rinsed Artemia nauplii to seawater in a 100 mL beaker, and bring
to a volume of 80 mL.  Aerate or swirl the suspension to equally distribute
the nauplii while withdrawing individual 2 mL portions of the Artemia nauplii
suspension by pipette or adjustable syringe to transfer to each replicate test
chamber.  Because the nauplii will settle and concentrate at the tip of the
pipette during the transfer, limit the volume of concentrate withdrawn each
time to a 2-mL portion for one test chamber helps ensure an equal distribution
to the replicate chambers.  Equal distribution of food to the replicates is
critical for successful tests.
                                      180

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13.10.5.4  On days 3-6, transfer 6 g wet weight or 6 mL of the Artemia nauplii
concentrate to seawater in a 100 mL beaker.  Bring to a volume of 80 mL and
dispense as described above.
                                                          I
13.10.5.5  If the larvae survival rate in any replicate on any day falls below
50%, reduce the volume of Artemia nauplii suspension added to that test
chamber by one-half (i.e., reduce from 2 ml to 1 ml) and continue feeding
one-half the volume through day 6.  Record the time of feeding on the data
sheets.                                                   |
                                                          I
13.10.6  DAILY CLEANING OF TEST CHAMBERS

13.10.6.1  Before the daily renewal of test solutions, uneaten and dead
Artemia, and other debris are removed from the bottom of the test chambers
with a siphon hose.  Alternately, a large pipet (50 mL), fitted with a safety
pi pet filler or rubber bulb, can be used.  If the test chambers illustrated in
Figure 1 are used, remove only as much of the test solution from the chamber
as  is necessary to clean, and siphon the remainder of the test solution from
the sump area.  Because of their small size during the first few days of the
test, larvae are easily drawn into a siphon tube when cleaning the test
chambers.  By placing the test chambers on a light box, inadvertent removal of
larvae can be greatly reduced because they can be more easily seen.  If the
water siphoned from the test chambers is collected in a white plastic tray,
the live larvae caught up in the siphon can be retrieved, and returned by
pipette to the appropriate test chamber and noted on data sheet.  Any
incidence of removal of live larvae from the test chambers by the siphon
during cleaning, and subsequent return to the chambers should be noted in the
test records.

13.10.7  OBSERVATIONS DURING THE TEST

13.10.7.1  Routine Chemical and Physical Determinations

13.10.7.1.1  DO is measured at the beginning and end of each 24-h exposure
period in one test chamber at all test concentrations and in the control.

13.10.7.1.2  Temperature, pH, and salinity are.measured at the end of each
24-h exposure period in one test chamber at all test concentrations and in the
control.  Temperature  should also be monitored continuously or observed and
recorded daily for at  least two locations  in the environmental control system
or  the samples.  Temperature should be measured in a sufficient number of test
chambers at least the  end of the test to determine the temperature variation
in  the environmental chamber.

13.10.7.1.3  The pH  is measured  in the effluent sample each day before new
test solutions are made.

13.10.7.1.4  Record  all measurements on the data sheet  (Figure 3).

13.10.7.2  Routine Biological Observation
                                      181

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                                                        183

-------
13.10.7.2.1  The number of live larvae in each test chamber are recorded daily
(Figure 3), and the dead larvae are discarded.

13.10.7.2.2  Protect the larvae from unnecessary disturbances during the test
by carrying out the daily test observations, solution renewals, and removal of
dead larvae.  Make sure the larvae remain immersed at all times during the
performance of the above operations.

13.10.8  TEST SOLUTION RENEWAL

13.10.8.1  The test solutions are renewed daily using freshly prepared
solutions, immediately after cleaning the test chambers.  The water level in
each chamber is lowered to .a depth of 7-to-10 mm, leaving 10 to 15% of the
test solution.  New test solution is added slowly by refilling each chamber
with the appropriate amount of test solution without excessively disturbing
the larvae.  If the modified chamber is used (Figure 1), renewals should be
poured into the sump area using a narrow bore (approximately 9 mm ID) funnel.

13.10.8.2  The effluent or receiving water used in the test is stored in an
incubator or refrigerator at 4°C.  Plastic containers such as 8-20 L
cubitainers have proven suitable for effluent collection and storage.  For
on-site toxicity studies no more than 24 h should elapse between collection of
the effluent and use in a toxicity test (see Section 8, Effluent and Receiving
Water Sampling, Sample Handling, and Sample Preparation for Toxicity Tests).

13.10.8.3  Approximately 1 h before test initiation, a sufficient quantity of
effluent or receiving water sample is warmed to 25 ± 1°C to prepare the test
solutions.  A sufficient quantity of effluent should be warmed to make the
daily test solutions.

13.10.8.3.1  An illustration of the quantities of effluent and seawater needed
to prepare test solution at the appropriate salinity is provided in Table 2.

13.10.9  TERMINATION OF THE TEST

13.10.9.1  The test is terminated after 7 days of exposure.  At test
termination dead larvae are removed and discarded.  The surviving larvae in
each test chamber (replicate) are counted, and immediately prepared as a group
for dry weight determination, or are preserved in 4% formalin or 70% ethanol.
Preserved organisms are dried and weighed within 7 d.  For safety, formalin
should be used under a hood.

13.10.9.2  For immediate drying and weighing, siphon or pour live larvae onto
a 500 /im mesh screen in a large beaker to retain the larvae and allow Artemia
to be rinsed away.  Rinse the larvae with deionized water to remove salts that
might contribute to the dry weight.  Sacrifice the larvae in an ice bath of
deionized water.

13.10.9.3  Small aluminum weighing pans can be used to dry and weigh larvae.
An appropriate number of aluminum weigh pans (one per replicate) are marked
for identification and weighed to 0.01 mg, and the weights are recorded
(Figure 4) on the data sheets.

                                      184

-------
Test Dates:
                        Species:.
    Pan
    No.
Cone.
  &
 Rep.
Initial
  Wt.
Final
 Wt.
 (mg)
Diff.
 (mg)
 No.
Larvae
Av. Wt./
 Larvae
  (mg)
Figure 4.      Data form for the inland silverside,  Menidia beryllina, larval
               survival  and growth test.  Dry weights of larvae (from USEPA,
               1987b).
                                      185

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 13.10.9.4  Immediately prior to  drying,  the  preserved  larvae  are  in  distilled'
 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  (Figure 4}  the weights.   Divide  the dry
 weight  by the number of original larvae  per  replicate  to determine the average
 dry weight,  and record (Figures  4  and  5)  on  the  data sheets.   For  the
 controls,  also calculate the mean  weight per surviving fish in the test
 chamber to evaluate if weights met test  acceptability  criteria (see
 Subsection 13.1).   Complete  the  summary  data sheet (Figure  5)  after
 calculating the average measurements and statistically analyzing the dry
 weights and percent survival  for the entire  test.  Average  weights should  be
 expressed to the nearest 0.001 mg.

 13.11   SUMMARY OF  TEST CONDITIONS  AND  TEST ACCEPTABILITY CRITERIA

 13.11.1  A summary of test conditions  and test acceptability criteria is
 listed  in Table 3.  '

 13.12   ACCEPTABILITY OF TEST RESULTS

 13.12.1  Test  results  are acceptable if  (1)  the  average  survival of control
 larvae  is  equal  to  or  greater than 80%,  and  (2)  where  the test starts with
 7-day old  larvae,  the  average dry weight  per  surviving control larvae, when
 dried immediately  after test  termination, is  equal to  or greater than 0.50 mg,
 or the  average  dry  weight of  the control  larvae  preserved not  more than 7 days
 in 4% formalin  or  70%  ethanol  equals or  exceeds  0.43 mg.

 13.13   DATA  ANALYSIS

 13.13.1   GENERAL

 13.13.1.1  Tabulate  and summarize the  data.

 13.13.1.2  The  endpoints of toxicity tests using the inland silverside are
 based on the adverse effects  on survival  and growth.   The LC50, the IC25,  and
the IC50 are calculated using  point estimation techniques (see Section 9,
Chronic Toxicity Test  Endpoints and Data Analysis).  LOEC and NOEC values,  for
survival and growth, are obtained using a hypothesis testing approach such as
Dunnett's Procedure  (Dunnett,  1955) or Steel's Many-one Rank Test  (Steel,
1959;  Miller, 1981)  (see Section 9).    Separate analyses are performed for the
estimation of the LOEC  and NOEC endpoints and for the estimation of the LC50,
 IC25,  and IC50.  Concentrations at which there is no survival   in any of the
test chambers are excluded from the statistical  analysis  of the NOEC and LOEC
for survival and growth but included in the estimation  of the LC50, IC25,  and
IC50.   See the Appendices for examples of the manual  computations and examples
of data input and program output.
                                      186

-------
Test Dates:
Species:
Effluent Tested:
TREATMENT
NO. LIVE
LARVAE
SURVIVAL
(%)
MEAN DRY WT/
LARVAE (MG)
± SD
SIGNIF. DIFF.
FROM CONTROL
(o)
MEAN
TEMPERATURE
CO
± SD
MEAN SALINITY
%0
+ SD
AVE. DISSOLVED
OXYGEN
(MG/L) ± SD
















































COMMENTS:
 Figure  5.      Data form for the inland silverside,  Mem'dia beryllina,  larval
               survival and growth test.  Summary of test results (from USEPA,
               1987c).
                                      187

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TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
         THE  INLAND SILVERSIDE, MENIDIA BERYLLINA, LARVAL SURVIVAL AND
         GROWTH TEST WITH EFFLUENTS AND RECEIVING WATERS
1.  Test type:

2.  Salinity:


3.  Temperature:

4.  Light quality:

5.  Light intensity:


6.  Photoperiod:

7.  Test chamber size:

8.  Test solution volume:


9.  Renewal of test solutions:

10. Age of test organisms:


11. No. larvae per test
     chamber:

12. No. replicate chambers
     per concentration:

13. No. larvae per concentration:

14. Source of food:
15. Feeding regime:
16. Cleaning:
Static renewal

5%o to 32%o (± 2%o of the selected
test salinity)

25 ± 1°C

Ambient laboratory illumination

10-20 /*E/m2/s  (50-100 ft-c)  (Ambient
laboratory levels)

16 h light, 8 h darkness

600 mL - 1 L containers

500-750 mL/replicate (loading and DO
restrictions must be met)

Daily

7-11 days post hatch; 24-h range in
age


15 (minimum of 10)


4 (minimum of 3)

60 (minimum of 30)

Newly hatched Artemia nauplii
(survival of 7-9 days old Menidia
beryl Una larvae improved by feeding
24 h old Artemia)

Feed 0.10 g wet weight Artemia nauplii
per replicate on days 0-2; Feed 0.15 g
wet weight Artemia nauplii per
replicate on days 3-6

Siphon daily,  immediately before test
solution renewal and feeding
                                    188

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TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY  CRITERIA  FOR
         THE  INLAND SILVERSIDE, MENIDIA BERYLLINA,  LARVAL SURVIVAL AND
         GROWTH TEST WITH  EFFLUENTS AND RECEIVING WATERS  (CONTINUED)
17. Aeration:
18. Dilution water:
19. Test concentrations:



20. Dilution factor:


21. Test duration:

22. Endpoints:

23. Test acceptability criteria:
24. Sampling requirement:
25. Sample volume required:
None, unless DO concentration falls
below 4.0 mg/L, then aerate all
chambers.  Rate should be less than
100 bubbles/min.
       ;
Uncontaminated source of natural sea
water, artificial seawater; deionized
water mixed with hypersaline brine or
artificial sea salts (HW Marinemix®,
FORTY FATHOMS®, GP2 or equivalent)

Effluent:  Minimum of 5 and a control
Receiving Waters: 100% receiving water
or minimum of 5 and a control
Effluents:  > 0.5
Receiving waters:

7 days
None, or > 0.5
Survival and growth (weight)

80% or greater survival in controls,
0.50 mg average dry weight of control
larvae where test starts with 7-days
old larvae and dried immediately after
test termination, or 0.43 mg or
greater average dry weight per
surviving control larvae, preserved
not more than 7 days in 4% formalin or
70% ethanol

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 for Toxicity Tests,
Subsection 8.5.4)

6 L per day
                                    189

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13.13.1.3  The statistical tests described here must be used with a knowledge
of the assumptions upon which the tests are contingent.  The assistance of a
statistician is recommended for analysts who are not proficient in statistics.

13.13.2   EXAMPLE  OF  ANALYSIS  OF  INLAND  SILVERSIDE,  MENIDIA  BERYLLINA,
          SURVIVAL DATA

13.13.2.1  Formal statistical analysis of the survival data is outlined in
Figures 6 and 7.  The  response used in the analysis is the proportion of
animals surviving in each test or control chamber.  Separate analyses are
performed for the estimation of the NOEC and LOEC endpoints and for the
estimation of the LC50 endpoint.  Concentrations at which there is no survival
in any of the test chambers are excluded from statistical analysis of the NOEC
and LOEC, but included in the estimation of the 1C, EC, and LC endpoint.

13.13.2.2  For the case of equal numbers of replicates across all
concentrations and the control, the evaluation of the NOEC and LOEC endpoints
is made via a parametric test, Dunnett's Procedure, or a nonparametric test,
Steel's Many-one Rank Test, on the arc sine square root transformed data.
Underlying assumptions of Dunnett's Procedure, normality and homogeneity of
variance, are formally tested.  The test for normality is the Shapiro-Wilk's
Test, and Bartlett's Test is used to test for the homogeneity of variance.  If
either of these tests fails, the nonparametric test, Steel's Many-one Rank
Test, is used to determine the NOEC and LOEC endpoints.  If the assumptions of
Dunnett's Procedure are met, the endpoints are estimated by the parametric
procedure.

13.13.2.3  If unequal numbers of replicates occur among the concentration
levels tested, there are parametric and nonparametric alternative analyses.
The parametric analysis is a t test with the Bonferroni adjustment (see
Appendix D).  The Wilcoxon Rank Sum Test with the Bonferroni adjustment is the
nonparametric alternative.

13.13.2.4  Probit Analysis (Finney, 1971; see Appendix H) is used to estimate
the concentration that causes a specified percent decrease in survival from
the control.  In this analysis, the total mortality data from all test
replicates at a given concentration are combined.  If the data do not fit the
Probit model, the Spearman-Karber method, the Trimmed Spearman-Karber method,
or the Graphical method may be used (see Appendices H-K).

13.13.2.5  Example of Analysis of Survival Data

13.13.2.5.1  This example uses the survival data from the inland silverside
larval survival  and growth test.  The proportion surviving in each replicate
in this example must first be transformed by the arc sine transformation
procedure described in Appendix B.  The raw and transformed data, means and
variances of the transformed observations at each effluent concentration and
control are listed in Table 4.  A plot of the data is provided in Figure 8.
Since there is 100% mortality in all three replicates for the 50% and 100%
concentrations,  they are not included in this statistical analysis and are
considered a qualitative mortality effect.


                                      190

-------
            STATISTICAL ANALYSIS OF INLAND SILVERSIDE LARVAL
                       SURVIVAL AND GROWTH TEST

                      SURVIVAL HYPOTHESIS TESTING
                               SURVIVAL DATA
                            PROPORTION SURVIVING
                                  ARC SINE
                              TRANSFORMATION
                             SHAPIRO-WILKSTEST
                  NORMAL DISTRIBUTION
                                                 NON-NORMAL DISTRIBUTION
        HOMOGENEOUS
          VARIANCE
                              BARTLETTS TEST
                       HETEROGENEOUS
                          VARIANCE
              EQUAL NUMBER OF
                REPLICATES?
          NO
    T-TESTWITH
    BONFERRONI
    ADJUSTMENT
              EQUAL NUMEIER OF
                 REPLICATES?
        YES
           NO
STEEL'S MANY-ONE
   RANK TEST
  WILCOXON RANK SUM
      TEST WITH
BONFERRON1 ADJUSTMENT
                             ENDPOINT ESTIMATES
                                 NOEC, LOEC
Figure  6.   Flowchart for statistical analysis of the  inland silverside,
           Menidia beryllina,  survival data by hypothesis testing.
                                                        I

                                    191

-------
         STATISTICAL ANALYSIS OF INLAND SILVERSIDE LARVAL
                     SURVIVAL AND GROWTH TEST

                     SURVIVAL POINT ESTIMATION
      MORTALITY DATA
         #DEAD
       TWO OR MORE
    PARTIAL MORTALITIES?
                        NO
            YES
      IS PROBIT MODEL
       APPROPRIATE?
    (SIGNIFICANT X2 TEST)
NO
            YES
ONE OR MORE
PARTIAL MORTALITIES?
\
YES
r
NO _

  GRAPHICAL METHOD
       LC50
      PROBIT METHOD
     ZERO MORTALITY IN THE
     LOWEST EFFLUENT CONG.
    AND 100% MORTALITY IN THE
    HIGHEST EFFLUENT CONG.?
NO
                                     YES
                              SPEEARMAN-KARBER
                                  METHOD
                             TRIMMED SPEARMAN-
                               KARBER METHOD
                                LC50AND95%
                                CONFIDENCE
                                  INTERVAL
Figure 7.   Flowchart for statistical  analysis of the inland silverside,
           Mem'dia beryTTina, survival  data by point estimation.

                                   192

-------
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              193

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    TABLE 4.  INLAND SILVERSIDE, MENIDIA BERYLLINA, LARVAL SURVIVAL DATA
                                           Effluent Concentration^)
           Replicate     Control
              6.25
        12.5
25.0
50.0   100.0

RAW

ARC SINE
TRANS-
FORMED
Mean(Y,}
s?
i
A
B
C
A
B
C



0.80
0.87
0.93
1.107
1.202
1.303
1.204
0.010
1
0.73
0.80
0.87
1.024
1.107
1.202
1.111
0.008
2
0.80
0.33
0.60
1.107
0.612
0.886
0.868
0.061
3
0.40 0.0
0.53 0.0
0.07 0.0
0.685
0.815
0.268
0.589
0.082
4
0.0
0.0
0.0

-
-



13.13.2.6  Test for Normality

13.13.2.6.1  The first step of the test for normality is to center the
observations by subtracting the mean of all observations within a
concentration from each observation in that concentration.  The centered
observations are summarized in Table 5.
         TABLE  5.   CENTERED  OBSERVATIONS  FOR  SHAPIRO-WILK'S  EXAMPLE
   Replicate
Control
Effluent Concentration (%)

 6.25       12.5       25.0
A
B
C
-0.097
-0.002
0.099
-0.087
-0.004
0.091
0.239
-0.256
0.018
0.096
0.226
-0.321
                                      194

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13.13.2.6.2  Calculate the denominator, D, of the statistic:
                                D = £
                                    i=l
~v\ 2
     Where:  X,-  = the ith centered observation
             X  = the overall mean of the centered observations
             n  = the total number of centered observations
13.13.2.6.3  For this set of data,    n =12
                                      X = 	L(0.002) = 0.0
                                            12             i
                                      D - 0.3214
13.13.2.6.4  Order the centered observations from smallest to largest:
               X(1)
0.018
0.091
0.096
0.099
0.226
0.239
13.13.2.6.5  From Table 4, Appendix B, for the number of observations, n,
obtain the coefficients a,, a2,  ...  ak where k is n/2 if n  is even and  (n-l)/2
if n is odd.  For the data in this example, n = 12 and k =| 6.  The a,- values
are listed in Table 7.
                                      195

-------
13.13.2.6.6  Compute the test statistic, W, as follows:
                             .       j
                             D 1=1

The differences x(n"!+1} - X
- x(2>
- x<3)
- x<4)
- x(5>
- XC6)
13.13.2.6.7  The decision rule for this test is to compare W as calculated in
Subsection 13.2.6.6 to a critical value found in Table 6, Appendix B.  If the
computed W is less than the critical value, conclude that the data are not
normally distributed.  For the data in this example, the critical value at a
significance level of 0.01 and n = 12 observations is 0.805.  Since W = 0.945
is greater than the critical value, conclude that the data are normally
distributed.

13.13.2.7  Test for Homogeneity of Variance

13.13.2.7.1  The test used to examine whether the variation in survival is the
same across all effluent concentrations including the control, is Bartlett's
Test (Snedecor and Cochran, 1980).  The test statistic is as follows:
                      B =
                                          -  Ev^ in
    Where: V,


            P


           In
degrees of freedom for each effluent concentration and
control, V,- = (nf  -  1)

number of levels of effluent concentration including the
control
                                      196

-------
            i   = 1,  2,  ...,  p where p is the number of concentrations
                 including the control

           n,-   = the number of replicates for concentration i.
            sr =
                     t
             C = l+[3(p-l)]-1[El/IA-(
                                2=1       2=1  ~

13.13.2.7.2  For the data in this  example (See Table 4),  all  effluent
concentrations including the control  have the  same number of  replicates
(n, = 3  for all  i).   Thus,  Vf = 2 for all i.

13.13.2.7.3  Bartlett's statistic  is  therefore:
                 B = [(8)1^2(0.0402) -2 £ In (S*)] /1.2083
                                         2=1

                    =  [8(-3.21391)  - 2(-14.731)]/1.2083

                    =  3.7508/1.2083

                    =  3.104

13.13.2.7.4  B is approximately distributed as chi-square  with  p  -  1  degrees
of freedom, when the variances are in fact  the same.   Therefore,  the
appropriate critical value for this  test, at a significance  level of  0.01 with
three degrees of freedom,  is 11.345.  Since B = 3.104 is less than  the
critical  value of 11.345,  conclude that the variances are  not different.

13.13.2.8  Dunnett's Procedure

13.13.2.8.1  To obtain an  estimate of the pooled variance  for the Dunnett's
Procedure, construct an ANOVA table  as described in  Table  8.
                                     197

-------
                      TABLE 8.   ANOVA TABLE
Source
Between
Within
Total
df Sum of Squares
(SS.)
p - 1 SSB
N - p SSW
N - 1 SST
Mean Square(MS)
(SS/df)
SB i SSB/(p-l)
Sy = SSW/(N-p)

Where:   p  - number of SDS concentration  levels  including the control

         N  = total number of observations n, + n2 ... + np

         nf = number of observations  in concentration i


        SSB = ~ETi/ni-G2/N        Between Sum of Squares
       SST =
Total Sum of Squares
       SSW = SST-SSB
Within Sum of Squares
         G  =  the grand total of all  sample observations,   G = £ T±


         Tf =  the total  of the replicate measurements for
               concentration i

        u   =  the jth observation for concentration i (represents the
               proportion surviving for toxicant concentration  i  in test
               chamber j)
                                198

-------
13.13.2.8.2  For the data  in this example:
                         = n2 = n3 =  n4 = 3
                      N  =  12
                      T1 = Yn  +  Yia + Y13  =  3.612
                      T, = Y21  +  Y22 + Y23  =  3.333

                      T3 = Y31  +  Y32 + Y33  -  2-605
                      T4 = Y41  +  Y42 + Y43  =1.768


                      G  = T, + T2 + T3  +  T4 -  11.318
                    SSB = f^Tl/ni-G2/N
                    SST =
                            1  (34.067)  -  fll.318)z  = 0.681
                            3                 12
                            1.002
                           i-lj'-l

                        =  11.677  -  (11. 318V
                                        12
                    SSW = SST-SSB
                   = 1.002 - 0.681 = 0.321
                      SB = SSB/(p-l) = 0.681/(4-l) = 0.227


                      Sy = SSW/(N-p) = 0.321/(12-4) = 0.040


13.13.2.8.3  Summarize these calculations  in the ANOVA  table  (Table 9)
             TABLE 9.  ANOVA TABLE FOR DUNNETT'S  PROCEDURE  EXAMPLE
Source
Between
Within
df
3
8
Sum of Squares
(SS)
0.681
0.321
Mean Square(MS)
(SS/df)
i
0.227
0.040
      Total
11
1.002
                                      199

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13.13.2.8.4  To perform the individual comparisons, calculate the t statistic
for each concentration, and control combination as follows:
Where:  Y?  = mean proportion surviving for effluent  concentration  i

        Y,,  « mean proportion surviving for the control

        SM  » square root of the within mean square

        n.,  - number of replicates for the control

        nf  = number of replicates for concentration  i.

13.13.2.8.5  Table 10 includes the calculated t values for each concentration
and control combination.  In this example, comparing the 1.0% concentration
with the control the calculation is as follows:
   (1.204 - 1.111)

[0.020
                                                  = 0.570
                        TABLE 10.  CALCULATED T VALUES
             Effluent Concentration(%)
6.25
12.5
25.0
2
3
4.
0.570
2.058
3.766
13.13.2.8.6  Since the purpose of this test is to detect a significant
reduction in survival, a one-sided test is appropriate.  The critical  value
for this one-sided test is found in Table 5, Appendix C.  For an overall  alpha
level of 0.05, eight degrees of freedom for error and three concentrations
(excluding the control) the critical value is 2.42.  The mean proportion
surviving for concentration i is considered significantly less than the mean
proportion surviving for the control if t,- is greater than the critical value.
Therefore, only the 25.0% concentration has a significantly lower mean
proportion surviving than the control.  Hence the NOEC is 12.5% and the LOEC
is 25.0%.
                                      200

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13.13.2.8.7  To quantify the sensitivity of the test,  the minimum significant
difference (MSD) that can be detected statistically may be calculated.
                          MSD = d Sw

                                                         \
Where:  d  = the critical value for Dunnett's Procedure

        Su = the square root  of the  within  mean  square

        n  = the common number of replicates at  each concentration
             (this assumes equal replication at  each concentration)

        n1 = the number of replicates  in  the control.

13.13.2.8.8  In this example:

                       MSD = 2.42 (0.20)^(1/3)+(1/3)

                           = 2.42 (0.20)(0.817)

                           = 0.395

13.13.2.8.9  The MSD (0.395)  is in transformed units.   To determine  the MSD in
terms of percent survival, carry out the following conversion.

   1.   Subtract the MSD from the transformed control mean

                            1.204 -  0.395 = 0.809

   2.   Obtain the untransformed values for the control  mean and the
       difference calculated in step 1.

                          [ Sine (1.204)  ]* = 0.871

                          [ Sine (0.809)  ]2 = 0.524

   3.   The untransformed MSD (MSDJ  is determined  by subtracting  the
       untransformed values from step 2.                  I

                        MSDU  =  0.871 - 0.524 = 0.347

13.13.2.8.10  Therefore, for this set of data, the minimum difference in mean
proportion surviving between the control  and any effluent concentration that
can be detected as statistically significant is  0.347.    >
                                                         j
13.13.2.8.11  This represents a 40% decrease in  survival  from the control.
                                     201

-------
13.13.2.9  Calculation of the LC50

13.13.2.9.1  The data used for the Probit Analysis is summarized in Table 11,
To perform the Probit Analysis, run the USEPA Probit Analysis Program.  An
example of the program input and output is supplied in Appendix H.

                     TABLE 11.  DATA FOR-PROBIT ANALYSIS


                                      Eff1uent Concentration (%)	
                                Control  6.25   12.5   25.0   50.0   100.0
Number Dead
Number Exposed
6
45
9
45
19
45
30
45
45
45
45
45
13.13.2.9.2  For this example, the chi-square test for heterogeneity was not
significant.  Thus Probit Analysis appears to be appropriate for this set of
data.

13.13.2.9.3  Figure 9 shows the output data for the Probit Analysis of the
data from Table 11 using the USEPA Probit Program.

13.13.3  ANALYSIS OF INLAND SILVERSIDE, MENEDIA BERYLLINA, GROWTH DATA

13.13.3.1  Formal statistical analysis of the growth data is outlined in
Figure 10.  The response used in the statistical analysis is mean weight per
original organism for each replicate.  The IC25 and IC50 can be calculated for
the growth data via a point estimation technique (see Section 9, Chronic
Toxicity Test Endpoints and Data Analysis).  Hypothesis testing can be used to
obtain an NOEC and LOEC for growth.  Concentrations above the NOEC for
survival are excluded from the hypothesis test for growth effects.

13.13.3.2  The statistical analysis using hypothesis tests consists of a
parametric test, Dunnett's Procedure, and a nonparametric test, Steel's
Many-one Rank Test.  The underlying assumptions of the Dunnett's Procedure,
normality and homogeneity of variance, are formally tested.  The test for
normality is the Shapiro-Wilk's Test and Bartlett's Test is used to test for
homogeneity of variance.  If either of these test fails, the nonparametric
test, Steel's Many-one Rank Test, is used to determine the NOEC and LOEC
endpoints.  If the assumptions of Dunnett's Procedure are met, the endpoints
are determined by the parametric test.

13.13.3.3  Additionally, if unequal numbers of replicates occur among the
concentration levels tested there are parametric and nonparametric alternative
analyses.  The parametric analysis is a t test with the Bonferroni adjustment.
The Wilcoxon Rank Sum Test with the Bonferroni adjustment is the nonparametric
alternative.  For detailed information on the Bonferroni adjustment, see
Appendix D.


                                      202

-------
    Probit Analysis of Inland Silverside Larval Survival Data

Cone.
Control
6
12
25
50
100
Chi
Chi
.2500
.5000
.0000
.0000
.0000
- Square
- Square
Number
Exposed
45
45
45
45
45
45
for Heterogeneity
for Heterogeneity
Observed
Number Proportion
Resp. Responding
6
9
19
30
45
45
(calculated)
(tabular value)
0
0
0
0
1
1


.1333
.2000
.4222
.6667
.0000
.0000


Proportion
Responding
Adjusted for
Controls
0
0
0
0
1
1
-

.0000
.0488
.3130
.6037
.0000
.0000
4.149
7.815
    Probit Analysis of  Inland Silverside Larval Survival Data
           Estimated LC/EC Values and Confidence Limits
          Point
    LC/EC    1.00
    LC/EC  50.00
Exposure
Cone.
4.980
18.302
Lower Upper
95% Confidence Limits
2
13
.023
.886
7.789
22.175
Figure 9.  Output for USEPA Probit Analysis Program,, Version 1.5.
                                203

-------
             STATISTICAL ANALYSIS OF INLAND SILVERSIDE LARVAL
                         SURVIVAL AND GROWTH TEST

                                   GROWTH

GROWTH DATA
MEAN DRY WEIGHT

1
r
    POINT ESTIMATION
    HYPOTHESIS TESTING
(EXCLUDING CONCENTRATIONS
 ABOVE NOEC FOR SURVIVAL)
    ENDPOINT ESTIMATE
        IC25, IC50
    SHAPIRO-WILICS TEST
                    NORMAL DISTRIBUTION
                       NON-NORMAL DISTRIBUTION
        HOMOGENEOUS
          VARIANCE
                                BARTLETTSTEST
                             HETEROGENEOUS
                                VARIANCE
               EQUAL NUMBER OF
                 REPLICATES?
           NO
     T-TESTWITH
     BONFERRONI
     ADJUSTMENT
                     EQUAL NUMBER OF
                       REPLICATES?
              YES
           NO
      STEEL'S MANY-ONE
         RANK TEST
  WILCOXON RANK SUM
      TEST WITH
BONFERRONI ADJUSTMENT
                              ENDPOINT ESTIMATES
                                  NOEC, LOEC
Figure 10.   Flowchart for statistical  analysis  of  the  inland silverside,
            Menidia beryl Una, growth  data.

                                     204

-------
13.13.3.4  The data, mean and variance of the growth observations at each
concentration including the control are listed in Table 12.  A plot of the
data is provided in Figure 11.  Since there was no survival in the 50% and
100% concentrations, these are not considered in the growth analysis.
Additionally, since there is significant mortality in the 25% effluent
concentration, its effect on growth is not considered.   i


        TABLE 12.  INLAND SILVERSIDE, MENIDIA BERYLLINA, GROWTH DATA


                                	Effluent Concentration (%)
  Replicate    Control           6.25     12.5    25.0   50.0   100.0



Me
Si
i
A
B
C
an (Yf)


0
0
0
0
0
1
.751
.849
.907
.836
.0062

0
0
0
0
0
2
.737
.922
.927
.859
.0130

0
0
0
0
0
3
.722
.285
.718
.575
.063

0.
0.
1.
0.
0.
4
196
312
079
196
0136


-

.
-
5 6
13.13.3.5  Test for Normality
                                                         i
13.13.3.5.1  The first step of the test for normality is to center the
observations by subtracting the mean of all the observations within a
concentration from each observation in that concentration.   The centered
observations are summarized in Table 13.

          TABLE  13.   CENTERED  OBSERVATIONS  FOR  SHAPIRO-MILK'S  EXAMPLE


                                Effluent Concentration (%)

       Replicate        Control            6.25            12.5
           A            -0.085             0.147
           B             0.013            -0.290
0.00
0.166
           C             0.071             0.143          -0.117
                                      205

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                                   206

-------
13.13.3.5.2  Calculate the denominator,  D,  of the test statistic:


                                    i=l

   Where:   X,-  =  the  ith  centered  observation

            X  = the overal1 mean of the centered observations

            n  = the total number of centered observations.
                                                          •i
For this set of data,            n = 9

                                 X = _J_(-0.002) = 0.000
                                       9                  I

                                 D = 0.167

13.13.3.5.3  Order the centered observations from smallest to largest:

                  y(1)    y(2)      <  v(n)

                                                          I

Where XO)  is the  ith  ordered observation.  These ordered observations are
listed  in Table 14.

    TABLE  14.  ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WALK'S EXAMPLE
1
1
2
3
4
5
X<0
-0.290
-0.117
-0.085
0.000
0.013
i
6
7
8
9

X(i>
0.071
0.116
0.143
0.147
i
 13.13.3.5.4  From Table 4,  Appendix B,  for the  number  of  observations, n,
 obtain the coefficients a,, a?, ..., a, where k  is n/2  if  n is even and  (n-
 l)/2 if n is odd.  For the  data in this example,  n = 9 and k = 4.   The a,-
 values are listed in Table  15.
                                      207

-------
 13.13.3.5.5   Compute the test statistic,  W,  as  follows:
                         W=
                              D
The differences  x(n"i+1)  -  Xci> are listed in Table 15.   For this  set of data:
W =
                              1     (0.3997)2 = 0.964
                            0.1657

      TABLE 15.  COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE
1
2
3
4
0.5888
0.3244
0.1976
0.0947
0.437
0.260
0.201
0.071
x<9)
x<8)
XC7)
X(6>
- XC1)
- x<2)
- x<3)
- x<4>
13.13.3.5.6  The decision rule for this test is to compare W with the critical
value found in Table 6, Appendix B.  If the computed W is less than the
critical value, conclude that the data are not normally distributed.  For this
example, the critical value at a significance level of 0.01 and nine
observations (n) is 0.764.  Since W = 0.964 is greater than the critical
value, the conclusion of the test is that the data are normally distributed.

13.13.3.6  Test for Homogeneity of Variance

13.13.3.6.1  The test used to examine whether the variation in mean dry weight
is the same across all effluent concentrations including the control, is
Bartlett's Test (Snedecor and Cochran,  1980).  The test statistic is as
follows:
                      B =

                                                 In

    Where: Vf  = degrees of freedom for each effluent concentration and
                 control, V,-  = (nf  -  1)

            p  = number of levels of effluent concentration including the
                 control
                                     208

-------
            i   = 1,2,  ...,  p where p is the number of concentrations  including
                 the control

           In   = loge

           n,-   = number of replicates for concentration i
                     v±sl)
                    1=1
             C =


13.13.3.6.2  For the data in this example, (See Table 13)
concentrations including the control have the same number
3 for all i).  Thus, V5  = 2  for all  i.
13.13.3.6.3  Bartlett's statistic is therefore:
B =  [(6)ln(0.274) -2
                                       all  effluent
                                       of replicates  (n,-
                                                    1/1.25
                     =  [6(-3.5972)-2(ln(0.0062)+ln(0.013())+ln(0.0631))]/1.25

                     =  [-26.583 - (-24.378)]/1.25

                     =  2.236

13.13.3.6.4  B is approximately distributed as chi -square with p - 1 degrees
of freedom, when the variances are in fact the same.  Therefore, the
appropriate critical value for this test, at a significance level of 0.01 with
2 degrees of freedom, is 9.210.  Since B = 2.236 is less than the critical
value of 9.210, conclude that the variances are not different.
                                      209

-------
13.13.3.7  Dunnett's Procedure
13.13.3.7.1  To obtain an estimate of the pooled variance for the Dunnett's
Procedure, construct an ANOVA table as described in Table 16.
                            TABLE 16.  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)
SB = SSB/(p-l)
Sy = SSW/(N-p)

Where:  p  « number of effluent concentrations including the control
        N  « total number of observations n, + n2 ... + n
        nf  » number of observations  in  concentration  i
      SSB = £ Tl/n± - G2/N   Between Sum of Squares

      SST -
             i-U-i
                              Total  Sum of Squares
      SSW » SST-SSB
                              Within Sum of Squares
              the grand total of all sample observations,
                                                               i=1
              the total of the replicate measurements for concentration i
              the jth observation for concentration i (represents the mean
              dry weight of the fish for toxicant concentration i in test
              chamber j)
                                     210

-------
13.13.3.7.2  For the data in this example:
                     n,  = n2 = n3 = 3

                     N  = 9

                     T,  - Y,. + Y1P + Y13 = 0.751 + 0.849 + 0.907 = 2.507
                     T,  = Yp  + Y~ + Yp3 = 0.727 + 0.922 + 0.927 =2.576
                     T3  " Y3i + Y32 + Y33 = 0.722 + 0.285 + 0,718 = 1.725

                     G  = T,  + T2 + T3 = 6.808
                   SSB =
                           _1_(15.896) - (6.80S)2  = 0.1488
                    SST
                          5.463  -  (6. 80S)2  = 0.3131
                    SSW = SST-SSB     =  0.3131  -  0.1488  ==  0.1643
                                                          i
                     S2  = SSB/(p-l) = 0.1488/(3-l) = 0.0744

                     Sj  = SSW/(N-p) = 0.1643/(9-3) = 0.0274

 13.13.3.7.3   Summarize  these calculations in  the ANOVA table (Table 17)


             TABLE 17.   ANOVA TABLE FOR  DUNNETT'S PROCEDURE  EXAMPLE


       Source        df         Sum of Squares         Mean  Square(MS)
                                    (SS)         -        (SS/df)
Between
Within
2
6
0.1488
0.1643
p. 0744
0.0274
       Total          8            0.3131
                                       211

-------
 13.13.3.7.4  To perform the individual  comparisons,  calculate  the t  statistic
 for each concentration and control  combination  as  follows:
 Where:   Yf  = mean dry weight for effluent concentration i

         Y.,  - mean dry weight for the control

         Sw  - square root of the within mean square

         n,  - number of replicates for the control

         n,-  = number of replicates for concentration i.

 13.13.3.7.5  Table 18 includes the calculated t values for each concentration
 and  control combination.   In this example, comparing the 6.25% concentration
 with the control  the calculation is as follows:
                             (0.836  - 0.859)

                          [0.1655^(1/3)+(1/3)]
= -0.120
                        TABLE 18.  CALCULATED T VALUES
  Effluent Concentration  (%)
6.25
12.5
2
3
-0.170
1.931
13.13.3.7.6  Since the purpose of this test is to detect a significant
reduction in mean weight, a one-sided test is appropriate.  The critical  value
for this one-sided test is found in Table 5, Appendix C.  For an overall  alpha
level of 0.05, six degrees of freedom for error and two concentrations
(excluding the control) the critical value is 2.34.  The mean weight for
concentration i is considered significantly less than mean weight for the
control if tf is greater than the critical  value.   Therefore,  all  effluent
concentrations in this example do not have significantly lower mean weights
than the control.  Hence the NOEC and the LOEC for growth cannot be
calculated.

13.13.3.7.7  To quantify the sensitivity of the test, the minimum significant
difference (MSD) that can be detected statistically may be calculated.

                                      212

-------
                          MSD = d Sw ^(1/1^)+(1/12)

Where:  d  = the critical value for Dunnett's Procedure

        Sw = the square root of the within mean square
                                                          i
        n  = the common number of replicates at each concentration
             (this assumes equal replication at each concentration)

        n1 = the "number of replicates in the control.


13.13.3.7.8  In this example:
                      MSD = 2.34 (0.1655)V(1/3) + (1/3)

                          = 2.34 (0.1655)(0.8165)

                          = 0.316

 13.13.3.7.9  Therefore, for this set of data, the minimum difference that can
 be detected as  statistically  significant  is 0.316 mg.

 13.13.3.7.10  This  represents a 37.8% reduction  in mean weight  from the
 control.
                                                         !
 13.13.3.8  Calculation  of the ICp

 13.13.3.8.1  The  growth data  from  Tables  4 and  12  are  utilized  in  this
 example.   As seen in  Table  19 and  Figure  11,  the observed means are not
 monotonically non-increasing  with  respect to  concentration  (the mean response
 for  each higher concentration is not less than  or  equal to  the  mean response
 for  the previous  concentration,  and  the reponses between concentrations  do  not
 follow a linear trend). Therefore,  the means are  smoothed  prior to
 calculating the 1C.   In the following discussion,  the  observed  means are
 represented by  Y,- and the smoothed means  by M,-.
                               .
 13.13.3.8.2  Starting with  the control mean,  Y.,  = 0.836 and Y2 = 0.859, we see
 that Y, <  Y2.  Set M,- = Y,-.
                                                         j
 13.13.3.8.3  Calculate  the  smoothed  means:
                                                         i

                        M1 - M2 = (Y1  + Y2)/2 = °'847

 13.13.3.8.4  Since Y5 = 0 < Y4 = 0.196 < Y3 =  0.575 < M2, set  M3 =  0.575, M4 =
 0.196, and M5 = 0.
                                      213

-------
 13.13.3.8.5  Table 19 contains the response means and the smoothed means  and
 Figure 12 gives a plot of the smoothed response curve.


       TABLE 19.   INLAND SILVERSIDE MEAN GROWTH RESPONSE AFTER SMOOTHING
Ef f 1 uent
Cone.
(%)
Control
6.25
12.50
25.00
50.00


i
1
2
3
4
5
Response
Means,
Yj
(mg)
0.836
0.859
0.575
0.196
0.00
Smoothed
Means,
M-
(mg)
0.847
0.847
0.575
0.196
0.0
13.13.3.8.6  An IC25 and IC50 can be estimated using the Linear Interpolation
Method.  A 25% reduction in weight, compared to the controls, would result in
a mean dry weight of 0.627 mg, where M^l-p/100)  = 1.847(1-25/100).   A 50%
reduction in mean dry weight, compared to the controls, would result in a mean
weight of 0.418 mg.  Examining the smoothed means and their associated
concentrations (Table 20), the response, 0.627 mg, is bracketed by C, = 6.25%
effluent and C3 = 25.0% effluent.   The response (0.418) is  bracketed by C, =
12.5% and by C4 - 25% effluent.                                           3

13.13.3.8.7  Using the equation from Section 4.2 of Appendix L, the estimate
of the IC25 is calculated as follows:
                       ICp =
                      IC25 = 6.25 + [0.847(1 - 25/100) - 0.847](12.50 - 6.25)
                           = 11.1%.
                                                              (0.575 - 0.847)
                                     214

-------
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                           215

-------
 13.13.3.8.8  Using the equation from Section 4.2 of Appendix L,  the estimate
 of the IC50 is calculated as follows:  ,
                        ICp -Cj
                       IC50  =  6.25  + [0.847(1  -  50/100)  -  0.847]  (12.50  -  6.25)


                            =  17.5%.
(0.575 -  0.847)
 13.13.3.8.9   When  the  ICPIN  program was  used  to  analyze this  set of data,
 requesting 80 resamples,  the estimate  of the  IC25 was  11.1136%.  The empirical
 95% confidence interval for  the  true mean was 5.7119%  to  19.2112%.  The
 computer program output for  the  IC25 for this data  set is shown in Figure  13.

 13.13.3.8.10   When the ICPIN program was used to analyze  this set of data  for
 the IC50, requesting 80 resamples, the estimate  of  the IC50 was 17.4896%.  The
 empirical 95% confidence  interval for  the true mean was 6.4891% to 22.4754%
 effluent.  The computer program  output is shown  in  Figure 14.
13.14  PRECISION AND ACCURACY

13.14.1  PRECISION

13.14.1.1  Single-Laboratory Precision

13.14.1.1.1  Data on the single-laboratory precision of the inland silverside
larval survival and growth test using copper (CU) sulfate and sodium dodecyl
sulfate (SDS) as reference toxicants, in natural seawater and GP2 are provided
in Tables 20-22.  In Tables 20-21, the coefficient of variation for copper
based on the IC25 is 43.2% and for SDS is 43.2% indicating acceptable
precision.  In the five tests with each reference toxicant, the NOEC's varied
by only one concentration interval, indicating good precision.  The
coefficient of variation for all reference toxicants based on the IC50 in two
types of seawater (GP2 and natural) ranges from 1.8% to 50.7% indicating
acceptable precision.  Data in Table 22 show no detectable differences between
tests conducted in natural and artificial seawaters.

13.14.1.2  Multilaboratory Precision

13.14.1.2.1  Data on the multilaboratory precision of the inland silverside
larval survival and growth test are not yet available.

13.14.2  ACCURACY

13.14.2.1  The accuracy of toxicity tests cannot be determined.
                                      216

-------
Cone. ID

Cone. Tested
Response
Response
Response
1
2
3
1
0
.751
.849
.907
2
6.25
.727
.922
.927
3
12.5
.722
.285
.718
4
25
.196
.312
.079
5
50
0
0
0
6
100
0
0
0
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent: Effluent
Test Start Date:    Test Ending Date:
Test Species: Menidia beryl!ina
Test Duration:             7-d
DATA FILE: silver.icp
OUTPUT FILE: silver.i25
Cone.
ID
1
2
3
4
5
6
Number
Replicates
3
3
3
3
3
3
Concentration
%
0.000
6.250
12.500
25.000
50.000
100.000
Response Std.
Means Dev.
0.836 0.079
0.859 0.114
0.575 0.251
0.196 0.117
0.000 0.000
0.000 0.000
Pooled
Response Means
0.847
0.847
0.575
0.196
0.000
0.000
The Linear Interpolation Estimate:    11.1136   Entered P Value: 25

Number of Resamplings:   80
The Bootstrap Estimates Mean:  11.5341 Standard Deviation:     2.1155
Original Confidence Limits:   Lower:     8.5413 Upper:    14.9696
Expanded Confidence Limits:   Lower:     5.7119 Upper:    19.2112
Resampling time in Seconds:     1.43  Random Seed: -1912403737
                Figure 13.  ICPIN program output for the IC25.

                                      217

-------
Cone. ID

Cone. Tested
Response
Response
Response
1
2
3
1
0
.751
.849
.907
2
6.25
.727
.922
.927
3
12.5
.722
.285
.718
4
25
.196
.312
.079
5
50
0
0
0
6
100
0
0
0
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent: Effluent
Test Start Date:    Test Ending Date:
Test Species: Menidia beryllina
Test Duration:             7-d
DATA FILE: silver.icp
OUTPUT FILE: silver.i50
Cone.
ID
1
2
3
4
5
6
Number
Replicates
3
3
3
3
3
3
Concentration
%
0.000
6.250
12.500
25.000
50.000
100.000
Response
Means
0.836
0.859
0.575
0.196
0.000
0.000
Std. Pooled
Dev. Response Means
0.079
0.114
0.251
0.117
0.000
0.000
0.847
0.847
0.575
0.196
0.000
0.000
The Linear Interpolation Estimate:    17.4896   Entered P Value: 50

Number of Resamplings:   80
The Bootstrap Estimates Mean:  16.9032 Standard Deviation:     2.4973
Original Confidence Limits:   Lower:    12.2513 Upper:    19.8638
Expanded Confidence Limits:   Lower:     6.4891 Upper:    22.4754
Resampling time in Seconds:     1.43  Random Seed: -1440337465
                Figure 14.  ICPIN program output for the IC50.

                                      218

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TABLE  20.  SINGLE-LABORATORY PRECISION OF THE  INLAND SILVERSIDE,  MENIDIA
           BERYLLINA, SURVIVAL AND GROWTH TEST PERFORMED  IN  NATURAL
           SEAWATER,  USING LARVAE FROM FISH MAINTAINED AND  SPAWNED  IN
           NATURAL StAWATER, AND COPPER (CU) AS A REFERENCE
                   172'*'4'^6'7
Most
Test
Number





n:
Mean:
CV(%)
1
2
3
4
5


•
\ Data from USEPA
NOEC
Ug/L)
63
125
63
125
31
5
NA
NA
(1988a) and USEPA
IC25
U9/L)
96.2
207.2
218.9
177.5
350.1
5
209.9
43.7
(1991a)
IC50 , Sensitive
(jug/L) Endpoint
I
148.6
NC8
493.4
241.4
479.8
i 4
340.8
50.7
i
S
s
G
S
G




4
5
      i	 —^  —.	. j — ..Vii iuvii  \AIIVI  i_ i i o i* i w i *^ i i vs }  L.IX i_ ii *  UOi_rr\5
Narragansett, RI.
Three replicate exposure chambers with  10-15 larvae  were used for the
control and each copper concentration.  Copper concentrations were:  31,
63, 125, 250, and 500 Mg/L.
Adults collected in the field.
S = Survival effects.   G = Growth data  at  these toxicant concentrations
were disregarded because there was  a  significant reduction in survival.
NOEC Range: 31 - 125 /j.g/1 (this represents a difference  of two exposure
concentrations).
For a discussion of the precision of  data  from chronic toxicity tests
see Section 4, Quality  Assurance.
NC = No linear interpolation estimate could  be  calculated from the ata,
since none of the group response means  were  less  than 50 percent of  the
control  response mean.
                                    219

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TABLE 21.  SINGLE-LABORATORY  PRECISION OF THE INLAND SILVERSIDE,  MENIDIA
           BERYLLINA,  SURVIVAL AND GROWTH TEST PERFORMED IN NATURAL
           SEAWATER,  USING  LARVAE FROM FISH MAINTAINED AND SPAWNED IN
           NATURAL SEAWATER,  AND SODIUM DODECYL SULFATE (SDS)  AS  A
           REFERENCE  TOXICANT1'2'3'4'5'6'7
    Test
    Number
 NOEC
(mg/L)
 IC25
(mg/L)
 IC50
(mg/L)
Most
Sensitive
Endpoint
      1
      2
      3
      4
      5
 1.3
 •1.3
 1.3
 1.3
 1.3
0.3
1.6
1.5
1.5
1.6
 1.7
 1.9
 1.9
 1.9
 2.2
   S
   S
   S
   S
   S
n:
Mean:
CV(X):
5
NA
NA
5
1.3
43.2
5
1.9
9.4
I Data  from  USEPA  (1988a)  and  USEPA (1991a)
  Tests performed  by  George  Morrison and Elise Torello,  ERL-N,  USEPA,
  Narragansett,  RI.
  Three replicate  exposure chambers with 10-15 larvae were used for the
  control  and  each SDS  concentration.   SDS concentrations were: 0.3,  0.6,
  1.3,  2.5,  and  5.0 mg/L.
  Adults collected in the  field.
  S » Survival Effects.  Growth data at these toxicant concentrations
  were  disregarded because there was a significant reduction in survival.
6 NOEC  Range 1.3 mg/L.
  For a discussion of the  precision of data from chronic toxicity tests
  see Section  4, Quality Assurance.
                                    220

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TABLE 22.    COMPARISON OF THE SINGLE-LABORATORY PRECISION OF THE INLAND
             SILVERSIDE, MENIDIA BERYLLINA, LARVAL SURVIVAL (LC50) AND
             GROWTH (IC50) VALUES EXPOSED TO SODIUM DODECYL SULFATE (SDS)
             OR COPPER (CU) SULFATE IN GP2 ARTIFICIAL SEAWATER MEDIUM OR
             NATURAL SEAWATER  (NSW)1'2'3'4
           SDS (mg/L)
                                 Survival
GP2
NSW
                                   Growth
GP2
NSW
3.59
4.87
5.95
Mean 4.81
CV (%) 24.6
3.69 3.60
4.29 5.54
8.05
6.70
5.34 . 5.28
44.2
29.6
3.55
5.27
8.53
5.79
43.8
           Copper
GP2

247
215
268
 NSW

 256
 211
 240
 GP2

 260
 236C
  NC5
NSW

277
223
238
Mean
CV (%)
243
10.9
236
9.8
248
6.9
246
11.2
  Tests performed by George Morrison and Glen Modica, ERL-N, USEPA,
  Narragansett, RI.
  Three replicate exposure chambers with 10-15 larvae per treatment.
  Adults collected in the field.
  For a discussion of the precision of data from chronic toxicity tests
  see Section 4, Quality Assurance.                     !
  NC= No linear interpolation estimate could be calculated from the data,
  since none of the group response means were less than 50 percent of the
  control response mean.
                                   221

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                                  SECTION 14

                                  TEST METHOD

                      MYSID, HYSIDOPSIS BAHIA, SURVIVAL,
                          GROWTH, AND FECUNDITY TEST
                                 METHOD 1007.0
14.1  SCOPE AND APPLICATION

14.1.1  This method, adapted in part from USEPA (1987d), estimates the chronic
toxicity of effluents and receiving waters to the mysid, Mysidopsis bahia,
during a seven-day, static renewal exposure.  The effects include the
synergistic, antagonistic, and additive effects of all the chemical, physical,
and additive components which adversely affect the physiological and
biochemical functions of the test organisms.

14.1.2  Daily observations on mortality make it possible to also calculate
acute toxicity for desired exposure periods (i.e., 24-h, 48-h, 96-h LCSOs).

14.1.3  Detection limits of the toxicity of an effluent or pure substance are
organism dependent.

14.1.4  Brief excursions in toxicity may not be detected using 24-h composite
samples.  Also, because of the long sample collection period involved in
composite sampling and because the test chambers are not sealed, highly
volatile and highly degradable toxicants present in the source may not be
detected in the test.

14.1.5  This test is commonly used in one of two forms:  (1) a definitive
test, consisting of a minimum of five effluent concentrations and a control,
and (2) a receiving water test(s), consisting of one or more receiving water
concentrations and a control.

14.2  SUMMARY OF METHOD

14.2.1  Mysidopsis bahia 7-day old juveniles are exposed to different
concentrations of effluent, or to receiving water in a static system, during
the period of egg development. The test endpoints are survival, growth
(measured as dry weight), and fecundity (measured as the percentage of females
with eggs in the oviduct and/or brood pouch).

14.3  INTERFERENCES

14.3.1  Toxic substances may be introduced by contaminants in dilution water,
glassware, sample hardware, and testing equipment (see Section 5, Facilities,
Equipment, and Supplies).

14.3.2  Improper effluent sampling and handling may adversely affect test
results (see Section 8, Effluent and Receiving Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests).

                                      222

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14.3.3  The test results can be confounded by (1) the presence of pathogenic
and/or predatory organisms in the dilution water, effluent, and receiving
water, (2) the condition of the brood stock from which the test animals were
taken, (3) the amount and type of natural food in the effluent, receiving
water, or dilution water, (4) nutritional value of the brine shrimp, Artemia
nauplii, fed during the test, and (5) the quantity of brine shrimp, Artemia
nauplii, or other food added during the test, which may sequester metals and
other toxic substances, and lower the DO.

14.4  SAFETY                                              \

14.4.1  See Section 3, Health and Safety.

14.5  APPARATUS AND EQUIPMENT                             I

14.5.1  Facilities for holding and acclimating test organisms.

14.5.2  Brine shrimp, Artemia, culture unit -- see Subsection 14.6.12 below
and Section 4, Quality Assurance.                         ;

14.5.3  Mysid, Mysidopsis bahia, culture unit -- see Subsection 6 below.  This
test requires a minimum of 240 7-day old (juvenile) mysids.  It is preferable
to obtain the test organisms from an in-house culture unit.  If it is not
feasible to culture mysids in-house, juveniles can be obtained from other
sources, if shipped in well  oxygenated saline water in insulated containers.

14.5.4  Samplers -- automatic sampler, preferably with sample cooling
capability, that can collect a 24-h composite sample of 5 L.

14.5.5  Environmental chamber or equivalent facility with temperature control
(26 ± 1°C).
                                                          I
                                                          I
14.5.6  Water purification system -- Millipore Milli-Q®,  doionized water or
equivalent.
                                                          i

14.5.7  Balance -- Analytical, capable of accurately weighing to 0.00001 g.

14.5.8  Reference weights, Class S -- for checking performance of balance.
Weights should bracket the expected weights of the weighing pans and weighing
pans plus organisms.

14.5.9  Drying oven -- 50-105°C range, for drying organisms.

14.5.10  Desiccator -- for holding dried organisms.

14.5.11  Air pump -- for oil-free air supply.

14.5.12  Air lines,  and air  stones -- for aerating cultures, brood chambers,
and holding tanks, and supplying air to test solutions with low DO.

14.5.13  Meters,  pH and DO -- for routine physical  and chemical  measurements.
                                                 -
                            1
                                      223

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14.5.14  Tray -- for test vessels; approximately 90 X 48 cm to hold 56
vessels.

14.5.15  Standard or micro-Winkler apparatus -- for determining DO and
checking DO meters.

14.5.16  Dissecting microscope (350-400X magnification) -- for examining
organisms in the test vessels to determine their sex and to check for the
presence of eggs in the oviducts of the females.

14.5.17  Light box -- for illuminating organisms during examination.

14.5.18  Refractometer or other method -- for determining salinity.

14.5.19  Thermometers, glass or electronic, laboratory grade -- for measuring
water temperatures.

14.5.20  Thermometers, bulb-thermograph or electronic-chart type -- for
continuously recording temperature.

14.5.21  Thermometer, National Bureau of Standards Certified (see USEPA Method
170.1, USEPA, 1979b) -- to calibrate laboratory thermometers.

14.5.22  Test chambers -- 200 ml borosilicate glass beakers or non-toxic 8 oz
disposable plastic cups (manufactured by Falcon Division of Becton, Dickinson
Co., 1950 Williams Dr., Oxnard, CA 93030) or other similar containers.
Forty-eight (48) test vessels are required for each test (eight replicates at
each of five effluent concentrations and a control).  To avoid potential
contamination from the air and excessive evaporation of test solutions during
the test, the chambers should be covered with safety glass plates or sheet
plastic (6 mm thick).

14.5.23  Beakers or flasks -- six, borosilicate glass or non-toxic
plasticware, 2000 mL for making test solutions.

14.5.24  Wash bottles -- for deionized water, for washing organisms from
containers and for rinsing small glassware and instrument electrodes and
probes.

14.5.25  Volumetric flasks and graduated cylinders -- Class A, borosilicate
glass or non-toxic plastic labware, 50-2000 ml for making test solutions.

14.5.26  Separatory funnels, 2-1 -- Two-four for culturing Artemia.

14.5.27  Pipets, volumetric -- Class A, 1-100 ml.

14.5.28  Pipets, automatic -- adjustable, 1-100 ml.

14.5.29  Pipets, serological -- 1-10 ml, graduated.

14.5.30  Pipet bulbs and fillers -- PROPIPET®, or equivalent.
                                      224

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 14.5.31  Droppers, and glass tubing with fire polished edges, 4 mm ID -- for
 transferring organisms.

 14.5.32  Forceps -- for transferring organisms to weighing pans.
                                                                 ,
 14.5.33  NITEX® or stainless steel  mesh sieves (< 150 //m, 500-1000 /j.m, 3-5 mm)
 -- for concentrating organisms.  (NITEX® is available from Sterling Marine
 Products,  18 Label Street,  Montclair,  NJ 07042; 201-783-9800).

 14.5.34  Depression glass slides or depression spot plates -- two, for
 observing  organisms.

 14.6  REAGENTS AND CONSUMABLE MATERIALS

 14.6.1  Sample containers -- for sample shipment and storage (see Section 8,
 Effluent and Receiving Water Sampling,  Sample Handling,  and Sample Preparation
 for Toxicity Tests).

 14.6.2  Data sheets (one set per test)  --  for data recording (Figures 14,  15,
 and 16).

 14.6.3  Tape,  colored -- for labelling  test chambers.      !

 14.6.4  Markers,  waterproof --  for  marking containers, etc,
                                                           j
 14.6.5  Weighing  pans,  aluminum --  to determine the  dry  weight of organisms.

 14.6.6  Buffers,  pH 4,  pH 7,  and pH 10  (or as per instructions of instrument
 manufacturer)  --  for standards  and  calibration check (see USEPA Method 150  1
 USEPA,  1979b).

 14.6.7 Membranes  and filling solutions  --  for dissolved  oxygen  probe (see
 USEPA  Method 360.1,  USEPA,  1979b),  or reagents for modified  Winkler analysis.

 14.6.8  Laboratory quality  assurance samples  and  standards  --  for  the above
 methods.

 14.6.9  Reference  toxicant  solutions --  see Section  4, Quality Assurance.

 14.6.10  Reagent water  -- defined as distilled or deionized  water  that does
 not contain substances which are toxic to the  test organisms  (see  Section 5,
 Facilities, Equipment, and  Supplies).                      j

 14.6.11' Effluent,  receiving water, and  dilution  water --see  Section  7,
 Dilution Water, and  Section 8,  Effluent  and Receiving Water  Sampling, Sample
 Handling, and Sample  Preparation for Toxicity  Tests.  Dilution water
 containing organisms  that might prey upon or otherwise interfere with the test
 organisms should be  filtered through a fine mesh  net (with 150 u.m  or  smaller
 openings).

 14.6.11.1  Saline test and dilution water -- The  salinity oif the test water
must be in the range of 20% to 30%o.  The salinity should vary by no more

                                     225

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than ± 2%o among the chambers on a given day.  If effluent and receiving water
tests are conducted concurrently, the salinities of these tests should be
similar.

14.6.11.2  The overwhelming majority of industrial and sewage treatment
effluents entering marine and estuarine systems contain little or no
measurable salts.  Exposure of mysids to these effluents will require
adjustments in the salinity of the test solutions.  It is important to
maintain a constant salinity across all treatments.  In addition, it may be
desirable to match the test salinity with that of the receiving water.  Two
methods are available to adjust salinities -- a hypersaline brine (HSB)
derived from natural seawater or artificial sea salts.

14 6.11.3  HSB has several advantages that make it desirable for use in
toxicity testing.  It can be made from any high quality, filtered seawater by
evaporation, and can be added to the effluent or to deionized water to
increase the salinity.  Brine derived from natural seawater contains the
necessary trace metals, biogenic colloids, and some of the microbial
components necessary for adequate growth, survival, and/or reproduction of
marine  and estuarine organisms,  and may be stored for prolonged periods
without any apparent degradation.  However,  if 100%o HSB is used as a diluent,
the  maximum concentration of effluent that can be tested is 80% effluent at
30%o salinity  and  70% effluent  at 30%  salinity.

14 6 11 3  1  The ideal  container for making  brine from natural seawater  is one
that (1) has a high  surface to  volume ratio,  (2)  is made of  a  non-corrosive
material,  and  (3)  is easily cleaned  (fiberglass containers are ideal).
Special care should  be  used to  prevent  any toxic  materials from  coming  in
contact with the seawater being used to generate  the  brine.   If  a heater is
immersed directly  into  the  seawater, ensure  that  the  heater  materials do not
corrode or leach any substances that would contaminate the brine.   One
successful  method  used  is a thermostatically controlled  heat  exchanger  made
from fiberglass.   If aeration  is used,  only  oil-free  air compressors  should be
used to prevent contamination.

 14.6.11.3.2  Before  adding  seawater  to  the  brine  generator,  thoroughly  clean
the  generator, aeration supply tube,  heater,  and  any  other materials  that  will
 be  in direct  contact with the  brine.   A good quality  biodegradable  detergent
 should be  used,  followed by several  (at least three)  thorough deionized water
 rinses.

 14.6.11.3.3  High  quality (and preferably high salinity) seawater should be
 filtered to at least 10 ^m  before placing into the brine generator.  Water
 should be collected on an incoming tide to minimize the possibility of
 contamination.

 14.6.11.3.4  The temperature of the seawater is increased slowly to 40°C.   The
 water should be aerated to prevent temperature stratification and to increase
 water evaporation.  The brine should be checked daily (depending on the volume
 being enerated) to ensure that the salinity does not exceed 100%o and that.the
 temperature does not exceed 40°C.  Additional seawater may be added to the
 brine to obtain the volume of brine required.
                                       226

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14.6.11.3.5  After the required salinity is attained, the HSB should be
filtered a second time through a 1 /im filter and poured directly into portable
containers (20-L cubitainers or polycarbonate water cooler jugs are suitable).
The containers should be capped and labelled with the date the brine was
generated and its salinity.  Containers of HSB should be s;tored in the dark
and maintained under room temperature until used.

14.6.11.3.6  If a source of HSB is available, test solutions can be made by
following the directions below.  Thoroughly mix together the deionized water
and HSB before mixing in the effluent.
                                                          I
14.6.11.3.7  Divide the salinity of the HSB by the expected test salinity to
determine the proportion of deionized water to brine.  For example, if the
salinity of the brine is 100%o and the test is to be conducted at 20%o, 100%o
divided by 20%o = 5.0.  The proportion of brine is 1 part in 5 (one part brine
to four parts deionized water).  To make 1 L of seawater at 20%o salinity from
a HSB of 100%o, 200 ml of brine and 800 ml of deionized water are required.

14.6.11.3.8  Table 2 illustrates the composition of 1800 ml test solutions at
20%o if they are made by combining effluent (0%o), deioni2f.ed water and HSB of
100%o (only).  The volume (ml) of brine required is determined by using the
amount calculated above.  In this case, 200 ml of brine is; required for 1 L;
therefore, 360 ml would be required for 1.8 L of solution.  The volumes of HSB
required are constant.  The volumes of deionized water are determined by
subtracting the volumes of effluent and brine from the total volume of
solution:  1800 ml - ml effluent - ml brine = ml deionized water.

14.6.11.4  Artifical sea salts:  FORTY FATHOMS® brand sea salts (Marine
Enterprises, Inc., 8755 Mylander Lane, Baltimore, MD 21204; 301-321-1189) have
been used successfully to culture and perform life cycle tests with mysids
(Home, et al., 1983; ASTM, 1993) (see Section 7, Dilution Water).  HW
Marinemix® (Hawaiian Marine Imports, Inc., P.O. Box 218687, Houston, TX 77218;
713-492-7864 sea salts have been used successfully to culture mysids and
perform the mysid toxicity test (USEPA Region 6 Houston Laboratory; EMSL-
Cincinnati).  In addition, a slightly modified version of the GP2 medium
(Spotte et al., 1984) has been successfully used to perform the mysid
survival, growth, and fecundity test (Table 1).
                                                          i
14.6.11.4.1  Synthetic sea salts are packaged in plastic bags and mixed with
deionized water or equivalent.  The instructions on the package of sea salts
should be followed carefully, and the salts should be mixed in a separate
container -- not in the culture tank.  The deionized water used in hydation
should be in the temperature range of 21-26°C.  Seawater made from artificial
sea salts is conditioned (Spotte, 1973; Spotte, et al., 1984; Bower, 1983)
before it is used for culturing or testing.  After adding the water, place an
airstone in the container, cover, and aerate the solution mildly for 24 h
before use.

14.6.11.4.2  The GP2 reagent grade chemicals (Table 1) should be mixed with
deionized (DI) water or its equivalent in a container other than the culture
or testing tanks.  The deionized water used for hydration should be between
21-26°C.  The artificial seawater must be conditioned (aerated) for 24 h
                                                          i
                                      227

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  TABLE 1.    REAGENT GRADE CHEMICALS USED IN THE PREPARATION OF GP2
              ARTIFICIAL SEAWATER FOR THE MYSID, MYSIDOPSIS BAHIA, TOXICITY
              TEST1'*'*
Compound
NaCl
Na2S04
KC1
KBr
Na2B407 • 10 H20
MgCl2 • 6 H20
CaCl2 • 2 H20
SrCl2 • 6 H20
NaHC03
1 Modified GP2 from Spotte
™ Tl* s* >«nvif>t4**i4*ii«>\M4* <•»••» T 4« <•» ** *^f
Concentration
(9/L)
21.03
3.52
0.61
0.088
0.034
9.50
1.32
0.02
0.17
et al. (1984).
•1 y'ttf'tMSt/tM^XM'N^-vrVMf* I.I
Amount (g)
Required for
20L
420.6
70.4
12.2
1.76
0.68
190.0
26.4
0.400
3.40
j-ivtsN 4- •* \ff\v\ <£%A/tm 1 1C CD A
      (1990b).  The  salinity is  30.89  g/L.
      GP2  can  be diluted  with deionized  (DI)  water  to  the  desired  test
      salinity.
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 NaHC03 in 500 mL of
deionized water.  Add 2.5 mL of this stock solution for each liter of the GP2
artificial seawater.

14.6.12  BRINE SHRIMP, ARTEMIA, NAUPLII -- for feeding cultures and test
organisms.

14.6.12.1  Newly hatched Artemia nauplii are used for food for the stock
cultures and test organisms.  Although there are many commercial sources of
brine shrimp cysts, the Brazilian or Colombian strains are preferred because
the supplies examined have had low concentrations of chemical residues and

                                      228

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  TABLE 2.    QUANTITIES OF EFFLUENT, DEIONIZED WATER, AND HYPERSALINE BRINE
              (100%o) NEEDED TO PREPARE 1800 ML VOLUMES OF TEST SOLUTION
              WITH A SALINITY OF 20%o
     Effluent    Volume  of    Volume of       Volume  of
  Concentration   Effluent    Deionized      Hypersaline      Total  Volume
      (%)          (0%o)         Water            Brine            (mL)
                   (mL)          (ml)             (mL)
    80          1440              0             360             1800

    40           720            720             360             1800

    20           360           1080             360        i     1800

    10           180           1260             360             1800

     5            90           1350             360        i     1800

  Control         0            1440             360        i,     1800
                                                          i


                                                          I

  Total        2790           5850            2160        t    10800
produce nauplii of suitably small size.  For commercial sources of brine
shrimp, Artemia, cysts, see Table 2 of Section 5, Facilities, Equipment, and
Supplies); and Section 4, Quality Assurance.

14.6.12.2  Each new batch of Artemia cysts must be evaluated for size
(Vanhaecke and Sorgeloos, 1980, and Vanhaecke et al.,  1980) and nutritional
suitability (Leger, et al., 1985, Leger, et al., 1986) against known suitable
reference cysts by performing a side-by-side larval growth test using the
"new" and "reference" cysts.  The "reference" cysts used in the suitability
test may be a previously tested and acceptable batch  of cysts, 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 Artemis cycts should not be
used if the concentration of total organic chlorine exceeds 0.15 ^g/g wet

                                      229

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weight or the total concentration of organochlorine pesticides plus PCBs
exceeds 0.30 /*g/g wet weight (For analytical methods see USEPA, 1982).

14.6.12.2.1  Artemia nauplii are obtained as follows:

   1.   Add 1 L of seawater, or an aqueous  uniodized salt  (NaCl) solution
        prepared with 35 g  salt or artificial sea salts to 1 L of deionized
        water, to a 2-L separatory funnel,  or equivalent.
   2.   Add 10 ml Artemia cysts to the separatory funnel and aerate for 24 h
        at 27°C.  Hatching  time varies with  incubation temperature and the
        geographic strain of Artemia used (see USEPA, 1985a; USEPA, 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.  To prevent
        mortality, do not leave the concentrated nauplii at the bottom of the
        funnel more than 10 min without aeration.
   4.   Drain the nauplii into a beaker or  funnel fitted with a < 150 ^m
        NITEX® or stainless steel screen, and rinse with seawater or
        equivalent before use.

14.6.12.3  Testing Artemia  nauplii as food for toxicity test organisms.

14.6.12.3.1  The primary criteria for acceptability of each new supply of
brine shrimp, cysts is adequate survival, growth, and reproduction of the
mysids.  The mysids used to evaluate the acceptability of the brine shrimp
nauplii must be of the same geographical origin and stage of development (7
days old) as those used routinely in the toxicity tests.  Sufficient data to
detect differences in survival and growth should be obtained by using eight
replicate test chambers, each containing 5 mysids, for each type of food.

14.6.12.3.2  The feeding rate and frequency, test vessels,  volume of control
water, duration of the test, and age of the Artemia nauplii at the start of
the test, should be the same as used for the routine toxicity tests.

14.6.12.3.3  Results of the brine shrimp, Artemia, nauplii  nutrition assay,
where there are only two treatments, can be evaluated statistically by use of
a t test.  The "new" food is acceptable if there are no statistically
significant differences in  the survival, growth, and reproduction of the
mysids fed the two sources  of nauplii.

14.6.13  TEST ORGANISMS, Mysidopsis bahia (see Rodgers et al., 1986 and USEPA,
1993a for information on mysid ecology).

14.6.13.1  Brood Stock

14.6.13.1.1  To provide an  adequate supply of juveniles for a test, mysid,
Mysidopsis bahia, cultures  should be started at least four weeks before the
test animals are needed.  At least 200 mysids, Mysidopsis bahia, should be
placed in each culture tank to ensure that 1500 to 2000 animals will be
available by the time preparations for a test are initiated.


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14.6.13.1.2  Mysids, Mysidopsis bahia, may be shipped or otherwise transported
in polyethylene bottles or CUBITAINERS®.  Place 50 animals in 700 ml of
seawater in a 1-L shipping container.  To control bacterial growth and prevent
DO depletion during shipment, do not add food.  Before closing the shipping
container, oxygenate the water for 10 min.  The mysids, Mysidopsis bahia, will
starve if not fed within 36 h, therefore, they should be shipped so that they
are not in transit more than 24 h.

14.6.13.1.3  The identification of the Mysidopsis bahia stock culture should
be verified using the key from Heard (1982), Price (1978), Price, (1982),
Stuck et al. (1979a), and Stuck et al.-(1979b).  Records of the verification
should be retained along with a few of the preserved specimens.

14.6.13.1.4  Glass aquaria (120- to 200-L) are recommended for cultures.
Other types of culture chambers may also be convenient.  Three or more
separate cultures should be maintained to protect against loss of the entire
culture stock in case of accident, low DO, or high nitrite levels, and to
provide sufficient numbers of juvenile mysids, Mysidopsis bahia, for toxicity
tests.  Fill the aquaria about three-fourths full of seawater.  A flow-through
system is recommended if sufficient natural seawater is available, but a
closed, recirculating or static renewal system may be used.if proper water
conditioning is provided and care is exercised to keep the pH above 7.8 and
nitrite levels below 0.05 mg/L.

14.6.13.1.5  Standard aquarium undergravel filters should be used with either
the flow-through or recirculating system to provide aeration and a current
conducive to feeding (Gentile et al., 1983). The undergravel filter is covered
with a prewashed, coarse (2-5 mm) dolomite substrate, 2.5 cm deep for
flow-through cultures or 10 cm deep for recirculating cultures.

14.6.13.1.6  The recirculating culture system is conditioned as follows:

   1.    After the dolomite has been added, the filter is attached to the air
        supply and operated for 24 h.
   2.    Approximately 4 L of seed water obtained from a successfully
        operating culture is added to the culture chamber,
   3.    The nitrite level is checked daily with an aquarium test kit or with
        EPA Method 354.1 (USEPA, 1979b).
   4.    Add about 30 ml of concentrated Artemia nauplii every other day until
        the nitrite level reaches at least 2.0 mg/L. The nitrite will
        continue to rise for several days without adding more Artemia nauplii
        and will then slowly decrease to less than 0.05 mg/L,,
   5.    After the nitrite level falls below 0.05 mg/L, add another 30 mL of
        Artemia nauplii concentrate and check the nitrite concentration every
        day.
   6.    Continue this cycle until the addition of Artemia nauplii does not
        cause a rise in the nitrite concentration.  The culture chamber is
        then conditioned and is ready to receive mysids.
   7.    Add only a few (5-20) mysids at first, to determine if conditions are
        favorable. If these mysids are still doing well after a week, several
        hundred more can be added.
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14.6.13.1.7  It is important to add enough food to keep the adult animals from
cannibalizing the young, but not so much that the DO is depleted or that there
is a buildup of toxic concentrations of ammonia and nitrite.  Just enough
newly-hatched Artemia nauplii are fed twice a day so that each feeding is
consumed before the next feeding.

14.6.13.1.8  Natural seawater is recommended as the culture medium, but HSB
may be used to make up the culture water if natural seawater is not available.
EMSL-Cincinnati has successfully used FORTY FATHOMS® artificial sea salts for
culturing and toxicity tests of mysids, and USEPA, Region 6 has used HW
MARINEMIX® artificial sea salts.

14.6.13.1.9  Mysids, Mysidopsis bahia, should be cultured at a temperature of
26 ± 1°C. No water temperature control equipment is needed if the ambient
laboratory temperature remains in the recommended range, and if there are no
frequent, rapid, large temperature excursions in the culture room.

14.6.13.1.10  The salinity should be maintained at 30 ± 2%o, or at a lower
salinity (but not less than 20%o) if most of the tests will be conducted at a
lower salinity.

14.6.13.1.11  Day/n'ight cycles prevailing in most laboratories will provide
adequate illumination for normal growth and reproduction. A 16-h/8-h day/night
cycle in which the light is gradually increased and decreased to simulate dawn
and dusk conditions, is recommended.

14.6.13.1.12  Mysid, Mysidopsis bahia, culture may suffer if DOs fall below 5
mg/L for extended periods.  The undergravel filter will usually provide
sufficient DO.  If the DO drops below 5 mg/L at 25°C and 30%o, additional
aeration should be provided.  Measure the DO in the cultures daily the first
week and then at least weekly thereafter.

14.6.13.1.13  Suspend a clear glass or plastic panel over the cultures, or use
some other means of excluding dust and dirt, but leave enough space between
the covers and culture tanks to allow circulation of air over the cultures.

14.6.13.1.14  If hydroids or worms appear in the cultures, remove the mysids
and clean the chambers thoroughly, using soap and hot water.  Rinse once with
acid (10% HC1) and three times with distilled or deionized water.  Mysids with
attached hydroids should be discarded.  Those without hydroids should be
transferred by hand pipetting into three changes of clean seawater before
returning them to the cleaned culture chamber.  To guard against predators,
natural seawater should be filtered through a net with 30 /j,m mesh openings
before entering the culture vessels.

14.6.13.1.15  Mysids, Mysidopsis bahia, are very sensitive to low pH and
sudden changes in temperature.  Care should be taken to maintain the pH at 8.0
± 0.3, and to limit rapid changes in water temperature to less than 3°C.

14.6.13.1.1(5  Mysids, Mysidopsis bahia, should be handled carefully and as
little as possible so that they are not unnecessarily stressed or injured.
They should be transferred between culture chambers with long handled cups

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with netted bottoms.  Animals should be transferred to the test vessels with a
large bore pipette (4-mm), taking care to release the animals under the
surface of the water.  Discard any mysids that are injured during handling.

14.6.13.1.17  Culture Maintenance (Also See USEPA, 1993a)

14.6.13.1.17.1  Cultures in closed, recirculating systems are fed twice a day.
If no nauplii are present in the culture chamber after four hours, the amount
of food should be increased slightly.  In flow-through systems, excess food
can be a problem by promoting bacterial growth and low dissolved oxygen.

14.6.13.1.17.2  Careful culture maintenance is essential.  The organisms
should not be allowed to become too crowded.  The cultures should be cropped
as often as necessary to maintain a density of about 20 mysids per liter.  At
this density, at least 70% of the females should have eggs in their brood
pouch.  If they do not, the cultures are probably under stress, and the cause
should be found and corrected.  If the cause cannot be found, it may be
necessary to restart the cultures with a clean culture chamber, a new batch of
culture water, and clean gravel.

14.6.13.1.17.3  In closed, recirculating systems, about one third of the
culture water should be replaced with newly prepared seawater every week.
Before siphoning the old media from the culture, it is recommended that the
sides of the vessel be scraped and the gravel carefully turned over to prevent
excessive buildup of algal growth.  Twice a year the mysids should be removed
from the recirculating cultures, the gravel rinsed in clean seawater, the
sides of the chamber washed with clean seawater, and the gravel and animals
returned to the culture vessel with newly conditioned seawater.  No detergent
should be used, and care should b.e taken not to rinse all the bacteria from
the gravel.

14.6.13.2  Test Organisms

14.6.13.2.1  The test  is begun with 7-day-old juveniles.  To have the test
animals available and  acclimated to test conditions at the start of the test,
they must be obtained  from the stock culture eight days in advance of the
test.  Whenever possible, brood stock should be obtained from cultures having
similar salinity, temperature, light regime, etc., as are to be used in the
toxicity test.

14.6.13.2.2  Eight days before the test is to start, sufficient gravid females
are placed in brood chambers.  Assuming that 240 juveniles are needed for each
test, approximately half this number (120) of gravid females should be
transferred to brood chambers.  The mysids are removed from the culture tank
with a net or netted cup and placed in 20-cm diameter finger bowls.  The
gravid females are transferred from the finger bowls to the brood chambers
with a large-bore pipette or, alternatively, are transferred by pouring the
contents of the finger bowls into the water in the brood chambers.

14.6.13.2.3  The mysid juveniles may be collected for the toxicity tests by.
transferring gravid females from the stock cultures to netted  (1000 /^m)
flow-through containers  (Figure 1) held within 4-1 glass, wide-mouth

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                                   INFLOW
                                            OUTFLOW
                                             NETTED
                                             CHAMBER
                                        SEPARATORY
                                          FUNNEL
                                           NETTED
                                          CHAMBER
                                         CULTURE DISH
Figure 1.  Apparatus (brood chamber) for collection of juvenile mysids,
           Nysidopsis bahia.  From USEPA (1987d).


separatory funnels.  Newly released juveniles.can pass through the netting,
whereas the females are retained.  The gravid females are fed newly hatched
Artemia nauplii, and are held overnight to permit the release of young.  The
juvenile mysids are collected by opening the stopcock on the funnel and
collecting them in another container from which they are transferred to
holding tanks using a wide bore (4 mm ID) pipette.  The brood chambers usually
require aeration to maintain sufficient DO and to keep the food in suspension.

14.6.13.2.4  The temperature in the brood chamber should be maintained at the
upper acceptable culture limit (26 - 27°C), or 1°C higher than the cultures,
to encourage faster brood release.  At this temperature, sufficient juveniles
should be produced for the test.

14.6.13.2.5  The newly released juveniles (age = 0 days) are transferred to
20-L glass aquaria (holding vessels) which are gently aerated.  Smaller
holding vessels may be used, but the density of organisms should not exceed 10
mysids per liter.  The test animals are held in the holding vessel for six
days prior to initiation of the test.  The holding medium is renewed every
other day.

14.6.13.2.6  During the holding period, the mysids are acclimated to the
salinity at which the test will be conducted, unless already at that salinity.
The salinity should be changed no more than 2%o per 24 h to minimize stress on
the juveniles.

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14.6.13.2.7  The temperature during the holding period is critical to mysid
development, and must be maintained at 26 ± 1°C.  If the temperature cannot be
maintained in this range, it is advisable to hold the juveniles an additional
day before beginning the test.

14.6.13.2.8  During the holding period, just enough newly-hatched Artemia
nauplii are fed twice a day (a total of at least 150 nauplii per mysid per
day) so that some food is constantly present.
                                                          i
14.6.13.2.9  If the test is to be performed in the field, the juvenile mysids,
Mysidopsis bahia, should be gently siphoned into 1-L polyethylene wide-mouth
jars with screw-cap lids filled two-thirds full with clean seawater from the
holding tank.  The water in these jars is aerated for 10 min, and the jars are
capped and packed in insulated boxes for shipment to the test site.  Food
should not be added to the jars to prevent the development of excessive
bacterial growth and a reduction in DO.
                     .
14.6.13.2.10  Upon arrival at the test site (in less than 24 h) the mysids,
Mysidopsis bahia, are gently poured from the jars into 20-cm diameter glass
culture dishes.  The jars are rinsed with salt water to dislodge any mysids
that may adhere to the sides.  If the water appears milky, siphon off half of
it with a netted funnel  (to avoid siphoning the mysids) and replace with clean
salt water of the same salinity and temperature.  If no Artemia nauplii are
present in the dishes, feed about 150 Artemia nauplii per mysid.

14.7  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
                                                          I •
14.9.1  See Section 4, Quality Assurance.

14.9.2  The reference toxicant recommended for use with the mysid 7-day test
is copper sulfate or sodium dodecyl sulfate.

14.10  TEST PROCEDURES
                                                          l
                                                          I
14.10.1  TEST DESIGN
                                                                        '
14.10.1.1  The test consists  of at  least five effluent concentrations plus a
site water control and a reference  water treatment  (natural seawater or
seawater made up from hypersaline brine, or equivalent).

14.10.1.2  Effluent concentrations  are expressed as percent effluent.


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14.10.1.3  Eight replicate test vessels, each containing 5 to 7 day old
animals, are used per effluent concentration and control.

14.10.2  TEST SOLUTIONS

14.10.2.1  Receiving waters

14.10.2.1.1  The sampling point(s) is determined by the objectives of the
test.  At estuarine and marine sites, samples are usually collected at mid-
depth.  Receiving water toxicity is determined with samples used directly as
collected or with samples passed through a 60 urn NITEX® filter and compared
without dilution, against a control.  Using eight replicate chambers per test,
each containing 150 ml, and 400 ml for chemical analysis, would require
approximately 1.6 L or more of sample per test per day.

14.10.2.2  Effluents

14.10.2.2.1  The selection of the effluent test concentrations should be based
on the objectives of the study.  A dilution factor of 0.5 is commonly used.  A
dilution factor of 0.5 provides precision of ± 100%, and testing of
concentrations between 6.25% and 100% effluent using only five effluent
concentrations (6.25%, 12.5%, 25%, 50%, and 100%).  Test precision shows
little improvement as dilution factors are increased beyond 0.5 and declines
rapidly if smaller dilution factors are used.  Therefore, USEPA recommends the
use of the ^ 0.5 dilution factor.  If 100%o MSB is used as a diluent, the
maximum concentration of effluent that can be tested will be 80% at 20%o and
70% at 30%o salinity.

14.10.2.2.2  If the effluent is known or suspected to be highly toxic, a lower
range of effluent concentrations should be used (such as 25%, 12.5%, 6.25%,
3.12%, and 1.56%).  If high mortality is observed during the first l-to-2 h of
the test, additional dilutions at the lower range of effluent concentrations
should be added.

14.10.2.2.3  The volume of effluent required for daily renewal of eight
replicates per concentration for five concentrations of effluent and a
control, each containing 150 ml of test solution, is approximately 1200 ml.
Prepare enough test solution (approximately 1600 ml) at each effluent
concentration to provide 400 ml additional volume for chemical analyses.

14.10.2.2.4  Just prior to test initiation (approximately 1 h),  the
temperature of a sufficient quantity of the sample to make the test solutions
should be adjusted to the test temperature (26 ± 1°C) and maintained at that
temperature during the addition of dilution water.

14.10.2.2.5  Higher effluent concentrations (i.e., 25%, 50%, and 100%) may
require aeration to maintain adequate dissolved oxygen concentrations.
However, if one solution is aerated, all concentrations must be aerated.
Aerate effluent as it warms and continue to gently aerate test solutions in
the test chambers for the duration of the test.
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14.10.2.2.6  Effluent dilutions should be prepared for all replicates in each
treatment in one flask to minimize variability among the replicates.  The test
chambers (cups) are labelled with the test concentration and replicate number.
Dispense 150 ml of the appropriate effluent dilution to each test chamber.

14.10.2.3  Dilution Water

14.10.2.3.1  Dilution water may be uncontaminated natural seawater  (receiving
water), HSB prepared from natural seawater, or artifical seawater prepared
from FORTY FATHOMS® or GP2 sea salts (see Table 1 and Section 7, Dilution
Water).  Other artifical sea salts may be used for culturing mysid  and for the
survival, growth, and fecundity test if the control criteria for acceptability
of test data are satisfied.

14.10.3  START OF THE TEST                                \

14.10.3.1  The test should begin as soon as possible, preferably within 24 h
after  sample collection.  The maximum holding time following retrieval of the
sample from the sampling device should not exceed 36 h for off-site toxicity
tests  unless permission is granted by the permitting authority.  In no case
should the test be started more than 72 h after sample collection (see
Section 8, Effluent and Receiving Water Sampling, Sample Handling,  and Sample
Preparation for Toxicity Tests).
                                                          j
14.10.3.2  Begin the test by randomly placing five animals  (one at  a time) in
each test cup  of each treatment using a large bore (4 mm  ID) pipette (see
Appendix A for an example of randomization).  It  is easier to capture the
animals  if the volume of water in the dish is reduced and the dish  is placed
on a light table.  It is recommended that the transfer pipette  be rinsed
frequently because mysids tend to adhere to the inside surface.

14.10.4  LIGHT,  PHOTOPERIOD, SALINITY AND TEMPERATURE

14.10.4.1  The light quality and  intensity under  ambient  laboratory conditions
are generally  adequate.  Light intensity of 10-20 /iE/m/si or 50 to 100 foot
candles  (ft-c), with a  photoperiod of 16 h light  and 8 h  darkness.   It  is
critical that  the test  water temperature be maintained at 26 ±  1°C.  It  is
recommended that the test water  temperature be continuously recorded.  The
salinity should  vary no more than ±  2%o  among chambers on a given day.   If
effluent and  receiving  water tests are conducted  concurrently,  the  salinities
of these tests should be similar.
                                                          I
 14.10.4.1.1   If  a water bath  is  used to  maintain  the test temperature,  the
water  depth  surrounding the test cups should  be  at  least  2.5 cm deep.

 14.10.4.1.2   Rooms  or  incubators with high volume ventilation  should  be  used
with  caution  because  the volatilization  of the test  solutions  and evaporation
of dilution water may cause wide fluctuations  in  salinity.  Covering the test
 cups  with  clear  polyethylene  plastic may help  prevent  volatilization  and
 evaporation  of the  test solutions.
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 14.10.5   DISSOLVED OXYGEN (DO)  CONCENTRATION

 14.10.5.1  Aeration may affect  the  toxicity of effluents  and  should  be  used
 only as  a last  resort  to maintain a satisfactory  DO.   The DO  should  be
 measured on new solutions at  the start  of the  test  (Day 0)  and  before daily
 renewal  of test solutions on  subsequent days.   The  DO  should  not  fall below
 4.0  mg/L (see Section.8,  Effluent and Receiving Water  Sampling, Sample
 Handling,  and Sample Preparation for Toxicity  Tests).  If it  is necessary to
 aerate,  all  treatments  and the  control  should  be  aerated.   The  aeration rate
 should not exceed  100  bubbles/minute, using a  pipet with  a  1-2 mm orifice,
 such as  a 1-mL  KIMAX®  serological pipet No. 37033,  or  equivalent.  Care should
 be taken to ensure that turbulence  resulting from aeration  does not  cause
 undue stress on the mysid.

 14.10.6   FEEDING

 14.10.6.1   Artemia nauplii  are  prepared as described above.

 14.10.6.2   During  the test, the mysids  in  each  test chamber should be fed
 Artemia  nauplii, (less  than 24-h old),  at  the  rate  of  150 nauplii per mysid
 per  day.   Adding the entire daily ration  at a  single feeding  immediately after
 test solution renewal may result in  a significant DO depression.  Therefore,
 it is preferable to feed  half of the daily ration immediately after  test
 solution renewal,  and the second half 8 -  12 h  later.  Increase the  feeding if
 the  nauplii  are consumed  in less than 4 h.  It  is important that  the nauplii
 be washed  before introduction to the test  chamber.

 14.10.7  DAILY CLEANING  OF TEST  CHAMBERS

 14.10.7.1   Before  the daily renewal  of  test solutions, uneaten and dead
 Artemia, dead mysids and  other  debris are  removed from the  bottom of the test
 chambers with a pipette.   As  much of the uneaten Artemia as possible should be
 removed  from each  chamber to  ensure  that the mysids principally eat new
 hatched  nauplii.   By placing  the test chambers  on a light box, inadvertent
 removal  of  live  mysids  can be greatly reduced because they  can be more easily
 seen.  Any  incidence of removal of live mysids  from the test chambers during
 cleaning, and subsequent  return to the  chambers should be noted in the test
 records.

 14.10.8  OBSERVATIONS DURING  THE TEST

 14.10.8.1   Routine  Chemical and Physical Determinations

 14.10.8.1.1  DO  is  measured at the beginning and end of each 24-h exposure
period in one test  chamber at. each test concentration and in the control.

 14.10.8.1.2  Temperature, pH,  and salinity are measured at the end of each
24-h exposure period in one test chamber at each concentration and in the
control.  Temperature should  also be monitored continuously observed and
recorded daily for  at least two locations in the environmental control  system
or the samples.   Temperature  should  be measured in a sufficient number of test
                                      238

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chambers at least at the end of the test to determine temperature variation in
environmental chamber.

14.10.8.1.3  The pH is measured in the effluent sample each day before new
test solutions are made.

14.10.8.2  Routine Biological Observations

14.10.8.2.1  The number of live mysids are counted and recorded each day when
the test solutions are renewed (Figure 2).  Dead animals and excess food
should be removed with a pipette before test solutions are renewed.

14.10.8.2.2  Protect the mysids from unnecessary distrubance during the test
by carrying out the daily test observations, solution renewals, and removal of
the dead mysids, carefully.  Make sure the mysids remain immersed during the
performance of the above operations.

14.10.9  TEST SOLUTION RENEWAL
      •
14.10.9.1  Before the daily renewal of test solutions, slowly pour off all but
10 mL of the old test medium into a 20 cm diameter culture dish on a light
table.  Be sure to check for animals that may have adhered to the sides of the
test chamber.  Rinse them back into the test cups.  Add 150 mL of new test
solution slowly to each cup.  Check the culture dish for animals that may have
been poured out with the old media, and return them to the test chamber.

14.10.10  TERMINATION OF THE TEST

14.10.10.1  After measuring the DO, pH, temperature, and salinity and
recording survival, terminate the test by pouring off the test solution in all
the cups to a one cm depth and refilling the cups with clean seawater.  This
will keep the animals alive, but not exposed to the toxicant, while waiting to
be examined for sex and the presence of eggs.

14.10.10.2  The live animals must be examined for eggs and the sexes
determined within 12 h of the termination of the test.  If the test was
conducted in the field, and the animals cannot be examined on site, the live
animals should be shipped back to the laboratory for processing.  Pour each
replicate into a labelled 100 mL plastic screw capped jar, arid send to the
laboratory immediately.

14.10.10.3  If the test was conducted in the laboratory, or when the test
animals arrive in the laboratory from the field test site, the test organisms
must be processed immediately while still alive as follows:

14.10.10.3.1  Examine each replicate under a stereomicroscope (240X) to
determine the number of immature.animals, the sex of the mature animals, and
the presence or absence of eggs in the oviducts or brood sacs of the females
(see Figures 3-6). This must be done while the mysids are alive because they
turn opaque upon dying.  This step should not be attempted! by a person who has
not had specialized training in the determination of sex and presence of eggs

                                                          i
                                      239

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TEST:	

START DATE:.

SALINITY:

DAY1

DAY 2

DAYS

DAY 4

DAY 6

DAY 6

DAY 7



DAY 1

DAY 2

DAY 3

DAY 4

DAY 5

DAY 0

DAY?


TRTMT
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP

TRTMT
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP
REP

TEMP















TEMP















SALINITY















SALINITY















D.O.















D.O.















PH















pH















TRTMT















TRTMT















TEMP















TEMP















SALINITY















SALINITY















D.O.















D.O















pH















pH















































Figure 2.  Data form for the mysid, Mysidopsis bahia, water quality
           measurements.  From USEPA (1987d).
                                      240

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            MATURE FEMALE, EGGS IN OVIDUCTS
                         . eyestalk
  antennule
                                                              statocyst
                                                                telson
                                       developing brood sac

                                     oviducts with developing ova
Figure 3.   Mature female, mysid, Mysidopsis bahia, with eggs in oviducts.  From
           USEPA  (1987d).

in the oviduct.   NOTE:  Adult females without eggs in the oviduct  or  brood sac
look like  immature  mysids  (see  Figure 6).
              1 "                                         I
14.10.10.3.2  Record  the number of immatures, males, females  with  eggs  and
females without  eggs  on data sheets  (Figure 7).

14.10.10.3.3  Rinse the mysids  by pipetting them into a small  netted  cup  and
dipping the cup  into  a dish containing deionized water.  Using forceps, place
the mysids from  each  replicate  cup on tared weighing boats and dry at 60°C for
24 h or at 105°C for  at  least 6 h.
                                                        i
14.10.10.3.4  Immediately  upon  removal from the drying oven,  the weighing pahs
were placed in a dessicator until weighed, to prevent absorption of moisture
from the,air. Weigh  to  the nearest  mg.  Record weighing pans and subtract the

                                     241

-------
                MATURE FEMALE, EGGS IN BROOD SAC

                            . eyestalk
                                      carapace
    antennule

                antenna
                                                              statocyst
                                                                telson
                            brood sac with
                         developing embroyos
                                                      uropod
                                        brood sac with
                                        developing embryos
                                        oviducts with developing ova
                                                                   telson

                                                                    uropods
Figure 4.  Mature female  mysid,  Mysidopsis  bahia,  with eggs  in  oviducts  and
           developing embryos in the  brood  sac.   Above:  lateral  view.  Below-
           dorsal view.   From USEPA (1987d).

tare weight to determine the dry weight  of the mysid in each replicate.
Record the weights (Figure 8).   For each test chamber, divide the first  dry
?£&,*,! V5e numker °f ordinal  mys1ds  per  repiicate to determine the average
individual dry weight and record data.   For  the controls also calculate  the
tSt SiSnJaPi •JUrVl^ng."or?1d  1n  Jhe test  Chamber to evaluate if weights  met
test acceptability criteria (see Subsection  14.2).

Jf;l°;?;3A5  PiSCSS  °! alumi™m  foil  d-cm square) or small aluminum weighing
10 mg in weight       Y W619   analyses'  The weighing pans should not  exceed

             Num5er  ea
-------
                          MATURE MALE
                          eyestalk
  antennule
                          K^^^mtm^
               antenna
                                        gonad.
                                             t
                                          pleopods
                                                    uropod "
statocyst
   telson
Figure 5.  Mature male mysid, Mysidopsis  bahia.  From USEPA (1987d).


14.11  SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA

14.11.1  A summary of test conditions and test  acceptability criteria is
listed in Table  3.

14.12  ACCEPTABILITY OF TEST RESULTS
                                                       !
14.12.1  The minimum requirements for an  acceptable test are 80% survival  and
an average weight of at least 0.20 mg/mysid in  the controls.  If fecundity in
the controls is  adequate  (egg production  by 50% of females), fecundity should
be used as a criterion of effect in addition to survival and growth.

14.13  DATA  ANALYSIS

14.13.1  GENERAL
                                                       I
14.13.1.1  Tabulate and summarize the data.  Table 4  presents a sample set of
survival, growth, and fecundity data.

                                     243

-------
                                IMMATURE
                           , eyestalk
                                      carapace
   antennule
                antenna
statocyst
  telson
Figure 6.  Immature mysid, Mysidopsis bahia,  (A)  lateral  view,  (B) dorsal view.
           From USEPA (1987d).
14.13.1.2  The endpoints of the mysid 7-day chronic test are based on the
adverse effects on survival, growth, and egg development.  The LC50, the IC25,
and the IC50 are calculated using point estimation techniques (see Section 9,
Chronic Toxicity Test Endpoints and Data Analysis).  LOEC and NOEC values for
survival, growth, and fecundity are obtained using a hypothesis testing
approach such as Dunnett's Procedure (Dunnett, 1955) or Steel's Many-one Rank
Test (Steel, 1959; Miller, 1981) (see Section 9).  Separate analyses are
performed for the estimation of the LOEC and NOEC endpoints and for the
estimation of the LC50, IC25, and IC50.  Concentrations at which there is no
survival in any of the test chambers are excluded from the statistical
analysis of the NOEC and LOEC for survival, growth, and fecundity, but
included in the estimation of the LC50, IC25, .and IC50.  See the Appendices
for examples of the manual computations, and examples of data input and
program output.

14.13.1.3  The statistical tests described here must be used with a knowledge
of the assumptions upon which the tests are contingent.  The assistance of a
statistician is recommended for analysts who are not proficient in statistics.

                                     244

-------
TEST:
.START DATE:
SAI TNITY:

TREATMENT/
REPLICATE

2





8

2
3

5

7
8

2

*
B
6
7
8
DAY 1
# ALIVE
























DAY 2
* ALIVE
























DAY 3
* ALIVE

























DAY 4
* ALIVE
























DAY 5
* ALIVE
























DAY 6
* ALIVE
























DAY 7
* ALIVE

























FEMALES
W/EGGS
























FEMALES
NO EGGS
























MALES
























IMMATURES
























Figure 7.   Data form for the mysid, Mysidopsis  bahia,  survival  and  fecundity
            data.   From USEPA (1987d).
                                    245

-------
TEST:	
START DATE:.
SALINITY:
TMATMINT/
ItlrUCATC
1
2
I
«
f
a
7
•
t
2
3
4
S
9
7
I
1
2
3
4
t
a
7
t
DAYI
»AUVE
























DAY 2
»AUVE
























DAY 3
»AUVE
























DAY 4
* ALIVE
























DAY 5
* ALIVE
























DAY 8
* ALIVE
























DAY 7
* ALIVE
























FEMALES
W/ECGS
























FEMALES
NO EGGS
























MALES
























IMMATURES
























Figure 7.  Data  form  for the mysid, Mysidopsis  bahia,  survival  and fecundity
           data  (CONTINUED).  From USEPA  (1987d).
                                      246

-------
TEST:	

START DATE:.

SALINITY:
TREATMENT*
REPLICATE
1
2
3
4
C
5
6
7
8
1
2
3
4
1
6
7
8
1
2
3
4
2
5
6
7
8
PAN #
























TARE
WT.
























TOTAL
WT.
























ANIMAL
WT.










t













#OF
ANIMALS




.'



1 .



I

!


1
!





X WT./
ANIMAL
























 Figure 8.  Data
            From
form for the mysid, Mysidopsis bahia, dry weight measurements,
USEPA (1987d).
                                       247

-------
 TEST:	
 START DATE:.
 SALINITY:
Figures. Data^fo™ for  «. n^id, (g»**ps/s -Mi,, dry weight  neasurercents
                                  248

-------
TABLE 3    SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
           THE MYSID, MYSIDOPSIS BAHIA, SEVEN DAY SURVIVAL, GROWTH, AND
           FECUNDITY TEST WITH EFFLUENTS AND RECEIVING WATERS
 1. Test type:
 2. Salinity:

 3. Temperature:
 4. Light quality:
 5. Light intensity:

 6. Photoperiod:

 7. Test chamber:

 8. Test solution  volume:
 9.  Renewal  of test solutions:
 10.  Age of test organisms:
 11.  No. organisms  per test
      chamber:
 12.  No. replicate  chambers
      per concentration:
 13.  No. larvae per concentration:
 14.  Source of food:

 15.  Feeding regime:

 16. Cleaning:
Static renewal
20%o to 30%o (± 2%o of the selected
test salinity)
26 ± 1°C
Ambient laboratory illumination
10-20 ME/m2/s (50-100 ft-c.)
(ambient laboratory "levels)
16 h light, 8 h darkness, with phase
in/out period
8 oz plastic disposable cups, or 400
mL glass beakers
150 mL per  replicate
Daily                i
7 days

5  (minimum)
8  (minimum)
 40 (minimum)
 Newly hatched Artemia nauplii  (less
 than 24 h old)
 Feed 150 24 h old nauplii per mysid
 daily, half after test solution
 renewal and half after 8-12 h.
 Pipette excess food from cups daily
 immediately before test solution
 renewal and feeding,,
                                     249

-------
 TABLE 3.    SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
            THE MYSID, HYSIDOPSIS BAHIA,  SEVEN DAY SURVIVAL,  GROWTH,  AND
            FECUNDITY TEST WITH EFFLUENTS AND RECEIVING WATERS (CONTINUED)
 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:
 None unless DO falls below 4.0 mg/L,
 then gently aerate in all  cups

 Uncontaminated source of natural
 seawater,  deionized water mixed with
 hypersaline brine  or artificial sea
 salts (HW  Marinemix®,  FORTY FATHOMS®,
 GP2  or equivalent)

 Effluents:   Minimum of 5 and a control
 Receiving  waters:  100% receiving water
 or minimum of  5  and a control

 Effluents:   >  0.5  series
 Receiving  waters:   None,  or > 0.5

 7 days

 Survival,  growth,  and  egg  development

 80%  or  greater survival,  average dry
 weight  0.20  mg or greater  in  controls;
 fecundity may be used  if 50% or more
 of females  in controls  produce eggs

 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 for Toxicity Tests,
Subsection 8.5.4)

3 L per day
                                   250

-------
TABLE 4.   DATA FOR MYSIDOPSIS BAHIA  7-DAY  SURVIVAL,  GROWTH, AND FECUNDITY
           TEST1
Treatment
Control







50 ppb
r r






100 ppb







210 ppb







450 ppb







Replicate
Chamber
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
Total
Mysids
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
4
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
No. Total
Al i ve Femal es
4 1
4 2
5 3
5 1
5 2
5 5
5 2
4 3
4 2
5 3
4 3
4 0
5 5
5 2
4 4
5 3
3 3
5 2
5 1
5 2
5 3
3 1
4 4
4 0
5 1
4 2
1 1
4 3
3 1
4 2
4 1
4 3
0 0
1 0
0 0
1 0
0 0
0 0
0 0
2 1
Femal es Mean
w/Eggs Weight
1 0.146
2 0.118
2 0.216
1 0.199
2 0.176
4 0.243
2 0.213
3 0.144
1 0.154
1 0.193
2 0.190
0 0.190
2 0.256
1 0.191
1 0.122
1 0.177
1 0.114
1 0.172
0 0.160
1 0.199
2 0.165
0 0.145
1 0.207
0 0.186
0 0.153
0 0.094
0 0.017
0 0.122
0 0.052
0 0.154
0 0.110
0 0.103
0 - -
0 0.012
0 - -
0 0.002
0 - -
0 - -
0 - -
0 0.081
     Dat.a provided by Lussier, Kuhn and Sewall,  Environment
     Laboratory,  U.S. Environmental Protection Agency,
                                     251
    al Research
N^arreigansett, RI.

-------
 14.13.2   EXAMPLE  OF ANALYSIS  OF  MYSID, MYSIDOPSIS BAHIA, SURVIVAL DATA

 14.13.2.1   Formal  statistical  analysis of the  survival data  is outlined  in
 Figures  9  and  10.  The  response  used  in the  analysis  is the  proportion of
 animals  surviving  in each test or control chamber.  Separate analyses are
 performed  for  the  estimation  of  the NOEC and LOEC endppints  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.

 14.13.2.2   For the case of equal numbers of  replicates across all
 concentrations and the  control,  the evaluation of the NOEC and LOEC endpoints
 is made  via a  parametric test, Dunnett's Procedure, or a nonparametric test,
 Steel's  Many-one Rank Test, on the arc sine  square root transformed data.
 Underlying assumptions  of Dunnett's Procedure, normality and homogeneity of
 variance,  are  formally  tested.   The test for normality is the Shapiro-Milk's
 Test, and  Bartlett's. Test is  used to  test for  homogeneity of variance.   If
 either of  these tests fails, the nonparametric test, Steel's Many-one Rank
 Test, is used  to determine the NOEC and LOEC endpoints.  If the assumptions of
 Dunnett's  Procedure are met, the endpoints are estimated by the parametric
 procedure.

 14.13.2.3   If  unequal numbers  of replicates  occur among the concentration
 levels tested, there are parametric and nonparametric alternative analyses.
 The parametric analysis is a t-test with the Bonferroni adjustment (see
 Appendix D).   The  Wilcoxon Rank Sum Test with the Bonferroni adjustment is the
 nonparametric  alternative.

 14.13.2.4   Probit  Analysis (Finney, 1971; see Appendix 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 method,
 or the Graphical  method may be used (see Appendices H-K).

 14.13.2.5  The proportion of survival  in each replicate must first be
 transformed by the arc sine transformation procedure described in Appendix B.
The raw and transformed data,  means and variances of the transformed
 observations at each concentration including the control are listed in
Table 5.  A plot of the survival  data is provided in Figure 11.
                                      252

-------
                STATISTICAL ANALYSIS OF MYSIDOPSIS BAHIA
                 SURVIVAL, GROWTH, AND FECUNDITY TEST
                                                       i

                      SURVIVAL HYPOTHESIS TESTING
                               SURVIVAL DATA
                            PROPORTION SURVIVING
                                 ARC SINE
                              TRANSFORMATION
                             SHAPIRO-WILK-S TEST
                                                 NON-NORMAL DISTRIBUTION
                  NORMAL DISTRIBUTION
        HOMOGENEOUS
          VARIANCE
                               BARTLETTS TEST
                       HETEROGENEOUS
                          VARIANCE
              EQUAL NUMBER OF
                REPLICATES?
               EQUAL NUMBER OF
                 REPLICATES?
NO
                        YES
    T-TESTWITH
    BONFERRONI
    ADJUSTMENT
        YES
NO
STEEL'S MANY-ONE
   RANK TEST
                                           WILCOXON RANK SUM
                                               TEST WITH
                                         BONFIERRONI ADJUSTMENT
                             ENDPOINT ESTIMATES
                                 NOEC, LOEC
Figure  9.  Flowchart for statistical  analysis of mysid,  Mysidopsis
          survival data by hypothesis testing.

                                    253

-------
              STATISTICAL ANALYSIS OF MYSIDOPSIS BAHIA
               SURVIVAL, GROWTH, AND FECUNDITY TEST

                      SURVIVAL POINT ESTIMATION
MORTALITY DATA
#DEAD
1
r
       TWO OR MORE
    PARTIAL MORTALITIES?
             YES
      IS PROBIT MODEL  '
       APPROPRIATE?
    (SIGNIFICANT X2 TEST)
             YES
 NO
NO
ONE OR MORE
PARTIAL MORTALITIES?
i
YES
r
NO

                              GRAPHICAL METHOD
                                   LC50
PROBIT METHOD L
Af

H
- 	 hi
                             ZERO MORTALITY IN THE
                            LOWEST EFFLUENT CONG.
                           AND 100% MORTALITY IN THE
                           HIGHEST EFFLUENT CONC.?
                            NO
                                      YES
                              SPEARMAN-KARBER
                                  METHOD
                                L.C50AND95%
                                CONFIDENCE
                                  INTERVAL
« '
TRIMMED SPEARMAN-
KARBER METHOD


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

                                  254

-------
o
o
ec
o
u.
Ul
UJ
                                                                       CQ
                                                                       a.
                                                                       a.
                                                                       UJ
                                                                       o


                                                                       8
                                                                  -8
                                                                              O)
                                                                              >
                                                                              d)
                                                                              (XI

                                                                              CD
                                                                             Jd

                                                                              O
                                                                              (O
                                                                              O)
                                                                              to
                                                                             •r~
                                                                             •c:
                                                                              to
                                                                             -Q
                                                                              §•
                                                                             •fj

                                                                              o
                                                                              o.
                                                                              o

                                                                              a.
                                                                              in



                                                                              O

                                                                             +->
                                                                              o

                                                                             cZ
          O    O>    00

          ^    0    CD
                          r».       o!o    d
                          NOlldOdOdd IVAIAdnS
                                                                              CD
                                   255

-------
              TABLE 5.  MYSID, MYSIDOPSIS BAHIA, SURVIVAL DATA
Concentration (ODD)
Repl



RAW





ARC 'SINE
TRANS-
FORMED




Mean(Y,)
si
1
i
icate
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8


Control
0.80
0.80
1.00
1.00
1.00
1.00
1.00
0.80
1.107
1.107
1.345
1.345
1.345
1.345
1.345
1.107
1.256
0.015
1
50.0
0.80
1.00
0.80
0.80
1.00
1.00
0.80
1.00
1.107
1.345
1.107
1.107
1.345
1.345
1.107
1.345
1.226
0.016
2
100.0
0.60
1.00
1.00
1.00
1.00
0.60
0.80
0.80
0.886
1.345
1.345
1.345
1.345
0.886
1.107
1.107
1.171
0.042
3
210.0
1.00
0.80
0.20
0.80
0.60
0.80
0.80
0.80
1.345
1.107
0.464
1.107
0.886
1.107
1.107
1.107
1.029
0.067
4
450.0
0.00
0.20
0.00
0.20
0.00
0.00
0.00
0.40
0.225
0.464
0.225
0.464
0.225
0.225
0.225
0.685
0.342
0.031
5
14.13.2.6  Test for Normality

14.13.2.6.1  The first step of the test for normality is to center the
observations by subtracting the mean of all observations within a
concentration from each observation in that concentration.   The centered
observations are listed in Table 6.

14.13.2.6.2  Calculate the denominator, D, of the test statistic:

                                D = £ (x±-x)2
    Where:   X,-  = the ith centered observation

             X  = the overall mean of the centered observations

             n  = the total number of centered observations.

                                      256

-------
            TABLE  6.   CENTERED  OBSERVATIONS  FOR SHAPIRO-WILK'S  EXAMPLE
Concentration
Replicate

1
2
3
4
5
6
7
8
Control
(Site Water)
-0.149
-0.149
0.089
0.089
0.089
0.089
0.089
-0.149
50.0

-0.119
0.119
-0.119
-0.119
0.119
0.119
-0.119
0.119
100.0

-0.285
0.174
0.174
0.174
0.174
-0.285
-0.064
-0.064
210.0
1
1
0.316
0.078
-0.565
0.078
-0.142
0.078
0.078
0.078
450.0

-0.117
0.121
-0.117
0.121
-0.117
-0.117
-0.117
0.342
14.13.2.6.3  For this set of data,  n = 40
           X  = _L(-0.006)
                 40
                                                     0.0
                                   D = 1.197

14.13.2.6.4  Order the centered observations from smallest  to  largest:
                  X(1)
The differences x
example:
- X(1) are listed in Table 7.   For this data in this
                                      257

-------
       TABLE 7.   ORDERED  CENTERED  OBSERVATIONS  FOR  SHAPIRO-MILK'S EXAMPLE


                   i           X<0.               i            X(f)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
-0.565
-0.285
-0.285
-0.149
-0.149
-0.149
-0.143
-0.119
-0.119
-0.119
-0.119
-0.117
-0.117
-0.117
-0.117
-0.117
-0.064
-0.064
0.078
0.078
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
0.078
0.078
0.078
0.089
0.089
0.089
0.089
0.089
0.119
0.119
0.119
0.119
0.121
0.121
0.174
0.174
0.174
0.174
0.316
0.342
                     W =    1    (1.0475)2 = 0.9167
                          1.197

14.13.2.6.7  The decision rule for this test is to compare W as calculated in
Subsection 14.13.2.6.5 with the critical value found in Table 6, Appendix B.
If the computed W is less than the critical value, conclude that the data are
not normally distributed.  For this set of data, the critical value at a
significance level of 0.01 and n = 40 observations is 0.919.  Since W = 0.9167
is less than the critical value, conclude that the data are not normally
distributed.

14.13.2.6.8  Since the data do not meet the assumption of normality, Steel's
Many-one Rank Test will be used to analyze the survival data.

14.13.2.7  Steel's Many-one Rank Test

14.13.2.7.1  For each control and concentration combination, combine the data
and arrange the observations in order of size from smallest to largest.
Assign the ranks (1, 2, ...  , 16) to the ordered observations with a rank of 1
assigned to the smallest observation, rank of 2 assigned to the next larger

                                      258

-------
       TABLE 8.  COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE
i
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
observation, etc.
tied observation.
ai
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
If ties

win-i-nj _ wti
0.907
0.601
0.459
0.323
0.323
0.323
0.264
0.240
0.238
0.238
0.238
0.236
0.206
0.206
0.206
0.206
0.153
0.142
0.0
0.0
occur when ranking,

j
: xc4o> -
x«9> .
x<38) -
X(37) _
X(36) _
x<35) .
x(34) -
Y<33)
A
; Y<32>
' i A ~
x<31) -
X(30) .
x<29> _
Y(28)
A
,-• x(27> -
X(26) _
v(25)
A ""
XC24> _
Y<23>
! A ~
Y<22>
A ~
X(21> -
i •
assign the average


xd)
x<2)
X(3)
X(4)
X(5)
X(6)
x<7)
X(8)
x<9)
X(10)
XC11)
XC12)
X(13)
X(H>
x<15>
x<16>
X(17)
x<18)
X(19)
Y<20)
A
rank to each

14.13.2.7.2  An example of assigning ranks to the combined data for the
control and 50.0 ppb concentration is given in Table 9.   This ranking
procedure is repeated for each control/concentration combination.   The
complete set of rankings is summarized in Table 10.   The ranks are then summed
for each concentration level, as shown in Table 11.
                                                          i
14.13.2.7.3  For this example, determine if the survival in any of the
concentrations is significantly lower than the survival  in the control.  If
this occurs, the rank sum at that concentration would be significantly lower
than the rank sum of the control.  Thus compare the  rank sums for  the survival
at each of the various concentration levels with some "minimum" or critical
rank sum, at or below which the survival would be considered significantly
lower than the control.  At a significance level of  0.05,  the minimum rank sum
in a test with four concentrations (excluding the control) and eight
replicates is 47 (See Table 5, Appendix E).               i
                                                          i1
14.13.2.7.4  Since the rank sum for the 450 ppb concentration level  is less
than the critical value, the proportion surviving in that  concentration is
considered significantly less than that in the control.   Since no  other rank
                                                          i
                                      259                  !

-------
     TABLE 9.  ASSIGNING RANKS TO THE CONTROL AND 50 PPB CONCENTRATION LEVEL
               FOR STEEL'S MANY-ONE RANK TEST
                             Transformed Proportion
           Rank                of Total Mortality        Concentration
4
4
4
4
4
4
4
12
12
12
12
12
12
12
12
12
1.107
1.107
1.107
1.107
1.107
1.107
1.107
1.571
1.571
1.571
1.571
1.571
1.571
1.571
1.571
1.571
Control
Control
Control
50 ppb
50 ppb
50 ppb
50 ppb
Control
Control
Control
Control
Control
50 ppb
50 ppb
50 ppb
50 ppb
sums are less than or equal to the critical value, no other concentrations
have a significantly lower proportion surviving than the control.  Hence, the
NOEC and the LOEC are assumed to be 210.0 ppb and 450.0 ppb, respectively.

14.13.2.8  Calculation of the LC50

14.13.2.8.1  The data used for the Probit Analysis is summarized in Table 12.
For the Probit Analysis, run the USEPA Probit Analysis Program.  An example of
the program output is provided in Figure 12.

14.13.2.8.2  For this example, the chi-square test for heterogeneity was not
significant.  Thus Probit Analysis appears to be appropriate for this set of
data.

14.13.3  EXAMPLE OF ANALYSIS OF MYSID, MYSIDOPSIS BAHIA GROWTH DATA

14.13.3.1  Formal statistical, analysis of the growth data is outlined in
Figure 13.  The response used in the statistical analysis is mean weight per
original of males and females combined per replicate.  The IC25 and IC50 can
be calculated for the growth data via a point estimation technique (see
Section 9, Chronic Toxicity Test Endpoints and Data Analysis).  Hypothesis
testing can be used to obtain an NOEC and LOEC for growth.  Concentrations
above the NOEC for survival are excluded from the hypothesis test for growth effect

                                      260

-------
                         TABLE 10.  TABLE OF RANKS
                                                  1
                                            Concentration (ppb)
Repli-
cate
1
2.
3
4
5
6
7
8
1
1
1
1
1
1
1
1
Control
.107(4,
.107(4,
.345(12
.345(12
.345(12
.345(12
.345(12
.107(4,
5,6.
5,6.
,12,
,12,
,12,
,12,
,12,
5,6.
5,10)
5,10)
13.5,14)
13.5,14)
13.5,14)
13.5,14)
13.5,14)
5,10)
1
1
1
1
1
1
1
1
50
.107(4)
.345(12)
.107(4)
.107(4)
.345(12)
.345(12)
.107(4)
.345(12)

0
1
1
1
1
0
1
1
100
.886(1.5)
.345(12)
.345(12)
.345(12)
.345(12)
.886(1.5)
.107(5)
.107(5)
210
1.345(13.5)
1.107(6.5)
0.464(1)
1.107(6.5)
0.886(2)
1.107(6.5)
1.107(6.5)
1.107(6.5)

0
0
0
0
0
0
0
0
450
.225(3)
.464(6.5)
.225(3)
.464(6.5)
.225(3)
.225(3)
.225(3)
.685(8)
   Control  ranks are given in the order of the concentration with which
   they were ranked.
                               TABLE 11.  RANK SUMS
                    Concentration
Rank Sum
                          50
                         100
                         210
                         450
   64
   61
   49
   36
14.13.3.2  The statistical analysis using hypothesis tests; consists of a
parametric test, Dunnett's Procedure, and a nonparametric test, Steel's
Many-one Rank Test.  The underlying, assumptions of the Dunnett's Procedure,
normality and homogeneity of variance, are formally tested.  The test for
normality is the Shapiro-Wilk's Test and Bartlett's Test is used to test for
homogeneity of variance.  If either of these tests fails, the nonparametric
test, Steel's Many-one Rank Test, is used to determine the NOEC and LOEC
endpoints.  If the assumptions of Dunnett's Procedure are met, the endpoints
are determined by the parametric test.
                                                          I

14.13.3.3  Additionally, if unequal numbers of replicates occur among the
concentration levels tested, there are parametric and nonparametric
alternative analyses.  The parametric analysis is a t test with the Bonferroni
adjustment.  The Wilcoxon Rank Sum Test with the Bonferroni adjustment is the

                                      261

-------
 Probit Analysis of Mysidopsis bshia Survival  Data


Cone.
Control
50.0000
100.0000
210.0000
450.0000

Number
Exposed
40
40
40
40
40

Number
Resp.
3
4
6
11
36
Observed
Proportion
Responding
Proportion Adjusted for
Responding
0.0750
0.1000
0.1500
0.2750
0.9000
Controls
0.0000
-.0080
0.0480
0.1880
0.8880
 Chi  - Square for Heterogeneity (calculated)     =    0.725
 Chi  - Square for Heterogeneity (tabular value)  =    5.991
Probit Analysis of Mysidopsis bahia Survival Data

       Estimated  LC/EC Values  and  Confidence Limits
 Point

 LC/EC  1.00
 LC/EC 50.00
Exposure
  Cone.

 123.112
 288.873
   Lower       Upper
 95% Confidence Limits
 65.283
239.559
165.552
335.983
   Figure 12.  Output for USEPA Probit Analysis Program, version 1.5.
                                  262

-------
                     TABLE  12.  DATA FOR  PROBIT ANALYSIS
                  Control
50,0
                                                 Concentration (ppb)
100.0
210.0
450.0
No Dead
No Exposed
3
40
4
40'
6
40
'
11
40 i
36
40
nonparametric alternative.  For detailed information on the Bonferroni
adjustment, see Appendix D.

14.13.3.4  The data, mean and variance of the observations at each
concentration including the control for this example are listed in Table 13.
A plot of the data is provided in Figure 14.  Since there is significant
mortality in the 450 ppb concentration, its effect on growth is not
considered.
                                                          i
                TABLE 13.   MYSID, MYSIDOPSIS BAHIA,  GROWTH DATA
         Replicate   Control
                                           Concentration (»pb)
 50.0
  100.0
  210.0
   450.0








Mean(Y,)
ST
i
1
2
3
4
.5
6
7
8



0.146
0.118
0.216
0.199
0.176
0.243
0.213
0.144
0.182
0.00186
1
0.157
0.193
0.190
0.190
0.256
0.191
0.122
0.177
0.184
0.00145
2
0.114
0.172
0.160
0.199
0.165
0.145
0.207
0.186
0.168
0.00091
3
0.1153
0.071
0.017
0.112
0.052
0.154
0.110
0.103
0.101
0.00222
4

0.012
-
0.002
-
-
-
0.081
_
-
5
14.13.3.5  Test for Normality
                                                         , j
14.13.3.5.1  The first step of the test for normality is to center the
observations by subtracting the mean of all observations within a

                                      263

-------
                 STATISTICAL ANALYSIS OF MYSIDOPSIS BAHIA
                  SURVIVAL, GROWTH, AND FECUNDITY TEST

                                  GROWTH
    POINT ESTIMATION
   ENDPOINT ESTIMATE
        IC25, IC50
                                 GROWTH DATA
                               MEAN DRY WEIGHT
    HYPOTHESIS TESTING
(EXCLUDING CONCENTRATIONS
 ABOVE NOEC FOR SURVIVAL
    SHAPIRO-WILICS TEST
                   NORMAL DISTRIBUTION
        HOMOGENEOUS
           VARIANCE
                                BARTLETTSTEST
                        NON-NORMAL DISTRIBUTION
                             HETEROGENEOUS
                                VARIANCE
1

NO
r
EQUAL NUMBER OF
REPLICATES?

T-TESTWITH
BONFERRONI
ADJUSTMENT



i
YES
DUNNETTS
TEST




}

YES
r
EQUAL NUMBER OF
REPLICATES?

1 NO
STEEL'S MANY-ONE
RANK TEST



WILCOXON RANK SUM
TEST WITH
BONFERRONI ADJUSTMENT


                              ENDPOINT ESTIMATES
                                  NOEC, LOEC
Figure 13.    Flowchart for statistical  analysis of mysid, Mysidopsis bahia,
             growth data.

                                     264

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                          265

-------
 concentration  from each  observation  in  that  concentration.   The  centered
 observations are  listed  in  Table  14.
           TABLE  14.   CENTERED  OBSERVATIONS  FOR  SHAPIRO-WILK'S  EXAMPLE
Concentration
Replicate
1
2
3
4
5
6
7
8
Control
-0.036
-0.064
0.034
0.017
-0.006
0.061
0.031
-0.038
50.0
-0.030
0.009
0.006
0.006
0.072
0.007
-0.062
-0.007
100.0
-0.054
0.004
-0.008
0.031
-0.003
-0.023
0.039
0.018
(DDb)
210.0
0.052
-0.007
-0.084
0.021
-0.049
0.053
0.009
0.002
14.13.3.5.2  Calculate the denominator, D, of the statistic:

                                D =  £ (X^X)2  .



    Where:  X,- = the ith centered observation

            X  = the overall mean of the centered observations

            n  = the total number of centered observations

14.13.3.5.3  For this set of data,  n = 32

                                    X = _i_  (0.007) = 0.000
                                        32

                                    D = 0.0451

14.13.3.5.4  Order the centered observations from smallest to largest

               v<1) < v(2) <     < w(n)


where XCl) denotes the ith ordered observation.  The ordered observations for
this example are listed in Table 15.
                                      266

-------
      TABLE 15.   ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-MILK'S EXAMPLE
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
-0.084
-0.064
-0.062
-0.054
-0.049
-0.038
-0.036
-0.030
-0.023
-0.008
-0.007
-0.007
-0.006
-0.003
0.002
0.004
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
0.006
0.006
0.007
0.009
0.009
0.017
0.018
0.021
0.031
0.031
0.034
0.039
0.052
0.053
0.061
0.072
14.13.3.5.5  From Table 4, Appendix B, for the number of observations, n,
obtain the coefficients a,,  a2,  ...  ak where k is n/2 if n is even and (n-l)/2
if n is odd.  For the data in this example, n = 32 and k == 16.  The a,- values
are listed in Table 16.

14.13.3.5.6  Compute the test statistic, W, as follows:
                         W =
                             D
The differences x
-------
     TABLE 16.  COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-MILK'S EXAMPLE
                                          - X
                                             en
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
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
0.156
0.125
0.115
0.106
0.088
0.072
0.067
0.061
0.044
0.026
0.024
0.016
0.015
0.010
0.004
0.002
xc»>
X(S1>
X 0>
y(29)
,,(28)
x(27>
v C^o/
* ^
x(2)
XC4>
X(23)
x( >
y(21)
x<20)
v(19)
X(18)
X(17)
- x(l>
- x<2)
- X
- x<4)
- x(5)
- x(6)
- x^'
X\oJ
- x<9)
- x<10>
- x<11>
w(12)
- x^^'
- x<^
- x !
- x(16>
14.13.3.6  Test for Homogeneity of Variance

14.13.3.6.1  The test used to examine whether the variation in mean weight of
the mysids is the same across all concentration levels including the control,
is Bartlett's Test (Snedecor and Cochran, 1980).  The test statistic is as
fol1ows:
                      B =
  Where:  V,
           p  s

          In  =
          ni  =
degrees of freedom for each copper concentration and
control, V,- = (n,-  -  1)

number of concentration levels including the control

loge

1, 2, ..., p where p is the number of concentrations
including the control

the number of replicates for concentration i.
                                      268

-------
            , c =
                                i-i
14.13.3.6.2  For the data in this example (See Table 13), all  concentrations
including the control have the same number of replicates (n- =  8  for  all  i).
Thus, V,- = 7 for all  i.

14.13.3.6.3  Bartlett's statistic is therefore:
                  B- [(28)ln(0.0016) -
                     =  [28(-6.4315) - 7(-25.9357)]/1.06

                     =  [-180.082 - (-181.5499)]/1.06

                     =  1.385
                                                          i

14.13.3.6.4  B is approximately distributed as chi-square with p - 1 degrees
of freedom, when the variances are in fact the same.   Therefore, the
appropriate critical value for this test, at a significance level  of 0.01 with
three degrees of freedom,  is 9.210.  Since B = 1.385  is less than  the critical
value of 9.210, conclude that the variances are not different.

14.13.3.7  Dunnett's Procedure

14.13.3.7.1  To obtain an  estimate of the pooled variance for the  Dunnett's
Procedure, construct an ANOVA table as described in Table 17.,

                            TABLE 17.  ANOVA TABLE
Source
Between
Within
Total
df
P - 1
N - p
N - 1
Sum of Squares
(SS)
SSB
SSW
SST
Mean Square (MS)
(SS/df)
S* = SSB/(p-l)
S* = SSW/(N-p)

                                     269

-------
Where: p

       N

       n
                number of concentration levels including the control

                total number of observations n.,  + n2  ... + np

                number of observations in concentration i
SSB
     = 2^,
              Tl/n±-G2/N
                                    Between Sum of Squares
SST =
                 YJ.j-G2/N
                                    Total Sum of Squares
     SSW = SST-SSB
                                    Within Sum of Squares
       G  = the grand total of all sample observations,   G      ±
                                                                = £ T
           I,.  = the total of the replicate measurements for concentration i

          Y,-j  -  the jth observation for concentration i (represents the mean
                dry weight of the mysids for concentration i in test
                chamber j)

14.13.3.7.2  For the data in this example:

                     n, =  n2 = n3 = n4 = 8
                      N = 32
                     T  - Y  -i- V
                     J.1  ~ .'.11   ..12
                     ^4 - Y41 + Y42
                                       Y18 = 1.455
                                       Y28 = 1.473
                                       Y38 = 1.348
                                       Y,. = 0.805
                          T1  + T2 + T3 + T4 = 5-081
                   SSB = f,Tl/n±-G2/N
                       1_(6.752) - (5.08i
                       8              32
                                                 = 0.0372
                   SST =
                           0.889 - (5.08ir  = 0.0822
                                     32
                                      270

-------
                   SSW = SST-SSB    =  0.0822 - 0.0372 - 0.0450
                    S*  = SSB/(p-l) = 0.0372/(4-l) = 0.0124
                    S*  = SSW/(N-p) = 0.0450/(32-4) = 0.0016
14.13.3.7.3  Summarize  these calculations in the ANOVA table  (Table 18)

            TABLE 18.  ANOVA TABLE FOR DUNNETT'S PROCEDURE EXAMPLE
Source
Between
Within
Total
df
3
28
31
Sum of Squares
(SS)
0.0372
0.0450
0.0822
Mean Square (MS)
(SS/df)
0.0127
0.0016

 14  13.3.7.4   To  perform the  individual  comparisons,  calculate  the  t  statistic
 for each  concentration,  and  control  combination  as  follows:
 Where:  Y,   =  mean  dry  weight  for concentration  i
         Y,   =  mean  dry  weight  for the control
         Su   =  square root of the within mean square
         n,   =  number of replicates for the control        i
         n-   =  number of replicates for concentration i    i
 14 13 3 7 5  Table  19  includes the calculated t values for each concentration
 and control combination.  In this example, comparing the 50.0 ppb
 concentration with  the control the calculation  is  as follows:
                                       271

-------
                                    (0.182-0.184)
                                [0 . 04CVU/8) +(1/8) ]


                               -0.100
                        TABLE 19.   CALCULATED T VALUES
                   Concentration (ppb)
50.0
100.0
210.0
2
3
4
-0.150
0.700
4.050
 14.13.3.7.6  Since the  purpose  of  this  test  is to detect a significant
 reduction in  mean  weight,  a  one-sided test is appropriate.  The critical value
 for this  one-sided test is found in Table 5, Appendix C.  For an overall alpha
 level  of  0.05,  28  degrees  of freedom for error and three concentrations
 (excluding the  control)  the  approximate critical value is 2.15.  The mean
 weight for concentration "i"  is considered significantly less than the mean
 !S195r« «  tte  contro1  1f *! is grater than  the critical  value.   Therefore,
 the 210.0 ppb concentration  has significantly lower mean weight than the
 control.   Hence the NOEC and the LOEC for growth are 100.0 ppb and 210.0 ppb
 respectively.                                                            KK '

 14.13.3.7.7   To quantify the sensitivity of the test, the minimum significant
 difference  (MSD) that can be detected statistically may be calculated.
Where:  d

        S

        n
                      = d

    the critical value for Dunnett's Procedure

>w - the square root of the within mean  square

    the common number of replicates at  each concentration
    (this assumes equal replication at  each concentration)
        r\i  = the  number of replicates  in  the control.

14.13.3.7.8  In this example:

                      MSD = 2.15(0.04)^(1/8)+(1/8)


                           = 2.15  (0.04)(0.5)

                           = 0.043
                                     272

-------
14 13 3.7.9  Therefore, for this set of data, the minimum idifference that can
be detected as statistically significant is 0.043 mg.

14.13.3.7.10  This represents a 23.6% reduction in mean weight from the
control.

14.13.3.8  Calculation of the ICp

14 13 3 8 1  The growth data from Table 13 are utilized in this example.  As
seen in', the observed means are not monotonically non-increasing with respect
to concentration.  Therefore, it is necessary to  smooth the means prior to
calculating the 1C.   In the following discussion, the observed means  are
represented by Y,- and the smoothed means by Mr

14.13.3.8.2  Starting with the  control mean, Y, = 0.182 arid Y2 = 0.184, we see
 that Y1 < Y2
Calculate the smoothed means:

         M, = M2 = (Y, + Y2)/2 =  0.183
 14 13  3  8.3   Since Y5 = 0.025 < Y, - 0.101 < Y3 = 0.168 < M2,  set  M3 = 0.168
 and M  =0.101,  and M5 = 0.025.  Table 20 contains the smoothed means and
 Figure 15 gives a plot  of the  smoothed response curve.
TABLE 20. MYSID, MYSIDOPSIS BAHIA,
MEAN
GROWTH RESPONSE AFTER SMOOTHING
Toxicant
Cone.
(ppb)
Control
50.0
100.0
210.0
450.0

i
1
2
3
4
5
Response
Means
Y1 (mg)
0.182
0.184
0.168
0.101
0.012
Smoothed
Mean
M,- (mg)
0.183
0.183
0.168
0.101
0.012!
  14  13 3 8  4  An  IC25  and  IC50  can  be  estimated  using  the  Linear  Interpolation
  Method." A 25% reduction  in weight, compared  to the controls,  would  result  in
  a mean weight of 0.136  mg, where M^l-p/100)  =  0.183(1-25/100).  A 50%
  reduction  in mean dry weight,  compared  to  the controls, would  result in  a mean
  weight of  0.091  mg.   Examining the smoothed means  and their associated
  concentrations  (Table 20), the response, 0.136  mg,  is bracketed  by C3 =  100
  ppb and C, = 210 ppb.   The response,  0.091  mg,  is bracketed by C4 = 210 ppb
  and C5 = 450 ppb.
                                       273

-------
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                                 274

-------
14.13.3.8.5  Using the equation in Section 4.2 from Appendix L, the estimate
of the IC25 is calculated as follows:                     i
                   icp = Cj
                   IC25 = 100 + [0.183(1 - 25/100) - 0.166]   (210 - 100)
                        = 151 ppb.

14.13.3.8.6  Using Equation 1 from Appendix L, the estimai
calculated as follows:

                   ICp = C^ [M, (l-p/100) -Afj] \C(j^ ~_l
                                                             (0.101 - 0.168)
                                                          e of the IC50 is
                    IC50 = 210 +  [0.183(1 - 50/100) - 0.101]   (450 - 210)
                                                            (0.012 - 0.101)
                        = 236 ppb.
 14.13.3.8.7  When the  ICPIN program was used to analyze this set of data,
 requesting 80 resamples, the estimate of the IC25 was  147.170 ppb.  The
 empirical 95.0% confidence interval for the true mean  was 97.0905 ppb  and
 186.6383 ppb.  The  computer program output for the  IC25 for this data  set  is
 shown  in Figure 16.

 14.13.3.8.8  When the  ICPIN program was used to analyze this set of data for
 the  IC50, requesting 80  resamples, the estimate of  the IC50 was 230.755 ppb.
 The  empirical 95.7% confidence  interval for the true mean was (183.84  ppb  to
 277.9211 ppb).  The computer program output for the IC50 for this data set is
 shown  in Figure 17.

 14.13.4  EXAMPLE OF ANALYSIS OF MYSID, MYSIDOPSIS BAHIA, FECUNDITY DATA

 14.13.4.1   Formal statistical  analysis of the  fecundity data is outlined in
 Figure 18.   The response used  in the statistical analysis  is the proportion of
 females with eggs in each  test  or control chamber.   If no  females were present
 in a replicate, a response of  zero should not  be used.  Instead there  are  no
 data available for  that  replicate and the number of replicates for that level
 of concentration or the  control should be reduced by one.   Separate  analyses
 are  performed  for the  estimation.of the NOEC and LOEC  endpoints, and  for the
 estimation  of  the EC,  LC,  and  1C endpoints.  The data  for  a concentration  are
 excluded  from  the statistical  analysis of the  NOEC  and LOEC endpoints  if no
 eggs were produced  in  all  of the replicates  in which females existed.
 However,  all data are  included in the estimation of the  IC25 and  IC50.
                                      275

-------
Cone. ID

Cone. Tested
Response
Response
Response
Response
Response
Response
Response
Response
1
2
3
4
5
6
7
8
1
0
.146
.118
.216
.199
.176
.243
.213
.144
2
50
.154
.19
.193
.190
.190
.191
.122
.177
3
100
.114
.172
.160
.199
.165
.145
.207
.186
4
210
.153
.094
.017
.122
.052
.154
.110
.103
5
450
0
.012
0
.002
0
0
0
.081
***  Inhibition Concentration  Percentage  Estimate ***
Toxi cant/Eff1uent:
Test Start Date:    Test Ending Date:
Test Species: MYSID SHRIMP, Mysidopsis bahia
Test Duration:     growth test
DATA FILE: mysidwt.icp
OUTPUT FILE: mysidwt.i25
Cone.
ID
1
2
3
4
5
Number
Replicates
8
8
8
8
8
Concentration
M9/1
0.000
50.000
100.000
210.000
450.000
Response
Means
0.182
0.184
0.168
0.101
0.012
Std. Pooled
Dev. Response Means
0.043
0.038
0.030
0.047
0.028
0.183
0.183
0.168
0.101
0.012
The Linear Interpolation Estimate:   150.6446   Entered P Value: 25

Number of Resamplings:   80
The Bootstrap Estimates Mean: 147.1702 Standard Deviation:    23.7984
Original Confidence Limits:   Lower:  97.0905 Upper:   186.6383
Resampling time in Seconds:     0.11  Random Seed: -1623038650
                Figure 16.   ICPIN program output for the IC25.
                                     276

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Cone. ID

Cone. Tested
Response
Response
Response
Response
Response
Response
Response
Response
1
2
3
4
5
6
7
8
1
0
.146
.118
.216
.199
.176
.243
.213
.144
2
30
.154
.193
.190
.190
.256
.191
.122
.177
3
100
.114
.172
.160
.199
.165
.145
.207
.186
4
210
.153
.094
.017
.122
.052
.154
.110
.103
5
450
0
.012
0
.002
0
0
0
.081
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent:
Test Start Date:    Test Ending Date:
Test Species: MYSID SHRIMP, Mysidopsis bahia
Test Duration:     growth test
DATA FILE: mysidwt.icp
OUTPUT FILE: mysidwt.i50
Cone.
ID
1
2
3
4
5
Number
Replicates
8
8
8
8
8
Concentration
ug/1
0.000
50.000
100.000
210.000
450.000
Response
Means
0.182
0.184
0.168
0.101
0.012
Std. Pooled
Dev. : iResponse Means
0.043
0.0313
0.030
0.047
0.028
0.183
0.183
0.168
0.101
0.012
The Linear Interpolation Estimate:   234.6761   Entered P Value: 50
Number of Resamplings:   80
The Bootstrap Estimates Mean:
Original Confidence Limits:
Resampling time in Seconds:
230.7551 Standard Deviation:    30.6781
Lower:   183.8197 Upper:   277.9211
  0.16  Random Seed: -628896314
                Figure 17.  ICPIN program output for the IC50.
                                      277

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                   STATISTICAL ANALYSIS OF MYSIDOPSIS BAHIA
                     SURVIVAL, GROWTH, AND FECUNDITY TEST

                                   FECUNDITY
                                  FECUNDITY DATA
                          PROPORTION OF FEMALES WITH EGGS
      POINT ESTIMATION
     HYPOTHESIS TESTING
  (EXCLUDING CONCENTRATIONS
  ABOVE NOEC FOR SURVIVAL)
     ENDPO1NT ESTIMATE
         IC25, IC50
   ARC SINE TRANSFORMATION
                                SHAPIRO-WIIJCS TEST
                                                    NON-NORMAL DISTRIBUTION
                     NORMAL DISTRIBUTION
           HOMOGENEOUS
             VARIANCE
                                 BARTLETTSTEST
                               HETEROGENEOUS
                                  VARIANCE
                EQUAL NUMBER OF
                   REPLICATES?
             NO
YES
       T-TESTWITH
       BONFERRONI
       ADJUSTMENT
                                                        I
                       EQUAL NUMBER OF
                         REPLICATES?
YES
NO
        STEEL'S MANY-ONE
           RANKTEST
             WILCOXON RANK SUM
                 TEST WITH
           BONFERRONI ADJUSTMENT
                                ENDPOINT ESTIMATES
                                   NOEC, LOEC
Figure 18.    Flowchart for statistical analysis  of mysid,  Mysidopsis bahia,
              fecundity data.

                                      278

-------
14 13.4.2  For the case of equal  numbers of replicates across all
concentrations and the control, the evaluation of the NOEC and LOEC endpoints
is made via a parametric test, Dunnett's Procedure, or a nonparametric test,
Steel's Many-one Rank Test, on the arc sine square root transformed data.
Under!yinq assumptions of Dunnett's Procedure, normality and homogeneity of
variance, are formally tested.  The test for normality is the Shapiro-Wilk s
Test  and Bartlett's Test is used to test for homogeneity of variance.  If
either of these tests fails, the nonparametric test, Steel's Many-one Rank
Test  is used to determine the NOEC and LOEC endpoints.  If the assumptions of
Dunnett's Procedure are met, the endpoints are estimated by the parametric
procedure.                                                |

14 13 4 3  If unequal numbers  of replicates occur  among the concentration
levels tested, there are parametric and nonparametric alternative analyses.
The parametric analysis is a t test with the Bonferroni adjustment
(Appendix D).  The Wilcoxon Rank Sum Test with the Bonferrom adjustment is
the nonparametric alternative.

14 13 4  4  The proportion  of  female mysids, Mysidopsis bahia, with eggs  in
each  replicate must first  be  transformed by the  arc  sine  square root
transformation procedure described  in Appendix B.   Since  the  denominator of
the proportion of females  with eggs varies with  the  number  of females
occurring  in that replicate,  the  adjustment of the arc  sine square root
transformation  for  0% and  100% is  not used for this  data.   The raw and
transformed  data, means  and  variances of the  transformed  observations  at each
test  concentration  including  the  control are  listed in  Table  21.  Since  there
 is  significant  mortality in  the  450 ppb concentration,  its  effect on
 reproduction is  not considered.   Additionally, since no eggs  were produced  by
 females  in any  of the replicates  for  the 210  ppb concentration,  it  is  not
 included in  this statistical  analysis  and  is  considered a qualitative
 reproductive effect.   A plot of the mean proportion of female mysids  with  eggs
 is  illustrated  in Figure 19.

 14.13.4.5  Test for Normality
                                                          i
 14.13.4.5.1   The first step of the test for normality is to center the
 observations by subtracting the mean  of all  observations within  a
 concentration from each observation in that concentration.   The  .centered
 observations are listed in Table 22.

 14.13.4.5.2  Calculate the denominator, D, of the statistic:

                                 D = t(X,-X)2        .   !
     Where:  Xs = the ith centered observation

             X  = the overall mean of the centered observations

             n  = the total  number of centered observations
                                       279

-------
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                      280

-------
TABLE 21.  MYSID, MYSIDOPSIS  BAHIA,  FECUNDITY DATA:  PERCENT FEMALES WITH
           EGGS
Test Concentration (oob)
Repl



RAW





ARC SINE
TRANS-
FORMED




MeanCY,-)
?T
i
icate
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8



Control
1.00
1.00
0.67
1.00
1.00
0.80
1.00
1.00
1.57
1.57
0.96
1.57
1.57
1.12
1.57
1.57
1.44
0.064
1
50.0
0.50
0.33
0.67
-
0.40
0.50
0.25
0.33
0.78
0.61
0.96
_
0.68
0.78
0.52
0.61
0.71
0.021
2
100.0 210.0
0.33 0.0
0.50 0.0
0.00 0.0
0.50 0.0
0.67
0.00
0.25
-
0.0
0.0
0.0
0.0
0.61
0.78
0.00
0.78
0.96
0.00
0.52
-
0.52
0.147
3
..
..
„



-
4
                                   281

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          TABLE 22.  CENTERED OBSERVATIONS FOR SHAPIRO-MILK'S EXAMPLE
Test Concentration (ODD)
Replicate
1
2
3
4
5
6
7
8
Control
0.13
0.13
-0.48
0.13
0.13
-0.32
0.13
0.13
50.0
0.07
-0.10
0.25
-
-0.03
0.07
-0.19
-0.10
100.0
0.09
0.26
-0.52
0.26
0.44
-0.52
0.00

14.13.4.5.3  For this set of data, n = 22
                                            (0.000)  =  0.000
                                        22

                                  '  D  =  1.4412

14.13.4.5.4  Order the centered observations from  smallest  to largest:
where Xci> denotes the  ith  ordered  observation.   The ordered observations for
this example are listed in Table 23.

14.13.4.5.5  From Table 4, Appendix  B,  for  the  number of observations,  n,
obtain the coefficients an, 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 = 22 and k = 11.   The a,  values
are listed in Table 24.

14.13.4.5.6  Compute the test  statistic,  W,  as  follows:
                              1  Ji                   Z
                         fir —  -1- r > =>  I v(tt-i+l) _ y (i) \ ~\
                         W — — L " "i {•"•       •"•   I J


The differences xcn"i+1> - Xci>  are  listed in Table 24.  For the data in this
example:

               W =    1    (1.1389)2  =  0.900
                    1.4412
                                      282

-------
      TABLE 23.   ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-MILK'S EXAMPLE
i
1
2
3
4
5
6
7
8
9
10
11
x
0.74 XQ:J^
0.57 X
0.32 X(!,}
0.23
0.23
0.16
0.13
0.06
0.02
X
X\ 16)
._
x<15)
X
v(13)
x<12>

- x(1)
- X
- x^
- x<4)
- x(5)
- x<6)
X(7j
„
- x<8)
- X
- xc;;>
- x<11>
14.13.4.5.7  The decision rule for this test is to compare M as calculated in
Subsection 14.13.4.5.6 to a critical value found in Table 6, Appendix B.   If
the computed M is less than the critical value, conclude that the data are not
normally distributed.  For this set of data, the critical value at a
significance level of 0.01 and n = 22 observations is 0.878.  Since M = 0.900
is greater than the critical value, conclude that the data are normally
distributed.
                                      283

-------
 14.13.4.6  Test for Homogeneity of Variance

 14.13.4.6.1  The test used to examine whether the variation in proportion of
 female mysids with eggs is the same across all  concentration levels  including
 the control,  is Bartlett's Test (Snedecor and Cochran,  1980).   The test
 statistic is  as follows:
               [(
                              £ V  InS2  - £ v.
                       B =
Where:  V


        p

       In
 f
1  =
                    degrees  of freedom  for  each  copper  concentration  and
                    control,  Vf = (n,. -  1)

                    number of concentration  levels  including the control

                    loge


                    1, 2, ...,  p where  p  is  the  number  of concentrations
                    including  the control
           n,-  -   the number of replicates for concentration i.
             c =
                                i-i
14.13.4.6.2  For the data in this example (see Table 21), n.  = 8,  n? =  7  and
n3 - 7.   Thus,  the respective degrees of freedom are 7,  6 and 6.

14.13.4.6.3  Bartlett's statistic is therefore:


       B -  [(19)ln(0.077) - (7 ln(0.064) + 6 ln(0.021)  + 6 ln(0.147))]/1.07

         -  [19(-2.564) - (-53.925)]/1.07

         -  [-48.716 - (-53.925)1/1.07

         -  4.868
                                     284

-------
14.13.4.6.4  B is approximately distributed as chi-square with p -  1  degrees
of freedom, when the variances are in fact the same.   Therefore, the
appropriate critical value for this test,  at a significance level  of  0.01  with
two degrees of freedom, is 9.210.  Since B = 4.868 is less than the critical
value of 9.210, conclude that the variances are not different.
                                                          i
14.13.4.7  T test with the Bonferroni Adjustment

14.13.4.7.1  A t test with the Bonferroni  adjustment is used as an  alternative
to Dunnett's Procedure when, as in this set of data,  the number of replicates
is not the same for all concentrations.  Like Dunnett's Procedure,  it uses a
pooled, estimate of the variance, which is  equal to the error value  calculated
in an analysis of variance.  To obtain an  estimate of the pooled variance,
construct an ANOVA table as described in Table 25.
                            TABLE 25.  ANOVA TABLE
      Source
      Total
df
       Sum of Squares
            (SS)
 - 1
            SST
Mean Square(MS)
    (SS/df).

Between

Within

P

N

- 1

- P

SSB

SSW
2
SB =
2
s«-

SSB/(p-l)

SSW/(N-p)
  Where:  p  = number concentration levels including the control

          N  = total number of observations n1  + n2  ... +nf[

          nf = number of observations in concentration  i
         SSB - Y,T\/ni-Gz/N      Between Sum of Squares'
                i-l
         SST =
:-G2/N
         SSW = SST-SSB
                Total Sum of Squares
                Within Sum of Squares
                                      285

-------
          6  = the grand total of all sample observations,  G = £ T±
          TJ = the total  of the replicate measurements for
               concentration i

         Y,-j - the -jth observation for concentration i (represents the
               proportion of females with eggs for concentration
               i in test chamber j)

14.13.4.7.2  For the data in this example:

                    n, = 8  n2 =  7   n3 = 7
                    N  = 22
                    J2: Y2': v22
                    I3 - Y31 + Y32
Y18 =  11.5
Y27 =  4.94
Y37 =  3.65
                    G  = T, + T2 +  T3 = 20.09
                   SSB =
                           132.25 + 24.40 + 13.32
                             877
            403.61  =  3,57
              22
                   SST =
                        =  23.396 - 403.61  = 5.05
                                      22


                   SSW = SST-SSB.    =  5.05  - 3.57 =  1.48


                    SB  - SSB/(p-l)  = 3.57/(3-l) = 1.785

                    Sy  = SSW/(N-p)  = 1.48/(22-3) = 0.078
                                      286

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14.13.4.7.3  Summarize these calculations in the ANOVA table (Table 26).

   TABLE 26.  ANOVA TABLE FOR THE T TEST WITH BONFERRONI'S ADJUSTMENT EXAMPLE
      Source        df        Sum of Squares        Mean Square(MS)
                                   (SS)                 (SS/df)
Between
Within
Total
2
19
21
3.57
1.48
5.05
1.785
0.078
1
1
14.13.4.7.4  To perform the individual comparisons, calculate the t statistic
for each concentration, and control combination as follows:
Where:  Y,-  = mean proportion of females with eggs for. concentration i
        Y!  = mean proportion of females with 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.
14.13.4.7.5  Table 27 includes the calculated t values for each concentration
and control combination.  In this example, comparing the 50.0 ppb
concentration with the control the calculation is as follows:
                         ,_  _      (1.44  -  0.52)
                               [0.279
                            = 5.05
                                      287

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                        TABLE 27.  CALCULATED T VALUES
               Test Concentration (ppb)
50.0
100.0
2
3
5.05
6.37
14.13.4.7.6  Since the purpose of this test is to detect a significant
reduction in mean proportion of females with eggs, a one-sided test is
appropriate.  The critical value for this one-sided test is found in Table 5,
Appendix D, Critical Values for the t test with Bonferroni's adjustment.
For an overall alpha level of 0.05, 19 degrees of freedom for error and two
concentrations (excluding the control) the approximate critical value is
2.094.  The mean proportion for concentration "i" is considered significantly
less than the mean proportion for the control if t,  is greater than the
critical value.  Therefore, the 50.0 ppb and the 100.0 ppb concentrations have
significantly lower mean proportion of females with eggs than the control.
Hence the LOEC for fecundity is 50.0 ppb.

14.13.4.7.7  To quantify the sensitivity of the test, the minimum significant
difference (MSD) that can be detected statistically may be calculated.
                          MSD =  t SrfJCL/Ili) + (1/72)

Where:  t  = the critical value for the t test with Bonferroni's adjustment

       Sw  = the square root of the within mean square

        n  » the common number of replicates at each concentration
             (this assumes equal  replication at each concentration)

       n,  = the number of replicates in the control.

14.13.4.7.8  In this example:
                     MSD = 2.094 (0.279)^(1/8)+(1/7)


                          = 2.094 (0.279)(0.518)

                          = 0.303

14.13.4.7.9 • Therefore, for this set of data, the minimum difference that can
be detected as statistically significant is 0.30.

14.13.4.7.10  The MSD (0.30) is in transformed units.  To determine the MSD in
terms of percent of females with eggs, carry out the following conversion.

                                      288

-------
   1.   Subtract  the  MSD from the  transformed control  mean.

                          1.44 -  0.30 = 1.14
                                                          I
                                                          i
   2.   Obtain  the  untransformed values for the control  mean  and the
       difference  calculated in 4.10.1.

                          [ Sine  (1.44) J2  = 0.983
                              '
                          [ Sine  (1.14) ]2 = 0.823

   3.   The untransformed MSD (MSD )  is  determined by  subtracting the
       untransformed values from 14.4.8.10.2.

                          MSDU =  0.983 -  0.823 = 0.16

14.13.4.7.11  Therefore, for this set of data, the minimum difference in mean
proportion of females with eggs between the control  and any copper
concentration that can be detected as statistically significant is 0.16.
              .
14.13.4.7.12  This represents a 17% decrease in proportion of females with
eggs from the control.

14.13.4.8  Calculation of the ICp

14.13.4.8.1  The fecundity data in Table 4 are utilized in this example.
Table 28 contains  the mean proportion of females with eggs for each toxicant
concentration.  As can be seen, the observed means are monotonically
nonincreasing with respect to concentration.  Therefore, it is not necessary
to smooth the means prior to calculating the 1C.  Figure 20 gives a plot of
the response curve.                                        !
          TABLE 28.  MYSID,. MYSIDOPSIS BAHIA, MEAN PROPORTION
                     OF FEMALES WITH EGGS                 |
Toxicant
Cone.
(ppb)
Control
50.0
100.0
210.0
450.0

i
1
2
3
4
5
Response Si
Means, Y1 M<
noothed
jans, M.-
(proportion) (proportion)
0.934
0.426
0.934
0.426
0.317 0.317
0.000 0.000
0.010 0.000
14.13.4.8.2  An IC25 and IC50 can be estimated using the Linear Interpolation
Method.  A 25% reduction in mean proportion of females with eggs, compared to

                                      289

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the controls, would result in a mean proportion of 0.701, where M^(l-p/100) =
0.934(1-25/100).  A 50% reduction in mean proportion of females with eggs,
compared to the controls, would result in a mean proportion of 0.467.
Examining the means and their associated concentrations (Table 28), the
response, 0.701, is bracketed by C.,  = 0 ppb and C2  =  50  ppb.   The  response,
0.467, is bracketed by C, = 0 ppb and C2  =  50  ppb.         ;

14.13.4.8.3  Using the equation in Section 4.2 from Appendix  L, the estimate
of the IC25 is calculated as follows:
                   icp - Cj
                   IC25 = 0 + [0.934(1 - 25/100) - 0.934]     (50 - 0)
                                                           (0.426 - 0.934)

                        = 23 ppb.

14.13.4.8.4  Using the equation in Section 4.2 from Appendix L, the estimate
of the IC50 is calculated as follows:
                   ICp =
                                                   w+1)
                   IC50 = 0 + [0.934(1 - 50/100) - 0.934]    (50 - 0)
                                                         (0.426 - 0.934)
                        = 46 ppb.                         I

14.13.4.8.5  When the ICPIN program was used to analyze this set of data,
requesting 80 resamples, the estimate of the IC25 was 22.9745 ppb.
The empirical 95.0% confidence interval for the true mean was 20.0499 ppb to
30.5675 ppb.  The computer program output for the IC25 for this data set is
shown in Figure 21.  This value is extrapolated below the lowest test
concentration and data should be used cautiously.
                                                          I
14.13.4.8.6  When the ICPIN program was used to analyze this set of data for
the IC50, requesting 80 resamples, the estimate of the IC50 v/as 45.9490 ppb.
The empirical 95.0% confidence interval for the true mean was 40.1467 ppb to
63.0931 ppb.  The computer program output for the IC50 for this data set is
shown in Figure 22.
                                      291

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Cone. ID

Cone. Tested
Response
Response
Response
Response
Response
Response
Response
Response
1
2
3
4
5
6
7
8
1
0
1
1
.67
1
1
.8
1
1
2
50
.5
.33
.67
.4
.5
.25
.33
3
100
.3
.5
0
.5
.67
0
.25
4
210
0
0
0
0
0
0
0
0
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent:
Test Start Date:    Test Ending Date:
Test Species: MYSID SHRIMP, Mysidopsis bahia
Test Duration:       fecundity
DATA FILE: mysidfe.icp
OUTPUT FILE: mysidfe.i25
Cone.
ID
1
2
3
4
Number
Replicates
8
7
7
8
Concentration
ug/l
0.000
50.000
100.000
210.000
Response
Means
0.934
0.426
0.317
0.000
Std. Pooled
Dev. Response Means
0.127
0.142
0.257
0.000
0.934
0.426
0.317
0.000
                                                                                  flt
The Linear Interpolation Estimate:     22.9745   Entered  P Value:  25

Number of Resamplings:   80
The Bootstrap Estimates Mean:  23.8871 Standard Deviation:      3.0663
Original Confidence Limits:   Lower:     20.0499 Upper:     30.5765
Resampling time in Seconds:     1.37   Random Seed:  1918482350
                Figure 21.  ICPIN program output for the  IC25.
                                     292

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Cone. ID

Cone. Tested
Response
Response
Response
Response
Response
Response
Response
Response
1
2
3
4
5
6
7
8
1
0
1
1
.67
1
1
.8
1
1
2
50
.5
.33
.67
.4
.5
.25
.33

3
100
.3
.5
0
.5
.67
0
.25

4
210
0
0
0
0
0
0
0
0
***  Inhibition  Concentration  Percentage  Estimate  ***
Toxi cant/Eff1uent:
Test Start Date:    Test  Ending Date:
Test Species: MYSID SHRIMP, Mysidopsis bahia
Test Duration:       fecundity
DATA FILE: mysidfe.icp
OUTPUT FILE: mysidfe.i50
Cone.
ID
1
2
3
4
Number
Replicates
8
7
7
8
Concentration
ug/i
0.000
50.000
100.000
210.000
Response
Means
0.934
0.426
0.317
0.000
Std. Pooled
Dev. Response Means
0.127
0.142
0.257
0.000
0.934
0.426
0.317
0.000
The Linear Interpolation Estimate:    45.9490   Entered P Value: 50
	'-	
Number of Resamplings:   80
The Bootstrap Estimates Mean:  47.8720 Standard Deviation:     8 2908
Original Confidence Limits:   Lower:    40.1467 Upper:    63 0931
Resampling time in Seconds:     1.32  Random Seed: -391064242
                Figure 22.   ICPIN program output for the IC50.

                                     293

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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 mysid survival,
growth, and fecundity using copper (Cu) sulfate and sodium dodecyl sulfate
(SDS) in natural seawater and in artificial seawater (GP2) are shown in Tables
29-33.  Survival NOEC/LOEC pairs showed good precision, and were the same in
four of the six tests with Cu and SDS.  Growth and fecundity were generally
not acceptable endpoints in either sets of tests.  In Tables 29-30 the
coefficient of variation for the IC25, ranges from 18.0 to 35.0 and the IC50,
ranges from 5.8 to 47.8, indicating acceptable test precision.  Data in Tables
31-33 show no detectable differences between tests conducted in natural or
Artificial seawaters.

14.14.1.2  Multilaboratory Precision

14.14.1.2.1  The multilaboratory precision of the test has not yet been
determined.

14.14.2  ACCURACY    .

14.14.2.1  The  accuracy of toxicity tests  cannot be determined.
                                      294

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 TABLE 29.   SINGLE-LABORATORY PRECISION OF THE MYSID,  MYSIDOPSIS BAHIA
            SURVIVAL,  GROWTH, AND FECUNDITY TEST PERFORMED IN NATURAL
            SEAWATER,  USING JUVENILES FROM MYSIDS CULTURED AND SPAWNED IN
            NATURAL  §|AWATER, AND COPPER (CU)  SULFATE  AS ,A REFERENCE
            TOXICANT 'f'4'5'6
Test
Number
1
2
3
4
5
NOEC
(M9/L)
63
125
125
125
125
IC25
96.1
,138.3
156.3
143.0
157.7
. IC50
NC8
175.5
187.5
179.9
200.3
Most
Sensitive
Endpoint
S
S
S
S
S
n:
Mean:
CV(%):
5
NA
NA
5
138.3
18.0
4
185.8
5.8
1
2
3
7
8
   Data from USEPA (1988a) and USEPA (1991a).
   Tests performed by Randy Camel eo, ERL-N, USEPA, Narragansett, RI.
   Eight replicate exposure chambers, each with five juveniles, were used
   for the control and each toxicant concentration.  The temperature of
   the test solutions was maintained at 26 + 1°C.
   Copper concentrations in Tests 1-2 were: 8, 16, 31, 63, and 125
   Copper concentrations in Tests 3-6 were, 16, 31, 63, ]25, and 250
   NOEC Range:   63 -  125 /zg/L (this represents a difference of two
   exposure concentrations).
   For a discussion of the precision of data from chronic toxicity tests
   see Section  4,  Quality Assurance.
   Endpoints: G=Growth;  S=Survival.
   NC  = No  linear  interpolation  estimate could be calculated from the
   data,  since  none of the group response means were  less than 50 percent
   of  the control  concentrations.
                                   295

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TABLE 30.  SINGLE-LABORATORY PRECISION OF THE MYSID, MYSIDOPSIS BAHIA,
           SURVIVAL, GROWTH, AND FECUNDITY TEST PERFORMED IN NATURAL
           SEAWATER USING JUVENILES FROM MYSIDS CULTURED AND SPAWNED IN
           NATURAL SEAWATER, AND SODIUM DODECYL SULFATE (SDS) AS A
           REFERENCE TOXICANT1'2'3'4'5'6
      Test
      Number
                   NOEC
                   (mg/L)
IC25
(mg/L)
IC50
(mg/L)
Most
Sensitive
Endpoint
1
2
3
4
5
6
2.5
< 0.3
< 0.6
5.0
2.5
5.0
4.5
NC
NC8
7.8
3.6
7.0
NC9
NC
NC
NC9
•4.6
9.3
S
S
S
S
S
S
        n:
      Mean:
                      4
                      NA
                      NA
 4
 5.7
 35.0
2
6.9
47.8
 1
 2
 3
Data from USEPA (1988a) and USEPA (1991a).
Tests performed by Randy Cameleo, ERL-N, USEPA, Narragansett, RI.
Eight replicate exposure chambers, each with five juveniles, were used
for the control and each toxicant concentration.  The temperature of
the test solutions was maintained at 26 ± 1°C.
SDS concentrations in Tests 1-2 were: 0.3, 0.6, 1.3, 2.5, and 5.0 mg/L.
                   in Tests 3-4 were: 0.6, 1.3, 2.5, 5.0 and 10.0 mg/L.
                                      1.3, 2.5, 5.0, 10.0, and
    SDS concentrations
    SDS concentrations in Tests 5-6 were:
    20.0 mg/L.
    NOEC Range:
 7
 8
             < 0.3 - 5.0 mg/L  (this represents a difference of four
 exposure concentrations).
For a discussion of the precision of data from chronic toxicity tests
see Section 4, Quality Assurance.
Endpoints: G=Growth; S=Survival.
NC - No linear interpolation estimate could be calculated from the
data, since none of the group  response means were less than 75 percent
of the control response mean.
NC = No linear interpolation estimate could be calculated from the
data,  since none of the group  response means were  less than  50  percent
of the control response mean.
                                     296

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TABLE 31.  COMPARISON OF SURVIVAL (LC50)1,  GROWTH AND FECUNDITY (IC50)1
           RESULTS FROM 7-DAY TESTS WITH THE MYSID, MYSIDOPSIS BAHIA,
           USING NATURAL SEAWATER (NSW) AND ARTIFICIAL SEAWATER (GP2) AS
           DILUTION WATER AND SODIUM DODECYL SULFATE (SDS) AS A
           REFERENCE TOXICANT
Test
             Survival LC50
 NSW
GP2
                       Growth IC50
                                 Fecundity IC50
 NSW
 GP2
 NSW
 GP2
 1

 2

 3
16.2

20.5

   2
16.3

19.2

21.9
16.8

24.2
   2
16.3

23.3

24.4
12.0

20.1

   2
10.9

18.5

21.7
   All  LC50/IC50 values in mg/L.
   No test performed.
                                  297

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TABLE 32.  COMPARISON OF SURVIVAL (LC50)1,  GROWTH AND FECUNDITY (IC50)1
           RESULTS FROM 7-DAY TESTS WITH THE MYSID, NYSIDOPSIS BAHIA,
           USING NATURAL SEAWATER (NSW) AND ARTIFICIAL SEAWATER (GP2) AS
           DILUTION WATER AND COPPER (CU) SULFATE AS A REFERENCE
           TOXICANT

Test
GP2
1
125
2
142
3
186
Survival LC50
NSW GP2

177 182

--2 173

190 174

Growth
NSW

208

2

195

IC50
GP2

186

210

179

Fecundity IC50
NSW

177

_-2

168

I  All  LC50/IC50 values in
   No test performed.
                                  298

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TABLE 33.  CONTROL RESULTS FROM 7-DAY SURVIVAL, GROWTH, AND FECUNDITY
           TESTS WITH THE MYSID, MYSIDOPSIS BAHIA, USING NATURAL SEAWATER
           AND ARTIFICIAL SEAWATER (6P2) AS A DILUTION WATER


Test
1
2
3
4
5
6

Survival (%)
NSW GP2
98 93
80 90
--2 95
94 84
--2 94
80 75


Control 1
Growth (mq)
NSW GP2
0.32 0.32
0.40 0.43
--
2 0.40
0.34 0.37
--
2 0.36
0.40 0.41

Fecundity (%)
NSW GP2
73 77
100 95
--2 100
89 83
83
79 93
   Survival  as percent of mysids alive after 7 days;  growth as mean
   individual dry weight; fecundity as percent females With eggs.
   No test performed.
                                    299

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                                  SECTION 15

                                 TEST  METHOD

             SEA URCHIN, ARBACIA PUNCTULATA, FERTILIZATION TEST
                                 METHOD 1008.0


15.1  SCOPE AND APPLICATION

15.1.1  This method, adapted in part from USEPA (1987e), measures the toxicity
of effluents and receiving water to the gametes of the sea urchin, Arbacia
punctulata, during a 1 h and 20 min exposure.  The purpose of the sperm cell
toxicity test is to determine the concentration of a test substance that
reduces fertilization of exposed gametes relative to that of the control.

15.1.2  Detection limits of the toxicity of an effluent or chemical substance
are organism dependent.

15.1.3  Brief excursions in toxicity may not be detected using 24-h composite
samples.  Also, because of the long sample collection period involved in
composite sampling and because the test chambers are not sealed, highly
volatile and highly degradable toxicants in the source may not be detected in
the test.

15.1.4  This test is commonly used in  one of two forms: (1) a definitive test,
consisting of a minimum of five effluent concentrations and a control, and (2)
a receiving water test(s), consisting  of one or more receiving water
concentrations and a control.

15.2  SUMMARY OF METHOD

15.2.1  The method consists of exposing dilute sperm suspensions to effluents
or receiving waters for 1 h.  Eggs are then added to the sperm suspensions.
Twenty minutes after the eggs are added, the test is terminated by the
addition of preservative.  The percent fertilization is determined by
microscopic examination of an aliquot  from each treatment.  The test results
are reported as the concentration of the test substance which causes a
statistically significant reduction in fertilization.

15.3  INTERFERENCES

15.3.1  Toxic substances may be introduced  by contaminants in dilution water,
glassware, sample hardware, and testing equipment (see  Section 5,  Facilities,
Equipment, and Supplies).

15.3.2  Improper effluent sampling and handling may adversely affect test
results (see Section 8, Effluent and Receiving Water Sampling, Sample
Handling,  and Sample Preparation for Toxicity Tests).
                                      300

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 15.4  SAFETY

 15.4.1  See Section  3,  Health  and Safety.

 15.5.   APPARATUS  AND EQUIPMENT
                                                         !

 15.5.1  Facilities for  holding and acclimating  test  organisms.

 15.5.2  Laboratory sea  urchins,  Arbacia punctulata,  culture  unit  --  See
 Subsection  6.17,  culturing  methods below  and  Section 4,  Quality Assurance.   To
 test effluent  or  receiving  water toxicity,  sufficient eggs and sperm must be
 available.


 15.5.3  Samplers  --  automatic  sampler, preferably with sample cooling
 capability,  that  can collect a 24-h  composite sample of 5 L.

 15.5.4  Environmental chamber  or equivalent facility with temperature control
 (20  ±  1°C).


 15.5.5  Water  purification  system -- Millipore  Milli-Q®, deionized water (DI)
 or equivalent.


 15.5.6  Balance -- Analytical,  capable of accurately weighing to  0.00001 g.

 15.5.7  Reference weights,  Class  S  -- for checking performance of balance.
 Weights  should bracket  the  expected weights of  materials to be weighed.

 15.5.8  Air  pump  --  for oil-free  air supply.
                                                         i
 15.5.9   Air  lines, and  air  stones  -- for aerating water containing adults, or
 for  supplying  air to test solutions with low  DO.

 15.5.10  Vacuum suction device  --  for washing eggs.

 15.5.11  Meters, pH  and DO  --  for routine physical and chemical  measurements.

 15.5.12  Standard or micro-Winkler apparatus  -- for determining DO (optional).

 15.5.13  Transformer, 10-12 Volt, with steel electrodes -- for stimulating
 release  of eggs and sperm.
                                        "
 15.5.14  Centrifuge,  bench-top, slant-head,  variable speed -- for washinq
 eggs.
                                                      •

 15.5.15  Fume hood -- to protect the analyst from formaldehyde fumes.

 15.5.16  Dissecting microscope -- for counting diluted egg stock.

 15.5.17  Compound microscope --for examining and counting sperm cells and
fertilized eggs.
                                     301

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15.5.18  Sedgwick-Rafter counting chamber -- for counting egg stock and
examining fertilized eggs.
15.5.19  Hemacytometer, Neubauer -- for counting sperm.
15.5.20  Count register, 2-place --for recording sperm and egg counts.
15.5.21  Refractometer -- for determining salinity.
15.5.22  Thermometers, glass or electronic, laboratory grade --for measuring
water temperatures.
15.5.23  Thermometers, bulb-thermograph or electronic-chart type -- for
continuously recording temperature.
15.5.24  Thermometer, National Bureau of Standards Certified (see USEPA Method
170.1, USEPA, 1979b) -- to calibrate laboratory thermometers.
15.5.25  Ice bucket, covered -- for maintaining live sperm.
15.5.26  Centrifuge tubes, conical -- for washing eggs.
15.5.27  Cylindrical glass vessel, 8-cm diameter -- for maintaining dispersed
egg suspension.
15.5.28  Beakers -- six Class A, borosilicate glass or non-toxic plasticware,
1000 ml for making test solutions.
15.5.29  Glass dishes, flat bottomed, 20-cm diameter -- for holding urchins
during gamete collection.
15.5.30  Wash bottles -- for deionized water, for rinsing small glassware and
instrument electrodes and probes.
15.5.31  Volumetric flasks and graduated cylinders -- Class A, borosilicate
glass or non-toxic plastic labware, 10-1000 ml for making test solutions.
15.5.32  Syringes, 1-mL, and 10-mL, with 18 gauge, blunt-tipped needles  (tips
cut off) -- for collecting sperm and eggs.
15.5.33  Pipets, volumetric -- Class A, 1-100 ml.
15.5.34  Pipets, automatic -- adjustable 1-100 ml.
15.5.35  Pipets, serological -- 1-10 ml, graduated.
15.6.36  Pipet bulbs and fillers -- PROPIPET®, or equivalent.
15.6  REAGENTS AND CONSUMABLE MATERIALS
15.6.1  Sea Urchins, Arbacia punctuTata minimum  12 of each sex.

                                      302

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15.6.2  Food -- kelp, Laminaria sp., or romaine lettuce for the sea urchin,
Arbacia punctulata.

15.6.3  Standard salt water aquarium or Instant Ocean Aquarium (capable of
maintaining seawater at 15°C) -- with appropriate filtration and aeration
system.
                                                          I
15.6.4  Sample containers -- for sample shipment and storage (see Section 8,
Effluent and Receiving Water Sampling, Sample Handling, and Sample Preparation
for Toxicity Tests).

15.6.5  Scintillation vials, 20 ml, disposable -- to prepare test
concentrations.

15.6.6  Tape, colored -- for labelling tubes.

15.6.7  Markers, waterproof -- for marking containers, etc.

15.6.8  Parafilm --to cover tubes and vessels containing test materials.

15.6.9  Gloves, disposable; labcoat and protective eyewear -- for personal
protection from contamination.

15.6.10  Data sheets (one set per test) -- for data recording (see Figures 1,
2, and 3).

15.6.11  Acetic acid, 10%, reagent grade, in seawater -- for preparing killed
sperm dilutions.                                          ;
                                                          i
15.6.12  Formalin,  1%, in 2 ml of seawater -- for preserving eggs (see
Subsection 10.7 Termination of the Test).

15.6.13  Buffers, pH 4, pH 7, and pH 10 (or as per instructions of instrument
manufacturer) -- for standards and calibration check (see USIEPA Method 150.1,
USEPA, 1979b).                                            I

15.6.14  Membranes  and filling solutions for dissolved oxygen probe (see USEPA
Method 360.1, USEPA, 1979b), or reagents -- for modified Winkler analysis.

15.6.15  Laboratory quality assurance samples and standards •-- for the above
methods.

15.6.16  Reference  toxicant solutions -- see Section 4, Quality Assurance.

15.6.17  Reagent water -- defined as distilled or deionized water that does
not contain substances which are toxic to the test organisms.

15.6.18  Effluent,  receiving water, and dilution water -- see Section 7,
Dilution Water, and Section 8, Effluent and Receiving Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests.
                                      303

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TEST DATE:
SAMPLE:
COMPLEX EFFLUENT SAMPLE:
    COLLECTION DATE:
    SALINITY/ADJUSTMENT:
    PH/ADJUSTMENT REQUIRED: _
    PHYSICAL CHARACTERISTICS:
    STORAGE: 	
    COMMENTS: 	
SINGLE COMPOUND:
    SOLVENT (CONC):
    TEST CONCENTRATIONS:
    DILUTION WATER: 	
    CONTROL WATER: 	
    TEST TEMPERATURE:
    TEST SALINITY: _
    COMMENTS: 	
Figure 1.  Data form for (1) fertilization test using sea urchin,  Arbacia
           punctulata.

                                      304

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TEST DATE:

SAMPLE: _
SPERM DILUTIONS:

    HEMACYTOMETER COUNT, E:
                          x  104 = SPM SOLUTION E =
    SPERM CONCENTRATIONS:  SOLUTION E x 40 = SOLUTION A =
                           SOLUTION E x 20 = SOLUTION B =
                           SOLUTION E x  5 = SOLUTION D =
    SOLUTION SELECTED FOR TEST  (

    DILUTION:  SPM/(5 x  107)
                          =  5  x  107 SPM):

                                 DF
                 [(DF) x  10)  -  10  =

     FINAL SPERM  COUNTS = 	
                                          +  SW.,  ml.
 EGG  DILUTIONS:

                                              INITIAL EGG COUNT
     ORIGINAL  EGG  STOCK CONCENTRATION =  10X (INITIAL
                                              EGG COUNT)
     VOLUME  OF SW  TO ADD TO DILUTE EGG STOCK TO 2000/mL:
                                              (EGG COUNT) - 200
                             CONTROL WATER TO ADD EGG STOCK, mL
                                                FINAL EGG COUNT
                                                          ,1
 TEST TIMES:

     SPERM COLLECTED: 	:	

     EGGS COLLECTED:	

     SPERM ADDED:  	

     EGGS ADDED: 	.
     FIXATIVE ADDED:

     SAMPLES READ:
                                                           SPM
                                                           SPM
                                                           SPM
 Figure 2.
Data form (2) for fertilization test using sea urchin, Arbacia
punctulata.
                                       305

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 DATE TESTED:

 SAMPLE: .	
               TOTAL AND  UNFERTILIZED  EGG COUNT AT END OF TEST:
 EFFLUENT
 CONC  (%)
1
                   REPLICATE VIAL
            TOTAL-UNFERT     TOTAL-UNFERT    TOTAL-UNFERT    TOTAL-UNFERT
STATISTICAL ANALYSIS:

    ANALYSIS OF VARIANCE:

         CONTROL: 	
         DIFFERENT FROM CONTROL (P)
    COMMENTS:
Figure 3.  Data form (3) for fertilization test using sea urchin,  Arbacia
           punctulata.
                                     306

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15.6.18.1  Saline test and dilution water -- the salinity of the test water
must be 30%o.  The salinity should vary by no more than ± 2%o among the
replicates.  If effluent and receiving water tests are conducted concurrently,
the salinities of these tests should be similar.
                                                         |
15.6.18.2  The overwhelming majority of industrial and sewage treatment
effluents entering marine and estuarine systems contain little or no
measurable salts.  Exposure of sea urchin eggs and sperm to these effluents
will require adjustments in the salinity of the test solutions.  It is
important to maintain a constant salinity across all treatments.  In addition
it may be desireable to match the test salinity with that of the receiving
water.  Two methods are available to adjust salinities --hypersaline brine
(MSB) derived from natural seawater or artificial sea salts.

15.6.18.3  Hypersaline brine (MSB):  MSB has several advantages that make it
desirable for use in toxicity testing.  It can be made from any high quality,
filtered seawater by evaporation, and can be added to the effluent or to
deionized water to increase the salinity.  MSB derived from natural seawater
contains the necessary trace metals, biogenic colloids, and some of the
microbial components necessary for adequate growth, survival, and/or
reproduction of marine and estuarine organisms, and may be stored for
prolonged periods without any apparent degradation.  However, if 100%o HSB is
used as a diluent, the maximum concentration of effluent that can be tested
will be 80% at 20%o salinity and 70% at 30%o salinity.

15.6.18.3.1  The ideal container for making HSB from natural seawater is one
that (1) has a high surface to volume ratio, (2) is made of a noncorrosive
material, and (3) is easily cleaned (fiberglass containers are ideal).
Special care should be used to prevent any toxic materials from coming in
contact with the seawater being used to generate the brine.  If a heater is
immersed directly into the seawater, ensure that the heater materials do not
corrode or leach any substances that woul.d contaminate the brine.  One
successful method used is a thermostatically controlled heat exchanger made
from fiberglass.  If aeration is utilized, use only oil-free air compressors
to prevent contamination.                                j

15.6.18.3.2  Before adding seawater to the brine generator, thoroughly clean
the generator, aeration supply tube, heater, and any other materials that will
be in direct contact with the brine.  A good quality biodegradable detergent
should be used, followed by several (at least three) thorough deionized water
rinses.                                                                 !
           .
15.6.18.3.3  High quality (and preferably high salinity) seawater should be
filtered to at least 10 /j.m before placing into the brine generator.  Water
should be collected on an incoming tide to minimize the possibility of
contamination.

15.6.18.3.4  The temperature of the seawater is increased slowly to 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%o and that

        .
                                      307

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the temperature does not exceed 40°C.  Additional seawater may be added to the
brine to obtain the volume of brine required.

15.6.18.3.5  After the required salinity is attained, the MSB should be
filtered a second time through a 1 f*m filter and poured directly into portable
containers, (20 L cubitainers or polycarbonate water cooler jugs are
suitable).  The containers should be capped and labelled with the date the
brine was generated and its salinity.  Containers of HSB should be stored in
the dark and maintained under room temperature until used.

15.6.18.3.6  If a source of HSB is available, test solutions can be made by
following the directions below.  Thoroughly mix together the deionized water
and brine before mixing in the effluent.

15.6.18.3.7  Divide the salinity of the HSB by the expected test salinity to
determine the proportion of deionized water to brine.  For example, if the
salinity of the brine is 100%o and the test is to be conducted at 30%o, 100%o
divided by 30%o = 3.3.  The proportion of brine is 1 part in 3.3 (one part
brine to 2.3 parts deionized water).  To make 1 L of seawater at 30%o salinity
from a HSB of 100%o, 300 ml of brine and 700 ml of deionized water are
required.

15.6.16.3.8  Table 1 illustrates the preparation of test solutions at 30%o if
they are made by combining effluent (0%o),  deionized water and HSB (100%o),  or
FORTY FATHOMS® sea salts.

15.6.16.4  Artificial sea salts:  FORTY FATHOMS® brand sea salts (Marine
Enterprises, Inc., 8755 Mylander Lane, Baltimore, MD 21204; 301-3211189) have
been used successfully at the EMSL-Cincinnati, for long-term (6-12 months)
maintenance of stock cultures of sexually mature sea urchins and to perform
the sea urchin fertilization test.  GP2 seawater formulation (Table 2) has
also been used successfully at ERL-Narragansett, RI.

15.6.16.4.1  Synthetic sea salts are packaged in plastic bags and mixed with
deionized water or equivalent.  The instructions on the package of sea salts
should be followed carefully, and the salts should be mixed in a separate
container -- not in the culture tank.  The deionized water used in hydration
should be in the temperature range of 21-26°C.  Seawater made from artifical
sea salts is conditioned (Spotte, 1973; Spotte, et al., 1984; Bower,  1983).-

15.6.16.4.2  The 6P2 reagent grade chemicals (Table 1) should be mixed with
deionized (DI)  water or its equivalent in a container other than the culture
or testing tanks.  The deionized water used for hydration should be between
21-26°C.  The artificial seawater must be conditioned (aerated) for 24 h
before use as the testing medium.  If the solution is to be autoclaved, sodium
bicarbonate is added after the solution has cooled.  A stock solution of
sodium bicarbonate is made up by dissolving 33.6 g NaHC03 in 500 ml of
deionized water.  Add 2.5 mL of this stock solution for each liter of the GP2
artifical seawater.
                                      308

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TABLE 1.   PREPARATION OF TEST SOLUTIONS AT A SALINITY OF 30%o USING
           NATURAL SEAWATER, HYPERSALINE BRINE, OR ARTIFICIAL SEA SALTS
                                          Solutions To Be Combined
            Eff1uent
Effluent  Concentration
Solution
Volume of
Effluent
Solution
  (mL)
Volume of Diluent
Seawater (30%o)

        (mL)
1
2
3
4
5
Control
1001 840
50 420 Solution 1 + 420
25 420 Solution 2 + 420
12.5 420 Solutions
6.25 420 Solution 4
0.0
Total
+ 420
+ 420
420
2080
   This illustration assumes: (1) the use of 5 mL of test solution in each
   of four replicates (total of 20 mL) for the control and five
   concentrations of effluent, (2) an effluent dilution factor of 0.5, (3)
   the effluent lacks appreciable salinity, and (4) 400 mL of each test
   concentration is used for chemical analysis.  A sufficient initial
   volume (840 mL) of effluent is prepared by adjusting the salinity to
   30%o.  In this example, the salinity is adjusted by adding artificial
   sea salts to the 100% effluent, and preparing a serial dilution using
   30%o seawater (natural seawater, hypersaline brine, or artificial
   seawater).  Stir solutions 1 h to ensure that the salts dissolve.  The
   salinity of the initial 840 mL of 100% effluent is adjusted to 30%o by
   adding 25.2 g of dry artificial sea salts (FORTY FATHOMS®).  Test
   concentrations are then made by mixing appropriate volumes of salinity
   adjusted effluent and 30%o salinity dilution water to provide 840 mL of
   solution for each concentration.  If hypersaline brine alone (100%o) is
   used to adjust the salinity of the effluent, the highest concentration,
   of effluent that could be tested would be 70% at 30%o salinity.
                                    309

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  TABLE 2.   REAGENT GRADE CHEMICALS USED IN THE PREPARATION OF GP2
             ARTIFICIAL SEAWATER FOR THE SEA URCHIN, ARBACIA PUNCTULATA,
             TOXICITY TEST1'2'3
           Compound
Concentration
    (9/L)
  Amount (g)
Required for
    20 L
NaCl
Na2S04
KC1
KBr
Na2B407
MgCl2 .
CaCl2 .
SrCl2 .
NaHC03




. 10 H20
6 H20
2 H20
6 H20

] Modified GP2 from Spotte
The constituent salts and
(1990b). The salinity is
3 f\r\n *»••«* L* n ^J*!*lii4>n^J • • * 4» L* *\
21.03
3.52
0.61
0.088
0.034
9.50
1.32
0.02
0.17
et al. (1984).
concentrations
30.89 g/L.
420.6
70.4
12.2
1.76
0.68
190.0
26.4
0.400
3.40
were taken from USEP/
          salinity.
15.6.17  TEST ORGANISMS, SEA URCHINS, ARBACIA PUNCTULATA

15.6.17.1  Adult sea urchins, Arbacia punctulata, can be obtained from
commercial suppliers.  After acquisition, the animals are sexed by briefly
stimulating them with current from a 12 V transformer.  Electrical stimulation
causes the immediate release of masses of gametes that are readily
identifiable by color -- the eggs are red, and the sperm are white.

15.6.17.2  The sexes are separated and maintained in 20-L, aerated fiberglass
tanks, each holding about 20 adults.  The tanks are supplied continuously
(approximately 5 L/min) with filtered natural seawater, or salt water prepared
from commercial sea salts is recirculated.  The animals are checked daily and
any obviously unhealthy animals are discarded.

                                      310

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15.6.17.3  The culture unit should be maintained at 15 ± 3°C, with a water
temperature control device.                               ;

15.6.17.4  The food consists of kelp, Laminaria sp., gathered from known
uncontaminated zones or obtained from commercial supply houses whose kelp
comes from known uncontaminated areas, or romaine lettuce.  Fresh food is
introduced into the tanks at approximately one week intervals.  Decaying food
is removed as necessary.  Ample supplies of food should always be available to
the sea urchins.

15.6.17.5  Natural or artificial seawater with a salinity of 30% is used to
maintain the adult animals, for all washing and dilution steps, and as the
control water in the tests  (see Subsection 15.6.16).      i

15.6.17.6  Adult male and female animals used in field studies are transported
in separate or partitioned  insulated boxes or coolers packed with wet kelp or
paper toweling.  Upon arrival at the field site, aquaria  (or a single
partitioned aquarium) are filled with control water, loosely covered with a
styrofoam sheet and allowed to equilibrate to 15°C before animals are added.
Healthy animals will attach to the kelp or aquarium within hours.

15.6.17.7  To successfully  maintain  about 25 adult animals for 7 days at a
field site, a screen-partitioned, 40-L glass aquarium using  aerated,
recirculating, clean saline water  (30%o) and a gravel bed filtration system,
is housed within  a water bath, such  as FORTY FATHOMS® or  equivalent  (15°C).
The  inner aquarium is used  to avoid  contact of animals and water bath with
cooling coils.

15.7  EFFLUENT AND RECEIVING WATER COLLECTION, PRESERVATION, AND STORAGE

15.7.1  See Section 8,  Effluent  and  Receiving Water Sampling,  Sample Handling,
and  Sampling  Preparation for Toxicity Tests.
                                                          i
15.8  CALIBRATION AND STANDARDIZATION
                                                          i
15.8.1  See Section 4,  Quality Assurance.

15.9  QUALITY CONTROL
                                                          I

15.9.1   See Section 4,  Quality Assurance.                 \

15.10   TEST PROCEDURES

15.10.1  TEST SOLUTIONS

 15.10.1.1   Receiving  Waters
           -
 15.10.1.1.1   The  sampling  point  is determined  by the  objectives  of the  test.
At estuarine  and  marine sites,  samples  are  usually collected at  mid-depth.
 Receiving water toxicity is determined  with samples used directly as collected
 or with samples passed  through  a 60 yum  NITEX®  filter  and compared without
dilution against  a control.  Using four replicate chambers  per test,  each

                                      311

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 containing 5 ml,  and 400 mL for chemical  analysis,  would  require  approximately
 420 ml or more of sample per test.

 15.10.1.2  Effluents

 15.10.1.2.1  The  selection  of the  effluent  test  concentrations  should  be  based
 on  the objectives of the study.  A dilution factor  of 0.5 is  commonly  used.
 A dilution factor of 0.5 provides  precision of ± 100%,  and testing  of
 concentrations between  6.25% .and 100% effluent using  only five  effluent
 concentrations (6.25%,  12.5%,  25%,  50%,  and 100%).  Test  precision  shows
 little improvement as dilution factors are  increased  beyond 0.5 and declines
 rapidly if smaller dilution factors are  used.  Therefore,  USEPA recommends the
 use of the ;> 0.5  dilution factor.   If 100%o HSB  is  used as a  diluent,  the
 maximum concentration of effluent  that can  be  tested  will  be  80%  at 20%o  and
 70% at 30%o salinity.

 15.10.1.2.2  If the effluent is  known or  suspected  to be  highly toxic, a  lower
 range  of effluent concentrations should  be  used  (such as  25%, 12.5%, 6.25%,
 3.12%,  and 1.56%).

 15.10.1.2.3  Just prior to  test  initiation  (approximately  1 h), a sufficient
 quantity of the sample  to make the  test  solutions should  be adjusted to the
 test temperature  (20 ±  1°C)  and  maintained  at  that  temperature  during  the
 addition of dilution water.

 15.10.1.2.4  The  test should begin  as  soon  as  possible, preferably  within 24 h
 of  sample collection.   The  maximum  holding  time  following  retrieval of the
 sample  from the sampling device  should not  exceed 36  h  for off-site toxicity
 tests  unless  permission is  granted  by  the permitting  authority.   In no case
 should  the sample be used 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).

 15.10.1.2.5   Effluent dilutions  should be prepared  for  all replicates  in  each
 treatment  in  one  beaker to minimize variability  among the  replicates.  The
 test chambers are labelled with  the test concentration  and replicate number.
 Dispense  into the appropriate  effluent dilution  chamber.

 15.10.1.3   Dilution  Water

 15,10.1.3.1   Dilution water  may  be  uncontaminated natural   seawater  (receiving
water),  HSB prepared from natural seawater,  or artifical seawater FORTY
 FATHOMS®  or GP2 sea  salts (see Table 2 and  Section  7, Dilution Water).
 Prepare 3  L of  control  water at  30%o using  HSB or artificial  sea salts (see
Table 1).  This water is  used  in all washing and diluting  steps and as control
water in the test.   Natural   seawater and local waters may  be  used as
additional controls.

15.10.2  COLLECTION OF  GAMETES FOR THE TEST

15.10.2.1  Select four  females and place in  shallow bowls, barely covering the
shell with seawater.  Stimulate  the release  of eggs by touching the shell  with

                                      312

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steel electrodes connected to a 10-12 volt transformer (about 30 seconds each
time).  Collect the eggs from each female using a 10 ml disposable syringe
fitted with an 18-gauge, blunt-tipped needle (tip cut off).  Remove the needle
from the syringe before adding the eggs to a conical centrifuge tube.  Pool
the eggs.  The egg stock may be held at room temperature for several hours
before use.  Note:  Eggs should be collected first to eliminate possibility of
pre-fertilization.

15.10.2.2  Select four males and place in shallow bowls, biirely covering the
animals with seawater.  Stimulate the release of sperm as described above.
Collect the sperm (about 0.25 ml) from each male, using a 1-3 ml disposable
syringe fitted with an 18-gauge, blunt-tipped needle.  Pool the sperm.
Maintain the pooled sperm sample on ice.  The sperm must be used in a toxicity
test within 1 h of collection.                            !

15.10.3  PREPARATION OF SPERM DILUTION FOR USE IN THE TEST
                                                          I
15.10.3.1  Using control water, dilute the pooled sperm sample to a
concentration of about 5 X 10  sperm/mL (SPM).   Estimate the sperm
concentration as described below:

   1. Make a sperm dilutions of 1:50, 1:100, 1:200, and 1:400, using 30%o
      seawater, as follows:
                                                          i
      a. Add 400 //L of collected sperm to 20 ml of  seawater in Vial A. Mix by
         gentle pipetting using a 5-mL pipettor, or by inversion;
      b. Add 10 ml of sperm suspension from Vial A  to 10 ml of seawater in
         Vial B.  Mix by gentle pipetting using a 5-mL pipettor, or by
         inversion;
      c. Add 10 mL of sperm suspension from Vial B  to 10 mL of seawater in
         Vial C.  Mix by gentle pipetting using a 5-mL pipettor, or by
         inversion;
      d. Add 10 mL of sperm suspension from Vial C  to 10 mL of seawater in
         Vial D.  Mix by gentle pipetting using a 5-mL pipettor, or by
         inversion;
      e. Discard  10 mL  from Vial D.  (The volume of  all suspensions  is 10 mL).

   2. Make a 1:2000 killed sperm suspension  and determine the SPM.

      a. Add 10 mL 10%  acetic  acid  in seawater to Vial C.  Cap Vial C and  mix
         by  inversion.
      b. Add 1 mL of  killed  sperm from Vial  C to 4  mL of seawater  in Vial  E.
         Mix by gentle  pipetting with a  4-mL pipettor.
      c. Add sperm from Vial  E to both  sides of the Neubauer  hemacytometer.
         Let the  sperm  settle  15 min.
      d. Count  the number  of  sperm  in the central 400 squares on both sides  of
         the hemacytometer using  a  compound  microscope  (IGiOX). Average  the
         counts from  the two  sides.                       \
      e. SPM  in Vial  E  =  10  x  average count.             ;

   3. Calculate the SPM in all  other suspensions using  the  SPM  in  Vial  E
       above:

                                      313

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                 SPM  in Vial A  =  40 x SPM  in Vial  E
                 SPM  in Vial B  =  20 x SPM  in Vial  E
                 SPM  in Vial D  =   5 x SPM  in Vial  E
                 SPM  in original sperm sample  =  2000 x SPM in Vial E

   4. Dilute the sperm suspension with a SPM greater than 5 x 107 SPM to 5 x
      107 SPM.

                Actual SPM/(5 x 107)  = dilution factor (DF)

                [(DF) x 10] - 10 = mL of seawater to add to vial.

   5. Confirm the sperm count by sampling from the test stock.  Add 0.1 ml
      of test stock to 9.9 ml of 10% acetic acid in seawater, and count with
      the hemacytometer.  The count should average 50 ± 5.

15.10.4  PREPARATION OF EGG SUSPENSION FOR USE IN THE TEST  Note:  The egg
suspension may be prepared during the 1-h sperm exposure.

15.10.4.1  Wash the pooled eggs three times using control water with gentle
centrifugation (500xg for 3 minutes using a tabletop centrifuge).  If the wash
water becomes red, the eggs have lysed and must be discarded.

15.10.4.2  Dilute the egg stock, using control water, to about 2000 eggs/mL.

   1. Transfer the eggs to a glass beaker containing 200 ml of control  water
      ("egg stock").

   2. Mix the egg stock using an air-bubbling device.  Using a wide-mouth
      pi pet tip,  transfer 1 ml of eggs from the egg stock to a vial
      containing 9 ml of control water.   (This vial  contains an egg suspension
      diluted 1:10 from egg stock).

   3. Mix the contents of the vial  by inversion.   Using a wide-mouth pipet
      tip,  transfer 1 ml of eggs from the vial to a Sedgwick-Rafter counting
      chamber.  Count all  eggs in the chamber using a dissecting microscope at
      24X "egg count".

   4. Calculate the concentration of eggs in the  stock.  Eggs/mL = 10X  (egg
      count).  Dilute the egg stock to 2000 eggs/mL by the formula below.

      a. If the egg count is  equal  to or greater  than 200:
         (egg count) - 200 =  volume (mL)  of control  water to add to  egg stock.
      b. If the egg count is  less than 200,  allow the eggs to settle and
         remove enough control  water  to  concentrate  the eggs to greater
         than 200,  repeat the count,  and dilute the  egg stock as in  a.
         above.   NOTE:  It requires 24 mL of a egg stock solution for each
         test with  a control  and five exposure concentrations.
      c. Transfer 1 mL of the diluted egg stock to a vial  containing 9  mL  of
         control  water.   Mix  well,  then  transfer  1 mL from the  vial  to   a
         Sedgwick-Rafter counting chamber.   Count all  eggs  using a

                                     314

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         dissecting microscope.  Confirm that the final egg count =
         2000/mL (± 200).                                 i

15.10.5  LIGHT, PHOTOPERIOD, SALINITY AND TEMPERATURE
                                                          I
15.10.5.1  The light quality and intensity should be at ambient laboratory
levels 10-20 /iE/m /s (50-100 ft-c)  with a photoperiod of 16 h light and 8 h
darkness.  The water temperature in the test chambers should be maintained at
20 ± 1°C. ' The test salinity should be in the range of 28 to 32%o.  The
salinity should vary by no more that ± 2%o among the chambers on a given day.
If effluent and receiving water tests are conducted concurrently, the
salinities of these tests should be similar.

15.10.6  DISSOLVED OXYGEN (DO) CONCENTRATION

15.10.6.1  Aeration may affect the toxicity of effluent and should be used
only as a last resort to maintain a satisfactory DO.  The DO concentrations
should be measured on new solutions at the start of the test (Day 0).  The DO
should not fall below 4.0 mg/L (see Section 8, Effluent and Receiving Water
Sampling, Sample Handling, and Sample Preparation for Toxicity Tests).   If it
is necessary to aerate, all treatments and the control should be aerated.  The
aeration rate should not exceed 100 bubbles/minute, using a pipet with a 1-2
mm orifice, such as a 1 mL KIMAX® serological pipet No. 37033, or equivalent.

15.10.7  OBSERVATIONS DURING THE TEST
                                                          i

15.10.7.1  Routine Chemical and Physical Observations

15.10.7.1.1  DO is measured at the beginning of the exposure period in one
test chamber at each test concentration and in the control.
                                                          i-
15.10.7.1.2  Temperature, pH,  and salinity are measured at the beginning of
the exposure period in one test chamber at each concentration and in the
control.  Temperature should also be monitored continuously observed and
recorded daily for at least two locations in the environmental control system
or the samples.  Temperature should be measured in a sufficient number of test
chambers at least at the end of the test to determine temperature variation  in
environmental chamber.

15.10.7.1.3  The pH is measured in the effluent sample each day before new
test solutions are made.                                  i
                                                          i

15.10.7.1.4  Record all the measurements on the data sheet.

15.10.7.2  Routine  Biological  Observations

15.10.7.2.1  Fertilization will be determined by the presence of a
fertilization membrane surrounding the egg.
                                      315

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15.10.8  START OF THE TEST

15.10.8.1  Effluent/deceiving water samples are adjusted to salinity of 30%o.
Four replicates (minimum of three) are prepared for each test concentration,
using 5 ml of solution in disposable liquid scintillation vials.  A 50% (0.5)
concentration series can be prepared by serially diluting test concentrations
with control water.  Sufficient test solution is prepared at each effluent
concentration to provide additional volume for chemical analyses, at the high,
medium, and low test concentrations.

15.10.8.2  All test samples are equilibrated at 20°C ± 1°C before addition of
sperm.

15.10.8.3  Within 1 h of collection add 100 pL of appropriately diluted sperm
to each test vial.  Record the time of sperm addition.

15.10.8.4  Incubate all test vials at 20 ± 1°C for 1 h.

15.10.8.5  Mix the diluted egg suspension (2000 eggs/mL), using gentle
bubbling.  Add 1 ml of diluted egg suspension to each test vial using a wide
mouth pi pet tip.  Incubate 20 min at 20 ± 1°C.

15.10.9  TERMINATION OF THE TEST

15.10.9.1  Terminate the test and preserve the samples by adding 2 ml of 1%
formalin in seawater to each vial.

15.10.9.2  Vials should be evaluated within 48 hours.

15.10.9.3  To determine fertilization, transfer about 1 ml eggs from the
bottom of a test vial to a Sedgwick-Rafter counting chamber.  Observe the eggs
using a compound microscope (100X).  Count between 100 and 200 eggs/sample.
Record the number counted and the number unfertilized.  Fertilization is
indicated by the presence of a fertilization membrane surrounding the egg.
Note: adjustment of the microscope to obtain proper contrast may be required
to observe the fertilization membrane.  Because samples are fixed in formalin,
a ventilation hood is set up surrounding the microscope to protect the analyst
from prolonged exposure to formaldehyde fumes.

15.11  SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA

15.11.1  A summary of test conditions and test acceptability criteria is
listed in Table 3.

15.12  ACCEPTABILITY OF TEST RESULTS

15.12.1  The spermregg ratio routinely employed should result in fertilization
of 70%-90% of the eggs in the control chambers.
                                      316

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TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
         SEA URCHIN, ARBACIA PUNCTULATA, FERTILIZATION TEST WITH EFFLUENT
         AND RECEIVING WATERS
1.  Test type:

2.  Salinity:


3.  Temperature:

4.  Light quality:


5.  Light intensity:


6.  Test chamber size:



7.  Test solution volume:

8.  No. of sea urchins:
9.  No. egg and sp.erm cells
     per chamber:
10. No. replicate chambers
     per concentration:

11. Dilution water:
12. Effluent concentrations:
Static
                    I
30%o (± 2%o of the selected test
salinity)
                    i
20 ± 1°C

Ambient laboratory light during test
preparation         j

10-20 ME/m2/s,  or 50-100 ft-c (Ambient
laboratory levels)

Disposable (glass) liquid
scintillation vials (20 mL capacity),
presoaked in control water

5 mL

Pooled sperm from four males and
pooled eggs from four females are
used per test
About 2000 eggs and 5, 000,000 sperm
cells per vial
4 (minimum of 3)

Uncontaminated source of natural
seawater; deionized water mixed with
hypersaline brine or artificial sea
salts (HW Marinemix®, FORTY FATHOMS®,
6P2, or equivalent) !

Effluents: Minimum of 5 and a control
Receiving waters: 100% receiving
water or minimum of 5 and a control
                                    317

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TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
         SEA URCHIN, ARBACIA PUNCTULATA, FERTILIZATION TEST WITH EFFLUENT
         AND RECEIVING WATERS (CONTINUED)
13. Test dilution factor:


14. Test duration:

15. Endpoint:

16. Test acceptability
    criteria:


17. Sampling requirements:
18. Sample volume required:
Effluents:  > 0.5
Receiving waters:  None or > 0.5

1 h and 20 min

Fertilization of sea urchin eggs
70% - 90% egg fertilization in
controls

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,
Subsection 8.5.4)

1 L per test
                                    318

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15.13  DATA ANALYSIS
                                                          i
15.13.1  GENERAL                                          I

15.13.1.1  Tabulate and summarize the data.  Calculate the proportion of
fertilized eggs for each replicate.  A sample set of test data is listed in
Table 4.

15.13.1.2  The statistical tests described here must be used with a knowledge
of the assumptions upon which the tests are contingent.  The assistance of a
statistician is recommended for analysts who are not proficient in statistics,
                           '

    TABLE 4.   DATA FROM SEA URCHIN,  ARBACIA PUNCTULATA,  FERTILIZATION  TEST1
  Copper
  Concentration
               No. of Eggs     No. of Eggs    Proportion
Replicate         Counted         Fertilized    Fertilized
Control


A
B
C
100
100
100
85
78
87
0.85
0.78
0.87
   2.5
   5.0
  10.0
  20.0
  40.0
   A
   B
   C

   A
   B
   C

   A
   B
   C

   A
   B
   C

   A
   B
   C
100
100
100

100
100
100

100
100
100

100
100
100

100
100
100
81
65
71

63
74
78

63
66
51

41
41
37

12
30
26
0.81
0.65
0.71

0.63
0.74
0.78

0.63
0.66
0.51

0.41
0.41
0.37

0.12
0.30
0.26
     Tests performed by Dennis M. McMullen, Technology Applications, Inc.,
     EMSL,  Cincinnati,  OH.
15.13.1.3  The endpoints of toxicity tests using the sea urchin are based on
the reduction in proportion of eggs fertilized.  The IC25 and the IC50 are
calculated using the Linear Interpolation Method (see Section 9, Chronic
Toxicity Test Endpoints and Data Analysis).  LOEC and NOEC values for
fecundity are obtained using a hypothesis testing approach such as Dunnett's
Procedure (Dunnett, 1955) or Steel's Many-one Rank Test (Steel, 1959; Miller,
                                      319

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1981) (see Section 9).  Separate analyses are performed for the estimation of
the LOEC and NOEC endpoints and for the estimation of IC25 and IC50.  See the
Appendices for examples of the manual computations, and examples of data input
and program output.

15.13.2  EXAMPLE OF ANALYSIS OF SEA URCHIN, ARBACIA PUNCTULATA, FERTILIZATION
         DATA

15.13.2.1  Formal statistical analysis of the fertilization data is outlined
in Figure 4.  The response used in the analysis is the proportion of
fertilized eggs in each test or control chamber.  Separate analyses are
performed for the estimation of the NOEC and LOEC endpoints and for the
estimation of the IC25 and IC50 endpoints.  Concentrations at which there are
no eggs fertilized in any of the test chambers are excluded from statistical
analysis of the NOEC and LOEC, but included in the estimation of the IC25 and
IC50.

15.13.2.2  For the case of equal numbers of replicates across all
concentrations and the control, the evaluation of the NOEC and LOEC endpoints
is made via a parametric test, Dunnett's Procedure, or a nonparametric test,
Steel's Many-one Rank Test, on the arc sine square root transformed data.
Underlying assumptions of Dunnett's Procedure, normality and homogeneity of
variance, are formally tested.  The test for normality is the Shapiro-Wilk's
Test, and Bartlett's Test is used to test for homogeneity of variance.  If
either of these tests fails, the nonparametric test, Steel's Many-one Rank
Test, is used to determine the NOEC and LOEC endpoints.  If the assumptions of
Dunnett's Procedure are met, the endpoints are estimated by the parametric
procedure.

15.13.2.3  If unequal numbers of replicates occur among the concentration
levels tested, there are. parametric and nonparametric alternative analyses.
The parametric analysis is a t test with the Bonferroni adjustment (see
Appendix D).  The Wilcoxon Rank Sum test with the Bonferroni adjustment is the
nonparametric alternative.

15.13.2.4  Example of Analysis of Fecundity Data

15.13.2.4.1  This example uses toxicity data from a sea urchin, Arbacia
punctulata, fertilization test performed with copper.  The response of
interest is the proportion of fertilized eggs, thus each replicate must first
be transformed by the arc sine square root transformation procedure described
in Appendix B.  The raw and transformed data, means and variances of the
transformed observations at each copper concentration and control are listed
in Table 5.  The data are plotted in Figure 5.

15.13.2.5  Test for Normality

15.13.2.5.1  The first step of the test for normality is to center the
observations by subtracting the mean of all observations within a
concentration from each observation in that concentration.  The centered
observations are summarized in Table 6.
                                      320

-------
         STATISTICAL ANALYSIS OF SEA URCHIN FERTILIZATION TEST
                              FERTILIZATION DATA
                         PROPORTION OF FERTILIZED EGGS
      POINT ESTIMATION
          ARC SINE
      TRANSFORMATION
     ENDPOINT ESTIMATE
         IC25, IC50
     SHAPIRO-WILK-S TEST
                         NON-NORMAL DISTRIBUTION
                   NORMAL DISTRIBUTION
        HOMOGENEOUS
          VARIANCE
                               BARTLETTS TEST
                               HETEROGENEOUS
                                  VARIANCE
              EQUAL NUMBER OF
                REPLICATES?
                      EQUAL NUMBER OF
                         REPLICATES?
          NO
YES
     T-TESTWITH
     BONFERRONI
     ADJUSTMENT
I YES
                                                               NO
        STEEL'S MANY-ONE
           RANK TEST
              WILCOXON RANK SUM
                  TEST WITH
            BONFERRONI ADJUSTMENT
                             ENDPOINT ESTIMATES
                                 NOEC, LOEC
Figure 4.    Flowchart for statistical  analysis  of sea urchin, Arbacia
            punctulata, by point estimation.

                                    321

-------
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                           322

-------
         TABLE  5.  SEA URCHIN, ARBACIA PUNCTULATA, FERTILIZATION DATA
Copper Concentration (//g/L)
Repl
RAW
ARC SINE
TRANSFORMED
Mean (YJ
?T
i
TABLE 6
icate Control
A 0.
B 0.
C 0.
A 1.
B 1.
C 1.
85
78
87
173
083
202
1.153
0.004
1
. CENTERED
2.5
0.
0.
0.
1.
0.
1.
1.
0.
2
81
65
71
120
938
002
020
009
OBSERVATIONS
5.0
0.63
0.74
0.78
0.917
1.036
1.083
1.012
0.007
3
10.0
0.63
0.66
0.51
0.917
0.948
0.795
0.887
0.007
4
FOR SHAPIRO-MILK'S
20.0
0.41
0.41
0.3.7
0.695
0.695
0.654
0.681
0.001
!>
EXAMPLE
40.0
0.12
0.30
0.26
0.354
0.580
0.535
0.490
0.014
6

Copper Concentration (//g/L)
Replicate
A
B
C
Control
0.020
-0.070
0.049

0
-0
-0
2.5
.100
.082
.018


5.0
-0.095
0.024
0.071
10.0
0.030
0.061
-0.092
20.0
0.014
0.014
-0.027
40.0
-0.136
0.090
0.045
15.13.2.5.2  Calculate the denominator,  D,  of the statistic:


                                D = £ (x,-x)2
                                    i-l


    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  = 18              !


                                      X  = _L_ (0)  = 0
                                          18


                                      D  = 0.0822
                                     323

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15.13.2.5.4  Order the centered observations from smallest to largest
                         <  ... 
1
2
3
4
5
6
7
8
9
-0.136
-0.095
-0.092
-0.082
-0.070
-0.027
-0.018
0.014
0.014
10
11
12
13
14
15
16
17
18
0.020
0.024
0.030
0.045
0.049
0.061
0.071
0.090
0.100
 15.13.2.5.5   From Table'4,  Appendix B,  for  the  number  of observations,  n,
 obtain  the coefficients  a15 a2, ... ak where k is n/2 if n is even and (n-l)/2       *
 if n  is odd.   For the  data  in  this example,  n = 18 and k = 9.   The  a,- values       \\
 are listed in Table 8.

 15.13.2.5.6   Compute the test  statistic,  W,  as  follows:
                         W =
                              D i=i

 The differences,  x(n"i+1) -  X(i),  are listed in Table 7.  For the data in this
 example:

                W =    1     (0.2782)2 =  0.942
                    0.0822

 15.13.2.5.7  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 example, the critical value at a
 significance level of 0.01 and  n = 18 observations is 0.858.  Since W = 0.942
 is greater than the critical value,  conclude that the data are normally
 distributed.
                                       324

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                                                                                          1
     TABLE 8.  COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-MILK'S EXAMPLE
i
1
2
3
4
5
6
7
8
9
ai
0.4886
0.3253
0.2553
0.2027
0.1587
0.1197
0.0837
0.0496
0.0163
« _
XC12) .
X(11) .
X(10) _

XC1)
X(2)
X(3)
X(4>
X(5)
X(6)
J(7)
x<8>
X(9,
15.13.2.6  Test for Homogeneity of Variance


15.13.2.6.1  The test used to examine whether the variation in the proportion
of fertilized eggs is the same across all copper concentrations including the
control, is Bartlett's Test (Snedecor and Cochran, 1980). The test statistic
is as follows:
[(
in
                                      S2 - £
                      B =
                                              v, in Sf]
Where: V
         ,   =  degrees of freedom for each copper concentration and control,
               V,-  =  (n,- - 1)


         p   =  number of levels of copper concentration  including the control
       n

       In
         ,   =  the number of replicates  for concentration  1.

            =  loge
         i   =  1,2,  ...,  p where  p  is  the  number  of concentrations  including
               the  control
         C =
                            i=l
                                      1=1
                                     325

-------
15.13.2.6.2  For the data in this example (see Table 5),  all  copper
concentrations including the control have the same number of  replicates  (nf
3 for all i).  Thus, V,- = 2 for all  i .

15.13.2.6.3  Bartlett's statistic is,  therefore:
B =  [ (12) Irz (0.0007) -2
                                                    1/1.194
                    =  [12(-4.962) - 2(-31. 332)]/1.194

                    =  3.122/1.194

                    =  3.615

15 13.2.6.4  B is approximately distributed as chi -square with p-1 degrees of
freedom, when the variances are in fact the same.  Therefore, the appropriate
critical value for this test, at a significance level of 0.01 with 5 degrees
of freedom, is 15.09.  Since B = 2.615 is less than the critical value of
15.09, conclude that the variances are not different.

15.13.2.7  Dunnett's Procedure

15.13.2.7.1  To obtain an estimate of the pooled variance for the Dunnett's
Procedure, construct an ANOVA table as described in Table 9.
                            TABLE 9.  ANOVA TABLE
Source

Between
Within
Total
df

P - 1
N - p
N - 1
Sum of Squares
(SS)
SSB
SSW
SST
Mean Square(MS)
(SS/df)
SB = SSB/ (p-1)
Sy = SSW/(N-p)

   Where:  p  = number of copper concentrations including  the  control

           N  = total  number of observations n, + n2 ... + np

           n,-  - number of observations  in  concentration  i
                                      326

-------
        SSB =  2^TJ/n±-G2/N       Between  Sum  of  Squares
              i-l
        SST =  E EFy-GV-W        Total  Sum  of  Squares
        SSW =  SST-SSB             Within  Sum  of  Squares
                                                         I
          6  =  the grand  total  of  all  sample observations,   G = £ r,
                                                                i-i
          T,-  =  the total  of the replicate measurements for concentration i
         YJJ =  the jth  observation for concentration i (represents the
               proportion of fertilized eggs for copper concentration i in
               test chamber j)
15.13.2.7.2  For the data in this  example:
                   n1 = n2 = n3 = n4 =  n5 = n6 =  3
                   N = 18
                   T, = ¥„ + Y12 + Y13  = 3.458             |
                   T2 = Y21 + Y22 + Y23  = 3.060
                   T3 = Y31 + Y32 + Y33  = 3'036
                   T4 = Y41 + Y42 + Y43  = 2.660             ;
                   fc: fe t fc: fc:!:«
                   G = T,- + T2 + T3 +  T4 + T5 + T6  =  15.727
                 SSB =
                       i=l
                                            2
                      = (43.950)/3  -  (15.727)/18  = 0.909
                 SST =
                        14.732  -  (15.727)2/18 - 0.991
                 SSW = SST-SSB   = 0.991  -  0.909  =  0.082
                                     327

-------
                   SB = SSB/(p-l) = 0.909/(6-l) = 0.182

                   $„ = SSW/(N-p) = 0.082/(18-6) = 0.007

15.13.2.7.3  Summarize these calculations in the ANOVA table  (Table 10)
            TABLE 10.  ANOVA TABLE FOR DUNNETT'S PROCEDURE EXAMPLE
Source
Between
Within
Total
df
5
12
17
Sum of Squares
(SS)
0.909
0.082
0.991
Mean Square(MS)
(SS/df)
0.182
0.007

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 fertilized eggs for copper concentration i
        Y,  = mean proportion fertilized eggs for the control
        Sw  » square root of the within mean square
        n.,  - number of replicates for the control
        n,-  = number of replicates for concentration i.
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.
                                      328

-------
15.13.2.7.5  Table 11 includes the calculated t values for each  concentration
and control combination.   In this example,  comparing the £.5
concentration with the control the calculation is as follows:
                              (1.153-1.020)

                           [0 . 084v/(l/3) + (1/3)]
=1.939
                           TABLE 11.   CALCULATED T VALUES
              Copper Concentration (M9/L)
2.5
5.0
10.0
20.0
40.0
2
3
4
5
6
1.939
2.056
3.878
6.882
9.667
15.13.2.7.6  Since the purpose of this test is to detect a significant
decrease in the proportion of fertilized eggs, a one-sided test is
appropriate.  The critical value for this one-sided test is found in  Table  5,
Appendix D.  For an overall alpha level  of 0.05, 12 degrees of freedom  for
error and five concentrations (excluding the control)  the critical  value  is
2.50.  The mean proportion of fertilized eggs for concentration i is
considered significantly less than the mean proportion of fertilized  eggs for
the control if t5 is  greater than the  critical  value.   Therefore, the 10.0
//g/L, 20.0 /jg/L and 40.0 /Kj/L concentrations have a significantly lower mean
proportion of fertilized eggs than the control.  Hence the NOEC is  5.0  //g/L
and the LOEC is 10.0 /^g/L.

15.13.2.7.7  To quantify the sensitivity of the test,  the minimum significant
difference (MSD) that can be statistically detected may be calculated:
                          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)

        n1 =   the number of replicates in the control.

15.13.2.7.8  In this example,
                      MSD = 2.50 (0.084)^(1/3) + (1/3)


                           = 2.50 (0.084)(0.816)

                           = 0.171


                                     329

-------
15.13.2.7.9  The MSD (0.171) is in transformed units.  To determine the MSD in
terms of proportion of fertilized eggs, carry out the following conversion.

   1. Subtract the MSD from the transformed control mean.

                          1.153 - 0.171 = 0.982

   2. Obtain the untransformed values for the control mean and the difference
      calculated in step 1 of 13.2.7.9.

                        [ Sine (1.153) ]2 = 0.835

                        [ Sine (0.982) ]2 = 0.692

   3. The untransformed MSD (MSDJ is determined by subtracting the
      untransformed values from step 2 in 14.2.7.9.

                      MSDU = 0.835 - 0.692 = 0.143

15.13.2.7.10  Therefore, for this set of data, the minimum difference in mean
proportion of fertilized eggs between the control and any copper concentration
that can be detected as statistically significant is 0.143.

15.13.2.7.11  This represents a 17% decrease  in  the proportion of fertilized
eggs from the control.

15.13.2.8  Calculation of the ICp

15.13.2.8.1  The fertilization data in Table  4 are utilized  in this example.
Table 12 contains the mean proportion of fertilized eggs for each toxicant
concentration.  As can be seen, the observed  means are monotonically non-
increasing with respect to concentration.  Therefore, it is  not necessary  to
smooth  the means prior to calculating the  ICp;  (see Figure 5 for a plot of the
response curve).

15.13.2.8.2  An IC25 and  IC50 can be estimated using the Linear Interpolation
Method.  A 25% reduction  in mean  proportion of fertilized eggs, compared to
the  controls, would result  in a mean proportion  of 0.625, where M/l-p/100) =
0.833(1-25/100).  A 50% reduction in mean  proportion of  fertilized eggs,
compared to the controls, would result in  a mean proportion  of 0.417.
Examining the means and their associated concentrations  (Table 12), the
response, 0.625, is bracketed by  C3 = 5.0 ^g/L copper and C4 = 10.0 fj.g/1
copper.  The response, 0.417, is  bracketed  by C4 = 10.0 /j.g/1 copper and C5 =
20.0 /ig/L copper.
                                      330

-------
        TABLE 12.   SEA URCHIN,  ARBACIA PUNCTULAJA,  MEAN PROPORTION
                   OF FERTILIZED EGGS
Copper
Cone.
(M9/L)
Control
2.5
5.0
10.0
20.0
40.0


i
1
2
3
4
5
6
Response_
Means, Y^
(proportion)
0.833
0.723
0.717
0.600
0.397
0.227
Smoothed
Means, Mn-
(proportion)
0.833
0.723
0.717
0.600
0.397
0.227
i
15.13.2.8.3  Using the equation from Section 4.2 in Appendix L, the estimate
of the IC25 is calculated as follows:
                  IC25 = 5.0 + [0.833(1 - 25/100) - 0.717]  (10.0 - 5.0)
                       = 8.9
                                                          1(0.600 1- 0.717)
15.13.2.8.4  Using the equation from Section 4.2 in Appendix L, the estimate
of the IC50 is calculated as follows:
                  IC50 = 10.0 +  [0.833(1  - 50/100)  - 0.600]   (20.0  -  10.0)
                          19.0
                                                            (0.397  -  0.600)
15.13.2.8.5  When the  ICPIN program was  used to  analyze this  set  of  data,
requesting 80 resamples, the estimate of the IC25 was 8.9286  fj.g/1.   The
empirical 95.0% confidence interval for  the true mean was  3.3036  /^g/L  to
14.6025 Mg/L.  The computer program output for the  IC25 for this  data  set  is
shown  in  Figure 6.
     ,
15.13.2.8.6  When the  ICPIN program was  used to  analyze this  set  of  data,
requesting 80 resamples, the estimate of the IC50 was 19.0418 /^g/L.
                                                          i
                                      331

-------
Cone. ID

Cone. Tested
Response
Response
Response
1
2
3
1
0
.85
.78
.87
2
2.5
.81
.65
.71
3
5.0
.63
.74
.78
4
10.0
.63
.66
.51
5
20.0
.41
.41
.37
6
40.0
.12
.3
.26
*** Inhibition Concentration  Percentage  Estimate ***
Toxicant/Effluent: Copper
Test Start Date:    Test Ending Date:
Test Species: SEA URCHIN, Arbacia punctulata
Test Duration:
DATA FILE: urchin.icp
OUTPUT FILE: urchin.i25
Cone.
ID
1
2
3
4
5
6
Number
Replicates
3
3
3
3
3
3
Concentration
ug/1
0.000
2.500
5.000
10.000
20.000
40.000
Response
Means
0.833
0.723
0.717
0.600
0.397
0.227
Std. Pooled
Dev. Response Means
0.047
0.081
0.078
0.079
0.023
0.095
0.833
0.723
0.717
0.600
0.397
0.227
The Linear Interpolation Estimate:     8.9286   Entered P Value: 25
— .•••,... — — — — ___ — ____________________	__„_	_ _ _ _ _	_ _ _ _ _ _	_ _ _ .. _	_.
Number of Resamplings:   80
The Bootstrap Estimates Mean:   8.7092 Standard Deviation:     1.5324
Original Confidence Limits:   Lower:     6.2500 Upper:    11.6304
Expanded Confidence Limits:   Lower:     3.3036 Upper:    14.6025
Resampling time in Seconds:     1.59  Random Seed: 1834854321
                 Figure 6.   ICPIN  program  output  for  the  IC25.

                                     332

-------
The empirical 95.0% confidence interval for the true mean was; 16.1083 ng/l to
23.6429 M9/L-  The computer program output for the IC50 for this data set is
shown in Figure 7.

15.14  PRECISION AND ACCURACY

15.14.1  PRECISION                                        I

15.14.1.1  Single-Laboratory Precision

15 14.1.1.1  Single-laboratory precision data for the reference toxicants,
copper (Cu)  and sodium dodecyl sulfate  (SDS), tested in FORTY FATHOMS®
artificial seawater, GP2 artificial seawater, and natural seawater are
provided in  Tables 13-18.  The test results were similar  in the three types of
seawater.  The IC25 and IC50 for the reference toxicants  (copper and sodium
dodecyl sulfate)  are reported in Tables 13-16.  The coefficient of variation,
based on the IC25, is 28.7% to 54.6% for natural and FORTY FATHOMS® seawater,
indicating acceptable precision.  The  IC50 ranges from 23:3% to 48.2%,  showing
acceptable precision.

15.14.1.2  Multilaboratory Precision

15.14.1.2.1  No data are available on  the multilaboratory precision of  the
test.

15.14.2  ACCURACY

15.14.2.1 The  accuracy of toxicity tests cannot be determined.
                                       333

-------
Cone. ID

Cone. Tested
Response
Response
Response
1
2
3
1
0
.85
.78
.87
2
2.5
.81
.65
.71
3
5.0
.63
.74
.78
4
10.0
.63
.66
.51
5
20.0
.41
.41
.37
6
40.0
.12
.3
.26
***  Inhibition Concentration  Percentage Estimate ***
Toxicant/Effluent: Copper
Test Start Date:    Test Ending Date:
Test Species: SEA URCHIN, Arbacia punctulata
Test Duration:
DATA FILE: urchin.icp
OUTPUT FILE: urchin.i50
Cone.
ID
1
2
3
4
5
6
Number
Replicates
3
3
3
3
3
3
Concentration
ug/1
0.000
2.500
5.000
10.000
20.000
40.000
Response
Means
0.833
0.723
0.717
0.600
0.397
0.227
Std. Pooled
Dev. Response Means
0.047
0.081
0.078
0.079
0.023
0.095
0.833
0.723
0.717
0.600
0.397
0.227
The Linear Interpolation Estimate:    19.0164   Entered P Value: 50
-------------	.
Number of Resamplings:   80
The Bootstrap Estimates Mean:  19.0013 Standard Deviation:     0.8973
Original Confidence Limits:   Lower:    17.6316 Upper:    21.2195
Expanded Confidence Limits:   Lower:    16.1083 Upper:    23.6429
Resampling time in Seconds:     1.65  Random Seed: -823775279
                Figure 7.  ICPIN program output for the IC50.

                                     334

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TABLE 13.   SINGLE-LABORATORY PRECISION OF THE SEA URCHIN, ARBACIA
            PUNCTULATA, FERTILIZATION TEST PERFORMED  IN FORTY  FATHOMS®
            ARTIFICIAL SEAWATER, USING GAMETES FROM ADULTS MAINTAINED IN
            FORTY FATHOMS® ARTIFICIAL SEAWATER, OR OBTAINED DIRECTLY FROM
            NATURAL SOURCES, AND COPPER (CU) SULFATE  AS A REFERENCE
            TOXICANT1'2'3'4'5
     Test
    Number
LOEC
 IC25
(M9/L)
  IC50
(M9/L)
1 5.0 8.92
2 12.5 26.35
3 < 6.2 11.30
4 6.2 34.28
5 12.5 36.67
n: 4 5
Mean: NA 23.51
CV(%): NA • 54.60
1
2
2
3



4

5

Data from USEPA (1991a)
Tests performed by Dennis McMullen, Technology Applic
EMSL, Cincinnati, OH.
All tests were performed using FORTY FATHOMS® synthet
29.07
38.96
23.93
61.75
75.14
5
45.77
47.87
atiions, Inc.,
ic seawater.
Copper test solutions were prepared with copper sulfcite» Copper
concentrations in Test 1 were: 2.5, 5.0, 10.0, 20.0,
Copper concentrations in Tests 2-5 were: 6.25, 12.5,
100.0 fj,g/l.
and 40.0 jug/L.
25,0, 50.0, and

NOEC Range: < 5.0 - 12.5 nq/l (this represents a difference of one
exposure concentrations).

For a discussion of the precision of data from chronic toxicity tests
see Section 4, Quality Assurance.

                                    335

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TABLE 14.   SINGLE-LABORATORY  PRECISION  OF  THE  SEA URCHIN,  ARBACIA
            PUNCTULATA,  FERTILIZATION  TEST  PERFORMED  IN  FORTY  FATHOMS®
            ARTIFICIAL SEAWATER,  USING GAMETES  FROM ADULTS  MAINTAINED IN
            FORTY  FATHOMS® ARTIFICIAL  SEAWATER,  OR OBTAINED DIRECTLY  FROM
            NATURAL SOURCES, AND  SODIUM  DODECYL  SULFATE  (SDS)  AS  A
            REFERENCE TOXICANT1'2'3'4'5-6
     Test
    Number
 NOEC
(mg/L)
                                              IC25
                                             (mg/L)
 IC50
(mg/L)
      1
      2
      3
      4
      5
  0.9
  0.9
  1.8
  0.9
  1.8
                                              1.11
                                              1.27
                                              2.26
                                              1.90
                                              2.11
 1.76
 1.79
 2.87
 2.69
 2.78
n:
Mean:
CV(%):
4
NA
NA
5
1.73
29.7
5
2.38
23.3
   Data from USEPA ,(1991a)
   Tests performed by Dennis M. McMullen, Technology Applications,  Inc.,
   EMSL, Cincinnati, OH.
   All tests were performed using FORTY FATHOMS® synthetic seawater.
   NOEC Range: 1.2 -3.3 mg/L (this represents a difference of one exposure
5


6
                     0.9,  1.8,  3.6,  7.2,  and
concentration).
SDS concentrations for all tests were:
14.4 mg/L.
For a discussion of the precision of data from chronic toxicity tests
see Section 4, Quality Assurance.                       '
                                    336

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TABLE 15.   SINGLE-LABORATORY PRECISION OF THE SEA URCHIN, ARBACIA
            PUNCTULATA, FERTILIZATION TEST PERFORMED IN NATURAL SEAWATER,
            USING GAMETES FROM ADULTS MAINTAINED  IN NATURAL SEAWATER AND
            COPPER (CU) SULFATE AS A REFERENCE TOXICANT 1'^'*':3'6
     Test
    Number
                     NOEC
IC25
 IC50
(M9/L)
      1
      2
      3
      4
      5
                     12.2
                     12.2
                     24.4
                    < 6.1
                      6.1
14.2
32.4
30.3
26.2
11.2
 18.4
 50.8
 46.
 34.
.3
,1
 17.2
n:
Mean :
CV(%) :
4
NA
NA
5
22.8
41.9 !
5
29.9
48.2
1
2


3
4


5
6
Data from USEPA (1991a)
Tests performed by Ray Walsh and Wendy Greene, ERL-N, USEPA,
Narragansett, RI.
Copper concentrations were:   6.1, 12.2, 24.4, 48.7, and 97.4 /^g/L.
NOEC Range:  < 6.1 - 24.4 /^g/L (this represents a difference of two
exposure concentrations).
Adults collected in the field.
For a discussion of the precision of data from chronic toxicity tests
see Section 4, Quality Assurance.
                                    337

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TABLE 16.   SINGLE-LABORATORY PRECISION OF THE SEA URCHIN, ARBACIA
            PUNCTULATA, FERTILIZATION TEST PERFORMED IN NATURAL SEAWATER,
            USING GAMETES FROM ADULTS MAINTAINED IN NATURAL SEAWATER AND
            SODIUM DODECYL SULFATE (SDS) AS A REFERENCE TOXICANT ^•3>^-6



1
2
3
A
5
6
Test NOEC
Number (mg/L)
1 1.8
2 1.8
3 1.8
4 0.9
5 1.8
n: 5
Mean : NA
CV(%): NA
Data from USEPA (1991a).
Tests performed by Ray Walsh and
Narragansett, RI.
SDS concentrations were: 0.9, 1
NOEC Range: 0.9 - 1.8 mg/L (this
exposure concentration).
Adults collected in the field.
For a discussion of the precision
see Section 4, Quality Assurance.
IC25
(mg/L)
2.3
3.9
2.3
2.1
2.3
5
2.58
28.7
IC50
(mg/L)
2.7
5.1
2.9
2.6
2.7
5
3.2
33.3
Wendy Greene, ERL-N, USEPA,
.8, 3.6, 7.3, and 14.5 mg/L.
represents a difference of one
of data from chronic toxicity tests
                                   338

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TABLE 17.   SINGLE-LABORATORY PRECISION OF THE SEA URCHIN, ARBACIA
            PUNCTULATA, FERTILIZATION TEST PERFORMED IN GP2, USING GAMETES
            FROM ADULTS MAINTAINED IN GP2 ARTIFICIAL SEAWATER AND
            COPPER (CU) SULFATE AND SODIUM DODECYL SULFATE (SDS) AS
            REFERENCE TOXICANTS1'2'3'4'5

Test
1
2
3
4
5
Mean
SD
CV

LC50
29.1
47.6
32.7
78.4
45.6
46.7
19.5
41.8


27
44
29
73
41



Cu
CI
.3-31
.6-50
.8-35
.3-83
.0-50



luQ/L) SDS (ma/L)

.1
.8
.8
.9
.7



NOEC
6.3
25.0
6.3
50.0
12.5



LOEC
12.5
50.0
12.5
100.0
25.0



LC50
2
1
2
2
1
2
0
10
.1
.8
.2
.3
.8
.0
.2
.0
CI
2.0-2.1
1.8-1.9
2.1-2.2
2.2-2.4
1.7-2.8

'

NOEC
1.3
1.3
1.3
1.3
1.3



LOEC
2.5
2.5
2.5
2.5
2.5



   Tests performed by Pamela Corneleo, Science Application International
   Corp., ERL-N, USEPA, Narragansett, RI.
   All tests were performed using GP2 artificial seawater.
   Copper concentrations were: 6.25, 12,5, 25.0, 50.0 and 100 ng/L.
   SDS concentrations were: 0.6, 1.25, 2.5, 5.0, and 10.0 mg/L.  SDS stock
   (14.645 mg/mL) provided by EMSL, USEPA, Cincinnati, OH.
   For a discussion of the precision of data from chronic toxicity tests
   see Section 4, Quality Assurance.                    ,
                                    339

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TABLE 18.   SINGLE-LABORATORY PRECISION OF THE SEA URCHIN, ARBACIA
            PUNCTULATA, FERTILIZATION TEST PERFORMED  IN NATURAL SEAWATER,
            USING GAMETES FROM ADULTS MAINTAINED  IN NATURAL SEAWATER AND
            COPPER  (CU) SULFATE.ANP SODIUM DODECYL SULFATE (SDS) AS
            REFERENCE TOXICANTS1'2'"'*
Cu (ua/l)
Test
1
2
3
4
5
LC50
28.6
13.0
67.8
36.7
35.6
CI
26.7-30.6
11.9-14.2
63.2-72.6
33.9-39.8
33.6-37.7
NOEC
6.3
6.3
6.3
< 6.3
< 6.3
LOEC
12.5
12.5
12.5
6.3
6.3
LC50
2.2
1.9
2.2
3.3
2.9
SDS
CI
2.1-2.2
1.9-2.0
2.1-2.3
3.1-3.4
2.8-3.1
(ma/L)
NOEC
1.3
1.3
1.3
< 0.6
< 0.6

LOEC
2.5
2.5
2.5
0.6
0.6
Mean
SD
CV
36.3
20.0
55.1
2.5
0.58
23.2
1 Tests performed by Anne Kuhn-Hines,  Catherine  Sheehan, Glen Modica,  and
  Pamela Comeleo, Science Application  International Corp.,  ERL-N, USEPA,
  Narragansett,  RI.
  Copper concentrations were prepared  with  copper  sulfate.   Concentrations
  were 6.25,  12.5, 25.0, 50.0,  and  100 /*g/L.
  SDS concentrations were: 0.6,  1.25,  2.5,  5.0,  and 10.0 mg/L.   SDS  stock
  (14.64 mg/mL) provided by EMSL, USEPA,  Cincinnati, OH.
  For a discussion of the precision of data from chronic toxicity tests
  see Section 4, Quality Assurance.
                                    340

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                                                                                          1
                                  SECTION 16

                                 TEST METHOD

           RED MACROALGA, CHAMPIA PARVULA, SEXUAL REPRODUC1
                                 METHOD  1009.0
ION TEST
16.1  SCOPE AND APPLICATION

16.1.1  This method, adapted in part from USEPA (1987f) measures the effects
of toxic substances in effluents and receiving water on the sexual
reproduction of the marine red macroalga, Champia parvula.  The method
consists of exposing male and female plants to test substances for two days,
followed by a 5-7 day recovery period in control medium, during which the
cystocarps mature.

16.1.2  Detection limits of the toxicity of an effluent or chemical substance
are organism dependent.

16.1.3  Brief excursions in toxicity may not be detected us;ing 24-h composite
samples.  Also, because of the long sample collection period involved in
composite sampling, highly volatile and highly degradable toxicants present in
the source may not be detected in the test.

16.1.4  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.

16.2  SUMMARY OF METHOD

16.2.1  Sexually mature male and female branches of the red macroalga, Champia
parvula, are exposed in a static system for 2 d to different concentrations of
effluent, or to receiving water, followed by a 5 to 7 day recovery period in
control medium. The recovery period allows time for the development of
cystocarps resulting from fertilization during the exposure; period.  The test
results are reported as the concentration of the test substance which causes a
statistically significant reduction in the number of cystocarps formed.

16.3  INTERFERENCES

16.3.1  Toxic substances may be introduced by contaminants in dilution water,
glassware, sample hardware, and testing equipment (see Section 5, Facilities,
Equipment, and Supplies).
                                                           I
16.3.2  Improper effluent sampling and handling may adversely affect test
results (see Section 8, Effluent and Receiving Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests).

16.3.3  Adverse effects of high concentrations of suspended and/or dissolved
solids, and extremes of pH, may mask the presence of toxic substances.
                                      341

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16.3.4  Pathogenic and/or predatory organisms in the dilution water and
effluent may affect test organism survival, and confound test results.
16.4  SAFETY
16.4.1  See Section 3, Safety and Health.
16.5  APPARATUS AND EQUIPMENT
16.5.1  Facilities for holding and acclimating test organisms.
16.5.2  Laboratory red macroalga, Champia parvula, culture unit -- see
culturing methods below.  To test effluent or receiving water toxicity,
sufficient numbers of sexually mature male and female plants must be
available.
16.5.3  Samplers -- automatic samplers, preferably with sample cooling
capability, that can collect a 24-h composite sample of 1 L.
16.5.4  Environmental chamber or equivalent facility with temperature control
(23 ± PC).
16.5.5  Water purification system -- Mi 11ipore Milli-Q®, deionized water (DI)
or equivalent.
16.5.6  Air pump -- for oil-free air supply.
16.5.7  Air lines, and air stones -- for aerating cultures.
16.5.8  Balance -- Analytical, capable of accurately weighing to 0.00001 g.
16.5.9  Reference weights, Class S -- for checking performance of balance.
16.5.10  Meter, pH -- for routine physical and chemical measurements.
16.5.11  Dissecting  (stereoscope) microscope -- for counting cystocarps.
16.5.12  Compound microscope -- for examining the condition of plants.
16.5.13  Count register, 2-place -- for recording cystocarp counts.
16.5.14  Rotary shaker -- for incubating exposure chambers  (hand-swirling
twice a day can be substituted).
16.5.15  Drying oven  --to dry glassware.
16.5.16  Filtering apparatus -- for use with membrane filters  (47 mm).
16.5.17  Refractometer -- for determining salinity.
16.5.18  Thermometers, glass or electronic, laboratory grade  -- for measuring
water temperatures.
                                      342

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16.5.19  Thermometers, bulb-thermograph or electronic-chart type -- for
continuously recording temperature.

16.5.20  Thermometer, National Bureau of Standards Certified (see USEPA Method
170.1, USEPA, 1979b) -- to calibrate laboratory thermometers.

16.5.21  Beakers -- Class A, borosilicate glass or non-toxic plasticware, 1000
ml for making test solutions.                             ;
                                                          i
16.5.22  Erlenmeyer flasks, 250 ml, or 200 ml disposable polystyrene cups,
with covers -- for use as exposure chambers.

16.5.23  Bottles -- borosilicate glass or disposable polystyrene cups  (200-400
ml) for use as recovery vessels.                          |

16.5.24  Wash bottles  -- for deionized water, for rinsing small glassware and
instrument electrodes  and probes.

16.5.25  Volumetric flasks  and graduated cylinders -- Class A, borosilicate
glass  or non-toxic plastic  labware, 10-1000 ml for making test solutions.

16.5.26  Micropipettors, digital,  200  and 1000 nl -- to make dilutions.

16.5.27  Pipets, volumetric -- Class A,  1-100 ml.

16.5.28  Pipettor,  automatic  --  adjustable,  1-100 ml.

16.5.29  Pipets, serological  --  1-10 mL, graduated.       |
                                                          i
16.5.30  Pipet  bulbs  and  fillers --  PROPIPET®, or equivalent.
                                                          I
16.5.31  Forceps,  fine-point,  stainless  steel  --  for cutting  and  handling
branch tips.

16.6   REAGENTS  AND CONSUMABLE MATERIALS

16.6.1  Mature  red macroalga,  Champia  parvuTa, plants  --  see Subsection
16.6.14  below.

 16.6.2  Sample  containers -- for sample  shipment  and  storage (see Section  8,
Effluent and Receiving Water Sampling, Sample Handling,  and Sample Preparation
 for Toxicity Tests).

 16.6.3  Petri dishes, polystyrene -- to  hold plants  for cystocarp counts and
 to cut branch tips.  Other suitable containers may  be  used.
                                 .
 16.6.4  Disposable tips for micropipettors.              |

 16.6.5  Aluminum foil, foam stoppers,  or other closures -|- to cover culture
 and test flasks.
                                                          I

                                       343

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 16.6.6  Tape, colored -- for labelling test chambers.

 16.6.7  Markers, waterproof -- for marking containers, etc.

 16.6.8  Data sheets (one set per test) -- for data recording.

 16.6.9  Buffers, pH 4, pH 7, and pH 10 (or as per instructions of instrument
 manufacturer) for standards and calibration check (see USEPA Method 150.1,
 USEPA, 1979b).

 16.6.10  Laboratory quality assurance samples and standards for the above
 methods.

 16.6.11  Reference toxicant solutions see Section 4,  Quality Assurance.

 16.6.12  Reagent water -- defined as distilled or deionized water that  does
 not contain substances which are toxic to the test organisms (see Section 5,
 Facilities, Equipment, and Supplies).

 16.6.13  Effluent,  receiving water,  and dilution  water --  see  Section 7,
 Dilution  Water;  and Section 8,  Effluent and Receiving Water Sampling, Sample
 Handling,  and Sample Preparation for Toxicity Tests.

 16.6.13.1   Saline test and dilution  water --  the  use  of natural  seawater  is
 recommended for  this test.   A recipe for the  nutrients that must be  added to
 the natural  seawater is  given in Table 1.   The salinity of the test  water must
 be  30%p,  and vary no more than  ± 2%o among  the replicates.   If effluent and
 receiving  water  tests  are conducted  concurrently,  the salinity of these tests
 should be  similar.

 16.6.13.2   The overwhelming majority of industrial  and sewage  treatment
 effluents  entering  marine and estuarine systems contain  little or no
 measurable salts.   Therefore, exposure of the  red  macroalga, Champia parvula,
 to  effluents will  usually require  adjustments  in the  salinity  of the test
 solutions.   Although the  red macroalga,  Champia parvuTa, cannot  be cultured in
 100% artificial  seawater,  100%  artificial seawater can  be  used  during the two
 day  exposure period.   This  allows  100% effluent to be  tested.   It  is important
 to maintain  a constant salinity  across  all  treatments.   The  salinity of the
 effluent can be  adjusted  by adding hypersaline brine  (MSB)  prepared from
 natural seawater  (100%o), concentrated  (triple strength) salt  solution (GP2
 described  in  Table  2), or dry GP2 salts  (Table 2), to  the effluent to provide
 a salinity  of 30%o.  Control  solutions  should  be prepared with the same
 percentage  of. natural  seawater and at  the same salinity  (using deionized water
 adjusted with dry salts,  or  brine) as  used  for the effluent dilutions.

 16.6.13.3  Artificial  seawater -- A  slightly modified version of the GP2
medium  (Spotte,  et  al, 1984) has been  used  successfully to perform the red
macroalga sexual  reproduction test.  The preparation of artificial seawater
 (GP2)  is described  in Table  2.
                                      344

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TABLE 1.    NUTRIENTS TO BE ADDED TO NATURAL SEAWATER AND TO ARTIFICIAL
            SEAWATER (GP2) DESCRIBED IN TABLE 2.  THE CONCENTRATED
            NUTRIENT STOCK SOLUTION IS AUTOCLAVED FOR 15 MIN (VITAMINS ARE
            AUTOCLAVED SEPARATELY FOR 2 MIN AND ADDED AFTER THE NUTRIENT
            STOCK SOLUTION IS AUTOCLAVED).  THE pH OF THE SOLUTION IS
            ADJUSTED TO APPROXIMATELY pH 2 BEFORE AUTOCLAVIN6 TO MINIMIZE
            THE POSSIBILITY OF PRECIPITATION


                               Amount of Reagent Per Liter of Concentrated
                               	Nutrient Stock Solution	

                               Stock Solution For       Stock Solution For
                                Culture Medium             Test Medium


Nutrient Stock Solution1

                                                        i
NaN03                                  6.35 g                 1.58 g

NaH2P04 .  H20                           0.64 g                 0.16 g

Na2EDTA .  2 H20                      133 mg

Na3C6H507 . 2 H20                      51 mg                  12.8 mg

Iron2                                  9.75 mL                2.4 mL

Vitamins3                             10 mL             '      2.5 mL
   Add 10 mL of appropriate nutrient stock solution per liter of culture
   or test medium.
   A stock solution of iron is made that contains 1 mg iiron/mL.  Ferrous
   or ferric chloride can  be  used.
   A vitamin stock solution is made by dissolving 4.88 g thiamine HC1,
   2.5 mg biotin,  and 2.5  mg  B12  in 500 mL deionized water.  Adjust  to
   approximately pH 4 before  autoclaving 2 min.   It is convenient to
   subdivide the vitamin stock  into 10 mL volumes  in test  tubes prior to
   autoclaving.
                                    345

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TABLE 2.    REAGENT GRADE CHEMICALS USED IN THE PREPARATION OF GP2
            ARTIFICIAL SEAWATER FOR USE IN CONJUNCTION WITH NATURAL
            SEAWATER FOR THE RED MACROALGA, CHAMPIA PARVULA, CULTURING AND
            TOXICITY TESTING1'2'3'4'5'6'^
    Compound
Concentration
   (9/L)
 Amount (g)
Required for
   20 L









1
2
3
NaCl
Na2S04
KC1
KBr
Na2B407 • 10 H20
MgCl2 ' 6 H20
CaCl2 ' 2 H20
SrCl2 • 6 H20
NaHC03
Modified GP2 from Spotte
The constituent salts and
21.03
3.52
0.61
0.088
0.034
9.50
1.32
0.02
0.17
et al. (1984).
concentrations were taken
420.6
70.4
12.2
1.76
0.68
190.0
26.4
0.400
3.40
from USEPA (19
   salts separately to avoid precipitation.  However, if the sodium
   bicarbonate is autoclaved separately (dry), all of the other salts can
   be autoclaved together.  Since no nutrients are added until needed,
   autoclaving is not critical for effluent testing.  To minimize
   microalgal contamination, the artificial seawater should be autoclaved
   when used for stock cultures.  Autoclaving (120°C) should be for a
   least 10 min for 1-L volumes, and 20 min for 10-to-20 L volumes.
   Prepare in 10-L to 20-L batches.
   A stock solution of 68 mg/mL sodium bicarbonate is prepared by
   autoclaving  it as a dry powder, and then dissolving it in sterile
   deionized water.  For each liter of GP2, use 2.5 mL of this stock
   solution.
   Effluent salinity adjustment to 30%o can be made by adding the
   appropriate amount of dry salts from this formulation, by using a
   triple-strength brine prepared from this formulation, or by using a
   100%o salinity brine prepared from natural seawater.
   Nutrients listed in Table 1 should be added to the artificial seawater
   in the same concentration described for natural seawater.
                                    346

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16.6.14  TEST ORGANISMS RED MACROALGA, CHAHPIA PARVULA
16.6.14.1  Cultures

16.6.14.1.1  Mature plants are illustrated in Figure  1.  The  adult  plant  body
(thai 1 us) is hollow, septate, and highly branched.  New  cultures  can  be
propagated asexually from excised branches, making  it  possible  to maintain
clonal material indefinitely.
            tetrasporangia ——
                                                             spermatia
                                                          fertilization
                 TETRASPOROPHYTE
                                                          — cystocarp
                                         5mm
 Figure  1.    Life  history of the  red  macroalga,  Champia parvula.   Upper left:
             Size  and degree of branching in female branch tips used for toxicity
             tests.   From USEPA (1987f).

 16.6.14.1.2   Unialgal  stock cultures  of both males and females are maintained
 in  separate,  aerated 1000 ml Erlenmeyer flasks containing 800 ml of the
 culture medium.   All  culture glass must be acid-stripped in 15% HC1 and rinsed
 in  deionized water  after washing.  This is necessary since some detergents can
 leave  a residue  that is toxic to the  red macroalga, Champia parvula.
 Periodically (at  least every 6 months) culture glassware should be baked in a
 muffle  furnace to remove organic material that may build up on its surface.
 Alternately,  a few  ml of concentrated sulfuric acid can be rolled around the
 inside  of wet glassware.  Caution:  the addition of acid to the wet glassware
 generates heat.
                                                           i
                                       347

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 16.6.14.1.3   The  culture  medium  is  made  from  natural  seawater  to which
 additional nutrients  are  added.   The  nutrients  added  are  listed in Table  1.
 Almost  any nutrient recipe  can be used for  the  red macroalga,  Champia parvula,
 cultured  in  either natural  seawater or a 50-50  mixture  of natural and
 artificial seawaters.   Healthy,  actively growing  plants are  the goal, not  a
 standard  nutrient recipe  for  cultures.

 16.6.14.1.4   Several  cultures of both males and females should be maintained
 simultaneously to keep  a  constant supply of plant material available.  To
 maintain  vigorous growth, initial stock  cultures  should be started
 periodically with about twenty 0.5  to 1.0 cm  branch tips.  Cultures are gently
 aerated through sterile,  cotton-plugged,  disposable,  polystyrene 1 ml
 pipettes.  Cultures are capped with foam plugs  and aluminum  foil and
 illuminated  with ca.  75 pE/m/s  (500  ft-c) of cool-white fluorescent light on
 a  16:8 light:dark cycle.  Depending on the  type of culture chamber or room
 used, i.e.,  the degree  of reflected light,  the  light  levels  may have to be
 adjusted  downward.  The temperature is 22 to  24°C and the salinity 28-30%o.
 Media are changed once  a  week.

 16.6.14.1.5   Prior to use in  toxicity tests,  stock cultures  should be examined
 to determine  their condition.  Females can  be checked by  examining a few
 branch tips  under a compound  microscope  (100  X  or greater).  Several
 trichogynes  (reproductive hairs  to  which  the  spermatia  attach) should be
 easily seen  near the  apex (Figure 2).

 16.6.14.1.6   Male plants  should  be  visibly producing spermatia.  This can be
 checked by placing some male  tissue in a  petri  dish, holding it against a dark
 background and looking  for  the presence  of spermatial sori.  Mature sori can
 also be easily identified by  looking  along the  edge of  the thallus under a
 compound  microscope (Figures  3 and  4).

 16.6.14.1.7   A final, quick way  to  determine the  relative "health" of the male
 stock culture is to place a portion of a  female plant into some of the water
 from the  male culture for a few  seconds.  Under a compound microscope numerous
 spermatia should be seen  attached to  both the sterile hairs  and the
 trichogynes  (Figure 5).

 16.6.14.2  Culture medium prepared  from natural seawater  is  preferred
 (Table 1).  However, as much  as  50% of the natural seawater may be replaced by
 the artificial seawater (GP2) described  in Table  2.

 16.6.14.2.1   Seawater for cultures  is filtered  at least to 0.45 pm to remove
 most particulates and then  autoclaved for 30 minute at  15 psi (120°C).   Carbon
 stripping the seawater  may  be necessary before  autoclaving to enhance its
water quality (USEPA, 1990b).  This is done by  adding 2 g activated carbon per
 liter of  seawater and stirring on a stir  plate for 2 h.   After stirring filter
through a Whatman number 2  filter, then through a 0.45 membrane filter.   The
culture flasks are capped with aluminum foil and autoclaved dry,  for 10
minute.   Culture medium is made up by dispensing seawater into sterile  flasks
 and adding the appropriate nutrients  from a sterile stock solution.
                                      348

-------
                                         sterile hairs
                                            ^Mrichogynes
                             1 mm
Figure 2.   Apex  of  branch   of  female  plant,   showing  sterile  hairs  and
            reproductive  hairs  (trichogynes).    Sterile  hairs  are  wider and
            generally much longer than trichogynes, and appear hollow except at
            the tip.  Both types of hairs occur on the entire circumference of
            the  thai!us,   but are  seen  easiest  at  the  '[edges."   Receptive
            trichogynes occur only  near  the branch tips.   From USEPA (1987f).
                          1 cm
                                           spemnatial sorus
Figure 3.   A portion  of  the  male thai!us showing spermatial  sori.   The sorus
            areas are generally slightly thicker and somewhat lighter in color.
            From USEPA (1987f).
                                      349

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                                   cuticle
                         100 urn
                                         0  O

                                            ^spermatia
                                           f\f
\
thallus surface
Figure 4.   A magnified portion of a spermatial sorus.  Note the rows of cells
            that protrude from the thallus surface.  From USEPA (1987f).
                                              spermatia
Figure 5.   Apex  of a  branch  on  a  mature female  plant that  was exposed  to
            spermatia from a male plant.  The sterile hairs  and trichogynes  are
            covered with spermatia.  Note that few or no spermatia are attached
            to the older hairs (those more than  1 mm from the apex).  From USEPA
            (1987f).


16.6.14.2.2  Alternately, 1-L flasks  containing seawater can  be autoclaved.
Sterilization is used to prevent microalgal contamination,  and not to keep
cultures bacteria free.

16.7  EFFLUENT AND RECEIVING WATER COLLECTION,  PRESERVATION,  AND STORAGE

16.7.1  See Section 8,  Effluent and  Receiving Water  Sampling,  Sample  Handling,
and Sample Preparation  for Toxicity  Tests.
                                      350

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16.8  CALIBRATION AND STANDARDIZATION

16.8.1  See Section 4, Quality Assurance.

16.9  QUALITY CONTROL

16.9.1  See Section 4, Quality Assurance.

16.10  TEST PROCEDURES

16.10.1  TEST SOLUTIONS

16.10.1.1  Receiving Waters                               '

16.10.1.1.1  The sampling point is determined by the objectives of the test.
At estuarine and marine sites, samples are usually collected at mid-depth.
Receiving water toxicity is determined with samples used directly as collected
or with samples passed through a 60 urn NITEX® filter and compared without
dilution, against a control.  Using four replicate chambers per test, each
containing 100 ml, and 400 mL for chemical analysis, would require
approximately 800 mL or more of sample per test.

16.10.1.2  Effluents                                      [

16.10.1.2.1  The selection of the effluent test concentrations should be based
on the objectives of the study.  A dilution factor of 0.5 is commonly used.  A
dilution factor of 0.5 provides precision of ± 100%, and allows for testing of
concentrations between 6.25% and 100% effluent using only five effluent
concentrations (6.25%, 12.5%, 25%, 50%,  and 100%).  Test precision shows
little improvement as dilution factors are increased beyond 0.5 and declines
rapidly if smaller dilution factors are  used.  Therefore, USEPA recommends the
use of the > 0.5 dilution factor.                         |

16.10.1.2.2  If the effluent is known or suspected to be highly toxic, a lower
range of effluent concentrations should  be used (such as 25%, 12.5%, 6.25%,
3.12%, and 1.56%).

16.10.1.2.3  The volume of effluent required for the test using a 0.5 dilution
series is approximately 1800 mL.  Prepare enough test solution at each
effluent concentration (approximately 800 mL) to provide 100 mL of test
solution for each of  four  (minimum of three) replicate test chambers and
400 mL for chemical analyses and record  data (Figure 6).

16.10.1.2.4  Effluents can be tested at  100%.  A 100% concentration of
effluent can be  achieved  if the salinity of the effluent is adjusted to 30%o
by  adding the  GP2 dry salt formulation described in Table 2.
                                                          i
16.10.1.2.5  Just prior to test initiation  (approximately 1 h), the
temperature  of sufficient  quantity of the sample to make the test solutions
should be adjusted to the  test temperature  (25 ± 1°C) and maintained at the
temperature  during the addition of dilution water.


                                      351

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                        SITE.

                        COLLECTION DATE
TEST DATE

LOCATION













INITIAL
SALINITY












FINAL
SALINITY












SOURCE OF SALTS FOR1
SALINITY ADJUSTMENT












1Natural  seawater, GP2 brine, GP2 salts, etc. (include some indication of amount)

COMMENTS:
Figure 6.   Data form for the red macroalga, Champia parvula, sexual
            reproduction test.  Receiving water summary sheet.  From USEPA
            (1987f).
                                      352

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 16.10.1.2.6   Effluent  dilutions  should  be  prepared  for  all  replicated  in  each
 treatment  in  one  beaker  to  minimize  variability  among the  replicates.   The
 test  chambers are labelled  with  the  test concentration  and  replicate number.
 Dispense into the appropriate  effluent  dilution  chamber.

 16.10.1.3  Dilution Water

 16.10.1.3.1.   The formula for  the  enrichment  for natural seawater  is listed  in
 Table  1.   Both EDTA and  trace  metals  have  been omitted.  This  formula  should
 be used for the 2-day  exposure period,  but it is not critical  for  the  recovery
 period.  Since natural seawater  quality can vary among  laboratories, a more
 complete nutrient medium (e.g.,  the  addition  of  EDTA) may result in faster
 growth (and therefore  faster cystocarp  development) during  the recovery
 period.

 16.10.2  PREPARATION OF  PLANTS FOR TEST
                                                          I

 16.10.2.1  Once cultures are determined to be usable for toxicity  testing
 (have  trichogynes  and  sori  with  spermatia), plant cuttings  should  be prepared
 for the test,  using fine-point forceps, with the plants in  a little seawater
 in a petri dish.   For  female plants,  five  cuttings, severed 7-10 mm from the
 ends of the branch, should  be  prepared  for each  treatment chamber.  Try to be
 consistent in  the  number of branch tips on  each  cutting.  For  male plants, one
 cutting, severed  2.0 to  3.0 cm from the end of the branch,  is  prepared  for
 each test chamber.  Prepare the  female  cuttings  first, to minimize the  chances
 of contaminating  them with  water containing spermatia from the male stock
 cultures.

 16.10.3.  START OF TEST

 16.10.3.1  Tests  should  begin  as soon as possible after sample collection,
 preferably within  24 h.  The maximum  holding time following retrieval  of the
 sample from the sampling device  should  not exceed 36 h for off-site toxicity
 tests unless permission  is granted by the  permitting authority.  In no  case
 should the sample  be used in a test more than 72 h after sample collection
 (see Section 8, Effluent and Receiving Water Sampling, Sample Handling, and
 Sample Preparation for Toxicity Test, Subsection 8.5.4).  i

 16.10.3.2  Just prior to test  initiation (approximately 1 h),  the temperature
 of a sufficient quantity of the sample.to make the test solution should be
 adjusted to the test temperature (23 ±  1°C) and maintained at that temperature
 during the addition of dilution water.
                                                          i
 16.10.3.3  Label the test chambers with a marking pen.   Use of color coded
 tape to identify each treatment and replicate is helpful.   A minimum of five
effluent concentrations and a control are used for each effluent test   Each
treatment (including controls)  should have four  (minimum of three)  replicates.

 16.10.3.4  Randomize the position of test chambers at the beginning of the
test.

 16.10.3.5  Prepare test solutions and add to the test chambers.

                                      353                  i

-------
16.10.3.6  Add five female branches and one male branch to each test chamber.
The toxicant must be present before the male plant is added.                       I

16.10.3.7  Gently hand swirl the chambers twice a day, or shake continuously
at 100 rpm on a rotary shaker.

16.10.3.8  If desired, the media can be changed after 24 h.

16.10.4  LIGHT, PHOTOPERIOD, SALINITY, AND TEMPERATURE

16.10.4.1  The light quality and intensity should be at 75 ^l/m2/s, or 500
foot candles  (ft-c) with  a photoperiod of 16 h light and 8 h darkness.  The
water temperature in the  test chambers should be maintained at 23 ± 1°C.  The
test salinity should be in the  range of 28 to 32%o.  The salinity should vary
by no more than ± 2%o  among the chambers on a given day.   If effluent  and
receiving water tests  are conducted concurrently, the salinities of these
tests should  be similar.

16.10.5  DISSOLVED  OXYGEN (DO)  CONCENTRATION

16 10.5.1  Aeration may affect  the toxicity of effluent and should be  used
only as  a last resort  to  maintain  a  satisfactory DO.  The  DO concentrations
should be measured  on  new solutions  at the  start of the test  (Day  0)  and
should be measured  before renewal  of  the test  solution  after 24  h.  The DO
should not fall  below  4.0 mg/L  (see  Section 8, Effluent and Receiving  Water
Sampling, Sample  Handling,  and  Sample  Preparation  for Toxicity Tests)   If  it
is necessary to  aerate, all  treatments  and  the control  should  be aerated.   I he
aeration rate should  not  exceed 100  bubbles/minute,  using  a pi pet  with a
1-2 mm orifice,  such  as a ImL KIMAX® serological pipet  No.  37033,  or
equivalent.   Care should  be taken  to ensure that turbulence resulting  from the
aeration does not occur.

 16.10.6   OBSERVATIONS DURING THE TEST

 16.10.6.1  Routine Chemical  and Physical  Observations

 16.10.6.1.1   DO is measured at the beginning  and  end of each  24-h exposure
 period in one test chamber at each concentration  and in the control.

 16.10.6.1.2  Temperature, pH, and salinity are measured at the end of each
 24-h exposure period in one test chamber at each  concentration and in the
 control.  Temperature should also be monitored continuously,  observed and
 recorded daily for at least two locations in the environmental  control system
 or the samples.  Temperature should be measured in a sufficient number of test
 chambers at least at the end of the test to determine temperature variation in
 environmental chamber.

 16.10.6.1.3  The pH is measured in the effluent sample each day before new
 test solutions are made.

 16.10.6.1.4  Record all  the measurements on the data sheet.


                                       354

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16.10.6.2  Routine Biological Observations

16.10.6.2.1  Protect the red macroalga from unnecessary disturbance during the
test by carrying out the daily test observations and solution renewals
carefully.
                                                          I
                                                          i
16.10.7  TRANSFER OF PLANTS TO CONTROL WATER AFTER 48 H

16.10.7.1  Label the recovery vessels.  These vessels can t>e almost any type
of container or flask containing 100 to 200 mL of seawater and nutrients (see
Tables 1 and 2).  Smaller volumes can be used, but should be checked to make
sure that adequate growth will occur without having to change the medium.

16.10.7.2  With forceps, gently remove the female branches from test chambers
and place into recovery bottles.  Add aeration tubes and foam stoppers.

16.10.7.3  Place the vessels under cool white light (at the same irradiance as
the stock cultures) and aerate for the 5-7 day recovery period.  If a shaker
is used, do not aerate the solutions  (this will enhance the water motion).

16.10.8  TERMINATION OF THE TEST

16.10.8.1  At the end of the recovery period, count the number of cystocarps
(Figures 7, 8, and 9) per female and record the data (Figure 10).  Cystocarps
may be counted by placing females between the inverted halves of a polystyrene
petri dish or other suitable containers with a small amount of seawater  (to
hold the entire plant in one focal plane).  Cystocarps can be easily counted
under a stereomicroscope, and are distinguished from young branches because
they possess an apical opening for spore release  (ostiole) and darkly
pigmented spores.
                          1 mm
 Figure  7.   A mature cystocarp.  In the controls and lower effluent
            concentrations, cystocarps often occur in clusters of 10 or 12.
            From USEPA (1987f).
 16.10.8.2   One  advantage  of  this  test  procedure  is that  if there  is
 uncertainty about  the  identification of  an  immature cystocarp,  it  is necessary
 only  to  aerate  the plants a  little  longer in the recovery bottles.  Within 24

                                      355

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                                       young branch

                                        .cells
                                           immature
                                            cystocarp
                          1 mm
Figure 8.   Comparison  of a very young branch and an immature cystocarp.   Both
            can  have sterile hairs.   Trichogynes might or might not be present
          ,  on a young  branch,  but are never present on an immature cystocarp.
            Young branches  are  more pointed  at the apex  and  are made  up  of
            larger cells  than  immature cystocarps,  and never  have  ostioles.
            From USEPA  (1987f).
Figure 9.   An  aborted cystocarp.   A new branch will eventually develop at the
            apex.   From USEPA (1987f).


to 48 h, the presumed cystocarp will either look more like a mature cystocarp
or a young branch, or will have changed very little, if at all (i.e., an
aborted cystocarp).  No new cystocarps will form since the males have been
removed, and the plants will only get larger.  Occasionally, cystocarps will
abort, and these should not be included in the counts.  Aborted cystocarps are
easily identified by their dark pigmentation and, often, by the formation of a
new branch at the apex.

16.11  SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA

16.11.1   A summary of test conditions and test acceptability criteria is
listed in Table 3.
                                      356

-------
COLLECTION DATE
EXPOSURE BEGAN (date).

EFFLUENT OR TOXICANT
.RECOVERY  BEGAN (date).

	COUNTED (date)	
            TREATMENT (% EFFLUENT, /iG/L, or RECEIVING WATER SITES)
1 RFPI TCATFS
CONTROI







A 1
7
3
4
MFAN




































R 1

3
4
MFAN




































f. 1
2
3
4
MFAN




































ft\/ F R A 1 1
MF AN

Temperature
Salinity
Liaht







Source of Dilution Water
Figure  10.  Data form for the red macroalga, Champia parvula, sexual
            reproduction test.  Cystocarp data sheet.  From USEPA  (1987f)
                                      357

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TABLE 3.    SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
            THE RED MACROALGA,  CHAMPIA PARVULA, SEXUAL REPRODUCTION TEST
            WITH EFFLUENTS AND  RECEIVING WATERS
 1. Test type:
 2. Salinity:

 3. Temperature:
 4. Photoperiod:
 5. Light intensity:
 6. Light source:
 7. Test chamber size:

 8. Test solution volume:
 9. No. organisms
     per test chamber:
 10. No. replicate chambers
     per concentration:
 11. No. organisms per
     concentrations:
 12. Dilution water:
 13. Test  concentrations:
 14.  Test  dilution  factor:
Static, non-renewal
30%o (± 2  of the selected test
salinity)
23 ± 1°C
16 h light, 8 h darkness
75 /*E/m2/s (500 ft-c)
Cool-white fluorescent lights
200 mL polystyrene cups, or 250 mL
Erlenmeyer flasks
100 mL (minimum)

5 female branch tips and 1 male plant

4 (minimum of 3)

24  (minimum of 18)
30%o salinity natural seawater, or a
combination of 50% of 30%o salinity
natural seawater and 50% of 30%o
salinity GP2 artificial seawater (see
Section 7, Dilution Water)
Effluents:  Minimum of 5 and a control
Receiving waters:  100% receiving water
or minimum of 5 and a control
Effluents:  > 0.5
Receiving waters:  None or > 0.5
                                    358

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TABLE 3.    SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
            THE RED MACROALGA,  CHAMPIA PARVULA,  SEXUAL REPRODUCTION TEST
            WITH EFFLUENTS AND  RECEIVING WATERS (CONTINUED)
15. Test duration:
16. Endpoints:
17. Test acceptability
      criteria:
18.  Sampling requirements:
19.  Sample volume required:
 2.day exposure to effluent,  followed
 by 5 to 7-day recovery period in
 control medium for cyctocarp
 development        i
                    i
Reduction in cystocarp production
compared to controls
80% or greater survival, and an
average of 10 cystocarps per plant in
controls

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,
Sampling Handling, and Sample
Preparation for Toxicity Tests,
Subsection 8.5.4).
2 L per test
                                   359

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16.12  ACCEPTABILITY OF TEST RESULTS

16.12.1  The test is acceptable if (1) control survival equals or exceeds 80%
and (2) control plants average 10 or more cystocarps per plant.

16.12.2  If plants fragment in the controls or lower exposure concentrations,
it may be an indication that they are under stress.
16.13  DATA ANALYSIS

16.13.1  GENERAL

16.13.1.1  Tabulate and summarize the data.
is listed in Table 4.
A sample set of reproduction data
16.13.1.2  The endpoints of the red macroalga, Champia parvula, toxicity test
are based on the adverse effects on sexual reproduction as the mean number of
cystocarps.  The LC50, the IC25, and the IC50 are calculated using point
estimation techniques (see Section 9, Chronic Toxicity Test Endpoints and Data
Analysis). NOEC and LOEC values are obtained using a hypothesis testing
approach, such as Dunnett's Procedure (Dunnett, 1955) or Steel's Many-one Rank
Test (Steel, 1959; Miller, 1981) (see Section 9).  Separate analyses are
performed for the estimation of the NOEC and LOEC endpoints and for the IC25
and IC50.  See the Appendices for examples of the manual computations, program
listing, and example of data input and program output.

16.13.1.3  The statistical tests described here must be used with a knowledge
of the assumptions upon which the tests are contingent.  The assistance of a
statistician is recommended for analysts who are not proficient in statistics.

16.13.2     EXAMPLE OF ANALYSIS OF THE RED MACROALGA, CHAMPIA PARVULA,
            REPRODUCTION DATA

16.13.2.1  Formal statistical analysis of the data is outlined in Figure 11.
The response used in the analysis is the mean number of cystocarps per
replicate chamber.  Separate analyses are performed  for the estimation of the
NOEC and LOEC endpoints and for the estimation of the IC25 endpoint and the
IC50 endpoint.  Concentrations that have exhibited no sexual reproduction
(less than 5% of controls) are excluded from the statistical analysis of the
NOEC and LOEC, but included in the estimation of the 1C endpoints.

16.13.2.2  For the case of equal numbers of replicates across  all
concentrations and the control, the evaluation of the NOEC and LOEC endpoints
is made via a parametric test, Dunnett's Procedure,  or a nonparametric test,
Steel's Many-one Rank Test.  The assumptions of Dunnett's Procedures,
normality and homogeneity of variance are formally tested.  The test for
normality is the Shapiro-Wilk's Test and Bartlett's  Test is used to test for
homogeneity of variance.  Tests for normality and homogeneity  of variance are
included in Appendix B.   If either of these tests fails, the nonparametric
test, Steel's Many-one Rank Test is used to determine the NOEC and LOEC
endpoints.  If the assumptions of Dunnett's Procedure are met, the endpoints
are determined by the parametric test.

                                      360

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TABLE 4.    DATA FROM THE RED MACROALGA,  CHANPIA PARVULA,  EFFLUENT
            TOXICITY TEST.   CYSTOCARP COUNTS FOR INDIVIDUAL PLANTS AND
            MEAN COUNT PER TEST CHAMBER FOR EACH EFFLUENT CONCENTRATION1
Effluent
Concentration
(%)
Control


0.8


1.3


2.2


3.6


6.0


10.0


Replicate
Test
Chamber
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C

1
19
19
17
10
12
12
10
6
4
1
7
3
2
3
0
1
1
0
0
1
2

2
20
12
25
16
10
9
0
4
4
2
9
2
1
4
4
0
2
4
0
0
1
Plant
3
24
21
18
11
6
9
3
4
2
5
9
2
1
6
3
0
1
3
0
0
0

4 5
7 18
11 23
20 16
12 11
9 10
13 8
5 4
8 4
6 4
4 0
4 6
0 0
5 0
4 2
1 3
0 0
0 0
1 3
0
0 0
0 0
Mean
Cystocarp
Count
17.60
17.20
19.20
12.00
9.40
10.20
4.40
5.20
4.00
2.40
7.00
1.40
1.80
3.80
2.20
0.20
0.80
2.20
0.00
0.20
0.60
   Data provided by the ERL-N, USEPA, Narragansett, RI,
                                    361

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                 STATISTICAL ANALYSIS OF CHAMPIA PARVULA
                        SEXUAL REPRODUCTION TEST
                              REPRODUCTION DATA
                            MEAN CYSTOCARP COUNT
    POINT ESTIMATION
   ENDPOINT ESTIMATE
        IC25, IC50
SHAPIRO-WILK-S TEST
                    NON-NORMAL DISTRIBUTION
                   NORMAL DISTRIBUTION
        HOMOGENEOUS
          VARIANCE
                               BARTLETTS TEST
                         HETEROGENEOUS
                            VARIANCE
                                                     1

NO
r
EQUAL NUMBER OF
REPLICATES?

TWITH
•RRONI
TMENT


1 YES
DUNNETTS
TEST




i

YES
'
EQUAL NUMBER OF
REPLICATES?

1 N°
STEEL'S MANY-ONE
RANK TEST



WILCOXON RANK SUM
TEST WITH
BONFERRONI ADJUSTMENT


                              ENDPOINT ESTIMATES
                                 NOEC, LOEC
Figure 11.  Flowchart for statistical analysis of the  red macroalga, Champia
           parvula,  data.

                                    362

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 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
 (Appendix  D).   The  Wilcoxon Rank Sum Test with  the Bonferroni  adjustment  is
 the nonparametric alternative.
                                                         i
 16.13.2.4   Example  of  Analysis of Reproduction  Data

 16.13.2.4.1   In this example,  the data, mean and  standard deviation  of  the
 observatfons  at each concentration including the  control are listed  in
 Table 5.   The data  are plotted in Figure  12.  As  can be  seen from the data in
 the table, mean reproduction per chamber  in  the 10% effluent concentration is
 less than  5%  of the control.   Therefore the  10% effluent concentration  is not
 included in the subsequent  analysis.                     I

      TABLE 5.  RED  MACROALGA, CHAMPIA PARVULA,  SEXUAL REPRODUCTION  DATA
                                      Effluent Concentration  (%)
  Replicate    Control
0.8
1.3
2.2    3.6
6.0   10.0
A
B
C
Mean(Y,.)
S,-
i

17
17
19
18
1
1

.60
.20
.20
.00
12


12.
9.
10.
10.
1.
2

00
40
20
53
77


4.40
5.20
4.00
4.53
0.37
3

2.40
7.00
1.40
3.60
8.92
4

1
3
2
2
1
5

.80
.80
.20
.60
.12


0.20
0.80
2.20
1.07
1.05
6

0
0
0
0
0
7

.00
.20
.60
.27
.09


16.13.2.5  Test for Normality

16.13.2.5.1  The first step of the test for normality is to center the
observations by subtracting the mean of all the observations within a
concentration from each observation in that concentration.   The centered
observations are summarized in Table 6.
                                     363

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                                                S-'
                                                O)
                                                O-

                                                co
                                                Q-
                                                S-
                                                «
                                                0
                                                O
                                                S-
                                                U)
                                                CD
                                                O
                                                51
                                                 O)
                                                 s-
                                                 3
                                                 O)
     CM   O
I—    T-   T-

 SdyVOOlSAO dO 'ON
            364

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         TABLE 6.  CENTERED OBSERVATIONS FOR SHAPIRO-MILK'S EXAMPLE
Ef f 1 uent
Replicate
A
B
C
Control
-0.40
-0.80
1.20
0.8
1.47
-1.13
-0.33
1.3
-0.13
0.67
-0.53
Concentration^)
2.2
-1.20
3.40
-2.20
3.6 1
-0.80
1.20
-0.40
6.0
-0.87
-0.27
1.13
16.13.2.5.2  Calculate the denominator, D, of the test statistic:
                                D = £ (X±-X)2
                                    i=l
    Where:   X,-  = 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 = 18
                                   X = J^(O.Ol) = 0.00
                                       18                 j
                                   D = 28.7201            |
16.13.2.5.4  Order the centered observations from smallest to largest
                  X(1) 
Where X
-------
      TABLE 7.  ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-MILK'S EXAMPLE


                  i        Xcn                   i         X(0
1
2
3
4
5
6
7
8
9
-2.20
-1.20
-1.13
-0.87
-0.80
-0.80
-0.53
-0.40
-0.40
10
11
, 12
13
14
15
16
17
18
-0.33
-0.27
-0.13
0.67
1.13
1.20
1.20
1.47
3.40
16.13.2.5.5  From Table 4, Appendix B, for the number of observations, n,
obtain the coefficients a.,, a,,  ...,  a^ where k is n/2 if n  is even  and (n-
l)/2 if n is odd.  For the data in this example, n = 18 and k = 9.  The a,-
values are listed in Table 8.
      TABLE 8.  COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE
                 af             Xn'l   - Xci)
1
2
3
4
5
6
7
8
9
0.4886
0.3253
0.2553
0.2027
0.1587
0.1197
0.0837
0.0496
0.0163
5.60
2.67
2.33
2.07
1.93
1.47
0.40
0.13
0.07
X(8)
x<7>
XC17)
XC15)
Y<14>
A
X(13)
X(12)
X(11)
X(10)
- x< >
- X?
- xc*>
- XC4>
- x<5)
- x(6>
- x(7>
- X(8)
- X(9)
16.13.2.5.6  Compute the test statistic, W, as follows:
                         W=
                              D
                                      366

-------
The differences x(n"i+1) - X
-------
16.13.2.6.3  Bartlett's statistic is therefore:

                  B = [(12)ln(2.3917) -2 f In (Sf) ]/1.194
                                          i=l

                    -  [12(0.8720) - 2(ln(1.12)+ln(1.77)+...+ln(1.05))]/1.1944

                    =  (10.4640 - 4.0809)/1.1944

                    =  5.34

16.13.2.6.4  B is approximately distributed as chi-square with p - 1 degrees
of freedom, when the variances are in fact the same.   Therefore, the
appropriate critical value for this test, at a significance level of 0.01 with
five degrees of freedom, is 15.09.  Since B = 5.34 is less than the critical
value of 15.09, conclude that the variances are not different.

16.13.2.7  Dunnett's Procedure

16.13.2.7.1  To obtain an estimate of the pooled variance for the Dunnett's
Procedure, construct an ANOVA table as described in Table 9.


                             TABLE 9.  ANOVA TABLE
Source df Sum of Squares
(SS)
Between p - 1 SSB
Within N - p SSW
Mean Square(MS)
(SS/df)
SB = SSB/(p - 1)
$1 = SSW/(N - p)
Total N - 1 SST
   Where:   p  = number effluent  concentrations  including the control

           N  = total  number of  observations  n1 + n2 ... + np

           n-  - number of observations in concentration i
                                      368

-------
         SSB = &Tl/ni-G2/N     Between  Sum  of  Squares
              =      Y2.-G2/N     Total  Sum of Squares
SST =  2, 2,
                      .j
          SSW = SST-SSB
                         Within Sum of Squares
           G  =
         the grand total  of all  sample  observations,   G = S 2^
                                                         i
           T-   =    the total of the replicate measurements for concentration i
                   the  jth  observation for concentration i (represents the
                   mean (across  plants) number of cystocarps for effluent
                   concentration i  in test chamber j)
16.13.2.7.2  For the data in  this  example:
           n«  = n, = n,
                                       n5 = n6  =3
N


T!
                        = 18
              - Y
                             + Y12 + Y13
                             + Y22 + '23
                             : ?» : >
                             + Y52 +
                           ?3 =
                           *6S
            G   =
                    SSB =
17.6 + 17.2 + 19.2
12.0 +  9.4 + 10.2
 4.4 +  5.2 +  4.0
 2.4 +  7.0 +  1.4
 1.8 +  3.8 +  2.2
 0.2 +  0.8 +  2.2
                                             T5 + T6 = 121.0
                             1  (4287.24) - (121.O)2 =  615.69
                            T"                18
54
31.6
13.6
10.8
 7.8
 3.2
                     SST =
                         =  1457.8 - (121. O)2  = 644.41
                                        18

                     SSW = SST-SSB

                         =  644.41 - 615.69 = 28.72
                                      369

-------
                       SB  =  SSB/(p-l) = 615.69/(6-l) =  123.14


                       Sy  =  SSW/(N-p) = 28.72/(18-6) =  2.39

 16.13.2.7.3  Summarize these calculations  in  the  ANOVA table  (Table  10)

            TABLE 10.   ANOVA TABLE FOR  DUNNETT'S PROCEDURE  EXAMPLE
Source
Between
Within
Total
df
5
12
17
Sum of Squares
(SS)
615.69
28.72
644.41
Mean Square (MS)
(SS/df)
123.14
2.39

 16.13.2.7.4  To  perform  the  individual comparisons, calculate the t
 statistic  for  each  concentration,  and control combination as follows:
  Where:  Y,  =  mean number of cystocarps for effluent concentration i

          Y1  =  mean number of cystocarps for the control

          SM  =  square root of the within mean square

          n1  -  number of replicates for the control

          rif  «  number of replicates for concentration i

16.13.2.7.5  Table 11 includes the calculated t values for each concentration
and control combination.  In this example, comparing the 0.8% concentration
with the control the calculation is as follows:
                                 (18-10.53)
                            [1. 55v/(l/3) + (1/3)]
                                                  = 5.90
16.13.2.7.6  Since the purpose of this test is to detect a significant
reduction in cystocarp production, a one-sided test is appropriate.  The
critical value for this one-sided test is found in Table 5, Appendix C.   For
an overall alpha level of 0.05, 12 degrees of freedom for error and five
                                     370

-------
                        TABLE  11.   CALCULATED T VALUES
Effluent





Concentration(%)
0.8
1.3
2.2
3.6
6.0
i
2
3
4
5
6
*i
i . .
5.90
10.64
11.38
12.17
13.38
concentrations (excluding the control)  the critical  value  is  2.50.   Mean
cystocarp production for concentration  i  is considered  significantly less
control if t,  is  greater than the  critical  value.  Therefore, mean cystocarp
productions for all  effluent concentrations in this  example  have  significantly
lower cystocarp production than the control.   Hence  the NOEC  is 0.8% and the
LOEC is 0.8%.

16.13.2.7.7  To quantify the sensitivity of the test,  the  minimum significant
difference (MSD)  that can be statistically detected  may be calculated:
                          MSD = d S^fCL/nJ + (l/n)


  Where:  d  = the critical  value  for Dunnett's Procedure
                                                         I

          SH = the square root  of  the within mean  square
                                                         i
          n  = the common number of replicates at  each  concentration
               (this assumes equal .replication at each  concentration)

          n., = the number of replicates in the control.

 16.13.2.7.8   In this example,
                       MSD = 2.50(1.55)/(l/3) + (1/3)

                           = 2.50 (1.55)(.8165)

                           = 3.16
                                                                   ,

 16.13.2.7.9  Therefore, for this set of data, the minimum difference that can
 be detected  as statistically significant is 3.16 cystocarps.

 16.13.2.7.10 This represents a 17.6% reduction in cystocarp production from
 the  control.                                             •
                                      371

-------
16.13.2.8  Calculation  of the  ICp

16.13.2.8.1  The  sexual  reproduction data  in Table 5  are utilized in this
example.  Table 12  contains the mean number of cystocarps for each effluent
concentration.  As  can  be seen, the observed means are monotonically non-
increasing with respect  to concentration.  Therefore, it is not necessary to
smooth the means  prior  to calculating the  ICp.  Refer to Figure 10 for a plot
of the response curve.

                    TABLE 12.   RED MACROALGA, CHAMPIA  PARVULA,
                               MEAN NUMBER  OF CYSTOCARPS
Effluent
Cone.
(%)
Control
0.8
1.3
2.2
3.6
6.0
10.0


i
1
2
3
4
5
6
7
Response
Means
Y,- (mg)
18.00
10.53
4.53
3.60
2.60
1.07
0.27
Smoothed
Means
M, (mg)
18.00
10.53
4.53
3.60
2.60
1.07
0.27
16.13.2.8.2  An IC25 and IC50 can be estimated using the Linear Interpolation
Method.  A 25% reduction in mean number of cystocarps, compared to the
controls, would result in a mean number of 13.50 cystocarps, where M^l-p/100)
= 18.00(1-25/100).  A 50% reduction in mean number of cystocarps, compared to
the controls, would result in a mean number of 9.00 cystocarps.  Examining the
means and their associated concentrations (Table 12), the response, 13.50, is
bracketed by C., = 0.0% effluent and C,  =  0.8%  effluent.   The response,  9.00,
is bracketed by C2 - 0.8% effluent and  C3 -  1.3%  effluent.

16.13.2.8.3  Using the equation from Section 4.2 in Appendix L, the estimate
of the IC25 is calculated as follows:
                   ICp = Cj.
                   IC25 = 0.0 + [18.00(1 - 25/100) - 18.00]  (0.8 - 0.0)
                        = 0.5%.
                                                           (10.53 - 18.00)
16.13.2.8.4  Using the equation from Section 4.2 from Appendix L, the estimate
of the IC50 is calculated as follows:

                                     372

-------
                   IC50 = 0.8 + [18.00(1  -  50/100)  -  10.53]   (1.3 - 0.8)
                                                           (4.53 - 10.53)

                        = 0.9 %.

16 13.2.8.5  When the ICPIN program was used to analyze this set of data,
requesting 80 resamples, the estimate of the IC25 was 0.4821%.  The empirical
95.0% confidence interval for the true mean was 0.4013% to 0.6075%.  The
computer program output for the IC25 for this data set is shown in Figure 13.

16 13.2.8.6  When the ICPIN program was used to analyze this set of data,
requesting 80 resamples, the estimate of the IC50 was 0.9278%.  The empirical
95.0% confidence interval for the true mean was 0.7893% and 1.0576%.  The
computer program output for the IC50 for this data set is shown in Figure 14.

16.14  PRECISION AND ACCURACY                             !

16.14.1  PRECISION
                                                          I
16.14.1.1  Single-Laboratory Precision
                                                          i
16.14.1.1.1  The single-laboratory precision data from six tests with copper
sulfate  (Cu) and six tests with sodium dodecyl sulfate (SDS)  are listed in
Tables 13-16.  The NOECs with Cu differed by only one concentration interval
(factor  of two), showing good precision.  The precision of the  first four
tests with SDS was somewhat obscured by the choice of toxicant  concentrations,
but  appeared similar to  that of Cu in the last two tests.  The  IC25 and IC50
are  indicated in Tables  13-16.  The coefficient of variation, based on the
IC25 for these two reference toxicants in natural seawater and  a mixture of
natural  seawater and GP2,  ranged from 59.6% to 69.0%, and for the  IC50, ranged
from 22.9% to 43.7%.
                                                          I
16.14.1.2  Multilaboratory Precision

16.14.1.2.1  The multilaboratory precision  of  the test has not  yet been
determined.

16.14.2   ACCURACY                                         \

16.14.2.1  The  accuracy of toxicity  tests  cannot  be  determined.
                                      373

-------
Cone. ID

Cone. Tested
Response
Response
Response
Response
Response
Response
Response
Response
Response
Response
Response
Response
Response
Response
Response
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
1
0
19
20
24
7
18
19
12
21
11
23
17
25
18
20
16
2
.8
10
16
11
12
11
12
10
6
9
10
12
9
9
13
8
3
1.3
10
0
3
5
4
6
4
4
8
4
4
4
2
6
4
4
2.2
1
2
5
4
0
7
9
9
4
6
3
2
2
0
0
5
3.6
2
1
1
5
0
3
4
6
4
2
0
4
3
1
3
6
6
1
0
0
0
0
1
2
1
0
0
0
4
3
1
3
7
10
0
0
0
0
1
0
0
0
0
2
1
0
0
0
0
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent: effluent
Test Start Date:    Test Ending Date:
Test Species: RED MACROALGA, Champia parvula
Test Duration:
DATA FILE: champia.icp
OUTPUT FILE: champ1 a.i25
Cone.
ID
1
2
3
4
5
6
7
Number Concentration
Replicates
15
15
15
15
15
15
15
The Linear Interpolation
%
0.000
0.800
1.300
2.200
3.600
6.000
10.000
Estimate:
Response
Means
18.000
10.533
4.533
3.600
2.600
1.067
0.267
0.4821
Std.
Dev.
4.928
2.356
2.356
3.066
1.805
1.335
0.594
Pooled
Response Means
18.000
10.533
4.533
3.600
2.600
1.067
0.267
Entered P Value: 25
Number of Resamplings:   80
The Bootstrap Estimates Mean:
Original Confidence Limits:
Resampling time in Seconds:
  0.4947 Standard Deviation:      0.0616
Lower:     0.4013 Upper:     0.6075
  3.68  Random Seed:  703617166
                Figure 13.  ICPIN program output for the IC25.

                                     374

-------
Cone. ID

Cone. Tested
Response
Response
Response
Response
Response
Response
Response
Response
Response
Response
Response
Response
Response
Response
Response
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
1
0
19
20
24
7
18
19
12
21
11
23
17
25
18
20
16
2
.8
10
16
11
12
11
12
10
6
9
10.
12
9
9
13
8
3
1.3
10
0
3
5
4
6
4
4
8
4
4
4
2
6
4
4
2.2
1
2
5
4
0
7
9
9
4
6
3
2
2
0
0
5
3.6
2
1
1
5
0
3
4
6
4
2
0
4
3
1
3
6
6
1
0
0
0
0
1
2
1
0
0
0
4
3
1
3
7
10
0
0
0
0
1
0
0
0
0
2
1
0
0
0
0
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent: effluent
Test Start Date:    Test Ending Date:
Test Species: RED MACROALGA, Champia parvula
Test Duration:
DATA FILE: champia.icp
OUTPUT FILE: champia.iSO
Cone.
ID
1
2
3
4
5
6
7
Number
Replicates
15
15
15
15
15
15
15
Concentration
%
0.000
0.800
1.300
2.200
3.600
6.000
10.000
Response
Means
18.000
10.533
4.533
3.600
2.600
1.067
0.267
Std.
Dev. F
4.928
2.356
2.356
3.066
1.805
1.335
0.594
Pooled
Response Means
18.000
10.533
4.533
3.600
2.600
1.067
0.267
The Linear Interpolation Estimate:
         0.9278   Entered P Value:  50
Number of Resamplings:   80
The Bootstrap Estimates Mean:
Original Confidence Limits:
Resampling time in Seconds:
  0.9263 Standard Deviation:      0.0745
Lower:      0.7893 Upper:      1.0576
  3.63   Random Seed:  -1255453122
                Figure 14.  ICPIN program output for the IC50.

                                      375

-------
TABLE 13.   SINGLE-LABORATORY PRECISION OF THE RED MACROALGA, CHAMPIA
            PARVULA, REPRODUCTION TEST PERFORMED  IN A 50/50 MIXTURE OF
            NATURAL SEAWATER AND GP2 ARTIFICIAL SEAWATER, USING GAMETES
            FROM ADULTS CULTURED IN NATURAL SEAWATER,, THE REFERENCE
            TOXICANT USED WAS COPPER (CU) SULFATE1'2'3'4'5
Test
Number
1
2
3
4
5
6
n:
Mean:
CV(%):
1 Data from USEPA
NOEC
UgA)
1.0
1.0
1.0
1.0
0.5
0.5
6
NA
NA
(1991a).
IC25
(/•fl/L)
1.67
1.50
0.69
0.98
0.38
0.38
6
0.93
59.6

IC50
(M9/L)
2.37
1.99
1.53
1.78
0.76
0.75
6
1.5
43.7
M IICCDA
3
4
Narragansett, RI.  Tests were conducted at 22°C, in 50/50 GP2 and
natural seawater at a salinity of 30%o.
Copper concentrations were:  0.5, 1.0, 2.5, 5.0, 7.5, and 1.0 ^g/L.
NOEC Range: 0.5 - 1.0 /*g/L (this represents a difference of one
exposure concentration).
For a discussion of the precision of data from chronic toxicity tests
see Section 4, Quality Assurance.
                                    376

-------
TABLE 14.   SINGLE-LABORATORY PRECISION OF THE RED MACROALGA,  CHAMPIA
            PARVULA,  REPRODUCTION TEST PERFORMED IN A 50/50 MIXTURE OF
            NATURAL SEAWATER AND GP2 ARTIFICIAL SEAWATER,  USING GAMETES
            FROM ADULTS CULTURED IN NATURAL SEAWATER.  THE REFERENCE
            TOXICANT USED WAS SODIUM DODECYL SULFATE (-SDS)1""3'*'5
Test
Number
1
2
3
4
5
6
7
8
9
n:
Mean:
CV(%):
1 Data
2 TQC-+<
NOEC
(mg/L)
< 0.80
0.48
< 0.48
< 0.48
0.26
0.09
0.16
0.09
< 0.29
5
NA
NA
from USEPA (1991 a).
- navFnvmaH hu Cl on ThllV^hv ;
IC25
(mg/L)
1
0.6
0.7
0.4
0.2
0.2
0.1
0.2
0.1 ;
0.3
9
0.31
69.0
snH Mark Taaliabue.
IC50
(mg/L)
0.3
0.6
0.2
0.4
0.5
0.3
0.3
0.2
0.4
9
0.36
37.0
ERL-N. USEPA,
      Narragansett,  RI.  Tests were  conducted  at  22°C,  in  50/50  GP2  and
      natural  seawater  at  a  salinity of  30%o.
      SDS  concentrations for Test  1  were:   0.8, 1.3,  2.2,  3.6, 6.0,  and
      10.0 mg/L.   SDS concentrations for Tests 2,  3,  and 4 were:  0.48,
      0.8, 1.3,  2.2, 3.6,  and 6.0  mg/L.  SDS concentrations for  Tests  5
      and  6  were:  0.09, 0.16, 2.26,  0.43,  0.72, and  1.2 mg/L.
      NOEC Range:  0.09  - 0.48 mg/L (this represents  a difference of  two
      exposure concentrations).
      For  a  discussion  of  the precision  of data from chronic toxicity
      tests  see Section 4, Quality Assurance.           j
                                     377

-------
TABLE 15.   SINGLE-LABORATORY PRECISION OF THE RED MACROALGA, CHAHPIA
            PARVULA,  REPRODUCTION TEST IN NATURAL SEAWATER (30%o
            SALINITY),..  THE REFERENCE TOXICANT USED WAS COPPER (CU)
            SULFATE1/2'3

Test NOEC
1 1.00
2 0.50
3 0.50
4 0.50
n: 4
Mean : NA
CV(%): NA
Cu (UQ/L)
IC25
2.62
0.71
2.83
0.99
4
1.79
61.09

IC50
4.02
1.66
3.55
4.15
4
3.35
34.45
    Data from USEPA (1991a).
    Copper concentrations were 0.5, 1.0, 2.5, 5.0, 7.5, and 10 M9/L.
    Concentrations of Cu were made from a 100 pg/mL CuSO,  standard
    obtained from Inorganic Ventures, Inc.,  Brick, NJ.
    Prepared by Steven Ward and Glen Thursby, Environmental Research
    Laboratory, USEPA, Narragansett, RI.
                                   378

-------
TABLE 16.   SINGLE-LABORATORY PRECISION OF THE RED MACROALGA, CHAMPIA
            PARVULA,  REPRODUCTION TEST IN NATURAL SEAWATER (30%o
            SALINITY).   THE REFERENCE TOXICANT USED WAS SODIUM DODECYL
            SULFATE  (SDS)1'2'3
Test
1
2
3
4
n:
Mean:
CV(%):
1 Data from USEPA

NOEC
0.60
0.60
0.30
0.15
4
NA
NA
(1991a).
t«*f* i,ifsi*st A AO 7C f\ i
SDS (mq/L)
IC25
0.05
0.48
0.69
0.60
4
0.46
62.29
\tz. ft i c ft ft^ ft en a

IC50
0.50
0.81
0.89
0.81
4
0.75
22.92
nrl 1 Oft mn/l
   Concentrations of SDS were made  from  a 44.64 ± 3.33 mg/mL  standard
   obtained from the EMSL-USEPA, Cincinnati, OH.
   Prepared by Steven Ward and Glen Thursby, Environmental  Research
   Laboratory, USEPA, Narragansett, RI.
                                    379

-------
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APHA.  1992.  Part 8010E.4.D.   In: Standard methods for the examination of
      water and wastewater.  18th edition.  American Public Health Association,
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ASTM.  1993.  Standard practice for using brine shrimp nauplii as food for
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Benoit, D.A., F.A. Puglisi,  and D.L. Olson.   1982.  A fathead minnow,
      Pimephales promelas, early life state toxicity test method  evaluation
      and exposure to four organic chemicals.  Environ. Pollut. (Series A)
      28:189-197.

Bengtson, D.A.  1984.  Resource partitioning  by Menidia mem'dia and Menidia
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      18:21-32.Bengtson, D.A.S., A.D. Beck, S.M. Lussier, D. Migneault, and
      C.E. Olney. 1984.  International study  on Artemia. XXXI.  Nutritional
      effects in toxicity tests: use of different Artemia geographical
      strains.  In:Persoone, 6.,, E. Jaspers,  and C. Claus, eds.,
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Birge, W.J., J.A. Black, and B.A. Ramey.  1981.  The reproductive  toxicology
      of aquatic contaminants.  Hazard assessments of chemicals, current
      developments, Vol. 1, Academic Press, Inc., p. 59-114.

Birge, W.J., and R.A. Cassidy.  1983.  Importance of structure-activity
      relationships in aquatic toxicology.  Fundam. Appl. Toxicol. 3:359-368.

Birge, W.J., J.A. Black, and A.G. Westerman.  1985.  Short-term fish and
      amphibian embryo-larval tests for determining the effects of toxicant
      stress on early life stages and estimating chronic values for single
      compounds and complex effluents.  Environ. Tox. Chem. (4):807-821.

Bower, C.E.  1983.  The basic marine aquarium.  Charles C. Thomas, Publ.,
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Chernoff, B., J.V. Conner,  and C.F. Byran.  1981.  Systematics of the Menidia
      berylTina complex (Pisces:Atherinidae) from the Gulf of Mexico and its
      tributaries. Copeia 2:319-335.
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DeWoskin, R.S.  1984.  Good laboratory practice regulations: a comparison.
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Dunnett, C.W.  1955.  Multiple comparison procedure for comparing several
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Emerson, K., R.C. Russo, R'.E. Lund, and R.V. Thurston.  1975.: Aqueous ammonia
      equilibrium calculations; effect of pH and temperature.  J. Fish. Res.
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FDA.  1978.  Good laboratory practices for nonclinical laboratory studies.
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Finney, D.J.  1971.  Probit analysis.  Third Edition.  Cambridge Press, NY,
      NY.  668 pp.

Gentile, J.H., S.M. Gentile, G. Hoffman, J.F. Heltshe, and N.IS. Hairston, Jr.
      1983.  The effects of chronic mercury exposure on the survival,
      reproduction  and population dynamics of Mysidopsis bahia.  Environ.
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Goodman, L.R., D.J. Hansen, D.P. Middaugh, G.M. Cripe, and J.C. Moore.   1985.
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      and results with chloropyrifos.  In:  Cardwell, R.D., R, Purdy, and R.C.
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Heard,  R.W.   1982.  Guide to the common tidal marsh  invertebrates of the
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Hildebrand,  S.F.   1922.  Notes  on  habits  and development  of eggs and larvae
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Hildebrand,  S.F.,  and W.C. Schroeder.   1928.  Fishes of Chesapeake Bay.   Bull.
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Hodges,  J.L., Jr.,  and  E.L.  Lehmann.   1956.  The efficiency of  some
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Home,  J.D.,  M.A.  Swirsky, T.A.  Hollister,  B.R. Oblad, and  J.H. Kennedy.  1983.
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Jensen,  A.L.   1972.  Standard  error of LC50  and sample size  in  fish  bioassays.
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Johnson, M.S.   1975.  Biochemical systematics of the atherinid genus Menidia.
      Copeia 4:662-691.

Kuntz, A.  1916.  Notes on the embryology and larval development of five
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      "International Study on Artemia.  XXV.  Factors determining  the
      nutritional effectiveness of Artemia:  The relative impact of
      chlorinated hydrocarbons and essential fatty acids in San Francisco Bay
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      pp. 71-82.

Leger, P., Bengtson, D.A., Simpson, K.L. and Sorgeloos, P.  1986.  "The use
      and nutritional value of Artemia as a food source," M. Barnes (ed.),
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Lussier, S.M.,  J.H. Gentile, and J. Walker.  1985.  Acute and chronic effects
      of heavy  metals and cyanide on Mysidopsis bahia (Crustacea:  Mysidacea).
      Aquat. Toxicol. 7:25-35.

Macek, K.J., and B.H. Sleight.  1977.  Utility of toxicity tests with embryos
      and fry of fish in evaluating hazards associated with the chronic
      toxicity  of chemicals to fishes.  In:  Mayer, F. L., and J.  L. Hamelink,
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Martin, F.D. and G.E. Drewry.  1978.  Development of fishes of the Mid-
      Atlantic  Bight.  An atlas of eggs, larval, and juvenile stages.
      Biological Service Program, Fish and Wildlife Service, U.S.  Department
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McKim, J.M.  1977.  Evaluation of tests with the early life stages of fish for
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Middaugh, D.P., and T. Takita.  1983.  Tidal and diurnal spawning  cues in the
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Middaugh, D.P., and M.J. Hemmer.  1984.  Spawning of the tidewater silverside,
      Menidia peninsuTae (Goode and Bean) in response to tidal and lighting
      schedules in the laboratory.  Estuaries 7:137-146.

Middaugh, D.P., M.J. Hemmer, and Y. Lamadrid-Rose.  1986.  Laboratory
      spawning  cues in Menidia beryllina and M. pennisulae (Pices:
      Atherinidae) with notes on survival and growth of larvae at  different
      salinities.  Environ. Biol. Fish. 15(2):107-117.
Miller, R.G.  1981.  Simultaneous statistical inference.
      New York. 299 pp.
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Mount, D.I., and C.E. Stephan.  1967.  A method for establishing acceptable
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Norberg, T.J., and D.I. Mount.  1985.  A new fathead minnow  (Pi'mephales
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      IC30, and IC50 for Appendix A of the revised technical support document.
      Memorandum to M. Heber, USEPA, Washington, DC.

Price, W. W.  1978.  Occurrence of Mysidopsis aTmyra Bowman, M. bahia
      Molenock, and Bowmaniella brasiliensis Bacescu (Crustacea, Mysidacea)
      from the eastern coast  of Mexico.  Gulf Res. Repts. 6:173-175.

Price, W.W.   1982.   Key to the shallow water Mysidacea of the Texas coast
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      recent  determinations of the  solubility of oxygen  in seawater.   Limnol.
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       (rapporteur).  1986.  Mysidopsis sp.: life history and culture.  A
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       1986.   American  Petroleum Institute, Washington, DC.

Simmons,  E.G.   1957.   Ecological  survey of the Upper Laguna  Madre of  Texas.
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       of Artificial  Island.   Part V.  The fish of  four low-salinity tidal
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       edition.   Iowa State University  Press, Ames,  IA.   593  pp.


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Spotte, S.  1973.  Marine  aquarium  keeping.  John Wiley  and  Sons, NY, NY.

Spotte, S., G. Adams,  and  P.M.  Bubucis.   1984.   GP2  as an  artificial  seawater
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      control.  Biometrics  15:560-572.

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      Mysidacea of the North Central Gulf of Mexico.  Gulf Res. Rept.
      6(3):225-238.

Stuck, K.C., H.M. Perry, and R.W. Heard.  1979b.  Records  and range extensions
      of Mysidacea from coastal and shelf water of the Eastern  Gulf of Mexico.
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Tagatz, M.E., and D.L. Dudley.  1961.  Seasonal  occurrence of marine  fishes  in
      four shore habitats near  Beaufort, N.C.,  1957-1960.  U.S. Fish. Wild!.
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Taylor, O.K.  1987.  Quality assurance of chemical measurements.  Lewis Publ.,
      Inc., Chelsea, MI.

Thurston, R.V., R.C. Russo, and K.  Emerson. 1974.  Aqueous ammonia equilibrium
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USDA.   1989.  Methods which detect multiple residues.   Vol.  1.  Pesticide
      analysis
      manual.  U. S. Department of-Health and Human Services, Washington D.C.

USEPA.  1973. Biological field  and  laboratory methods for  measuring the
      quality of surface waters and effluents.   C. I. Weber  (ed.).  U. S.
      Environmental Protection Agency, Methods  Development and  Quality
      Assurance Research Laboratory, Cincinnati, OH 45268. EPA  600/4-73-001.

USEPA.  1975.  Methods for  acute toxicity tests  with fish, macroinvertebrates,
      and amphibians.  Environmental Research Laboratory,  U. S. Environmental
      Protection Agency, Duluth, MN 55804.  EPA/660/3-75/009.

USEPA.  1978. Life-cycle toxicity test using sheepshead  minnows (Cypn'nodon
      variegatus).  Hansen, D.J., P.R. Parrish,  S.C. Schimmel,  and L.R.
      Goodman.   In: Bioassay procedures for the  ocean disposal  permit program,
      U. S. Environmental Protection Agency, Environmental Research
      Laboratory, Gulf Breeze, FL 32561. EPA/600/9-78/010, pp.  109-117.

USEPA.  1979a.   Handbook for analytical quality  control  in water and
      wastewater laboratories.  U. S. Environmental Protection Agency,
      Environmental Monitoring and Support Laboratory, Cincinnati, OH 45268.
      EPA/600/4-79/019.
                                         384

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 USEPA.   19795.   Methods  for chemical  analysis of water and wastes.
       Environmental  Monitoring  and Support Laboratory, U.  S.  Environmental
       Protection Agency,  Cincinnati,  OH 45268.   EPA-600/4-79/020,  revised
       March  1983.

 USEPA.   1979c.   Interim  NPDES compliance biomonitoring inspection  manual.
       Office of  Water  Enforcement,  U.  S.  Environmental Protection  Agency,
       Washington,  DC 20460.  (MCD-62).

 USEPA.   1979d.   Good laboratory practice standards  for health effects.
       Paragraph  772.110-1,  Part 772 -  Standards  for development of test data.
       Fed. Reg.  44:27362-27375,  May 9,  1979.
                                                              i
 USEPA.   1980a.   Appendix  B  - Guidelines for Deriving Water Quality  Criteria
       for the Protection  of Aquatic Life and  Its Uses.  Federal  Register, Vol.
       45, No. 231, Friday,  November 28,  1980.

 USEPA.   1980b.   Proposed  good laboratory practice guidelines  for toxicity
       testing.   Paragraph 163.60-6.   Fed.  Reg. 45:26377-26382,  April  18,  1980.

 USEPA.   1980c.   Physical, chemical,  persistence,  and ecological  effects
       testing; good  laboratory  practice standards (proposed rule).  40  CFR
       772, Fed.  Reg. 45:77353-77365, November 21, 1980.

 USEPA.   1981.  In  situ acute/chronic toxicological  monitoring of industrial
      effluents  for  the NPDES biomonitoring program using  fish  and  amphibian
      embryo/larval  stages  as test  organisms.  Birge,  W.J., and  J.A.  Black.
      Office  of  Water Enforcement  and  Permits, U. S.  Environmental  Protection
      Agency, Washington, DC 20460. OWEP-82-001.

 USEPA.   1982.  Methods for organic  chemical analysis  of municipal and
       industrial wastewater.  Environmental Monitoring and Support  Laboratory,
      U. S. Environmental Protection Agency, Cincinnati, OH 45268.
      EPA/600/4-82/057.
                                                              I
                                                              i
 USEPA.   1983.  Guidelines and format for  EMSL-Cincinnati methods.   Kopp,  J.F.
      Environmental Monitoring  and  Support  Laboratory, U. S.  Environmental
      Protection Agency,  Cincinnati, OH 45268. EPA/600/8-83/020.

USEPA.   1984.  Effluent and ambient toxicity testing  and instream community
      response on the Ottawa River, Lima, Ohio.  Mount, D.I., N.A.  Thomas,
      T.J. Norberg, M.  T. Barbour, T.H. Roush, and W.F. Brandes.
      Environmental Research Laboratory, U. S. Environmental  Protection
      Agency, Duluth, MN  55804.  EPA/600/3-84/080.

USEPA.   1985a.  Methods for measuring the acute toxicity of effluents to
      freshwater and marine organisms.  Third Edition. Peltier, W., and C.I.
      Weber,  eds.  Environmental Monitoring and Support Laboratory, U. S.
      Environmental Protection Agency, Cincinnati, OH 45268.  EPA/600/4-85/013.
                                        385

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USEPA.  1985b.  Short-term methods for estimating the chronic toxicity of
      effluents and receiving waters to'freshwater organisms.  Horning, W.B.,
      and C.I. Weber (eds). Second edition.  Environmental Monitoring and
      Support Laboratory, U. S. Environmental Protection Agency, Cincinnati,
      OH 45268. EPA/600/4-85/014.

USEPA   1985c.  Validity of effluent and ambient toxicity tests for predicting
      biological impact, Scippo Creek, Circleville, Ohio.  Mount, D.I., and
      T J  Norberg-King  (eds.).   Environmental Research Laboratory, U. S.
      Environmental Protection Agency, Duluth, MN 55804. EPA/600/3-85/044.

USEPA   1985d.  Validity of effluent and ambient toxicity testing for
      predicting biological impact on  Five  Mile Creek, Birmingham, Alabama.
      Mount,  D.I., A.E.  Steen, and T.J. Norberg-King  (eds.).  Environmental
      Research  Laboratory, U.  S.  Environmental Protection Agency, Duluth, MN
      55804.  EPA/600/8-85/015.

USEPA.  1985e.  Validity of effluent and ambient toxicity tests for predicting
      biological impact, Ohio  River, near Wheeling, West Virginia.  Mount,
      D.I., A.  E.  Steen, and T.J. Norberg-King  (eds.).   Environmental  Research
      Laboratory,  U. S.  Environmental  Protection Agency, Duluth,  MN 55804.
      EPA/600/3-85/071.

USEPA.  1986a.  Validity of effluent and  ambient toxicity tests for predicting
      biological impact, Back  River, Baltimore  Harbor, Maryland.  Mount,  D.I.,
      A.  E. Steen,  and T.  Norberg-King (eds.).   Environmental  Research
      Laboratory,  U. S.  Environmental Protection  Agency, Duluth, MN  55804.            IBHi
      EPA/600/8-86/001.                                                                ^1"

USEPA.   1986b.  Validity of effluent  and  ambient  toxicity  tests  for  predicting
      biological  impact, Skeleton Creek,  Enid,  Oklahoma.  Norberg, T.J.,  and
      D.I.  Mount  feds.)-  Environmental  Research  Laboratory, U.  S.
       Environmental  Protection Agency, Duluth,  MN  55804.  EPA/600/8-86/002.

USEPA.   1986c.   Validity of effluent  and  ambient toxicity tests for  predicting
       biological  impact, Kanawha River,  Charleston, West Virginia.   Mount,
       D.I., and T. Norberg-King  (eds.).   Environmental  Research Laboratory, U.
       S.  Environmental Protection Agency,  Duluth,  MN 55804. EPA/600/3-86/006.

 USEPA.   1986d.   Validity of effluent and ambient toxicity tests for predicting
       biological  impact, Naugatuck River,  Connecticut.   Mount, D.I.,  T.
       Norberg-King, and A.E.  Steen (eds.).  Environmental  Research Laboratory,
       U.  S. Environmental  Protection Agency, Duluth, MN. 55804
       EPA/600/8-86/005.

 USEPA.   1986e.   Occupational  health and safety manual.  Office of
       Administration,  U. S. Environmental  Protection Agency, Washington,
       DC 20460.
                                          386

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USEPA.  1987a.  Users guide to the conduct and interpretation of complex
      effluent toxicity tests at estuarine/marine sites.  Schimmel, S.C., ed.
      Environmental Research Laboratory, U. S. Environmental Protection
      Agency, Narragansett, RI 02882.  Contribution No. 796.,j265 pp.
                                                             "I            ,
USEPA.  1987b.  Guidance manual for conducting complex effluent and receiving
      water larval fish growth-survival studies with the sheepshead minnow
      (Cypn'nodon van'egatus).  Contribution No. X104.  Hughes, M.M., M.A.
      Heber, S.C. Schimmel, and W.J. Berry.  In:  Schimmel, S.C., ed.  Users
      guide to the conduct and interpretation of complex effluent toxicity
      tests at estuarine/marine sites.  Environmental Research Laboratory, U.
      S. Environmental Protection Agency, Narragansett, RI 02882.
      Contribution  No. 796., 265 pp.

USEPA.  1987c.  Guidance manual for rapid chronic toxicity tests on effluents
      and receiving waters with larval inland silversides (Metm'dia beryllina).
      Contribution No. 792.  Heber, M.A., M.M. Hughes, S.C. Schimmel, and D.A.
      Bengtson.  In:  Schimmel, S.C. ed., Users guide to the conduct and
      interpretation of complex effluent toxicity tests at estuarine/marine
      sites.  Environmental Research Laboratory, U. S. Environmental
      Protection Agency, Narragansett, RI 02882. Contribution No. 796., 265
      PP-                                                    ;

USEPA.  1987d.  Guidance manual for conducting seven day mysid
      survival/growth/reproduction study using the estuarine mysid, Mysidopsis
      bahia.  Contribution No. X106.  Lussier, S.M., A. Kuhn, and J. Sewall.
      In:  Schimmel, S. C., ed.  Users guide to the conduct and interpretation
      of complex effluent toxicity tests at estuarine/marine sites.
      Environmental Research Laboratory, U. S. Environmental Protection
      Agency, Narragansett, RI 02882.  Contribution No. 796., 265 pp.

USEPA.  1987e.  Guidance manual for conducting sperm cell tests with the sea
      urchin, Arbacia punctulata, for use in testing complex effluents.
      Nacci, D., R. Walsh, and E. Jackim.  Contribution No. X105.  In:
      Schimmel, S.C., ed.  Users guide to the conduct and interpretation of
      complex effluent toxicity tests at estuarine/marine site. Environmental
      Research Laboratory, U. S. Environmental Protection Agency,
      Narragansett, RI 02882.  Contribution No. 796., 265pp.

USEPA.  1987f.  Guidance manual for conducting sexual reproduction test with
      the marine macroalga Champia parvula for use in testing complex
      effluents.  Contribution No. X103.  Thursby, G.B., and R.L. Steele.  In:
      Schimmel, S. C., ed.  Users guide to the conduct and interpretation of
      complex effluent toxicity tests at estuarine/marine sites.
      Environmental Research Laboratory, U. S. Environmental Protection
      Agency, Narragansett, RI 02882.  Contribution No. 796., 265 pp.
                                         387

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USEPA.  1987g.  Methods for spawning, culturing and conducting toxicity-tests
      with early life stages of four antherinid fishes: the inland silverside,
      Hem'dia beryllina, Atlantic silverside, M. menidia, tidewater
      silverside, M. pem'nsulae, and California grunion, Leuresthes tenuis.
      Middaugh, D.P., M.J. Hemmer, and L.R. Goodman.  Office of Research and
      Development, U. S. Environmental Protection Agency, Washington, DC
      20460. EPA/600/8-87/004.

USEPA.  1988a.  Short-term methods for estimating the chronic toxicity of
      effluents and receiving waters to marine and estuarine organisms.
      Weber, C.I., W.B. Horning, II, D.J. Klemm, T.W. Neiheisel, P.A. Lewis,
      E.L. Robinson, J. Menkedick, and F. Kessler (eds.). Environmental
      Monitoring and Support Laboratory, U. S. Environmental Protection
      Agency, Cincinnati, OH 45268. EPA/600/4-87/028.

USEPA.  1988b.  NPDES compliance inspection manual.  Office of Water
      Enforcement and Permits  (EN-338), U. S. Environmental Protection Agency,
      Washington, DC 20460.

USEPA.  1988c.  Methods for aquatic toxicity  identification evaluations:
      Phase  I toxicity characterization procedures.  D.I. Mount and  L.
      Anderson-Carnahan.  Environmental Research Laboratory, U. S.
      Environmental Protection Agency, Duluth, MN 55804. EPA-600/3-88/034.

USEPA.  1988d.  An  interpolation estimate for chronic toxicity: The  ICp
      approach.  Norberg-King, T.J.  Technical Report 05-88, National Effluent
      Toxicity Assessment Center,  Environmental Research Laboratory,  U.  S.
      Environmental Protection Agency, Duluth, MN 55804.

USEPA.  1989a.  Short-term methods for estimating the chronic toxicity of
      effluents and receiving  waters to freshwater  organisms.   Second Edition.
      Weber,  C.I.,  W.H. Peltier, T.J. Norberg-King,  W.B. Horning,  II, F.A.
      Kessler, J.R. Menkedick, T.W.  Neiheisel, P.A.  Lewis,  D.J. Klemm, Q.H.
      Pickering, E.L. Robinson, J.M. Lazorchak, L.J. Wymer, and R.W.  Freyberg
      (eds.)-  Environmental Monitoring Systems Laboratory, U.  S.
      Environmental  Protection Agency, Cincinnati,  OH 45268.  EPA/600/4-89/001.

USEPA.  1989b.  Toxicity  reduction evaluation protocol  for  municipal
      wastewater treatment plants.   J.A.  Botts, J.W. Braswell,  J.  Zyman, W.L.
      Goodfellow,  and S.B. Moore  (eds.).  Risk Reduction Engineering
      Laboratory,  U. S. Environmental  Protection Agency, Cincinnati,  OH  45268.
      EPA/600/2-88/062.

USEPA.  1989c.   Generalized methodology for  conducting  industrial  toxicity
      reduction  evaluations  (TREs).  J.A.  Fava, D.  Lindsay, W.H.  Clement,  R.
      Clark,  G.M.  DeGraeve, J.D.  Cooney,  S.  Hansen,  W.  Rue, S.  Moore, and  P.
      Lankford (eds.).  Risk  Reduction  Engineering  Laboratory,  U.  S.
      Environmental  Protection Agency,  Cincinnati,  OH  45268.  EPA/600/2-88/070.
                                         388

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USEPA.  1989d.  Methods for aquatic toxicity identification evaluations:
      Phase II toxicity identification procedures.  D.I. Mount, and L.
      Anderson-Carnahan.  Environmental Research Laboratory, U. S.
      Environmental Protection Agency, Duluth, MN 55804. EPA-600/3-88/035.

USEPA.  1989e.  Methods for aquatic toxicity identification evaluations:
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                                        398

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                                  APPENDICES
                                                                          Page
A.     Independence,  Randomization,  and Outliers	  403
       1.  Statistical  Independence	403
       2.  Randomization	403
       3.  Outliers     	i,	408
B.     Validating Normality  and Homogeneity of Variance Assumptions .  .  .  409
       1.  Introduction	-.	409
       2.  Tests  for  Normal  Distribution  of Data	409
       3.  Test for Homogeneity of Variance    	417
       4.  Transformations of  the Data    	I	417
C.     Dunnett's  Procedure   	  419
       1.  Manual  Calculations	419
       2.  Computer Calculations  	I	426
D.     T test with the Bonferroni Adjustment   	':	432
E.     Steel's Many-one Rank Test     	I	438
F.     Wilcoxon Rank  Sum Test with  the Bonferroni  Adjustment   	443
G.     Single Concentration  Toxicity Test - Comparison of Control  with
          100% Effluent or  Receiving Water	450
H.     Probit Analysis  	'•.	454
I.     Spearman-Karber Method	457
J.     Trimmed Spearman-Karber Method    	  	  462
K.     Graphical  Method   	[..	467
L.     Linear Interpolation  Method	471
                                                           !
       1.  General  Procedure	471
                                      401

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                     APPENDICES (CONTINUED)
                                                                   Page
2.  Data Summary and Plots	471
3.  Monotonicity	471
4.  Linear Interpolation Method   	  472
5.  Confidence Intervals    	  473
6.  Manual Calculations	  .  473
7.  Computer Calculations   	  477
Cited References	482
                               402

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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 test organisms are placed serially into test chambers, or
where all replicates for  a test concentration are located  adjacent to one
another, which  could introduce bias into the test results.
                             .
2.2  An example of randomization of the distribution  of test organisms  among
test chambers,  and an example of randomization of arrangement of treatments
and replicate chambers are described  using the Sheepshead  Minnow Larval
Survival and  Growth  test.  For the purpose of the example, the test design  is
as follows:   Five  effluent concentrations are tested  in addition to the
control.  The effluent concentrations  are as  follows:  6.25%,  12.5%, 25.0%,
50.0%,  and  100.0%.   There are four replicate  chambers  per  treatment.  Each
replicate chamber  contains ten fish.                       j

2.3   RANDOMIZATION OF FISH TO REPLICATE  CHAMBERS  EXAMPLE
                                                           I
2.3.1   Consider first the random  assignment  of the  fish to the replicate
chambers.   The  first step is  to  label  each  of the  replicate chambers with  the
control  or  effluent  concentration  and the replicate number.  The next step  is
to assign each  replicate chamber  four double-digit  numbers:.  An example of
this  assignment is provided  in Table  A.I.   Note  that  the  double digits  00  and
97 through  99 were not  used.


                                      403

-------
      TABLE A.I.    RANDOM ASSIGNMENT OF FISH TO REPLICATE CHAMBERS EXAMPLE
                    ASSIGNED NUMBERS FOR EACH REPLICATE CHAMBER
Assigned
01,
02,
03,
04,
05,
06,
07,
08,
09,
10,
11,
12,
13,
14,
15,
16,
17,
18,
19,
20,
21,
22,
23,
24,
25,
26,
27,
28,
29,
30,
31,
32,
33,
34,
35,
36,
37,
38,
39,
40,
41,
42,
43,
44,
45,
46,
47,
48,
Numbers
49,
50,
51,
52,
53,
54,
55,
56,
57,
58,
59,
60,
61,
62,
63,
64,
65,
66,
67,
68,
69,
70,
71,
72,
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96




6.25%
6.25%
6.25%
6.25%
12.5%
12.5%
12.5%
12.5%
25.0%
25.0%
25.0%
25.0%
50.0%
50.0%
50.0%
50.0%
Replicate Chamber
Control ,
Control ,
Control ,
Control ,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
100.0% effluent,
100.0% effluent,
100.0% effluent,
100.0% effluent,
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
2.3.2  The random numbers used to carry out the random assignment of fish to
replicate chambers are provided in Table A.2.  The third step is to choose a
starting position in Table A.2, and read the first double digit number.   The
first number read identifies the replicate chamber for the first fish taken
from the tank.  For the example, the first entry in row 2 was chosen as  the
starting position.  The first number in this row is 37.  According to Table
A.I, this number corresponds to replicate chamber 1 of the 25.0% effluent
concentration.  Thus, the first fish taken from the tank is to be placed in
replicate chamber 1 of the 25.0% effluent concentration.

2.3.3  The next step is to read the double digit number to the right of  the
first one.  The second number identifies the replicate chamber for the second
fish taken from the tank.  Continuing the example, the second number read in
row 2 of Table A.2 is 54.  According to Table A.I, this number corresponds to
replicate chamber 2 of the 6.25% effluent concentration.  Thus, the second
fish taken from the tank is to be placed in replicate chamber 2 of the 6.25%
effluent concentration.
                                      404

-------
TABLE A.2.  TABLE OF RANDOM NUMBERS (Dixon and Massey, 1983)
10 09 73
37 54 20
08 42 26
99 01 90
12 80 79
66 06 57
31 06 01
85 26 97
63 57 33
73 79 64
98 52 01
11 80 50
83 45 29
88 68 54
99 59 46
65 48 11
80 12 43
74 35 09
69 91 62
09 89 32
91 49 91
80 33 69
44 10 48
12 55 07
63 60 64
61 19 69
15 47 44
94 55 72
42 48 11
23 52 37
04 49 35
00 54 99
35 96 31
59 80 80
46 05 88
32 17 90
69 23 46
19 56 54
45 15 51
94 86 43
98 08 62
33 18 51
80 95 10
79 75 24
18 63 33
74 02 94
54 17 84
11 66 44
48 32 47
69 07 49
25 33
48 05
89 53
25 29
99 70
47 17
08 05
76 02
21 35
57 53
77 67
54 31
96 34
02 00
73 48
76 74
56 35
98 17
68 03
05 05
45 23
45 98
19 49
37 42
93 29
04 46
52 66
85 73
62 13
83 17
24 94
76 54
53 07
83 91
52 36
05 97
14 06
14 30
49 38
19 94
48 26
62 32 •
04 06
91 40
25 37
39 02
56 11
98 83
79 28
41 38
76 52 01 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
24
23
38
64
36
35
68
90
35
22
50
13
36
91
58
45
43
36
46
46
70
32
12
40
51
59
54
16
68
45
96
33
83
77
05
15
40
43
34
44
89
20
69
31
97
05
59
02
35
67 35
80 52
20 90
31 13
03 23
69 73
30 34
66 57
55 35
80 83
10 94
72 56
74 67
76 66
82 60
04 77
31 82
23 60
93 68
42 75
16 28
29 73
97 92
86 07
21 95
92 43
36 78
62 24
86 84
93 59
86 25
11 96
35 13
60 94
28 14
56 70
95 66
41 92
66 79
88 88
99 90
43 54
15 12
86 10
01 02
79 01
33 51
38 17
29 53
58 40
43 76
40 37
25 60
11 65
66 53
61 70
26 14
48 18
75 48
42 82
05 58
82 48
00 78
79 51
89 28
69 74
23 74
02 10
72 03
67 88
35 54
41 35
65 75
46 97
25 63
37 29
38 48
44 31
87 67
14 16
10 25
38 96
54 62
97 00
40 77
70 07
00 00
15 85
45 43
15 53
88 '96
85 81
33 87
25 91
46 74
71 19
29 69
15 39
68 70
44 01
80 95
20 63
15 95
88 67
98 95
65 81
86 79
73 05
28 46
60 93
60 97
29 40
18 47
90 36
93 78
73 03
21 11
45 52
76 62
96 29
94 75
53 14
57 60
96 64
43 65
65 39
82 39
91 19
03 07
26 25
61 96
54 69
77 97
13 02
93 91
86 74
18 74
66 67
59 04
01 54
39 09
88 69
25 01
74 85
05 45
52 52
56 12
09 97
32 30
10 51
90 91
61 04
33 47
67 43
11 68
33 98
90 74
38 52
82 87
52 03
09 34
52 42
54 06
47 64
56 13
95 71
57 82
16 42
11 39
77 88
08 99
03 33
04 08
48 94
17 70
45 95
61 01
04 25
11 20
22 96
27 93
28 23
45 00
12 48
08 36
31 71
39 24
43 68
79 00
03 54
47 34
54 19
62 52
22 05
56 14
75 80
71 92
33 34
75 75
82 16
17
02
64
97
77
85
39
47
09
44
33
01
10
93
68
86
53
37
90
22
23
40
81
39
82
93
18
92
59
63
35
91
24
92
47
57
23
06
33
56
07
94
98
39
27
21
55
40
46
15
39
00
35
04
12
11
23
18
83
35
50
52
68
29
23
40
14
96
94
54
37
42
22
28
07
42
33
92
25
05
65
23
90
78
70
85
97
84
20
05
35
37
94
00
77
80
36
88
15
O'l
29 27
82 29
08 03
43 62
27 17
19 92
40 30
62 38
49 12
27 38
50 07
77 56
71 17
60 91
47 83
21 81
38 55
28 60
40 05
38 21
08 92
05 08
22 20
70 72
20 73
58 26
21 15
92 74
70 14
52 28
33 71
28 72
10 33
56 52
61 74
39 41
11 89
96 28
82 66
01 45
44 13
54 87
62 46
38 75
93 89
81 45
04 09
46 12
02 00
84 87
49 45
16 65
36 06
76 59
68 33
91 70
97 32
85 79
56 24
84 35
39 98
78 51
78 17
10 62
41 13
65 44
37 63
26 55
64 18
45 98
00 48
23 41
64 13
58 15
17 90
05 27
94 66
59 73
66 70
25 62
24 72
95 29
93 33
01 06
29 41
18 38
63 38
52 07
95 41
11 76
18 80
30 43
11 71
95 79
19 36
17 48
03 24
33 56
99 94
69 38
                              405

-------
2.3.4  Continue in this fashion until all the fish have been randomly assigned
to a replicate chamber.  In order to fill each replicate chamber with ten
fish, the assigned numbers will be used more than once.  If a number is read
from the table that was not assigned to a replicate chamber, then ignore it
and continue to the next number.  If a replicate chamber becomes filled and a
number is read from the table that corresponds to it, then ignore that value
and continue to the next number.  The first ten random assignments of fish to
replicate chambers for the example are summarized in Table A.3.

       TABLE A.3.      EXAMPLE OF RANDOM ASSIGNMENT OF FIRST TEN FISH TO
                       REPLICATE CHAMBERS
Fish
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
Assignment
25.0% effluent,
6.25% effluent,
50.0% effluent
100.0% effluent,
6.25% effluent,
25.0% effluent,
50.0% effluent,
100.0% effluent,
50.0% effluent,
100.0% effluent,

repl
repl
repl
repl
repl
repl
repl
repl
repl
repl

icate
icate
icate
icate
icate
icate
icate
icate
icate
icate

chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber

1
2
4
4
1
4
1
3
2
4
2.3.5  Four double-digit numbers were assigned to each replicate chamber
(instead of one, two, or three double-digit numbers) in order to make
efficient use of the random number table (Table A.2).  To illustrate, consider
the assignment of only one double-digit number to each replicate chamber:  the
first column of assigned numbers in Table A.I.  Whenever the numbers 00 and 25
through 99 are read from Table A.2, they will be disregarded and the next
number will be read.

2.4  RANDOMIZATION OF REPLICATE CHAMBERS TO POSITIONS EXAMPLE

2.4.1  Next consider the random assignment of the 24 replicate chambers to
positions within the water bath (or equivalent).  Assume that the replicate
chambers are to be positioned in a four row by six column rectangular array.
The first step is to label the positions in the water bath.  Table A.4
provides an example layout.

2.4.2  The second step is to assign each of the 24 positions four double-digit
numbers.  An example of this assignment is provided in Table A.5.  Note that
the double digits 00 and 97 through 99 were not used.

2.4.3  The random numbers used to carry out the random assignment of replicate
chambers to positions are provided in Table A.2.  The third step is to choose
a starting position in Table A.2, and read the first double-digit number.  The
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

                                      406

-------
  TABLE A.4.   RANDOM ASSIGNMENT OF REPLICATE CHAMBERS TO POSITIONS:
              LABELLING THE POSITIONS WITHIN THE WATER BATH
  TABLE A.5.   RANDOM ASSIGNMENT OF REPLICATE CHAMBERS TO POSITIONS:
              ASSIGNED NUMBERS FOR EACH POSITION

EXAMPLE
1
7
13
19
2
8
14
20
3
9
15
21
4
10
16
22
i5 •
11
17
2.3
6
12
18
24
EXAMPLE
Assigned
01,
02,
03,
04,
05,
06,
07,
08,
09,
10,
11,
12,
13,
14,
15,
16,
17,
18,
19,
20,
21,
22,
23,
24,
25,
26,
27,
28,
29,
30,
31,
32,
33,
34,
35,
36,
37,
38,
39,
40,
41,
42,
43,
44,
45,
46,
47,
48,
Numbers Position
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 1
74
2
75 3
76 4
77 5
78
79
80
81 •
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
6
7
8
9
10
11 ,
12
13
14
15
16
17
18
19
20
21
22
23
24
chosen as the starting position.  The first number in this row was 73.
According to Table A.5, this number corresponds to position 1.  Thus, the
first replicate chamber for the control will be placed in position 1.
                                      407

-------
2.4.4  The next step is to read the double-digit number to the right of the
first one.  The second number identifies the position for the second replicate
chamber of the control.  Continuing the example, the second number read in row
10 of Table A.2 is 79.  According to Table A.5, this number corresponds to
position 7.  Thus, the second replicate chamber for the control will be placed
in position 7.

2.4.5  Continue in this fashion until all the replicate chambers have been
assigned to a position.  The first four numbers read will identify the
positions for the control replicate chambers, the second four numbers read
will identify the positions for the lowest effluent concentration replicate
chambers, and so on.  If a number is read from the table that was not assigned
to a position, then ignore that value and continue to the next number.  If a
number is repeated in Table A.2, then ignore the repeats and continue to the
next number.  The complete randomization of replicate chambers to positions
for the example is displayed in Table A.6.


    TABLE A.6.  EXAMPLE OF RANDOM ASSIGNMENT OF REPLICATE CHAMBERS TO
                POSITIONS:  ASSIGNMENT OF ALL 24 POSITIONS
Control
Control
100.0%
50.0%
100.0%
12.5%
50.0%
50.0%
6.25%
Control
100.0%
25.0%
6.25%
25.0%
Control
50.0%
6.25%
12.5%
100.0%
12.5%
12.5%
25.0%
25.0%
6.25%
2.4.6  Four double-digit numbers were assigned to each position (instead of
one, two, or three) in order to make efficient use of the random number table
(Table A.2).  To illustrate, consider the assignment of only one double-digit
number to each position:  the first column of assigned numbers in Table A.5.
Whenever the numbers 00 and 25 through 99 are read from Table A.2, they will
be disregarded and the next number will be read.

3.  OUTLIERS

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).
                                      408

-------
                                 APPENDIX  B

        VALIDATING NORMALITY  AND HOMOGENEITY OF  VARIANCE ASSUMPTIONS


1.  INTRODUCTION
                                                           i
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
                                                           i
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 Kollmogorov "D"
statistic (Stephens, 1974) is recommended.  An example of the Shapiro-Wilk's
test is provided below.

2.2  The example uses growth data from the Sheepshead Minnow Larval  Survival
and Growth Test.  The same data are used in the discussion of the homogeneity
of variance determination in  Paragraph 3 and Dunnett's Procedure in  Appendix
C.  The data, the mean and variance of the observations  at each concentration,
including the control, are listed in Table B.I.

2.3  The first step of the test for normality is to center the observations by
subtracting the mean of all observations within  a concentration from each
observation in that concentration. The centered  observations are listed  in
Table B.2.

2.4  Calculate the denominator, D, of the test statistic:

                                     •A     .   ^
                                r\ —  \^ / v   v\ 2
                                U —  Z~t \J\.j~~'4\)


    Where:  X,- =  the centered  observations, X is the overall mean of the
                  centered observations, and  n is the total number of the
                  centered observations.   For this  set of data, X =  0,
                  and D = 0.1589.

                                      409

-------
   TABLE B.I.  SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, LARVAL GROWTH
               DATA (WEIGHT IN MG) FOR THE SHAPIRO-WILK'S TEST
                                     Effluent Concentration (%)
  Replicate
 Control
   6.25
 12.5
25.0
50.0
1
2
3
Mean(Yf)
?i
i
1.017
0.745
0.862
0.875
0.019
1
1.157
0.914
0.992
1.021
0.015
2
0.998
0.793
1.021
0.937
0.016
3
0.837
0.935
0.839
0.882
0.0031
4
0.715
0.907
1.044
0.889
0.027
5
     TABLE B.2.  EXAMPLE OF SHAPIRO-WILK'S TEST:  CENTERED OBSERVATIONS
                                    Effluent Concentration (%)
  Replicate
Control
6.25
12.5
25.0
 50.0
1
2
3
0.142
- 0.130
- 0.013
0.136
- 0.107
- 0.029
0.061
- 0.144
0.084
- 0.009
0.053
- 0.043
- 0.174
0.018
0.155
2.4.1  For this set of data,

            n = 15
                _
                  15

            D = 0.1589
                      (.0) = 0.0
                                      410

-------
2.5  Order the centered observations from smallest to largest,

                           X(1)     v(2)  .,        .,  vWt
                               <  A    <  .  .  .  <  A         I

where X(1> denote the ith order statistic.  The ordered  observations  are
listed in Table B.3.

       TABLE B.3.  EXAMPLE OF THE SHAPIRO-WILK'S TEST:  ORDERED OBSERVATIONS
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
- 0.174
- 0.144
- 0.130
- 0.107
- 0.043
- 0.029
- 0.013
- 0.009
0.018
0.053
0.061
0.084
0.136
0.142
0.155
 2.6   From  Table  B.4,  for  the  number of observations,  n,  obtain the
 coefficients  a,, a2, ...,  ak,  where k is n/2 if n is even,  and (n-l)/2 if n is
 odd.   For  the data  in  this  example,  n  = 15,  k = 7,  and the a,-  values  are
 listed in  Table  B.5.

 2.7   Compute  the test  statistic,  W,  as follows:
                          WD
     The differences,  X
                       Cn-i+1)
- x(f),
are listed in Table B.5.
 2.8  The decision rule for this test is to compare the critical  value from
 Table B.6 to the computed W.   If the computed value is less than the critical
 value,  conclude that the data are not normally distributed.  For this example,
 the critical value at a significance level of 0.01 and 15 observations (n) is
 0.835.   The calculated value, 0.9516, is not less than the critical value.
 Therefore conclude that the data are normally distributed.
                                      411

-------
TABLE B.4. COEFFICIENTS FOR THE SHAPIRO-MILK'S TEST (Conover, 1980)

n
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
-
•
Number of
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








n
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


-
1
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
                                412

-------
TABLE B.4. COEFFICIENTS FOR THE SHAPIRO-WILK'S TEST (CONTINUED)
\
A-
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
0.4156
0.2876
0.2451
0.2137
0.1880
0.1660
0.1463
0.1284
0.1118
0.0961
0.0812
0.0669
0.0530
0.0395
0.0262
0.0131
0.0000
-
-
-

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
O.CI733
0.0622
O.CI515
O.CI409
O.CI305
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

\
A-
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
0.3894
0.2684
0.2334
0.2078
0.1871
0.1695
0.1539
0.1398
0.1269
0.1149
0.1035
0.0927
0.0824
0.0724
0.0628
0.0534
0.0442
0.0352
0.0263
0.0175
0.0087
0.0000
-
-
~

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
0.0211
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
0.1548
0.1415
0.1293
0.1180
0.1073
0.0972
0.0876
0.0783
0.0694
0.0607
0.0522
0.0439
0.0357
0.0277
0.0197
0.0118
0.0039
-
*
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
0.1312
0.1205
0.1105
0.1010
0.0919
0.0832
0.0748
0.0667
0.0588
0.0511
0.0436
0.0361
0.0288
0.0215
0.0143
0.0071
0.0000
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
                                413

-------
                 TABLE B.5.  EXAMPLE OF THE SHAPIRO-WILK'S TEST:
                             TABLE OF COEFFICIENTS AND DIFFERENCES
                                                 _  w
1
2
3
4
5
6
7
0.5150
0.3306
0.2495
0.1878
0.1353
0.0880
0.0433
0.329
0.286
0.266
0.191
0.104
0.082
0.031
X(H)
X(3)
*'
* >
X(10)
X(09>
X<08)
- x< >
- x«}
- *?
- x<*>
- *T
- x(«
- x(7)
2.9  In general, if the data fail the test for normality, a transformation
such as to log values may normalize the data.  After transforming the data,
repeat the Shapiro Milk's Test for normality.

3.  TEST FOR HOMOGENEITY OF VARIANCE

3.1  For Dunnett's Procedure and the t test with Bonferroni's adjustment,  the
variances of the data obtained from each toxicant concentration and the
control are assumed to be equal.  Bartlett's Test is a formal test of this
assumption.  In using this test, it is assumed that the data are normally
distributed.

3.2  The data used in this example are growth data from a Sheepshead Minnow
Larval Survival and Growth Test, and are the same data used in Appendices C
and D.  These data are listed in Table B.7, together with the calculated
variance for the control and each toxicant concentration.

3.3  The test statistic for Bartlett's Test (Snedecor and Cochran, 1980) is as
fol1ows:
In
                                                 In
                      B =
Where:    Mi  = degrees of freedom for each effluent concentration and
                control, (Vf = nf  -  1)

           p  = number of levels of toxicant concentration
                including the control

          In  = loge
                                      414

-------
TABLE B. 6,
n 0.01
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
. QUANTILES OF THE SHAPIRO MILK'S TEST STATISTIC (Conover,
0.02 0.05 0.10 0.50 0.90 0.95 0.98 0.99
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
0.985
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
i.ooo
0.996
0.991
0.986
0.985
0.984
0.984
0.983
0.984
0.984
0.984
0.984
0.984
0.985
0.985
0.986
0.986
0.986
0.987
0.987
0.987
0.987
0.988
0.988
0.988
0.988
0.988
0.988
0.988
0.988
0.989
0.989
0.989
0.989
0.989
0.989
0.989
0.989
0.989
0.989
0,990
0,.990
0.990
0.990
0..990
0..990
0,,990
0..990
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
415

-------
           i   « 1,  2,  ...,  p  where  p  is  the  number  of  concentrations
                including  the control

          n,.   » the number of replicates for concentration  i.
              ;_  i-1
             C=l+ [3 (p-1) ] -1 [   I/ V± - (
  TABLE B.7.   SHEEPSHEAD MINNOW,  CYPRINODON VARIEGATUS,  LARVAL  GROWTH  DATA
              (WEIGHT IN MG)  USED FOR BARTLETT'S TEST FOR HOMOGENEITY  OF
              VARIANCE

Replicate
1
2
3
Mean
Si
i1

Control
1.017
0.745
0.862
0.875
0.019
1
Effl
6.25
1.157
0.914
0.992
1.021
0.015
2
uent Concentration
12.5
0.998
0.793
1.021
0.937
0.016
3
25.0
0.837
0.935
0.839
0.882
0.0031
4
m
50.0
0.715
0.907
1.044
0.889
0.027
5
3.4  Since B is approximately distributed as chi-square with p-1 degrees of
freedom when the variances are equal, the appropriate critical  value is
obtained from a table of the chi-square distribution for p-1  degrees of
freedom and a significance level of 0.01.  If B is less than the critical
value then the variances are assumed to be equal.

3.5  For the data in this example, V, = 2,  p = 5,  S2  =  0.0158,  and  C  = 1.2.
The calculated B value is:
                           2 [5 (In 0.0.158) -
                       B =
                                       1.2
                           2 [5(- 4.1477) - (- 22.1247)]
                                       1.2
                           2.3103
                                      416

-------
3.6  Since B is approximately distributed as chi-square with p  - 1 degrees of
freedom when the variances are equal, the appropriate critical  value for the
test is 13.3 for a significance level of 0.01.  Since B is less than 13.3, the
conclusion is that the variances are not different.

4.  TRANSFORMATIONS OF THE DATA                           j

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-Milk's and
Bartlett's tests should be performed on the transformed observations to
determine whether the assumptions of normality and/or homogeneity of variance
are met.
                                                          i

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  ?. 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 Pf  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 J p  ) transformation is commonly used for such data to stabilize
the variance and satisfy the normality requirement.

4.2.2  Arc sine transformation consists of determining the angle (in radians)
represented by a sine value.  In the case of arc sine square root
transformation of mortality data, the proportion of dead (or affected)
organisms is taken as the sine value, the square root of the sine value is
determined, and the angle (in radians) for the square root of the sine value
is determined.  Whenever the proportion dead is 0 or 1, a special  modification
of the arc sine square root transformation must be used (Bartlett,  1937).  An
explanation of the arc sine square root transformation and the modification is
provided below.
                                                          I
4.2.3  Calculate the response proportion (RP) at each effluent concentration,
where:
                                                          ! .
RP = (number of surviving or unaffected organisms)/(number exposed).

    Example:   If 12 of 20 animals in a given  treatment replicate survive:

              RP = 12/20

                 = 0.60
                                      417

-------
4.2.4  Transform each RP to its arc sine square root,  as follows:
4.2.4.1  For RPs greater than zero or less than one:
         Angle (radians) =  ^
         Example: If RP = 0.60:
                  Angle = arc sine
/0.60
                        = arc sine 0.7746
                        = 0.8861 radians
4.2.4.2  Modification of the arc sine square root when RP = 0.
         Angle  (in radians) = arc sine
         Where: N = Number of animals/treatment replicate
         Example: If 20 animals are used:
                                     a/so
                  Angle = arc sine
                        - arc sine 0.1118
                        « 0.1120 radians
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
                                      418

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                                  APPENDIX C

                              DUNNETT'S PROCEDURE
1.  MANUAL CALCULATIONS
1.1  Dimnett's Procedure (Dunnett, 1955; Dunnett, 1964) is; used to compare
each concentration mean with the control mean to decide if any of the
concentrations differ from the control.  This test has an overall error rate
of alpha, which accounts for the multiple comparisons with the control.  It is
based on the assumptions that the observations are independent and normally
distributed and that the variance of the observations is homogeneous across
all concentrations and control.  (See Appendix B for a discussion on
validating the assumptions).  Dunnett's Procedure uses a pooled estimate of
the variance, which is equal to the error value calculated in an analysis of
variance.  Dunnett's Procedure can only be used when the same number of
replicate test vessels have been used at each concentration and the control.
When this condition is not met, the t test with Bonferroni's adjustment is
used (see Appendix D).
                                                          I

1.2  The data used in this example are growth data from a Sheepshead Minnow
Larval  Survival and Growth Test, and are the same data used in Appendices B
and D.   These data are listed in Table C.I.
          TABLE C.I.  SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS,  LARVAL
                      GROWTH DATA (WEIGHT IN MG) USED FOR DUNNETT'S
                      PROCEDURE
Effluent
Cone (%)
Control
6.25
12.5
25.0
50.0
i

1
2
3
4
5


1
1
0
0
0
Replicate
1
.017
.157
.998
.873
.715
Test

0
0
0
0
0
2
.745
.914
.793
.935
.907
Vessel
3
0.
0.
1.
0.
1.
i
Total

862
992
021
839
044
(
2
3
2
2
2
i
.624
.063
.812
.647
,666
Mean
c
0
i
0
0
0
f,-)
.875
.021
.937
.882
.889
                                     419

-------
1.3  One way to obtain an estimate of the pooled variance is to construct an
ANOVA table including all sums of squares, using the following formulas:
     Where:  p  =  number of effluent concentrations including the control:
                   the total sample size;
            n- =  the number of replicates for concentration "i"
                                   Total Sum of Squares
            SSB=LTi2/n±-G2/N     Between Sum of Squares
                ^L Y±J2-G2/N
            SSW=SST-SSB
                                    Within Sum of Squares
              6   =    the  grand  total  of  all  sample observations;  G=2^Ti

             T-   =    the  total  of the replicate measurements for  concentration
                     i
              N
                    the total  sample  size;
            n-  =    the number of replicates  for  concentration  i
           Yr  =    the jth observation for concentration  i
1.4  For the data in this example:
            n,  -  n2 = n3 = n4 = n5 = 3
            N = 20
            Ti  -  Y-, + Y12 + Y13 = 2.624
            T2  -  Y21 + Y22 + Y23 - 3.063
                 YSI
                     + Y   + Y   = 2.812
                 Y  -f
                                  2.647
             TS -    +     + Y53 - 2.666
             G = T, + T2 +  T3 + T4 + T5 = 13.812
                                       420

-------
                 12.922  -  (13.812)715
                 0.204
               = 12.763  -  (13.812)715
               = 0.045
           SSW=SST-SSB

               = 0.204 - 0.045
               = 0.159
1.5  Summarize  these data  in the ANOVA table (Table C.2).  j

               TABLE C.2.  ANOVA TABLE FOR DUNNETT'S PROCEDURE
Source
Between
Within
Total
df Sum of Mean Square (MS)
Squares (SS) (SS/df)
P - 1 SSB SB = SSB/(p-l)
N - p SSW Sj = SSW/(N-p)
N - 1 SST

                                    421

-------
1.6  Summarize data for ANOVA (Table C.3).
       TABLE C.3.  COMPLETED ANOVA TABLE FOR DUNNETT'S PROCEDURE
  Source
  Total
df
                                              SS
14
0.204
               Mean Square
Between
Within
5-1=4
15 - 5 = 10
0.045
0.159
0.011
0.016
1.7  To perform the  individual comparisons, calculate the t statistic for each
concentration  and control combination,  as follows:
                                           I- (1/fll)
     Where:  Yf =  mean for each concentration i.
            Y, -  mean for the control
            SH =  square root of the within mean square
            n1 =  number of replicates  in the control.
            n- -  number of replicates  for  concentration  i
                                       422

-------

1.8  Table C.4  includes the calculated t values for each concentration and
control combination.

                        TABLE C.4.  CALCULATED T VALUES

            Effluent
            Concentration         i                   t.
6.25
12.5
25.0
50.0
2
3
4
5
- 1.414
- 0.600
- 0.068
- 0.136 !
1.9  Since the purpose of the test is only to detect a decrease in growth from
the control, a one-sided test is appropriate.  The critical value for the
one-sided comparison (2.47), with an overall alpha level of 0.05, 10 degrees
of freedom and four concentrations excluding the control is read from the
table of Dunnett's "T" values (Table C.5; this table assumes an equal number
of replicates in all treatment concentrations and the control).  Comparing
each of the calculated t values in Table C.4 with the critical  value, no
decreases in growth from the control  were detected.   Thus the NOEC is 50.0%.

1.10  To quantify the sensitivity of the test, the minimum significant
difference (MSD) may be calculated.   The formula is  as follows:
                          MSD =
   Where:  d  =  critical  value for the Dunnett's Procedure

          Sw  =  the square root of the within mean square

           n  =  the number of replicates at each concentration,  assuming  an
                 equal  number of replicates at all  treatment  concentrations

          n1  =  number of replicates in the control

   For example:

           AffiD=2. 47 (0.126) [V(1/3)+(1/3)] =2.47(0.126)

              = 2.47 (0.126)(0.816)

              = 0.254
                                     423

-------
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-------
1.11  For this set of data, the minimum difference between the control mean
and a concentration mean that can be detected as statistically significant is
0.254 mg.  This represents a decrease in growth of 29% from the control.

1.11.1  If the data have not been transformed, the MSD (and the percent
decrease from the control mean that it represents) can be reported as is.

1.11.2  In the case where the data have been transformed, the MSD would be in
transformed units.  In this case carry out the following conversion to
determine the MSD in untransformed units.

1.11.2.1  Subtract the MSD from the transformed control moan.  Call this
difference D.  Next, obtain untransformed values for the control mean and the
difference, D.                                           !

          MSDU =  controlu -  Du                           I
                                                         I
Where:    MSDU = the minimum significant difference for untransformed data

      Controlu = the untransformed control  mean

            Du = the untransformed difference            |
                      •
1.11.2.2  Calculate the percent reduction from the control  that MSDU
represents as:

                                 MSDU                    !
         Percent Reduction =	  X  100
                               Controlu                  i
                                                         i
1.11.3  An example of a conversion of the MSD to untransformed units, when the
arc sine square root transformation was used on the data, follows.

   Step 1.  Subtract the MSD from the transformed control mean.  As an
            example, assume the data in Table C.I were transformed by the arc
            sine square root transformation.  Thus:

                            0.875 - 0.254 = 0.621        i
                                                         i
   Step 2.  Obtain untransformed values for the control mean (0.875) and the
            difference (0.621) obtained in Step 1, above.

                   [ Sine  (0.875)]2  =  0.589

                   [ Sine  (0.621)]2  =  0.339            j
                                                         I
   Step 3.  The untransformed MSD (MSDU) is determined by subtracting the
            untransformed values obtained in Step 2.

                  MSDU  =  0.589 - 0.339  =  0.250
                                      425

-------
In this case, the MSD would represent a 42% decrease in survival from the
control [(0.250/0.589)(100)].

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 4526.8.  A compiled
version of the program can be obtained from EMSL-Cincinnati by  sending a
diskette with a written request.

2.6  DATA INPUT AND OUTPUT

2.6.1  Data on the number of surviving mysids, Mysidopsis bahia, from a
survival, growth and fecundity test (Table C.6) are used to illustrate the
data input and output for this program.

2.6.2  Data Input

2.6.2.1  When the program is entered, the user is asked to select the type of
data to be analyzed:

   1. Response proportions, like survival or fertilization proportions data.
   2. Counts and measurements, like offspring counts, cystocarp and algal cell
      counts, weights, chlorophyll measurements or turbidity measurements.
                                      426

-------
2.6.2.2  After the type of analysis for the data is chosen,  the user has the
following options:
                                                          i
   1. Create a data file
   2. Edit a data file
   3. Perform analysis on existing data set
   4. Stop                                                '     .     .
                                                          i
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.)                           ;
                                                          I
2.6.2.4  After the data have been entered, the user may save the file on a
disk, and the program returns to the 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),
                                                    ,
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.
                                      427

-------
TABLE C.6.  SAMPLE DATA FOR DUNNETT'S PROGRAM FOR SURVIVING MYSIDS,
            MYSIDOPSIS BAHIA
Treatment
1 Control







2 50 ppb







3 100 ppg







A 210 ppb







5 450 ppb







Replicate
Chamber
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
Total
Mysids
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
4
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
No.
Alive
4
4
5
5
5
5
5
4
4
5
4
4
5
5
4
5
3
5
5
5
5
3
4
4
5
4
1
4
3
4
4
4
0
1
0
1
0
0
0
2
                                  428

-------
                      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                                           j
                                                          j

Number of concentrations, including control  ? 5

Number of replicates for cone. 1 (the control) ? 8
                                                          i
replicate    number of organisms exposed    number of organisms responding
                                             (organisms surviving, eggs
                                                  fertilized, etc.)
  1
  2
  3
  4
  5
  6
  7
  8
5
5
5
5
5
5
5
5
Number of replicates for cone.  2 ? 8

Do you wish to save the data on disk ? y

Disk file for output   ?  mysidsur.dat
 4
'4
 Figure  C.I.  Sample  Data  Input  for Dunnett's  Program for  Survival  Data from
             Table C.6.
                                      429

-------
                      EMSL Cincinnati:  Dunnett Software
                                 Version 1.5
     1) Create a data file
     2) Edit a data file
     3) Analyze an existing data set
     4) Stop

Your choice ? 3

File name   ?  mysidsur.dat

Available Transformations

    1)  no transform
    2)  square root
    3)  loglO
    4)  arcsine square root
Your choice ? 4

Dunnett's test  as  implemented in this  program is a one-sided  test.  You must
specify the direction the test is to be run; that is, do you expect the means for
the test concentrations to  be less than or greater than the mean  for the control
concentration.

Direction for Dunnetts test : L=less than, 6=greater than ? 1
  Cone.
  Summary  Statistics  for  Raw Data

n           Mean         s.d.
cv%
1




» control
2
3
4
5
8
8
8
8
8
.9250
.9000
.8500
.7250
.1000
.1035
.1069
.1773
.2375
.1512
11.2
11.9
20.9
32.8
151.2
Hysid Survival Example with Data in Table C.6
Figure C.2. Example  of Choosing Option  3  from the  Main  Menu of  the Dunnett
            Program.
                                      430

-------
                Mysid Survival Example with Data in Table C.6

                         Summary  Statistics and ANOVA    |

                    Transformation = Arcsine Square Root

                                                         I
                               Mean	s.d.             cv%
1 = control
2
3
4*
5*
8
8
8
8
8
1.2560
1.2262
1.1709
1.0288
.3424
.1232 9.8
.1273 10.4
.2042
17.4
.2593 25.2
.1752 51.2
*) the mean for this cone, is significantly less than
    the control mean at alpha = 0.05 (1-sided) by Dunnett's test
                                                          i
                                                          i
Minimum detectable difference for Dunnett's test =        -.208074
This corresponds to a difference of        -.153507 in original units
This difference corresponds to   -16.98 percent of control
                                                          i
 *'                               •                         i
Between concentrations
sum of squares    =         4.632112 with  4 degrees of freedom.

Error mean square =          .034208 with 35 degrees of freedom.

Bartlett's test p-value for equality of variances =    .257


Do you wish to restart the program ?                      i
 Figure  C.3.  Example  of  Program Output  for the  Dunnett's  Program  Using  the
             Survival  Data in  Table  C.6.
                                      431

-------
                                   APPENDIX D

                      T TEST WITH BONFERRONI'S ADJUSTMENT
 1.   The t test with Bonferroni's adjustment is used as an alternative to
 Dunnett's Procedure when the number of replicates is not the same  for all
 concentrations.  This test sets an upper bound of alpha on the  overall  error
 rate,  in contrast to Dunnett's Procedure,  for which the overall  error rate  is
 fixed  at alpha.  Thus,  Dunnett's Procedure is a more powerful test.

 2.   The t test with Bonferroni's adjustment is based on the same assumptions
 of  normality of distribution and homogeneity of variance as Dunnett's
 Procedure (See Appendix B for testing  these assumptions),  and,  like  Dunnett's
 Procedure,  uses a pooled estimate of the variance,  which is equal  to the error
 value  calculated in an  analysis of variance.

 3.   An example of the use of the t test  with  Bonferroni's  adjustment is
 provided below.  The data used in the  example are the same as in Appendix C,
 except that the third replicate from the 50% effluent treatment  is presumed  to
 have been lost.  Thus,  Dunnett's Procedure cannot be used.   The  weight data
 are presented in Table  D.I.
  TABLE D.I.  SHEEPSHEAD MINNOW, CYPRINODON  VARIEGATUS, LARVAL GROWTH
              DATA  (WEIGHT  IN MG) USED  FOR THE T TEST WITH BONFERRONI'S
              ADJUSTMENT
Ef f 1 uent
Cone {%)
Control
6.25
12.5
25.0
50.0
i

1
2
3
4
5
Replicate Test Vessel
1
1.017
1.157
0.998
0.873
0.715
2
0.745
0.914
0.793
0.935
0.907
3
0.862
0.992
1.021
0.839
(Lost)
Total
(T,-)
2.624
3.063
2.812
2.647
1.622
Mean

-------
               n,- = the number of replicates for  concentration i
                                   Total Sum of Squares
                                   Between Sum  of Squares
               SSW=SST-SSB
                   Within Sum of Squares
  Where:
      The grand  total of all sample observations;
              T,-  =   The total  of the replicate measurements for
                      concentration i

             YfJ.  =   The jth  observation for concentration i

3.2  For the data  in this example:
        ni  =
= n3 = n4 = 3
        N   -  20
        ill
 + Y12 + Y13 = 2.624
 + Y22 + Y23 = 3.063
 + Y32 + Y33 = 2.812
 + Y,, + Y7, = 2.647

           = 1.622
        !  =  T1 + T2 + T3 + T4 + T5 = 12.768

         SSB=ETi2/ni-G2/N
             ±1
                11.709 - (12.768)714

                0.064

               Y±J2-G2/N
                 11.832 - (12.768)714

                 0.188



                                   433

-------
          SSW=SST-SSB


              =   0.188 - 0.064

              =   0.124

3.3  Summarize these data in the ANOVA table (Table  D.2):


               TABLE D.2.  ANOVA TABLE FOR BONFERRONI'S ADJUSTMENT
Source df
Between p - 1
Within N - p
Total N - 1
Sum of
Squares (SS)
SSB
SSW
SST
Mean Square (MS)
(SS/df)
SB = SSB/(p-l) .
S* = SSW/(N-p)

3.4  Summarize these calculations in the ANOVA table (Table  D.3):
       TABLE D.3.  COMPLETED ANOVA TABLE FOR THE T TEST WITH  BONFERRONI'S
                       ADJUSTMENT
Source
Between
Within
df
5-1=4
14 - 5 = 9
SS
0.064
0.124
Mean Square
0.016
0.014
  Total
13
0.188
                                     434

-------
3.5  To perform the individual comparisons, calculate the 't statistic for each
concentration and control combination, as follows:
   Where:  Y, =  mean for concentration i

           Y, =  mean for the control

           Sw =  square root of the within mean square

           n1 =  number of replicates in the control.

           n,- =  number of replicates for concentration i.

3.6  Table D.4   includes the calculated t values for each concentration and
control  combination.                                      !
                        TABLE D.4.   CALCULATED T VALUES
             Effluent
             Concentration
6.25
12.5
25.0
50.0
2
3
4
5
- 1.511
- 0.642
- 0.072
- 0.592
 3.7  Since the purpose of the test is only to detect a decrease in growth from
 the control,  a one-sided test is appropriate.  The critical  value for the
 one-sided comparison (2.686), with an overall alpha level  of 0.05, nine
 degrees of freedom and four concentrations excluding the control, was obtained
 from Table D.5.  Comparing each of the calculated t values in Table D.4 with
 the critical  value, no decreases in growth from the control  were detected.
 Thus the NOEC is 50.0%.
                                       435

-------
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                                                      437

-------
                                   APPENDIX  E

                           STEEL'S  MANY-ONE  RANK TEST

 1.   Steel's  Many-one  Rank  Test  is  a  nonparametric  test  for  comparing
 treatments with  a  control.   This test  is  an alternative to  Dunnett's
 Procedure, and may be applied to data  when  the  normality  assumption has not
 been met.  Steel's Test  requires equal  variances across the treatments and the
 control,  but it  is thought to be fairly insensitive to  deviations from this
 condition  (Steel,  1959).   The tables for  Steel's Test require an equal number
 of  replicates at each concentration.   If  this is not the  case, use Wilcoxon's
 Rank Sum  Test, with Bonferroni's adjustment (See Appendix F).

 2.   For an analysis using  Steel's  Test, for each control  and concentration
 combination,  combine  the data and  arrange the observations  in order of size
 from smallest to largest.   Assign  the  ranks to  the ordered  observations (1 to
 the smallest, 2  to the next smallest,  etc.).  If ties occur in the ranking,
 assign the average rank to  the  observation.  (Extensive ties would invalidate
 this procedure).   The sum  of the ranks  within each concentration and within
 the control  is then calculated.  To  determine if the response in a
 concentration is significantly  different  from the response  in the control, the
 minimum rank sum for  each  concentration and control combination is compared to
 the significant  values of  rank  sums  given later in the  section.  In this
 table, k equals  the number  of treatments  excluding the  control and n equals
 the number of replicates for each  concentration and the control.

 3.   An example of  the use  of this  test  is provided below.   The test employs
 survival data from a  mysid  7-day,  chronic test.  The data are listed in Table
 E.I.  Throughout the  test,  the  control  data are taken from  the site water
 control.  Since  there is 0% survival for  all eight replicates for the 50%
 concentration, it  is  not included  in this analysis and  is considered a
 qualitative  mortality effect.

 4.   For each control  and concentration  combination, combine the data and
 arrange the  observations in  order  of size from smallest to  largest.   Assign
 the  ranks (1, 2, 3, ..., 16) to the ordered observations  (1 to the smallest,  2
 to  the next  smallest,  etc.).  If ties occur in the ranking, assign the average
 rank to each tied  observation.

 5.   An example of  assigning  ranks  to the  combined data  for  the control  and
 3.12% effluent concentration is given in  Table E.2.  This ranking procedure is
 repeated for each  control and concentration combination.  The complete set of
 rankings is listed  in  Table  E.3.   The ranks are then summed for each effluent
 concentration, as  shown in Table E.4.

6.   For this set of data, determine if the  survival in  any  of the effluent
concentrations is  significantly lower than the survival  of the control
 organisms.   If this occurs, the rank sum  at that concentration would be
 significantly lower than the rank  sum of  the control.   Thus, compare the rank
 sums for the survival   at each of the various effluent concentrations with  some
 "minimum"  or critical  rank sum,  at or below which the survival  would be
considered to be significantly lower than  the  control.   At a probability level

                                     438

-------
TABLE E.I.  EXAMPLE OF STEEL'S MANY-ONE RANK TEST:
            MYSIDOPSIS BAHIA, 7-DAY CHRONIC TEST
DATA FOR MYSID,
Effluent
Concentration
Start of Test


Control

-------
TABLE E.2.  EXAMPLE OF STEEL'S MANY-ONE RANK TEST:  ASSIGNING
            RANKS TO THE CONTROL AND 3.12% EFFLUENT CONCENTRATIONS
Rank
1
6.5
6.5
6.5
6.5
6.5
6.5
6.5
6.5
6.5
6.5
14
14
14
14
14
Number of Live
My s ids, Mysidopsis bahia
3 .
4
4
4
4
4
4
4
4
• 4
4
5
5
5
5
5
Control or % Effluent
3.12
Control
Control
Control
Control
Control
3.12
3.12
3.12
3.12
3.12
Control
Control
Control
3.12
3.12
                     TABLE E.3.   TABLE OF RANKS
Repl
icate
Chamber Control
1
2
3
4
5
6
7
8
4 (6.5,6,6.5,5)
4 (6.5,6,6.5,5)
5 (14,13.5,13.5,12.5)
4 (6.5,6,6.5,5)
5 (14,13.5,13.5,12.5)
4 (6.5,6,6.5,5)
4 (6.5,6,6.5,5)
5 (14,13.5,13.5,12.5)
Effluent Concentration (%)
3
4
4
4
5
4
4
5
3
.12
(6.5)
(6.5)
(6.5)
(14)
(6.5)
(6.5)
(14)
(1)

3
4
5
4
4
4
5
5
6.25
(1)
(6)
(13.5)
(6)
(6)
(6)
(13.5)
(13.5)
12.5
5
4
5
3
5
4
4
3
(13
(6.
(13
(1.
(13
(6.
(6.
(1.
.5)
5)
.5)
5)
.5)
5)
5)
5)
5
5
5
5
3
5
4
4
25.0
(12
(12
(12
(12
(1)
12.
(5)
(5)
.b)
.5)
.5)
.5)

5)


 Control  ranks  are given in the order  of the concentration with which
 they were ranked.
                                 440

-------
                               TABLE E.4.  RANK SUMS
                      Effluent
                    Concentration
Rank Sum
                       3.12
                       6.25
                      12.50
                      25.00
  61.5
  65.5
  63.0
  73.5
of 0.05, the critical rank sum in a test with four concentrations and eight
replicates per concentration, is 47 (see Table F.4).
                                                          i
7.  Of the rank sums in Table E.4, none are less than 47.  Therefore, due to
the qualitative effect at the 50% effluent concentration, the NOEC is 25%
effluent and the LOEC is 50% effluent.
                                     441

-------
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

20


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
282
339
315
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
278
333
310
number of
4
10
17
--
25
21
35
30
47
41
61
54
76
68
93
84
112
102
133
121
155
142
180
165
206
190
234
217
264
245
296
275
330
307
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
273
327
305
6
10
16
--
24
--
34
29
46
40
59
52
74
66
91
82
110
99
130
119
153
140
177
162
203
187
231
213
260
241
292
272
325
303
(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
270
323
301
control )
8
--
16
--
24
--
33
29
45
39
58
51
73
65
90
81
108
98
129
117
151
138
175
160
200
185
228
211
257
239
288
268
322
300
9
--
15
--
23
--
33
29
44
39
58
51
72
65
89
80
108
98
128
117
150
137
174
160
199
184
227
210
256
238
287
267
320
299
                            442

-------
                                  APPENDIX F              i

                            WILCOXON RANK SUM TEST
                                                          i

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.
                                                          j
2.  The use of this test may be illustrated with fecundity data from the mysid
test in Table F.I.  The site water control and the 12.5% effluent •
concentration each have seven replicates for the proportion of females bearing
eggs, while there are eight replicates for each of the remaining three
concentrations.

3.  For each concentration and control combination, combine the data and
arrange the values in order of size, from smallest to largest.  Assign ranks
to the ordered observations (a rank of 1 to the smallest, 2 to the next
smallest, etc.).  If ties in rank occur, assign the average rank to each tied
observation.                                              ;
                                                          i
4.  An example of assigning ranks to the combined data for the control and
effluent concentration 3.12% is given in Table F.2.  This ranking procedure is
repeated for each of the three remaining control versus test concentration
combinations.  The complete set of ranks is listed in Tab'le F.3.  The ranks
are then summed for each effluent concentration, as shown in Table F.4.

5.  For this set of data, determine if the fecundity in any of the test
concentrations is significantly lower than the fecundity in the control.  If
this occurs, the rank sum at that concentration would be significantly lower
than the rank sum Thus, compare the rank sums for fecundity of each of the
various effluent concentrations with some "minimum" or critical rank sum, at
or below which the fecundity would be considered to be significantly lower
than the control.  At a probability level of 0.05, the critical rank in a test
with four concentrations and seven replicates in the control is 44 for those
concentrations with eight replicates, and 34 for those concentrations with
seven replicates  (see Table F.5, for K = 4).              i

6.  Comparing the rank sums in Table F.4 to the appropriate critical rank,
only the 25% effluent concentration does not exceed its critical value of 44.
Thus, the NOEC and LOEC for fecundity are 12.5% and 25%, respectively.
                                      443

-------
TABLE F.I. EXAMPLE OF WILCOXON'S RANK SUM TEST:  FECUNDITY DATA FOR
           MYSID, MYSIDOPSIS BAHIA, 7-DAY CHRONIC TEST
Effluent
Concentration
Control
(Site Water)






Control
(Brine &
Dilution Water)





3.12%







6.25%








2.5X






25. OX







50.0X







Replicate
Chamber
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
Number of
Mysids at
Start of Test
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
Number of
Live Mysids
at End of Test
4
4
5
4
5
4
4
5
3
5
3
3
4
4
3
3
4
4
4
5
4
4
5
3
3
4
5
4
4
4
5
5
5
4
5
3
5
4
4
3
5
5
5
5
3
5
4
4
0
0
0
0
0
0
0
0
Proportion
of Females
with Eggs
0.50

0.75
0.67
0.67
0.50
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.50
0.50
0.50
1.00
0.50
0.67
1.00
0.50
1.00
1.00
0.00
0.50
0.00
0.75
1.00
1.00
1.00
0.67
0.67
0.33
0.50
1.00

1.00
0.00
0.33
0.50
0.00
0.50
0.13
0.00
0.50
0.00
0.50
0.50

	
	
	
	
	
	
	
                                   444

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TABLE F.2.  EXAMPLE OF WILCOXON'S RANK SUM TEST:  ASSIGNING RANKS TO THE
            CONTROL AND 3.12% EFFLUENT CONCENTRATIONS
             Rank
Proportion of
Females W/Eggs
Site Water Control
   or Effluent
1
3.5
3.5
3.5
3.5
7
7
7
9
12.5
12.5
12.5
12.5
12.5
12.5
0.00
0.50
0.50
0.50
0.50
0.67
0.67
0.67
0.75
1.00
1.00
1.00
1.00
1.00
1.00
3.12
Control
Control
3.12
3.12
Control
Control
3.12
Control
Control
Control
3.12
3.12
3.12
3.12
                                   445

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                     TABLE F.3.  TABLE OF RANKS
                                                  1
Rep
1
2
3
4
5
6
7
8
Proper- Site Water
tlon Control Rank '
0.50

0.75
0.67
0.67
0.50
1.00
1.00
(3.5,3,5.5,7.5)
— —
(9,9.5,10,13)
(7,6.5,8.5,11.5)
(7,6.5,8.5,11.5)
(3.5,3,5.5,7.5)
(12.5,13,12.5,14.5)
(12.5,13,12.5,12.5)
Effluent Concentration (%V
3.12
1.00 (12.5)
0.50 (3.5)
0.67 (7)
1.00 (12.5)
0.50 (3.5)
1.00 (12.5)
1.00 (12.5)
0.00 (1)
6.25
0.50 (3)
0.00 (1)
0.75 (9.5)
1.00 (13)
1.00 (13)
1.00 (13)
0.67 (6.5)
0.67 (6.5)
12.5
0.33 (2.5)
0.50 (5.5)
1.00 (12.5)
'
1.00 (12.5)
0.00 (1)
0.33 (2.5)
0.50 (5.5)
25.0
0.00 (2)
0.50 (7.5)
0.33 (4)
0.00 (2)
0.50 (7.5)
0.00 (2)
0.50 (7.5)
0.50 (7.5)
'Control ranks are given in the order of the concentration with which they, were ranked.
                         TABLE F.4.   RANK  SUMS
    Effluent
  Concentration
Rank Sum
 No. of
Replicates
Critical
Rank  Sum
3.12
6.25
12.50
25.00
65
65.5
42
40
8
8
7
8
44
44
34
44
                                   446

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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 Reolicates Per Effluent
in Control

1 3
4
5
6
7
8
9
10
2 3
4
5
6
7
8
9
10
3 3
4
5
6
7
8
9
10
4 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
..
--
--
.6
6
7
7
7

4
10
11
12
13
14
15
16
17

10
11
12
13
14
14
15
„
10
11
11
12
13
13
14

--
10
11
12
12
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

15
16
17
18
19
20
21

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
21
22
23
24
26
27
28
30

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
28
30
31
33
34
36
38
40

8
39
41
44
46
49
51
54
56
38
40
42
44
46
49,
51
• 53
37
39
41
43
45
47
49
51
37
38
40
42
44
46
48
50
Concentration

9
49
51
54
57
60
63
66
69
47
49
52
55
57
60
62
65
46
48
51
53
56
58
61
63
46
48
50
52
55
57
60
62

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
56
59
•61
64
67
69
72
75
                                   447

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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 Reolicates Per Effluent
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
8 3
4
5
6
7
8
9
10

3

__
__
__
6
6
7
7

_.
__
__
6
6
6
7

__
--
-_
_-
6
6
7

__
--
--
__
6
6
6

4

-_
10
11
11
12
13
13

_-
10
11
11
12
12
13

--
--
10
11
11
12
13

_-
--
10
11
11
12
12

5

15
16
17
18
19
20
21

15
16
16
17
18
19
20

--
15
16
17
18
19
20

--
15
16
17
18
19
19

6

22
23
24
25
27
28
29

21
22
24
25
26
27
.29
_.
21
22
23
25
26
27
28

21
22
23
24
25
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
..
29
30
31
33
34
36
37

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
36
37
39
40
42
44
46
48
Concentration

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
45
47
49
51
53
55
57
59

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
55
57
59
62
64
67
69
72
                                    448

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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. Replicat
in Control

9 3
4
5
6
7
8
9
10
10 3
4
5
6
7
8
9
10
es No. of Reolicates Per Effluent

3 4
..
__
__
10
10
11
6 11
6 12
..
__
__
10
10
11
6 11
6 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

' IB
I
37
39
40
42
44
46
47
1_
37
38
40
42
43
45
47
.
Concentration

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
                                   449

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                                  APPENDIX G

          SINGLE CONCENTRATION TOXICITY TEST - COMPARISON OF CONTROL
                    WITH 100% EFFLUENT OR RECEIVING WATER
1.  To statistically compare a control  with one concentration,  such as  100%
effluent or the instream waste concentration,  a t test is the recommended
analysis.  The t test is based on the assumptions that the observations are
independent and normally distributed and that the variances of the
observations are equal between the two groups.

2.  Shapiro-Wilk's test may be used to test the normality assumption (See
Appendix B for details).  If the data do not meet the normality assumption,
the nonparametric test, Wilcoxon's Rank Sum Test, may be used to analyze the
data.  An example of this test is given in Appendix F.  Since a control and
one concentration are being compared, the K = 1 section of Table F.5 contains
the needed critical values.

3.  The F test for equality of variances is used to test the homogeneity of
variance assumption.  When conducting the F test, the alternative hypothesis
of interest is that the variances are not equal.

4.  To make the two-tailed F test at the 0.01 level of significance, put the
larger of the two variances in the numerator of F.
                           F =
                                si
where
 5.  Compare  F with the 0.005 level of a tabled F value with n., - 1 and n2  -  1
 degrees of freedom, where n, and n2 are  the number  of replicates for  each  of
 the two groups.

 6.  A  set of mysid growth data  from an effluent (single concentration) test
 will be used to  illustrate  the  F test.  The raw data, mean and  variance for
 the control  and  100%  effluent are  given in Table 6.1.

 7.  Since the variability of the 100% effluent is greater than  the variability
 of the control,  S  for the  100% effluent concentration is placed in the
 numerator of the F statistic and S for the control is placed in the
 denominator.

                            F=  °-00131  =1.52
                                 0.000861
 8.   There are 8 replicates  for  the  effluent  concentration and 8 replicates for
 the control.   Thus,  both  numerator  and  denominator degrees of freedom are
 equal  to 7.   For a two-tailed test  at the  0.01  level of  significance, the

                                      450

-------
  TABLE 6.1.  MYSID, MYSIDOPSIS BAHIA, GROWTH DATA  FROM AM  EFFLUENT  (SINGLE
              CONCENTRATION) TEST
                                    Replicate
               1
                                          8
  Control   0.183 0.148 0.216 0.199 0.176 0.243 0.213 0.180 0.195  0.000861
  100%            	  --..,.. ,..:-•.                 ;
   Effluent 0.153 0.117 0.085 0.153 0..086 0.193 0.137 0.129 0.132  0.00131
critical F value is obtained from a table of the F distribution  (Snedecor and
Cochran, 1980).  The criticalF value for this test is 8.89.  Since  1.52 is
not greater thani 8.89, the conclusion is that the variances of the control and
100% eff1uent are homogeneous.                          .              ;   ,

9.  Equal Variance T Test,                              ...  !         .;.'.-
               ,,.,..•                 .             i

9.1  To perform the t test, calculate the following test statistic:
Where:
           : t =
                         n2
=  mean for the control

=  mean for the effluent concentration
            SP =
                        ni+n2-2
           S^  =  estimate of the variance for the control
                                 -- '    '.               . ,     i
           S2  =  estimate of the variance for the effluent
                  concentration                           |

           n1  =  number of replicates  for the control

           n2  =  number of replicates  for the effluent
                  concentration
                                     451

-------
9 2  Since we are usually concerned with a decreased response from the
control, such as a decrease in survival or a decrease in reproduction, a.
one-tailed test is appropriate.  Thus, you would compare the calculated t with
a critical t, where the critical t is at the 5% level of significance with n,
+ n2 - 2 degrees of freedom.   If the calculated t exceeds  the critical  t,  the
mean responses are declared different.

9.3  Using the data from Table G.I to illustrate the t test, the calculation
of t is as follows:
                          t =  0.195-0.132  = 3>83
                               0.0329*  -=•+-=•
                   _ ,/(8-l)0.000861+(8-l) 0.00131  ^n
Where:
 9 4  For an  0.05  level  of significance test with  14 degrees of freedom, the
 critical  t is  1.762  (Note:   Table  D.5 for  K =  1 includes the critical t values
 for comparing  two groups).   Since  3.83 is  greater than  1.762, the cone usion
 is that the  growth for  the 100% effluent concentration  is  significantly lower
 than growth  for the  control.

 10.  UNEQUAL VARIANCE T TEST.

 10 1  If the F test  for equality of variance  fails, the t  test is still a
 valid test.   However, the denominator of the  t statistic  is adjusted  as
 follows:
      Where:
Y,  =
mean for the control

mean for the effluent concentration

estimate of the variance for the control

estimate of the variance for the effluent
concentration

number of replicates for the control

number of replicates for the effluent
concentration

               452

-------
                                                          I
10.2  Additionally, the degrees of freedom for the test are adjusted using the
following formula:                                        I

                         df'=-
                                 -l) C2+(i-c)
         Where:
                   C =
                        ni   n2                           ;
                                                          |

10.3  The modified degrees of freedom is usually not an integer.   Common
practice is to round down to the nearest integer.         i

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.
                                     453

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                                  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  Data input is illustrated by a set of mortality data (Figure H.I) from a
sheepshead minnow embryo-larval survival and teratogenicity test.  The program
begins with a request for the following information:

   1. Desired output of abbreviated (A) or full  (F) output?   (Note: only
      abbreviated output is shown below.)
   2. Output designation (P = printer, D = disk  file).
   3. Title for the output.
   4. The number of exposure concentrations.
   5. Toxicant concentration data.

2.2  The program output for the abbreviated output  includes  the  following:

   1. A table of the observed proportion responding and  the  proportion
      responding adjusted  for the  controls  (see  Figure H.2)
   2. The calculated chi-square statistic  for  heterogeneity  and  the
      tabular value. This  test  is  one indicator  of  how well  the  data  fit
      the model.   The  program will  issue  a warning  when  the  test
       indicates  that the data do  not  fit  the model.
   3. The estimated  LCI  and  LC50  values  and associated  95%  confidence
       intervals  (see Table H.2).
                                       454

-------
 Do you wish abbreviated (A) or full (F) input/output? A
 Output to  printer (P) or disk file (D)? P
 Title ? Example of Probit Analysis

 Number responding in the control group = ? 17
 Mumbep of  animals exposed in the concurrent control  group = ? 100
 Number of  exposure concentrations, exclusive of controls ? 5

 Input data starting with the lowest exposure concentration

 Concentration = ? 6.25
 Number responding = ? 14
 Number exposed = ? 100

 Concentration = ? 12.5
 Number responding =' ? 16
 Number exposed = ? 102

 Concentration = ? 25.0
 Number responding = ? 35
 Number exposed = ? 100

 Concentration = ? 50.0
 Number  responding = ? 72
 Number  exposed = ? 99

 Concentration = ? 100
 Number  responding = ? 99
 Number  exposed = ? 99
    Number
                 Cone.

                6.2500
               12.5000
               25.0000
               50.0000
              100.0000
Number
Resp.
  16
  35
  72
  99
 Number
Exposed

  100
  102
  100
   99
   99
Do you wish to modify your data ? N


The number of control animals which  responded
The number of control animals exposed  =  100
Do you wish to modify these values ? N
                   =  17
Figure H.I.   Sample  Data Input  for USEPA Probit Analysis  Program,
                 Version  1.5.
                                               455

-------
Example of Probit Analysis
    Cone.

   Control
    6.2500
   12.5000
   25.0000
   50.0000
  100.0000
 Number
Exposed

   100
   100
   102
   100
    99
    99
Number
Resp.

  17
  14
  16
  35
  72
  99
 Observed
Proportion
Responding

 0.1700
 0.1400
 0.1569
 0.3500
 0.7273
 1.0000
                                                            Proportion
                                                            Responding
                                                            Adjusted for
                                                              Controls
0.0000
0.0201
0.0001
0.2290
0.6765
1.0000
Chi  - Square  for  Heterogeneity  (calculated)
Chi  - Square  for  Heterogeneity
         (tabular  value  at  0.05  level)
                                   3.472

                                   7.815
 Example of Probit Analysis

       Estimated LC/EC Values and Confidence Limits
 Point

 LC/EC  1.00
 LC/EC 50.00
     Exposure
       Cone.

       12.917
       37.667
                                              Lower          Upper
                                             95% Confidence Limits
              8.388
             32.898
                16.888
                42.081
 Figure H.2.  USEPA Probit Analysis Program used for Calculating LC/EC
              Values, Version 1.5.
                                     456

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                                   APPENDIX I

                             SPEARMAN-KARBER METHOD

                                                          i
 1.   The  Spearman-Karber  Method  is  a nonparametric statistical  procedure for
 estimating  the  LC50  and  the  associated 95% confidence interval  (Finney,  1978).
 The  Spearman -Karber  Method estimates the  mean  of the  distribution  of the 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 Rrobit  model.

 4.   To calculate the LC50 using the Spearman-Karber Method, the  following must
 be true:  1) the smoothed adjusted proportion  mortality  for the  lowest
 effluent concentration (not  including the control) must  be zero, and 2)  the
 smoothed adjusted  proportion mortality for the highest effluent  concentration
 must be  one.

 5.   To calculate the 95% confidence interval for the  LC50 estimate,  one  or
 more of  the smoothed adjusted proportion  mortalities  must be between zero  and
 one.

 6.  The  Spearman-Karber  Method is  illustrated  below using a set  of mortality
 data from a Sheepshead Minnow Larval  Survival   and Growth  test.   These data are
 listed in Table  I.I.
                                                          •I

 7.  Let  p0,  p1}  ...,  pk denote the  observed response proportion mortalities
 for the  control   and  k  effluent concentrations.   The first step is to  smooth
 the p.  if they do not satisfy p0  <  p1  < ...  < pk.  The  smoothing process
 replaces any adjacent  pt's  that  do  not conform to p0 < p,  <  ... < p. with their
 average.   For example, if p.  is  less than  p,.,  then:
                           P/-I = Pi =
Where:   p?    =  the smoothed observed proportion mortality for effluent
                  concentration i.

7.1  For the data in this example, because the observed mortality proportions
for the control and the 6.25% effluent concentration are greater than the
observed response proportions for the 12.5% and 25.0% effluent concentrations,
the responses for these four groups must be averaged:

                                      457

-------
             s    s    s
            Po  =Pi =Pa =
                         0.05 + 0.05+0.00+0.00  _  0.10 _
                                                  -- --
       TABLE  I.I.    EXAMPLE  OF  SPEARMAN-KARBER METHOD:  MORTALITY DATA FROM
                    A SHEEPSHEAD  MINNOW  LARVAL SURVIVAL AND GROWTH TEST (40
                    ORGANISMS PER CONCENTRATION)
Ef f 1 uent
Concentration
Control
6.25
12.5
25.0
50.0
100.0
Number of
Mortalities
2
2
0
0
26
40
Mortality
Proportion
0.05
0.05
0.00
0.00
0.65
1.00
7.2  Since p.  = 0.65 is  larger than  pj, set p^ = 0.65.   Similarly,  p5 =  1.00  is
larger than p£, so set p| = 1.00.  Additional smoothing is not necessary.  The
smoothed observed proportion mortalities  are shown in  Table  1.2.

  TABLE 1.2.  EXAMPLE OF SPEARMAN-KARBER  METHOD:   SMOOTHED,  ADJUSTED
              MORTALITY  DATA FROM A SHEEPSHEAD MINNOW  LARVAL SURVIVAL AND
              GROWTH TEST

Effluent
Concentration
01
/o
Control
6.25
12.5
25.0
50.0
100.0


Mortality
Proportion
0.05
0.05
0.00
0.00
0.65
1.00

Smoothed
Mortality
Proportion
0.025
0.025
0.025
0.025
0.650
1.000
Smoothed,
Adjusted
Mortality
Proportion
0.000
0.000
0.000
0.000
0.641
1.000
8.  Adjust the smoothed observed proportion mortality in each effluent
concentration for mortality in the control group using Abbott's formula
(Finney,  1971).  The adjustment takes the form:

Where:    pj - (p' -  p*) /  (1 - p?)
                                      458

-------
          PO = the smoothed observed proportion mortality for the control
                                                         i
          pf = the smoothed observed proportion mortality for effluent
              concentration i.
                                                         i

 8.1   For  the data in this example, the data for each effluent concentration
 must  be adjusted for control mortality using Abbott's formula,  as follows:
                            =  0.650-0.025 =  0.0625
                                 1-0.025
0.975

                 a _ P5S~PoS  _ 1.000-0.025    0.975
                   -- —
1-P0S
                                  1-0.025
0.975
                                                            nnn
                                                       = 1.000
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 =
Where:  p° =  the  smoothed  adjusted proportion mortality at concentration i

        Xf =  the  Iog10 of concentration i

        k  =  the  number of effluent  concentrations tested, not including the
             control.                                     i

9.1  For this example,  the Iog10 of the estimated LC50,  m,  is  calculated  as
follows:

      m = [(0.000 - 0.000)  (0.7959 + 1.0969)]/2 +         '
          [(0.000 - 0.000)  (1.0969 + 1.3979)]/2 +
          [(0.641 - 0.000)  (1.3979 + 1.6990)]/2 +
          [(1.000 - 0.641)  (1.6990 + 2.0000)]/2

        = 1.656527
                                     459

-------
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460

-------
 10.  Calculate the estimated variance of m as follows:


                  '     v(a)  -
                               1-2
Where:  X,- = the Iog10 of concentration i

        n,- = 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
f ol 1 ows :
      V(m) = (0.000)(1.000)(1.3979 - 0.7959)?/4(39)
             (0.000)(1.000)(1.6990 - 1.0969)74(39)
             (0.641)(0.359)(2.0000 - 1.3979)74(39)
rf^jy;
      +
           = 0.00053477

                                                         I

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:
          •   1.656527 ±2^0.00053477 =  (1.610277, 1.702777)



12.  The estimated LC50 and a 95% confidence interval  for  the estimated  LC50
can be found by taking base10 antilogs of the above values.

12.1  For this example, the estimated LC50 is calculated as  follows:

             LC50 = antilog(m)  = antilog(l.656527)  =  45.3%.

12.2  The limits of the 95% confidence interval  for the estimated LC50 are
calculated by taking the antilogs of the  upper and  lower limits of the 95%
confidence interval for m as follows:                     I
                                                         j

            lower limit:   antilog(l.610277)  = 40.8%

            upper limit:   antilog(l.702777)  = 50.4%
                                     461

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                                  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 log1Q 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.

E.  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 PO, p«, ..., pk denote  the observed proportion mortalities  for  the
control and the k effluent concentrations.  The  first step  is  to  smooth the  p5
if they do  not satisfy p0  < p, <  ... < pk.  The  smoothing process  replaces any
adjacent  p^s that  do  not  conform to  p0 < p, < ...  < pk,  with their average.
For example,  if p,-  is  less than pM then:
 Where:
Pi-1

Pi
-  Pi = (Pi + Pi-i)/2

=  the smoothed observed proportion mortality for effluent
   concentration i.
 7.   Adjust the smoothed observed proportion mortality in each effluent
 concentration for mortality in the control group using Abbott's formula
 (Finney, 1971).  The adjustment takes the form:
 Where:
          Po
      -  (Pi - Po)  /  (1  -  Po)

      -  the smoothed observed proportion mortality for the control

      =" the smoothed observed proportion mortality for effluent
         concentration  i.
                                       462

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                                                                                          1
8.  Calculate the amount of trim to use in the estimation of the LC50 as
follows:

Where:   Trim  =  max(p^,  1-pj!)
                                                          I
         p^    =  the smoothed, adjusted proportion mortality for the lowest
                  effluent concentration, exclusive of the control

         pj^    =  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 calculated for each data set rather
than using a fixed amount of trim for each data set.
                                    1
9.  Due to the intensive nature of the calculation for the estimated LC50 and
the calculation of the associated 95% confidence interval using the Trimmed
Spearman-Karber Method, it is recommended that the data be analyzed by
computer.

10.  A computer program which estimates the LC50 and associated 95% confidence
interval using the Trimmed Spearman-Karber Method, can be obtained through the
EMSL, 3411 Church Street,  Cincinnati, OH 45244.  The program can be obtained
from EMSL-Cincinnati by sending a written request to the above address.

11.  The Trimmed Spearman-Karber program automatically performs the following
functions:                                                i
                    ,
     a.  Smoothing.
     b.  Adjustment for mortality in the control.
     c.  Calculation of the necessary trim.               i
     d.  Calculation of the LC50.
     e.  Calculation of the associated 95% confidence interval.

12.  To illustrate the Trimmed Spearman-Karber method using the Trimmed
Spearman-Karber computer program, a set of data from a Sheepshead Minnow
Larval Survival and Growth test will be used.  The data are listed in
Table J.I.                                                I

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.     i
      c.  The estimated LC50 and the associated 95% confidence interval.


                                      463

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TABLE J.I.  EXAMPLE OF TRIMMED SPEARMAN-KARBER METHOD:  MORTALITY
            DATA FROM A SHEEPSHEAD MINNOW LARVAL SURVIVAL AND
            GROWTH TEST (40 ORGANISMS PER CONCENTRATION)
      Eff1uent
    Concentration
 Number of
Mortalities
         "I
         fa
Mortality
Proportion
      Control
        6.25
       12.5
       25.0
       50.0
      100.0
     2
     0
     2
     0
     0
    32
   0.05
   0.00
   0.05
   0.00
   0.00
   0.80
                             464

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A:>TSK

TRIMMED SPEARMAN-KARBER METHOD.  VERSION 1.5

ENTER DATE OF TEST:
1
ENTER TEST NUMBER:
2
WHAT IS TO BE ESTIMATED?.
(ENTER "L" FOR LC50 AND "E" FOR EC50)
L
ENTER TEST SPECIES NAME:
Sheepshead minnow
ENTER TOXICANT  NAME:
eff1uent
ENTER UNITS FOR EXPOSURE CONCENTRATION OF TOXICANT :
y
E°NTER THE NUMBER OF INDIVIDUALS IN THE CONTROL:
40
ENTER THE NUMBER OF MORTALITIES IN THE CONTROL:
2
ENTER THE NUMBER OF CONCENTRATIONS
(NOT INCLUDING THE CONTROL;  MAX = 10):
5
ENTER THE  5 EXPOSURE CONCENTRATIONS (IN INCREASING ORDER):
6.25  12.5  25  50  100
ARE THE NUMBER OF INDIVIDUALS AT EACH EXPOSURE CONCENTRATION EQUAL(Y/N)?
y
ENTER THE NUMBER OF INDIVIDUALS AT EACH EXPOSURE CONCENTRATION:
40
ENTER UNITS FOR DURATION OF EXPERIMENT
(ENTER "H" FOR HOURS, "D" FOR DAYS, ETC.):
Days
ENTER DURATION OF TEST:
7
ENTER THE NUMBER OF MORTALITIES AT EACH EXPOSURE CONCENTRATION:
0 2 0 0 32
WOULD YOU LIKE THE AUTOMATIC TRIM CALCULATION(Y/N)?
y
        Figure J.I.  Example input for Trimmed Spearman-Karber Method.


                                      465                I

-------
TRIMMED SPEARMAN-KARBER METHOD.  VERSION 1.5
DATE: 1
TOXICANT:
SPECIES:
RAW DATA:








TEST
eff 1 uent
sheepshead minnow
Concentration
[0/\
(/o)
.00
6.25
12.50
25.00
50.00
100.00
NUMBER: 2


Number
Exposed
40
40
40
40
40
40
D


Mortalities

2
0
2
0
0
32
                                                      DURATION:
                                  7 Days
  SPEARMAN-KARBER TRIM:

  SPEARMAN-KARBER ESTIMATES:
   20.41%

LC50:          77.28
        95% CONFIDENCE LIMITS
        ARE NOT RELIABLE.
 NOTE:   MORTALITY PROPORTIONS WERE NOT MONOTONICALLY INCREASING.
        ADJUSTMENTS WERE MADE PRIOR TO SPEARMAN-KARBER ESTIMATION.
       Figure 0.2.  Example output for Trimmed Spearman-Karber Method.
                                     466

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                                  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 its use is only recommended when there are
no partial mortalities.  The only requirement for the Graphical Method is that
the observed percent mortalities bracket 50%.

2.  For an analysis using the Graphical Method the data must first be smoothed
and adjusted for mortality in the control replicates.  The procedure for
smoothing and adjusting the data is detailed in the following steps.
                                                         i
3.  The Graphical Method is illustrated below using a set of mortality data
from an Inland Silverside Larval Survival and Growth test.  These data are
listed in Table K.I.                                     !
         TABLE K.I.    EXAMPLE OF GRAPHICAL METHOD:  MORTALITY DATA FROM AN
                       INLAND SILVERSIDE LARVAL SURVIVAL AND GROWTH TEST (40
                       ORGANISMS PER CONCENTRATION)
Ef f 1 uent
Concentration
Control
6.25
12.5
25.0
50.0
100.0
Number of Mortality
Mortalities
Proportion
2 0.05
0 0.00
0
0
0.00
0.00
40 1.00
40
1.00
4.  Let p0, p.,  ...,  p^ denote the observed proportion mortalities  for  the
control and tne k effluent concentrations.  The first step is to smooth the p,-
if they do not satisfy p0 < p. < ...  < pk.  The smoothing  process replaces  any
adjacent p/s that do not conform to p0 < p1 <  ... < pk with their  average.
For example, if p? is less than p,-^ then:
Where:
P/-I = Pi =
                                              -i) /2
                  the smoothed observed proportion mortality for effluent
                  concentration  i.
                                      467

-------
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:
       PcT
^s   ^s     s    0.05+0.00+0.00+0.00
Pi  - P2  - Ps = 	-.	:	
                                                        0.05
  = 0.0125
4.2  Since p4 - p5 = 1.00 are larger then 0.0125,  set p^ = p!j = 1.00.
Additional smoothing is not necessary.   The  smoothed observed proportion
mortalities are shown  in Table  K.2.

5.  Adjust the smoothed observed proportion  mortality in each effluent
concentration for mortality in  the control group using Abbott's formula
(Finney, 1971).  The adjustment takes  the  form:
Where:
                           Pi =  (P/-Pos)  /d-Pos)
          PQ = the smoothed  observed proportion mortality for the control

          p? = the smoothed  observed proportion mortality for effluent
               concentration i.

5.1  Because the smoothed observed proportion mortality for the control group
is greater than zero, the responses must  be  adjusted using Abbott's formula,
as follows:
                              a _  s
      ^a_^a_^a   „ a  _  Pi  Po
      Po - Pi  ~ Pz = P3	—
                             1-PO
                          0.0125-0.0125 =   0.0
                            1  -  0.0125       0.9875
       = 0.0
                        1-PO
                                 1.00 -0.0125
                                   1 -0.0125
                                      0.9875
                                      0.9875
= 1.00
A table of the smoothed,  adjusted response  proportions for the effluent
concentrations are shown  in Table K.2.

5.2  Plot the smoothed,  adjusted data on  2-cycle  semi-log graph paper with the
logarithmic axis (the y axis)  used for percent  effluent concentration and the
linear axis (the x axis)  used  for observed  percent mortality.  A plot of the
smoothed, adjusted data is shown in Figure  K.I.

                                     468

-------
         TABLE K.2.  EXAMPLE OF GRAPHICAL METHOD:  SMOOTHED, ADJUSTED
                     MORTALITY DATA FROM AN INLAND SILVERSIDE LARVAL
                     SURVIVAL AND GROWTH TEST
Ef f 1 uent
Concentration
%
Control
6.25
12.5
25.0
50.0
100.0
Mortality
Proportion
0.05
0.00
0.00
0.00
1.00
1.00
Smoothed
Mortality
Proportion
0.0125
0.0125
0.0125
0.0125
1.0000
1.0000
Smoothed,
Adjusted
Mortality
Proportion
0.00
0.00
0.00
0.00
1.00
1.00
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.
                                     469

-------
   100
    50
LLJ
S
Z>

£
LLI


LL.
LL
LL1

z:
LLJ
O
DC
111
Q_
10
      1
        0  10  20  30  40  50 60 70 80 90 100

                PERCENT MORTALITY
 Figure K.I.  Plot of the smoothed adjusted response proportions for
           inland silverside, Menidia beryllina,  survival data.
                         470

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                                 APPENDIX  I-

                          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 v/as designed for
general applicability in the analysis of data from short-term chronic toxicity
tests, and the generation of an endpoint from a continuous; model that allows a
traditional quantitative assessment of the precision of the endpoint, such as
confidence limits for the endpoint of a single test, and a mean and
coefficient of variation for the endpoints of multiple tests-
1.2  The Linear Interpolation Method assumes that the responses (1)
monotonically non-increasing, where the mean response for each higher
concentration is less than or equal to the mean response for the previous
concentration, (2) follow a piecewise linear response function, and (3) are
from a random, independent, and representative sample of test data.  If the
data are not monotonically 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.    i

2.  DATA SUMMARY AND PLOTS
                                                             •
2.1  Calculate the mean responses for the control and each toxicant
concentration, construct a summary table, and plot the data.

3.  MONOTONICITY

3.1  If the assumption of monotonicity of test results is met, the observed
response means (Yj) should stay the same or decrease as the toxicant
concentration increases.   If the means do not decrease monotonically, the
responses are "smoothed" by averaging (pooling) adjacent means.

3.2  Observed means at each concentration are considered in order  of
increasing concentration,  starting with the control mean  (Y.,).  If the mean
observed response  at the lowest toxicant concentration (Y;>) is equal to or
smaller than the control mean  (Y,,), it is used as the response.  If it is
larger than the control mean,  it is averaged with the control, and this
average is used for both the control  response (M,,) and the lowest  toxicant
concentration response  (M?) .  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


                                      471                 I

-------
 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
                                                             Where Y,  decrease
monotonicity may require an additional step of smoothing).
monotonically, the Y,- become Mf without smoothing.

4.  LINEAR INTERPOLATION METHOD

4.1  The method assumes a linear response from one concentration to the next.
Thus, the ICp is estimated by  linear interpolation between two concentrations
whose responses bracket the response of interest, the  (p) percent reduction
from the control.

4.2  To obtain the estimate, determine the concentrations Cj and CJ+1 which
bracket the response M, (1  - p/100), where M,  is  the  smoothed control  mean
response and p is the percent  reduction in response relative to the control
response.  These calculations  can easily be done by hand or with a computer
program as described below.    The linear interpolation estimate is calculated
as follows:
              ICp = Cj. + [ Mx (l - p/ioo) -
                                                       "J +• 1
                                                            - CT)
                                                     (M.7
                                                         + i
Where:    Ca            -      tested  concentration  whose  observed  mean  response
                              is  greater  than  M.,(l  - p/100).

          Cj+1         =      tested  concentration  whose  observed  mean  response
                              is  less than M,,(l - p/100).

          M.,            =      smoothed mean  response for  the  control.

          Mj            =      smoothed mean  response for  concentration  J.

          Mj + 1         =      smoothed mean  response for  concentration  J +  1.

          p             =      percent reduction in  response relative to the
                              control  response.

          ICp           =      estimated concentration at  which there is a
                              percent reduction from the  smoothed  mean  control
                              response.  The ICp is reported  for the test,
                              together with the 95% confidence interval
                              calculated by the ICPIN.EXE program  described
                              below.

4.3  If the Cj is the highest concentration tested, the ICp would be specified
as greater than C,.   If the response at the lowest concentration tested is
used to extrapolate the ICp value, the ICp should be expressed as a 7ess than
the lowest test concentration.
                                      472

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 5.  CONFIDENCE  INTERVALS

 5.1  Due to the use of  a  linear  interpolation technique to calculate an
 estimate of the ICp,  standard statistical methods for calculating confidence
 intervals are not applicable for the  ICp.  This limitation is avoided by use a
 technique known as the  bootstrap method as proposed by Efron (1982) for
 deriving point estimates  and confidence intervals.

 5.2  In the Linear Interpolation Method, the smoothed response means are used
 to obtain the ICp estimate reported for the test.  The bootstrap method is
 used to obtain the 95%  confidence interval for the true mean.  In the
 bootstrap method, the test data YM  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 of the data.  When confidence intervals are
wide, the reliability of the 1C estimate is in question.   However, narrow
 intervals do not necessarily indicate that the estimate is highly reliable,
 because of undetected violations of assumptions and the fact that the
 confidence limits based on the empirical quantiles of a bootstrap distribution
 of 80 samples may be unstable.
                                                          i
 5.4  The bootstrapping method of calculating confidence intervals is
computationally intensive.  For this reason,  all of the calculations
associated with determining the confidence intervals for the ICp estimate have
been incorporated into a computer program.  Computations are most easily done
with a computer program such as the revision of the BOOTSTRP program (USEPA,
 1988; USEPA,  1989) which is now called "ICPIN" which is described below in
subsection 7.

6.  MANUAL CALCULATIONS

6.1  DATA SUMMARY AND PLOTS

6.1.1  The data used in this example are the mysid growth  data  used  in  the
example in Section 14.  The data is presented as the mean  weight  per original
number of organisms.   Table L.I  includes the raw data and  the mean growth for
each concentration.   A plot of the data is provided in Figure L.I.
                                     473

-------
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                                                          474

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           TABLE L.I.  MYSID, MYSIDOPSIS BAHIA, GROWTH DATA
  Replicate  Control
                                     Toxicant Concentration  (ppb)
50
100
210
450
1
2
3
4
5
6
7
8
0.146
0.118
0.216
0.199
0.176
0.243
0.213
0.144
0.154
0.193-
0.190
0.190
0.256
0.191
0.122
0.177
0.114
0.172
0.160
0.199
0.165
0.145
0.207
0.186
0.153 0
0.094 0.012
0.017 0
0.122 0.002
0.052
0.154
0
0
0.110 0
0.103 0.081
Mean (Y;) 0.182 0.184 0.168 0.101
i 1 2 3 4

0.012
5

6.2  MONOTONICITY
                                                          I
6.2.1  As can be seen from the plot in Figure L.I, the observed means are not
monotonically non-increasing with respect to concentration.  Therefore, the
means must be smoothed prior to calculating the 1C.       i

6.2.2_ Starting with the control mean Y., = = 0.186 and Y~2 = 0.184,  we see  that
Y-, < Y2  .  Calculate  the  smoothed means:

                        ML = M2 = (Ti + T2) /2 = 0.193    •
6.2.3  Since Y5 = 0.025 < Y,  =  0.101  <  Y3 = 0.168 < M2, set, M3 = 0.168 and M,
0.101, and M5 = 0.025.   Table L.2 contains the smoothed means and Figure L.l
gives a plot of the smoothed response curve.

6.3  LINEAR INTERPOLATION                                 \
                                                          i
6.3.1  Estimates of the IC25 and IC50 can be calculated using the Linear
Interpolation Method.  A 25% reduction in mean weight, compared to the
controls, would result in a mean weight of 0.139, where M^l-p/100)  =
0.185(1-25/100).  A 50% reduction in mean weight, compared to the controls,
would result in a mean weight of 0.093 mg.  Examining the smoothed means and
their associated concentrations (Table L.2), the two effluent concentrations.
bracketing the mean weight per original of 0.139 mg are C3 = 100 ppb and C4 =


                                     475

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  TABLE L.2.   MYSID,  MYSIDOPSIS BAHIA, MEAN GROWTH RESPONSE AFTER SMOOTHING
Toxicant
Cone.
(ppb)
Control
50
100
210
450


i
1
2
3
4
5
Smoothed
Mean
Mf (mg)
0.183
0.183
0.168
0.101
0.025
210 ppb.   The two effluent concentrations bracketing a response of 0.093 mg
per total  original  number of organisms  are C4 = 210 ppb and C5  = 450 ppb.

6.3.2  Using the equation from section  4.2, the estimate of the IC25 is
calculated as follows:

  ICp = Cj. + [ Mx (i - p/100) - Mj ;
                                         (Mj
                                               - AfT)
 IC25 - 100  +  [0.193  (1 - 25/100)  - 0.164]

       = 151 ppb
                                                /ft        »\
                                                (0 . 101  -  0 . 164)
6.3.3  Using Equation 1 from 4.2,  the  estimate of the IC50 is calculated as
fol1ows:
                                         /1-<      /-< \
   ICp = Cj + [ ML (1 - p/100)  - Mj ]
                                         (MJ + 1 -
                                                  (450  - 210)
                                                (0.028  - 0.101)
 JC50 = 210  + [0.193  (1 - 50/100)  - 0.101]

       - 239 ppb

6.4  CONFIDENCE INTERVALS

6.4.1  Confidence intervals for the  ICp  are derived using the bootstrap
method.  As described above,  this  method involves randomly resampling the
individual  observations  and recalculating the  ICp at least 80 times, and
determining the mean ICp, standard deviation,  and empirical 95% confidence
intervals.   For this reason,  the confidence intervals are calculated using a
computer program called  ICPIN.   This program is described below and is
available to carry out all  the  calculations of both the interpolation estimate
(ICp) and the confidence intervals.
                                     476

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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 (Y-)  (response means);   2)  it
calculates the standard deviations;  3) checks the responses for monotonicity;
4) calculates smoothed means (M,-)  (pooled  response means)  if necessary;  5)
uses the means, Mp  to calculate the initial  ICp of choice by linear
interpolation; 6) performs a user-specified number of bootstrap resamples
between 80 and 1000 (as multiples of 40);  7) calculates the mean and standard
deviation of the bootstrapped ICp estimates; and 8) provides an original 95%
confidence intervals to be used with the initial ICp when the number of
replicates per concentration is over six and provides both original and
expanded confidence intervals when the number of replicates per concentration
are less than seven (Norberg-King, 1993).

7.3  For the ICp calculation, up to twelve treatments can be input (which
includes the control).  There can be up to 40 replicates per concentration,
and the program does not require an equal  number of replicates per
concentration.  The value of p can range from 1% to 99%.

7.4  DATA INPUT

7.4.1  Data is entered directly into the program onscreen. ' A sample data
entry screen in shown in Figure L.2.  The  program documentation provides
guidance on the entering and analysis of data for the Linear Interpolation
Method.                                                    i

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).;
                                      477

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                                                 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











































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.
                                    478

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7.5  DATA OUTPUT

7.5.1  The program output includes the following (Figures L.3 and L.4)

   1. A table of the concentration identification, the concentration tested
      and raw data response for each replicate and concentration.
   2. A table of test concentrations, number of replicates, concentration
      (units), response means (Y,.),  standard deviations for each response
      mean, and the pooled response means (smoothed means; M,-).
   3. The linear interpolation estimate of the ICp using the means (M,-).
      Use this value for the ICp estimate.
   4. The mean ICp and standard deviation from the bootstrap resampling.
   5. The confidence intervals calculated by the bootstrap method for the ICp.
      Provides an original 95% confidence intervals to be used with the
      initial ICp when the number of replicates per concentration is over six
      and provides both original and expanded confidence intervals when the
      number of replicates per concentration are less than seven.

7.6  ICPIN program output for the analysis of the mysid growth data in Table
L.I is provided in Figures L.3 and L.4.

7.6.1  When the ICPIN program was used to analyze this set of data, requesting
80 resamples, the estimate of the IC25 was 147.1702 (ppb).  The empirical 95%
confidence intervals for the true mean was 97.0905 to 186.6383 (ppb).

7.6.2  When the ICPIN program was used to analyze this set of data, requesting
80 resamples, the estimate of the IC50 was 233.3311 (ppb).  The empirical 95%
confidence intervals for the true mean were 184.8692 to 283.3965 (ppb).
                                     479

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Cone. ID

Cone. Tested
Response
Response
Response
Response
Response
Response
Response
Response
1
2
3
4
5
6
7
8
, 1
0
.146
.118
.216
.199
.176
.243
.213
.144
2
50
.154
.193
.190
.190
.256
.191
.122
.177
3
100
.114
.172
.160
.199
.165
.145
.207
.186
4
210
.153
.094
.017
.122
.052
.154
.110
.103
5
450
0
.012
0
.002
0
0
0
.081
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent:
Test Start Date:    Test Ending Date:
Test Species: MYSID SHRIMP, Mysidopsis bahia
Test Duration:     growth test
DATA FILE: mysidwt.icp
OUTPUT FILE: mysidwt.i25
Cone.
ID

1
2
3
4
5
Number
Replicates

8
8
8
8
8
Concentration
ug/1

0.000
50.000
100.000
210.000
450.000
Response
Means

0.182
0.184
0.168
0.101
0.012
Std.
Dev.
Means
0.043
0.038
0.030
0.047
0.028
Pooled
Response

0.183
0.183
0.168
0.101
0.012
The Linear Interpolation Estimate:   133.5054   Entered P Value: 25

Number of Resamplings:   80
The Bootstrap Estimates Mean: 147.1702 Standard Deviation:    23.7984
Original Confidence Limits:   Lower:    96.8623 Upper:   186.6383
Resampling time in Seconds:     0.16  Random Seed: -1623038650
          Figure L.3.  Example of ICPIN program output for the IC25.
                                      480

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Cone.  ID

Cone.  Tested
            50
           100
           210
             5

           450
Response   1
Response   2
Response   3
Response   4
Response   5
Response   6
Response   7
Response   8
.146
.118
.216
.199
.176
.243
.213
.144
.154
.193
.190
.190
.256
.191
.122
,177
.114
.172
.160
.199
.165
.145
.207
.186
.153
.094
.017
.122
.052
.154
.110
.103
   0
.012
   0
.002
   0
   0
   0
.081
*** Inhibition Concentration Percentage  Estimate ***
Toxi cant/Eff1uent:
Test Start Date:    Test Ending Date:
Test Species: MYSID SHRIMP, Mysidopsis bahia
Test Duration:     growth test
DATA FILE: mysidwt.icp
OUTPUT FILE: mysidwt.iSO
Cone.
ID
1
2
3
4
5
Number
Replicates
8
8
8
8
8
Concentration Response Std. Pooled
ug/1 Means Dev. Response Means
0.000
50.000
100.000
210.000
450.000
0.182
0.184
0.168
0.101
0.012
0.043
0.038
0.030
0.047
0.028
0.183
0.183
0.168
0.101
0.012
The Linear Interpolation Estimate:   234.6761   Entered P Value: 50

Number of Resamplings:   .80                                :
The Bootstrap Estimates Mean: 233.3311 Standard Deviation:    28.9594
Original Confidence Limits:   Lower:   184.8692 Upper:   283.3965
Resampling time in Seconds:     0.11  Random Seed:  1103756486
         Figure L.4.   Example of ICPIN program output for the IC50.
                                     481

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                                        483
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