540/09-87-198
                     PESTICIDE ASSESSMENT GUIDELINES

                              SUBDIVISION E

                            HAZARD EVALUATION:

                      WILDLIFE AND AQUATIC ORGANISMS

                           Series 72-1 to 72-5

Aquatic Testing for Marine/Estuarine and Freshwater Fish and  Invertebrates

                     ADDENDUM 2 ON DATA REPORTING
                               Prepared by:

                             Curtis E.  Laird
                        Ecological Effects Branch
                        Hazard Evaluation Division
                                Edited by:

                           Karen E.  Warkentien
                        Hazard Evaluation Division
                             Project Manager:

                       Elizabeth M.  K.  Leovey,  PhD
                        Hazard Evaluation Division
                       Office of Pesticide Programs
                    US Environmental Protection Agency

                           December 1986

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                                         -1-
               Subdivision E - Aquatic Testing for Marine/Estuarine and
                          Freshwater Fish and Invertebrates
                            Table of Contents of Addendum


 Discussion                                                              Page

 Introduction                                                              1

 Response to Public Conuients                                               1


 Guidelines

- Acute Toxicity Test for Freshwater Fish                                   4

 Acute Toxicity Test for Freshwater Aquatic Invertebrates                 10

 Acute Toxicity Test for Shrimp                                           16

 Acute Toxicity Test for Estuarine and Marine Fish                        22

 Oyster Embryo Test                                                       28

 Shell Deposition Study for Oyster                                        34

 Fish Early Life-Stage                                                    40

 Aquatic Invertebrate Life-Cycle                                          49

 Life-Cycle Tests for Fish                                                56

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                                        -11-






           Table of Contents  for Acute Toxicity Test for Freshwater Fish






GUIDELINE                                                               Page



         Title/Cover Page                                             ~    4



         Table of Contents                                                4



     I.  Summary/Introduction                                            4



    II.  Materials/Methods                                                5



   III.  Discussion and Results                                           7



    IV.  Conclusions                                                      7



         Tables                                                           8
   Table of Contents for Acute Toxicity Test for Freshwater Aquatic Invertebrates






GUIDELINE                                                               Page



         Title/Cover Page                                                10



         Table of Contents                                               10



     I.  Summary/Introduction                                            10



    II.  Materials/Methods                                               11



   III.  Discussion and Results                                          13



    IV.  Conclusions                                                     13



         Tables                                                          14

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                                        -111-






                 Table of  Contents  for Acute Toxicity Test for Shrimp






 GUIDELINE                                                              Page



          Title/Cover  Page                                               16



          Table of Contents                                               16



      I.   Summary/Introduction                                            16




     II.   Materials/Methods                                               17



    III.   Discussion and Results                                          19



     IV.   Conclusions                                                     19



          Tables                                                          20
      Table of Contents for Acute Toxicity Test for Estuarine and Marine Fish





GUIDELINE                                                               Page



         Title/Cover Page                                                22



         Table of Contents                                               22



     I.  Summary/Introduction                                            22



    II.  Materials/Methods                                               23



   III.  Discussion and Results                                          25



    IV.  Conclusions                                                     25



         Tables                                                          26

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                                        -IV-
                      Table of Contents for Oyster Embryo Test
GUIDELINE
Page
         Title/Cover Page                                                28



         Table of Contents                                               28



     I.  Summary/Introduction                                            28



    II.  Materials/Methods                                               29



   III.  Discussion and Results                                          31



    IV.  Conclusions                                                     31



         Tables                                                          32
              Table of Contents for Shell Deposition Study for Oyster






GUIDELINE                                                               Page




         Title/Cover Page                                                 34



         Table of Contents                                                34



     I.  Summary/Introduction                                             34




    II.  Materials/Methods                                                35



   III.  Discussion and Results                                           37



    IV.  Conclusions                                                      37



         Tables                                                           38

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                                        -V-






                    Table of Contents  for Fish Early Life-Stage






GUIDELINE                                                               Page



         Title/Cover  Page                                                40



         Table of Contents                                               40



      I.  Summary/Introduction                                            40



    II.  Materials/Methods                                               41



   III.  Discussion and Results                                          43



    IV.  Conclusions                                                     43



         Tables                                                          44
               Table of Contents for Aquatic Invertebrate Life-Cycle






GUIDELINE



         Title/Cover Page                                                49



         Table of Contents                                               49



     I.  Summary/Introduction                                            49



    II.  Materials/Methods                                               50



   III.  Discussion and Results                                          52




    IV.  Conclusions                                                     52



         Tables                                                          53

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                                        -VI-






                  Table of Contents for Life-Cycle Tests for Fish






GUIDELINE                                                               Page




         Title/Cover Page                                                56




         Table of Contents                                               56




     I.  Summary/Introduction                                            56




    II.  Materials/Methods                                               57




   III.  Discussion and Results                                          59




    IV.  Conclusions                                                     59




         Tables                                                          60

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                           PESTICIDE  ASSESSMENT GUIDELINES

                            WILDLIFE  AND AQUATIC ORGANISMS

      AQUATIC TESTING FOR MARINE/ESTUARINE AND FRESHWATER FISH AND INVERTEBRATES

                          Subdivision E, Series 72-1 to 72-5

                                   DATA REPORTING



 INTRODUCTION

      A.    Purpose

      To provide a data  reporting format for  fish and invertebrate studies.


      B.    Objective

      These Data Reporting Guidelines are  designed to aid the petitioner/registrant
      in generating reports which are compatible with the Agency's review process.
      Data  submitters  are encouraged  to  submit complete reports  following this
      guidance for efficient review by the Agency.  This guidance pertains  to the
      substance of the data report.   PR  Notice 86-5, effective on November  1, 1986
      (available from the Registration Support and Emergency Response Branch, Office
      of Pesticide Programs, US EPA),  pertains to the physical formating of reports
      (which  are referred to as "studies") and submittal packages.  Some of the re-
      quirements in PR Notice 86-5  are mandatory.


RESPONSE TO  PUBLIC COMMENTS

      The purpose of  this seciton is  to  acknowledge and address  the concerns ex-
pressed in the letters  of comments received  by the Agency  in response  to the public
notice in  the FEDERAL REGISTER (51 FR 18660) of May 21, 1986.

      This  addendum to the Pesticide  Assessment Guidelines  [Subdivision E]  is to be
considered an all-encompassing document.  The Ecological Effects Branch (EEB) has
reviewed the ten (10) comments submitted  by  the registrants and/or committees in
regard to  the Data Reporting Guidelines (DRGs).  These Guidelines are  not  intended
to create  new data requirements, but to provide for consistent  reporting of the
necessary  aquatic data  required to perform a scientifically sound hazard assessment.
This  approach should  eliminate most,  if not  all, of the recycling of submissions
between EEB  scientists  and the registrant.

     The submitted comments pertain  to  either items common to all studies  or a study
specific item.  Hence,  this discussion  has been arranged to consider the general
comments first, followed by the specific  comments.

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                                        -2-
kGeneral  Comments

 1.   Location of a  reporting  item -

     Considering the  need to  maintain a consistent format, revisions were made to the
     DRGs in response to these comments Whenever possible.

 2.   Composition of the test  substance -
         is asking  for the  "Identity and Composition" of the test substance instead
    of  the "Identity and Composition" of the impurities.  Under the present guide-
    lines, these data are  required under Subdivision E, Test Substance 70-4(2) (i).

 3.  Ingredient  information from testing laboratories -

    It  appears  some  sponsors do not make this information available to testing lab-
    oratories.  In such a  situation, the sponsor bears the responsibility since he
    is  submitting  the data for registration.

 4.  Telephone number of individual who can provide details of test -

    This item has  been deleted from the DRG.

 5.  Time to  implement new  format -

    The registrant will be given sufficient time to implement the necessary changes
    in  order to meet the recommended format standard.

 6.  Limited to  DRG -

    Additional  data  can and should be reported, providing it is meaningful and use-
    ful in making  a  risk assessment.

 7.  Public comments  -

    Ecological  Effects Branch (EEB) is only addressing the comments relating  to  the
    nine aquatic studies.

 Study-Specific  Comments

 1.  The following  were changed as a result of comments.

    A.  The shell  deposition value has been changed from IC$Q to ECsg in ppm.

    B.  The fish early life-cycle value has been changed from EC5Q to MATC in ppm.

    C.  The aquatic  invertebrate life-cycle value has been changed from  EC5Q  to  MATC
        in ppm.

    D.  Total and  un-ionized ammonia has been deleted from fish early life-stage,
        aquatic invertebrate life-cycle, and fish life-cycle test.

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                                        -3-
2.  The following were not changed as a result of comments.

    A.   The water hardness,  alkalinity, and conductivity are required under Subdivi-
        sion E, Sections 70-4(6)(ii)(E)(F) which calls for "[t]he source of dilution
        water, its chemical characteristics,  and description of any pretreatment"
        and the "[m]ethods used for, and results of, all chemical analysis of water
        and all toxicant concentrations at beginning, during, and at end of tests,
        including validation studies and reagent blanks, if there is reason to sus-
        pect that the concentrations administered to the test water do not approxi-
        mate the actual concentrations."

    B.   The no-observed-effect level (NOEL) for aquatic studies is required, except
        if the LC5o or £€50 has not been determined, as allowed by the specific
        testing requirement.

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                                       -4-


                         PESTiaEE ASSESSMENT GUIDELINES

                          WILDLIFE AND AQUATIC ORGANISMS

                     Acute Toxicity Test for Freshwater Fish

                            Subdivision E, Series 72-1

                                  DATA REPORTING

GUIDELINE

      The following describes the order and format for a study report item by item.

      TITLE/COVER PAGE

      Refer to PR Notice 86-5.

      TABLE OF CONTENTS

      The Table of Contents should indicate the overall organization of the study,
      including tables and figures.  It must follow the title, data confidentiality,
      and GLP (if appropriate) pages as described in PR Notice 86-5.

I.    SUMMARY/INTRODUCTION

      A.  Sponsor:  (name of study owner);

      B.  Name:   (person(s) who can provide details of test procedures);

      C.  Location of study;

      D.  Location of raw data and final report;

      E.  Material:  (comnDn/trade name);

      F.  Subject:  (final or draft report, acute 96-hour IC^Qi and species tested);

      G.  Test Doses:  (control, concurrent vehicle control, #, #, #, #, # ppm);

      H.  Test Dates:  (initiation - date test started, termination and date of
          last day of observation);

      I.  Length of Study:  (hours);

      J.  Results:  (LC5Q value, 95% confidence limits, and no-observed-effect
          level);

      K.  Tested Material:  (chemical name, formulation, and percent active
          ingredient);

      L.  Test Species/Strain:  (both common and scientific name);

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                                         -5-


       M.  Source of Organisms:  (company and address);

       N.  Weight/Length and Physical Condition at the Initiation of  Study.

II.    MATERIAL/METHODS

       This section is a narrative,  which would include the following items.

       A.  Test Substance

           1.   Identification (this  information may be provided by the laboratory or
               sponsor):

               a.   Chemical name;
               b.   Composition (qualitative and quantitative description);
               c.   Percent active  ingredient;
               d.   Molecular structure;
               e.   Source, lot number, or code;
               f.   Identity and percent  composition of impurities in the test
                   substance.

           2.   Preparation of  test solution (control/treatment):

               a.   Any vehicle used  to dissolve test material;
               b.   Amount of vehicle added to control  (if used);
               c.   Description of  method used to get test material into solution;
               d.   Water source (reconstituted - if so,  how prepared);
               e.   Solubility of test material in ppm;
               f.   Total amount of test  material used;
               g.   Temperature of  water.

       B.   Test Fish

           1.   Rationale for selection of species if the species used is
               different than  that preferred in Subdivision E.

               a.   Test species name (both scientific  and common);
               b.   History of  test organisms (strain,  diseases, and treat-
                   ment).

           2.   Description of  any  pretest conditioning:

               a.   Health:

                   1.   Sickness;
                   2.   Injuries;
                   3.   Abnormalities;
                   4.   Name of medication (if used);
                   5.   Pretest diet.

           3.   Size/age/physical condition:

               a.   Age (if known);
               b.   Size (weight in grams and length in mm).

-------
    4.  Source/acclimation:

        a.  Complete name and address of test fish supplier;
        b.  Source of food and dilution water;
        c.  Size of test container [in milliliters (mL)];
        d.  Temperature, pH, and dissolved oxygen, and name of equipment used
            to measure water quality;
        e.  Feeding schedule, holding, and acclimation period;
        f.  Test organisms from the same source (yes or no);
        g.  Number of days held.
C.  Method
    1.  Test Vessels:

        a.  Material type;
        b.  Volume [in liters (L)];
        c.  Depth of test solution [in centimeters (cm)];
        d.  Size [in liters (L)].

    2.  Test system:

        a.  Source of dilution water;
        b.  If flow-through, description of system and flow rate/day;
        c.  Procedures used to prepare toxicant stock solution;
        d.  Criteria used to determine effects.

    3.  Test design:

        a.  Method used in assigning test organisms to test and control
            groups, and the number of replicates used;
        b.  Number of fish per dose level and control group;
        c.  Name of protocol followed during this test;
        d.  Loading (weight per unit volume of water);
        e.  Number of treatment levels used (nominal or measured);
        f.  Length of exposure period;
        g.  Type of control (positive, negative, or solvent control);
        h.  Temperature;
        i.  pH (when checked - see table 1);
        j.  Lighting (time in hours and intensity in footcandles);
        k.  Dissolved oxygen (see table 1);
        1.  Water hardness (expressed in mg/L as CaC03 - see table 1);
        m.  Alkalinity (expressed in mg/L as CaCC>3 - see table 1);
        n.  Conductivity (umhos/cm - see table 1);
        o.  Range finding test results, concentrations, and mortality (if
            used);
        p.  Aeration (yes or no);
        q.  Water physical characteristic at the end of test:
            a.  Water depth and volume;
            b.  Temperature, pH, and dissolved oxygen;
        r.  Concentration analysis (see table 1);
        s.  Examination of fish physiology, locomotion, behavior and pathology;
        t.  Mortalities during the test;

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                                          -7-


        D.  Statistical Analysis

            1.  Cite references (author,  title,  journal,  number,  page,  etc.).

III.     DISCUSSION AND RESULTS

        A.  Include LCso value in ppm with 95% confidence limits  (graphs,  printouts,
            and other calculations should be attached to report).  Provide raw mor-
            tality data (see table 2).

        B.  Discuss the relationship,  if  any,  between the physical factors,  toxicant,
            and observation, and describe any precipitation and solubility problems.

        C.  Provide statistical method used (cite references,  author,  etc.).

        D.  Observation - Provide data on the following:                            v

            1.  Signs of intoxication - temporal onset,  duration, and concentrations
                that showed effects;

            2.  The no-observed-effect level.

 IV.     CONCLUSIONS

        This is a summation of the above  section - Discussion/Results.

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                               -8-


                             TABLE 1

                     WATER QUALITY PARAMETERS
Client       :
Test Material:
Test Organism:
Test Water   :
                          WATER QUALITY
Conductivity Hardness Alkalinity Temperature
pH umhos/cm mg/L as CaCC-3 mg/L as CaCO-} °C
X
X
X X
X
Nominal Concentration
Parameter
Dissolved
Oxygen
(mg/L)
pH
Time
Initial
48 hours
96 hours
Initial
96 hours
(mg/L)
Control XXX
Hig_h Medium
XX X
XX X
XX X
XX X
XX X

X X
Low
X
X
X
X
X

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                -9-


              TABLE 2

PERCENT MORTALITIES AND
                                            VALUES
Client:
Test Material:
Test Organism:
Test Water:
                        PERCENT MORTALITY

               TEST MATERIAL NOMINAL CONCENTRATION
                              (mg/L)
         Control
 24 Hour  #(#/#)    #(#/#)   #(#/#)    #(#/#)    #(#/#)   #(f/#)
 48 Hour  #(#/#)    #(#/#)   #(#/#)    #(#/#)    #(#/#)   #(#/#)
 96 Hour  !(#/#)    #(#/#)   #(#/#)    #(#/#)    #(#/#)   #(#/#)
                            24 hour
                            48 hour
96 hour
LC50 (mg/L) X
95% Low X
Confidence
Limits High X
X X
X X
X X

Observed Effects
  a floating
  b swimming at surface
  c lying on bottom
  d turned dark
                          e loss of equilibrium
                          ^ g\olping of air
                          9 etc.
  £/ How the percent of mortality table works.

  1.   #(#/#)a.
  2.   # = the total number of organisms tested per dose level.
  3.   f(#/#) = % mortality [No. dead/No, treated, e.g., 10% (1/10)]
  4.   a = the observed effects.

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                                        -10-


                          PESTICIDE ASSESSMENT GUIDELINES

                           WILDLIFE AND AQUATIC ORGANISMS

              Acute Toxicity Test for Freshwater Aquatic Invertebrates_

                             Subdivision E, Series 72-2

                                   DATA REPORTING

GUIDELINE

      The following describes the order and format for a study report item by item.

      TITLE/COVER PAGE

      Refer to PR Notice 86-5.                                                   v

      TABLE OF CONTENTS

      The Table of Contents should indicate the overall organization of the study,
      including tables and figures.  It must follow the title, data confidentiality,
      and GLP (if appropriate) pages as described in PR Notice 86-5.

I.    SUMMARY/INTRODUCTION

      A.  Sponsor:  (name of study owner);

      B.  Name:  (person(s) who can provide details of test procedures);

      C.  Location of study;

      D.  Location of raw data and final report;

      E.  Material:  (common/trade name);

      F.  Subject:  (final or draft report, 48-hour LCso/ and species tested);

      G.  Test Doses:  (control, concurrent vehicle control, #, #, f, #, # ppm);

      H.  Test Dates:  (initiation, date test started, termination, and date of
          last day of observation);

      I.  Length of Study:  (hours);

      J.  Results:  (LC5Q value, 95% confidence limits, and no-observed-effect level);

      K.  Tested Material:  (chemical name, formulation, and percent active
          ingredient);

      L.  Test Species/Strain:  (both common and scientific name);

      M.  Source of Organisms:  (company and address);

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                                  -11-


N.  Age of Organisms at the Initiation of Study:  (hours or life stage).

MATERIAL/METHODS

This section is a narrative, which would include the following items.

A.  Test Substance

    1.  Identification (this information may be provided by the laboratory or
        sponsor):

        a.  Chemical name;
        b.  Composition (qualitative and quantitative description);
        c.  Percent active ingredient;
        d.  Molecular structure;
        e.  Source, lot number, or code;
        f.  Identity and percent composition of impurities in the test
            substance.

    2.  Preparation of test solution (control/treatment):

        a.  Any vehicle used to dissolve test material;
        b.  Amount of vehicle added to control (if used);
        c.  Description of method used to get test material into solution;
        d.  Vfeter source (reconstituted - if so, how prepared);
        e.  Solubility of test material in ppm;
        f.  Total amount of test material used;
        g.  Temperature of water.

B.  Test Organisms

    1.  Rationale for selection of species if the species used is different
        than that preferred in Subdivision E (if 1st instar were not used,
        then give stage of life in which organisms were tested):

        a.  Test species name (both scientific and common);
        b.  History of test organisms (strain, diseases, and treatment).

    2.  Description of any pretest conditioning:

        a.  Health:

            1.   Sickness;
            2.   Injuries;
            3.   Abnormalities;
            4.   Name of medication (if used);
            5.   Pretest diet;
            6.   Number of days brood organisms were quarantined.

    3.  Size/age/physical condition:

        a.  Age (in hours);
        b.  Date of spawn.

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                                  -12-
    4.  Source/acclimation:

        a.  Complete name and address of test species supplier;
        b.  Source of food and dilution water;
        c.  Volume of test container [in milliliters (mL)];
        d.  Photoperiod and lighting;
        e.  Temperature, pH, dissolved oxygen, and name of equipment used to
            measure water quality;
        f.  Chemical treatment (if used);
        g.  Feeding schedule, holding and acclimation period;
        h.  Test organisms from the same source (yes or no) ;
        i.  Number of days adults were held.
C.  Method
    1.  Test Vessels:

        a.  Material type;
        b.  Volume of test solution [in milliliters (mL)];
        c.  Depth of test solution [in centimeters (cm)];
        d.  Size of test containers [in milliliters (mL)].

    2.  Test system:

        a.  Source of dilution water;
        b.  If flow-through, description of system and flow rate/day;
        c.  Procedures used to prepare toxicant stock solution;
        d.  Criteria used to determine effects.

    3.  Test design:

        a.  Method used in assigning test organisms to test and control
            groups, and the number of replicates used;
        b.  Number of organisms per dose level and control group;
        c.  Name of protocol followed during this test;
        d.  Loading (number of organism per unit volume of water);
        e.  Number of treatment levels used (nominal or measured);
        f.  Length of exposure period;
        g.  Type of control (positive, negative, or solvent control);
        h.  Temperature;
        i.  pH (when checked - see table 1);
        j.  Lighting (time in hours and intensity in footcandles);
        k.  Dissolved oxygen (see table 1);
        1.  Water hardness (expressed in mg/L as CaCC>3 - see table 1);
        m.  Alkalinity (expressed in mg/L as CaCD3 - see table 1);
        n.  Conductivity (umhos/cm - see table 1);
        o.  Range finding test results, concentrations, and mortality (if
            used);
        p.  Aeration (yes or no);
        q.  Water physical characteristic at the end of test:
            1.  Water depth and volume;
            2.  Temperature, pH, and dissolved oxygen;

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                                          -13-


                r.  Percent of death/effects at each dose level;
                s.  Concentration analysis (see table 1);
                t.  Examination of invertebrate physiology,  locomotion,  and behavior;
               • u.  Mortalities during the test.

        D.  Statistical Analysis

            1.  Cite references (author, title, journal, number,  page, etc.).

III.    DISCUSSION AND RESULTS

        A.  Provide LCso value in ppm with 95% confidence limits (graphs, printouts,
            and other calculations should be attached to report).  Provide raw mortality
            data (see table 2).

        B.  Discuss the relationship,  if any, between the physical factors, toxicant,
            and observation, and describe any precipitation and solubility problems".

        C.  Provide statistical method used (cite references, author, etc.).

        D.  Observation - Provide the following data:

            1.  Signs of intoxication - temporal onset and duration, concentrations
                that showed effects;

            2.  The no-observed-effect level.

 IV.    CONCLUSIONS

        This is a summation of the above section - Discussion/Results.

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Client        :
Test Material:
Test Organism:
Test Water    :
                               -14-


                             TABLE 1

                     WATER QUALITY PARAMETERS
                          WATER QUALITY
          Conductivity    Hardness      Alkalinity    Temperature
            umhos/cm    mg/L as CaCO-^  mg/L as CaCO^	°C	
Parameter    Time
Control
Nominal Concentration
	(mg/L)	

X    X    X    X    X
Dissolved
Oxygen
(mg/L)
pH

Initial
48 hours
Initial
48 hours
X
X
X
X
High
X
X
X
X
Medium
X
X
X
X
Low
X
X
X
X

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                               -15-


                             TABLE 2

               PERCENT MORTALITIES AND IC$Q VALUES
Client       :
Test Material:
Test Organism:
Test Water   :
                        PERCENT MORTALITY

         TEST MATERIAL NOMINAL OR MEASURED CONCENTRATION
                              (mg/L)
Control X
24 Hour #(#/#) #(#/#)
48 Hour #(#/#) #(#/#)

LC50 (mg/L)
X
#(#/#)
#(#/#)
24 hour
X
X X
#(#/#) #(#/#)
#(#/#) #(#/f)
48 hour
X
X
#(#/#)
#(#/#)


95%
Confidence
Limits
Low

High
X

X
X

X

Observed Effects

a floating
b lying on bottom
                                c swimming near surface
                                d etc.
£/ How the percent of  mortality table works.

1.   #(#/#)a.
2.   # = the total number of organisms tested per dose level.
3.   #(#/#) = % mortality [No.  dead/No, treated,  e.g.,  10% (1/10)].
4.   a = the observed effects.

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                                        -16-


                          PESTICIDB ASSESSMENT GUIDELINES

                           WILDLIFE AND AQUATIC ORGANISMS

                           Acute Toxicity Test for Shrimp

                             Subdivision E, Series 72-3

                                   DATA REPORTING

GUIDELINE

      The following describes the order and format for a study report item by item.

      TITLE/COVER PAGE

      Refer to PR Notice 86-5.                                                   v

      TABLE OF CONTENTS

      The Table of Contents should indicate the overall organization of the study,
      including tables and figures.  It must follow the title, data confidentiality,
      and GLP (if appropriate) pages as described in PR Notice 86-5.

I.    SUMMARY/ INTRODUCTION

      A.  Sponsor:  (name of study owner);

      B.  Name:  (person(s) who can provide details of test procedures;

      C.  Location of study;

      D.  Location of raw data and final report;

      E.  Material:  (ccmmon/trade name);

      F.  Subject:  (final or draft report, 96-hour IC$Q, and species tested);

      G.  Test Doses:  (control, concurrent vehicle control, #, #, #, #, # ppm);

      H.  Test Dates:  (initiation, date test started, termination, and date of
          last day of observation);

      I.  Length of Study:  (days);

      J.  Results:  (LCso value, 95% confidence limits, and no-observed-effect level);

      K.  Tested Material:  (chemical name, formulation, and percent active
          ingredient);

      L.  Test Species/Strain:  (both common and scientific name);

      M.  Source of Organisms:  (company and address);

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                                         -17-


       N.   Size/Age of Organisms at the Initiation of  Study:   (hours).

II.     MATERIAL/METHODS

       This section is a narrative, Which would include the following  items.

       A.   Test Substance

           1.   Identification (this information may be provided by the laboratory or
               sponsor):

               a.   Chemical name;
               b.   Composition (qualitative and quantitative description);
               c.   Percent active ingredient;
               d.   Molecular structure;
               e.   Source,  lot number,  or code;
               f.   Identity and percent composition of impurities  in the test
                   substance.

           2.   Preparation of  test solution (control/treatment):

               a.   Any vehicle used to  dissolve test material;
               b.   Amount of vehicle added to control  (if used);
               c.   Description of itethod used to get test material into solution;
               d.   Water  source (reconstituted - if so,  how prepared);
               e.   Solubility in ppm;
               f.   Total  amount of test material used;
               g.   Salinity (o/oo).

       B.   Test Shrimp

           1.   Rationale  for selection  of species if the species used  is different
               than that  preferred in Subdivision E:

               a.   Test species name (both scientific  and cannon);
               b.   History of  test organisms (strain,  diseases, and treatment).

           2.   Description of  any pretest conditioning:

               a.   Health:

                   1.   Sickness;
                   2.   Injuries;
                   3.   Abnormalities;
                   4.   Pretest diet.

           3.   Size/age/physical condition:

               a.   Age;
               b.   Size;
               c.   Date hatched (if known).

-------
                                  -18-
    4.  Source/acclimation;

        a.  Complete name and address of test species supplier;
       .b.  Source of food and dilution water;
        c.  Volume of test container [in liters (L)];
        d.  Photoperiod and lighting;
        e.  Temperature, pH, and dissolved oxygen, name of equipment used to
            measure water quality;
        f.  Chemical treatment (if used);
        g.  Percent mortality;
        h.  Feeding schedule;
        i.  Test organisms from the same source (yes or no);
        j.  Acclimation period (in days).
C.  Method
    1.  Test Vessels:

        a.  Material type;
        b.  Volume [in liters (L)];
        c.  Depth of test solution [in centimeters (cm)];
        d.  Size [in centimeters (cm)].

    2.  Test system:

        a.  Source of dilution water;
        b.  If flow-through, description of system and flow rate/day;
        c.  Procedures used to prepare toxicant stock solution;
        d.  Criteria used to determine effects.

    3.  Test design:

        a.  Method used in assigning test organisms to test and control
            groups;
        b.  Number of organisms per dose level and control group;
        c.  Number of days food was withheld prior to test;
        d.  Loading (weight of organism per unit volume of water);
        e.  Number of treatment levels used (nominal or measured);
        f.  Length of treatment period;
        g.  Type of control (positive, negative, or solvent control);
        h.  Temperature;
        i.  pH (when checked - see table 1);
        j.  Lighting (time in hours and intensity in footcandles) ;
        k.  Dissolved oxygen (see table 1);
        1.  Range finding test results, concentrations, and mortality (if
            used);
        m.  Aeration (yes or no);
        n.  Water physical characteristic at the end of test:
            a.  Water depth and volume;
            b.  Temperature, pH, and dissolved oxygen;
        o.  Percent of death/effects at each dose level;
        p.  Name of protocol followed during the test;
        q.  Salinity (o/oo).

-------
                                          -19-


        D.   Statistical Analysis

            1.  Cite references (author,  title,  journal,  number,  page,  etc.).

III.     DISCUSSION AND RESULTS

        A.   Provide IC$Q value in ppm with 95% confidence limits  (graphs,  printouts,
            and other calculations should be attached to report).  Provide raw mortality
            data (see table 2).

        B.   Discuss the relationship if any, between the physical factors, toxicant,
            and observation, and describe any precipitation and solubility problems.

        C.   Provide statistical method used (cite references,  author,  etc.).

        D.   Observation - Provide the following data:
                                                                                  v
            1.  Signs of intoxication - temporal onset,  duration, and concentrations
                that showed effects;

            2.  The no-observed-effect level.

 IV.     CONCLUSIONS

        This is a summation of the above section - Discussion/Results.

-------
                               -20-


                             TABLE 1

                     WATER QUALITY PARAMETERS
Client       :
Test Material:
Test Organism:
Test Water   :
                          WATER QUALITY

Temperature
6 hours, 12 hours, etc.
Nominal Concentration

Parameter

Dissolved
Oxygen
(mg/L)
pH

Salinity
o/oo

Time

Initial
48 hours
96 hours
Initial
96 hours
Initial
96 hours

Control

X
X
X
X
X
X
X

X
High
X
X
X
X
X
X
X
(mg/L)
X
Medium
X
X
X
X
X



X
Low
X
X
X
X
X



-------
                               -21-


                             TABLE 2

               PERCENT MORTALITIES AND LC50 VALUES
Client       :
Test Material:
Test Organism:
Test Water   :
                        PERCENT MORTALITY

               TEST MATERIAL NOMINAL CONCENTRATION
                              (mg/L)
         Control     X
 24 Hour  #(#/#)    #(#/#)   #(#/#)    #(#/#)     #(#/#)    #(#/#)
 48 Hour  #(#/#)    #(#/#)   #(#/#)    #(#/#)    #(#/#)   #(#/#)
 96 Hour  #(#/#)    #(#/#)   #(#/#)    #(#/#)    #(#/#)   #(#/#)
                            24 hour
                                          48 hour
96 hour
  LC50 (mg/L)
95%
Confidence
Limits
Low

High
X

X
X

X
X

X

Observed Effects

a floating
b swimming at surface
c lying on bottom
                                        ^ turned dark
                                        e etc.
£  How the percent of mortality table works.

1.   #(#/#)a.
2.   I = the total number of organisms tested per dose level.
3.   #(#/#) = % mortality [No. dead/No, treated, e.g., 10% (1/10)]
4.   a = the observed effects.

-------
                                        -22-


                          PESTICTDE ASSESSMENT GUI KLINES

                           WILDLIFE AND AQUATIC ORGANISMS

                 Acute Toxicity Test for Estuarine and Marine Fish

                             Subdivision E, Series 72-3

                                   DATA REPORTING

GUIDELINE

      The following describes the order and format for a study report item by item.

      TITLE/COVER PAGE

      Refer to PR Notice 86-5.

      TABLE OF CONTENTS

      The Table of Contents should indicate the overall organization of the study,
      including tables and figures.  It must follow the title, data confidentiality,
      and GLP (if applicable) pages as described in PR Notice 86-5.

I.    SUMMARY/ INTRODUCTION

      A.  Sponsor:  (name of study owner);

      B.  Name:  (person(s) who can provide details of test procedures);

      C.  Location of study;

      D.  Location of raw data and final report;

      E.  Material:  (common/trade name);

      F.  Subject:  (final or draft report, acute 96-hour LCso* and, species tested);
      G.  Test Doses:  (control, concurrent vehicle control, #, f, #, f, #, and
          # ppm);

      H.  Test Dates:  (initiation - date test started, termination - date of last
          day of observation);

      I.  Length of Study:  (hours);

      J.  Results:  (LC5Q value, 95% confidence limits, and no-observed-effects
                     level);

      K.  Tested Material:  (chemical name, formulation, and percent active
          ingredient);

      L.  Test Species/Strain:  (both common and scientific name);

-------
                                         -23-


       M.   Source of Organisms:   (company and address);

       N.   Weight/Length and Physical Condition at  the  Initiation of Study.

II.     MATERIAL/METHODS

       This section is a narrative,  which would include  the following  items.

       A.   Test Substance

           1.   Identification (this  information may be provided by  the laboratory or
               sponsor):

               a.   Chemical name;
               b.   Composition (qualitative and quantitative description);
               c.   Percent active  ingredient;
               d.   Molecular structure;
               e.   Source, lot number, or code;
               f.   Identity and percent  composition of  impurities in the test
                   substance.

           2.   Preparation of  test solution (control/treatment):

               a.   Any vehicle used  to dissolve test material;
               b.   Amount of vehicle added to control  (if used);
               c.   Description of  method used to get test material  into solution;
               d.   Water source  (reconstituted - if so,  how prepared);
               e.   Solubility of test material in ppm;
               f.   Total amount of test  material used;
               g."   Temperature of  water;
               h.   Salinity (o/oo).

       B.   Test Fish

           1.   Rationale for selection of species if the species used  is
               different than  that preferred in Subdivision E:

               a.   Test species name (both scientific and common);
               b.   History of  test organisms (strain, diseases, and treatment).

           2.   Description of  any  pretest conditioning:

               a.   Health:

                   1.   Sickness;
                   2.   Injuries;
                   3.   Abnormalities;
                   4.   Name of medication (if used);
                   5.   Pretest diet;
                   6.   Number  of days brood fish were quarantined;

-------
                                  -24-
    3.  Size/age/physical condition:

        a.  Age (if known);
        b.  Size (weight in grams and length in mm).

    4.  Source/acclimation:

        a.  Complete name and address of test fish supplier;
        b.  Source of food and dilution water;
        c.  Size of test container [in milliliters (mL)];
        d.  Temperature, pH, dissolved oxygen, and name of equipment used to
            measure water quality;
        e.  Feeding schedule, holding, and acclimation period;
        f.  Test organisms from the same source (yes or no);
        g.  Number of days held;
        h.  Salinity (o/oo).
C.  Method
    1.  Test Vessels:

        a.  Material type;
        b.  Volume [in liters (L)];
        c.  Depth of test solution [in centimeters (cm)];
        d.  Size [in liters (L)].

    2.  Test system:

        a.  Source of dilution water;
        b".  If flow-through, description of system and flow rate/day;
        c.  Procedures used to prepare toxicant stock solution;
        d.  Criteria used to determine effects.

    3.  Test design:

        a.  Method used in assigning test organisms to test and control
            groups, and the number of replicates used;
        b.  Number of fish per dose level and control group;
        c.  Name of protocol followed during this test;
        d.  Loading (weight per unit volume of water);
        e.  Number of treatment levels used (nominal or measured);
        f.  Length of exposure period;
        g.  Type of control (positive, negative, or solvent control);
        h.  Temperature;
        i.  pH (when checked - see table 1);
        j.  Lighting (time in hours and intensity in footcandles);
        k.  Dissolved oxygen (see table 1);
        1.  Range finding test results, concentrations, and mortality (if
            used);
        m.  Aeration (yes or no);
        n.  Water physical characteristic at the end of test:
            1.  Water depth and volume;
            2.  Temperature, pH, and dissolved oxygen;

-------
                                          -25-


                o.  Concentration analysis;
                p.  Examination of fish physiology,  locomotion,  behavior and pathology;
                q.  Mortalities during the test.

        D.  Statistical Analysis

            1.  Cite references (author, title, journal, number, page, etc.).

III.    DISCUSSION AND RESULTS

        A.  Provide LCso value in ppm with 95% confidence limits (graphs, printouts,
            and other calculations should be attached to report).  Provide raw mortality
            data (see table 2).

        B.  Discuss the relationship,  if any, between the physical factors, toxicant,
            and observation, and describe any precipitation and solubility problems.
                                                                                   v
        C.  Provide statistical method used (cite references,  author, etc.).

        D.  Observation - Provide data on the following:

            1.  Signs of intoxication - temporal onset, duration, and concentrations
                that showed effects;

            2.  The no-observed-effect level.

|IV.     CONCLUSIONS

        This is a summation of the above section - Discussion/Results.

-------
                               -26-


                             TAELE 1

                     WATER QUALITY PARAMETERS
Client:
Test Material:
Test Organism:
Test Water:
                          WATER QUALITY



Dissolved
Oxygen
(mg/L)
PH

Temperature

Initial
48 hours
96 hours
Initial
96 hours
CO 6
Control
X
X
X
X
X
hrs, 12
High
X
X
X
X
X
hrs, etc
Medium
X
X
X
X
X

Low
X
X
X
X
X

-------
                               -27-


                             TABLE 2

               PERCENT MORTALITIES AND LCs0 VALUES
Client       :
Test Material:
Test Organism:
Test Water   :
                        PERCENT MORTALITY

               TEST MATERIAL NOMINAL CONCENTRATION
                              (mg/L)
         Control
X
 24 Hour  #(#/#)    #(#/#)   #(#/#)    #(#/#)     #(#/#)    #(#/#)
 48 Hour  #(#/#)    #(#/#)   #(#/#)     #(#/#)     #(#/#)    #(#/#)
 96 Hour  #(#/#)    #(#/#)   #(#/#)     #(#/#)     #(#/#)    #(#/#)
24 hour 48 hour 96 hour
LC5o (mg/L)
95% Low
Confidence
Limits High
X XX
X XX
X XX

Observed Effects
 a floating
 b swimming at surface
 c lying on bottom
 ^ turned dark
e loss of equilibrium
f gulping of air
9 etc.
£/ How the percent of mortality table works.

1.   #(#/#)a.
2.   # = the total number of organisms tested per dose level.
3.   #(#/#) = % mortality [No.  dead/No, treated, e.g., 10% (1/10)]
4.   a = the observed effects.

-------
                                        -28-


                          PESTICICE ASSESSMENT GUIDELINES

                           WILDLIFE AND AQUATIC ORGANISMS

                                 Oyster Embryo Test

                             Subdivision E,  Series 72-3

                                   DATA REPORTING

GUIDELINE

      The following describes the order and format for a study item by item.

      TITLE/COVER PAGE

      Refer to PR Notice 86-5.

      TABLE OF CONTENTS

      The Table of Contents should indicate the overall organization of the study,
      including tables and figures.  It must follow the title, data confidentiality,
      and GLP (if appropriate) pages as described in PR Notice 86-5.

I.    SUMMARY/INTRODUCTION

      A.  Sponsor:  (name of study owner);

      B.  Name: (person(s) who can provide details on test procedures);

      C.  Location of study;

      D.  Location of raw data and final report;

      E.  Material:  (common/ trade name);

      F.  Subject:  (final or draft report,  48-hour ECso and species tested);

      G.  Test Doses:  (control, concurrent vehicle control, #, f, #, #, and # ppm);

      H.  Test Dates:  (initiation, date test started, termination and date of last
          day of observation);

      I.  Length of Study:  (days);

      J.  Results:  (ECso value, 95% confidence limits, and no-observed-effect level);
      K.   Tested Material:  (chemical name, formulation, and percent active
          ingredient);

      L.   Test Species/Strain:  (both common and scientific name);

      M.   Source of Organisms:  (company and address);

-------
                                         -29-


       N.  Stage of Organisms at the Initiation of Study:   (hours).

II.     MATERIAL/METHODS

       This section is a narrative,  which would include  the following items.

       A.  Test Substance

           1.   Identification (this  information may be provided by the  laboratory or
               sponsor):

               a.   Chemical name;
               b.   Composition (qualitative  and quantitative description);
               c.   Percent active ingredient;
               d.   Molecular structure;
               e.   Source,  lot number, or code;
               f.   Identity and percent  composition of impurities  in the test
                   substance.

           2.   Preparation of  test solution  (control/treatment):

               a.   Any vehicle used  to dissolve test material;
               b.   Amount of vehicle added to control  (if  used);
               c.   Description of method used to get test  material into solution;
               d.   Water  source (reconstituted - if so,  how prepared);
               e.   Solubility in ppm;
               f.   Total  amount of test  material used;
               g.   Salinity (o/oo).

       B.   Test Oyster

           1.   Rationale  for selection of species if the species used is different
               than that  preferred in Subdivision E:

               a.   Test species name (both scientific  and  common);
               b.   History of  test organisms (strain,  diseases, and treatment).

           2.   Description of  fertilization  process and  method used to  collect
               embryos.

           3.   Size/age:

               a.   Stage  of life at  initiation of test;
               b.   Date of  spawn;
               c.   List of  abnormalities (if any occurred).

           4.   Source/acclimation:

               a.   Complete name and address of test species supplier;
               b.   Source of food and water;
               c.   Volume of test container  [in liters (L)];
               d.   Photopericd and lighting;
               e.   Temperature,  pH,  dissolved oxygen,  and  name of  equipment used to
                   measure water quality;

-------
                                  -30-


         f.  Chemical  treatment  (if used);
         g.  Percent mortality;
         h.  Feeding schedule;
         i.  Test organisms  from the same source (yes or no);
         j.  Salinity  (o/oo).

C.  Method

    1.   Test vessels:
                                  /
         a.  Material  type;
         b.  Volume [in  liters (L)];
         c.  Volume of test  solution [in centimeters (cm)];
         d.  Size [in  centimeters  (cm)].

    2.   Test system:

         a.  Source of dilution water;
        b.  If flow-through, description of system and flow rate/day;
         c.  Procedures  used to  prepare toxicant stock solution;
         d.  Criteria used to determine effects;
         e.  Duplication of  treatment levels (yes or no).

    3.   Test design:

         a.  Method used in assigning test organisms to test and control
            groups;
        b.  Number of fertilized  eggs per liter of test solution;
        c.  Loading (weight of organism per unit volume of water);
         d.  Number of treatment levels used (nominal or measured);
        e.  Length of treatment period;
         f.  Type of control (positive, negative, or solvent control);
        g.  Temperature (prior  to and during spawning);
        h.  pH (when checked -  see table 1);
         i.  Lighting  (time  in hours, intensity in footcandles, and  transition
            period between light  and dark);
         j.  Dissolved oxygen (see table 1);
        k.  Range finding test  results (if used);
        1.  Aeration  (yes or no);
        m.  Water physical characteristics at the end of  test:
            1.  Water depth and volume;
            2.  Temperature, pH,  dissolved oxygen, and salinity;
        n.  Name of protocol used during the test;
        o.  How embryos collected and preserved;
        p.  Number of normal developed larvae at the end  of observation;
        q.  Number of deformed  larvae at the end of observation period.

D.  Statistical Analysis

    A.  Cite references (author,  title, journal, number,  page, etc.).

-------
I1-
                                         -31-
       DISCUSSION AND RESULTS

       A.  Provide £€50 value in ppm with 95% confidence limits (graphs, printouts,
           and other calculations should be attached to report).  Provide raw mortality
           data (see table 2).

       B.  Discuss the relationship, if any, between the physical factors, toxicant,
           and observation, and describe any precipitation and solubility problems.

       C.  Provide statistical method used (cite references, author, etc.).

       D.  Observation - Provide the following data:

           1.  Signs of intoxication - temporal onset and duration, and concentrations
               that showed effects?

           2.  The no-observed-effect level.

IV.     CONCLUSIONS

       This is a summation of the above section - Discussion/Results.

-------
Client       :
Test Material:
Test Organism:
Test Water   :
                               -32-


                             TABLE 1

                     WATER QUALITY PARAMETERS
                          WATER QUALITY
Temperature
Dissolved
Oxygen
(mg/L)
PH
(°C)
Initial
48 hours
Initial
48 hours
6 hrs, 12 hrs, etc.
High
X X
X X
X X
X X

Medium
X
X
X
X

Low
X
X
X
X

-------
                               -33-
                     TABLE 2

PERCENT REDUCTION OF NORMAL LARVAE AND
                                                   VALUES
Client      :
Test Material:
Test Organism:
No. Per Cone.:
Test Water   :
                PERCENT REDUCTION OF NORMAL LARVAE

         TEST MATERIAL NOMINAL OR MEASURED CONCENTRATION
                              (mg/L)
Control
24 Hour #(#/#)
48 Hour #(#/#)
X X X X
#(#/#) #(#/#) #(#/#) #(#/#)
#(#/#) #(#/#) #(#/#) #(*/*)
X
*(*/#)
#(#/#)
24 hour 48 hour
EC50 (mg/L)
95% Low
Confidence
Limits High
X X
X X
X X



Observed Effects
a floating
b dead
c lying on bottom
d etc.

£/ How the percent of mortality table works.

 1.  #(f/#)a.
 2.  # = the total number of organisms tested per dose level.
 3.  #(#/#) = % mortality [No. dead/No, treated, e.g., 10% (1/10)].
 4.  a = the observed effects.

-------
                                        -34-


                          PESTICIDE ASSESSMENT GUIDELINES

                           WILDLIFE AND AQUATIC ORGANISMS

                         Shell Deposition Study for Oyster

                             Subdivision E,  Series 72-3

                                   DATA REPORTING

GUIDELINE

      The following describes the order and format for a study report item by item.

      TITLE/COVER PAGE

      Refer to PR Notice 86-5.

      TABLE OF CONTENTS

      The Table of Contents should indicate the overall organization of the study,
      including tables and figures.  It must follow the title, data confidentiality,
      and GLP (if appropriate) pages as described in PR Notice 86-5.

I.     SUMMARY/INTRODUCTION

      A.  Sponsor:  (name of study owner);

      B.  Name(s) of principal investigator(s);

      C.  Location of study;

      D.  Location of raw data and final report;

      E.  Material:  (common/trade name);

      F.  Subject:  (final or draft report,  96-hour £€50, and species tested);

      G.  Test Doses:  (control, concurrent vehicle control, #, #, #, #, # ppm);

      H.  Test Dates:  (initiation, date test started, termination, and date of
          last day of observation);

      I.  Length of Study:  (days);

      J.  Results:  (EC$Q value, 95% confidence limits, and no-observed-effect level);

      K.  Tested Material:  (chemical name,  formulation, and percent active
          ingredient);

      L.  Test Species/Strain:  (both common and scientific name);

      M.  Source of Organisms:  (company and address);

-------
                                         -35-


       N.   Size of Organisms at the Initiation of Study:   (mm).

II.     MATERIAL/METHODS

       This section is a narrative, which would include the following  items.

       A.   Test Substance

           1.   Identification (this information may be provided by the laboratory or
               sponsor):

               a.   Chemical name;
               b.   Composition (qualitative and quantitative description)?
               c.   Percent active  ingredient;
               d.   Molecular structure;
               e.   Source,  lot number, or code;
               f.   Identity and percent  composition of impurities  in the test
                   substance.

           2.   Preparation of test solution (control/treatment):

               a.   Any vehicle used to dissolve test material;
               b.   Amount of vehicle added to control  (if used);
               c.   Description of  method used to get test material into solution;
               d.   Water  source (reconstituted - if so, how prepared);
               e.   Solubility of test material in ppm;
               f.   Total  amount of test  material used;
               g.   Salinity (o/oo).

       B.   Test Oyster

           1.   Rationale  for selection of species if the  species used  is
               different  than that preferred in Subdivision E:

               a.   Test species name (both scientific  and common);
               b.   History of test organisms (strain,  diseases, and treatment).

           2.   Description of any  pretest conditioning:

               a.   Health:

                   1.   Sickness;
                   2.   Injuries;
                   3.   Abnormalities;
                   4.   Pretest diet;

           3.   Size/age/physical condition:

               a.   Height (in mm);
               b.   Abnormalities (if any occurred).

-------
                                  -36-
    4.  Source/acclimation:

        a.  Complete name and address of test species supplier;
        b.  Source of food and water;
        c.  Size of test container [in liters (L)];
        d.  Photoperiod and lighting;
        e.  Temperature, pH, dissolved oxygen, and name of equipment used to
            measure water quality;
        f.  Chemical treatment (if used);
        g.  Percent mortality;
        h.  Feeding schedule;
        i.  Test organisms from the same source (yes or no);
        j.  Salinity and acclimation period in days.
C.  Method
    1.  Test Vessels:

        a.  Material type;
        b.  Volume [in liters (L)];
        c.  Depth of test solution [in centimeters (on)];
        d.  Size [in liters (L)].

    2.  Test system:

        a.  Source of dilution water;
        b.  If flow-through, description of system and flow rate/day;
        c.  Procedures used to prepare toxicant stock solution;
     '  d.  Criteria used to determine effects.

    3.  Test design:

        a.  Method used in assigning test organisms to test and control
            groups;
        b.  Number of organisms per dose level;
        c.  Loading (weight of organism per unit volume of water);
        d.  Number of treatment levels used (nominal or measured);
        e.  Length of treatment period;
        f.  Type of control (positive, negative, or solvent control);
        g.  Temperature (how often measured);
        h.  pH (when checked - see table 1);
        i.  Lighting (time in hours, intensity in footcandles, and transition
            period between light and dark);
        j.  Dissolved oxygen (see table 1);
        k.  Range finding test results (if used - include sample size, test
            levels, and mortality data);
        1.  Aeration (yes or no);
        m.  Water physical characteristic at the end of test:
            1.  Water depth and volume;
            2.  Temperature, pH, and dissolved oxygen;
        n.  Percent of death/effects at each dose level;
        o.  Name of protocol followed during the test;
        p.  How peripheral valve edge removed;
        q.  Method used to measure oyster size.

-------
                                          -37-


        D.  Statistical Analysis

            1.  Cite references (author, title,  journal,  number, page,  etc.)*

III.     DISCUSSION AND RESULTS

        A.  Provide ECso value in ppm with 95% confidence limits (graphs,  printouts,
            and other calculations should be attached to report).  Provide raw mortality
            data (see table 2).

        B.  Discuss the relationship,  if any,  between the physical factors, toxicant,
            and observation, and describe any precipitation and solubility problems.

        C.  Provide statistical method used (cite references, author, etc.).

        D.  Observation - Provide the following data:

            1.  Signs of intoxication - temporal onset and duration, and concentrations
                that showed effects;

            2.  The no-observed-effect level.

 IV.     CONCLUSIONS

        This is a summation of the above section - Discussion/Results.

-------
Client       :
Test Material:
Test Organism:
Test Water   :
                               -38-


                             TABLE 1

                     WATER QUALITY PARAMETERS
                          WATER QUALITY
Temperature
Parameter
Dissolved
Oxygen
(mg/L)
pH
Salinity
0/00
6 hours,
Time
Initial
48 hours
96 hours
Initial
96 hours
Initial
96 hours
12 hours, etc.
Control
X
X
X
X
X
X
X

Nominal
X X
High
X
X
X
X
X
X
X


Concent rat ion
(mg/L)
X X
Medium
X
X
X
X
X

X
Low
X
X
X
X
X


-------
                               -39-


                             TABLE 2

          PERCENT CHANGE IN SHELL GROWTH AND EC$Q VALUES
Client       :
Test Material:
Test Organism:
Test Water   :
                  PERCENT CHANGE IN SHELL GROWTH

         TEST MATERIAL NOMINAL OR MEASURED CONCENTRATION
                              (mg/L)
           Control
 24 Hour  #(#/#)    #(#/#)   #(#/#)     #(#/f)     #(#/#)    #(#/#)
 48 Hour  #(#/#)     #(#/#)    #(#/#)     #(#/#)     #(#/#)    #(#/#)
 96 Hour  #(#/#)     #(#/#)    #(#/#)     #(#/#)     #(#/#)    #(#/#)

EC50 (mg/L)
95% Low
Confidence
Limits High
24 hour
X
X
X
48 hour 96 hour
X X
X X
X X

Observed Effects
a floating
b dead
c no growth
d etc.

 V How the percent of change in shell growth table works.

 1.   #(#/#)a-
 2.   # = the total number of organisms tested per dose level.
 3.   #(#/#) = % mortality [No. dead/No, treated,  e.g., 10% (1/10)]
 4.   a = the observed effects.

-------
                                        -40-


                          PESTICICE ASSESSMENT GUIDELINES

                           WILDLIFE AND AQUATIC ORGANISMS

                               Fish Early Life-Stage

                             Subdivision E, Series 72-4

                                   DATA REPORTING

GUIDELINE

      The following describes the order and format for a study report item by item.

      TITLE/COVER PAGE

      Refer to PR Notice 86-5.

      TABLE OF CONTENTS

      The Table of Contents should indicate the overall organization of the study,
      including tables and figures.  It must follow the title, data confidentiality,
      and GLP (if appropriate) pages as described in PR Notice 86-5.

I.    SUMMARY/INTRODUCTION

      A.  Sponsor:  (name of study owner);

      B.  Name(s) of principal investigator(s)

      C.  Location of study;

      D.  Location of raw data and final report;

      E.  Material:  (common/trade name);

      F.  Subject:  (final or draft report, fish early life-stage, and species
          tested);

      G.  Test Doses:  (control, concurrent vehicle control, #, #, #, #, # ppm);

      H.  Test Dates:  (initiation, date test started, termination and date of last
          day of observation);

      I.  Length of Study:  (days);

      J.  Results:  (MATC value ppm and no-observed-effects level);

      K.  Tested Material:  (chemical name, formulation, and percent active
          ingredient);

      L.  Test Species/Strain:  (both common and scientific name);

      M.  Source of Eggs:  (company and address);

-------
N.  Stage of Eggs development at the Initiation of Study.

MATERIAL/METHODS

This section is a narrative, which would include the following items.

A.  Test Substance

    1.  Identification (this information may be provided by the laboratory or
        sponsor):

        a.  Chemical name;
        b.  Composition (qualitative and quantitative description);
        c.  Percent active ingredient;
        d.  Molecular structure;
        e.  Source, lot number, or code;
        f.  Identity and percent composition of impurities in the test
            substance.

    2.  Preparation of test solution (control/treatment):

        a.  Any vehicle used to dissolve test material;
        b.  Amount of vehicle added to control (if used);
        c.  Description of method used to get test material into solution;
        d.  Water source (reconstituted - if so, how prepared);
        e.  Solubility in ppm;
        f.  Total amount of test material used.

B.  Test Fish

    1.  Rationale for selection of species if the species used is different
        than that preferred in Subdivision E (if eggs were not used, give
        stage of life cycle in which organisms were tested):

        a.  Test species name (both scientific and common);
        b.  History of test organisms (strain, diseases, and treatment).

    2.  Description of any pretest conditioning:

        a.  Health:

            1.   Sickness;
            2.   Injuries;
            3.   Abnormalities;
            4.   Name of medication (if used);
            5.   Pretest diet;
            6.   Number of days brood organisms were quarantined.

    3.  Size/age/physical condition:

        a.  Age of unfertilized eggs and milt at the beginning of test;
        b.  Description of procedures used to strip eggs and milt;
        c.  Description of the procedures used in storage and handling of  eggs;
        d.  Description of the fertilization process.

-------
                                  -42-
    4.  Source/acclimation:

        a.  Complete name and address of test species supplier;
        b.  Source of food and dilution water;
        c.  Size of test container [in liters (L)];
        d.  Number of females used per batch;
        e.  Temperature, pH, and dissolved oxygen;
        f.  Description of methods used initially to determine different stages
            of development;
        g.  Percent mortality (brood fish);
        h.  Feeding schedule, holding and acclimation period;
        i.  Test organisms from the same source (yes or no);
        j.  Number of males used per batch of eggs.
C.  Method
    1.  Test Vessels:

        a.  Material type;
        b.  Volume [in milliliters (mL)];
        c.  Depth of test solution [in centimeters (cm)];
        d.  Size of vessels [in centimeters (cm)].

    2,  Test system:

        a.  Source of dilution water;
        b.  If flow-through, description of system construction material,
            system, and flow rate/day;
        c.  Procedures used to prepare toxicant stock solution;
        d.  Criteria used to determine effects.

    3.  Test design:

        a.  Method used in assigning test organisms to test and control
            groups;
        b.  Number of eggs per dose level;
        c.  Name of protocol followed during this test;
        d.  Loading (number of organism per unit volume of water);
        e.  Number of treatment levels used (nominal or measured);
        f.  Length of exposure period;
        g.  Type of control (positive, negative, or solvent control);
        h.  Temperature;
        i.  pH (when checked - see table 2);
        j.  Lighting (time in hours and intensity in footcandles);
        k.  Dissolved oxygen (see table 2);
        1.  Water hardness (expressed in mg/L as CaCC>3 - see table 2);
        m.  Alkalinity (expressed in mg/L as CaCC>3 - see table 2);
        n.  Conductivity (umhos/cm - see table 2);
        o.  Range finding test results, concentrations, and mortality (if
            used);
        p.  Aeration (yes or no);

-------
                q.  Water physical characteristic at the end of test:
                    1.  Water depth and volume;
                    2.  Temperature, pH, dissolved oxygen, and name of equipment
                        used to measure the water quality;
                r.  Period food was withheld prior to termination;
                s.  Number of normal fry at the end of test (see table 3);
                t.  Concentration analysis (see table 1);
                u.  Examination of fish physiology, locomotion, and behavior;
                v.  Length and weight (see table 5);
                w.  Mortalities during the test;
                x.  Fry survival and growth;
                y.  The larvae fish released from the retaining cup when batch was
                    ( ) % complete or ( ) hours has elapsed;
                z.  Description of method used to keep eggs clean.

        D.  Statistical Analysis

            1.  Cite references (author, title, journal, number, page, etc.).

III.     DISCUSSION AND RESULTS

        A.  Provide MATC value in ppm (graphs, printouts, and other calculations
            should be attached to report).  Provide raw mortality data (see table 3).

        B.  Discuss the relationship, if any, between the physical factors, toxicant,
            and observation.

        C.  Provide statistical method used (cite references, author, etc.).

        D.  Observation - Provide the following data:

            1.  Signs of intoxication - temporal onset and duration;

            2.  The no-observed-effect level.

 IV.     CONCLUSIONS

        This is a summation of the above section - Discussion/Results.

-------
                         TABIE  1

        MEASURED CONCENTRATION  OF TEST COMPOUND IN
    WATER TO WHICH  (Test fish name)  (Scientific Name)
                 WERE EXPOSED FOR #  DAYS
Nominal
Concent rat ion
(mg/L) Mean + SD

Number of
Range Samples
Water Control
                  11 -- "  (Detected)  "
Solvent Control
                      Detected or  not

-------
                              BIS-
             WATER QUALITY CHARACTERISTICS MEASURED
                 DURING EXPOSURE OF (TEST FISH)
             EMBRYOS AND lARVAE TO (TEST COMPOUND)
 Water Quality                                      Number of
Characteristic       Mean + SD          Range      Measurements
 Temperature (°C)      "  +  "

 Dissolved Oxygen      "  +  "
     (mg/L)

 pH                    "~  -'

 Total Hardness        "  +  "
 (mg/L as
 Total Alkalinity
 (mg/L as CaC03)

 Specific Conductance
     (umhos/cm)

-------
                          -46-
                        TABLE 3

SURVIVAL OF (TEST FISH NAME) AT HATCH  (DAY #), AT  # DAYS
  POST-HATCH, AFTER EXPOSURE TO SEVERAL (XINCENTRATIONS
                   OF (TEST COMPOUND)
Mean
Measured
Concentration
(mg/L) Tank
#

#

#

#

#

Water Control

Solvent
Control
#
#
#
I
#
#
#
#
#
#
#
#
#
#
Percent
Larvae
Survival
At Hatch on
Day 1 (Mean)
* I
#
* #
#
* I
#
* #
#
* #
#
* #
#
* #
#
Percent
Survival
At # Days
Post-Hatch
(Mean)
* #
#
* #
#
* #
#
* #
#
* I
#
* #
#
* #
#
Percent
Survival
At # Days
Post-Hatch
(Mean)
* #
#
* 1
#
* I
#
* #
#
* # -
1
* #
1
* #
#

-------
                               -47-
                             TABLE 4

            LENGTH AND WEIGHT CF  [TEST FISH NAME] AT #
            DAYS  POST-HATCH AFTER EXPOSURE TO SEVERAL
                CONCENTRATIONS OF (TEST COMPOUND)
    Mean                                          Weight  (g)
  Measured              Total Length  (mm)        Replicate  (N)
Concentration             Mean + SD  (N)            Average
    (mg/L)      Tank    (Treatment Mean + SD)   (Treatment Average)
#


#


#


#


#


ter Control


•Ivent Control


#
#

#
#

#
#

#
#

#
#

f
#

*
#

# +
* +
(# T
t +
# +
(# +
f +
# +
(# +
# +
1 +
(t ±
# +
f +
(#±
f +
# +
(f 7
# +
# +
(# +
#
#
*)
#
f
f)
#
#
#)
*
#
f)
#
#
f)
#
f
#)
#
#
f)
# +
# +
(# +
# +
# +
<# ±
# +
# +
(#i
f +
# +
(# +
# +
# +
<*±
# +
* +
(f i
t +
# +
(t +
#
#
#)
#
#
f)
#
#
i)
#
#
t)
#
f
#)
#
#
f)
f
f
t)
a The mean length of these fish was/was not  significantly less
  than the mean length of fish in solvent control  group according
  to a (statistic name) test  (P < 0.05).

-------
                               -48-
                             TABLE 5

            LENGTH AND WEIGHT CF [TEST FISH NAME] AT #
            DAYS  POST-HATCH AFTER EXPOSURE TO SEVERAL
                CONCENTRATIONS OF (TEST COMPOUND)
    Mean                                            Weight  (g)
  Measured                Total Length  (mm)       Replicate (N)
Concentration               Mean + SD (N)           Average
    (mg/L)       Tank     (Treatment Mean + SD)  (Treatment Average)
# #
ft

# ft
#

# #
ft

# #
#

\
# #
#

Water Control #
f

Solvent Control ft
ft

ft
ft
(#
#
#
(ft
#
#
(ft
#
#
(#

#
#
(#
ft
#
(ft
#
ft
(ft
+ #
+ ft
T ft)
+ ft
+ ft
T ft)
+ ft
+ ft
7 ft)
+ ft
+ ft
± *)

+ ft
+ ft
±*>
+ ft
+ ft
± *)
+ ft
+ ft
+ ft)
(#)
(ft)

(ft)
(ft)

(ft)
(ft)

(ft)
(ft)


(ft)
(ft)

(ft)
(ft)

(ft)
(ft)

f +
ft +
(ft 7
ft +
ft +
(ft +
ft +
ft +
(ft 7
ft +
ft +
(ftT

ft +
ft +
(ft +
ft +
ft T
(ft +
ft +
ft +
(# +
#
ft
ft)
ft
ft
ft)
ft
ft
ft)
ft
ft
ft)

ft
ft
ft)
ft
ft
ft)
ft
ft
ft)
(#)
(ft)

(ft)
(ft)

(ft)
(ft)

(ft)
(ft)


(ft)
(ft)

(ft)
(ft)

(ft)
(ft)

a Mean lengths and mean weights of the groups of  fish exposed to
  (test compound) were/were not significantly different  than the
  mean length of fish in water and solvent control groups  accord-
  ing to a (statistic name) test  (       ).

-------
                          PESTICIDE ASSESSMENT GUIDELINES

                           WILDLIFE AND AQUATIC ORGANISMS

                          Aquatic Invertebrate Life-Cycle

                             Subdivision E, Series 72-4

                                   DATA REPORTING

GUIDELINE

      The following describes the order and format for a study report item by item.

      TITLE/COVER PAGE

      Refer to PR Notice 86-5.

      TABLE OF CONTENTS

      The Table of Contents should indicate the overall organization of the study,
      including tables and figures.  It must follow the title, data confidentiality,
      and GLP (if appropriate) pages as described in PR Notice 86-5.

I.     SUMMARY/INTRODUCTION

      A.  Sponsor:  (name of study owner);

      B.  Name:   (person(s) who can provide details of test procedures);

      C.  Location of study;

      D.  Location of raw data and final report;

      E.  Material:  (common/trade name);

      F.  Subject:  (final or draft report, invertebrate life-cycle, and species
          tested);

      G.  Test Doses:  (control, concurrent vehicle control, #, #, #, #, # ppm);

      H.  Test Dates:  (initiation, date test started, termination, and date of last
          day of observation);

      I.  Length of Study:  (days);

      J.  Results:  (MATC value in ppm and no-observed-effects level);

      K.  Tested Material:  (chemical name, formulation, and percent active
          ingredient);

      L.  Test Species/Strain:  (both common and scientific name);

-------
                                         -50-


       M.  Source of Organisms:  (company and address);

       N.  Age of Organisms at the Initiation of Study:   (hours).

II.     MATERIAL/METHODS

       This section is a narrative which would include the following items.

       A.  Test Substance

           1.   Identification (this information may be provided by the laboratory or
               sponsor):

               a.  Chemical name;
               b.  Composition (qualitative and quantitative description);
               c.  Percent active  ingredient;
               d.  Molecular structure;
               e.  Source, lot number, or code;
               f.  Identity and percent  composition of impurities  in the test
                   substance.

           2.   Preparation of  test solution (control/treatment):

               a.  Any vehicle used to dissolve test material;
               b.  Amount of vehicle added to control (if used);
               c.  Description of  method used to get test material into solution;
               d.  Water source (reconstituted - if so,  how prepared);
               e.  Solubility in ppm;
               .f.  Total  amount of test  material used;

       B.  Test Invertebrate

           1.   Rationale for selection of species if the species used is
               different  than that preferred in Subdivision E:

               a.  Test species name (both scientific and common);
               b.  History of  test organisms (strain, diseases, and treatment).

           2.   Description of  any  pretest conditioning:

               a.  Health:

                   1.   Sickness;
                   2.   Injuries;
                   3.   Abnormalities;
                   4.   Mortalities (%);
                   5.   Pretest diet.

           3.   Size/age/physical condition:

               a.  Age at initiation of  test (in hours);
               b.  Date spawned;
               c.  List of any abnormalities.

-------
                                  -51-
    4.  Source/acclimation:

        a.  Complete name and address of test species supplier;
        b.  Source of food and dilution water;
        c.  Size of test container [in milliliters (mL)];
        d.  Temperature, pH, and dissolved oxygen;
        e.  Percent mortality;
        f.  Feeding schedule;
        g.  Test organisms from the same source (yes or no);
        h.  Number of days adults were held in laboratory prior to use of
            offspring.

C.  Method                                          Materials Belong To-     " ""*
                                                    O.PPT Lr-n-y    '  '
    1.  Test Vessels:                               4C"!. V H-t:-.r.-.-.i.' P-T (\*'z.rf?i\
        a.  Material type;
        b.  Volume [in milliliters (mL)];
        c.  Volume of test solution [in centimeters (cm)];
        d.  Size of vessel [in milliliters (mL)].

    2.  Test system:

        a.  Source of dilution water;
        b.  If flow-through, description of system and flow rate/day;
        c.  Procedures used to prepare toxicant stock solution;
        d.  Criteria used to determine effects.

    3.  Test design:

        a.  Method used in assigning test organisms to test and control
            groups;
        b.  Number of organisms per dose level;
        c.  Name of protocol followed during this test;
        d.  Loading (number of organisms per unit volume of water);
        e.  Number of treatment levels used (nominal or measured);
        f.  Length of exposure period;
        g.  Type of control (positive, negative, or solvent control);
        h.  Temperature;
        i.  pH (When checked - see table 1);
        j.  Lighting (time in hours and intensity in footcandles);
        k.  Dissolved oxygen (see table 1);
        1.  Water hardness (expressed in mg/L as CaCC>3 - see table  1);
        m.  Alkalinity (expressed in mg/L as CaCC>3 - see table 1);
        n.  Conductivity (umhos/cm - see table 1);
        o.  Range finding test results, concentrations, and mortality  (if
            used);
        p.  Aeration (yes or no);
        q.  Water physical characteristic at the end of test:
            1.  Water depth and volume;
            2.  Temperature, pH, and dissolved oxygen;
        r.  Number of normal organisms at the end of test  (see table 3);
        s.  Concentration analysis (see table 1);

-------
                                          -52-


                t.  Examination of invertebrate physiology,  locomotion, behavior,  and
                    pathology;
                u.  Organisms count/measurement (see table 3);
                v.  Mortalities during the test;
                x.  If static system was used, how often test solution renewed.

        D.  Statistical Analysis

            1.  Cite references (author, title, journal, number, page, etc.).

III.    DISCUSSION AND RESULTS

        A.  Provide MATC value in ppm (graphs, printouts, and other calculations
            should be attached to report).  Provide raw mortality data (see table 3).

        B.  Discuss the relationship, if any,  between the physical factors, toxicant,
            and observations.

        C.  Provide statistical method used (cite references, author, etc.).

        D.  Observation - Provide the following data:

            1.  Detailed record of spawning, fertility, and fecundity;

            2.  Signs of intoxication - temporal onset and duration;

            3.  The no-observed-effect level and reproductive effects.

 IV.    CONCLUSIONS

        This is a summation of the above section - Discussion/Results.

-------
                              -53-
                            TABLE 1

             WATER QUALITY CHARACTERISTICS MEASURED
              DURING EXPOSURE OF (TEST ORGANISMS)
                       TO (TEST COMPOUND)
Water Quality
Characteristic
Mean + SD
Range
 Number of
Measurements
Temperature (°C)

Dissolved Oxygen
     (mg/L)

PH

Total Hardness
(mg/L as CaC03)

Total Alkalinity
(mg/L as CaCO3)

Specific Conductance
   (umhos/cm)

-------
                               -54-


                             TABLE 2

               PERCENT MORTALITIES AND MATC VALUES
Client       :
Test Material:
Test Organism:
Test Water   :
                        PERCENT MORTALITY

               TEST MATERIAL NOMINAL CONCENTRATION
                              (mg/L)
         Control
 Day #    #(#/#)    #(#/#)   #(#/#)    #(#/#)    #(#/#)   #(#/#)
 Day #    #(#/#)    #(#/#)   #(#/#)    #(#/#)    #(#/#)   #(#/#)
                             Day #
                                             Day #
 Chronic Results
 MATC (mg/L)

 No effect level
 95%
 Confidence
 Limits
             Low
             High
Observed Effects

a floating
k swimming near surface
c lying on bottom
                                        ^ turned dark
                                        e etc.
 £  How the percent of mortality table works.

 1.   f(#/#)a.
 2.   # = the total number of organisms tested per dose level.
 3.   #(#/#) = % mortality [No. dead/No, treated, e.g., 10% (1/10)]
 4.   a = the observed effects.

-------
                           -55-


                         TABLE 3

              INVERTEBRATE MEASUREMENT/COUNT
                                     I OF
CONCENTRATIONS    t OF ADULTS   TOUNG/ALIVE/DEAD   LENGTH

     #               #           #     #     #       #
     #               #           #     f     #       #
     #               *           #     #     f       #
CONTROLS

     #               #           #     #     #      #
     #               #           *     #     #      f

-------
                                        -56-


                          PESTICIDE ASSESSMENT GUIDELINES

                           WILDLIFE AND AQUATIC ORGANISMS

                             Life-Cycle Tests for Fish

                             Subdivision E, Series 72-5

                                   DATA REPORTING

GUIDELINE

      The following describes the order and format for a study report item by item.

      TITLE/COVER PAGE

      Refer to PR Notice 86-5.

      TABLE OF CONTENTS

      The Table of Contents should indicate the overall organization of the study,
      including tables and figures.  It must follow the title, data confidentiality»
      and GLP (if appropriate) pages as described in PR Notice 86-5.

I.    SUMMARY/INTRODUCTION

      A.  Sponsor:  (name of study owner);

      B.  Name: (person(s) who can provide details of test procedures);

      C.  Location of study;

      D.  Location of raw data and final report;

      E.  Material:  (common/trade name);

      F.  Subject:  (final or draft report, fish life-cycle, and species tested);

      G.  Test Doses:  (control, concurrent vehicle control, #, #, #, #, # ppm);

      H.  Test Dates:  (initiation, date test started, termination, and date of
          last day of observation);

      I.  Length of Study:  (days);

      J.  Results:  (MATC value and no-observed-effects level);

      K.  Tested Material:  (chemical name, formulation, and percent active
          ingredient);

      L.  Test Species/Strain:  (both common and scientific name);

      M.  Source of Organisms:  (company and address);

-------
                                         -57-


       N.  Stage of Organisms at the Initiation of Study:   (hours).

11.     MATERIAL/METHODS

       This section is a narrative,  which would include  the following Items.

       A.  Test Substance

           1.   Identification (this  information may be provided by the  laboratory or
               sponsor):

               a.   Chemical name?
               b.   Composition (qualitative and quantitative description);
               c.   Percent active  ingredient;
               d.   Molecular structure;
               e.   Source, lot number, or code;
               f.   Identity and percent  composition of impurities  in the test
                   substance.

           2.   Preparation of  test solution (control/treatment):

               a.   Any vehicle used  to dissolve test material;
               b.   Amount of vehicle added to control  (if  used);
               c.   Description of  method used to get test  material into solution;
               d.   Water source (reconstituted - if so,  how prepared);
               e.   Solubility of test material in ppm;
               f.   Total amount of test  material used.

       B.   Test Fish

           1.   Rationale for selection of species if the species used is different
               than that preferred in Subdivision E (if  eggs were  not used,  give
               stage of life in which organisms were tested):

               a.   Test species name (both scientific  and  cannon);
               b.   History of  test organisms (strain,  diseases, and  treatment).

           2.   Description of  any  pretest conditioning:

               a.   Health:

                   1.   Sickness;
                   2.   Injuries;
                   3.   Abnormalities;
                   4.   Pretest diet;
                   5.   Number  of days brood fish were  quarantined.

           3.   Size/age/physical condition:

               a.   Age of unfertilized eggs and milt  (in hours);
               b.   Description of  the procedures used  to strip the eggs and milt;
               c.   Description of  the procedures used  in handling  and storage of
                   eggs;
               d.   Description of  the fertilization procedures.

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                                  -58-
    4.  Source/acclimation:

       . a.  Complete name and address of test species supplier;
        b.  Source of food and dilution water;
        c.  Size of test container [in liters (L)];
        d.  Number of females used per batch;
        e.  Temperature, pH, dissolved oxygen, and name of equipment used to
            measure water quality;
        f.  Description of methods used initially to determine different
            stages of development;
        g.  Percent mortality (brood fish);
        h.  Feeding schedule, holding, and acclimation period;
        i.  Test organisms from the same source (yes or no);
        j.  Number of males used per batch of eggs.
C.  Method
    1.  Test vessels:

        a.  Material type;
        b.  Volume [in milliliters (mL)];
        c.  Volume of test solution [in milliliters (mL)];
        d.  Size [in centimeters (cm)].

    2.  Test system:

        a.  Source of dilution water;
        b.  If flow-through, description of system construction material,
            system, and flow rate/day;
        c.  Procedures used to prepare toxicant stock solution;
        d.  Criteria used to determine effects.

    3.  Test design:

        a.  Method used in assigning test organisms to test and control
            groups and number of replicates used;
        b.  Number of eggs per dose level;
        c.  Name of protocol followed during this test;
        d.  Loading (number of eggs per unit volume of water - if known);
        e.  Number of treatment levels used (nominal or measured);
        f.  Length of exposure period;
        g.  Type of control (positive, negative, or solvent control);
        h.  Temperature;
        i.  pH (when checked - see table 2);
        j.  Lighting (time in hours and intensity in footcandles);
        k.  Dissolved oxygen (see table 2);
        1.  Water hardness (expressed in mg/L as CaC03 - see table 2);
        m.  Alkalinity (expressed in mg/L as CaCO3);
        n.  Conductivity (umhos/cm - see table 2);
        o.  Range finding test results, concentrations, and mortality  (if
            used);
        p.  Aeration (yes or no);

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                                          -59-


                q.  Water physical characteristic at the end of test:
                    1.  Water depth and volume;
                    2.  Temperature, pH, and dissolved oxygen;
                r.  Period food was withheld prior to termination;
                s.  Number of normal fry at the end of test (see tables" 3a and 3b);
                t.  Concentration analysis (see table 1);
                u.  Examination of fish physiology, locomotion, and behavior;
                v.  Organisms length and weight (see table 5);
                w.  Mortalities and effects during the test;
                x.  If static system was used, how often test solution renewed;
                y.  Fry survival, growth, and deformities.

        D.  Statistical Analysis

            1.  Cite references (author, title, journal, number, page, etc.).

III.     DISCUSSION AND RESULTS

        A.  Provide maximum acceptable toxic concentration (MATC) expressed as the
            range between the lowest observed effect concentration and the highest
            observed no effect concentration in ppm.

        B.  Discuss the relationship, if any, between the physical factors, toxicant,
            and observation.

        C.  Provide statistical method used (cite references, author, etc.)

 IV.     CONCLUSIONS

        This is a summation of the above section - Discussion/Results.

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                           -60-
                         TABIE 1

        MEASURED CONCENTRATION OF TEST COMPOUND IN
    WATER TO WHICH  (Test fish name)  (Scientific Name)
                 WERE EXPOSED FOR #  DAYS
   Nominal
Concentration
  (mg/L)
                Mean + SD
                Range
Number of
Samples
                     +

                     T
Water Control
"	" (Detected) "	"
Solvent Control
    Detected or not

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                              -61-
                            TABLE 2

             WATER QUALITY" CHARACTERISTICS MEASURED
                 DURING EXPOSURE OF  (TEST FISH)
             EMBRYOS AND LARVAE TO (TEST COMPOUND)
 Water Quality                                      Number of
Characteristic       Mean + SD          Range      Measurements
Temperature  (eC)      "  +

Dissolved Oxygen      "  +
    (mg/L)

pH

Total Hardness        "  +
(mg/L as CaC03)

Total Alkalinity      "  +
(mg/L as CaCO3)

Specific Conductance  "  +
   (umhos/cm)

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                          -62-
                     TABLE 3a
SURVIVAL OF (TEST FISH NAME) AT HATCH (DAY #), AT # DAYS
   POST-HATCH, AT # DAYS POST-HATCH AND SWIM-UP AFTER  -
 EXPOSURE TO SEVERAL CONCENTRATIONS OF (TEST COMPOUND)
Mean
Measured
Concentration
(mg/L) Tank
ft *
ft
# *
#
# *
#
# *
#
# *
#
Water Control ft
t
Solvent ft
Control #
Percent Percent Percent
Larvae Survival Survival
Survival At # Days At # Days
At Hatch on Post-Hatch Post-Hatch
Day # (Mean) (Mean) (Mean)
#
#
#
#
#
#
ft
#
I
ft
#
#
ft
#
# *
#
# *
ft
# *
ft
t *
#
t #
ft
# *
#
t *
ft
# # #
#
# # #
ft
# # #
ft
# * #
#
# # #
ft
ft * ft
ft
ft # ft
#
Percent
At Swim-up
At # Days
Post-Survival
(Mean)
#
ft
#
#
#
ft
#
#
#
#
#
#
#
ft
ft

#

ft

#

#

ft

#


-------
                          -63-
                     TABLE 3b (F2)

SURVIVAL OF (TEST FISH NAME) AT HATCH  (DAY #), AT # DAYS
   POST-HATCH, AT + DAYS POST-HATCH AND SWIM-UP AFTER
 EXPOSURE TO SEVERAL CONCENTRATIONS OF (TEST COMPOUND)
Mean
Measured
Concent rat ion
(mg/L) Tank
#

#

#

#

#

Water Control

Solvent
Control
#
#
#
#
#
#
#
#
#
#
#
#
#
#
Percent Percent
Larvae Survival
Survival At # Days
At Hatch on Post-Hatch
Day # (Mean) (Mean)
#
#
#
#
#
*
#
#
#
#
#
#
#
#
# # #
#
* # #
#
# # #
ft
1 # t
#
# # f
#
f # #
#
# # 1
#
Percent Percent
Survival At Swim-up
At # Days At # Days
Post-Hatch Post-Survival
(Mean) (Mean)
* #
#
* #
#
* #
f
# #
f
* #
#
* #
#
* #
f
#
#
#
#
f
#
#
#
#
#
#
#
#
#
#

#

#

#

#

#

#


-------
                               -64-
                             TABLE 4

             LENGTH AND WEIGHT CF  [TEST FISH NAME] AT  #
             DAYS  POST-HATCH AFTER EXPOSURE  TO SEVERAL
                CONCENTRATIONS CF (TEST COMPOUND)
    Mean                                          Weight  (g)
  Measured               Total Length  (mm)       Replicate  (N)
Concentration              Mean + SD (N)           Average
  (mg/L)         Tank   (Treatment Mean + SD)   (Treatment  Average)
#


#


ft


#


#


Water Control


Solvent Control


#
#

#
ft

ft
#

ft
#

#
ft

ft
ft

ft
ft

# +
# +
(# T
f +
ft +
(ft +
ft +
# +
(# +
ft +
ft +
(ft +
ft +
ft +
(ft +
ft +
ft +
(ft +
ft +
ft +
(ft +
#
ft
ft)
ft
ft
ft)
ft
ft
ft)
ft
ft
ft)
ft
ft
ft)
ft
ft
ft)
ft
ft
ft)
# +
# +
ft +
ft +
ft +
(ft +
ft +
ft T
(ft +
ft +
ft +
(ft +
ft +
ft +
(ft ?
ft +
ft +
(ft +
ft +
ft +
(ft +
#
ft
ft
ft
ft
ft)
ft
ft
ft)
ft
ft
ft)
ft
ft
ft)
ft
ft
ft)
ft
ft
ft)
a The mean length of these fish was/was not  significantly less
than the mean length of fish in solvent control group according
to a (statistic name) test (P < 0.05).

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                               -65-
                             TABLE 5

            LENGTH AND WEIGHT CF [TEST FISH NAME] AT f
            DAYS POST-HATCH AFTER EXPOSURE TO SEVERAL
                CONCENTRATIONS OF (TEST COMPOUND)
Mean
Measured
Concentration
(mg/L) Tank
f


#


#


#


#


ter Control


ilvent Control


#
#

#
#

#
#

f
#

#
f

f
#

#
#

Total Length (mm)
Mean + SD (N)
(Treatment Mean + SD)
# + #
f + #
(# ± #)
# + #
# + #
(# ± #)
# + t
f + #
(# ±#)
# + #
# + #
(# + #)
t + #
t + f
(# + f)
# + #
# + #
(# + #)
# + #
# + #
(# + t)
(#)
(#)

(#)
(#)

(#)
(#)

(#)
(#)

(#)
(#)

(#)
(#)

(#)
(#)

Weight (g)
Replicate (N)
Average
(Treatment Average)
# + #
t + #
(# ± #)
# + #
# + t
(# + #
# + #
# + f
(# ±f)
f + #
# +
(# + # )
# + #
# + #
(t ±# )
t + 1
# + #
(#±D
# + #
# + 1
(# + #)
(#)
(#)

(#)
(#)

(#)
(#)

(t)
(#)

(#)
(#)

(t)
(t)

(t)
(#)

a The mean length of these fish was/was not significantly less
than the mean length of fish in solvent control group according
to a (statistic name) test (P < 0.05).

-------