SUBDIVISION N


                 ENVIRDNMENTAL FATE

                    Series 165-4

Laboratory Studies of Pesticide Accumulation in Fish

                    Prepared by:

                 E. Brinson Conerly
                 Samuel M. Creeger
             Exposure Assessment Branch
             Hazard Evaluation Division
            Office of Pesticide Programs
                  Project Manager:

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

Subdivision N - Laboratory Studies of Pesticide Accumulation in Fish

                   Table of Contents of Addendum

   Introduction                                                2

   Response to Public Comments                                 2


          Title/Cover Page                                     7

          Table of Contents                                    7

      I.  Abstract                                             7

     II.  Introduction                                         7

    III.  Materials/Methods                                    7

     IV.  Results/Discussion                                   9

      V.  Conclusion                                          10

     VI.  Certification                                       10

    VII.  Tables/Figures                                      10

   VIII.  References                                          10

     IX.  Appendix(es)                                        10


                               ENVIRONMENTAL FATE

              Laboratory Studies of Pesticide Accumulation in Fish

                          Subdivision N, Series 165-4

                                 DATA REPORTING


     A.   Purpose

          The laboratory study of pesticide accumulation in fish is used by the
          Agency to determine if pesticide residues accumulate in fish used as
          human food sources, and to determine the extent of the pesticide
          residues in edible portions of these fish.  These data are used to
          establish label restrictions, or tolerance levels, or action levels
          when necessary.

     B.   Objective

          This Data Reporting Guideline (DRG) is provided to aid the petitioner/
          registrant in generating reports which are compatible with the Agency's
          review process.  Data subnitters are encouraged to use it to produce
          complete reports for efficient review by the Agency.  It pertains to
          organizing and presenting the substance of the data report.  PR
          Notice 86-5, effective on November 1, 1986, available from the Office
          of Pesticide Programs, US EPA, pertains to physical formatting of
          reports (which are referred to as "studies") and submittal packages.
          Some of its requirements are mandatory.


     A common comment was that the Data Submission Guidelines are being used as
a mechanism to impose new data requirements.  This document does not impose new
data requirements but mentions the types of information submitted by registrants
in the past with their fish bioaccumulation studies.  The purpose of this document
is to recommend the organizational format of that information.  If the registrant
feels some of the information will not aid in the efficient review of the study,
then he may decide not include it with the study.  Conversely, if the registrant
feels the information will aid in the efficient review of the study, then it
would be advantageous to include it with the study based on this recommended

     After considering all of the comments, it was obvious that many commenters
do not understand how some information impacts heavily on the efficient review
of a study.  Therefore, in addressing the comments, an attempt has been made to
explain how the information does aid in the efficient review of a study and that
it will be in the registrant's interest to include that material with the study.

Conments are addressed below:

    1.  Comment;

        BCF's (Bio-Concentration Factors) for fillet and whole fish are the
        most relevant numbers.  Measuring and reporting BCF's for the inedible
        portion should be optional.  The inedible portion is not usually an
        area of concern„  It can be accounted for by the whole fish value minus
        the edible value.


        The BCF figure for the inedible portion may be a useful validation of
        the other two figures and should be included.

    2.  Comment;

        It is inappropriate to calculate BCF value for any metabolites formed.
        The definition of BCF is Cfish/Cwater.  The fish are not exposed to the
        metabolites, but to the parent compound.  The metabolites are a result
        of the exposure to the parent compound, not direct uptake from water.
        They should only be expressed as a definite quantity and/or a percent
        of total residue.


        The sentence has been clarified.  The applicant should provide both
        figures, quantity and percentage for metabolites.

    3.  Comment;

        EPA should define the term "edible"... It is suggested that they use
        the definition...used by FDA in its Pesticide Analytical Manual,
        Volume I.


        "Edible" varies with the species, as noted by the Agency.  The registrant
        should state in the study which portions are considered edible for the
        specific test fish used.  The PAM definition is an acceptable one.

    4.  Comment;

        Name and phone number of contact person would be better placed on cover


        This information is optional.  The applicant may place it on the cover

    5.  Comment;

        [The ABSTRACT] section might be better termed SUMMARY...


    In the interest of uniformity in the Data Reporting Guidelines for
    Subdivision N, the Agency prefers the term "ABSTRACT."

6.  Comment;

    The units for describing the activity of radiolabeled material is
    stipulated as cures [sic]/mole and disintegrations per minute per gram
    (dpm/g).  The practice of most specialists has been to express activity
    as millicuries per millimole (mCi/famol) and disintegrations per minute
    (dpm).  We, therefore, suggest the language in this section...accomodate
    this usual practice.


    The applicants may use these units if they so desire, however, units
    should be clearly specified and used consistently throughout the report.

7.  Comment:

    There should be no need to state the actual dates samples of fish were
    sampled, frozen, thawed, and analyzed.  It should be adequate to state
    that fish were sampled, frozen, and analyzed within a given number of
    days.  Specific calender dates the fish were sampled, frozen, thawed,
    and analyzed would be in the original data.


    We agree.

8.  Comment;

    The Agency should clarify what type of storage stability data is
    acceptable.  Since the nature of the residue is not known, the registrant
    can only identify chromatographic patterns of metabolites as soon as
    possible after sacrifice.  By reanalysis at the end of the study, some
    demonstration of storage stability can be made.  It would be helpful to
    know if this, or other methods, would be acceptable to address the
    stability question.  This points again to the fact that the issuance of
    a DRG before a Standard Evaluation Procedure (SEP) raises questions
    which should be settled if the reverse sequence were followed.


    The ccmmenter has suggested one valid approach.  "Field spiking" is
    another possibility.  The Agency cannot dictate a priori what stability
    data might be appropriate in a specific case.  An applicant may at any
    time consult with the Agency as to the data which might satisfy this


 9.  Comment;

     [Storage stability] is a new data requirement.  A primary purpose in a
     fish accumulation study is to isolate and identify metabolites, not
     monitor their stability.


     This document imposes no new data requirements.  We agree that storage
     stability is not the prime purpose of this study.  However, for proper
     evaluation of study results, it is necessary to have a good understanding
     of the behavior of the test chemical and degradates.

10.  Comment;

     The implication [in Test Method D. 1. g.]  is that retention of samples
     need not be discussed if the registrant plans no further work.
     Clarification of this would be helpful.


     An appropriate clarification has been made in the text.

11.  Comment;

     This request for method validation recovery and sensitivity data,
     quality control procedure and results, would appear to relate to unlabeled
     (i.e. "cold") methods of analysis involving GLC or HPLC.  If so, it
     should be so stated.


     Appropriate method validation data would relate either to "cold" or "hot"
     methods.  The applicant should provide sufficient evidence to demonstrate
     that whatever analytical results are reported are reliable.

12.  Comment;

     Likewise, the inclusion of a "material balance" is not clearly under-
     stood and needs further clarification.


     Material balance, or "Recovery of Applied Material," allows the reviewer
     to assess whether the starting material is properly accounted for or
     was mostly lost during the experiment.

13.  Comment;

     Radiolabeled material balances are normally provided in the RESULTS/
     DISCUSSION Section.  Hence, it would not be appropriate to include it
     in the Analytical Method section.


     It may be placed in RESULTS.

14.  Comment t

     "Figures/photographs of specialized equipment used" is a reasonable
     requirement.  "Figures/photographs of the equipment used" is an excessive


     We agree.

15.  Comment;

     Flow diagram can be adequately placed in Section [TABLES/FIGURES].
     Inclusion [in Section III, under Analytical Method(s)] should be


     The applicant may place it in Section VII if desired.

16.  Comment;

     The request to place tables, figures and graphics in  [the RESULTS/
     DISCUSSION section] is confusing.  A separate Section [TABLES/FIGURES]
     has been set aside for this data.  Clarification is needed.


     Short tables, figures, and graphics may be incorporated into the
     RESULTS/DISCUSSION section.  Lengthy material should be placed in the
     separate Section so designated.

17.  Comment;

     ...detailed information on fish weight, graphic representations of results
     would be better placed in Section [TABLES/FIGURES].


     See above response.

18.  Comment;

     Summaries of structures/chemical names are located in Section III.  To
     include them in Section IV would be redundant.


     This material need only be furnished once in any given study.



       Title page and additional documentation requirements (i.e., requirements for
       data submission and statement of data confidentiality claims), if relevant
       to the study report, must precede the content of the study formatted below.
       These requirements are described in PR Notice 86-5.


       This page should indicate the overall organization of the study, including
       tables and figures.  The Table of Contents must follow the title, data con-
       fidentiality, and GLP (if appropriate) pages.


       This section should contain the overall summary of the study addressing
       the following points:

       A.   A brief summary of exposure conditions and intervals;

       B.   A brief summary of observations such as fish behavior or mortality
            during the study;

       C.   A concise statement of results obtained, including the bioconcen-
            tration factors for edible, inedible, and whole fish samples for parent
            compound; and concentrations of significant metabolites.  A definition
            of "edible" should be included here, since it varies from species to

       D.   A discussion of unexpected problems (such as technical difficulties
            or unusual fish behavior) resulting in necessary deviations from the
            intended test protocol, and a description of the effects of these
            deviations on the results of the study; and

       E.   Provide a name and a phone number of a contact person in case the
            reviewer has technical questions about the study.  [This is optional.
            Provision of this information will facilitate efficient review in
            case of questions.]


       This section should open with a description of the purpose of the study,
       what requirement it is intended to satisfy and (if applicable) how it
       supports the position of the registrant.  Background and historical
       information relative to the study should be placed in this section.


       The applicant may elect to describe materials and methods in separate
       sections or combine into a single section.  In this DRG, they are

combined into one section.

This section should be in narrative form.  All details with regard to
materials, equipment, experimental design, test animals and containers,
procedures used in conducting the study, and drawings and photographs of
the experimental specialized equipment should be placed in this section.
In addition, the following are to be included, when appropriate:

A.   Chemical

     1.   If using radiolabeled material, provide the purity of the material,
          its activity in Curies/mole or other standard units, disintegrations
          per minute per gram (dpm/g), and the site of radiolabeling;

     2.   Provide the chemical purity of any non-radiolabelled material
          and state method of analysis if appropriate; and

     3.   The means of delivery of the test chemical, including any special
          apparatus required.

B.   Water

     Describe the source, if appropriate, and give results of analyses for
     characteristics including pH, hardness, pesticidal contaminants,
     chlorine, dissolved oxygen, dissolved salts, temperature, etc.

C.   Test Fish

     A description including such items as:

          1.   Species;
          2.   Source;
          3.   Age;
          4.   Size and weight before and after the study;
          5.   How acclimated before initiation of the study; and
          6.   How they were maintained, including type and amount of food
               and feeding intervals.

D.   Test Method

     1.   General

          a.   The technique of aquarium preparation prior to pesticide
               exposure of the fish, including set-up and equilibration;

          b.   A description of the exposure method and protocol;

          c.   A description of the depuration conditions and protocol;

          d.   A description of the sampling protocol, for both fish and

          e.   A description of the methods used to observe behavior and

                f.   A description of the handling of the fish and water samples/
                     from the tine of taking of the samples until analysis, with
                     special attention to the conditions under which they were
                     stored and the thawing procedure (if frozen); in addition,
                     the storage stability data to be used in determining if the
                     pesticide residues are stable under the storage conditions;
                     length of tiine between taking of samples and analysis;

                g.   Information on how long samples will be retained and the
                     conditions under which they will be retained (in the event
                     additional analytical work is necessary); and

                h.   An elaboration on any difficulties or special problems that
                     arose during the study which necessitated deviation from the
                     intended test protocol and on the effects the deviations
                     had on the results.

           2.   Analytical Method(s)

                a.   The full description of each method used in this study
                     should be placed in this section.  Note that methods for
                     degradation products, when appropriate, are included.  This
                     section should also include the method validation data,
                     recovery and limit of detection data, quality control
                     procedures and results, sample chromatograms, sample calcu-
                     lations and a material balance.  The detailed description
                     of the procedures used in preparation and handling of the
                     sample throughout the method should also be placed here.

                b.   The identity of the instrumentation, equipment and reagents
                     used and the operating conditions of the instrumentation; and

                c.   Figures/photographs of any special or unusual equipment
                     used in the methods; and flow diagrams of particularly
                     complex extraction/clean-up procedures are to be included


      A.   This section should contain the scientific results of the study.
           Narrative and tables describing the steps taken in determining the
           pesticide residues in the fish and water samples should be presented

      B.   Results of the observations of behavior and mortality during the
           acelimatation, study, and post-study periods;

      C.   The results of analyses of all fish and water samples, including
           separate analyses of edible portions, viscera and whole fish for
           parent compound and significant degradates should be included here.
           Graphical presentations of the data (accompanied by the tables of
           actual values from which the graphs were constructed) may be
           appropriate.  Proportional or actual weights of the edible and viscera


             portions and whole fish should be included; and

        D.   This section should contain the table of structures and chemical names/
             designations for the parent compound and degradation products discussed
             in the study.

        This section should contain the discussion of the degree and significance of
        the residues of parent and degradate residues found in the test animals.


        A.   Signatures of each of the senior scientific personnel responsible for
             the study; and

        B.   Certification by the applicant that the report is a complete and
             unaltered copy of the report provided by the testing facility
             (except for title page changes required by PR Notice 86-5).


        It is recommended that tables and figures be numbered using arabic numerals
        for figures and reman numerals for tables.



        At the registrant's option, reprints of methods and other studies/ raw
        data, copies of relevant letters/memos and other material which will help
        support the registrant's conclusions, but do not fit in any of the other
        sections, should be placed in an appendix.