540/09-88-051 PESTICIDE ASSESSMENT GUIDELINES SUBDIVISION N CHEMISTRY: ENVIRDNMENTAL FATE Series 165-4 Laboratory Studies of Pesticide Accumulation in Fish ADDENDUM 8 ON DATA REPORTING Prepared by: E. Brinson Conerly and 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 Discussion Introduction 2 Response to Public Comments 2 Guideline 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 -1- ------- PESTICIDE ASSESSMENT GUIDELINES ENVIRONMENTAL FATE Laboratory Studies of Pesticide Accumulation in Fish Subdivision N, Series 165-4 DATA REPORTING INTRODUCTION 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. RESPONSE TO PUBLIC COMMENTS 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 format. 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. -2- ------- -3- 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. Response; 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. Response; 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. Response; "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 page. Response; This information is optional. The applicant may place it on the cover page. 5. Comment; [The ABSTRACT] section might be better termed SUMMARY... ------- -4- Response; 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. Response; 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. Response; 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. Response; 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 request. ------- -5- 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. Response; 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. Response; 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. Response; 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. Response; 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. ------- -6- Response; 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 requirement. Response; We agree. 15. Comment; Flow diagram can be adequately placed in Section [TABLES/FIGURES]. Inclusion [in Section III, under Analytical Method(s)] should be optional. Response; 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. Response; 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]. Response; See above response. 18. Comment; Summaries of structures/chemical names are located in Section III. To include them in Section IV would be redundant. Response; This material need only be furnished once in any given study. ------- -7- GUIDELINE TITLE/COVER PAGE 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. TABLE OF CONTENTS 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. I. ABSTRACT 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 species; 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.] II. INTRODUCTION 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. III. MATERIALS/METHODS The applicant may elect to describe materials and methods in separate sections or combine into a single section. In this DRG, they are ------- -8- 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 water; e. A description of the methods used to observe behavior and mortality; ------- -9- 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 here. IV. RESULTS/DISCUSSION 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 here; 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 ------- -10- 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. V. CONCLUSION ! This section should contain the discussion of the degree and significance of the residues of parent and degradate residues found in the test animals. VI. CERTIFICATION 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). VII. TABLES/FIGURES It is recommended that tables and figures be numbered using arabic numerals for figures and reman numerals for tables. VIII. REFERENCES IX. APPENDIX(ES) 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. ------- |