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
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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
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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.
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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...
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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.
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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.
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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.
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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
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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;
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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
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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
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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.
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