United States       Prevention, Pesticides     EPA712-C-95-188
          Environmental Protection    and Toxic Substances     August 1995
          Agency         (7101)
&EPA    Residue Chemistry
          Test Guidelines
          OPPTS 860.1850
          Confined Accumulation
          in Rotational Crops
                'Public Draft"

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                           INTRODUCTION
     This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.

     The Office of Prevention,  Pesticides and Toxic Substances (OPPTS)
has  developed this guideline through a  process of harmonization that
blended the testing  guidance and requirements that existed in the  Office
of Pollution Prevention and Toxics  (OPPT) and appeared in Title 40,
Chapter I,  Subchapter R of the Code of Federal Regulations  (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical  Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).

     The purpose of harmonizing these guidelines into a single  set of
OPPTS  guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic  Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide,  Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).

     Public Draft Access Information: This draft guideline is part of a
series of related harmonized guidelines that  need to  be considered as a
unit. For copies: These guidelines are available electronically from the
EPA Public Access Gopher (gopher.epa.gov) under the heading "Environ-
mental Test Methods and Guidelines" or in paper by contacting the OPP
Public    Docket    at    (703)    305-5805    or    by    e-mail:
guidelines@epamail.epa.gov.

     To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs, Field  Operations Division (7506C), Environmental
Protection Agency,  401  M  St.  SW.,  Washington, DC 20460. In  person:
bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Ar-
lington, VA. Comments may also be submitted  electronically by sending
electronic mail (e-mail) to: guidelines@epamail.epa.gov.

     Final  Guideline Release:  This document is available  from the U.S.
Government Printing Office,  Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet:  federal.bbs.gpo.gov
3001, or call 202-512-1530 for disks or  paper  copies.  This guideline is
available in ASCII and PDF (portable document format).

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OPPTS 860.1850 Confined accumulation in rotational crops.
     (a) Scope.

     (1) Applicability. This guideline is intended to meet testing require-
ments  of both the Federal Insecticide,  Fungicide,  and Rodenticide Act
(FIFRA) (7 U.S.C. 136, et seq.) and the Federal Food, Drug, and Cosmetic
Act(21U.S.C. 301, etseq.)

     (2) Background. The source material used in developing this har-
monized OPPTS test guideline is the OPP guideline 165-1. This  OPPTS
guideline  should be used in conjunction with OPPTS guideline 860.1000,
Background,  which provides general information and overall guidance for
the 860 series on Residue Chemistry.

     (b) Purpose.  Data from confined accumulation studies on rotational
crops will enable the Agency to determine the nature and amount of pes-
ticide residue uptake in rotational crops. Such data are used to establish
realistic crop  rotation restrictions (time  from application to a time  when
crops can be rotated) or to provide information  for determining if limited
field trials are needed for rotational crops.

     (1) Historical.

Studies on confined rotational  crops are conditionally  required under 40
CFR 158  for uses  of pesticides on terrestrial food crops and aquatic food
crops.  A rotational crop use is any field-vegetable  crop use, aquatic crop
use or any other site use on which it is reasonably foreseeable that any
food or feed crop  may be produced after application of a  pesticide. The
purpose of these studies is to determine the nature and amount of pesticide
residue uptake into rotational crops. The confined  study uses radioactive
material applied to a small plot.  Results of these ' 'hot'' studies are used
to determine  whether field studies (using non-radioactive  pesticide)  are
needed. Based on these data appropriate crop rotation restrictions  (time
from application to  planting of rotational crop) may be established and
the need for tolerances on the rotated crops determined.

     (2) Scientific  considerations.

Confined  rotational crop studies are essentially metabolism  studies;  there-
fore, guideline 860.1300, Nature of the residue  plants, livestock, should
be consulted before conducting a confined rotational crop study. (It should
be noted that, in the case of confined rotational crop studies, the applica-
tion rate is IX, not an exaggerated rate.) This section will refer frequently
to that document when discussing  various phases  of the subject experi-
ments. A flow diagram describing the approach discussed in this guideline
is provided in Figure 1.

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   Figure 1.
                   Pesticide is applied to crept that can be rotated.
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                              14-C Study-1x Application Rate
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(12 months max).
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No tolerances required. A
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may be needed.
                                                 £0.01 ppm In craps after 12 month
                                                 plant-bat* (a shorter Interval»
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                                                Characterize/Identify
                                                (See QukMne BS0.1300).
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                                    Un*ed Held TrWe (Cold)
                 Residues of concern
                 at QuanWacw levels.
                       Extended Field TrUs
                 Tolerances Required
                (See Guideline 880.1600).
Resfdues of concern at non-
quanMablelevsla.


No tolerances required. Plant-tuck
restriction may be needed.
observed.



No tolerances requked.
PfenKbsck restrtctfcn may be
needed.
The following should be considered when a confined rotational crop study
is to be conducted. The test material should be the pure active ingredient
radiolabeled (PAIRA) with 14C in a non-labile position (e.g., in a ring).
The parent compound only should be applied to the  appropriate  soil type
(usually a sandy loam) at  the maximum seasonal label rate (IX) and the
required three rotated crops (small grain [e.g., wheat, barley], leafy vegeta-
ble [e.g.,  spinach, lettuce],  and root crop [e.g., radish, table beets, carrots])
should be planted at appropriate soil aging intervals (e.g.,  1, 4, 7 or 9,
and 12 months). The representative root crop should not be a bulb vegeta-
ble such  as onions or garlic. It is acceptable to substitute soybeans for
a leafy vegetable due to the importance of this crop in rotational practices.
Growing  a primary crop in the soil  during the aging period is not precluded
provided  the  soil is treated prior to planting.  Sampling of the soil is not
required and  need only be performed  at  the Registrant's discretion. The
three rotated  crops should be harvested  and  the appropriate plant parts
(see Table I in OPPTS guideline 860.1000) should be sampled and com-
busted to determine  the total  radioactive  residue (TRR). At this point, if
each of the three crops demonstrate a TRR of <0.01 ppm in edible portions
at one of  the plantback or soil aging intervals then the Chemistry Branches
will conclude that no further work and no tolerances are needed. An appro-
priate rotational crop restriction can be set at the shortest interval where
no  TRR is (replacement note for greater than less than symbol) 0.01 ppm,
provided  that the Registrant is willing to  place this interval on the label.
If the  TRR is  <0.01 ppm in all three crops  at the one month interval,
then no plantback restriction will be needed on the label. If, however, in
the three  confined studies, the minimum intervals at  which  the TRR is
<0.01 ppm differ, then the rotational crop  restrictions will be set at the

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interval appropriate to each tested  crop group with  the longest interval
being applied to all other (untested) rotated crops. The following example
should be considered:

    The TRR for leafy vegetables is <0.01 ppm at the 1 month plantback inter-
val, the  root crop TRR is <0.01 ppm at the 4 month interval and the grain crop
TRR is <0.01  ppm at the 9 month interval. The rotational crop restrictions would
be 1 month for leafy vegetables, 4 months for root crops and 9  months for
small grains and all other rotated crops. It is the Registrant's prerogative to per-
form additional confined rotational crop studies on other crops to establish less
restrictive intervals based on levels of radioactivity.

In rare  cases, the toxicologists may have concerns regarding the presence
of a pesticide or metabolite  at levels  <0.01 ppm.  Determination of the
presence (or  absence) of specific metabolites  of concern at levels <0.01
ppm may be required in these cases.

If any of the plants in the confined studies exceed the trigger value  (0.01
ppm) at the  12 month interval,  then the nature of the  residue in  those
test crops having a TRR >0.01 ppm must be determined. The registrant
is referred to the guidance provided in guideline 860.1300, Nature of the
residue - plants,  livestock (however, see also the above comment regarding
the application rate for confined  rotational crop  studies)  for a description
of the procedures which need to be followed to accomplish this determina-
tion.

If any one of the  three crops shows <0.01 ppm at a given interval but
the Registrant desires  a shorter interval on the label  for that  crop where
the TRR is >0.01 ppm, then the composition  of the  TRR in  that rotated
crop (at the desired interval) should be determined as  described above for
the crop parts where the trigger value (i.e., 0.01 ppm) was  exceeded. If
several  samples of the  crop are available at the  desired interval, the sample
having  the highest  TRR should be  utilized. In either  of the above cases,
this information is needed in order that the Agency can make a conclusion
as to whether the residue is an inadvertent residue of no concern or wheth-
er cold (i.e.,  not radiolabeled) field trials are needed  to  make  that deter-
mination.

If the metabolism in rotated  crops appears to be different than that in the
primary crop, that is, if different metabolites are  observed in rotated  crops
than in primary crops, the Agency will make a determination as to whether
the different  rotational crop  metabolites  are of concern at the levels ob-
served.  If necessary, the  HED Metabolism Committee will be consulted.

The following are examples of the situations described above:

The primary  (target) crop metabolism studies indicate that the  parent and
metabolites A, B, C, D and E are present in the plant.  The Agency  decides
that only the  parent and metabolite  B need to be regulated in the tolerance

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expression. The following three scenarios might be encountered regarding
rotational crops:

     (i) The confined rotational crop studies indicate that the TRR is >0.01
ppm and that parent and metabolites A, B, C and D are present. Limited
rotational crop  field trials will normally be required with analysis for par-
ent and metabolite B if it is determined that these residues could be present
at detectable levels. If, however,  metabolites A,  C and D are present at
much higher levels  in the rotational crops  than in the primary  crop, the
HED Metabolism Committee may be  consulted  as to whether the  other
metabolites need to be  quantitated.

     (ii) The confined  rotational crop studies show that the TRR  is >0.01
ppm and that the  radioactive residue consists of only metabolites D and
E. In this case  the Agency would normally conclude  that this is  an  inad-
vertent  residue of no  concern  situation and no  field trials would be re-
quired.  A rotational crop restriction (i.e., plantback interval) may be nec-
essary.  As above  however, if metabolites D  and E  are  present  at much
higher levels in the rotational  crops than in the  primary crop, the  HED
Metabolism Committee may be consulted as to  whether these  metabolites
need to be quantitated.

     (iii) The confined rotational crop studies  indicate that the TRR is
>0.01 ppm and that there is no parent  present but that the major portion
of the TRR consists of a new metabolite F. This will require a decision,
as to whether there  is  toxicological concern over the new metabolite. At
this point the HED Metabolism Committee may be consulted for an expe-
dited decision.  If  it  is  concluded that the metabolite is  of concern at the
levels likely to be present,  then F should be analyzed for in  the limited
rotational crop  field trials. If it is decided that F is of no concern  then,
as in (ii) above, this is an inadvertent residue of no concern situation and
no field trials are necessary. However, a rotational  crop restriction may
be necessary.

It is recommended that the confined studies be  submitted to the Agency
as soon after completion as possible, so that the Agency can make a con-
clusion as to whether  there  is  a potential inadvertent residue  of concern
(i.e., will limited  field trials be  needed?).  This  will  allow the registrant
to design the field trials in a more efficient manner (i.e., what  compounds
require  analysis in the field trials).

     (c) Test standards.

     (1) Test substance.  This  study shall be conducted using the radio-
actively-labeled analytical grade  of the active ingredient.  If multiple ring
structures are present,  separate studies reflecting labeling of each ring will
normally be required.

     (2) Test procedures.

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     (i) This study should be  performed using a  sandy loam  soil which
has been treated with the test substance  applied  at a  rate equivalent to
the maximum seasonal rate (IX) under actual field use conditions. How-
ever,  if the label instructions of the product limit  its use to one soil  type
other than the sandy  loam,  then the study should be  run with the  soil
type specified on the label. In either case, the soil should not be sterilized.
Also, if the maximum seasonal application rate can only  be  attained by
multiple treatments under actual use conditions (e.g., many foliar insecti-
cides and fungicides),  the radiolabeled material may be  applied to the soil
in a similar manner. In other words,  the entire dose for the season need
not be applied at once for such  active ingredients.

     (ii)  The study may be  performed either in a greenhouse or on an
outdoor plot or container.  Following  application to the soil the pesticide
may be incorporated into the soil if the product label so instructs or this
represents typical agricultural practice.

     (iii) Following soil treatment, the pesticide should be aged under aero-
bic conditions in the soil for a  time approximating the anticipated agricul-
tural practice (e.g., 1 year for  crops rotated the following  year, 120  days
for crops rotated immediately after harvest, and 30 days for assessing cir-
cumstances of crop failure). Growing a primary crop  in the soil during
the aging period is not precluded provided the soil is treated prior to plant-
ing.

     (iv) Crops planted in the  treated and aged soil should include those
expected in the proposed rotational schedule and, where possible, be rep-
resentative of each of the following crop groupings: root and tuber vegeta-
ble (e.g., radish, table beets, carrots), small grain (e.g., wheat, barley), and
leafy vegetable  (e.g.,  spinach,  lettuce).  Soybeans  may  be  substituted for
a leafy vegetable due to the importance of this crop in rotational practices.
The selected crops shall be  analyzed for residues at appropriate harvest
intervals. (Residue analyses should be performed on selected crops at mul-
tiple intervals if both immature and mature crops  are normally harvested
in the course of usual agricultural practices.)

     (d)  Reporting and evaluation of data. In addition to the applicable
reporting requirements specified in OPPTS  860.1000,  the  following  data
should be reported:

     (1) The registrant should characterize and, when feasible, identify and
provide analytical values for significant residues in the crops  tested.  Sig-
nificant  residues include  parent  compound,  closely-related  degradates,
metabolites and/or their conjugates in the  crop. In cases where identifica-
tion of residues is  not feasible due to  insufficient sample, then pooling
of samples obtained from replicate experiments conducted  simultaneously
should be carried out to enable residue identification to be achieved. From
the results of this  study, the Agency will  determine whether additional

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     studies to measure the accumulation of pesticide residues in rotated crops
     under actual field conditions are needed. If such field studies are needed,
     the registration applicant will need to determine whether to conduct the
     limited field studies described in OPPTS 860.1900 (two trials each on rep-
     resentative crop(s)) or to carry  out a  complete set  of crop  field trials as
     described in OPPTS  860.1500 necessary to support the establishment of
     a tolerance in the rotated crop(s).

          (2) Depending on the  crop tested,  separate analyses  should be con-
     ducted on those  portions of the plant considered to be raw agricultural
     commodities (see Table I in 860.1000). However, even if the aerial portion
     for the particular root crop is not in Table I, both the aerial and root por-
     tions of root crops should be analyzed.

          (3) There is no need  to account for all applied radioactivity  since
     soil analyses are not required.

          (4) A  description  of the growing conditions should be reported.  If
     the study is conducted outdoors, rainfall data, temperature monitoring data,
     and general climatic conditions should be reported for the test period.

          (e) Format  of data report. The following  describes the order and
     format for a study report item by item.

          (1) Title /cover page. Title page and  additional  documentation re-
     quirements  (i.e., requirements for data submission  and  statement of data
     confidentiality of data) if relevant to the study reported  should precede
     the content of the study formatted below. These requirements are described
     in PR Notice 86-5 (see paragraph (f)(2)).

          (2) Table of contents. The Table of Contents must follow the title,
     data confidentiality, and GLP pages. This page should indicate the overall
     organization of the study, including tables and figures.

          (3) Abstract. This section should contain the overall summary of the
     study addressing the following points:

          (i) The chemical (use the same name throughout the report), the for-
     mulation  and the mode of application.  Structures of the  chemical and
     metabolites may be included in this section.

          (ii) Maintenance of the  treated plot.

          (iii) A table similar to the following with an appropriate title:

ays a er pp ica ion


Total

Residues (ppm)
Parent


Metabolites1 plant tissue

1Metabolites may need to be named and quantitated individually.

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     (iv) A discussion of unexpected problems (such as technical difficul-
ties or unusual weather) which necessitated deviations from the intended
test protocol and a  description of the  effects of these  deviations on the
results of the study; and

     (v) Provide a name and a phone number of a contact person in the
event the reviewer has technical  questions about the study. [This is op-
tional. However, providing this information will facilitate efficient review
in case of questions.]

     (4) 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.

     (5) Materials/methods. The registrant may elect to describe materials
and methods in separate sections  or combine into a single section.  The
following format combines  the two into one section.  This section should
be in narrative form. All details (including drawings and photographs) with
regard to the materials, equipment, experimental design, test plots or con-
tainers, procedures used in  conducting the study, and of different phases
of the study should be placed in this  section. In addition,  the following
are to be included, when appropriate:

     (i) Chemical.  Provide the purity of the material, its activity in Curies/
mole,  disintegrations  per minute per  gram (dpm/g),  and the  site of
radiolabeling.

     (ii) Site.

     (A) If test plots are used:

     (1) Provide a map describing location, topography and size, and loca-
tion and size of any control plots in relation to the test plot; and an indica-
tion of whether the test plot contains a subsurface drainage system;

     (2) Describe the means by which the test material is confined in the
areas surrounding the test plantings -  e.g.,  aluminum pipe sunk  in the
ground;

     (3) Provide the soil characteristics  (% sand,  % silt, % clay, % organic
matter, pH,  cation exchange capacity, and moisture capacity) of the plot;

     (4) Provide a complete record of daily temperature, daily rainfall and
pan evaporation data throughout the study and how they compare to aver-
age temperature and rainfall at the  test site based on records from the
nearest weather station; and

     (5) Include crop  and pesticide use history  on the  plot for the three
year period preceding the study.

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     (B) If test containers are used, provide information on the size  of
the containers; amount of soil,  its type and characteristics as detailed  in
"A" above, and other distinctive details.

     (iii) Test method.

     (A) General.

     (1)  The date  and technique of plot or container preparation prior  to
pesticide application;

     (2)  A description of how  and when the pesticide was applied; the
ambient conditions at the time of application; the application rate and the
application technique; also, similar descriptions for each of any additional
applications made; information  on how much pesticide  was applied  in
comparison to actual use rates; and if application technique differed from
label recommendations;

     (3)  The identity of the  treated crop, if applicable, and a description
of how and when the treated crop was planted;

     (4) A description of any post-treatment crop maintenance such as use
of fertilizers and other pesticides, irrigation  (when applied, how much, and
its source), tilling,  weeding, etc.;

     (5) A description of the crop  sampling technique, procedure and de-
vices used; and the date of harvest of the treated crop;

     (6)  A description of what was done to the plot after harvest  of the
treated crop before planting  of the rotational crop, during planting  of the
rotational crop, and during its growth;

     (7)  Identity of the rotational crops planted should be placed in this
section. Also, provide a description of the procedure used in planting the
rotational crops; the number  of days between treatment of the initial crop
with the pesticide  and planting of the rotational crop; and a description
of all procedures used in the maintenance of the rotational crops  (as done
for the  treated crop), sampling method and how many samples/replicates
were taken. All dates should  be provided in terms of "days from pesticide
application."

     (8) A description of the handling  of the crop samples from  the time
of taking of the samples until analysis with special attention to the  condi-
tions under which they were  stored and the thawing procedure (if frozen);
in addition, the storage stability data to be used in determining if the pes-
ticide residues are stable under the storage conditions; and the  dates the
samples were stored/frozen, thawed, and analyzed;

                                   8

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

     (10) An elaboration on any difficulties or special problems that arose
during the study which necessitated deviation from the intended test proto-
col and on the effects the deviations had on the results.

     (B) Analytical method.

     (1) 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 vali-
dation data, recovery and limit of detection data, quality control procedures
and results, sample chromatograms, and sample calculations. The detailed
description of the procedures used in preparation and handling  of the sam-
ple throughout the method should also be placed here;

     (2) The identity of the instrumentation, equipment and reagents used
and the operating conditions of the instrumentation;

     (3) Figures/photographs of any special equipment used in the meth-
ods; and flow diagrams of particularly complex extraction/clean-up proce-
dures are to be included here; and

     (4) The results of analyses of all crop samples.

     (6) Results/discussion.

     (i) This section should contain the scientific results of the study, for
instance;

     (A)  Narrative and tables  describing the  steps  taken in determining
the pesticide residues in the crop samples in addition to the graphical pres-
entations of the data (accompanied by the tables of the actual values from
which the graphs were constructed);

     (B) Results of the analysis of any control plots. The registrant should
note that the crop samples for both control and treated plots and/or contain-
ers are to be analyzed for levels of radioactive residues;

     (C)  Significant  residues should be characterized further, with special
attention to known degradates from plant metabolism, soil metabolism, hy-
drolysis and photolysis.

     (ii) This  section should contain the table of structures and chemical
names/designations for the parent compound and degradation products dis-
cussed in the study.

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     (7) Conclusion. This section should contain the discussion of the na-
ture, magnitude and toxicological significance  of the residues of parent,
metabolites and degradates found in the rotational crops.

     (8) Certification.

     (i) Signatures of each of the senior scientific  personnel  responsible
for the study; and

     (ii) Certification by the  registrant 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).

     (9) Tables/figures. It is recommended that tables and figures be num-
bered using arabic numerals  for  figures and roman numerals for tables.

     (10) References.

     (11) Other. At the registrant's option, reprints of methods and other
studies, raw data, copies of relevant letters/memos and 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.

     (f) References. The source material for this guideline is taken directly
from the following set of documents.

     (1) U.S.  Environmental  Protection  Agency,  Pesticide  Assessment
Guidelines, Subdivision O, Residue  Chemistry. EPA Report  No. 540/9-
82-023, October, 1982, (Available from National Technical Information
Service, Springfield, VA).

     (2) U.S. Environmental Protection Agency, Pesticide Registration No-
tice PR 86-5,  Standard Format for Data Submitted  under the FIFRA  and
Certain Provisions of the Federal Food, Drug, and Cosmetic Act (FFDCA),
May 3,1986.
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