&EPA
               United States
               Environmental Protection
               Agency
               Office of Pesticide Programs
               Washington, DC 20460
EPA 540/9-86-132
June 1986
Hazard Evaluation Di
Standard  Evaluation I  Support Document 38
               Non-Target Plants: Seed Gerhriination/
               Seedling Emergence - Tiers 1  and 2

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                                              EPA 540/9-86-132
                                              June 1986
                          HAZARD EVALUATION DIVISION

                        STANDARD EVALUATION PROCEDURE

                               NON-TARGET PLANTS:

                     SEED GERMINATION/SEEDLING EMERGENCE

                                 TIERS 1 AND 2
                                  Prepared by

                             Robert W. Hoist, Ph.D.
                 Standard Evaluation  Procedures Project Manager
                               Stephen L.  Johnson
                           Hazard Evaluation Division
                          Office of Pesticide Programs
                 United States Environmental Protection Agency
                          Office of Pesticide Programs
                            Washington, D.C.  20460
*

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                          TABLE OF CONTENTS
  I.   INTRODUCTION
                                                        Paqe
       A.  Purpose of the Standard Evaluation
           Procedure	    1
       B.  Background Information	    1
       C.  Objective of Seed Germination/Seedling
           Emergence Tests 	 	    1
            1.  Tier 1 Test	    1
            2.  Tier 2, Test . .	    2


 II.   INFORMATION TO BE SUPPLIED .	    2


III.   DATA INTERPRETATION'...		. ...    2


 IV.   THE DATA EVALUATION PROCESS

       A.  Identify Data Gaps	    3
       B.  Assess the Appropriateness and  Adequacy
           of the Data .'	    3
       D.  Report Preparation	    4
       D.  Conclude if the Requested Action is
           Supportable	••••	    4


  V.   APPENDICES

       Appendix 1:  Information Requested  of the
                    Registrant	    5

       Appendix 2:  Specific Questions  for the
                    Reviewer	    9

       Appendix 3:  Sample Standard Format for
                    Preparation of Scientific
                    Reviews	   12


  REFERENCES	   13

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                         NON-TARGET PLANTS:

        SEED GERMINATION/SEEDLING EMERGENCE - TIERS 1 AND 2


I.   INTRODUCTION

     A.  Purpose of the Standard Evaluation Procedure

     This Standard Evaluation Procedure is designed to aid Ecologi-
cal Effects Branch (EEB) data reviewers in their evaluations of
preliminary (Tier 1) laboratory seed germination/seedling emergence
studies submitted by registrants in the assessment of pesticide
effects on non-target plants.  This document is also designed to aid
EEB reviewers in their evaluations of laboratory/greenhouse/small
field plot (Tier 2) seed germination/seedling emergence studies sub-
mitted by registrants for the same purpose.

     B.  Background Information

     Seed germination/seedling emergence studies are designed to pro-
vide phytotoxicity data on a pesticide.  These phytotoxicity data are
needed to evaluate the effect of the level of pesticide exposure to
non-target and terrestrial plants and to assess the impact of pesti-
cides on endangered and threatened plants as noted under the Endan-
gered Species Act.  The preliminary level (Tier 1) study evaluates
the effect of the maximum exposure level while the greenhouse/labora-
tory/small field plot (Tier 2) study evaluates the effects of differ-
ing exposure levels.  Where a phytotoxic effect is noted in one or
more plants, further seed germination/seedling emergence studies may
be required.  These studies are required by 40 CFR § 158.150 to sup-
port the registration of any pesticide  intended for outdoor use under
the Federal insecticide, Fungicide and  Rodenticide Act  (FIFRA), as
amended.

     Pesticides with outdoor use patterns that do not readily release
the pesticide to the environment do not have to be evaluated using
this phytotoxicity test.  These use patterns include tree injection,
subsurface soil applications, recapture systems, wick applications,
and swimming pool uses.  If any of these use patterns do readily
expose non-target plants to the pesticide, as through vapors, the
pesticide phytotoxicity potential may need to be evaluated.

     C.  Objective of Seed Germination/Seedling Emergence Tests

          1.  Tier 1 Test

     The objective of the Tier 1 seed germination/seedling emergence
test is to determine if a pesticide exerts a detrimental effect  to
plants during critical  stages  in their  development.  The test  is
performed on species from a cross-section of the non-target terres-
trial  plant population  that have been historically used for this  type

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  of testing and, therefore, have known types of responses.  This is
  a maximum dose test designed to quickly evaluate the phytotoxic
  effects of the pesticide at the one dose.

            2.   Tier 2 Test

       The objective of the Tier 2 seed germination/seedling emergence
  test is to determine if a pesticide exerts a detrimental effect to
  plants during critical stages in their development.  The test is per-
  formed on species from a cross-section of the non-target terrestrial
  plant population that have been historically used for this type of
  testing arid,  therefore, have known types of responses.  This is a
  multiple dose test designed to evaluate the phytotoxic effects of
  the pesticide over a wide range of anticipated pesticide quantiti.es
  as may be found in the environment.


 II.  INFORMATION TO BE SUPPLIED

       The registrant's report on preliminary seed germination/seed-
  ling emergence studies should include all information necessary to
  provide:  1)  a complete and accurate description of the laboratory/
  greenhouse treatments and procedures, 2) sampling data and phytotox-
  icity rating, 3) data on storage of the plant materials until analy-
  sis, if so performed, 4) any chemical analysis of the plant material-
  as to chemical content, if so performed, 5) reporting of the data,
  rating system and statistical analysis, and 6) quality control mea-
  sures/precautions taken to ensure the fidelity of the operations.

       A guideline of specific information that should be included in
  the registrant's report on seed germination/seedling emergence
  studies is provided in Appendix 1 of this document.  The lists of
  requested information and reviewer aids are derived from the Pesti-
  cide Assessment Guidelines, Subdivision J;  Hazard Evaluation of
  Non-Target Plants, which is complemented by this Standard Evaluation
  Procedure.


III.  DATA INTERPRETATION

       The acceptability of the study results will depend upon whether
  the test requirements/standards are followed.  If a deviation is
  made, a determination must be made as to whether the deviation has
  changed the quality of the results in such a manner that the results
  cannot be extrapolated to the natural environment.  There should be
  little or no deviation from the liberal standards prescribed in this
  study.

       The results of the pesticide phytotoxicity tests with respect
  to the quantity of material applied to or near the seed are important.
  The concentration of the chemical in the carrier is important in that
  even slightly stronger concentrations than normally used can lead to

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                                 -3-
 stunting and necrosis.  Subtoxic concentrations, on the other hand,
 may cause unwanted rapLd growth.

      Plants can recover from certain types of injury with little or
 no resulting effect on the esthetic or economic value of the plant(s)
 tested or upon which an evaluation is made.  Therefore, it is impor-
 tant that a minimum of two weeks of observations be made after appli-
 cation of the pesticide to evaluate seedling emergence.  If seed ger-
 mination is evaluated, the extent of germination (percentage of seed
 showing root and shoot emergence) should be evaluated at least five
 days after imbibition.

      A decision point to proceed to the next higher test is a 25%
 detrimental effect, i.e., a 25% change in the average germination
 or plant growth or injury as compared to untreated controls.  This
 level is considered to be that point at which the plants will not
 recover to their full esthetic value, economic value or reproductive
 potential as in the case of the maintenance of the endangered or
 threatened species.


IV.  THE DATA EVALUATION PROCESS

      Upon careful examination of the information/data supplied by
 the registrant in his submission to the Agency, the reviewer shall
 evaluate the data as follows.

      A.  Identify Data Gaps

      Using Appendix 1 of this document as a guide, the reviewer
 should then look for data gaps - omissions in the information sup-
 plied by the registrant  in his report.  These should be duly noted
 in the reviewer's report, and a judgment made as to which are con-
 sidered significant enough to adversely affect the review process.
 Those so identified should be communicated back to the registrant
 by the Product Manager for corrective action.

      B.  Assess the Appropriateness and Adequacy of the Data

      The data reviewer then considers the appropriateness, i.e., the
 intended use pattern, and adequacy of the data/information that has
 been supplied.  Appendix 1 of this document is a useful guide to the
 various parameters that  need to be considered.  Appendix 2 provides
 specific questions that  should be answered by the reviewer during
 the study evaluation process.  Statistical treatments of the data
 should be independently  verified and the quality control precautions
 noted.

      As an adjunct to these, the reviewer should draw upon the tech-
 nical guidance in  the reviewer aids materials that are available.
 (see also the recommended references  in Subdivision J - Hazard Eval-
 uation;  Non-Target Plants.)  A  listing of additional source materials
 is located in the  References section of this document.

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                                -4-
     In addition to the data gaps noted above, any perceived  defici-
encies in the data/information supplied should also be  identified.
A statement as to these deficiencies should be made in  the reviewer's
report and corrective action to resolve them should be  provided.
This information can be relayed to the registrant by the Product
Manager for appropriate action.

     C.  Report Preparation        <

     The Agency reviewer prepares a standard review report following
the standard format for preparation of scientific reviews as  provided
in Appendix 3 of this document.  All important information provided
by the registrant including the methodology and results should be
summarized in order that future evaluations can be made.  The
results may be expressed in the form of tables where specific
values are related.  Figures (graphs) may be provided but are not
to be the sole source of the values needed for future evaluations.

     D.  Conclude if the Requested Action is Supportable

     Lastly, the reviewer considers the results of the  seed germi-
nation/seedling emergence studies and makes a judgment  as to  whether
they support the requested registration action of the data submitter.
If the data are not supportive, possible alternative action(s) that
may be taken by the registrant, such as label modifications,  are sug-
gested.  If deficiencies/omissions exist in the submitted data, the
reviewer may have to defer judgment until such time as  appropriate
corrective action has been rendered by the registrant.

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                                 -5-
                              APPENDIX 1

               INFORMATION REQUESTED OF THE REGISTRANT


      The registrant's  report on preliminary seed germination/seed-
 ling  emergence studies  should include all information necessary to
 provide:  1)  a complete and accurate description of the laboratory/
 greenhouse/small field  plot treatments and procedures,  2)  sampling
 and phytotoxicity rating, 3) data on storage of the plant material
 until analyzed,  if so performed, 4) any chemical analysis of the
 plant material as to chemical content, if so performed, 5) reporting
 of  the data,  rating  system and statistical analysis,  and 6) quality
 control measures/precautions taken to ensure the fidelity of the
 operations.

      Specifically, each laboratory/greenhouse/small field plot seed
 germination/seedling emergence report should include  the following
 information.


 I.  General

      0  Cooperator or  researcher (name and address),  test location
 (county and  state; country, if outside of the U.S.A.),  and date of
 study;

      0  Name (and signature), title, organization, address, and
 telephone number of  the person(s) responsible for planning/supervising/
 monitoring and,  for the field plot studies, applying the pesticide;

      0  Trial identification number;

      0  Quality  assurance indicating:  control measures/precautions
 followed to  ensure the  fidelity of the phytotoxicity determinations;
 record-keeping procedures and availability of logbooks; skill of
 the laboratory personnel; equipment status of the  laboratory or
 greenhouse;  degree of  adherence to good laboratory practices; and
 degree of adherence to good agricultural practices in maintaining
 healthly plants; and

      0  Other information the registrant considers appropriate and
 relevant to  provide a complete and thorough description of the test
 procedures and results.


II.   Test Substance (Pesticide)

      0  Identification of the test pesticide active  ingredient  (ai)
 including chemical name, common name  (ANSI, BSI,  ISO,  WSSA), and
 Company developmental/experimental name;

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                                  -6-
       0   Active  ingredient percentage in the technical grade material
  or  in the  manufacturing-use product, if the technical grade material
  is  unavailable  for test purposes;
  cide  is
Solvent used
insoluble in
to dissolve and apply the pesticide
water or other" intended carrier;
                                                           if the pesti-
       0   Dose  rate(s)  in terms of
  or concentration as applied;
                         active  ingredient per  area of  land
       0   For Tier 1,  dose rate(s)  in terms of the maximum label rate,
  or if the  registrant has shown that the maximum quantity that will
  be present in the non-target area is significantly less than the
  maximum label rate,  the dose equal to or no less than three times
  that maximum environmental quantity;

       0   For Tier 2,  dose rate(s)  in terms of less than the maximum
  label rate, with dosages in a geometrical progression of no more
  than two-fold and with subtoxic (< £C$Q level) and non-toxic {no-
  observable-effect- level ) concentrations;

       0   Method of application including equipment type; and

       0   Number of applications.
III.   Plant Species

       0  For Tier 1, identification of the six dicotyledoneae species
  and four monocotyledoneae species with family identification.  The
  six dicots are to be of at least four different families and the
  moncots of at least two families.  Soybeans, corn, and a dicot root
  crop like carrot are the required species.  The proposed species
  and families as originally provided in Subpart J of the proposed
  guidelines [FR notice of 3 November 1980] are given below and are
  acceptable for the laboratory/greenhouse seed germination/seedling
  emergence test:
     Family

    Solanaceae
    Cucurbitaceae
    Compositae
    Leguminosae
    Cruciferae
    Umbelliferae
    Gramineae
    Gramineae
                Species

               Lycopers icon esculentum
               Cucumis sativus
               Lactuca sativa
                                 Commo n

                                Tomato
                                Cucumbe r
                                Lettuce
                                Soybean
              Glycine max
               (Innoculation  with  Rhizoblum  japonicum is
              unnecessary)
               Brassica oleracea
               Daucus  carota
               Avena sativa
               Lolium  perenne
                                 Cabbage
                                 Carrot
                                 Oat
                                 Perennial  Ryegrass

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t
    Family              Species                         Common

   Gramineae           Zea mays                        Corn
   Amaryllidaceae      Allium cepa                    Onion

 Seeds  of  plants with a low or variable  germination potential should
 be avoided for the seed germination study.

      0  For Tier 2,  identification of  the plant species tested in-
 cluding those phytotoxically affected  in the Tier 1 test;

      0  Identification of the cultivar(s) of the plant species or
 assignment of an identification number  to the cultivar used and
 seed or plant source;

      0  Identification of the number of replicates and the number
 of plants per replicate per dose; and

      0  Identification of the date of  planting or imbibition, date
 of pesticide application, and date of  phytotoxicity rating or
 harvest and analysis.


IV.  Site of the Test

      0  Site description of the seed germination/seedling emergence
 study such as the type of growth chamber, greenhouse, or field
 (small field plots);

      0  Location of the test site;

      0  Climatological data during the test (records of applicable
 conditions for the type of site, i.e.,  temperature and thermoperiod,
 rainfall or watering regime, light regime - intensity and quality,
 relative humidity, wind speed);

      0  Field lay-out (for small field plots), e.g., size and number
 of control and experimental plots; number of plants per plot/unit
 area;

      0  Pot, plant or row density of seeds or plants;

      9  Cultural practices such as cultivation and irrigation; and

      0  Substrate characteristics  (name/designation of soil type and
 its physical and chemical properties,  including pH and percent
 organic matter).

 V.  Results

      0  Reporting of percent germination/emergence, root length or
 other growth parameters that may have been measured to ascertain

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                                 -8-
 toxic effects of the pesticide upon the plants with dates of obser-
 vations;

      0  Phytotoxici ty rating (including a description of the rating
 system) for each plant or population in the test;  and

      0  Statistical analysis of the results including an environ-
 mental or effective concentration (EC) value.   (Note, for Tier 1,
 there will be only a percent effect level at a specific concentration
 which is then compared to 25% of the growth [mass or rate]  of the
 control.)

VI.  Evaluation

      0  For Tier 1 studies,  determination as to whether Tier 2
 studies would be required due to phytotoxic effects noted in one or
 more of the tested species.

      0  For Tier 2 studies,  determination as to whether Tier 3 tests
 (terrestrial field study) would be required due to phytotoxic effects
 noted in one or more of the tested species.

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                                     -9-



                                  APPENDIX  2

                     SPECIFIC QUESTIONS FOR THE  REVIEWER


          The  following questions are  provided  to  aid  the  reviewer in
     performing the standard evaluation procedure  in a scientific manner
     and  in acquiring the necessary  information  to complete  a  standard
     format for preparation of  scientific  reviews.

     I.   General

          0  Was  the name of the cooperator or  researcher  (name  and
     address), test location (county and state;  country,  if  outside  of
     the  U.S.A.),  and date of study  provided?

          0  Was  the name (and  signature),  title,  organization,  address,
     and  telephone number of the person(s)  responsible for planning/super-
     vising/monitoring and, for small  field plot studies,  applying the
     pesticide provided?

          0  Was  the trial identification  number provided?

          0  Were quality assurance  control measures/precautions indicated?

          0  Was  the Tier 1 seed germination/seedling  emergence  study done
     as a separate study?  If not, were the doses  and  plant  species  re-
     quired by Tier 1  included  in  the  Tier 2  study?


     II.   Test  Chemical

          0  Is  the test chemical  being used  the technical grade, or if
     not  available, the manufacturing-use  product with the highest
     percentage  of active ingredient?

          0  Is  the active  ingredient  percentage or degree of  purity of
     the  chemical given?

          0  If  a solvent was used,  was  it used  at concentrations  that
     are  not phytotoxic and was a  solvent  control used?

          0  Is  the dose given  in  quantity per  unit area (of plant  or
      land surface) or  in tank concentration?

          0  For Tier  1, was the dose  equal to  or greater than the  maxi-
     mum  label rate, or  if  the  registrant  has shown that the maximum
     quantity  that will  be present in  the  non-target area is significantly
      less than the maximum  label rate, was the  dose equal to or  no  less
      than three  times  that maximum environmental quantity?
t

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                                  -10-
       0  For Tier 2, was the maximum dose less than the maximum label
  rate?

       0  For Tier 2, were the additional dosages of a geometric pro-
  gression of no more than two-fold, e.g., 0.1, 0.2, 0.4, 0.8, 1.6 kg/ha?

       0  For Tier 2, were a subtoxic (< EC5Q level) and a non-toxic
  (no-observable-effeet-level) concentration evaluated?


III.   Test Species

       0  For Tier 1, were at least ten different species tested with
  species names provided?

       0  For Tier 1, were the ten species split between monocots and
  dicots, four and six, respectively?

       0  For Tier 1, were the ten species from six different families
  and the family names provided?

       0  For Tier 1, were two of the species tested soybeans and corn
  and was the third species a dicot root crop?

       0  For Tier 2, were at least those species that were phytotox-
  ically affected in Tier 1 tested?            "

       0  Where various cultivars could be used, such as in the case
  of most agronomic and horticultural plants, were cultivar or varietal
  names provided?

       0  Were seed and plant sources provided?

       0  Were at least three replicates used with ten seeds per repli-
  cate for each dose level?

       0  Were some of the seeds pretested for germination and emer-
  gence potential?  Seeds of plants with a low or variable potential
  should be avoided.

       0  Were endangered or threatened plant species not used?


 IV.  Test Procedures

       0  Was the test site specified, i.e., greenhouse, growth cham-
  ber, or small field plot?

       0  Were the environmental conditions that prevailed during the
  test (temperature and thermoperiod, light regime - intensity and
  quality, rainfall or watering regime, relative humidity, wind)
  provided as appropriate for the site?

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                                 -11-
      0  Were the environmental conditions that prevailed during the
 test those most favorable and most typical to the growth of the
 plants used?  Were these conditions referenced?

      0  Was the test duration for seedling emergence at least two
 weeks in length or for seed germination at least five days in length?

      0  Were observations taken at least weekly for seedling emer-
 gence and after the five days for seed germination?

      0  Was the method of pesticide application including the type
 of application equipment employed given?


 V.  Reporting

      0  Were the detrimental effects reported as severity of phyto-
 toxicity (rating or percentage), percent germination or percent
 emergence?

      0  If a rating system was used, was an explanation provided?

      0  Were abnormal changes in growth, development and/or morpho-
 logy reported with comparisons to the controls or "normal" plants?

      0  Though not required, were direct measurements of root
 length or seedling length provided?

      0  Were the results statistically analyzed?  Note that care
 should be taken in interpreting the statistical results where the
 sample size is small.


VI.  Evaluation

      0  Were the results tabulated to indicate a percentage effect
 level (EC value) for each species as compared to the untreated
 control plants?

      0  For Tier 1 studies, was a determination made as to whether
 Tier 2 tests should be performed if any of the Tier 1 species were
 detrimentally affected (greater than 25% detrimental effect on
 growth)?

      0  For Tier 2 studies, were 25 and 50 percent detrimental effect
 levels determined  for those plant species of Tier 1 that showed a
 phytotoxic effect  to the chemical?

      0  For Tier 2 studies, was a determination made as to whether
 Tier 3 tests (terrestrial field study) should be performed  if any of
 the Tier 2 species were detrimentally affected  (greater than 25% de-
 trimental effect on growth)?

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                                -12-
                             APPENDIX 3

    SAMPLE STANDARD FORMAT FOR PREPARATION OF SCIENTIFIC REVIEWS


     The following format shall be used in documenting the review
of the Subdivision J - Hazard Evaluation;  Non-Target Plants - Seed
Germination/Seedling Emergence'Tier 1 and Tier 2 Studies.


Chemical:     (Common Name)

Formulation:  (percent Active Ingredient)

Study/Action: (Purpose of the Submission)

Study Identification:

              (Subdivision J Test Title)
              (Reference or Registrant Data Information with
               Study Number)
              (EPA Accession Number)

Reviewer:     (Name and Address of Reviewer; Date of Review)

Approval:     (Quality Control Reviewer)
                                                          •
Conclusions:  (Summary and Conclusion of Tests)

Acceptability and Recommendatipns:

              (Decide as to (1) the scientific validity of the study
              and (2) compliance to the Subdivision J - Seed Germi-
              nation Tier 1 and Tier 2 Studies.)

Background:   (introductory Information and Directions for Use)

Discussion:   1. Study Identification
              2. Materials and Methods
              3. Reported Results
              4. Reported Conclusions
              5. Reviewer's Interpretation of Results and Conclusion

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                                 -13-
                             REFERENCES


Bewley, J. D.  1983.  Physiology  and  Biochemistry of Seeds in Relation
     to Germination.

Khan, A. A.  1977.  Physiology  and  Biochemistry of Seed Dormancy and
     Germination.

Mayer, A. M.  1982.  Germination  of Seeds.

Truelove, B., ed.   1977.  Research  Methods  in Weed Science.  Southern
     Weed Science Society.  Auburn,  AL:   Auburn Printing, Inc.

U.S. Department of  Agriculture.   1952.   Manual for Testing Agricul-
     tural and Vegetable  Seeds.   Agriculture Handbook No. 30.


     Other scientific articles  of seed  germination may be found in
the following journals:

     Agronomy Journal
     Environmental  Science  and  Technology
     Journal of Environmental Quality
     Soil Science and Plant Nutrition
     Weed Science
                       * U.3. GOVERNMENT PMNTINQ OFFICE:  1986 - 621-735 - 1302/60519

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