&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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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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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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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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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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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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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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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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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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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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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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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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