tnvironmental Protection
Agency
Washington, Ulj
June IJKJO
&EPA
Hazard Evaluation
Standard Evaluation F
OF
0
Support Document 37
Non-Target Plants: Growth and
Reproduction of Aquatic Plants -
Tiers 1 and 2
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EPA 540/9-86-134
June 1986
HAZARD EVALUATION DIVISION
STANDARD EVALUATION PROCEDURE
NON-TARGET PLANTS:
GROWTH AND REPRODUCTION OF AQUATIC PLANTS
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
Page
I. INTRODUCTION
A. Purpose of the Standard Evaluation
Procedure 1
B. Background Information 1
C. Objective of the Growth and Reproduction of
Aquatic Plants Test 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
C. 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 8
Appendix 3: Sample Standard Format for
Preparation of Scientific
Reviews 12
REFERENCES 13
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NON-TARGET PLANTS;
GROWTH AND REPRODUCTION OF AQUATIC PLANTS - 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 growth and reproduction studies of
aquatic plants submitted by registrants in the assessment of pesti-
cide effects on non-target plants. This document is also designed
to aid EEB data reviewers in their evaluations of laboratory (growth
chamber) (Tier 2) growth and reproduction studies of aquatic plants
submitted for the same purpose.
B. Background Information
Growth and reproduction studies of aquatic plants are designed
to provide phytotoxicity data on a pesticide. These phytotoxicity
data are needed to evaluate the effect of the level of pesticide
exposure to non-target aquatic plants and to assess the impact of
pesticides on endangered and threatened plants as noted under the
Endangered Species Act. The preliminary (Tier 1) study evaluates
the effect of the maximum exposure level while the laboratory (growth
chamber) (Tier 2) study evaluates the effects of differing exposure
levels. Where a phytotoxic effect is noted in one or more plants,
further growth and reproduction of aquatic plants studies may be
required. These studies are required by 40 CFR § 158.150 to support
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 phytotoxici ty test. These use patterns include tree
injection, subsurface soil applications, recapture systems, and
wick applications. If any of these use patterns do readily expose
non-target plants to the pesticide, the pesticide phytotoxicity poten-
tial may need to be evaluated.
C. Objective of the Growth and Reproduction of Aquatic
Plants Test
1. Tier 1 Test
The objective of the Tier 1 growth and reproduction study of
aquatic plants 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
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aquatic plant population that have been historically used for this
type of testing and, therefore, have known types of responses.
This is a maximum dose test designed to quickly evaluate the phyto-
toxic effects of the pesticide at the one dose.
2. Tier 2 Test
The objective of the Tier 2 growth and reproduction study of
aquatic plants 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
aquatic plant population that have been historically used for this
type of testing, and, 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
quantities as may be found in the environment.
II. INFORMATION TO BE SUPPLIED
The registrant's report on growth and reproduction studies of
aquatic plants should include all information necessary to provide:
1) a complete and accurate description of the laboratory (growth
chamber) 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.
Guidelines of specific information that should be included in
the registrant's report of growth and reproduction studies of aquatic
plants are 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 liberalized standards prescribed in
these studies.
The results of the phytotoxicity study of the chemical with re-
spect to the quantity applied to the waters of the aquatic plant are
important. The concentration of the chemical in the water column is
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important in that even slightly stronger concentrations than normally
used can lead to stunting and necrosis. Subtoxic concentrations may
also cause unwanted rapid 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 plant growth and reproduction
of Lemna. Algal studies must be evaluated for a minimum of five days
for this same purpose.
After Tier 1 and Tier 2 growth and reproduction of aquatic
plants studies, respectively, have been performed, a decision point
to perform Tier 2 or Tier 3 aquatic plant growth tests, respectively,
is a 50% detrimental effect, i.e., a 50% change in plant growth or
injury as compared to untreated controls. This level is considered
to be that point at which the aquatic 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 look for data gaps - omissions in the information supplied
by the registrant in his report. These should be duly noted in the
reviewer's report, and a judgment made as to which are considered
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 pro-
vides 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 pre-
cautions noted.
As an adjunct to these, the reviewer should draw upon the tech-
nical guidance in the reviewer aids materials that are available.
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(See also the recommended references in Subdivision J — Hazard Eval-
uation; Non-Target Plants.) A listing of additional source materi-
als is located in the References section of this document.
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 re-
lated. 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 growth and
reproduction of aquatic plants 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 modifi-
cation, are suggested. If deficiencies/omissions exist in the sub-
mitted 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 growth and reproduction
studies of aquatic plants should include all information necessary
to provide: 1) a complete and accurate description of the labora-
tory (growth chamber) treatments and procedures, 2) sampling data
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 (growth chamber) growth and
reproduction of aquatic plants 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/super-
vising/monitoring;
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 and
growth chambers; 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;
0 Solvent used to dissolve and apply the pesticide if the pesti-
cide is insoluble in water or other intended carrier;
0 Dose rate(s) in terms of active ingredient per area of land
or final concentration in the test waters;
0 For Tier 1, dose rate(s) in terms of the maximum label rate
as though it were applied directly to the surface of a 15-cm or
6-inch water column, 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 egual 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 as though it were applied directly to the surface of a
15-cm or 6-inch water column or in terms of less than the one-fold
concentration as tested in Tier 1 with dosages in a geometrical
progression of no more than two-fold and with subtoxic (< ECso
level) and non-toxic (no-observable-effeet-level) concentrations;
0 Method of application including the equipment type; and
0 Number of applications.
III. Plant Species
0 Identification of the test aquatic plant species as noted
below:
Species Name
Lemna gibba
Skeletonema costaturn
Anabaena flos-aquae
Selenastrum capricornuturn
(Unspecified species)
Common Name
Duckweed
Marine diatom
Blue-green alga
Freshwater green alga
Freshwater diatom
Selenastrum capricornuturn shall be tested regardless of the intended
outdoor use pattern. The other aquatic plant species shall be
tested where the use pattern is for those other than swimming pools.
The Organization for Economic Cooperation and Development (OECD)
guidelines for the Tier 1 algal study also gives Chlorella vulgaris
and Scenedesmus subspicatus as suitable species. They may be
substituted for Selenastrum only where this species is not readily
available.
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0 Identification of the number of replicates and the number
of plants per replicate per dose; and
0 Identification of the date of the plant addition to the
growth media without test chemical (for stabilization of plant
growth, if necessary), date of pesticide application, and date of
phytotoxicity rating or harvest and analyses.
IV. Site of the Test
0 Site description of the aquatic plant growth and reproduc-
tion study such as the type of growth chamber;
0 Location of the test site;
0 Climatological data during the test (records of applicable
conditions for the type of site, i.e., temperatures, thermoperiods,
rainfall or watering regime, light regime - intensity and quality,
relative humidity);
0 Cultural practices such as flow-through or static test; and
0 Substrate characteristics (type of growth media including
its physical and chemical properties, including pH).
V. Results
0 Phytotoxicity rating (including a description of the rating
system) for each plant or plant population in the test;
0 Weight, size (Lemna), or other growth parameters that may
have been measured to ascertain toxic effects of the pesticide upon
the plants with the dates of the observations; 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 50% of the growth [mass or rate] of the
control.)
VI. Evaluation
0 For Tier 1 studies, determination as to whether Tier 2 tests
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 aqua-
tic field studies 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 forpreparation of scientific reviews.
I. General
0 Were 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 Were the name (and signature), title, organization, address,
and telephone number of the person(s) responsible for planning/super-
vising/monitoring and applying the pesticide provided?
0 Was the trial identification number provided?
0 Were quality assurance control measures/precautions indicated?
0 For Tier2,"was the Tier 1 growth and reproduction study of
aquatic plants done as a separate study? In not, were the dose and
plant species required by Tier 1 included in the Tier 2 study?
II. Test Chemical
0 Was the test chemical used the technical grade, or if not
available, the manufacturing-use product with the highest percen-
tage of active ingredient?
0 Was the active ingredient percentage or degree of purity of
the chemical given?
0 If a solvent was used, was it used at concentrations that
^re not phytotoxic and was a solvent control used?
0 Was the dose given in quantity per unit area (of plant or
\ surface) or in tank concentration (parts per million)?
0 For Tier 1, was the dose equal to or greater than the maxi-
>bel rate as though it were applied directly to the surface of
•n or 6-inch water column? An application of 1 Ib active in-
t per acre or 1.12 kg per hectare is equal to 735 parts per
(ppb) in a 6-inch or 15-cm water column. If registrant has
\t the maximum quantity that will be present in the non-target
ignificantly less than the maximum label rate, was the dose
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equal to or no less than three times that maximum environmental
quantity?
0 For Tier 2, was the dose less than the maximum label rate
as though it were applied directly to the surface of a 15-cm or
6-inch water column or was it less than the one-fold concentration
as tested in Tier 1? If registrant has shown that the maximum quan-
tity that will be present in the non-target area is significantly
less than the maximum label rate (Tier 1), was the maximum dose
less than three times that maximum environmental quantity (as com-
pared to dosage in Tier 1)?
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
per 15-cm water column?
0 For Tier 2, were a subtoxic (< EC5g) and a non-toxic (no-
observable-effeet-level) concentration evaluated?
III. Test Species
0 For Tier 2, were at least those plant species of Tier 1
which exhibited phytotoxic effects tested?
0 Was Selenastrum capricornutum tested regardless of the out-
door use pattern?
0 If the use pattern was for aquatic pest control at sites
other than swimming pools, were the following species also tested:
Lemna gibba Duckweed
Skeletonema costatum Marine diatom
Anabaena flos-aquae Blue-green alga
(Unspecified species) Freshwater diatom
0 For Tier 2, were the above species tested if they were af-
fected in the Tier 1 test?
0 If OECD recommended species other than Selenastrum
capricornutum be used, was justification provided as to its use?
0 Where various culture types could be used, such as in the case
of most cultured algae and Lemna, were culture types and sources
ident if ied?
0 Were at least three replicates used with five plants per
replicate for each dose tor the vascular aquatic plant - Lemna
gibba?
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0 Were the initial cell concentrations for the algal tests
approximately:
Selenastrum capricornutum 3000 cells/mL
Skeletonema costatum 10000 cells/mL
Anabaena flos-aquae 3000 cells/mL
(Unspecified species) 3000 cells/mL
0 Were endangered or threatened plant species not used?
IV. Test Procedures
0 Was the test site specified, i.e.-, growth chamber?
0 Were the environmental conditions that prevailed during the
test those that were given in § 122-30 of Subdivision J?
Species
Selenastrum
Skeletonema
capricornutum
costatum
Anabaena flos-aquae
Unspecif ied
Lemna gibba
diatom species
Temp.
<°C)
24
20
24
24
25
Light
In tens i ty
Uux[K])
4.0
4.0
2.0
4.3
5.0
Photo-
Period
Continuous
16/8 hr D/N
Continuous
Continuous
Continuous
Medium
pH
( approx. )
7.5
8.0
7.5
7.5
5.0
0 If modifications to the environmental conditions were used
and reported, were their uses substantiated?
0 Were the culture conditions for the test species those that
were given in § 122-30 of Subdivision J? If modifications were
made, were they reported?
0 Was the test duration for Lemna growth at least two weeks
in length and, for algal growth, at least five days in length?
0 Were observations taken at least every three days for Lemna
growth?
0 Were observations taken at least daily for the algal growth?
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)?
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0 If a rating system was used, was an explanation provided?
0 Were abnormal changes in growth, development, and/or mor-
phology reported with comparisons to the controls or "normal"
plants?
0 Was the growth of- Lemna expressed as the number of original
plants and fronds and the number of additional plants and/or fronds?
0 Was the growth of the algae expressed as the cell count
per mL, biomass per volume, or degree of growth as determined by
spectrop ho tome trie means? If spectrophotometric means were used,
was some attempt made to equate the absorbance readings to number
of cells or biomass?
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 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 would be performed if any of the Tier 1 species were
detrimentally affected (greater than 50% 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 aquatic field tests would be performed if any of the Tier 2
species were detrimentally affected?
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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 - Growth
and Reproduction of Aquatic Plants - 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 Recommendations:
(Decide as to (1) the scientific validity of the
study and (2) compliance to the Subdivision J -
Growth and Reproduction of Aquatic Plants Tier 1
or Tier 2 Study guidelines). '
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
Davis, J. A. 198.1. "Comparison of static-replacement and flow-
through bioassays using duckweed, Lemna gibba G-3." EPA Report
No. EPA 560/6-81-003
Hoist, R. W., J. H. Yopp, and G. Kapusta. 1982. "Effect of several
pesticides on the growth and nitrogen assimilation of Azolla-
Anabaena symbiosis." Weed Science 30:54-58
Sculthorpe, C. D. 1967. The Biology of Aquatic Vascular Plants.
London: Edward Arnold (Publ.) Ltd.
Other scientific articles on growth and pesticide effects of
aquatic plants may be found in the following scientific journals:
Aquatic Botany
Botanica Marina
Canadian Journal of Fisheries and Aquatic Sciences
Chemosphere
Environmental Pollution
Environmental Science and Technology
Hydrobiologia
Journal of Environmental Quality
Microbios Letters
Phycologia
Physiologia Plantarum
Water Research
fl US. GOVERNMENT PRINTING OFFICE: 1986 - 621-735 - 1302/60519
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