xvEPA
Environmental Protection
Agency
Washington, DC 20460
June 1986
Hazard Evaluation Division
Standard Evaluation Procedure
Non-Target Plants: Aquatic Field Testing
' I6r. Support Document #36
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EPA 540/9-86-136
June 1986
HAZARD EVALUATION DIVISION
STANDARD EVALUATION PROCEDURE
NON-TARGET PLANTS:
AQUATIC FIELD TESTING - TIER 3
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 Aquatic Field Testing
Tier 3 Test . 1
II. INFORMATION TO BE SUPPLIED 1
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 3
D. Conclude if the Requested Action
is Supportable 3
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; AQUATIC FIELD TESTING - TIER 3
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
Tier 3 aquatic field testing studies submitted by registrants in
the assessment of pesticide effects on non-target plants.
B. Background Information
Aquatic field testing studies (Tier 3) 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. Where a phytotoxic effect is noted in one or more
plants, further field aquatic field testing studies may be required.
These studies are required by 40 CFR § 158.150 to support the regis-
tration of any pesticide intended for outdoor use under the Federal
Insecticide, Fungicide and Rodenticide Act (FIFRA), as amended.
Pesticides with outdoor use patterns thatdo 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, and wick applications,
If any of these use patterns do readily expose non-target plants to
the pesticide, the pesticide phytotoxicity potential may need to be
evaluated.
C. Objective of the Aquatic Field Testing Tier 3 Test
The objective of the Tier 3 aquatic field testing study 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 pop-
ulation. It is also performed under natural conditions and in the
environment in which the pesticide is to be applied. By this pro-
cedure, direct assessments) can be made as to the potential phyto-
toxicity of the pesticide. 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 aquatic field testing studies should
include all information necessary to provide: 1) a complete and
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accurate description of the treatments and procedures, 2) sampling
data and phytotoxicity rating, 3) data on storage of the plant mate-
rials until analysis, if so performed, 4) any chemical analysis of
the plant material as to chemical content, if so performed, 5) re-
porting of the data, rating system and statistical analysis, 6) and
quality control measures/precautions taken to ensure the fidelity
of the operations.
A guideline of specific information that should be included in
the registrant's report of aquatic field testing studies is provided
in Appendix 1 of this document. The l^sts of requested information
and reviewer aids are derived from the Pesticide Assessment Guide-
lines, 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 of this study.
The results of the phytotoxicity test of the chemical with re-
spect to the quantity applied to the water body near the aquatic plant
are important. The concentration of the chemical in the water
column is important in that stronger concentrations than normally
used can lead to stunting and necrosis or unwanted growth. Subtoxic
concentrations, on the other hand, may 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
important that a minimum of two weeks of observations be made after
application of the pesticide to record higher plant (vascular and
Bryophyta) growth and development. A minimum of five days of obser-
vations to record the algal growth and development should be allowed.
A decision point to perform additional Tier 3 aquatic plant
growth tests 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
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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
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 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.
(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.
In addition to the data gaps noted above, any perceived deficien-
cies 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 aquatic field
testing studies and makes a judgment as to whether they support the
requested registration action of the data submitter. If the data
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are not supportive, possible alternative action(s) that may be taken
by the registrant, such as label modification, are suggested. 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 aquatic field testing studies
should include all information necessary to provide: 1) a complete
and accurate description of the field treatments and procedures/
2) sampling data and phytotoxicity rating, 3) data on storage of
the plant material, if so performed, 4) any chemical analysis of
the plant material as to chemical content, if so performed, 5) re-
porting of the data, rating system and statistical analysis and
6) quality control measures/precautions taken to ensure the fidel-
ity of the operations.
Specifically, each aquatic field testing 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 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;
0 Active ingredient percentage in the end-use product or a
representative end-use product from the same major formulation
category for that general use pattern;
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0 Additional solvents or adjuvants used to dissolve and
apply the pesticide if the pesticide is insoluble in or immiscible
with water;
0 Dose rate(s) in terms of active ingredient per area of
land or final concentration in the test waters;
0 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 (< EC5Q level) and non-toxic
(no-observable-effeet-level) concentrations;
0 Method of application including equipment type, nozzles,.
pressure, etc.; and
0 Number of applications.
III. Plant Species
0 Identification of the test aguatic plant species as noted
below:
Plant Groups
Dicotyledonae (dicots)
Monocotyledonae (monocots)
Vascular Cryptogamae
(ferns and allies)
Bryophyta (mosses) or
Hepatophyta (liverworts)
Algae (including Cyanobacteria)
Number of
Family Representatives
1
3
Each Division Represented (5)
0 Identification of the cultivar(s) of the plant species used
and source, where available;
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.
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IV. Site of the Test
0 Site description of the aquatic field testing study as to
the type of system, e.g., enclosed, controlled areas of a lake,
pond, swamp, or stream, or artificial water systems such as aquaria,
or large tubs;
0 Location of the test site(s) as to whether the test was
performed in the following general geographical regions in which
the pesticide is to be used;
Northeastern temperate deciduous
Southeastern temperate deciduous
Northern grassland (cool prairie)
Southern grassland (warm prairie)
Northwestern (and Alaskan) conifer forest and high desert
Southwestern chaparral Mediterranean and low desert
Hawaiian and Caribbean semi-tropical and tropical regions
0 Climatological data during the test (records of applicable
conditions for the type of site, i.e., temperatures, thermoperiods,
rainfall or water regime, light regime - intensity and quality,
relative humidity, wind speed, etc.);
0 Physical environment characteristics such as tidal action,
water turbidity, flow rates, salinity, and degree of exposure of
the environment; and
0 Substrate characteristics (type of growth media including
its physical and chemical properties, including pH) including soil
type of bottom muds.
V. Results
0 Phytotoxicity rating (including a description of the rating
system) for each plant or plant population (individual container)
in the test;
0 Weight, size (vascular plants) or other growth parameters
that may have been measured to ascertain toxic effects of the
pesticide upon the plants; and
0 Statistical analysis of the results including environmental
or effective concentration (EC) values.
VI. Evaluation
0 Determination as to whether further aquatic field testing
with aquatic species is necessary.
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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 the scienti-
fic review report.
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/
supervising/monitoring and applying the pesticide provided?
0 Was the trial identification number provided?
— ,-- ... "Were quality assurance control measures/precautions indicated?
II. Test Chemical
0 Is the test chemical being used the end-use product or a
representative end-use product from the same major formulation
category for that general use pattern?
0 Is the active ingredient percentage of the chemical given?
0 If a solvent or adjuvant was used where the pesticide
end-use was immiscible with water, was it used at concentrations
that were not phytotoxic and was a solvent carrier used?
0 Is the dose given in quantity per unit area (of plant or
land surface) or in tank concentration (parts per million)?
0 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 the natural habitat or was it less than the one-fold concen-
tration as tested in Tier 1? An application of 1 Ib active ingredient
per acre or 1.12 kg per hectare is equal to 735 parts per billion
(ppb) in a 6-inch or 15-cm water column. If registrant has shown that
the maximum quantity that will be present in the non-target area is
significantly less than the maximum label rate (Tier 1), was the
maximum dose tested less than three times that maximum environ-
mental quantity?
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0 Were the additional dosages of a geometric progression 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 Were a subtoxic (< EC50) and a non-toxic concentration
evaluated?
III. Test Species
0 Were the test aquatic plant species tested representative of
the families or divisions noted below?
Number of
Plant Groups Family Representatives
Dicotyledonae (dicots) 1
Monocotyledonae (monocots) 3
Vascular Cryptogamae (ferns and allies) 3
Bryophyta (mosses) or Hepatophyta
(liverworts) (for wetland use-patterns,
i.e., forests) 1
Algae (including Cyanobactria) Each Division Represented (5)
0Where various culture types could be used, such as in the case
of most cultured algae, were culture types and sources identified?
0 Were species names of all test organisms given?
0 Were at least three replicates used with five plants per
replicate (where appropriate test conditions are available such as
in artificial test systems) for each dose level for the vascular
aquatic plants?
0 Were the initial cell concentrations for the algal tests
similar to those below for the various algal divisions tested?
Chlorophyta 3000 cells/mL
Chrysophyta (marine) 10000 cells/mL
Cyanobacteria 10000 cells/mL
Chrysophyta (freshwater) 3000 cells/mL
0 Were the date of the plant addition to the growth media
without test chemical (for stabilization of plant growth, if neces-
sary) , date of pesticide application, and date of phytotoxicity
rating or harvest and analyses identified?
0 Were endangered or threatened plant species not used?
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IV. Site of the Test
0 Was the site of the aquatic field testing study identified
as to the type of enclosed, controlled areas of a lake, pond, swamp,
or stream or in artificial water systems such as aquaria, or large
tubs?
0 Were the locations of the test site(s) within the following
general geographical regions in which the pesticide is to be used
provided?
Northeastern temperate deciduous
Southeastern temperate deciduous
Northern grassland (cool prairie)
Southern grassland (warm prairie)
Northwestern (and Alaskan) conifer forest and high desert
Southwestern chaparral Mediterranean and low desert
Hawaiian and Caribbean semi-tropical and tropical regions
0 Were the environmental conditions that prevailed during the
test (temperatures, thermoperiods, light regime - intensity and
quality, rainfall or watering regime, relative humidity, wind
speed, etc.) provided as appropriate for the site?
0 Were the physical environment characteristics such as tidal
action, water turbidity, flow rates, salinity, and degree of exposure
of the environment provided? ;
0 Were the substrate characteristics (type of growth media
including its physical and chemical properties, including pH)
including soil type of bottom muds provided?
0 Where an artificial culture media was used, was it identi-
fied?
V. Test Procedures
0 If modifications in the environmental conditions were used
and reported, was their use substantiated?
0 Was the test duration for vascular plant (including Bryo-
phyta) growth two weeks to four weeks in length?
0 Were observations to note vascular plant growth and re-
sponse to the pesticide taken at least twice weekly with the last
observation occurring at least two weeks after the last application?
0 Was the test duration for non-vascular plant growth (pri-
marily the algae) at least five days in length with at least one
observation during the middle of the test?
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0 Was the method of pesticide application including the
type of application equipment employed given?
VI. Reporting
0 Were the detrimental effects such as severity of phyto-
toxicity (rating or percentage), percent plant kill, or percent
dieback reported?
0 If a rating system was used, was an explanation pro-
vided?
0 Were abnormal changes in growth, development, and/or mor-
phology reported with comparisons to the controls?
° Was the growth of higher plants expressed as the number
of original plants or plant parts and the number of additional
plants and/or parts?
0 Was the growth of the algae expressed as cell count per mL,
biomass per volume, or degree of growth as determined by spectrophoto-
metric means? If spectrophotometrie means were used, was some attempt
made to equate the absorbance readings to number of cells or biomass?
VII. Evaluation
0 were the results tabulated to indicate a percentage effect
level for each species as compared to the untreated control plants?
0 Were 25 and 50 percent detrimental effect levels (EC25 and
EC50) determined for those plant species that showed a phytotoxic
effect to the chemical?
0 Was a determination made as to whether additional aquatic
field phytotoxicity tests were necessary to evaluate the effects of
the pesticide on non-target plants?
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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 - Tier 3 -
Aquatic Field Testing study.
Chemical: (Common Name)
Formulation: (Percent Active Ingredient)
Study/Action: (Purpose of the Submission)
Study Identification:
Reviewer:
Approval:
Conclusions:
(Subdivision J Test Title)
(Reference or Registrant Data Information with
Study Number)
(EPA Accession Number)
(Name and Address of Reviewer; Date of Review)
(Quality Control Reviewer) •- -
(Summary and Conclusion of Tests)
Acceptability and Recommendations:
Background:
Discussion:
(Decide as to (1) .the scientific validity of the
study and (2) compliance to the Subdivision J -
Aquatic Field Testing guidelines)
(Introductory Information and Directions for Use)
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. 1981. "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
Little, T. M., and e. J. Hills. 1978. Agricultural Experimentation
Design and Analysis. New York: John Wiley and Sons.
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
6 US. GOVERNMENT PRINTING OFFICE: 1986 - 621-735 - 1302/60519
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