United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA 712-C-96-157
April 1996
&EPA Ecological Effects Test
Guidelines
OPPTS 850.4450
Aquatic Plants Field
Study, Tier III
I
Public Draft"
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Introduction
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7 U.S.C. 136, etseq.).
Public Draft Access Information: This draft guideline is part of a
series of related harmonized guidelines that need to be considered as a
unit. For copies: These guidelines are available electronically from the
EPA Public Access Gopher (gopher.epa.gov) under the heading "Environ-
mental Test Methods and Guidelines" or in paper by contacting the OPP
Public Docket at (703) 305-5805 or by e-mail:
guidelines@epamail.epa.gov.
To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs, Field Operations Division (7506C), Environmental
Protection Agency, 401 M St. SW., Washington, DC 20460. In person:
bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Ar-
lington, VA. Comments may also be submitted electronically by sending
electronic mail (e-mail) to: guidelines@epamail.epa.gov.
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202-512-0135 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from the EPA Public Access Gopher
(gopher.epa.gov) under the heading "Environmental Test Methods and
Guidelines."
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OPPTS 850.4450 Aquatic plants field study, Tier III.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of the Federal Insecticide, Fungicide, and Rodenticide
Act (FIFRA) (7 U.S.C. 136, et seq.).
(2) Background. The source material used in developing this har-
monized OPPTS test guideline is OPP 124-2 Aquatic Field Testing (Pes-
ticide Assessment Guidelines, Subdivision J—Hazard Evaluation;
Nontarget Plants) EPA report 540/09-82-020, 1982.
(3) Test objective. This guideline should be used in conjunction with
OPPTS guideline 850.4000, Background—Nontarget plant testing, which
provides general information and overall guidance for the nontarget plants
test guidelines.
(i) General. (A) This guideline should be used in conjunction with
OPPTS guideline 850.4000, Background—Nontarget plant testing, which
provides general information and overall guidance for Group D of the 850
series. Aquatic field testing studies (Tier III) 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 nontarget 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 registration of any pesticide intended for outdoor use under
the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA), as
amended.
(B) 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, and wick applications. However, if any
of these use patterns do readily expose nontarget plants to the pesticide,
the pesticide phytotoxicity potential may need to be evaluated.
(ii) Objective of the aquatic field testing Tier III test. (A) The
objective of the Tier III 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
nontarget aquatic plant population. It is also performed under natural con-
ditions and in the environment in which the pesticide is to be applied.
By this procedure, direct assessments can be made as to the potential
phytotoxicity of the pesticide. This is a multiple dose test designed to
evaluate the phytotoxic effects of the pesticide over as wide a range of
anticipated pesticide quantities as may be found in the environment.
(B) The Tier III aquatic nontarget plant phytotoxicity field studies
are required if greater than 50 percent adverse effects on plant growth
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for aquatic plants are expected to occur when the product is used as di-
rected by the label (the estimated environmental concentration exceeds the
EC50 aquatic values). The Tier III tests are expected to provide informa-
tion on detrimental effects to plants during critical stages of development.
The typical end-use product (TEP) is used to assess effects on a broader
range of nontarget plant species in a number of geographic areas.
(b) Test standards. In addition to the general test standards set forth
in OPPTS 850.4000, the test standards for this guideline are the same as
those in OPPTS 850.4400, with the following modifications:
(1) Test substance. The test substance is to be the end-use product
or a representative end-use product from the same major formulation cat-
egory for that general use pattern such as wettable powders, emulsifiable
concentrates, and granulars. (If the manufacturing-use product is usually
formulated into end-use products comprising two or more major formula-
tion categories, a separate study must be performed with a typical end-
use product for each category.) The test substance should contain the high-
est percentage of active ingredient (AI) and/or be the most widely used
(total pounds AI).
(2) Application levels. The dosages tested should be the same as
those specified in the Tier II aquatic test standards in guideline OPPTS
850.4400.
(3) Species, (i) Aquatic plant representatives of the following plant
groups are to be tested:
(A) Dicotyledonae (dicots), one representative.
(B) Monocotyledonae (monocots), representatives of three families.
(C) Vascular Cryptogamae (ferns and allies), representatives of three
families.
(D) Algae (including Cyanophyta), a representative of each division.
(E) Bryophyta (mosses) or Hepatophyta (liverworts), one representa-
tive (not required for true aquatic use patterns, rather for wetland use pat-
terns).
(ii) Plant species used for testing Tiers I and II can be used to satisfy
the monocot and dicot test plant requirements of this guideline.
(iii) Additional plant species may be required if the general selectivity
of the pesticide cannot be readily identified.
(4) Environmental conditions, (i) Plants may be grown in either na-
tive soil, water, or other substrate of similar nature to that of the indige-
nous area or under other conditions similar to the natural habitat.
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(ii) Reduction of light intensity by natural or constructed light shade
may be necessary to simulate the reduced light intensities found with cer-
tain plant communities such as deeply submerged sites or shaded waters.
(iii) Other natural conditions should also be maintained where plants
are removed from their natural habitat. Soil, water, and air temperatures
should approximate those of the natural habitat. For estuarine and marine
habitats, the following conditions should, to the extent possible, simulate
the natural environment: Tidal action, water turbidity, flow rates, salinity,
and degree of exposure.
(iv) Tests should be performed either in enclosed, controlled areas
of a lake, pond, or swamp, or in large water cultures such as aquaria or
plastic wash tubs. Tests are not to be performed in dynamic or flowing
water where the release of the chemical cannot be contained or its escape
prevented.
(v) The field studies should be conducted using acceptable protocols
as may be found in paragraph (e)(5) of this guideline Truelove, B., 1977,
Research Methods in Weed Science, 2nd Ed. Southern Weed Science Soci-
ety, Auburn Printing Inc., Auburn, AL or a protocol with prior approval
of the Agency.
(5) Duration. The test duration should be of sufficient length to as-
sess multiple applications directed by the label. Observations should con-
tinue for the entire life cycle of test plants, with observations every 2 to
4 weeks.
(6) Season of application. The test substance is to be applied over
the period of time or season according to the proposed label instructions.
(7) Test locations. The pesticide should be tested in those geographic
locations where it is expected to be used, as based on proposed label use
sites. Where important species diversity and physiographic differences
occur within a region of intended application, regional testing may be inad-
equate, and testing at a more specific region or biome level may be re-
quired. United States regional areas of potential testing include North-
eastern temperate deciduous, Southeastern temperate deciduous, Northern
grassland (prairie), Southern grassland (prairie), Northwestern (and Alas-
kan) conifer forest and high desert, Southwestern chaparral Mediterranean
and low desert, and Hawaiian and Caribbean tropical regions.
(c) Reporting. In addition to the information required by OPPTS
850.4000 and 850.4400, the test report should include the test conditions
(including soil, water, and environmental conditions) and the determination
of the 50 percent detrimental effect level.
(d) Data reporting. (1) The registrant's report on aquatic field testing
studies should include all information necessary to provide:
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(1) A complete and accurate description of the field treatments and
procedures.
(ii) Sampling data and phytotoxicity rating.
(iii) Data on storage of the plant material, if so performed.
(iv) Report any chemical analysis of the plant material.
(v) Reporting of the data, rating system and statistical analysis.
(vi) Quality control measures/precautions taken to ensure the fidelity
of the operations.
(2) Each aquatic field testing report should include the following in-
formation:
(1) General. (A) Cooperator or researcher (name and address), test
location (county and state; country, if outside of the United States), and
date of study.
(B) Name (and signature), title, organization, address, and telephone
number of the persons responsible for planning/supervising/monitoring
and, for field plot studies, applying the pesticide.
(C) Trial identification number.
(D) Quality assurance measures employed:
(7) Control measures/precautions followed to ensure the fidelity of
the phytotoxicity determinations.
(2) Record-keeping procedures and availability of logbooks.
(3) Skill of the laboratory personnel.
(4) Equipment status of the laboratory or greenhouse.
(5) Degree of adherence to good laboratory practices.
(6) Degree of adherence to good agricultural practices in maintaining
healthly plants.
(E) 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). (A) Identification of the test pesticide
AI including chemical name, common name (ANSI, BSI, ISO, WSSA),
and company developmental/experimental name.
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(B) Percentage of AI in the end-use product or a representative end-
use product from the same major formulation category for that general
use pattern.
(C) Additional solvents or adjuvants used to dissolve and apply the
pesticide if the pesticide is insoluble in or immiscible with water.
(D) Dose rates in terms of AI per area of land or final concentration
in the test waters.
(E) Dose rates in terms of less than the maximum label rate as though
it were applied directly to the surface of a 15-cm or 6-in water column
or in terms of less than the onefold concentration as tested in Tier I with
dosages in a geometrical progression of no more than twofold and with
subtoxic (
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(B) Location of tbe test sites 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 de-
ciduous, Northern grassland (cool prairie), Southern grassland (warm prai-
rie), Northwestern (and Alaskan) conifer forest and high desert, Southwest-
ern chaparral Mediterranean and low desert, Hawaiian and Caribbean
semi-tropical and tropical regions.
(C) Climatological data during the test (records of applicable condi-
tions for the type of site, i.e., temperatures, thermoperiods, rainfall or water
regime, light regime—intensity and quality, relative humidity, wind speed,
etc.).
(D) Physical environment characteristics such as tidal action, water
turbidity, flow rates, salinity, and degree of exposure of the environment.
(E) Substrate characteristics (type of growth media including its phys-
ical and chemical properties, including pH) including soil type of bottom
muds.
(v) Results. (A) Phytotoxicity rating (including a description of the
rating system) for each plant or plant population (individual container) in
the test.
(B) Weight, size (vascular plants) or other growth parameters that
may have been measured to ascertain toxic effects of the pesticide upon
the plants.
(C) Statistical analysis of the results including environmental or effec-
tive concentration (EC) values.
(vi) Evaluation, (i) Determination as to whether further aquatic field
testing with aquatic species is necessary.
(e) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) Davis, J.A. Comparison of static-replacement and flow-through
bioassays using duckweed, Lemna gibba G-3. EPA Report No. EPA 560/
6-81-003 (1981).
(2) Hoist, R.W. et al. Effect of several pesticides on the growth and
nitrogen assimilation of Azolla-Anabaena symbiosis. Weed Science 30:54-
58 (1982).
(3) Little, T.M., and F.J. Hills. Agricultural Experimentation—Design
and Analysis. Wiley, NY (1978).
(4) Sculthorpe, C.D. The Biology of Aquatic Vascular Plants. Lon-
don. Arnold Publishers (1967).
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(5) Truelove, D. Research Methods in Weed Science, 2nd Ed., South-
ern Weed Science Society, Auburn Printing Inc., Auburn, AL (1977).
(6) Voshell, J.R. Jr. Using Mesocosms to Assess the Aquatic Ecologi-
cal Risk of Pesticides: Theory and Practice. Miscellaneous Publication
No. 75, 34th Annual Meeting of the Entomological Society of America,
Nov. 29-Dec. 3, 1987MPPEAL 75: 1-88 (1987).
(7) Touart, L. W. and M. W. Slimak. Mesocosm Approach for As-
sessing the Ecological Risk of Pesticides. Miscellaneous Publication No.
75, 34th Annual Meeting of the Entomological Society of America, Nov.
29—Dec. 3, 1987. MPPEAL 75: 1-88 (1987).
(8) DeNoyelles, F. Jr. et al. Use of Experimental Ponds to Assess
the Effects of A Pesticide on the Aquatic Environment. Miscellaneous
Publication No. 75, 34th Annual Meeting of the Entomological Society
of America, Nov. 29 - Dec. 3, 1987. MPPEAL 75: 1-88 (1987).
(9) Siefert, R. E. et al. Littoral Enclosures for Aquatic Field Testing
of Pesticides: Effects of Chlorpyrifos on a Natural System. Miscellaneous
Publication No. 75, 34th Annual Meeting of the Entomological Society
of America, Nov. 29—Dec. 3,1987. MPPEAL 75: 1-88 (1987).
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