&EPA
United States
Environmental Protection
Agency
Office of Chemical Safety
and Pollution Prevention
(7101)
EPA712-C-011
January 2012
Ecological Effects
Test Guidelines
OCSPP 850.4150:
Vegetative Vigor
-------
NOTICE
This guideline is one of a series of test guidelines established by the United States
Environmental Protection Agency's Office of Chemical Safety and Pollution Prevention
(OCSPP) for use in testing pesticides and chemical substances to develop data for
submission to the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601,
et seq.), the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, et
seq.), and section 408 of the Federal Food, Drug and Cosmetic (FFDCA) (21 U.S.C. 346a).
Prior to April 22, 2010, OCSPP was known as the Office of Prevention, Pesticides and Toxic
Substances (OPPTS). To distinguish these guidelines from guidelines issued by other
organizations, the numbering convention adopted in 1994 specifically included OPPTS as
part of the guideline's number. Any test guidelines developed after April 22, 2010 will use
the new acronym (OCSPP) in their title.
The OCSPP harmonized test guidelines serve as a compendium of accepted scientific
methodologies and protocols that are intended to provide data to inform regulatory decisions
under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the
test, and is also used by EPA, the public, and the companies that are subject to data
submission requirements under TSCA, FIFRA, and/or the FFDCA. As a guidance
document, these guidelines are not binding on either EPA or any outside parties, and the
EPA may depart from the guidelines where circumstances warrant and without prior notice.
At places in this guidance, the Agency uses the word "should." In this guidance, the use of
"should" with regard to an action means that the action is recommended rather than
mandatory. The procedures contained in this guideline are strongly recommended for
generating the data that are the subject of the guideline, but EPA recognizes that departures
may be appropriate in specific situations. You may propose alternatives to the
recommendations described in these guidelines, and the Agency will assess them for
appropriateness on a case-by-case basis
For additional information about these test guidelines and to access these guidelines
electronically, please go to http://www.epa.qov/ocspp and select "Test Methods &
Guidelines" on the left side navigation menu. You may also access the guidelines in
http://www.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-
0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.
-------
OCSPP 850.4150: Vegetative vigor.
(a) Scope—
(1) Applicability. This guideline is intended to be used to help develop data to submit to
EPA under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the
Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and
the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 U.S.C. 346a).
(2) Background. The source materials used in developing this harmonized OCSPP test
guideline include the OPP 122-1 Seed Germination/Seedling Emergence and Vegetative
Vigor (Tier I) and 123-1 Seed Germination/Seedling Emergence and Vegetative Vigor
(Tier II) guidelines (Pesticide Assessment Guidelines Subdivision J—Hazard Evaluation:
Nontarget Plants); OPP Standard Evaluation Procedure Nontarget Plants Vegetative
Vigor (Tier I and 2); OECD 208 Terrestrial Plants, Growth Test; OECD 227 Terrestrial
Plant Test: Vegetative Vigor Test, and ASTM E 1963-02, Standard Guide for Conducting
Terrestrial Plant Toxicity Tests. This guideline incorporates what were formerly Public
Drafts OCSPP 850.4150 and OCSPP 850.4250 (April, 1996).
(b) Purpose. This guideline is intended for use in developing data on the toxicity of chemical
substances and mixtures ("test chemicals" or "test substances") subject to environmental effects
test regulations. The objective of the vegetative vigor test is to measure the effect of a foliar
applied test substance upon terrestrial plants during the vegetative growth period of their
development. Procedures for testing a single exposure concentration (i.e. Tier I testing for
pesticides) as well as procedures for testing multiple exposure concentrations (i.e. Tier II testing
for pesticides) are described. This guideline should be used in conjunction with OCSPP
850.4000, which provides general information and overall guidance for the guidelines in OCSPP
Series 850, Group D. The Environmental Protection Agency will use data from vegetative vigor
tests in assessing the hazard a test substance may present in the terrestrial environment.
(c) Definitions. The definitions in OCSPP 850.4000 apply to this guideline. In addition, the
more specific definitions in this paragraph also apply:
Biomass is defined as all portions of the plant above the soil surface (i.e., does not
include roots).
Shoot height is defined as the length of the above-ground vegetation from the soil surface
to the apical tip or highest aerial part of the plant.
(d) General considerations—
(1) Summary of the test. The vegetative vigor test is a test that evaluates the affect of a
test substance upon the growth of a number of plant species. Effects measured include
survival, plant height, and plant biomass. In addition, qualitative phytotoxic effects are
observed and evaluated. The test is designed to determine the quantity of test substance
required to cause a 25 percent (25%) inhibition in plant height and plant biomass (IC25
values and to determine the no observed effect concentration (NOEC) for these effect
measures. A number of crop and non-crop plant species may be used. It may be
Page 1 of20
-------
performed in a growth chamber, greenhouse, or in small field plots. A natural or
synthetic soil serves as the substrate and the test substance is sprayed on the foliage at test
initiation. The results are used to establish toxicity levels, evaluate hazards or risks to
terrestrial plants, and to indicate if further testing at a higher tier is necessary. Note
historically in OCSPP pesticide and industrial chemical guidelines the term ECX was used
to cover both the current OCSPP 850.4000 definition of ECX (concentration where x% of
the population exhibit the effect (e.g., survival)) and ICX (concentration resulting in an x%
decrease or inhibition effect on an attribute of the population (e.g., plant yield)).
(2) General test guidance. The general guidance in OCSPP 850.4000 applies to this
guideline, except as specifically noted herein.
(3) Range-finding test. A range-finding test is usually conducted to establish the
appropriate test concentrations to be used for the definitive test. In the range-finding test,
the test organisms are exposed to a series of widely-spaced concentrations of the test
substance (e.g., 1, 10, 100 pounds per acre (Ibs/acre)). The details of the range-finding
test do not have to be the same as for definitive testing in that there are no replicates, and
the number of test organisms used, and the duration of exposure may be less than in
definitive testing. In addition, the types of observations made on test organisms are not
as detailed or as frequently observed as that of a definitive test and results are analyzed
using nominal concentrations. However, the range-finding test will be more useful the
greater the similarity between the range-finding and the definitive test.
(4) Definitive test. The goal of the definitive test is to determine for each measure of
effect (shoot height and shoot biomass) its concentration-response curve and NOEC (and
lowest observed effect concentration (LOEC)); the shoot height and weight IC25 values
(with 95% confidence intervals and standard errors). If possible the slopes of the
concentration-response curves, associated standard errors, and the 95% confidence
intervals of the slopes should also be determined. However, at a minimum, the full
concentration-response curve (concentration range covers ICos to ICgo) is determined for
the most sensitive measure of effect using a minimum of five concentrations of the test
chemical, plus appropriate controls. Recommend adding one or two additional test
concentrations in the lower tail of the concentration-response curve for the most sensitive
endpoint to ensure bracketing of both the most sensitive NOEC (or ICos) and IC25 values.
For a satisfactory test, the lowest treatment concentration is below both the shoot height
and biomass IC25 values. A summary of test conditions is provided in Table 2 and
validity elements for an acceptable definitive test are listed in Table 3.
(5) Limit test. In some situations, it is only necessary to ascertain that the EC25 value
and the shoot height and shoot biomass IC25 values for a given plant species occur above
a certain limit concentration, and that at this limit concentration there is no observable
adverse effect. In a vegetative vigor limit test, at least 40 seedlings (divided into at least
4 replicates of 10 plants each) are exposed to a single "limit concentration," with the
same number of replicates and organisms in appropriate controls. The multiple-
concentration definitive test may be waived for a given test species if the following two
conditions are met for survival, shoot height, shoot weight, and phytotoxic ranking. First,
the "limit" treatment response is both statistically less than a 25% decrease from the
Page 2 of20
-------
control response (i.e., ECzs and IC25 values > limit concentration), and second, the limit
treatment responses are not significantly reduced (or inhibited) as compared to the control
responses (i.e., NOEC > limit concentration). The limit concentration is 3 times the
estimated environmental concentration for industrial chemicals or for pesticides the limit
concentration is based upon the maximum label rate, e.g. the maximum recommended
amount of active ingredient (a.i.), in the recommended minimum quantity of carrier (such
as water), to be used per land area. Results are reported in grams or pounds of a.i. per
acre. Except for the number of treatment groups, an acceptable limit test follows the
same test procedures, is the same duration, and has the same number of controls as the
definitive test. Acceptable limit tests like definitive tests include analytical confirmation
of the applied dose.
(e) Test standards—
(1) Test substance. For pesticides the substance to be tested is the typical end-use
product (TEP). If there is more than one TEP with the same inert substances, the one
with the highest percent a.i. and/or the one most commonly used should be tested. If
there is more than one TEP with different inert substances, a TEP representative of each
different inert substance should be tested in the range-finding test and at a minimum the
most sensitive one tested in the definitive or limit test. In addition, if an adjuvant is
recommended for use on a TEP label, the adjuvant is added with the TEP at the label rate
to constitute the test substance. If a TEP is not available (i.e. new pesticide), the
technical grade a.i. is used with a representative formulation, which should include any
adjuvant that will be recommended for use on the label. If products are applied in a tank
mixture, dosages of each a.i. should be reported with identification and formulation for
each product in the tank mix. For industrial chemicals the substance to be tested should
be technical grade, unless the test is designed to test a specific formulation or mixture.
OCSPP 850.4000 lists the type of information that should be known about the test
substance before testing.
(2) Test duration. The limit and definitive tests last for 21 days after test substance
application. If phytotoxic symptoms first start to appear between day 14 and 21 post-
application, the test is extended to 28 days post-application.
(3) Test organisms—
(i) Species. The test is performed using crop and/or non-crop terrestrial plant
species selected from a cross-section of the terrestrial plant species that have been
historically used for this type of testing. A list of crop and non-crop plant taxa
that have been used in toxicity tests can be found in Table 1 of this guideline and
Table 2 of the OCSPP 850.4100 guideline. Endangered or threatened species as
determined by the Endangered Species Act of 1973 (Public Law 93-205) may not
be used without permission from the Fish and Wildlife Service.
(A) Number of species tested. For testing industrial chemicals, the
specific plant(s) used are selected on a case-by-case basis. For pesticide
Page 3 of20
-------
testing, at a minimum ten plant species from the plant groups and families
identified in paragraphs (e)(3)(i)(A)(7y) through (e)(3)(i)(A)(%) are tested:
(1) Dicotyledoneae: Six species of at least four families, one
species of which is soybean (Glycine max).
(2) Monocotyledoneae: Four species of at least two families, one
species of which is corn (Zea mays).
(3) At least one test species should be a root crop (either a monocot
such as onion or dicot such as carrot, table beet or sugar beet).
(B) Crop species. A representative list of crop species which are
acceptable test species are listed in Table 1.
Table 1.—Crop species and families acceptable for use in the vegetative vigor test
Family
Species
Common name
Dicots
Monocots
Solanaceae
Cucurbitaceae
Asteraceae
Fabaceae1
Brassicaceae
Apiaceae
Chenopodiaceae
Asteraceae
Fabaceae
Brassicaceae
Brassicaceae
Fabaceae
Malvaceae
Polygonaceae
Poaceae
Poaceae
Poaceae
Liliaceae
Lycopersicon esculentum or
Solarium lycopersicum
Cucumis sativus
Lactuca sativa
Glycine max
Brassica oleracea
Caucus carota
Beta vulgaris
Helianthus annuus
Pisum sativum
Brassica rapa
Brassica napus
Phaseolus vulgaris
Gossypium spp.
Fagopyrum esculentum
Avena sativa
Lolium perenne
Zea mays
Allium cepa
Tomato
Cucumber
Lettuce
Soybean
Cabbage
Carrot
Sugar beet or table beet
Sunflower
Pea
Field mustard, Canola
Turnip, Rape
Garden bean
Cotton
Buckwheat
Oat
Perennial ryegrass
Corn
Onion
Inoculation with Rhizobiumjaponicum is unnecessary.
(C) Non-crop plant species. When selecting plant species other than the
three crop species (soybean, corn, and a root crop (onion, carrot, table beet
or sugar beet)), which are tested at a minimum for pesticide testing, the
use of sensitive non-crop plant species is recommended. Table 2 of
OCSPP 850.4100, which is based on a table from the reference in
paragraph (j)(5) of this guideline, provides a list of recommended non-
crop species.
Page 4 of20
-------
(ii) Source. Within a given test, all seedlings of a given species, including the
controls, should be from the same source and lot number, and same cultivation
group.
(iii) Age or growth stage and size. Seedlings used in testing are selected from
groups of plants grown from seed to the 2 to 4 true leaf stage. Selected seedlings
should also be of uniform size.
(iv) Condition. Healthy plants should be used. For cultivation of seedlings from
seed, healthy seeds should be used with reported germination rate for a seed lot of
70% or better. Seeds should be examined before use and sorted to remove broken
or damaged seeds.
(v) Care and handling. Seeds, used to cultivate seedlings for this test, should be
stored in a desiccator and refrigerated until needed. Pesticide treated seeds should
be avoided except for approved seed treatments. The Agency should be consulted
prior to test initiation if seed treatments other than a weak hypochlorite solution
(recommended by Environment Canada), captan, or thiram are used. Captan and
thiram seed treatments (nonsystemic mode of action; see paragraph (j)(4) of this
guideline) are the only Agency-approved pesticide seed treatments. When
unapproved pesticide seed treatments are used in a study, the test should be
designed to demonstrate no synergistic or antagonistic interactions occur between
or among the seed treatment and test substance. Steam sterilization of soil is
recommended as a non-pesticide alternative for killing pathogens, fungi, and
insects in soil media.
(4) Administration of test substance. Within 48 hours prior to the application of the
test substance, seedlings of similar size and condition (morphological symptoms) are
selected for use in the test, and the height and condition of each plant is determined and
recorded. At test initiation, test substance is applied to the seedling foliage as a spray.
(i) Preparation of spray solutions. The amount of water used in the spray as a
carrier is equivalent to the recommendation on the pesticide label. For example,
vegetation within a given pot (test container) is sprayed with x milligrams of TEP
mixed into 10 milliliters of water, where the 10 milliliters of water per the pot
area is equivalent to the minimum number of gallons per acre specified on the
label. For a satisfactory test, all pots in all treatment levels and controls have the
same equivalent volume of water applied (e.g., in the example just given this is 10
milliliters).
(ii) Treatment levels.
(A) For a given plant species, five treatment levels are tested at a
minimum. A range-finding test can be used to establish the appropriate
test doses for the definitive test (see paragraph (d)(3) of this guideline).
For scientifically sound estimates of a given point estimate (e.g., IC25,
ICos), test substance concentrations should immediately bracket the point
Page 5 of20
-------
estimate(s). The concentrations should be a geometric progression of
twofold at a minimum (e.g., 0.1, 0.2, 0.4, 0.8, and 1.6 Ib/acre). While a
twofold progression is preferred, threefold and fourfold progressions are
acceptable. If a fourfold series progression is used, the rationale for using
this large an interval between concentrations and the effect on the
accuracy and reproducibility of the point estimate or NOEC should be
provided. For an acceptable study for a given plant species, the lowest test
treatment level should be lower than the IC25 values for shoot height and
biomass for that plant species. The NOEC should be determined by
hypothesis testing for each effect measure. The lack of a NOEC for an
effect measure is not critical as long as the response-curve for the effect
measure is acceptable for calculation of the 5% inhibition concentration
(ICos). It is recommended that one or two additional test concentrations in
the lower tail of the concentration-response curve of the most sensitive
endpoint be added to insure bracketing of both the most sensitive IC25
value and the most sensitive NOEC (or ICos) value.
(B) For pesticides dosages should be expressed as mass of test substance
per unit of soil surface area in the pot or container (i.e. Ib/acre).
Additionally for pesticides, dosages should be expressed in unit mass of
a.i. or acid equivalent per unit of land area treated, as appropriate.
(C) The use of pesticide treatments to control pests during the test should
be avoided. Mechanical, cultural, and biological pest control methods are
suggested. If other pesticides are used in the test for pest control, for a
satisfactory test a demonstration should be performed (i.e., additional test
data) documenting that the pesticide is not toxic to the test species and that
no synergistic or antagonistic interactions with the test substance exist
(additional test data).
(5) Controls.
(i) Every test includes controls consisting of the same support medium,
conditions, procedures, seedling source, seed source and lot, except that no test
substance is added. In addition, vehicle (solvent) controls are also included if a
solvent is used. However for pesticides, because TEP is typically tested, solvents
are generally not necessary.
(ii) If the negative control is contaminated with the test substance, the study is not
acceptable and should be repeated.
(iii) A test is not acceptable if at test termination less than 90% of control plants
survive.
(6) Number of test organisms and replicates.
(i) For each species, the minimum number of test organisms is 30 seedlings per
dose level (a minimum of six replicates, each with a minimum of 5 seedlings).
Page 6 of20
-------
Alternatively, the number of test organisms is 40 seedlings per dose level (a
minimum of four replicates, each replicate with a minimum of 10 seeds). A test is
not acceptable if a container is too small, resulting in overcrowding and
competition among plants in the container. The recommended loading in a 6 inch
container for corn, soybean, tomato, cucumber or sugar beets is one to two
seedlings; the loading for rape, onion, wheat, or other small seed species is three
to four seedlings. To prevent bias, seedlings are impartially or randomly assigned
to the containers (pots, flats) or plots. Within a given test, all test organisms of a
given species, including the controls, should be from the same source and
cultivation group. Note that a replicate in this case will consist of several pots
and/or flats and the integrity of the replicate should be maintained throughout the
duration of the study (i.e. pots/flats from one replicate are not moved to or mixed
with another replicate and if the replicate is moved all pots/flats composing the
replicate are moved as a unit). Do not mix species within a replicate.
(ii) Randomization.
(A) Placement of the flats or pots within the greenhouse or arrangement of
the field plots should follow a random or randomized block design. To
minimize spatial differences, which can have a significant impact upon
plant growth, the placement of replicates should be randomized during the
test (at least every three to five days).
(B) Alternative placement should be used with volatile test substances to
prevent cross contamination. For example, in a greenhouse setting use
positive air flow throughout the duration of the study with placement of
controls and treatment levels such that air flows first across controls and
then from the lowest treatment to the highest treatment. In addition,
include another set of controls which are placed in a separate greenhouse
for comparison with control results from the greenhouse containing the
treatments. Where tests are conducted using growth chambers where
positive air flow can not be achieved, the controls and each treatment level
should be placed in separate growth chambers.
(7) Facilities, apparatus and supplies—
(i) Reliability. All equipment used in conducting the test, including equipment
used to prepare and administer the test substance, and equipment to maintain and
record environmental conditions, should be of such design and capacity that tests
involving this equipment can be conducted in a reliable and scientific manner.
Equipment should be inspected, cleaned, and maintained regularly, and be
properly calibrated.
(ii) Application equipment. The application equipment used in testing products
in small field plot studies should be designed to simulate conventional farm
equipment using the basic components of commercial application equipment in
the design of the small-plot equipment. For example, nozzle types, sizes, and
Page 7 of20
-------
arrangements on small plot sprayers should be identical to those used by growers
on commercial ground sprayers. The application equipment used in greenhouse
or growth chamber tests (such as a spray chamber or computerized belt sprayer)
should provide uniform coverage and should be calibrated at spray volumes (to
the extent practical) that are representative of field conditions. Acceptable foliar
spray methods are found in the reference in paragraph (j)(8) of this guideline.
The amount of water used in spray as a carrier is to be equivalent to the
recommendation on the label.
(iii) Facilities. Vegetative vigor tests should be conducted under controlled
conditions in greenhouses and growth chambers, or under ambient conditions in
small field plots.
(iv) Test containers. Test containers should be nonporous so that the test
substance is not absorbed or does not react in any way with the container. Glass
or stainless steel containers with drainage holes can be used as plant pots.
Polyethylene plastic pots or flats that have not been previously used are
acceptable test containers, provided they are free of toxic materials. Do not use
clay or peat containers. Containers should be thoroughly cleaned prior to use. A
dichromate solution should not be used to clean containers. The volume of the
pot should be large enough so that seedling growth is not restricted during the
test, see paragraph (e)(6)(i) of this guideline. Containers should be consistent
within species.
(v) Support medium. Plants may be grown in a natural soil (free of pesticide
contamination) or synthetic soil. Growth chamber and greenhouse tests are
performed using a sterilized standardized soil that consists primarily of sandy
loam, loamy sand, loamy clay, or clay loam soil that contains up to 3% organic
matter (up to 1.5% organic carbon). Commercial potting soil or synthetic soil
mixes may be used as the soil medium provided that the organic matter does not
exceed 3%. Clay soils should not be used if the test substance is known to have a
high affinity for clay. Field soils should be sieved to remove coarse (greater than
2 millimeters) particles and recommend pasteurizing or heat treating the soil to
reduce the effect of soil pathogens. The soil pH may be adjusted to the optimum
growing range of 6.0 to 7.5 by the addition of a basic substance (calcium
carbonate) or an acidic substance (gypsum, ammonium sulfate, or sulfuric acid).
A slow-release fertilizer may be added to the soil to provide nutrients for plant
growth.
(vi) Plant supply. Seeds are planted and cultivated ahead of time to provide
sufficient seedlings at the 2- to 4-true leaf stage of growth (typically one to four
weeks post-emergence) and of uniform size to initiate the test for a given test
species on the same day. Thinning the seedlings to uniform size and condition is
performed before use. For non-crop species guidance on the time to germination
may be found in Table 2 of OCSPP 850.4100.
Page 8 of20
-------
(8) Environmental conditions. For greenhouse and growth chamber studies, general
conditions in paragraphs (e)(8)(i) through (e)(8)(v) of this guideline are recommended;
however, excursions outside these recommendations do not invalidate the study if other
acceptance criteria are met. Additional guidance for non-crop species is provided in
Table 2 of OCSPP 850.4100.
(i) Temperature. Air temperature should be uniform throughout the greenhouse
or growth chamber. Air temperature during the day should be 25 ± 6 degrees
Celsius (°C) while temperature during the night should be 20 ± 6 °C.
(ii) Humidity. Humidity should be uniform throughout the greenhouse or growth
chamber. Relative humidity should approach 70 ± 15% during light periods.
(iii) Lighting and photoperiod. Luminance of 350 ± 50 |imol/m2/sec, measured
at the top of the canopy, is desirable, on a photoperiod of 16 hours light and 8
hours darkness. Artificial lighting may be used to lengthen short-day periods or
to supplement natural sunlight on overcast days. Care should be taken to ensure
that plants are not affected from the heat generated from supplemental lighting.
(iv) Watering. For greenhouse and growth chambers, top watering below the
canopy is used at the first watering after the test substance has been applied to
initiate the capillary movement of water for bottom watering. Bottom watering of
test containers is used for the duration of the study in order to prevent washing the
chemical off the foliage and through the soil during watering. The watering
method should prevent the test substance from washing off the foliage or leaching
out of the soil or the pots/flats.
(v) Nutrients. Nutrients may be supplied during the study by using a nutrient
solution of defined chemical composition, such as half-strength modified
Hoagland nutrient solution, to water the plants. Alternatively, nutrients may be
supplied by amending the test soil at the start of the test with fertilizer including
standard nutrients for the species tested.
(9) Observations—
(i) Measurement of test substance. The dosing solution (i.e. spray tank) is
sampled at the start of and end of application and analyzed for the test substance
concentration (in a.i. or acid equivalent units for pesticides). The total volume of
dosing solution used, and the total soil surface area per pot or container in a
replicate and treatment level should be recorded. The analytical methods used to
measure the amount of test substance in a sample are validated before beginning
the test, as described in OCSPP 850.4000.
(ii) Support medium. Characteristics of a batch of soil representative of that
used in the study or the native soil (for tests done in field plots) are determined,
including soil type and texture, pH, particle size distribution, and organic matter
content.
Page 9 of20
-------
(iii) Environmental conditions. Environmental conditions to be monitored in
greenhouses and growth chambers include air temperature, humidity, and light
intensity. For field plots, environmental conditions should be monitored at the
field site during and after test substance application and daily throughout the
duration of the study. Environmental information to be collected in the field
should include air temperature, precipitation, relative humidity, wind speed, light
intensity at the canopy, and cloud cover. Source of environmental field condition
data can be taken from a weather station that is within 5 miles of the field.
(A) Air temperature and humidity. The air temperature and humidity
during the study should be recorded at representative locations throughout
the area in which the test plants are growing. Measurements are made
preferably continuously, but alternatively as maximum and minimum
values over each 24-hour period.
(B) Light intensity. Light intensity at the canopy should be determined
every 3 to 5 days at representative locations throughout the area in which
the test plants are growing. A photosynthetically active radiation (PAR)
sensor should be used to measure light quality. For field plots, ideally
PAR measurements should be taken between eleven in the morning and
two in the afternoon. Additional information on the use of lighting in
plant toxicity tests can be found in the references given in OCSPP
850.4000.
(C) Watering and precipitation. Frequency of watering should be
recorded for greenhouse and growth chamber studies, and observations of
moisture stress should be made and recorded daily. Frequency of watering
and frequency and amount of precipitation should be recorded for field
studies, and observations of moisture stress should be recorded daily.
(D) Pests. Daily observations should be made on pest pressure using an
index of the extent of infestation. Pest infestation may affect the
interpretation of study results and therefore should be adequately
described. Frequency, methods, and rates used for treating an insect or
disease should be recorded.
(iv) Phytotoxic Effects
(A) Observations on the number of alive and dead plants, and visual
morphological symptoms of phytotoxicity are made and recorded at least
on days 7, 14 and 21 post-application, and if the test is extended (see
paragraph (e)(2) of this guideline) on day 28. Shoot height for each
individual surviving plant is recorded at a minimum on days 14 and 21
post-application, and if the test is extended on day 28. At test termination
after shoot heights are measured, plants are dried (constant weight at 70
°C) and the total plant biomass (i.e. dry weight) in each replicate is
measured and recorded. Biomass measurements should be made for each
Page 10 of 20
-------
replicate not each individual plant.
(B) Seedling condition is determined using a standard morphological
phytotoxicity rating scale. Observations on morphological symptoms of
phytotoxicity should include all variations, either inhibitory or stimulatory,
between the treated and the untreated organisms. Such variations may be
phytotoxic symptoms (e.g., chlorosis, necrosis, pigmentation, leaf curling
and wilting) and formative (e.g., leaf and stem deformation) effects.
Observations should include the treatment level and replicate, stage of
development and dates when adverse results occurred, subsided or
recovered, and counts for each plant affected. Uniform scoring procedures
should be used to evaluate the observable toxic responses. Such data
should include the actual values used to determine any percentages of
effects. Any lack of effects by the test substance should also be reported.
(f) Treatment of results—
(1) Summary statistics—
(i) Environmental conditions. Air temperature, humidity, and light intensity
data should be summarized in tabular form, showing the mean, standard
deviation, and range during the test at each measurement location. For field
studies precipitation events and the amount of precipitation should be summarized
in tabular form by date of occurrence, and total precipitation calculated. Watering
frequency and duration should be summarized in tabular form by date of
occurrence.
(ii) Test substance concentration. Compare the initial test substance dosing
solution concentration with test substance concentration in the dosing solution at
end of application. If the substance was not stable calculate a rate of decline of
the test substance. The total volume of dosing solution used, total area treated,
and the time application started and ended for a given treatment level summarized
in tabular form.
(iii) Shoot height. Calculate and plot the average shoot height and standard
deviation of shoot height for each replicate and the treatment level mean shoot
height and standard deviation at each observation period (including test
initiation).
(iv) Shoot biomass. Calculate and plot the average plant dry weight (total dry
weight biomass divided by number of surviving plants) for each replicate and the
treatment level mean shoot weight and standard deviation.
(v) Survival. Calculate and plot the percent survival (number of surviving
seedlings divided by the number of seedlings at test initiation) for each replicate
and the treatment level mean and standard deviation percent survival at each
observation time. For each treatment level calculate mean percent survival as a
percentage of the control mean percent survival at test termination.
Page 11 of 20
-------
(vi) Phytotoxic effects. Morphological symptoms of plant injury should be
summarized in tabular form by time of observation, treatment, and replicate.
Definition of any index values used for morphological symptoms, indicating the
severity of the symptom(s), should be provided.
(2) Percent inhibition. For shoot height and shoot biomass calculate the percent
inhibition (%I) at each treatment level using Equation 1 .
0/T
%/ = -i - ii — L Equation 1
where:
C = the control mean response value (shoot height or shoot biomass); and
X = the treatment mean response value (shoot height or shoot biomass,
respectively). Stimulation is reported as negative %I.
(3) Limit test—
(i) ECis and ICis values. To ascertain that the survival £€25 value and shoot
height and shoot biomass IC25 values for a given plant species occur above the
"limit" concentration, a one-sided test which compares the difference between
two sample groups to a fixed value (or difference) is performed for each of these
response measures. For a comparison of sample means, the difference defining
the EC25 and IC25 compared to controls is operationally defined as a 25%
reduction or inhibition from the control sample mean (Equation 2). The null
hypothesis (Ho) stated in terms of true population parameters is that the difference
of the "limit" treatment mean response (niimit) from the control mean response
(^control) is greater than or equal to a 25% inhibition or reduction, compared to the
control (i.e.., H0: |icontroi - Hiimit > So). The alternative hypothesis (HA) is that this
difference is less than a 25% reduction, compared to the control (i.e.., HA: ^control -
Hiimit < So). An example of a parametric two-sample comparison test of this is the
Student's t-test. If the null hypothesis is rejected, the effect level or inhibition
level for the given response measure (i.e., survival, shoot height and biomass) in
the limit treatment as compared to the control is declared to be less than 25%
(e.g., IC25 > limit dose). If the null hypothesis is not rejected, the effect level or
inhibition level in the limit treatment as compared to the control response is
declared to be 25% or greater (e.g., IC25 < limit dose).
S0=(xamtml)^{p/I00) Equation 2
where:
So = difference between two parameters, defined in this case as ap percent
reduction from the control sample mean;
Page 12 of 20
-------
^control = control sample mean response (e.g., survival, shoot height and
biomass); and
p = percent reduction from the control sample mean, which is 25 in the
case of the EC25 and IC25.
(ii) NOEC. To ascertain that there is no observable effect at the limit treatment
(i.e., NOEC > limit dose) for a given response measure (survival, shoot height and
biomass), the limit treatment response is compared to the control treatment
response using a one-sided two-sample parametric or nonparametric test, as
appropriate (see OCSPP 850.4000). The minimum significant difference
detectable by the test or a similar estimate of the sensitivity of the test should be
determined and reported.
(iii) Multiple-dose definitive testing.
(A) A multiple-dose definitive test is performed for a given test species if
either the effect or inhibition level for one or more response measures (i.e.,
survival, shoot height and biomass) in the limit treatment as compared to
the control response at test termination are declared to be 25% or greater
effect (i.e., the null hypothesis is not rejected) or the NOEC is less than the
limit concentration.
(B) Multiple-dose definitive testing may be waived for a given test species
if at test termination the "limit" treatment response is both statistically less
than a 25% decrease from the control response and there is no observable
adverse effect from the control response for all measures of effect
(survival, shoot height and biomass).
(4) Multiple-dose definitive test —
(i) Dose-response curves, slopes and ICis and EQs values —
(A) Shoot height and shoot biomass. For dose-response-response tests
the IC25 value (standard error and 95% confidence interval) is calculated
for each of shoot height and shoot biomass (see OCSPP 850.4000 and
references in paragraphs (j)(2) and (j)(9) of this guideline for statistical
guidance). If a dose-response curve was fit to the data to determine the
IC25, the model parameters (e.g., slope) and their uncertainty estimates
(e.g., standard error) should be recorded. Where the dose-response range
tested does not result in the determination of a definitive IC25 value for a
given response measure, test and document that the IC25 value is above the
highest treatment level tested (see the statistical guidance in OCSPP
850.4000 and in paragraph (f)(3) of this guideline). Such an event may
arise if one of the other response measures is much more sensitive, and
while the full response curve for that response measure is captured too
many additional treatments would be needed to capture the full response
Page 13 of 20
-------
relationship for the other less sensitive response measure(s). Methods,
assumptions, and results of the statistical approaches used should be
recorded.
(B) Percent survival. The EC25 value (standard error and 95%
confidence interval) is calculated for percent survival (see OCSPP
850.4000 for statistical guidance). If a dose-response curve was fit to the
data to determine the EC25 the model parameters (e.g., slope) and their
uncertainty estimates (e.g., standard error) should be recorded. Where the
dose-response range tested does not result in the determination of a
definitive £€25 value, test and document that the EC25 value is above the
highest treatment level tested (see statistical guidance in OCSPP 850.4000
and in paragraph (f)(3) of this guideline). Such an event may arise if one
of the other response measures is much more sensitive, and while the full
response curve for that response measure is captured too many additional
treatments would be needed to capture the full response relationship or a
definitive EC25 for survival. Methods, assumptions, and results of the
statistical approaches used should be recorded.
(ii) NOEC. The NOEC (and LOEC) for each response measure (survival, shoot
height and shoot weight) is determined (see OCSPP 850.4000 and the reference in
paragraph (j)(3) of this guideline). If a NOEC value can not be determined for a
given response measure, as appropriate, the dose at which there is a 5% inhibition
(i.e., an ICos value for shoot height and shoot weight) or the dose at which there is
a 5% reduction in survival of the exposed population (i.e., an ECos value) is
estimated and used in place of the given NOEC. The standard error and 95%
confidence interval should also be calculated for the ICos and ECos values.
Methods, assumptions, and results of the statistical approaches used should be
recorded.
(g) Tabular summary of test conditions. Table 2 lists the important conditions that should
prevail during the definitive test. Except for the number of test treatments, Table 2 also lists the
important conditions that should prevail during a limit test. Meeting these conditions will greatly
increase the likelihood that the completed test will be acceptable or valid.
Page 14 of 20
-------
Table 2.—Summary of Test Conditions for Vegetative Vigor Test
Test type
Test duration
Substrate
Nutrients
Temperature
Relative humidity
Light intensity
Photoperiod
Watering
Test chamber (pot) size
Number of plants per test chamber
Number of replicates per test treatment
Number of plants per test treatment
Test treatment levels
Test substance application method
Measures of effect or measurement endpoints
Vegetative vigor test
Minimum of 21 days after test substance application
(extended to 28 days post-application if phytotoxic
symptoms initially show between days 14 and 21 post-
application).
Natural or synthetic soil with 3% organic matter and pH
6.0-7.5
As naturally available, or supplemented with either a
soil fertilizer or watered with nutrient solution
25/20 °C (daytime/nighttime) ± 6 °C
70% (daytime) ±15%
350 ± 50 umol/m2/sec at the top of the canopy
16 hours light: 8 hours dark (for non-crop species see
Appendix 1 of this guideline)
For greenhouse and growth chamber, the initial
watering event is by top watering below the canopy and
bottom watering is used throughout the remainder of
the study. In field plots top watering below the canopy
is used.
Varies with plant species selected. Six-inch diameter
plastic pots are typical. Flats are also encouraged.
Varies with species and test chamber (pot) size.
6 (a minimum of 4 if increase number of seedlings per
replicate to 1 0)
30 plants (a minimum of 40 seedlings if there is only 4
replicates)
Unless performing limit test, minimum of 5 treatment
levels plus appropriate controls
Applied to foliage
IC25 and NOEC (or IC05) for each of shoot height and
shoot weight
EC25 and NOEC (or EC05) for survival
(h) Test validity elements. This test would be considered to be unacceptable or invalid if one or
more of the conditions in Table 3 did not occur or one or more performance objectives in Table 3
were not met. This list should not be misconstrued as limiting the reason(s) that a test could be
found unacceptable or invalid. However, except for the conditions listed in Table 3 and in
OCSPP 850.4000, it is unlikely that a study will be rejected when there are slight variations from
guideline environmental conditions and study design unless the control organisms are
significantly affected, the precision of the test is reduced, the power of a test to detect differences
is reduced, and/or significant biases are introduced in defining the magnitude of effect on
measurement endpoints as compared to guideline conditions. Before departing significantly
from this guideline, the investigator should contact the Agency to discuss the reason for the
departure and the effect the change(s) will have on test acceptability. In the test report, all
Page 15 of 20
-------
departures from the guideline should be identified, reasons for these changes given, and any
resulting effects on test endpoints noted and discussed.
Table 3.—Test Validity Elements for the Vegetative Vigor Test
1. Mean control plant survival is at least 90% at test termination.
2. Control seedlings do not contain any visible phytotoxic symptoms during the test that are the same as
due to the test substance.
3. For a given species, all seedlings in a test are from the same cultivation group and source.
4. All test chambers used for a particular species should be identical and should contain the same
amount of soil from the same source.
5. A negative (untreated) control [and solvent (or vehicle) control, when a solvent was used] is included in
the test.
6. For a given plant species, the lowest test concentration level was below both the shoot height and
biomass IC25 values for the plant species.
7. If pesticides are used for pest control during the test, additional test data was submitted to document
that the pesticide used was not toxic to the test species and that there were no synergistic or antagonistic
interactions with the test substance.
8. The water carrier used at the time of test substance application did not excessively exceed the amount
of water on the label (gallon per acre).
(i) Reporting—
(1) Background information. Background information to be supplied in the report
consists at a minimum of those background information items listed in paragraph (j)0) of
OCSPP 850.4000.
(2) Guideline deviations. Provide a statement of the guideline or protocol followed.
Include a description of any deviations from the test guideline or any occurrences which
may have influenced the results of the test.
(3) Test substance.
(i) Identification of the test substance: common name, IUPAC and CAS names,
CAS number, structural formula, source, lot or batch number, chemical state or
form of the test substance, and its purity (i.e. for pesticides, the identity and
concentration of active ingredient(s)).
(ii) Storage conditions of the test chemical or test substance and stability of the
test chemical or test substance under storage conditions if stored prior to use.
(iii) Methods of preparation of the test substance and the treatment doses used in
the range-finding and definitive test, or limit test.
Page 16 of 20
-------
(iv) If a vehicle (e.g., solvent, dust) is used to prepare stock or test substance
provide: the name and source of the vehicle, the nominal concentration(s) of the
test substance in the vehicle in stock solutions or mixtures, and the vehicle dose(s)
used in the treatments.
(4) Plant test species.
(i) Scientific and common name, plant family, and variety.
(ii) History of the young test plants including source, name of supplier, batch or
lot number of the seed.
(iii) Test date of germination rating and germination percentage.
(iv) Date of planting.
(v) Pre-test planting and seedling cultivation conditions, grow out conditions and
handling including pest and disease treatments, for the young plants before use in
the test.
(vi) Height and morphological condition (i.e., any abnormalities, pigmentation,
wilting, etc.) of seedlings selected for the test.
(5) Test system and conditions. Description of the test system and conditions used in
the definitive or limit test, and any preliminary range-finding tests.
(i) Location of testing, and if not a field plot study the type of growth chamber or
greenhouse.
(ii) For small field plot testing describe the plot design: size of field plots, number
of control and experiment plots, the number of plots per treatment and control, the
plot lay-out, the number of plants in each plot.
(iii) For greenhouse or growth chambers the description of the containers, pots
and flats: type, material, dimensions, and the soil surface area per pot or test
container.
(iv) Number of seedlings per pot/flat.
(v) Number of pots or flats per replicate, and number of replicates per treatment
level.
(vi) Description of the support medium: source, soil type designation, soil
composition, pH, percent organic matter, type and amounts of soil amendments.
(vii) Methods used for treatment randomization and impartial assignment of
plants to test plots, pots, and/or flats.
Page 17 of 20
-------
(viii) Method of test substance application: equipment type, equipment design,
method for calibrating the application equipment, dose levels, volume of dosing
solution applied.
(ix) Date of test substance application and test duration (date plants were
harvested at test termination).
(x) Culture practices during the test such as cultivation, pest control, and irrigation
practices (type and watering schedule or regime).
(xi) The photoperiod and light source.
(xii) Methods and frequency of environmental monitoring performed during the
definitive or limit study for air temperature, humidity, and light intensity and
additionally for field plot tests rainfall and cloud cover.
(xiii) For the definitive, or limit test, all analytical procedures should be
described. The accuracy of the method, method detection limit, and limit of
quantification should be given.
(6) Results.
(i) Environmental monitoring data results (air temperature, humidity and light
intensity) in tabular form (provide raw data for measurements not made on a
continuous basis), and descriptive statistics (mean, standard deviation, minimum,
maximum).
(ii) For preliminary range-finding tests, if conducted, the number of surviving
plants, and shoot and biomass, if measured, at each dose level and in the
control(s). A description and count of visual phytotoxic effects, if recorded, at
each dose level and in the control(s).
(iii) For a limit test, tabulate for the limit concentration and the control by
replicate, the number of seedlings exposed and their shoot height at test initiation,
the number of surviving and number of dead seedlings at each observation time,
and the shoot height and biomass at test termination (provide the raw data).
(iv) For the definitive test, tabulation by treatment and replicate the number of
seedlings exposed and their shoot height at test initiation, the number of surviving
and number of dead seedlings at each observation time, and the shoot height for
each individual plant and total plant biomass at test termination (provide the raw
data).
(v) For the limit and definitive tests, tabulation by treatment of the percent
reduction in mean height, biomass, and survival as compared to control plants at
test termination.
Page 18 of 20
-------
(vi) For the limit and definitive test, a description of visual (morphological) signs
of phytotoxicity including: time of onset, duration, severity (e.g. rank), and
number affected at each dose level and control(s) (provide the raw data). A
description of the phytotoxicity rating system used should be included.
(vii) Graphs of the dose-response data for shoot height and weight at test
termination.
(viii) For a limit test, provide the results of hypothesis tests.
(ix) For the limit test, provide a description of the statistical methods used
including software package, and the basis for the choice of method.
(x) For the definitive study and for those effect measures (shoot height and
weight) with data sufficient to fit a concentration-response relationship, tabulation
of the slope of the dose-response curve and its standard error and 95% confidence
limits and any goodness of fit results.
(xi) For the definitive test, tabulation of IC25 values for plant height and plant
biomass.
(xii) For the definitive test, a tabulation of the NOEC and LOEC for each measure
of effect (plant height, and plant biomass). The ECos or ICos, as applicable,
should be reported for effect measure data where an NOEC could not be
determined.
(xiii) Description of statistical method(s) used for point estimates, including
software package, for determining IC25 values, fitting the dose-response model,
and the basis for the choice of method. Provide results of any goodness-of-fit
tests.
(xiv) Description of statistical method(s) used for NOEC and LOEC
determination, including software package, and the basis for the choice of
method. If an ICos value is used in place of a NOEC provide a description of
statistical method(s) used for point estimates, including software package, for
determining ICos values, fitting the dose-response model, and the basis for the
choice of method. Provide results of any goodness-of-fit tests.
(j) References. The following references should be consulted for additional background
material on this test guideline.
(1) American Society for Testing and Materials. ASTM E 1963-02. Standard guide for
conducting terrestrial plant toxicity tests. In Annual Book of ASTM Standards, Vol.
11.06, ASTM, West Conshohocken, PA. Current edition approved December 10, 2002.
(2) Bruce, R.D. and DJ. Versteeg, 1992. A statistical procedure for modeling continuous
toxicity data. Environmental Toxicology and Chemistry 11:1485-1494.
Page 19 of 20
-------
(3) Gulley, D.D. etal., 1989. Toxstat Release 3.0. University of Wyoming, Laramie, WY.
(4) Hatzios, K.K. and D. Penner, (1985). Interactions of herbicides with other
agrochemicals in higher plants. Rev. Weed Sci. 1:1-63.
(5) Organization for Economic Cooperation and Development (OECD). 2006. New Test
Guideline (Section 2- Effects On Biotic Systems), 227, Terrestrial Plant Test: Vegetative
Vigour Test, 21pp. Adopted on 19 July 2006 and published as part of the 17th
Addendum to the OECD Guidelines for the Testing of Chemicals, 972006131, ISBN 92-
64-01553.
(6) OECD. 2006. Revised Test Guideline (Section 2), 208, Terrestrial Plant Test:
Seedling Emergence and Seedling Growth Test, 21pp. Adopted on 19 July 2006 and
published as part of the 17th Addendum to the OECD Guidelines for the Testing of
Chemicals, 972006131, ISBN 92-64-01553-1.
(7) U.S. Environmental Protection Agency, 1982. Pesticide Assessment Guidelines
Subdivision J, Hazard Evaluation: Nontarget Plants. Office of Pesticides and Toxics,
Washington, D.C. EPA 540/9-82-020, October 1982.
(8) U.S. Environmental Protection Agency, 1986. Hazard Evaluation Division Standard
Evaluation Procedure, Nontarget Plants: Vegetative Vigor -Tiers 1 and 2. Office of
Pesticides Programs, Washington, D.C. EPA 540/9-86-133, June 1986.
(9) VanEwijk, P.H. and J.A. Hoekstra, 1993. Calculation of the EC50 and its confidence
interval when a subtoxic stimulus is present. Ecotoxicology and Environmental Safety
25:25-32.
Page 20 of 20
------- |