
United States
Environmental Protection Agency
   Office of Chemical Safety
   and Pollution Prevention
   (7101)
EPA 712-C-011
January 2012


Ecological Effects
Test Guidelines



OCSPP 850.4150:

Vegetative Vigor




                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
  
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  NOTICE
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  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.
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  	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
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  	For additional information about these test guidelines and to access these guidelines electronically, please go to http://www.epa.gov/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.
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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 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 (lbs/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 IC05 to IC90) 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 IC05) 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 control response (i.e., EC25 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 testing, at a minimum ten plant species from the plant groups and families identified in paragraphs (e)(3)(i)(A)(1) through (e)(3)(i)(A)(2) 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
Solanaceae
Lycopersicon esculentum or Solanum lycopersicum
Tomato

Cucurbitaceae
Cucumis sativus
Cucumber

Asteraceae
Lactuca sativa
Lettuce

Fabaceae[1]
Glycine max
Soybean

Brassicaceae
Brassica oleracea
Cabbage

Apiaceae
Daucus carota
Carrot

Chenopodiaceae
Beta vulgaris
Sugar beet or table beet

Asteraceae
Helianthus annuus
Sunflower

Fabaceae
Pisum sativum
Pea

Brassicaceae
Brassica rapa
Field mustard, Canola

Brassicaceae
Brassica napus
Turnip, Rape

Fabaceae
Phaseolus vulgaris
Garden bean

Malvaceae
Gossypium spp.
Cotton

Polygonaceae
Fagopyrum esculentum

Buckwheat
Monocots
Poaceae
Avena sativa
Oat

Poaceae
Lolium perenne
Perennial ryegrass

Poaceae
Zea mays
Corn

Liliaceae
Allium cepa
Onion
[1] Inoculation with Rhizobium japonicum 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.
            (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, IC05), test substance concentrations should immediately bracket the point 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 lb/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 (IC05).  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 IC05) 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. lb/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).  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 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.
      (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 ([o]C) while temperature during the night should be 20 +- 6 [o]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 umol/m[2]/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.
            (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 [o]C) and the total plant biomass (i.e. dry weight) in each replicate is measured and recorded.  Biomass measurements should be made for each 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.
            (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. 
		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) EC25 and IC25 values.  To ascertain that the survival EC25 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 (H0) stated in terms of true population parameters is that the difference of the "limit" treatment mean response (ulimit) from the control mean response (ucontrol) is greater than or equal to a 25% inhibition or reduction, compared to the control (i.e., H0: ucontrol - ulimit > δ0).  The alternative hypothesis (HA) is that this difference is less than a 25% reduction, compared to the control (i.e., HA: ucontrol - ulimit < δ0).  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).
		Equation 2
            where:
                  δ0 = difference between two parameters, defined in this case as a p percent reduction from the control sample mean;
                   = 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 IC25 and EC25 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 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 EC25 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 IC05 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 EC05 value) is estimated and used in place of the given NOEC.  The standard error and 95% confidence interval should also be calculated for the IC05 and EC05 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.

	Table 2. -- Summary of Test Conditions for Vegetative Vigor Test
Test type
Vegetative vigor test
Test duration
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).
Substrate
Natural or synthetic soil with 3% organic matter and pH 6.0 - 7.5
Nutrients
As naturally available, or supplemented with either a soil fertilizer or watered with nutrient solution 
Temperature 
25/20 [o]C (daytime/nighttime) +- 6 [o]C
Relative humidity
70% (daytime) +- 15%
Light intensity
350 +- 50 umol/m[2]/sec at the top of the canopy
Photoperiod
16 hours light: 8 hours dark (for non-crop species see Appendix 1 of this guideline)
Watering
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.
Test chamber (pot) size
Varies with plant species selected.  Six-inch diameter plastic pots are typical.  Flats are also encouraged.
Number of plants per test chamber
Varies with species and test chamber (pot) size.
Number of replicates per test treatment
6 (a minimum of 4 if increase number of seedlings per replicate to 10)
Number of plants per test treatment
30 plants (a minimum of 40 seedlings if there is only 4 replicates)
Test treatment levels
Unless performing limit test, minimum of 5 treatment levels plus appropriate controls
Test substance application method
Applied to foliage
Measures of effect or measurement endpoints
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 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
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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.

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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)(1) 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.
            (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.
      	(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.
      	(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 EC05 or IC05, 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 IC05 value is used in place of a NOEC provide a description of statistical method(s) used for point estimates, including software package, for determining IC05 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 D.J. Versteeg, 1992. A statistical procedure for modeling continuous toxicity data. Environmental Toxicology and Chemistry 11:1485-1494.
	(3) Gulley, D.D. et al., 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.
