United States

Environmental Protection 

Agency

Office of Chemical Safety

and Pollution Prevention

(7101)

EPA 712-C-002

January 2012

Ecological Effects

Test Guidelines

OCSPP 850.4800:

Plant Uptake and 

Translocation Test
i

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.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.
Page 1 of 24

OCSPP 850.4800: Plant uptake and translocation test.

(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 material used in developing this harmonized OCSPP test 

guideline is the OPPT guideline under 40 CFR 797.2850 Plant Uptake and Translocation 

Test.

(b) Purpose. This guideline is intended for use in developing data on the uptake and 

translocation, by terrestrial plants, of chemical substances and mixtures (“test chemicals” or “test 

substances”) subject to environmental effects test regulations. This guideline describes tests 

using commercially important terrestrial plants to develop data on the quantity of test substances 

incorporated in plant tissues and the potential for entry into food chains with resultant indirect 

human and/or wildlife exposure. The test design is a composite of prior plant experiments in 

which the uptake of heavy metals, pesticides, or organic chemicals has been investigated (see 

paragraphs (j)(2) to (j)(12) of this guideline). Test substances of concern may be transported to 

various sites as gases or dust, solubilized in preparation or in irrigation water, or may be 

encountered in soils. It is well known that plants readily take up, translocate, accumulate, and 

metabolize chemicals that are non-essential for plant growth and development. Such uptake and 

incorporation often represents the first step in the transport of these substances within terrestrial 

food webs. The uptake and translocation test addresses this concern of entry into food webs. 

This test is not concerned with toxicity to plants; it deals with hazard to animals and the potential 

for indirect human exposure. The Environmental Protection Agency will use data from this test 

to assess the hazard or risks a test substance may present in the terrestrial environment. This 

guideline should be used in conjunction with the OCSPP 850.4000 guideline, Background and 

special considerations for conducting ecological effects tests with terrestrial and aquatic plants, 

cyanobacteria, and terrestrial soil core microcosms.

(c) Definitions. The definitions in the OCSPP 850.4000 guideline apply to this test guideline. 

In addition, the following more specific definitions also apply:

Mass balance refers to a quantitative accounting of the distributions of test substance in 

plant components, support media, and test solutions. For gas fumigation with the test 

substance, it also refers to a quantitative determination of uptake as the difference 

between the quantity of gas entering an exposure chamber, the quantity leaving the 

chamber, and the quantity adsorbed to the chamber walls.

Translocation is the transference or transport of chemical from the site of uptake to other 

plant components.

Uptake refers to a process by which substances are transferred into a terrestrial plant.
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(d) General considerations―

(1) Summary of the test. In preparation for the test, seeds are planted in potting

containers (or in cotton or glass-wool plugs supported in hydroponic solution) and, after 

germination, seedlings are thinned, by pinching the stem at the support surface to 1 to 3 

plants per pot, depending on the species, and discarding the pinched off seedlings. 

Potting mixtures of sand, glass beads or soil are subirrigated with nutrient solution. The 

test substance is applied to the plants via nutrient solution or adsorbed to the support 

media to produce a root exposure scenario; or applied to the plants by either spraying or 

dusting the foliage or exposing the plants to gas in a fumigation chamber to produce a 

foliar exposure scenario. An alternative root exposure scenario involves application of 

the test substance to soil after the seeds have been planted but prior to germination. 

Plants are then harvested when there is either adequate material for analysis or until fruit 

or seeds are mature. If plants are fumigated, either rates of uptake and surface adsorption 

should be calculated, or the plants may be harvested and analyzed for test chemical and 

residues. The primary endpoints for the plant uptake and translocation test are the 

concentrations of free parent test substance, metabolites and soluble residues, and bound 

residues in pooled plant organs and pooled whole plants. For gaseous test substances, in 

addition to or in lieu of these measurements, calculated measurements of photosynthesis, 

transpiration, and stomatal conductance before, during, and after exposure to the test 

substance are made.

(2) General test guidance. The general guidance in the OCSPP 850.4000 guideline 

applies to this test 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.

(i) Because of the different mechanisms involved in root and leaf uptake, and to 

more closely define the chemical concentrations to be used in the uptake test, the 

definitive early seedling growth test (see the OCSPP 850.4230 guideline) is 

recommended as the range-finding test for tests with industrial chemicals. The 

exposure technique used in the early seedling growth test (root or foliar exposure) 

should reflect the anticipated fate of the test substance. The concentration 

selected as the upper limit for the uptake and translocation test should be near to 

but below the threshold of visible injury (e.g., NOEC). Short exposure periods to 

gas in fumigation chambers are not expected to promote visible injury or gross 

reductions in growth but may alter stomatal resistance, transpiration, or 

photosynthesis. Absorption and adsorption rates may be calculated and gas 

concentrations for definitive testing selected based on the calculated sorption 

rates.

(ii) Alternatively, a seed germination or root elongation test or other appropriate 

phytotoxicity test (see paragraph (j)(1) of this) may be used to establish the 

appropriate upper concentration for testing. For tests with pesticides, which are 

applied to surface soil, the seedling emergence and seedling growth test (see the 

OCSPP 850.4100 guideline) is recommended as the range-finding test.
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(4) Definitive test. The purpose of the definitive uptake and translocation test is to 

determine the propensity for a test substance to accumulate in plants or plant parts. The 

test substance should be added to the hydroponic or nutrient solution, coated on glass 

beads, or applied to the soil surface (for the root uptake test); or sprayed, dusted, or 

gassed directly on the foliage (for the foliage uptake test). Two different plant species are 

used. The test consists of one run for each of the two specified plant species. For a 

particular chemical, a run is defined as exposure of the plant species to three 

concentrations of the test chemical with a minimum of six replicate pots per treatment 

level and appropriate controls. Exposure is followed by extraction and analysis for parent 

compound, metabolites, and bound residues in plant tissues for solid and liquid test 

substances and by calculating these values and/or rates of absorption and adsorption for 

gas test substances. Analytical confirmation of the test concentrations in exposure media 

is performed as described in the OCSPP 850.4000 guideline. The elements of an 

acceptable or valid definitive test are given in Table 3 of this guideline.

(e) Test standards―

(1) Test substance. For industrial chemicals, the substance to be tested should be 

technical grade unless the test is designed to test a specific formulation, mixture, or enduse product. 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 active ingredient (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 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. The OCSPP

850.4000 guideline lists the type of information that should be known about the test 

substance before testing, and discusses methods for preparation of test substances.

(2) Test duration. The duration of the exposure period, for solid and liquid test 

substances, should be equal to the length of time required for the particular test species to 

achieve sufficient biomass for analysis or until fruit or seeds are mature. Carrot, lettuce, 

onion, cabbage, and ryegrass may be harvested whenever there is adequate plant material 

for chemical analysis. Cucumber, corn, soybean, tomato, and oats should be grown until

fruit or seed are mature. The test duration for gaseous test substances should be similar, 

or may be reduced to the length of time required to make the specified gas exchange 

measurements if these measurements are used as the test endpoints.

(3) Test organism―

(i) Species. 

(A) The test is performed using two plant species of potentially differing 

sensitivity, such as a monocotyledonous and a dicotyledonous species. It 
Page 4 of 24

is further suggested that the test plants selected should be of different 

growth forms, e.g., a root crop and a leaf crop. Plant species and groups 

recommended for testing are listed in Table 1.

(B) A listing of some additional plant taxa that have been used in toxicity

tests can be found in the reference in paragraph (j)(1) of this guideline. 

Use of these alternative species should be submitted to and reviewed by

the Agency prior to conduct of the study. 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.

Table 1.—Species and families acceptable for the plant uptake and translocation test

Family Species Common name

Dicots Solanaceae Lycopersicon esculentum or 

Solanum lycopersicum

Tomato

Cucurbitaceae Cucumis sativus Cucumber

Asteraceae Lactuca sativa Lettuce

Fabaceae1 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 Rape, Turnip

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

(ii) Source. Within a given test, all test organisms of a given species, including 

the controls, should be from the same source and lot number.

(iii) Growth stage at test initiation. 

(A) Except for surface soil applications, newly germinated plants are used 

at test initiation for root (applied in nutrient solution or sorbed to support 

medium) and foliage exposures. The plants should be exposed from 

seedlings through maturation when monitoring residues in fruit or seeds.
Page 5 of 24

(B) Where the test substance is applied or deposited on the soil surface, 

the test is initiated using seeds which have been planted but have not yet

germinated.

(iii) Condition. Healthy seeds should be used to generate seedlings. Within a 

given test, all test organisms of a given species, including the controls, should be 

selected to have uniform size. Seeds should be selected from a single class size 

within each species. The seeds of most test species are sorted by the supplier 

according to size; however, if necessary, the seeds should be further sorted prior 

to use to provide a more uniform size class that reduces the potential for bias from 

testing different sizes of seed. In addition broken or damaged seeds should be 

separated from those used in the test and discarded.

(iv) Care and handling. 

(A) Seeds should be stored in a dessicator and refrigerated until needed.

(B) Pesticide treated seeds should be avoided. 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 are the only approved 

pesticide seed treatments (non-systemic with most other pesticides; see 

paragraph (j)(9) of this guideline). Steam sterilization of soil is 

recommended as a non-pesticide alternative for killing pathogens, fungi, 

and insects in soil media. 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.

(C) If plants are to be grown hydroponically seeds should be planted in 

plugs of cotton or glass-wool supported in the tops of the containers. 

When sand or glass beads are used, the recommended planting procedure 

is to fill potting containers with sand or glass beads to within 2.5 

centimeters (cm) of the top and to sow seeds directly on the support 

medium.

(D) When soil is used, for pesticides, the recommended planting procedure 

is to fill potting container with soil medium (see the OCSPP 850.4100 or 

OCSPP 850.4150 guidelines) to within 2.5 cm of the top and sow seeds in 

the soil.

(E) A sufficient number of seeds should be planted (e.g. 30) to provide 

more seedlings than called for in the test. After 50 percent (50%) of the 

seeds have germinated, the seedlings should be thinned, by pinching the 

stem at the support surface, to the desired number (typically, one to four 

per pot, depending upon the species). Any plants subsequently emerging 

are also pinched off.
Page 6 of 24

(F) Alternate planting methods may be required when the test substance is 

highly volatile. An impervious barrier of polyethylene film, a 

modification of the double pot method, a glass plate, or other appropriate 

apparatus should be used to prevent volatilization from the root zone. 

Seeds should be germinated in the dark at 25 degrees Celsius (

oC) and 

seedlings with radicle length in the median range transplanted into the 

potting containers. The seedlings should be positioned such that their 

roots are exposed to the support media while the shoots pass through holes 

in the barrier. A ring of inert, non phytotoxic, pliable putty should be used 

to seal the holes around the stems. Control pots should be handled 

identically except there is no exposure to the test substance. This 

transplanting procedure, without the volatilization barrier, is also 

recommended when the test substance is adsorbed to the support medium.

(4) Administration of test substance―

(i) Exposure pathway. The choice of exposure technique is dependent upon the 

expected route of exposure of plants in the environment and the form of the test 

substance.

(A) Root exposure. 

(1) If the anticipated fate of the test substance is incorporation into 

soil or soil-water, and the mechanism of concern is root uptake, the 

test substance should be applied in nutrient solution to the root 

support media, or coated on sand or glass beads (for non-watersoluble test substances).

(2) For pesticides where the test substance is expected to be 

applied or deposited on the soil surface, the test substance should 

be applied to the soil surface prior to germination of seed but after 

the seeds have been planted.

(B) Foliar exposure. With a test substance whose anticipated mode of 

exposure to plants is surface deposition by atmospheric transport or 

irrigation water, the appropriate testing method may be foliar application 

(accomplished by spraying, dusting, or fumigating the plants), allowing 

subsequent movement into the rooting zone with watering.

(ii) Preparation of exposure treatment―

(A) Root exposure. Except for test substances that are purposely applied 

to surface soil (such as pesticides), root exposure is accomplished by

adding the test substance to the nutrient medium (or hydroponic solution) 

used to subirrigate the plants, as described in paragraphs (e)(4)(ii)(A)(1)

and (e)(4)(ii)(A)(2) of this guideline for soluble and insoluble test 

substances, respectively, or by sorbing the test substance to the sand or 

glass beads used as support medium, as described in paragraph 
Page 7 of 24

(e)(4)(ii)(A)(3) of this guideline. The test substance is repeatedly applied 

through the subirrigation scenario, while the test substance is only applied 

once when it is sorbed on the support medium. For hydroponic solutions 

the entire test solution should be replaced weekly or earlier if the 

concentration of test substance in the test or nutrient solution varies by 

more than 20% of that specified. Methodology for surface soil 

applications for pesticides are described in paragraph (e)(4)(ii)(A)(4) of 

this guideline.

(1) Test substances that are soluble in water should be dissolved in 

the nutrient solution just prior to the beginning of the test. Reagent 

water should be used in making stock solutions of the test 

substance. Sufficient quantities of each concentration should be 

made up as needed to minimize storage time and disposal volume. 

The OCSPP 850.1000 and OCSPP 850.4000 guidelines contain 

additional information on preparation of stock solutions.

(2) Test substances that are insoluble in water but which can be 

placed in aqueous suspension with a vehicle should be added to the 

nutrient solution. The vehicle should be soluble in water, 

relatively nontoxic to plants, and should be used in the minimum 

amount required to dissolve or suspend the test substance. There 

are no preferred vehicles; however, acetone, gum arabic, 

polyethylene glycol, ethanol, and others have been used 

extensively in testing herbicides, plant growth regulators, 

fungicides, and other chemicals that affect plants. Vehicle controls 

should be included in the experimental design and tested 

simultaneously. The OCSPP 850.4000 guideline contains 

additional information on preparation of stock solutions using a 

vehicle.

(3) Water-insoluble test substances for which no nontoxic, watersoluble vehicle is available, should be dissolved in an appropriate 

volatile solvent. The solution should be mixed with the sand or 

glass beads which are then placed in a rotary vacuum apparatus 

and evaporated, leaving a uniform coating of test substance on the 

sand or beads. A weighed portion of sand or beads should be 

extracted with the same organic solvent and the concentration of 

test substance determined before the potting containers are filled. 

Solvent controls are included in the experimental design and tested 

simultaneously.

(4) For the surface soil exposure scenario, the test substance is 

applied to the soil surface after planting the seeds and prior to 

germination. For pesticides, this is typically accomplished using a 

properly calibrated sprayer after the seeds have been planted. At 

test initiation, test substance is applied to surface soil immediately 
Page 8 of 24

after the seeds have been planted using a properly calibrated 

sprayer. 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 of water 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). Additional 

information can be found in the OCSPP 850.4100 guideline.

(B) Foliar exposure. Foliar exposure is accomplished by spraying the 

plants, as described in paragraph (e)(4)(ii)(B)(1) of this guideline by

dusting the plants, as described in paragraph (e)(4)(ii)(B)(2) of this 

guideline or by exposing the plants to gas in a fumigation chamber, as 

described in paragraph (e)(4)(ii)(B)(3) of this guideline. The test substance

is applied weekly via spraying or dusting, but only applied once when the 

fumigation scenario is used.

(1) Water soluble test substances should be dissolved in reagent 

water just prior to use. Test substances that can be suspended in an 

aqueous solution by a vehicle should be prepared as described in 

paragraph (e)(4)(ii)(A)(1) of this guideline except using reagent 

water not nutrient solution. Sufficient quantities of each test 

concentration should be made up as needed. These solutions 

should be applied at weekly intervals. Plants should be placed in 

an exhaust hood and the test substance applied to the foliage. A

plastic sleeve may be fitted over the top of the pot, and the foliage 

sprayed with specific quantities of test solution at known 

concentrations. The plastic sleeve, confining the test substance to 

plant and pot, facilitates expression of test substance dosage as 

quantity per pot area (i.e., micrograms per square meter). Shoots 

of control plants should also be sprayed, in an identical manner, 

with reagent water. In addition, a vehicle control group is included 

if a vehicle was used to prepare the test substance. Alternatively, a 

miniature compressed-air sprayer may be mounted on a pendulum 

and equipped to automatically spray a plant positioned directly 

beneath the center of its arc of swing. Track or belt sprayers may 

also be used (see the OCSPP 850.4000 and OCSPP 850.4100 

guidelines for additional information). When radioisotope-labeled 

test substances are applied, health and safety considerations 

prohibit spray application. Instead, specific quantities of labeled 

chemical should be applied directly to leaves in single drops.

(2) Water-insoluble test substances, existing as solids, may be 

prepared for testing by grinding or other reduction methods to 
Page 9 of 24

particles of smaller than 200 micrometers (μm) in diameter. These 

test substances should be applied at weekly intervals. Plants 

should be placed in an exhaust hood, a plastic sleeve fitted over the 

top of the pot, and a specific quantity of test substance sprinkled 

uniformly over them. Prior to test substance application, plants 

should be misted with water to promote foliar retention of the test 

substance. Control plants are also misted with reagent water at 

each treatment date and dusted with an inert substance of the same 

particle size as the test substance. Applications are expressed as 

quantity per unit pot area (i.e., micrograms per square meter).

(3) Test substances existing in gaseous form at normal ambient 

temperatures and pressures can be generated for use as needed or 

stored under pressure. The bottled gas may be 100% pure test 

substance or may be mixed with an inert carrier, such as nitrogen, 

to known concentrations. Controlled or measured concentrations 

of test substance should be metered into the exposure chamber, 

uniformly mixed about the plants, and exhausted through the outlet 

port where the flow rate and concentration are again measured. 

Use of this system design provides an alternate method of analysis 

if the quantity of test substance sorbed by plants is less than that 

required for chemical analysis. Plants should be fumigated 

whenever they have reached sufficient size for measurement of 

photosynthesis and transpiration rates, assuming equivalent 

detection sensitivity of carbon dioxide, water vapor, and chemical 

analyzers. The appropriate size is a function of the gas exchange 

system and constitutes an area of expert judgment.

(iii) Treatment levels. 

(A) For this determination, at least three concentrations of the test 

substance, exclusive of controls, are tested. Recommended concentrations 

would be a descending geometric progression from the upper 

concentration tested (i.e. 100, 50, 25 milligrams per liter (mg/L)). The 

concentration selected as the upper limit for the uptake and translocation 

test should be near to but below the threshold of visible injury (e.g., 

NOEC). Short exposure periods to gas in fumigation chambers are not 

expected to promote visible injury or gross reductions in growth but may 

alter stomatal resistance, transpiration, or photosynthesis. Absorption and 

adsorption rates may be calculated and the upper limit for the uptake and 

translocation test for definitive testing selected based on the calculated 

sorption rates.

(B) The use of pesticide treatments to control pests during the test should 

be avoided. Mechanical, cultural, and biological pest control methods are 

suggested. If pesticide treatments are used in the test for pest control, for a 
Page 10 of 24

satisfactory test there should be a negative control and a control with only 

the treatments used for infestation purposes.

(iv) Introduction of test organisms. Seedlings used in testing are produced by 

planting seeds of the desired species in the selected support medium and allowing 

them to germinate, at which time they are thinned to 1-4 plants of similar size and 

condition, such that there is no crowding or competition among the plants at 

harvest or maturity. Within a given test, all test organisms of a given species, 

including the controls, must be from the same source and seed lot. For a 

satisfactory test, a system of impartial or random assignment of the seeds to the 

pots and the pots to test and control groups is used to prevent bias.

(5) Controls. 

(i) Every test includes controls consisting of the same support medium, 

conditions, procedures, and test population, except that no test substance is added. 

In addition, vehicle (solvent) controls are also included if a solvent is used. 

(ii) Seed germination standards. 

(A) For a satisfactory test, seed germination percentage in each of the 

controls should be at least 65%; or the percentages in minimum acceptable 

United States Department of Agriculture (USDA) seed germination

standards listed in paragraph (e)(5)(ii) of this guideline. For native species 

that do not have germination standards, sufficient numbers of seeds should 

be used to insure the identified number of seedlings per pot for test 

initiation.

(B) The following minimum acceptable USDA seed germination (control) 

standards for vegetable crops (as described in the Federal Seed Act 

Regulation, 7 CFR parts 201-202) and other available standards for 

agronomic crops will be used: Field corn (85%), pop corn (75%), sweet 

corn (75%), carrot (55%), onion (70%), tomato (75%), field-garden bean 

(70%), pea (80%), cabbage (75%), lettuce (55%), mustard—all types 

(75%), soybean (75%), sugar beet (55%), small grains—wheat, oats, 

barley, rice (80%), ryegrass (75%), and rape (75%). Refer to regulation 

for additional vegetable crops.

(6) Number of test organisms and replicates. 

(i) For each species, the minimum number of replicates is six pots per 

concentration, with each pot containing from one to four seedlings. If techniques 

other than radioisotopes are used to determine uptake, more replicates may be 

needed to provide sufficient plant materials for analysis. The number of seedlings 

per replicate depends on the species tested, the size of the containers, and the size 

to which the plants will grow. When plants are grown hydroponically, one plant 

per pot will probably be the preferred method. The number of plants selected 

should provide sufficient biomass for analytical procedures. A greater number of 
Page 11 of 24

plants may be required depending on species tested, duration of test, and 

analytical procedures. Too many plants in a container may actually reduce the 

growth and biomass; therefore, more replicate pots rather than increasing plants 

per pot may be required to obtain sufficient biomass.

(ii) A randomized complete block design is recommended for this test, with 

blocks delineated within the growth chambers or over greenhouse benches and 

randomization of treatments occurring within the blocks. If there exists 

inadequate space within chambers for blocking because of very large pots and 

plants, total randomization within chambers is acceptable. This design is also 

appropriate for the growth of plants to be used for foliar exposure with gas. 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).

(iii) 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 replicate 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) Greenhouse, environmental chamber, or growth room. Greenhouses, 

environmental chambers, or growth rooms should provide adequate 

environmental controls to meet the carbon dioxide, humidity, irradiation, 

photoperiod, and temperature specifications. Chambers should be designed to 

prevent escape of internal air into the external environment other than through 

appropriate filtering material or media to prevent contamination of the external 

environment with radioactivity and/or test substances. Environmental chambers 

and growth rooms are preferred over greenhouses because in the first two, 

environmental conditions are more easily controlled.

(ii) 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 pots that have not been previously used are acceptable test 

containers, provided they are free of toxic materials. Do not use clay or peat 

containers. For a satisfactory test, pots sufficiently large to grow (e.g., large 

enough to effectively not result in competition among the plants for space, 

lighting, or nutrition) the desired number of test plants for at least 28 days or up to 

maturity. It is equally acceptable to use small, individual containers if plants are 

grown in hydroponic solution. Potting containers used in each experiment should 
Page 12 of 24

be of equal size and volume and possess the same configuration. Potting

containers should be covered with opaque polyethylene bags to exclude light and 

minimize volatilization of test chemical.

(iii) Cleaning and disposal. 

(A) Potting and receiving containers, nutrient storage containers, and root 

support medium should be cleaned before use. All equipment should be 

washed to remove any residues remaining from manufacturing or use. 

Dichromate solution should not be used for cleaning beads or pots.

(B) Quartz sand rooting medium, but not glass beads, should be discarded 

at the end of the experiment. Disposal should conform to existing 

regulations. Glass beads can be re-used provided they are not 

contaminated with previous test substances.

(iv) Support medium. 

(A) For tests with industrial chemicals, an artificial support medium, 

consisting of quartz sand or glass beads, is used. Alternatively, a 

hydroponic system may be used, in which seeds are planted in plugs of 

cotton or glass wool supported at the top of the solution. Perlite, 

vermiculite, natural (native) soils, or any formulated soils should not be 

used as root support medium in this test. Additional information is found 

in the OCSPP 850.4320 guideline.

(B) For tests with pesticides, a natural soil (free of chemical 

contamination) or a synthetic soil may be used. Additional information on 

natural and synthetic soil is found in the OCSPP 850.4100 and OCSPP

850.4150 guidelines.

(v) Nutrient media. A nutrient solution of defined chemical composition, such 

as half-strength modified Hoagland nutrient solution (see the reference in 

paragraph (j)(5) of this guideline), should be utilized as nutrient medium for this 

test. Hydroponic solutions should be aerated and sand or glass filled potting 

containers should be periodically filled with nutrient solution and drained to 

provide aeration. For root exposure tests, the test substance should be added to 

the nutrient solution or directly to substrate. The entire test solution should be 

replaced weekly or earlier if the concentration of test substance in the test or 

nutrient solution varies by more than 20% of that specified. The volume of 

solution added should be recorded. An automated system design is 

recommended. For tests with pesticides, any commonly recommended fertilizer 

can be added to the soil at recommended rates.

(8) Environmental conditions. Controlled environmental conditions are necessary to 

maintain uniform growth and ensure reliable data. Standardization of environmental 

conditions is essential because it has been demonstrated that differences in environmental 

conditions influence the response of plants exposed to chemicals. For growth chamber 
Page 13 of 24

studies, the general conditions listed in paragraphs (e)(8)(i) through (e)(8)(v) of this 

guideline should be maintained. If greenhouses or large growth rooms are used, 

comparability of the environment between small groups of plants is not as critical and 

environmental variability specifications may be relaxed on a case-by-case basis.

(i) Temperature. Air temperature should be uniform throughout the greenhouse 

or growth chamber or greenhouse. In growth chambers air temperature during the 

day should be 25 ± 3 oC while temperature during the night should be 20 ± 3 oC. 

Temperature in greenhouses is more difficult to control. Under these 

circumstances, temperature during the day should be 25 ± 6 oC while temperature 

during the night should be 20 ± 6 oC.

(ii) Humidity. Humidity should be uniform throughout the greenhouse or growth 

chamber or greenhouse. In growth chambers relative humidity should approach 

70 ± 5% during light periods and 90 ± 5% during dark periods. Humidity in 

greenhouses is more difficult to control. Under these circumstances, relative 

humidity should approach 70 ± 15% during light periods. 

(iii) Lighting and photoperiod. Luminance of 350 micromoles per square meter 

per second (μmol/m2/s), measured at the top of the canopy, is desirable. Artificial 

lighting may be used to lengthen short-day periods or to supplement natural 

sunlight on overcast days. A photoperiod of 16 hours light and 8 hours darkness, 

assuming an average wavelength of 400 to 700 nanometers (nm), should be used 

for all species except soybean which should be provided with 11 hours light and 

13 hours darkness prior to flowering.

(iv) Watering. Nutrient media (containing test substance, if this is the selected 

application route) is used to subirrigate the plants, i.e., frequent enough to 

maintain good health (should not see nutrition deficiency in controls) and 

moisture. Pots should be irrigated regularly (for example, 30 minutes per hour 

(min/h)) with nutrient solution, for example using a system in which water flows 

from the bottom of the pot upward (level should reach at least half way up the 

pot). An automatic system design is recommended such as the one described in 

the OCSPP 850.4600 guideline. For soil applications, top watering is used at the 

first watering after the test substance has been applied to initiate the capillary 

movement of water for bottom watering (subirrigation).

(v) Carbon dioxide. Carbon dioxide concentration at 350 ± 50 parts per million 

(ppm) (678 + 97 milligrams per cubic meter (mg/m3) of air).

(9) Observations―

(i) Measurement of test substance. For a satisfactory test, analytical 

confirmation of the concentration of test substance in the vehicle used to expose 

the plants (nutrient medium, glass beads or sand, spray, dust or gas) should be 

performed. Additionally, analytical confirmation of the amount of test substance

in plant tissues is determined at test termination as discussed in (e)(9)(iv)(A) of 
Page 14 of 24

this guideline. Analytical methods, validated before test initiation, are used to 

measure the amount of test substance in a sample matrix, as described in the 

OCSPP 850.4000 guideline.

(A) Nutrient solution. Where test substance was introduced by addition 

to the nutrient medium, chemical analysis should be conducted at a 

minimum for each treatment level and controls at test initiation (0-hour) in 

the bulk solutions and test termination in at least three replicates per 

treatment and controls at test termination when nutrient solutions are not 

renewed. Where nutrient solution is renewed during the study, chemical 

analysis should be conducted at a minimum for each treatment level and 

controls at test initiation (0-hour) in the bulk solutions and at the end of 

this first cycle in the used nutrient solution, at the beginning (fresh 

solution) and end (used solution) of the longest renewal cycle and the last 

renewal cycle. It is also recommended that measurements be made at the 

beginning of each renewal cycle. 

(B) Quartz sand or glass bead support medium. Where test substance

was introduced by addition to the quartz sand or glass bead support 

medium, chemical analysis should be conducted at a minimum in the bulk 

support medium for each treatment level and control at test initiation and 

from at least three pots for each treatment level and control at test 

termination. 

(C) Foliar spray. Where test substance is applied by foliar spray and a 

single batch of test solution for each treatment level and control is used 

throughout the study, chemical analysis in the spray solutions should be 

conducted at a minimum at test initiation and termination for each 

treatment level and control batch. If one or more spray solution batches 

are prepared during the study, chemical analysis of spray solutions should 

be conducted at a minimum for each treatment level and controls at test 

initiation (0-hour) and at the end of the use of this nutrient solution batch, 

at the beginning (fresh solution) and end (used solution) of the longest 

time between batches and the last batch. It is also recommended that 

measurements be made at the beginning of each batch.

(D) Foliar dusting. The amount of test substance to be applied to a 

replicate should be weighed prior to application and the weight recorded. 

The sleeve placed around the pot during application should be weighed 

before and after application if there will be difficulties expected with test 

substance not landing in the pot or on the plants or remaining on the 

sleeve.

(E) Gas application. For the gas exposure system, at the time of 

fumigation the air temperature, dew point temperature or water vapor 

pressure of incoming and outgoing air streams, air speed within chamber, 
Page 15 of 24

carbon dioxide concentration at inflow and outflow ports, and gas flow 

rate into and out of exposure system are measured and recorded.

(ii) Environmental conditions―

(A) Temperature and humidity. The air temperature and humidity 

during the study should be recorded continuously in growth chambers, but 

alternatively as maximum and minimum values over each 24-hour period, 

especially in greenhouses at representative locations throughout the area in 

which the test plants are growing.

(B) Carbon dioxide. In environmental chambers, carbon dioxide 

measurements should be made at the top of the plant canopy, preferably 

on a continuous basis.

(C) Light intensity. Light intensity should be determined daily at 

representative locations throughout the area in which the test plants are 

growing, and should be measured at the top of the foliage. A

photosynthetically active radiation (PAR) sensor should be used to 

measure light quality. Additional information on the use of lighting in 

plant toxicity tests can be found in the references given in the OCSPP

850.4000 guideline.

(D) Watering and evapotranspiration. The amount of nutrient solution 

depleted each week should be recorded, to observe changes in 

evapotranspiration rates which may indicate stress. Furthermore, these 

data will be used to compute chemical uptake per volume of water 

transpired for the uptake test.

(E) 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.

(F) Nutrient media pH. The pH of nutrient solutions are measured prior 

to use in bulk solutions and in used solutions at test termination in each 

replicate. If more than one batch of nutrient solution is used throughout 

the study the pH of each nutrient solution treatment concentration and 

control(s) are measured in the nutrient solution batch prior to use, and at 

replacement in the used solution.

(iii) Appearance and condition. Plants are observed on a weekly basis for any 

abnormalities and phytotoxic symptoms. All abnormalities (visible effects 

including stunting of growth, discoloration, chlorosis and/or necrosis of the 

leaves, decreased moisture content, or morphological abnormalities, etc.) should 

be recorded. Uniform scoring procedures should be used to evaluate any 

observable toxic response. Observations should include the treatment level and 
Page 16 of 24

replicate, stage of development and dates when adverse results occurred, subsided 

or recovered, and counts for each plant affected. Any lack of effects by the test 

substance should also be recorded. Observations of plants should be made at least 

once every seven days.

(iv) Uptake and translocation. When sufficient plant biomass for analysis has 

been obtained, or when fruits or seeds are mature, the plants are harvested and the 

various plant organs are analyzed to establish the mass balance of the test 

substance. Carrot, lettuce, onion, cabbage, and ryegrass may be harvested 

whenever there is adequate plant material for chemical analysis. Cucumber, corn, 

soybean, tomato, and oats should be grown until fruit or seed are mature. An 

alternative method can be used for the gaseous exposure system, where direct 

calculation of uptake rates can be made based upon measurements of 

photosynthesis, transpiration and stomatal conductance, obviating the need for 

chemical analysis of the plant tissues.

(A) Plant residue measurements―

(1) Preparation of plant material. The entire plant should be 

harvested, rinsed with a minimum amount of reagent water (which 

is returned to the nutrient solution), and separated into its 

respective organs as follows: carrot—root peels, peeled roots, and 

tops; cucumber—fruit, vines plus leaves, and roots; corn—kernels, 

husk plus cob, stalk plus leaves, and roots; lettuce—tops and roots; 

onion—bulb and tops; ryegrass—tops and roots; soybean—grain, 

chaff plus tops, and roots; oats—grain, chaff plus tops, and roots; 

tomato—fruit, vines, and roots; cabbage—head and roots. Plants 

from two pots in each treatment may be pooled, giving three 

replicate sample pools per treatment.

(a) Fresh weight, dry weight, and percent moisture. 

After the fresh weights of the plant organs are obtained, 

they should be subsampled for percent moisture 

determinations by drying, at 70 degrees Celsius (

oC) for 24 

hours in a forced-air drying oven, and weighing. Percent 

moisture determined from these subsamples is used to 

correct for dry weight of the fresh samples.

(b) Extraction. Each sample is then homogenized and 

extracted in organic and aqueous solvents. A suggested 

extraction procedure appropriate for many organic 

chemicals is as follows: plant material (1 gram (g)) is 

homogenized with 1 g of solvent-washed anhydrous 

sodium sulfate in 4 milliliters (mL) of hexane or 

acetonitrile. The homogenate is then filtered or 

centrifuged, the solid residue rinsed with an appropriate 

organic solvent, and the filtrate or supernatant combined 
Page 17 of 24

with the rinse. The solid residue should be extracted by

sequentially (one) homogenizing in water, (two) 

centrifuging and decanting the supernatant, (three) 

extracting of the pellet with 6 Normal (N) hydrochloric acid 

at 60 oC for 10 hours, (four) subsequently digesting with 10 

N potassium hydroxide, and (five) combining supernatants.

(2) Analytical methods. If radioisotopes are used, the amount of 

test substance in each extract should be determined by liquid or 

solid scintillation; otherwise, the amount of test substance should 

be determined by standard methods. The organic extract should be 

evaporated under vacuum to a sufficiently small volume for thin 

layer chromatography (TLC) and co-chromatographed on silica gel 

plates with known standards of the parent test substance. If 

radioisotopes were used, the chromatographs could be scanned for 

radioactive substances on a radiochromatogram scanner. 

Alternatively, zones may be removed from the plates, extracted, 

and the quantity of test substance from each zone determined by

liquid scintillation spectrometry or gas liquid chromatography 

(GLC) methodology. The unextractable test substance in the 

remaining residue may be determined by oxidizing the residue in a 

complete combustion oxidizer.

(B) Gaseous test substances―

(1) Direct calculation of uptake rates. A gas exposure system 

yields requisite data for a direct calculation of uptake rates. At 

steady state, test substance uptake may be determined by a mass 

balance calculation. Correction for adsorption to surfaces of the 

exposure chamber should be made by operating the system without 

plants. Pots filled with hydroponic solution or support media 

should be included in the system adsorption calibration. 

Consequently, chemical analysis of plant tissues exposed to 

gaseous chemicals may not be required in order to demonstrate and 

quantitate uptake rates.

(2) Physiological rates. Altered rates of net photosynthesis, 

transpiration, and stomatal conductance are anticipated as a result 

of test substance uptake. Rates of these physiological processes 

before, during, and after exposure to the gaseous chemical should 

be determined. Data required for these calculations are available 

as a consequence of maintaining the specified environmental 

conditions within the fumigation chamber.

(3) Plant surface area mass measurements. For each pot 

measure the plant surface area mass.
Page 18 of 24

(C) Root support medium measurements. At test completion, the root 

support material should be washed in organic and then aqueous solvent 

and analyzed for test substance before discarding.

(f) Treatment of results―

(1) Data summary―

(i) For liquid and solid test substances. 

(A) Means and standard deviations of the fresh weight and dry weight 

mass of plant organs and by summation, the mass of the whole plant are

calculated for each treatment (includes controls).

(B) Means and standard deviations of the concentration of free parent test 

substance, metabolites and soluble residues, and bound residues in plant 

organs and whole plants by treatment. Concentrations should be 

expressed in appropriate weight units per grams of dry plant material.

(C) Mean evapotranspiration rate per plant.

(D) Initial and final total concentration of the test substance (total 

residues: parents plus degradates) in the support media.

(E) Mean and standard deviations of the mass balance of test substance by

treatment.

(ii) For gaseous test substances. 

(A) Measurements as described in paragraphs (f)(1)(i)(A) through 

(f)(1)(i)(E) of this guideline for solid and liquid test substances; 

alternatively, the measurements described in paragraphs (f)(1)(ii)(B) 

through (f)(1)(ii)(D) of this guideline.

(B) Mass of leaves and stems and surface area (one side of leaves) in the 

exposure system.

(C) Steady state rates of photosynthesis, transpiration, and test substance

uptake before, during, and after fumigation.

(D) Stomatal conductance or leaf diffusion resistance before, during, and 

after fumigation.

(2) Definitive test. Mass balance of the test substance and evapotranspiration rate of the 

plants is subjected to analysis of variance. An analysis of variance, followed by a means 

separation test, is performed to determine which test substance treatments resulted in 

residues from uptake/translocation or evapotranspiration rates that are significantly 

different from those in the control(s).
Page 19 of 24

(g) Tabular summary of test conditions. Table 2 lists the important conditions that should 

prevail during the definitive test. Meeting these test conditions will greatly increase the 

likelihood that the completed test will be acceptable or valid. These test conditions apply mainly

to growth chambers and growth rooms; they are preferred to greenhouses where for the latter it 

may be more difficult to meet or control these conditions.Table 2.—Summary of Test 

Conditions for Plant Uptake and Translocation Test1

Test duration To provide sufficient biomass (or to allow gas exchange 

measurements), or until fruit or seeds are mature.

Substrate Quartz sand or glass beads. Hydroponic system may also be 

used. (For pesticides, natural or synthetic soil may also be used.)

Nutrients Watered with nutrient solution (half-strength modified Hoagland’s 

medium)

Temperature 25/20 oC (daytime/nighttime) ± 3 oC (applicable to growth 

chambers)

Relative humidity 70/90% (daytime/nighttime) ± 5% (applicable to growth chambers)

Carbon dioxide 350 ± 50 ppm (applicable to growth chambers)

Light quality Fluorescent or representative of natural sunlight

Light intensity 350 ± 50 μmol/m2/sec 

Photoperiod 16 hours light: 8 hours dark for all species except soybean with 11 

hours light: 13 hours dark prior to flowering

Watering Bottom watering as needed, using nutrient solution

Test chamber (pot) size Varies with plant species selected.

Number of organisms per test 

chamber

Typically, 1 - 4 seedlings of one species per pot

Number of replicate chambers per 

test treatment

6 (minimum) 

Number of organisms per test 

treatment

6 - 24 (minimum)

Test treatment levels Minimum of 3 treatment levels plus appropriate controls

Test substance application method Root exposure (via watering with nutrient solution or sorbed onto 

the substrate) or foliar exposure (via spray, dust or gas) or soil 

surface exposure.

Measures of effect (Measurement 

endpoints)

Mass balance on test substance, including concentrations of free 

parent test substance, metabolites and soluble residues, and 

bound residues in plant organs and whole plants. (Alternatively, for 

gaseous test substances, measurements of photosynthesis, 

transpiration, and stomatal conductance, and mass of plant surface 

area).

1 Constraints on environmental conditions are mainly applicable to growth chamber studies 

(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 the 
Page 20 of 24

OCSPP 850.4000 guideline, it is unlikely 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 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 plant uptake and translocation test

1. For any species, all organisms in a test are from the same source.

2. All test chambers used for a particular species are identical and contain the same amount of substrate 

from the same source.

3. An untreated (or negative) control [and solvent (or vehicle) control, when a solvent was used] is 

included in the test.

4. Seed germination percentage in each of the controls is at least 65% for the recommended test species; 

or the minimum acceptable USDA seed germination standards listed in paragraph (e)(5)(ii)(B) of this 

guideline. For native species that do not have germination standards, sufficient numbers of seeds, are 

used to provide statistically valid measures of effect.

5. There are no signs of stress from competition or crowding of plants.

(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 

the OCSPP 850.4000 guideline.

(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)), radiolabeling if any, location of label(s), 

and radiopurity.

(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.
Page 21 of 24

(iii) Methods of preparation of the test substance and the treatment doses used in 

the range-finding and definitive 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) Test date of germination rating and determining percent germination.

(iii) History of the seed: source, name of supplier, seed year or growing season 

collected, batch or lot number, seed treatment(s), and storage conditions.

(iv) Seed size class.

(v) Description of handling and processing of seeds, pre-test planting, production, 

and handling of test seedlings before use in test.

(vi) Height and condition of seedlings selected for the test.

(5) Test system and conditions. Description of the test system and conditions used in 

the definitive test, and any preliminary range-finding tests.

(i) A description of the test system, including type of growth chamber, growth 

room, or greenhouse.

(ii) The description of pots or test containers: type, material, dimensions.

(iii) Number of seedling plants per pot or test container.

(iv) Number of pots per replicate, and number of replicates per treatment level.

(v) Description of the support medium or hydroponic system: source, type, 

material.

(vi) Volume of support medium per pot or test container.

(vii) Methods used for treatment randomization.

(viii) Methods for preparing the test treatments: exposure route (root or foliar 

exposure), application methods (including equipment type and method for 

calibrating the application equipment), information about any solvent used to 

dissolve and apply the test substance, and the concentrations (concentration of the 

test substance in nutrient solution or in the foliar application vehicle, or the 

quantity of test substance per unit weight of root support material when it is 
Page 22 of 24

coated on the support material, the concentration of test substance applied to the 

soil surface; the concentration of test substance in the dust or spray; or the 

concentration of gaseous test substances at inflow and outflow ports) and the 

amounts used.

(ix) The method for calibrating the application equipment.

(x) Number of applications and dates applied.

(xi) Culture practices during the test such as pest control, and irrigation practices 

(type and watering schedule or regime).

(xii) The photoperiod and light source.

(xiii) Methods and frequency of environmental monitoring performed during the 

definitive test for air temperature, humidity, and light intensity.

(xiv) For the definitive 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 (carbon dioxide concentrations, 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) Tabulation of the watering schedule, the volume of nutrient solution (or 

hydroponic solution) used and depleted each week by replicate within a treatment 

and control and the evapotranspiration rates calculated from the data.

(iii) For the gas exposure system, tabular summary of the air temperature, dew 

point temperature or water vapor pressure of incoming and outgoing air streams, 

air speed within chamber, carbon dioxide concentration at inflow and outflow 

ports, and gas flow rate into and out of exposure system (provide raw data for 

measurements not made on a continuous basis).

(iv) For the 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.

(v) For the definitive test, tabulation of the fresh weight, dry weight, and percent 

moisture of plant organs (e.g., carrot—root peels, peeled roots, and tops; 

cucumber—fruit, vines plus leaves, and roots; oats--grain, chaff plus tops, and 

roots) by replicate and treatment (provide the raw data). For gaseous test 
Page 23 of 24

substances, in addition to or in lieu of these measurements, tabulation of the plant 

surface area mass by replicate and treatment.

(vi) For the definitive test, tabulation of the concentrations of parent test 

substance, metabolites, soluble residues and bound residues in plant organs and 

whole plants by replicate (pooled replicate, if applicable) and treatment. For 

gaseous test substances, in addition to or in lieu of these measurements, tabulation 

of the calculated measurements of photosynthesis, transpiration, and stomatal 

conductance before, during, and after exposure to test chemicals by replicate and 

treatment (provide raw data).

(vii) For the definitive test, tabulation of the mass balance of test substance by

replicate and treatment.

(viii) For the definitive test, means and standard deviations of mass balance of test 

substance by treatment.

(ix) For the definitive test, plots of the means and standard deviations of mass 

balance of test substance by treatment.

(x) For the definitive test, analysis of variance, F-test, means, and standard 

deviation about the mean for the measured endpoints. 

(xi) Methods of statistical analyses, including software used, should be reported.

(j) References. The references in this paragraph 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) Beall, M.L., Jr. and Nash, R.G. 1969. Crop seedling uptake of DDT, dieldrin, endrin, 

and heptachlor from soil. Agronomy Journal 61:571-575.

(3) Bennett, J.H., Hill, C., and Gates, D.M. 1973. A model for gaseous pollutant 

sorption by leaves. Journal of the Air Pollution Control Association 23:957-962.

(4) Chou, S.F., Jacobs, L.W., Penner, D., and Tiedje, J.M. 1978. Absence of plant 

uptake and translocation of polybrominated biphenyls (PBBs). Environmental Health 

Perspectives 23:9-12

(5) Cole, L.K., Sanborn, J.R., and Metcalf, R.L. 1976. Inhibition of corn growth by

aldrin and the insecticide's fate in the soil, air, crop, and wildlife of a terrestrial model 

ecosystem. Environmental Entomology 5:583-589.

(6) Downs, R.J. 1975. Controlled Environments for Plant Research. Columbia Univ. 

Press, NY.
Page 24 of 24

(7) Downs, R.J. and Helmers, H. 1975. Environment and Environmental Control of 

Plant Growth. Academic Press, NY. 

(8) Fuhr, F. and Mittelstaedt, W. 1980. Plant experiments on the bioavailability of 

unextracted (carbonyl-14C) methabenzthiazuron residue from soil. Journal of 

Agriculture and Food Chemistry 28:122-125.

(9) Hatzios, K.K. and D. Penner. 1985. Interactions of herbicides with other 

agrochemicals in higher plants. Rev. Weed Sci. 1:1-63.

(10) Kelly, J.M., Parker, G.R., and McFee, W.W. 1980. Heavy metal accumulation and 

growth of seedlings of five forest species as influenced by soil cadmium level. Journal of 

Environmental Quality 8:361-364. [

(11) Tibbits, T.W. and Kozlowski, T.T. (eds.). 1979. Controlled Environment 

Guidelines for Plant Research. Academic Press, NY

(12) Wickliff, C., Evans, H.J., Carter, K.R. and Russell, S.A., 1980. Cadmium effects on 

the nitrogen fixation system of red alder. Journal of Environmental Quality 9:180-184.