United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA712-C-96-159
April 1996
&EPA Ecological Effects Test
Guidelines
OPPTS 850.4800
Plant Uptake and
Translocation Test
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Introduction
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7 U.S.C. 136, etseq.).
Public Draft Access Information: This draft guideline is part of a
series of related harmonized guidelines that need to be considered as a
unit. For copies: These guidelines are available electronically from the
EPA Public Access Gopher (gopher.epa.gov) under the heading "Environ-
mental Test Methods and Guidelines" or in paper by contacting the OPP
Public Docket at (703) 305-5805 or by e-mail:
guidelines@epamail.epa.gov.
To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs, Field Operations Division (7506C), Environmental
Protection Agency, 401 M St. SW., Washington, DC 20460. In person:
bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Ar-
lington, VA. Comments may also be submitted electronically by sending
electronic mail (e-mail) to: guidelines@epamail.epa.gov.
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202-512-0135 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from the EPA Public Access Gopher
(gopher.epa.gov) under the heading "Environmental Test Methods and
Guidelines."
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OPPTS 850.4800 Plant uptake and translocation test.
(a)	Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).
(2) Background. The source material used in developing this har-
monized OPPTS test guideline is 40 CFR 797.2850 Plant Uptake and
Translocation Test.
(b)	Purpose. The guideline in this section is intended for use in devel-
oping data on the uptake and translocation of chemical substances and
mixtures ("chemicals") by terrestrial plants subject to environmental ef-
fects test regulations. This guideline prescribes tests using commercially
important terrestrial plants to develop data on the quantity of chemical
substances incorporated in plant tissues and the potential for entry into
food chains with resultant indirect human exposure. EPA will use data
from these tests in assessing the hazard of a chemical to the environment.
(c)	Definitions. The definitions in section 3 of the Toxic Substances
Control Act (TSCA), and 40 CFR Part 792—Good Laboratory Practice
Standards apply to this test guideline. The following definitions also apply
to this guideline:
EC X means the experimentally derived chemical concentration that
is calculated to effect X percent of the test criterion.
Mass balance means a quantitative accounting of the distributions of
chemical in plant components, support medium, and test solutions. It also
means 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.
Support media means the sand or glass beads used to support the
plant.
Translocation means the transference or transport of chemical from
the site of uptake to other plant components.
(d)	Test procedures—(1) Summary of the test—(i) Root exposure.
In preparation for the test, seeds are planted in the potting containers (or
in cotton or glass-wool plugs supported in hydroponic solution) and, after
germination, seedlings thinned, by pinching the stem at the support sur-
face. Potting mixtures of sand or glass beads should be subirrigated with
nutrient solution. Chemicals are applied to the plants via nutrient solution
or adsorbed to the support media. Carrot, lettuce, onion, cabbage, and rye-
grass 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.
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(ii) Foliar exposure. The foliar exposure test is identical to the root
exposure test except that chemicals are applied to plants by either spraying
or dusting the foliage or exposing the plants to gas in a fumigation cham-
ber. 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.
(2) Chemical application—(i) Root exposure. (A) Chemicals that
are soluble in water should be dissolved in the nutrient solution just prior
to the beginning of the test. Deionized or glass-distilled water should be
used in making stock solutions of the test chemical. Sufficient quantities
of each concentration should be made up as needed to minimize storage
time and disposal volume.
(B)	Chemicals that are insoluble in water but which can be placed
in aqueous suspension with a carrier should be added, with the carrier,
to the nutrient solution. The carrier should be soluble in water, relatively
nontoxic to plants, and should be used in the minimum amount required
to dissolve or suspend the test chemical. There are no preferred carriers;
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. Carrier controls shoud
be included in the experimental design and tested simultaneously.
(C)	Water-insoluble chemicals for which no nontoxic, water-soluble
carrier 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 chemical on the sand or beads. A weighed portion of beads
should be extracted with the same organic solvent and the chemical as-
sayed before the potting containers are filled. Solvent controls should be
included in the experimental design and tested simultaneously.
(ii) Foliar exposure. (A) Water soluble chemicals should be dis-
solved in deionized or glass distilled water just prior to use. Sufficient
quantities of each concentration should be made up as needed. These solu-
tions should be applied at weekly intervals. Plants should be placed in
an exhaust hood and the chemical 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, con-
fining the chemical to plant and pot, facilitates expression of chemical
dosage as quantity per pot area (i.e., micrograms per square meter). Shoots
of control plants should be sprayed in an identical manner with deionized
or distilled water. 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. When radioiso-
tope-labelled chemicals are applied, health and safety considerations pro-
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hibit spray application. Instead, specific quantities of labelled chemical
should be applied directly to leaves in single drops.
(B)	Water-insoluble chemicals, existing as solids, may be prepared
for testing by grinding or other reduction to particles of <200 (im diameter.
These chemicals 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 chemical sprinkled uniformly over them. Prior
to chemical application, plants should be misted with water to promote
foliar retention of the chemical. Control plants also should be misted with
deionized or distilled water at each treatment date and dusted with an inert
material of the same particle size. Applications should be expressed as
quantity per unit pot area (i.e., micrograms per square meter).
(C)	Chemicals existing in gaseous form at normal ambient tempera-
tures and pressures should be generated for use as needed or stored under
pressure. The bottled gas may be 100 percent pure chemical or mixed
with an inert carrier, such as nitrogen, to known concentrations. Chemicals
of controlled or measured concentrations should be metered into the expo-
sure chamber, uniformly mixed about the plants, and exhausted through
the outlet port where the flow rate and concentration are again measured.
Use of this systems design provides an alternate method of analysis if
the quantity of chemical 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 ex-
change system and constitutes an area of expert judgment.
(3) Range-finding test, (i) A range-finding test should be conducted
to establish the chemical concentrations used in the uptake and
translocation test.
(ii) 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 is recommended
as the range-finding test. Seeds should be germinated directly in containers
filled with sand or glass beads or in cotton or glass-wool plugs supported
in hydroponic solution. When 50 percent of the seedlings have germinated,
the seedlings should be thinned (by pinching) to the 10 most uniform per
container and exposed to a concentration series of test chemical. The low-
est concentration in the series, exclusive of controls, should be at or below
the EC 10 while the upper concentration should be at or above the EC90.
If the anticipated fate of the chemical is soil or soil-water, and the mecha-
nism of concern is root uptake, the chemical should be applied in nutrient
solution to the root support media (or coated on sand or glass beads for
non-water-soluble chemicals). With a chemical whose anticipated mode
of exposure to plants is surface deposition by atmospheric transport or
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irrigation water, the appropriate testing method may be foliar application
allowing subsequent movement into the rooting zone with watering. Effect
is assessed as growth reduction. The concentration selected as the upper
limit for the uptake and translocation test should be near the threshold
of visible injury. 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. Absorp-
tion and adsorption rates may be calculated and gas concentrations for
definitive testing selected based on the calculated sorption rates.
(iii) Alternatively, the seed germination/root elongation test or other
appropriate phytotoxicity test may be used to establish the appropriate
upper concentration for testing.
(4) Definitive test, (i) The purpose of the uptake and translocation
test is to determine the propensity for a chemical's accumulation in plants
or plant parts.
(ii)	At least three concentrations of chemical, exclusive of controls,
should be used in the uptake test. Recommended concentrations would
be a descending geometric progression from the upper concentration tested
(i.e. 100, 50, 25 mg/L). A minimum of six replicate pots per concentration,
each containing from one to four seedlings, should be used. If techniques
other than radioistopes are used to determine uptake, more replicates may
be required to provide sufficient plant materials for analysis. Test chemi-
cals should be added to the hydroponic or nutrient solution or coated on
glass beads for the root uptake test; or sprayed, dusted, or gassed directly
on the foliage in the foliage uptake tests. Only untreated seed (not treated
with fungicides, repellants, etc.) taken from the same lot, and year or sea-
son of collection should be used in a given test.
(iii)	Control pots should be included in the experimental design and
should be used in each run. In addition, a carrier control should be used
for those chemicals that need to be solubilized.
(iv)	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 cm
of the top and to sow seeds directly. After germination, the seedlings
should be thinned by pinching the stem at the support surface. From one
to four seedlings per potting container are required depending on 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 prob-
ably be the preferred method. The number of plants selected should pro-
vide sufficient biomass for analytical procedures. A greater number of
plants may be required depending on species tested, duration of test, and
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analytical procedures. Too many plants in a container may actually reduce
the growth and biomass.
(v)	Alternate planting methods may be required when the chemical
is highly volatile. An impervious barrier of polyethylene film, a modifica-
tion 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 °C 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 to be no expo-
sure to the test chemical. This transplanting procedure, without the vola-
tilization barrier, is also recommended when the test chemical is adsorbed
to the support medium.
(vi)	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 chemical
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
chemical in the test or nutrient solution varies by more than 20 percent
of that specified. The volume of solution added should be recorded.
(vii)	The test consists of one run for each of two specified plant spe-
cies. The duration of a run, for solid and liquid chemicals, should be equal
to the length of time required for the particular test variety to achieve
sufficient biomass for testing. The duration of a run for gaseous chemicals
should be the length of time required to make the specified gas exchange
measurements. For a particular chemical, a run is defined as exposure of
the plant species of three concentrations of test chemical with a minimum
of six replicate pots and appropriate controls. Exposure is followed by
extraction and analysis for parent compound, metabolites, and bound resi-
dues in plant tissues, and in the whole plants for solids, liquids, and gasses
or by calculating rates of absorption and adsorption of gasses.
(viii)	Visible effects (stunting of growth, discloration, chlorosis and/
or necrosis of the leaves, decreased moisture content, or morphological
abnormalities, etc.) should be recorded.
(ix)	A randomized complete block design is recommended for this
test, with blocks delineated within the chambers or over greenhouse bench-
es and randomization of treatments occurring within the blocks. If, because
of very large pots and plants, there exists inadequate space within cham-
bers for blocking, total randomization within chambers is acceptable. This
design is also appropriate for the growth of plants to be used for foliar
exposure with gas.
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(x)	Irradiation measurements should be taken to the top of the plant
canopy and the mean, plus a maximum and a minimum value, determined
over the plant-growing area. These measurements should be taken at the
start of the test, at biweekly intervals during the test, and at test termi-
nation. If the test is conducted in a greenhouse facility, hourly measure-
ments of irradiation should be recorded and presented as daily total irradi-
ance plus representative hourly curves for clear sky conditions and cloudy
days.
(xi)	Temperature and humidity measurements should be measured
daily at the top of the plant canopy during each light and dark period.
(xii)	Measurements of carbon dioxide concentration should be made
at the top of the plant canopy (of chamber-growth plants) on a continuous
basis.
(xiii)	The amount of water and 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 chem-
ical uptake per volume of water transpired for the uptake test.
(5) Analytical measurements—(i) Solid or liquid test chemicals.
(A) Stock solutions should be diluted with glass distilled or deionized
water to obtain the test solutions. Standard analytical methods, if available,
should be used to establish concentrations of these solutions and should
be validated before beginning the test. An analytical method is not accept-
able if likely degradation products of the chemical, such as hydrolysis and
oxidation products, give positive or negative interference. The pH of these
solutions should also be measured prior to use.
(B) The entire plant should be harvested, rinsed with a minimum
amount of 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 treat-
ment may be pooled, giving three replicate sample pools per treatment.
After the fresh weights of the plant organs are obtained, each pool of or-
gans should be subsampled for percent moisture determinations by drying,
at 70 °C for 24 h in a forced-air drying oven, and weighing. Percent mois-
ture determined from these subsamples is used to correct for dry weight
of the fresh samples which should then be homogenized and extracted
in organic and aqueous solvents. If radioisotopes are used, the amount
of test chemical in each extract should be determined by liquid or solid
scintillation depending on the type of radiation; otherwise, the amount of
chemical should be determined by standard methods. At test completion,
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the root support material should be washed in organic and then aqueous
solvent and analyzed for test chemical before discarding.
(C) A suggested extraction procedure appropriate for many organic
chemicals is as follows: Plant material (lg) should be homogenized with
lg of solvent-washed anhydrous sodium sulfate in 4 mL of hexane or ace-
tonitrile. The homogenate should be filtered or centrifuged, the solid resi-
due rinsed with an appropriate organic solvent, and the filtrate or super-
natant combined with the rinse. The solid residue should be extracted by
sequentially (7) homogenizing in water, (2) centrifuging and decanting the
supernatant, (3) extracting of the pellet with 6N hydrochloric acid at 60
°C for 10 h, (4) subsequently digesting with ION potassium hydroxide,
and (5) combining supernatants. The resulting solution should be analyzed
by liquid scintillation spectrometry or gas liquid chromatography (GLC)
methodology. 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
chemical. 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
chemical from each zone determined by liquid scintillation spectrometry
or GLC methodology. The unextractable chemical in the remaining residue
may be determined by oxidizing the residue in a complete combustion
oxidizer.
(ii)	Gaseous test chemicals. (A) A gas exposure system yields req-
uisite data for a direct calculation of uptake rates. At steady state, chemical
uptake may be determined by a mass balance calculation. Correction for
adsorption to surfaces of the exposure chamber should be made by operat-
ing the system without plants. Pots filled with hydroponic solution or sup-
port media should be included in the sytem adsorption calibration. Con-
sequently, chemical analysis of plant tissues exposed to gaseous chemicals
may not be required in order to demonstrate and quantitate uptake rates.
(B) Altered rates of net photosynthesis, transpiration, and stomatal
conductance are anticipated as a result of chemical 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 con-
ditions within the fumigation chamber.
(iii)	Numerical. Mass of pooled plant organs and pooled whole plants
should be measured for the uptake and translocation test and subjected
to chemical analysis (above) to quantify free parent test chemicals, its
metabolites and soluable and bound residues. Mass balance of the test
chemical and evapotranspiration rates of the plants are also determined.
Means and standard deviations should be calculated and plotted for each
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of the above for every treatment and control. The data should also be
subjected to an analysis of variance.
(e) Test conditions—(1) Test species, (i) Test plants recommended
for the uptake test include:
(A)	Lycopersicon esculentum (tomato).
(B)	Cucumis sativus (cucumber).
(C)	Lactuca sativa (lettuce).
(D)	Glycine max (soybean).
(E)	Brassica oleracea (cabbage).
(F)	Avena sativa (oat).
(G)	Lolium perenne (perennial ryegrass).
(H)	Allium cepa (common onion).
(I)	Daucus carota (carrot).
(J) Zea mays (corn).
(ii) Other species of economic or ecologic importance to the region
of impact, may also be appropriate and selected for testing. Two species
of potentially differing sensitivity should be selected such as monocotyle-
donous and a dictyledonous species. It is further suggested that the test
plants selected should be of different growth forms, e.g., a root crop and
a leaf crop.
(2) Facilities—(i) Apparatus. Greenhouses, environmental chambers,
or growth rooms should provide adequate environmental control 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 radioactive and/or test substances. Laboratory facilities for plant ex-
tractions and chemical determinations should include nonporous floor cov-
ering, absorbent bench covering with non-porous backing, and adequate
disposal facilities to accommodate plant nutrient, test, and wash solutions
containing radioisotope and/or test chemical at the end of each run, and
any bench covering, lab clothing, or other contaminated materials.
(ii) Containers and support media. For testing purposes, at least
24 polyethylene pots sufficently large to grow at least five plants up to
28 days or one to three plants to maturity are required. If plants are grown
hydroponically, one plant per pot may be the preferred method. If a carrier
control is needed, 30 pots are used. Potting containers used ine ach experi-
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ment should be of equal size and volume and possess the same configura-
tion. When sand or glass beads are used the potting containers should be
filled to within 2.5 cm of their tops with sand or glass beads. Perlite,
vermiculite, native soils, etc., should not be used for root support. Potting
containers should be covered with opaque polyethylene bags to exclude
light and minimize volatilization of test chemical.
(iii)	Cleaning and sterilization. Potting containers, nutrient storage
containers, and root support medium should be cleaned before use. All
equipment should be washed according to good standard laboratory proce-
dures to remove any residues remaining from manufacturing or use. A
dichromate solution should not be used for cleaning beads or pots. Rooting
media other than glass beads should be discarded at the end of the experi-
ment. Disposal should conform to existing regulations.
(iv)	Nutrient media. Half-strength modified Hoagland nutrient solu-
tion should be utilized as nutrient media for this test. Hydroponic solution
should be aerated and sand or glass beads potting containers should be
filled with nutrient solution and drained periodically. An automated system
design is recommended.
(3) Test parameters. Environmental conditions should be maintained
as specified below:
(i)	Clean dioxide concentrations at 350 ± 50 ppm.
(ii)	Relative humidity approaching 70 ± 5 percent during light periods
and 90 percent during dark periods.
(iii)	Irradiation, measured at 1 meter from the source, at 350 ± 50
(lE/m2 sec at 400 to 700nm.
(iv)	Photoperiod of 16 h light and 8 h darkness for all species except
soybean which should be provided with 11 h light and 13 h darkness prior
to flowering.
(v)	Day/night temperatures at 25/20 ± 3 °C.
(f) Reporting. Reporting requirements of 40 CFR Part 792—Good
Laboratory Practice Standards apply to this guideline. Concentrations
should be expressed in appropriate weight units per grams of dry plant
material and of water lost by evapotranspiration. Data should also include
initial and final total concentration of the test chemical in the growth
media. These data will be used to compute mass balance. The following
should be reported for each of the species tested in tabular form:
(l)Solid and liquid test chemicals, (i) Concentration of chemical in
nutrient solution and root support material when chemical is soluble in
water or solubilized with a carrier compound, as well as the concentration
of carrier compound in nutrient solution when carrier is used, or the quan-
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tity of chemical per unit weight of root support material when it is coated
on the material.
(ii)	The quantity of chemical, the concentration at which it was ap-
plied, and the number of applications for those chemicals applied to the
foliage.
(iii)	Environmental conditions (day/night temperatures, relative hu-
midity, light intensity, carbon dioxide concentration, and photoperiod) and
the occurrence and extent of any disruption of environmental control facili-
ties.
(iv)	Mass of each pool of plant organs and by summation, the mass
of whole plants (dry weight after 24 h at 70 °C).
(v)	Concentration of free parent test chemical, metabolites and soluble
residues, and bound residues in pooled plant organs and pooled whole
plants.
(vi)	Mass balance of chemical.
(vii)	Means evapotranspiration rate per plant.
(viii)	Visible effects of chemical, if any, on the intact plants.
(ix)	Analysis of variance, F-test, means, and standard deviation about
the mean are calculated under paragraphs (f)(l)(iv), (f)(l)(v), (f)(l)(vi),
and (f)(l)(vii) of this guideline.
(2) Gaseous test chemicals, (i) Concentration of gaseous test chemi-
cal at inflow and outflow ports.
(ii)	Environmental conditions within gas exposure system (air tem-
perature, dew point temperature or water vapor pressure of incoming and
outgoing air streams, light intensity, air speed within chamber, carbon di-
oxide concentration at inflow and outflow ports, gas flow rate into and
out of exposure system).
(iii)	Mass (dry weight after 24 h at 70 °C) of leaves and stems and
surface area (one side of leaves) in the exposure system.
(iv)	Calculated measurements of photosynthesis, transpiration, and
stomatal conductance before, during, and after exposure to test chemicals.
(v)	Visible effects of chemical, if any, on the plants.
(vi)	Analysis of variance, F-test, means, and standard deviation about
the mean are calculated for each of the following:
(A) Steady state rates of photosynthesis, transpiration, and chemcial
uptake before, during, and after fumigation.
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(B) Stomatal conductance or leaf diffusion resistnace before, during,
and after fumigation.
(vii) If uptake is determined by direct chemical analysis of plant tis-
sues, then the reporting requirements also include:
(A)	Concentration of free parent test chemical, metabolites and
soluable residues, and bound residues in pooled plant organs and pooled
whole plants.
(B)	Mass balance of the chemical.
(C)	Analysis of variance, F-test, means and standard deviation about
the mean under paragraphs (f)(2)(vi)(A) and (f)(2)(vi)(B) of this guideline.
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