United States Prevention, Pesticides EPA712-C-98-047
Environmental Protection and Toxic Substances January 1998
Agency (7101)
&EPA Fate, Transport and
Transformation Test
Guidelines
OPPTS 835.1210
Soil Thin Layer
Chromatography
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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
(7U.S.C. I36,etseq.).
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 202-512-0132 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from EPA's World Wide Web site
(http://www.epa.gov/epahome/research.htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelines/OPPTS Harmonized Test
Guidelines."
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OPPTS 835.1210 Soil thin layer chromatography.
(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 796.2700 Soil Thin Layer Chro-
matography.
(b) Introduction—(1) Background and purpose, (i) Leaching of
chemicals through soil is an important process which affects a chemical's
distribution in the environment. If a chemical is tightly adsorbed to soil
particles, it will not leach through the soil profile but will remain on the
soil surface. If a chemical is weakly adsorbed, it will leach through the
soil profile and may reach ground waters and then surface waters. Knowl-
edge of the leaching potential is essential under certain circumstances for
the assessment of the fate of chemicals in the environment.
(ii) Chemical leaching also affects the assessment of ecological and
human health effects of chemicals. If a chemical reaches ground water,
deleterious human health effects may arise due to the consumption of
drinking water. If a chemical remains at the soil surface, deleterious envi-
ronmental and human health effects may arise due to an increased con-
centration of the chemical in the zone of plant growth, possibly resulting
in contamination of human food supplies.
(iii) Soil thin layer chromatography (TLC) is a qualitative screening
tool suitable for obtaining an estimate of a chemical's leaching potential.
This test is one of several tests which can be used in obtaining a rough
estimation of a chemical's leaching potential.
(2) Definitions and units, (i) Cation exchange capacity (CEC) is the
sum total of exchangeable cations that a soil can adsorb. The CEC is ex-
pressed in milliequivalents of negative charge per 100 g (meq/100 g) or
milliequivalents of negative charge per gram (meq/g) of soil.
(ii) Particle size analysis is the determination of the various amounts
of the different particle sizes in a soil sample (i.e., sand, silt, clay) usually
accomplished by sedimentation, sieving, micrometry, or combinations of
these methods. The names and size limits of these particles as widely used
in the United States are:
Name
Very coarse sand
Coarsesand
Medium sand
Fine sand
Very fine sand
Diameter range
(mm)
2 0 to 1 0
1.0 to 0.5
0.5 to 0.25
025 to 0 125
0.125 to 0.062
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Name
Silt
Clay
Diameter range
(mm)
0 062 to 0 002
<0.002
(iii) Rf is the furthest distance traveled by a test material on a TLC
plate divided by the distance traveled by a solvent front (arbitrarily set
at 10.0 cm in soil TLC studies).
(iv) Soil is the unconsolidated mineral material on the immediate sur-
face of the earth that serves as a natural medium for the growth of land
plants; its formation and properties are determined by various factors such
as parent material, climate, macro- and microorganisms, topography, and
time.
(v) Soil aggregate is the combination or arrangement of soil separates
(sand, silt, clay) into secondary units. These units may be arranged in the
profile in a distinctive characteristic pattern that can be classified on the
basis of size, shape, and degree of distinctness into classes, type, and
grades.
(vi) Soil classification is the systematic arrangement of soils into
groups or categories. Broad groupings are made on the basis of general
characteristics, subdivisions, on the basis of more detailed differences in
specific properties. The soil classification system used today in the United
States is the 7th Approximation Comprehensive System. The ranking of
subdivisions under the system is: order, suborder, great group, family, and
series.
(vii) Soil horizon is a layer of soil approximately parallel to the land
surface. Adjacent layers differ in physical, chemical, and biological prop-
erties or characteristics such as color, structure, texture, consistency, kinds
and numbers of organisms present, and degree of acidity or alkalinity.
(viii) Soil order is the broadest category of soil classification and is
based on general similarities of physical/chemical properties. The forma-
tion by similar genetic processes causes these similarities. The soil orders
found in the United States are: Alfisol, aridisol, entisol, histosol, inceptisol,
mollisol, oxisol, spodosol, ultisol, and vertisol.
(ix) Soil organic matter is the organic fraction of the soil; it includes
plant and animal residues at various stages of decomposition, cells and
tissues of soil organisms, and substances synthesized by the microbial pop-
ulation.
(x) Soil pH is the negative logarithm to the base 10 of the hydrogen
ion activity of a soil as determined by means of a suitable sensing elec-
trode coupled with a suitable reference electrode at a 1:1 soil to water
ratio.
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(xi) Soil series is the basic unit of soil classification and is a subdivi-
sion of a family. A series consists of soils that were developed under com-
parable climatic and vegetational conditions. The soils comprising a series
are essentially alike in all major profile characteristics except for the tex-
ture of the "A" horizon (i.e. the surface layer of soil).
(xii) Soil texture refers to the classification of soils based on the rel-
ative proportions of the various soil separates present. The soil textural
classes are: clay, sandy clay, silty clay, clay loam, silty clay loam, sandy
clay loam, loam, silt loam, silt, sandy loam, loamy sand, and sand.
(3) Principle of the test method, (i) Before 1968, methods of inves-
tigating the mobility of nonvolatile organic chemicals within soils were
based on the use of field analysis, soil adsorption isotherms, and soil col-
umns. In 1968, Helling and Turner introduced soil TLC as an alternative
procedure; it is analogous to conventional TLC, with the use of soil instead
of silica gels, oxides, etc., as the adsorbent phase.
(ii) Papers by Helling (1968, 1971) under paragraphs (e) (5), (6), and
(7) of this guideline and Helling and Turner (1968) under paragraph (e)(3)
of this guideline were the basis of this test guideline. The soil and colloid
chemistry literature and the analytical chemistry literature substantiate the
experimental conditions specified in the guideline.
(iii) Soil TLC offers many desirable features. First, mobility results
are reproducible. Mass transfer and diffusion components are distinguish-
able. The method has relatively modest requirements for chemicals, soils,
laboratory space, and equipment. It yields data that are amenable to statis-
tical analyses. A chemical extraction-mass balance procedure to elicit in-
formation on degradation and chemical transformations occurring at col-
loid interfaces can be incorporated into this test. The ease with which the
Rf and mass balance are performed will depend upon the physical/chemical
properties of the test chemical and the availability of suitable analytical
techniques for measuring the chemical.
(4) Applicability and specificity, (i) Soil TLC can be used to deter-
mine the soil mobility of sparingly to infinitely water soluble chemicals.
In general, a chemical having a water solubility of less than 0.5 ppm need
not be tested since the literature indicates that these chemicals are, in gen-
eral, immobile (see Goring and Hamaker (1972) under paragraph (e)(l))
of this guideline. However, this does not preclude future soil adsorption/
transformation testing of these chemicals if more refined data are needed
for the assessment process.
(ii) Soil TLC may be used to test the mobility of volatile chemicals
by placing a clean plate over the spotted soil TLC plate and then placing
both plates in a closed chromatographic chamber.
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(iii) Soil TLC was originally designed for use with soils. The lit-
erature shows no published use of this method with sediments as the ab-
sorbent phase, probably due to the fact that sediment surface properties
change significantly during air drying. It is extremely important that the
TLC plate coated with the absorbent be air dried before leaching studies
can be undertaken.
(c) Test procedures—(1) Test conditions, (i) Equipment required—
distilled-deionized water adjusted to pH 7 by boiling to remove CCh, clean
glass plates (TLC plates), glass rods or a variable thickness plate spreader,
masking tape, closed chromatographic chambers, and analytical instrumen-
tation necessary and appropriate for the detection and quantitative analysis
of the test chemical.
(ii) The test procedure may be run at 23+5 °C.
(iii) It is recommended that three replicate plates for each soil be
used.
(2) Test procedures, (i) To reduce aggregate size before or during
sieving, crush and grind the air-dried soil very, very gently.
(ii) Sieve air-dried soils with a 250 (im sieve.
(iii) Add water to the sieved soil until a smooth, moderately fluid
slurry is attained (approximately 0.75 mL H2O added for each gram of
soil).
(iv) Spread the slurry evenly and quickly across the clean glass plate
using a variable thickness plate spreader, a glass rod, or other available
method. If a glass rod is used, control the layer thickness by affixing mul-
tiple layers of masking tape along the plate edges. Soil layer thickness
should be 0.50-0.75 mm.
(v) Air dry the plates at 25 °C for a minimum of 24 h after uniform
slurry application is achieved.
(vi) Scribe a horizontal line 11.5 cm above the base through the soil
layer down to the glass to stop solvent movement.
(vii) Spot the test chemical, in solution, 1.5 cm above the base. For
radiolabeled materials, 0.5-5 (ig containing 0.01-0.03 (iCi of 14C-labeled
compound may be used.
(viii) If the compound is volatile, it is extremely important that a
clean plate be placed over the soil TLC plate to impede volatilization.
(ix) Immerse the plate with the base down at some angle from the
vertical in a closed chromatographic chamber containing water at a height
of 0.5 cm.
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(x) Allow the solvent front to migrate to the 11.5 cm line before
removing the plates from the chamber.
(xi) Determine the Rf values. Zonal extraction, plate scanning, or any
other method or combination of methods suitable for detection of the par-
ent test chemical may be used.
(xii) Determine the amount of the parent test chemical on the entire
soil TLC plate after test chemical migration. Any method or combination
of methods suitable for the extraction and quantitative detection of the
parent test chemical may be used.
(d) Data and reporting. Report the following information. (1) Tem-
perature at which the test was conducted.
(2) Amount of the test chemical applied and amount recovered from
the plates.
(3) Detailed description of the analytical technique used in the Rf
determination, the chemical extraction, and the quantitative recovery and
analysis of the parent chemical.
(4) The mean frontal Rf value with the standard deviation for each
soil tested.
(5) A photograph or diagram of the TLC plate which shows the entire
leaching pattern (from 1.5 to 11.5 cm).
(6) Soil information: Soil order, series, texture, sampling location, ho-
rizon, general clay fraction mineralogy.
(7) Soil physical/chemical properties: Percent sand, percent silt, and
percent clay (particle size analysis); percent organic matter; pH (soil-to-
water ratio, 1:1); and cation exchange capacity.
(e) References. For additional background information on this test
guideline the following references should be consulted:
(1) Goring, C.A.I, and Hamaker, J.W. Organic Chemicals in the Soil
Environment. Vol. I & II. Marcel Dekker, NY (1972).
(2) Helling, C.S. Pesticide mobility investigations using soil thin layer
chromatography. American Society for Agronomy Abstracts (1968).
(3) Helling, C.S. and Turner, B.C. Pesticide mobility: Determination
by soil thin layer chromatography, Science 162:562 (1968).
(4) Helling, C.S., Movement of S-triazine herbicides in soils, Residue
Review 32:175-210 (1970).
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(5) Helling, C.S. Pesticide mobility in soils I. Parameters of soil thin
layer chromatography. Soil Science Society of America Proceedings
35:732-737 (1971).
(6) Helling, C.S. Pesticide mobility in soils II. Applications of soil
thin layer chromatography. Soil Science Society of America Proceedings
35:737-743 (1971).
(7) Helling, C.S. Pesticide mobility in soils III. Influence of soil prop-
erties, Soil Science of America Proceedings, 35:743-748 (1971).
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