Toxic Substance!
Washington, DC 20460
Laboratory Trials
and Critical
Evaluations of
Soil TLC Tests
Final
Report
1PA
560/
11-
80-002
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LABORATORY TRIALS AND CRITICAL EVALUATIONS
OF SOIL TLC TESTS
by
Edith G. Leighty and Carl A. Alexander
BATTELLE
Columbus Laboratories
505 King Avenue
Columbus, Ohio 43201
FINAL REPORT
00
or>
U.S. Envirorjacmtal Protection Ae*ncy
Library. Room 2404 ?M-:m-A
401 M .Slreot, S.V,;.
TiC 204[-0
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U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
WASHINGTON, D.C. 20460
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r
DISCLAIMER
This report has been reviewed by the Office of Pesticides and Toxic
Substances, U. S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily reflect
the views and policies of the U. S. Environmental Protection Agency, nor does
mention of trade names or commercial products constitute endorsement or
recommendation for use.
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TABLE OF CONTENTS
Page
Scope of Work 2
Materials and Method 2
Results and Discussion 6
Evaluation of Soil TLC Test Procedure 7
TABLES
Number
1 Soils Used to Prepare TLC Plates 3
2 l4C-Compounds Evaluated on Soil TLC Plates 4
3 Matrix of Laboratory Trials 5
4 RF Values (a) of 14C-Chemicals on Soil TLC Plates 6
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TABLE 2. 14C-COMPOUNDS EVALUATED ON SOIL TLC PLATES
14C -Com pound
2,6-14C-Pyridine
2-14C-Imidazole
Carboxyl-14C-Nicotinic Acid
Carboxyl-14C-DL-Histidine
2-14C-Thymine
Water solubil ity
Infinite
Very soluble
Slightly soluble
Low
SI ightly soluble
Classification
Weak base
Weak base
Acid
Weak acid
Weak acid
Pyridine and imidazole are parent structures for four of the com-
pounds and are highly soluble in water. The two derivatives are carboxylic
acids and are much less soluble. Thymine is a very different compound which,
in its tantomeric forms, shows substantial aromatic character.
Soil TLC plates, 5 x 20 cm, were prepared and the mobility of the
five chemicals on the plates was determined according to the following proto-
col, as described by Helling(l~4):
(1) Soils were used with a pH between 4 and 8, an organic matter
content between 1 and 8 percent, a cation exchange capacity
greater than 1 meq/100 g and less than 70 percent sand.
(2) Soils were prepared by sieving to remove stones, coarser sand
fractions, and large plant fragments. Any crushing or grinding
involved reduced soil aggregate size but did not create more
fine particles (silt and clay) than originally present. Soils
were sieved at 250 m to remove coarse (500 to 2,000 m) and
medium (250 to 500 m) sand fractions.
(3) Water used was distilled, deionized H20 adjusted to pH 7 by
boil ing to remove C02-
(4) Water was added to the sieved soil until a smooth, moderately
fluid slurry was attained. Approximately 0.75 ml HgO was
added for each gram of soil, providing a moderately fluid
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slurry. The soil slurry was added quickly to clean glass plates
to prevent particle size segregation.
(5) The soil slurry was spread evenly across the plate using a
variable thickness plate spreader.
(6) The plates were air dried for a minimum of 24 hours after slurry
application.
(7) The purest grade of test ^C-chemicals available was used in
this study.
(8) At least four replicate plates were used for each soil. Approx-
imately 5 yg (0.05 yd) of the l4C-labeled test solution was
spotted 1.5 cm from the edge of the plate.
(9) The plate was developed in a closed chromatographic chamber con-
taining 0.5 cm HgO for a distance of 11.5 cm.
(10) Radioactive spots on the plates were detected with a Radiochro-
matogram Scanner (Packard). Mobility Rf values of the test
l^c-chemicals were determined by dividing the distance that
the chemical travelled on the plate by the distance of the
solvent front.
Overall, four replicate trials of a chemical and soil were made to
examine the validity of each result. The trials were performed according to
the matrix presented in Table 3.
For one chemical, 14C-nicotinic acid, replication of 3 x 3 or 9
trials was made for each soil to check for reproducibility of the soil TLC
plates.
TABLE 3. MATRIX OF LABORATORY TRIALS
Chemicals
1
Z
3
4
5
A
3x3
4
4
4
4
B
3x3
4
4
4
4
Soils
C
3 x 3
4
4
4
4
D
3x3
4
4
4
4
E
3x3
4
4
4
4
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RESULTS AND DISCUSSION
The mobilities of the five test ^C-chemicals on the five different
soil TLC plates are given in Table 4.
TABLE 4. Rf VALUES (a) of ^C-CHEMICALS ON SOIL TLC PLATES
14C-Chemical
Pyridine
. Imidazole
Nicotinic Acid
Histidine
Thymi ne
Hagerstown No. 20
soil
0.46+0. 02(b)
0.16+0.03
0.91+0.05
0.14*3.04
0.80+0.05
Ritzville
soil
0.22+.02(fa)
0.16+0.04
0.97+0.03
0.22+0.05
0.57+0.08
Crosby
soil
0.17+0.02
0.20+0.04
0.78+0.06
0.15+0.03
0.76+10.06
Brookston
soil
0.22+0.05
0.15+0.05
0.83+0.04
0.16+0.02
0.38+0.03
Zynbar
soil
0.21+0.02
0.37+0.05
0.54+0.06
0.18+0.04
0.56+0.02
+Standard deviation.
Flate developed in buffer pH 4 solution.
e could not be detected on the Hagerstown No. 20 and
Ritzville soil TLC plates when they were developed in water. This was ap-
parently due to pyridine evaporating from the plates because of its azeotropic
properties with water under the conditions of the two soils. We were, how-
ever, able to develop ^C-pyridine on these soil plates when an acid buffer
was used as the developing solvent. This azeotropic property of l^C-pyri-
dine was not a major problem with the other three types of soils.
Histidine was strongly adsorbed on all five types of soil and showed
very little migration on any of the plates. Imidazole and pyridine were like-
wise strongly adsorbed, with imidazole showing only a slight migration on the
Zynbar soil plates and pyridine on the Hagerstown No. 20 plates when developed
in a pH 4 buffer solution. The mobility of nicotinic acid was extensive on
all five types of soil plates, with slightly less being observed on the Zynbar
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soil plates. Thymine, a weak acid, was the only test chemical to show
selective mobility on the five different soil plates. It showed extensive
migration on the Hagerstown No. 20 and Crosby soil plates, slightly less on
the Ritzville and Zynbar soil plates, and low migration on the Brookston soil
plates. Hagerstown No. 20 and Crosby are low-pH and high-silt and -clay
soils. Although Zynbar is also a low-pH soil its other properties are not
known. Ritzville and Brookston are more neutral soils, with Ritzville soil
containing a high percentage of sand and a low percentage of clay and
Brookston soil containing a low percentage of sand and a high percentage of
clay.
EVALUATION OF SOIL TLC TEST PROCEDURE
No difficulties were found with the written soil TLC test procedure
with respect to clarity of the directions, adequacy of detail, and suggested
data formats. There were also no difficulties in making soil TLC plates from
five different soils with no modification of the procedure.
Each chemical was evaluated on four plates of each of the different
soils except for one chemical which was evaluated on nine plates. The stan-
dard deviation of the data was low, with all of the chemicals and plates
indicating good reproducibility. It is recommended, therefore, that each
determination be repeated on four to five plates for statistical validity.
The only difficulty experienced in this evaluation was that due to
the evaporation of 14c_pyridine fr0m two different types of soil TLC plates
because of its azeotropic properties with water. This was corrected by devel-
oping these plates in an acidic buffer rather than water.
Except for modifications due to the difficulty with pyridine, the
same TLC method was used for all five types of soil and for all five chemi-
cals. The ease of operation and general utility of the method is therefore
good. In order to measure the mobility of different structured chemicals in
the system, however, it may be necessary to use a buffered solvent system with
a slightly higher or lower pH than deionized water. For example, histidine
did not migrate on any of the five soil TLC plates using water as the solvent
system. It may, however, migrate at a different pH than that of water.
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8
The chemical being tested may also not be available, or cannot be
synthesized readily, as a radioactive compound. This may necessitate using
another spot-detecting procedure rather than radioactivity scanning for
measuring chemical migration on the plate. This may be done with detecting
reagents for specific classes of chemicals. The natural brown color of soil
TLC plates, however, may cause some problems with this visual method of spot
detection. As a last resort, zonal extraction of the plate may be necessary
to remove the chemical from the plate, with subsequent spectrophotometric, GC,
or HPLC analyses of the extract. This, of course, would be a much more time-
consuming and expensive method of analysis.
An estimated cost for performing a radioactive chemical TLC routine
evaluation using the above procedure, is shown below:
Radioactive Chemical - $150
Man-Hour Costs - 150
Supplies - 10
Equipment Costs - 8
Space Costs - 1
Capital Costs - !_
Total $320
CONCLUSION
Our conclusion from this study is that the Soil Thin-Layer Chroma-
tography Test described above may be a cost-effective screening test for
giving soil mobility data on certain chemicals.
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REFERENCES
1. Helling, C. S. 1971a. Pesticide Mobility in Soils I. Parameters of Thin
Layer Chromatography. Soil Sci. Soc. Am. Proc. 35:723-737.
2. Helling, C. S. 1971b. Pesticide Mobility in Soils II. Applications of Soil
Thin Layer Chromatography. Soil Sci. Soc. Am. Proc. 35:737-743.
3. Helling, C. S. 1971c. Pesticide Mobility in Soils III. Influence of Soil
Properties. Soil Sci. Soc. Am. Proc. 35:743-748.
4. Helling, C. S., and 8. C. Turner, 1968, Pesticide Mobility Determination
by Soil Thin Layer Chromatography. Science 162:562-563.
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TECHNICAL REPORT DATA
(Please read Instructions on the rcvcne before completing)
1. REPORT NO. 2.
EPA-560/1 1-80-002
4. TITLI AND SUBTITLi
Laboratory Trials and Critical Evaluatic
of Soil TLC Tests
7. AUTMOR(S)
E. 6. Leighty and C. A. Alexander
9, PERFORMING ORGANIZATION NAME AND ADDRESS
Battelle-Col unbus Laboratories
505 King Avenue
Columbus, OH 43201
12. SPONSORING AGENCY NAME AND ADDRESS
EPA, Office of Pesticides and Toxic Subs
401 M Street, S.W.
Washington, D.C. 20460
3. RECIPIENT'S ACCESSION NO.
5. REPORT DATE
m* February 1980
6. PERFORMING ORGANIZATION CODE
8. PERFORMING ORGANIZATION REPORT NO.
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
68-01-5043
13. TYPE OF REPORT AND PERIOD COVERED
tanrp- Fina1 ReP°rt>> 5/79 ' 2/80
14. SPONSORING AGENCY CODE
EPA-560/1 1
15. SUPPLEMENTARY NOTES
EPA project officer for this report is Ronald A. Stanley (202)755-4860
16. ABSTRACT
This report describes the performance of laboratory trials and critical
evaluations of the soil thin-layer chroma tog raphy test described in the
discussion in Section 5 of the Toxic Substances Control Act (Federal
Register, March 16, 1979, pp. 16257-16259). The overall purpose was to
validate this test, or modifications of it, as a cost-effective screen-
ing test. Results indicate that this test may be a cost-effective
method for giving soil mobility data on certain chemicals.
17. KEY WORDS AND DOCUMENT ANALYSIS
a. DESCRIPTORS
Soil thin-layer chroma tog raphy
Chemical mobility
Laboratory evaluations
18. DISTRIBUTION STATEMENT
Unl imited
b.lOENTIFIERS/OPEN ENDED TERMS C. COSATI Held/Group
Mobility of toxic com-
pounds
Soil
19. SECURITY CLASS (This Report) 21. NO. OF PAGES
Unclassified 9
20. SECURITY CLASS (This page) 22. PRICE
Unclassified
EPA Form 2220-1 (R>v, 4-77) PREVIOUS EDITION is OBSOLETE
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