JESZHCIDS .AITAI.yp-FS Bx GAS C3EOMATOGPAPHY
AT THE LA::3 MICHIOA:i BASH; OFFICE*
by
W. D, Johnson, F, D. Fuller and L. E. Scarce*'^
Hecords of organic pesticide usage by farmers show that, in IS66,
37 percent were herbicides^ 29 percent insecticides, h percent fungicides,
e.nd 8 percent miscellaneous pesticides. More than 150 million pounds of
pesticides are purchased by urban dwell er^j hk to 109 pounds of LOT per
acre was used to control Dutch Elra disease in Wisconsin urban co.rj-iunit.Les,
This high urbar usage of pesticides contributes a large pollution load to
lakes and streams. The evolutionary growth of gas chromatography is meeting
the Federal Water Pollution Control Administration (FrfPCA) laboratories' ne.-^d
.to measure low level and changing pesticide characteristics.
The PvJTCA's Lake Michigan Bab in Office (LMEO) has the responsibility
for conducting pesticide surveillance program in the Great; Lakes and the
Central Missis sitroi River Basins waters. Assistance and consultation Is also
i
vided to federal; strata, and local agencies for pesticide analysis by gs.s
chromatography.
This paper elaborates on analytical methods employed for chlorinated
end thiophosphated pesticides, types of samples analyzed, tyrleal con-
centrations, and the significance of findings.
•^Presented e.t the National Meeting of vhe 20th Mid-Amerie'r Symposiur1 on
Spectroscopy? May 13; 19^9; ir^ 'Jiiicago, Illinois
'^Orgardc Chemist» .Supervisor^ Che,mi?try Sect: on jana Chi?:\, Leboratc -y
Services Branch, respective] y; U.S. D&ps-^'c jsnt of the I~~crior_, Federal
Water Pollution Control Adniniso.-v.'.-M . La!-;e Jiicbig
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INTRODUCTION
The Lake Michigan Basin Office (LMBO) is responsible for conducting
pesticide surveillance programs in the Great Lakes and the Central Mississippi River
Basins waters. Assistance and consultation is also provided to federal, state,
and local agencies for pesticide analysis "by gas chromatography. Pesticide
pollution of these regions presents a potential hazard for aquatic organisms,
fish, birds, vildlife, and man. The chlorinated pesticides are the most
persistent,while the thiophosphates are more highly toxic.
" Records of organic pesticide usage by farmers show that, in 19^6,
37 percent were herbicides, 29 percent insecticides, h percent fungicides,
and 8 percent miscellaneous pesticides. More than 150 million pounds of
pesticides vere'purchased by urban dwellers; ^ to 109 pounds of DDT par acre
"was used to control Dutch Elm disease in Wisconsin urban coomunities. This
high urban usage of pesticides contributes a large pollution load to lakes
and streams. Total. U-.S. production of pest control chemicals in 1967 was
approximately 1.25 billion pounds having a market value of about 800 million
dolls;rs. The total U.S. consumption was over one billion pounds of active
-pesticide chemicals (1). Of all the insecticides used today, 75$ is applied
to less than 2$ of the land, and of the lj-57 million acres of farmland in the
United States, it is estimated that only 15$ of total crop acreage receive
pesticides (2).
Thousands'of pounds of chlorinated, thiophosphated, and other pesticides
run off into lakes and streams yearly. The application of pesticides has
been so extensive that DDT is found in antarctic penguins and arctic life
forms, such as lichens. Chlorinated pesticides are so persistent that
toxaphene was found ten years after application in Wisconsin lakes (3)•
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The concentration of pesticides are determined by several factors such
as kind of pesticide, sorption ability and climate. As a general statement.,
the concentration of pesticides in waters is "based upon the amount directly
received, that portion from runoff, and the stability of molecules towards
physicochemical ' effects.- For example, aldrin decomposes to form its
epoxide, dieldrin; DDT generally decomposes to its isomers/ pp'-DDT, op-DDT,
DDE, etc,; and heptachlor is converted to heptachlor epoxide.
>
The Initial, higher concentrations of chlorinated and thiophosphated
pesticides after spraying, dusting or runoff have the most detrimental
effect on fish and wildlife. Dosages necessary to produce immediate kills
vary from species to species'but generally 0.1 mg/1 will seriously sffg-st
or kill most game fish and benthic fauna. Numerous fish and fowl kills
have been reported in and around Lake Michigan. (4).
This paper presents some highlights on how pesticides are routinely
eoaalyzed at the Lake Michigan Basin Office by gas chromatography with
electron capture and microcoulometric titration detection, thin layer .
chromatography, and positive identification by infrared spectroscopy (iR).
"FWPCA's Lake Michigan Basin Office routinely analyzes for the following
chlorinated pesticides: lindane, heptachlor, aldrin, heptachlor epoxide,
dieldrin, endrin, op-DDT and pp' DDT. Analyses have "been performed according to
the U.S. Public Health Service revised methods (5)".
The Lake Michigan Enforcement Conference Pesticides Committee recommends th
following compounds be determined in the water, fish and clams of Lake
Michigan:_ DDT, dieldrin, DDD, DDE, methoxychlor, chlordane, and endrin (6).
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SAMPLING PROCEDURE
The type of samples analyzed at ths 1,-aka iiiehigaa Basin Office ej.-e; vater,
bottom sediments, algae or aquatic plants, soils,and fish.
The vater samples are generally grab samples collected in a modified
Kemmerer Sampler. The bottom samples are taken vith an Efcasit or Peters en
dredge or by core sampling. Algae or small aquatic plants are collected
through the use of a plankton net. Soil samples are taken from top soil in
areas having been treated vith pesticides.
The containers are h to 10 liter glass bottles and quart jars cleaned
vith dicihr ornate cleaning solution and rinsed vith distilled water, alcohol,
acetone] ether, then chloroform. The caps are lined vith aluminum foil (3 )•
I
&
Carbon adsorption filters may be used as a means of collecting or con-
"~ *_.-_-». _ v _ _ _ _
centrating pesticides from vater. This method involves the filtering of
300 to 5,000 gallons of vater through a cartridge (3 x 18 inches in size)
packed vith granular carbon at the rate of 0.03-0.5 gallons per minute. The
cartridge is filled vith 4.5 inches of 4 x 10 mesh carbon, folloved bylnine
inches of 30 mesh carbon and finally 4.5 inches of 4 x 10 mesh carbon,
. A rapidly analyzed grab sample of vater, bottom sediments, algae, aquatic
plants and fish may be more reliable since it permits less degradation of
pesticides. Grab sampling also offers a savings in manpower and equipment.
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Coraposite sampling of the above may give a more representative picture
of the pesticide content over a period of 2 or 3 days. However, some
pesticides, such as thiophosphated and carbemates, may be degraded before
the analysis can be performed.
All types of samples, except water, may be preserved for a few days or
months by freezing depending upon type of pesticide. However, when collecting
water samples in the winter months, it is necessary to add sodium chloride or
ethyl alcohol to the water sample to prevent freezing and breakage of the
sample containers while in the field. Water samples are stored at 5°C
until analyzed.
ANALYTICAL PROCEDURES
Sanrole and ReaŁent_Prep_aration.
All water samples analyzed for pesticides by the Leke Sichlgan Basin Office are
subjected to liquid-liquid extraction, using redistilled chloroform as
the solvent. One gallon of sample is placed in a ^--liter separatory
funnel and 25 ml of saturated sodiun sulfate solution and 5 ^l of 1:1
hydrochloric acid are added for each liter of sample. The sample is
extracted three times using 100 ml of redistilled chloroform for each
extraction. The extract is dried by pouring over a 2-inch column of
anhydrous sodium sulfate. The extract is cleaned-up by passing through
a column of Florisil topped with 1-inch of anhydrous sodium sulfate.
The extract is then evaporated to a volume of 0.5 ml. Further clean-up may
be accomplished by thin-layer chromatography separation or trapping
of specific peaks by gas chromatography.
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Bottom sediments are spread in aluminum lined pans approximately
12 x 2h x 2 inches,, covered with gauze, then air dried by forcing air
currents across the surface of the sediment until dry enough to grind to
approximately 30 mesh. This requires from one to four days. Pesticides
sorted on particulate matter may "be deserted by a 2k- hour continuous Soxhlet
extraction of a 25-200 gram sample with chloroform. The bottom sediments
extracts are cleaned- up in the same manner described in the paragraph above,
Algae, aquatic plants and soil samples are dried, ground, extracted
end" the extracts cleaned-up in the same manner as bottom sediment samples.
Fish and other aquatic organisms are ground in s. blender end. an aliquot
removed for analysis. The aliquot is dried vith anhydrous sodiun sulfate,
extracted with chloroform and the extract cleaned-up in the same manner as
described above.
Carbon filter samples are dried at 40°C, then extracted vith redistilled
chloroform in a Soxhlet apparatus for 35 hours. The chloroform extract is
then concentrated and cleaned-up in preparation for further analyses (7).
All solvents are redistilled or commercially available redistilled
solvents are employed. Reliability of solvent purity is checked by running
a blank.
Analysis of Samples
Water analyses are generally performed according to procedures in the
Federal Water Pollution Control Administration' s Interim Official ''isthods for
Chj.orina.ted I^drj3carbon_ .Pesti^cid.es in Water and Wjyy^'^at&r by Gas_ Chro^atosjranhy.
es recommended by the Pesticide Committee of the Lake Michigan Enforcement
Conference
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Bottom sediment samples are analyzed In accordance with the procedures
described in the F^PCA Great Lakes Region's Ch^mistr,/ Jghorator^ Manual -
Bottom §e3j.ments_ (9)-
The analyses of fish samples are performed according to procedures
described by the Food and Drug Administration (lO).
Samples of water, bottom sediments, algae, aquatic plants and fish are
analyzed by thin layer chromatography and gas chromatography with electron
capture and microcoulonetric titration. The Lake Michigan Basin Office
•utilizes gas ehromatographs with an electron capture Nickel-63 detector,
flame ionization and microcoulometric halogen and sulfur titration cells.
The operating parameters of the gas ehromatographs and microcoulometrlc
o
titration have bean as follows:
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1. Electron Capture
a. Volume injected: 0.5-4.0 ul -
b. Inlet temperature: 225 °C
c. Column temperature: 175°c
d. Detector temperature: 195°C
' 2
e. Attenuator setting: 32 x 10
2. Microcoulometrlc Titration
a. Volume injected: 5-0 to 50.0 |al
b. Column temperature: 195"c
c. Inlet temperature: 250°C
d. Furnace temperature: 850°G
e. Sensitivity setting: 500 ohms
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The following columns are used, in gas chromatography vith electron
capture detector.
Material Suport
l) Aluminum 5 ft. 1/4" 100/120 Mesh Gas Chrom Q 7^ OV-17
9^ QF-1
2) Aluminum 5 ft. - 1/4" 80/100 Mesh Gas Chrom Q 10$ DC-200
1556 QP-1
For Biicrocoulometric detection, the gas chromatography column used is as follows;
Stainless Steel 5 ft 1/4" 70-80 M8sh IXDusted Gas 5$ DC-200
Pack
«OV-17 - Methyl Phenyl Silicone
QP-1 = Pluorosilicone
DC-200 s Dow Corning Silicone Oil 200 (l2, ^00 centistokes)
Thin layer chroratography involves spotting microliter portions of an
extract from a pesticide sample on a 200 x 200 mm glass plate coated with a
0.25 mm silica gel or alumina layer impregnated with silver nitrate. This
procedure is used to semi -quant it ate, separate, or clean-up a sample. " The
prepared plate is developed in a closed developing tank containing a 1 cm
depth of carbon tetrachloride . The plate is permitted to develop to a 10 cm
finish line. The plate is removed and placed under strong ultraviolet (U.Y.)
light to develop the chlorinated pesticides which, if present, react with
the silver nitrate to precipitate silver as "black spots. If TLC is employed
for clean-up, plain silica gel or alumina plates are developed with rhodamine 3
solution and the spots are identified by the JL-< value (ratio of spot travel
to the travel of the solvent front). Samples are semi -quant it at ed by comparing
visually with standards .
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I^truinentati^on
Infrared spectrophotornetry is used for identification vhen pesticides
are present in relatively high concentrations.
Gas chromatography results may-be'coupled with mass spectroscopy (MS),
IR, UV, Nuclear Magnetic Resonance (111®) or Differential Thermal Analysis(DTA)
The resulting data can be fed into a computer for extended studies and data
processing. This multiple instrumentation is available commercially for
use vhen tnalysis or studies require further confirmation.
DISCUSSION
Clean-up of. samples, is a major problem, in pesticide analysis. Water
samples relatively free of oil may be cleaned-up by passing through a k-6 inch
Fierisil-1-inch-anhydrous sodium sulfate column after extraction. If the
sample is oily^ acetonitrile partitioning is used to separate the pesticides
from the oil. Bottom sediment, algae, aquatic plants, and fish extracts are
cleaned-up in the sane manner as oily water samples. If further clean-up is
necessary, the extracts are passed through a Florisil-arihydrous sodium sulfate
column. After concentrating the cleaned-up samples, containing a drop of
0.0015$ paraffin oil (a pesticide holding agent), the samples are ready for
analysis by gas chromatography, thin layer chromatography or infrared
spectroscopy (3).
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Gas chromatography with electron capture is used as a rapid, quantitative
method for screening pesticides at the Lake Michigan Basin Office. "By employing
shorter columns (approximately 2~5 feet), the retention time has been shortened;
however, if better resolution is desired, longer columns are employed. Two or
more columns of different polarities are used to change the retention time
of the standard and unknown, thereby obtaining a means for tentative
identification
There are numerous problems associated with the gas chromatograph. The
electron capture detector may give improper response due to low standing
current. This condition may be corrected by cleaning the detector, unless
this effect is due to a slow bleeding column; this latter condition may be
corrected by replacing tHe old column with one that has an immobile phase with
a higher boiling point. Carrier gas filter, poor voltage, defective detector,
and gas leaks in the system cause low standing voltage.
Poor resolution can be corrected by employing the proper column substrate,
temperature, replacing defective columns, correcting gas flow and improving
or correcting injection techniques.
Thin layer chromatography is a useful tool that L?ISQ employs to isolate,
clean-up, and semi-quantitatively or qualitatively analyze for pesticides.
This is one of the most rapid methods of analysis and clean-up procedures. However,
the sensitivity of this procedure is much less than by gas chromatography
and less quantitative.
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Since TLC is approximately 1,000 times less sensitive than gas chrorta-
tography (GO), TLC is useful only when pesticide concentrations are present in
microgram quantities. Visual estimation of concentrations'makes' this method
only semi-quantitative. However,.what -seems to "be a problem "becomes an asset
if the purified spots are employed in GC or IR for confirmatory or positive
identification.
Infrared spectroscopic identification is employed to give positive
identification of questionable pesticides. This method relies upon obtaining
purified saaples so that there are a minimum number of overlapping
absorption peaks in the fingerprint. Approximately 50 to 100 ug of sample
i.
are required for infrared identification. A purified sample is obtained
from a column-chromatography fraction, thin layer chromatography s-oot or gas
chromatography trapping. The large sample requirement prevents the maximum
use of ordinary infrared spectroscopy, since many water saaples have less
than 50 ug of material after preparation and purification.
Infrared spectroseopy is one of the most povcrful scientific tools for
giving positive identification, but presents the problem of obtaining
10,OOO times the amount of residue required for gas chromatography, even when
employing a beam condenser and scale expansion. For good fingerprints the
sample extract must be thoroughly cleaned-up and separated into relatively
pure compounds prior to analysis.
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Mierocoulometric tltration after gas chromatogra-phy is a specific method
for determining halogenated or thiophosphated pesticides. The sensitivity
of the method is approximately 1/3 to 1/20 of that obtained in gas
chromatography with electron capture. -
Microcoulometric gas chronatography combines the principles of gas
chromatography, combustion, and eoulometry into one operation. The problem
of separation of extract into pure fractions is handled through the use of
an appropriately packed column of the correct length and gas flow to give
good resolution. The problem of incomplete combustion is minimized by
adjusting furnace temperature, oxygen or hydrogen and nitrogen flow rate.
Also, resistance is adjusted 'so that one nanograin of sulfur or chloride
* o
produces at 3,east 5 percent of a full recorder scale deflection during
microcoulometric titration.
Maintaining a uniform amount of electrically generated silver ion in
the coulometer cell is insured by maintaining the correct amount of silver
plate on the electrodes, .flushing electrodes vith electrolyte, and quickly
cleaning with dilute nitric acid and rinsing vith distilled water. When
sulfur dioxide is being titrated with the triiodide ion, the platinum
electrodes may become coated, reducing the sensitivity. This difficulty can
be corrected by cleaning the electrodes with dilute hydrochloric acid followed
by a rinsing with distilled water, and/or changing the electrolyte.
SUMMARY
*
The lake Michigan Basin Office has the responsibility for conducting
pesticide surveillance programs in the Great Lakes and the Central Mississippi
River Basins waters.
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The detection of pesticides at low levels in water is made
possible through the use of gas chromatography and other sophisticated
analytical instrumentation,
Grab sampling and analysis of water, "bottom sediment,, algae, aquatic
plants, soils, and fish are used for the rapid assessment of pesticide
3.evels in streams and lakes.
Composite sampling over a period of 2 or 3 clays, 'however, is more
representative of pesticide loadings.
After sample preparation, extraction, clean-up and concentration,
i
samples are analyzed by the following methods:
Gas chromatography vith a HI-63 electron capture detector and micro-
coulornetric titration is employed with special columns to separate
end analyze pesticide samples.
Thin layer chromatography is a practical vay to semi-quanti-
tatively screen pesticide samples; this method may also be
employed to clean-up or purify samples for infrared spectroscopy
end gas chromatography.
Infrared spectroscopy is employed as a more absolute method for
identifying unknown pesticides.
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CONCLUSIOKS
1. Pesticide concentrations in lakes and streams may "be kept under
surveillance through the use of gas chromatography with electron
capture and microcoulometric detection, vith infrared and
thin layer chromatography as associated or hack-up methods.
2« The analysis ofvater, bottom sediments, algae, aquatic plsxrbs, soils,
and fish gives information required to adequately assess pesticide
• levels of lakes and streams.
Disclaimer:
Mention of products and manufacturers is for- identification only and does
not imply endorsement by the Federal Water Pollution Control Administration
or the U, S. Dept. of the Interior.
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REFERENCES
1, D, E. H. Frear, Pesticide Handbook .- Entoma, 20th Ed,, College Science
Pub., State College, Perm. (1968),, pp. 27-31.
2, R. F. Gould. Advance in Chemist.ry Series, Organic Pesticides in the
Environment, (1966), p. 3*
3. W. D. Johnson, G. F. Lee and D« Spyridakis, Persistence of Toxaphene
in Treated Lakes. (Thesis, Univ. Wisconsin) (1965) and Air and Water
Pollution, Int. J. Pergarnon Press 10, (1966) pp. 55-560. Printed in
Great Britain; also CA 66sl8l6b, (196?).
li; J. J. Hiekey, J, Keith and F. B» Coon. An Exploration of Pesticides
in a Lake Michigan Ecosystem. J. Appl. Ecol. 3, (1966), pp. liu-153.
5?o H. P. Burchfield, D, W. Johnson and E. E. Storra. Guide to the
Analysis of Pesticide Residues, Yols. I and II. U. S. Dept. of
Health, Education, and Welfare, Washington, D. C. Supt. of Documents,
• U. S. Government Printing Office, I, (l)-VIir.S,3.d«,(l). (196Ł).'
6. U, S. Dept. of the Interior, F,=/PCA. Report on Insecticides in L^ke
Michigan, prepared by Pesticide Com.'nittee of the Lake Michigan Enforce-
ment Conference (Donald I. Mount, Ph.D., Chairman, National Water
Quality Laboratory, Duluth, Minn.), (1968), pp. 1-kk.
7. A. W. Briedenbach', J. J. Lichtenberg, C. F. Henke, D, J. Smith,
" J. W. Eichelberger, Jr., and H. Stierl. The Identification and
Measurement of Chlorinated Hydrocarbon Pesticides in Surface Waters,
U.S. Dept. of the Interior, WP-22, (1966), pp. 1-70.
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8. (Organic Pesticide Subcommittee on Methods Validation and Analytical
Quality Control. FWPCA Interim Official Methods for Chlorinated
i
Hydrocarbons Pesticides in Water and Waste Water by Gas Chromatography,
(1968), pp. 1-21.
5>. U. S. Dept. of the Interior, F/JPCA, GLR, LMBO. Great La.kes Region
Committee on Analytical Methods, Chemistry Laboratory Manual, Bottom
Sediments, (1963) pp. $0-73.
10. R. E. Duggan, H. C. Barry, H, Y. Johnson, and Sidney 'v'illiarr.s. Pesticide
Analytical Manual, Vol. I: Methods Which Detect Multiple Residues)
Vol. lit Methods for Individual Pesticide Residues, U.S. DKEW, Food
a'nd Drug Administration, (revised 1?67 and 1968),
11. A1, A. Rosen. Chemical Analysis - A Weapon Against Water Pollution,
J. Anal. Cheni. 39, (196?) pp. 26A-33A.
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