EPA-600/3-76-076
August 1976
Ecological Research Series
ANALYSIS AND GC-MS CHARACTERIZATION OF
TOXAPHENE IN FISH AND WATER
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into five series. These five broad
categories were established to facilitate further development and application of
environmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal
species, and materials. Problems are assessed for their long- and short-term
influences. Investigations include formation, transport, and pathway studies to
determine the fate of pollutants and their effects. This work provides the technical
basis for setting standards to minimize undesirable changes in living organisms
in the aquatic, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-76-076.
August 1976
ANALYSIS AND GC-MS CHARACTERIZATION
OF TOXAPHENE IN FISH AND WATER
by
David L. Stalling
James N. Huckins
Fish-Pesticide Research Laboratory
Fish & Wildlife Service
United States Department of the Interior
Columbia, Missouri 65201
Contract No. EPA-IAG-0153 (D)
Project Officer
Leonard H. Mueller
Environmental Research Laboratory
Duluth, Minnesota 55804
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
ENVIRONMENTAL RESEARCH LABORATORY
DULUTH, MINNESOTA 55804
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DISCLAIMER
This report has been reviewed by the Environmental Research Laboratory*
Duluth, Minnesota, U.S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily re-
flect the views and policies of the U.S. Environmental Protection Agency,
nor does mention of trade names or commercial products constitute endorse-
ment or recommendation for use.
ii
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ABSTRACT
Sensitive methods for the detection and identification of toxaphene
in water and fish were described. Polyurethane foam, gel permeation
and silicic acid chromatography were utilized to permit accurate
quantitation of multi-component toxaphene residues. A method for
characterization of changes in isomer composition of toxaphene
residues in fish was reported. The chemical composition of toxaphene
was examined by electron impact and chemical ionization mass
spectrometry. Chemical ionization gas chromatography-mass spec-
trometry was particularly applicable to the analysis and confirmation
of toxaphene residues in environmental samples.
iii
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CONTENTS
SECTIONS Page
I CONCLUSIONS 1
II RECOMMENDATIONS 2
III INTRODUCTION 3
IV EXPERIMENTAL 4
A. Reagents 4
B. Apparatus 4
V RECOMMENDED METHODS 6
A. Sample Preparation 6
B. Extraction 8
C. Sample cleanup 9
D. PCB-Toxaphene separation 10
E. Toxaphene quantitation 11
VI RESULTS AND DISCUSSION 13
A. Water analysis 13
B. Fish analysis 13
C. Gas chromatography 14
D. Difference chromatography 15
E. Gas chromatography - Mass spectrometry 15
VII REFERENCES 39
VIII LIST OF PUBLICATIONS 41
IX GLOSSARY OF ABBREVIATIONS 42
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LIST OF FIGURES
No. Page
1 Water sampling apparatus. 7
2 Difference chromatograms obtained by subtracting
individual chromatograms (GC curves of cleaned up
extracts from fish exposed to toxaphene 156 days and
from fish 14 and 56 days after cessation of exposure
to toxaphene) from a toxaphene standard. 16
3 Difference chromatogram obtained by subtracting a
toxaphene standard from the difference of a 20 liter
toxaphene water sample and a similar 20 liter control
sample. 17
4 Large volume sample injection system for GC-MS. 18
5 Characteristic EI-MS spectra of four major toxaphene
constituents. 20
6 Mass chromatogram of combined ion intensities of m/e
291 + m/e 293 from 151 continuous GC-EI-MS scans. 21
7 Mass chromatograms of m/e 83 (top) , m/e 117 (bottom)
from 151 continuous GC-EI-MS scans. 22
8 Toxaphene detection utilizing a "SOL-VENT" injection
system and a computer generated mass chromatogram. 23
9 Isobutane-direct probe CI-MS of toxaphene. Direct
probe CI-TIC plots of toxaphene for all masses
(left) and for masses 400-500 (right). 24
10 Theoretical Cl isotope patterns for substitution of
1-10 Cl. CI-MS of a toxaphene GC component whose
principal constituents were CioH^Clg (M-C1-305) and
C10H11C15 (M-C1-271). 29
"11 CI-MS of toxaphene constituents consisting of a mixture
of C^EgClj and CioH^Cly. Another CI-MS of toxaphene
constituents consisting mainly of CiQHCl. 30
12 CI-MS of toxaphene constituents consisting of a 1:20
mixture of C^HgCls and CioHlQCls- Another CI-MS of
toxaphene constituents with a 20:1 mixture of
C10H9C19 and C10H11C19- 31
13 RGC-TIC for GC-CI-MS of toxaphene obtained and plotted
using Finnigan-System Industries computer system. 32
vi
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No. Page
14 GC-EI-MS-TIC histogram of toxaphene obtained and
plotted using Digital Equipment Corporation's MASH
computer system. 33
15 CI-MS of toxaphene constituents
having an atypical mass fragment at 243
Another CI-MS of toxaphene constituents having an
atypical mass fragment CgHCl^ + (m/e=245). 34
16 RGC's of m/e 243 and m/e 245 from CI-MS of toxaphene. 35
17 RGC-CI-MS-TIC of an extract from a brook trout exposed
to toxaphene for 141 days. 36
18 RGC-CI-MS of m/e 343 from an extract of a brook trout
exposed to toxaphene for 141 days. Also, RGC of m/e
343 from CI-MS of toxaphene. 37
19 RGC of m/e 339 from CI-MS of toxaphene. Also,
RGC-CI-MS of m/e 339 from an extract of a brook
trout exposed to toxaphene for 141 days. 38
vn
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LIST OF TABLES
No. Page
1 Relative concentration ratios of chlorinated
toxaphene constituents determined from the direct
probe CI-MS. 25
2 Empirical formula of toxaphene isomers from CI-MS. 28
viii
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ACKNOWLEDGMENTS
The assistance of James L. Johnson and Jerry D. Troyer In developing methods
for the analysis of toxaphene in fish and water samples was appreciated.
Also, we gratefully acknowledge the cooperation of Dr. Henry Fales and
Dr. Craig Shew in obtaining chemical ionization-mass spectra of toxaphene.
Finally, we give thanks to Dr. Foster L. Mayer, Jr. for his assistance
throughout the project.
ix
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SECTION I
CONCLUSIONS
1. Toxaphene residue analysis in fish using gas chromatography with
electron capture detectors required multiple cleanup techniques
for reliable analysis. Gel permeation, Florisilv;} and silicic
acid chromatography permitted efficient separation of toxaphene
from coextracted lipids and PCBs . Computer difference chroma-
tography allowed direct comparisons of a toxaphene gas chromato-
graph standard with environmental residues.
2. Concentrations of toxaphene residues in water ranging from 10 to
500 ng/1 were quantitively extracted with a column of polyure-
thane foam.
3. Environmental analysis of toxaphene residues was best accomplished
using chemical ionization mass spectrometry combined with gas
chromatography. Specific detection of toxaphene was only feasible
by using chemical ionization mass spectrometry and specific ion
monitoring techniques.
4. From chemical ionization mass spectra toxaphene was found to be
compose'd of several homologous series of chlorinated camphenes
containing 5 to 10 chlorines per molecule. For each degree of
chlorination there were numerous isomers derived from three empirical
formulas which differ by two hydrogen atoms, i.e., C10H(14-N)C%»
C10H(16-N)C-Hl' an<* C10H(18-N)^%> T^ie first empirical formula
represents replacement of 4 hydrogen atoms with 4 chlorine atoms.
This reaction may also produce 4 molecules of HCL. The second and
third empirical formulas are most likely produced by the addition
to camphene of 2 and 4 molecules of HCL respectively. One or two
additional series of compounds existed which had atypical chemical
ionization mass spectra. These compounds were characterized by
a base peak of mass 243 and 245. Ion fragments from these com-
pounds represented CgHyCl^ and CgHgC^ respectively, and may have
reflected a structural rearrangement of camphene during synthesis
of toxaphene.
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SECTION II
RECOMMENDATIONS
1. Gel permeation and silicic acid chromatography should be
utilized for the separation of lipids and PCBs from
toxaphene.
2. Concentrations of toxaphene residues in water ranging from
10 to 500 ng/1 should be extracted with polyurethane foam
columns.
3. Environmental residues of toxaphene should be analyzed
or confirmed by chemical ionization mass spectrometry.
4. Computer difference chromatography should be utilized as
a direct means to detect change in isomer composition of
environmental residues of toxaphene.
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SECTION III
INTRODUCTION
Analysis and chemical characterization of toxaphene is challenging to
pesticide analysts due to the extreme complexity of the material.
Toxaphene is a mixture of compounds produced by the chlorination of
camphene; characterized by having an average empirical formula of C-^Q
H10c-^8 atu* a corresponding 35ci molecular weight of 410. Analytical
techniques for toxaphene residues were reviewed by Zweig^ who regarded
gas chromatography (GC) as the most useful of the chromatographic tech-
niques. However the utility of GC in toxaphene residue analysis is limited
by the multiplicity of constituents. Over 40 components have been resolved
by the use of a support-coated open tubular (SCOT) GC column-*. Many of
these peaks are due to multiple components that are not separated with a
SCOT column*. The complex isomer composition decreases toxaphene's GC
detection limit because of the multi-component nature of chromatograms.
In addition, widespread contamination from ubiquitous polychlorinated
biphenyls (PCBs), which are also complex multi-isomer chemicals, often
interfere with toxaphene analysis.
Chronic laboratory studies of fish exposed to toxaphene require very low
concentrations (10-500 ng/1) and therefore sensitive residue analysis
is necessary. Sample preparation schemes should not introduce interfering
contaminants and cleanup techniques should remove any other interfering
materials. Methods used for residue recovery must be quantitative for
individual toxaphene isomers to permit detection of any alteration in
isomer ratios.
The objectives of this study were: 1) to improve techniques for toxaphene
quantitation in water samples, 2) to refine or develop analytical metho-
dology for sample preparation, cleanup, and quantitation of residues and
shifts in isomeric composition, 3) to characterize the composition of
toxaphene using both electron impact (El) and chemical ionization (CI)
gas chromatography-mass spectrometry (GC-MS), and 4) to determine the
applicability of GC-MS for the confirmation or analysis of toxaphene in
environmental fish samples.
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SECTION IV
EXPERIMENTAL
REAGENTS
(a) Solvents - pesticide grade, redistilled in glass.
(b) White, porous polyurethane plugs - Gaymar Identi-plugs, fits
24 to 35 mm opening, order no. L 800.
(c) FlorisilR - 60-100 mesh, activated at 130 C.
(d) Silicic acid - 100 mesh analytical reagent (Mallinckrodt
No. 2847).
APPARATUS
(a) Silicic acid extraction column - glass, 460 mm x 85 mm id,
fitted with a 5 mm bore teflon stopcock.
(b) Silicic acid chromatography column - glass, constructed with
a 300 mm x 22 mm id Kimax column (order #17800) having a
removable teflon stopcock. The Kimax column was joined to a
250 ml reservoir which was fitted with a 24/40 standard
female ground glass joint. A 24/40 male joint was attached
to an air outlet (air is filtered through charcoal) and was
held in place on the column with a Thomas standard taper
clamp. The system could maintain a pressure of 5 Ibs.
(c) Gel permeation chromatograph - an automated system having a
22 sample capacity**. Separation was achieved by a column
(2.5 cm x 23 cm) of BioBeads S-X2 and cyclohexane at a flow
rate of 5 ml/min.
(d) Gas chromatographs - Perkin-Elmer Model 881, equipped with
Tracer 6%i-electron capture detector. Operational parameters:
213 cm (71) x 2.0 mm id coiled glass column packed with 3%
(w/w) OV-7 on chromosorb W-hp; nitrogen carrier gas flow rate
40 ml/min; temperatures (C) - injection port 230, detector
330, column 180. Beckman GC-5, equipped with Tracer 6%i-
electron capture detector. Operational parameters: same as
those above, only change was a higher column temperature of
200 C.
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(e) Gas chromatograph-electron Impact-mass spectrometer (GC-EI-MS).
Perkin-Elmer model 270 double focusing, low resolution El mass
spectrometer coupled through Watson-Bieman molecular separator
to a temperature programmed gas chromatograph. The GC-EI-MS
system was interfaced to a Digital Equipment Corporation PDP-12
LDP (8K) computer.
(f) Gas chromatograph-chemical ionization-mass spectrometers
(GC-CI-MS). A modified MS-9 CI-MS7 interfaced to a PDP-8I
minicomputer. Ionizing gas was isobutane and sample access
was with direct probe. Sample spectra were obtained in coop-
eration with Dr. Henry Fales, National Heart and Lung Institute,
National Institute of Health, Bethesda, Md.
(g) A Finigan quadrapole GC-CI-MS combined with system industry
"System-150" PDF 8-m computer was employed to examine GC peaks
of toxaphene. The gas used for ionization was methane. Toxaphene
spectra from this system were obtained in cooperation with
Dr. Craig Shew, Kerr Research Center, EPA, Ada, Oklahoma.
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SECTION V
RECOMMENDED METHODS
SAMPLE PREPARATION
(a) Fish - Each yearling or adult sample was prepared according to the
method described by Benville and Tindle . A frozen fish was cut
into small pieces, 1" x 1", and ground with an equal amount of dry
ice in a Waring industrial blender, until.a homogenous mixture was
obtained. Then the fish and ice mixture were loosely sealed in a
polyethylene bag and placed in a freezer (-12 C) overnight. After
the dry ice sublimed, a 20 g sample of the ground fish was mixed
with 80 g of anhydrous Na2SO^ in a beaker. Due to contaminants,
all Na2SO^ was heated in a muffle furnace overnight at 550 C be-
fore use. To prevent hardening the sample was occasionally stirred
with a glass rod until a dry mixture was obtained.
Fry or egg samples (2 g or less) were placed in 70 ml capacity
porcelain evaporating dishes, each with 8 g sodium sulfate. If
an individual fry was larger than 1 g it was dissected with a
scalpel before proceeding. A stainless steel rod (16 cm x 0.9
cm od) for each sample was employed to crush the tissue with NaS04-
Occasional stirring was necessary to prevent hardening while drying.
(b) Water - White, porous polyurethane plugs were cleaned by solvent
extraction in a 2 liter stainless steel beaker with a mixture of
250 ml of acetone-petroleum ether (1:1; v/v). A smaller 1.0 liter
steel beaker with several holes in the bottom was used with a piston
like action on the plugs to facilitate contaminant extraction by the
solvent mixture. After several minutes of washing, the contaminanted
solvent mixture was discarded and the process repeated, until chroma-
tographic analysis of the solvent mixture showed no significant GC
peaks. Three clean polyurethane plugs were pushed into a 1" id
clean copper column (Fig. 1). The column was constructed from a 12"
piece of copper tubing with a 1" sweat union on top and a 1" to 1/2"
sweat reducer on bottom. A copper plug with 8 mm hole was inserted
into the reduced end of column and silver soldered in place. A
Teflon buret tip (8 mm od) was inserted into the bottom of the column
with the stopcock open. Then 50 ml of acetone was poured into the
column and allowed to elute.
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Figure 1. Water sampling apparatus consisting of a 1" id copper
column (center) and siphon arm (perimeter). The left
side of siphon arm from elbow down is aluminum.
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This rinse was followed by 100 ml of methanol and finally,
100 ml of distilled water. Afterwards , the buret tip was
removed and a siphon shown in Fig. 1 was securely coupled
to the column. A small piece (2" x 2") of wire screen was
inserted into the aluminum arm of the siphon to filter
large particulates in the water. Aluminum pipe was used
for the aquarium arm of the siphon because of the toxicity
of copper to fish. The column and attached siphon were
filled with water and corks were placed in both ends. Then
the completed assembly was transferred to the aquaria to be
sampled and the siphon was started by removing the two corks.
A large wash tub, or other suitable container was calibrated
to a 20 liter volume for receiving the sample eluate.
EXTRACTION
(a) Fish - After drying the yearling or adult sample - Na2SO/
(20 g/80 g) mixture was gently packed in a 19 mm id column
of similar design to that reported by Hesselberg and Johnson".
Toxaphene was eluted quantitatively with 200 ml of 5% diethyl
ether in petroleum ether. The elution rate should be 3 to 8
ml/min, and was controlled by packing tightness and sample
texture. Then 5 ml of cyclohexane was added to the extract
and it was concentrated to a 5 ml volume by evaporation on a
hot plate set at 75 C in a fume hood. An additional 10 ml of
cyclohexane was added and the solution re-evaporated to a
5 ml volume.
The dry fry or egg sample - Na2S04 (2 g/8 g) mixture was
gently packed in a 10 mm id extraction column of similar
design to that used for yearling and adult fish. Toxaphene
was eluted quantitatively with 100 ml of 5% diethyl etfoer
in petroleum ether. The extract was concentrated to a 2 ml
volume as described for yearling and adult samples.
(b) Water - The elution rate for the siphon-column extraction
system should be 300 to 400 ml/min. However, extraction of
toxaphene was quantitative up to 500 ml/min. After 20 liters
of eluate had been collected the siphon was broken and the
column was removed. A buret stopcock was again inserted
into the bottom of the column and 25 ml of acetone was added,
carefully washing down the sides of the column. Acetone
removed water from the plugs which interfered with efficient
extraction. The elution rate was adjusted to 5 ml/min and the
eluate collected in a 250 ml separatory funnel. Then, 100 ml
of petroleum ether was added to the column, and eluted into
the separatory funnel at 5 ml/min, which completed extraction
of the plugs.
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The biphasic eluate consisted of petroleum ether on the top
and acetone-H2<3 on the bottom. The toxaphene in the acetone-
H20 phase was partitioned into the petroleum ether phase upon
shaking of the separatory funnel. After separation of the two
phases, the acetone-t^O phase was drained into a 2nd separatory
funnel. Then an additional 100 ml of petroleum ether was added
to the acetone-H20 phase and the partitioning step repeated.
The two petroleum ether extracts were combined in a 400 ml
beaker and dried with 20 g of anhydrous Na2SO^. The combined
extract was then transferred to a 250 ml casserole and concen-
trated to 2 ml on a hot plate set at 80 C in a fume hood.
SAMPLE CLEANUP
(a) Fish - An automated gel permeation system as reported by
Tindle and Stalling" was used for initial cleanup of yearling
or adult samples. The sample was diluted to 20 ml volume in
cyclohexane and 5 ml (equivalent to 5 g tissue) was loaded
on the gel system. The 0 to 100 ml eluate containing lipids
was discarded and toxaphene was recovered quantitatively in
the 100 to 325 ml eluate. The toxaphene eluate was concentrated,
as previously described, to a 5 ml volume. Changes in toxaphene
isomer ratios were negligible with this method. Less than 0.5%
of the original lipid remained in the collected toxaphene fraction.
Additional cleanup of yearling and adult samples was achieved
by a 19 mm glass column, identical to the extraction column,
with 10 g of activated (130 C) Florisi3© The sample was
applied to the dry column and followed by two 5 ml washes
of petroleum ether. Toxaphene was eluted with 180 ml of 5%
diethyl ether in petroleum ether. The flow rate was 30 ml/min
or higher and toxaphene recovery was 95-100%. The eluate was
concentrated to 5 ml and silicic acid chromatography was employed
if PCBs were present.
Fry or egg samples (2 g or less) contained less total lipid
than yearling or adult samples and were normally purified in
one step. A 10 mm glass column, used for sample extraction
described earlier, was loaded with 4 g of activated Florisil
followed by 0.5 g of anhydrous Na2S04« The column was washed
with 20 ml of petroleum ether and the sample was added when
the solvent reached the top of the ^280^ layer. Toxaphene
was eluted with 50 ml of 5% diethyl ether in petroleum ether.
The eluate was collected in a porcelain evaporating dish
(70 ml capacity) and concentrated to 5 ml on a hot plate
set at 75 C. Then the eluate was transferred to a culture
tube, rinsing with 5 ml of petroleum ether. The sample was
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concentrated with a stream of nitrogen to an appropriate
volume in a liquid bath module blok heater set at 55 C.
The sample was ready for GC unless PCB contamination was
present, in which case silicic acid chromatography was
employed.
(b) Water - Polyurethane foam was not specific for toxaphene, and
sample cleanup was usually necessary. Florisil chromatography
was utilized and the same cleanup procedure as described for
fry or egg samples was used with the following exceptions:
Only 2 g of activated Florisil was required and toxaphene was
eluted with 45 ml of 5% diethyl ether in petroleum ether.
PCB-TOXAPHENE SEPARATION
(a) Extraction and activation - 400-500 g of non-activated silicic
acid was slurred, as received from manfacturer, with 1 liter
of 40% acetonitrile in dichloromethane and poured into an 85 mm
id x 45 cm extraction column (glass wool used as column plug)
with Teflon stopcock closed. After adding an additional 2 liters
of the same solvent, the flow rate was adjusted to 10 ml/min and
aluminum foil was placed over top of column. Solvent percolation
was completed overnight. Next, the adsorbent was transferred
to a 5 liter evaporating dish and placed under a fume hood. The
solvent was allowed to evaporate at ambient temperature. When
dry, the crust formed on surface of silicic acid was broken with
a glass stirring rod and spread to a depth of 1". The silicic
acid was transferred to an oven at 160 C and heated for a minimum
of 48 hrs. Care was used in making certain all solvent volatilized
before heating. After activation, the adsorbent was placed in
a large jar and deactivated with 2% water (98 g silicic acid + 2
ml water). Finally, the jar was sealed and tumbled on a jar mill
for 1 hr. The deactivated silicic Bcid was allowed to equilibrate
for 24 hours before use.
(b) Column preparation - 20 g of deactivated silicic acid was weighed
into a 250 ml beaker. Next, the silicic acid was slurried with
70 hi of petroleum ether and poured into a 22 mm id chromato-
graphic column with stopcock open. The column was not permitted
to drain dry. Silicic acid remaining in the beaker was rinsed
into the column with additional petroleum ether and the sides
of the column were rinsed. Air pressure was applied (2-3 psi)
and the silicic acid was allowed to settle. 2 g of Na2S04 was
added to the top of the silicic acid and air pressure was applied
until solvent level in column was even with top of Na2S04 layer.
10
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(c) Chromatographic separation - The sample extract, previously
cleaned up with gel permeation chromatography and/or Florisil,
was added in not more than 5 ml of petroleum ether and pressure
was applied until sample level was even with Na2SO^. The sides
of the column were washed twice with 5 ml of petroleum ether
and the solvent level was brought even with Na2SO^. A 275
ml capacity porcelain casserole dish was placed under the
column and 250 ml of 1% benzene in petroleum ether (PCB eluate)
was added. Air pressure was applied until a flow rate of
4 ml/min was achieved. The PCB eluate was collected until
solvent level was 1 cm from top of Na2SOA layer and elution was
stopped. Another casserole was quickly placed under the column
and 200 ml of 20% diethyl ether in benzene was added to column.
The separation was completed by elution of the toxaphene fraction
and the casserole dish containing toxaphene was transferred to an
explosion proof hot plate (75 C) in a fume hood. With hood fan
on, the toxaphene fraction was evaporated to a 5 ml volume and
rinsed into a culture tube using 5 ml of petroleum ether. Then
the sample was placed in a water bath module blok with heater
set at 55 C. Using a stream of N^, the toxaphene fraction was
evaporated to a 5 ml volume.
(d) NaOH partition of pesticide fraction - If early eluting GC
peaks with retention tine less than p_,p_'-DDE were encountered in
the analysis of the toxaphene fraction, an additional cleanup
step was necessary. The contaminant was sometimes observed at
higher GC temperatures as a broad solvent peak. To remove this
contaminant, 1 ml of 1 N NaOH was added to the concentrated
toxaphene fraction in a culture tube and shook thoroughly in a
super (vortex) mixer for 30 seconds. The two layers were allowed
to settle for 15 minutes, centrifugation was optional, and the
toxaphene sample was ready for GC analysis.
TOXAPHENE QUANTISATION
(a) Total residues - Toxaphene residues were calculated by measuring
three major GC peaks having retention times well separated and
representative of the total residue. The GC peaks routinely used
had retention times relative to p_,p_'-DDE of 1.48, 1.90, and 2.66.
After drawing a full baseline (injection point through last sample
peak), heights of the respective peaks were measured and summed.
The sum of the GC peaks of three toxaphene standards covering the
range of sample peak heights, were also calculated. The sum of
the standard peak heights were correlated with injected quantities
using a programmable Olivetti-Underwood 101 calculator to
determine a linear regression curve. This standard curve was
then compared with the sums of sample peak heights. Correctipns
for injection amounts, volume changes, and recovery were made
11
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by the program, and the resulting residue concentration computed
from the regression curve. Residues were expressed as ug/g.
(b) Isomer changes - Eight prominent peaks in a sample were selected
having GC retention times representative of the total toxaphene
residue. A full base line was drawn as described earlier and
the heights of the eight selected peaks were measured. The
heights of the same eight peaks from each of four toxaphene
standards were measured. The quantity of toxaphene used for
standards covered the range of sample peaks heights. Using the
programmed calculator, peak heights of a sample and the standards
were separately summed and individual peaks were expressed as
percent of total peak height. A standard was selected whose
summed peak heights was nearest that of the sample being
analyzed. Corresponding peaks (% of total peak height) of the
standard were then subtracted from those of the sample and
results were given in + percent deviation from a toxaphene
standard.
12
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SECTION VI
RESULTS AND DISCUSSION
The recommended methods used in this report were developed in
conjunction with an EPA financed study on the residue dynamics
of toxaphene in brook trout (Salvelinus fontinalis)^. Uptake,
elimination and changes in isomer ratios of toxaphene residues
in brook trout were elucidated and discussed.
WATER ANALYSIS
Various sources of PCS and phthalate ester contaminants were
major obstacles in the measurement of low concentrations of
toxaphene (10-500 ng/1) residues in water. These contaminants
originated from solvents, adsorbents, and other unidentified
sources. However, PCB and phthalate ester contamination were
reduced by using redistilled solvents and solvent extraction of
reagents.
The initial use of large volumes of distilled organic solvents for
partition extraction of toxaphene water samples resulted in
significant GC interference when concentrated to small volumes.
Later we modified a column extraction method utilizing polyurethane
plugs for sampling large volumes of water . A rigid siphon with
an attached column was used for sampling water in aquaria. The
attachment of a siphon eliminated pouring large volumes of water
through the polyurethane column. Recoveries of toxaphene with the
siphon-polyurethane column system were as follows: 100 ng/l«100%,
50 ng/l=80%, 25 ng/l=50%. These recoveries were based on the mean
of duplicate determinations for each of the three toxaphene con-
centrations. Organic solvent required for extraction of toxaphene
utilizing polyurethane plugs was only 200 ml. Thus, the contami-
nation from large volumes of organic solvents was decreased and
time required for toxaphene analysis was greatly reduced.
FISH ANALYSIS
There were several reported techniques used for the extraction of
organochlorine pesticides from fish samples for residue analysis.
However, most methods were subject to serious limitations at low
concentrations and often required subsequent filtering and drying
steps which may introduce contaminants. The column extraction pro-
cedure described in the experimental section combined extraction,
filtration and drying^. Recoveries of toxaphene using the extrac-
tion column ranged from 97-100% with a mean of 98.5% for three
spiked samples.
13
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The separation of lipids from pesticides often has been the most
time-consuming process in pesticide analysis. Automated gel
permeation chromatography of lipid-toxaphene extracts resulted in
improved analytical precision, decreased manipulative sample
losses, and significant saving of labor6. In addition all GC
resolved toxaphene isomers were recovered with no apparent change
in ratios. Mean recovery for three toxaphene samples was 96% and
sample recoveries ranged from 95-98%. The remaining lipid or
polar contaminants (less than 0.5% of original lipid content)
were removed with Florisil column chromatography.
Low concentrations (0.05-0.1 ug/g) of PCBs were found in toxaphene
fish sample extracts. Toxaphene's GC sensitivity was considerably
less than PCBs which necessitated a PCB-toxaphene separation step.
We found that only silicic acid chromatography separated all
toxaphene components resolved by GC from interfering PCBs. However
recent lots of silicic acid as received from the supplier were often
contaminated with detectable levels of PCBs, phthalate esters, and
large amounts of an unidentified early eluting (GC) contaminant.
We modified the method of Stalling and Huckins . The modifications
eliminated many of the contaminants in silicic acid and provided
reproducible separations. A large volume column (85 mm id) extraction
procedure reduced PCBs and phthalates in silicic acid. The early GC
eluting silicic acid contaminant was removed by partitioning the
toxaphene fraction of the separation with 1 N NaOH. Additional
research into the nature of this contaminant is now being conducted
utilizing our GC-MS-computer system.
GAS CHROMATOGRAPHY
Research into GC solid supports and liquid phases demonstrated that
OV-7 on chromosorb W-hp provided good resolution and sensitivity
of toxaphene. Two GC column temperatures were used for most
toxaphene analysis. A temperature of 180 C appeared optimum for
isomer studies, exhibiting acceptable component resolution. However,
whole body fish residues and water samples were analyzed at 200 C
column temperature, a compromise of component resolution and long
retention times of toxaphene samples. The minimum detection limits
for toxaphene residues in fish and water utilizing the recommended
methods and GC conditions described were 0.05 ug/g and 0.010 Hg/1
respectively.
A gas chromatograph utilized for toxaphene isomer studies was
interfaced with our PDP-12 LDP computer. Multiple component
chromatograms were processed by the computer as two thousand
Sequential data points and stored on magnetic tape using the
program "CATACAL". The chromatogram data thus generated was
manipulated and plotted for graphical comparisons.
14
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DIFFERENCE CHROMATOGRAPHY
Quantitative data representing changes or differences between
complex GC curves are not readily presented. Toxaphene residues
were no exception due to the large number of constituents and
limited GC resolution. We devised a computer based method to
assist in characterization and presentation of toxaphene residue
chromatograms. The technique used our PDP-12 computer and the
program CATACAL to create a "difference chromatogram". A
difference chromatogram was created by subtracting a standard
toxaphene chromatogram from a sample chromatogram or subtraction
of sample from standard chromatogram after the sample chromatogram
was normalized by making its largest peak height equal to the
height of the corresponding toxaphene GC peak in the standard (Fig.
2 and 3). If the two chromatograms were identical, a straight line
was obtained; constituents whose relative concentration exceeds that
of the standard appeared as positive peaks. Constituents which
were not common to the standard were usually observed as discrete
positive or negative peaks. This technique easily enabled exami-
nation of toxaphene residues for changes in isomer composition or
weathering due to environmental conditions. The utility of the
method was demonstrated by the preferential elimination of early
eluting toxaphene components by fish 14 and 56 days after cessation
of toxaphene exposure.
GC-MASS SPECTROMETRY
Computerized GC-MS helped overcome many of the difficulties in
toxaphene characterization and detection. With our GC-EI-MS the
computer programs "MASH" (Digital Equipment Corp. Users Manual,
DEC-12-SQ-A-D) enabled rapid data acquisition, storage, and
reduction of complex multi-component samples. Detection sensitivity
was increased by using mass chromatograms generated by the computer
from several key toxaphene fragments in sequentially acquired spectra.
A large volume sample injection system (Fig. 4) which was a modified
version of SOL-VENT12, was adapted to the GC-EI-MS allowing injection
volumes of 50-100 ul without adverse effect. The system functioned
to trap less volatile constituents in the liquid phase of a large
diameter pre-column and vented the volatile solvents for a variable
time of 15-90 seconds. After venting and closing the toggle
valve, the trapped constituents were temperature programmed into
the analytical column and analysis proceeded normally. A SCOT GC
column was utilized when high resolution was necessary. However,
a 274 cm (9f) x 2 mm glass column with 3% OV-7 on chromosorb W-hp
was used for routine separations.
15
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Toxaphene Elimination-Difference Chroaatograas
Toxaphene
Figure 2. Difference chromatograms for toxaphene elimination from
brook trout. Curve A. Toxaphene standard chromatogram.
Curves B, C, and D were generated from computer subtracting
of three individual GC curves representing toxaphene
residues in fish after 156 day exposure, 14 day and 56
day post exposure.
16
-------
Minutes
Figure 3. Curve A. Difference chromatogram obtained by
subtracting toxaphene standard from the difference
of curves B and C. Curve B. Toxaphene chromatogram
from 20 liters of water containing 0.5 ug toxaphene/1.
Curve C. Background chromatogram from 20 liters of
water in control aquaria. All samples were equivalent
injections into the GC.
17
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CARBIEB OAS ,1.
COLUMN OVIN
CABBIE*
GAS1
Figure 4. Large volume sample Injection system for GC-MS.
A pre-column with a solenoid valve is used for
venting excess solvents.
18
-------
Toxaphene was initially examined using our computerized GC-EI-MS
to obtain spectra of GC peaks. The toxaphene peaks eluting during
temperature programming were scanned over the mass range of 18 to
550 in 4 seconds at 8-second intervals for 151 scans. Data
representing each spectrum was stored on magnetic tape. EI-MS
spectra from several major toxaphene components were examined for
characteristic ion fragments (Fig. 5). The mass chromatograms
constructed from the combined ion intensities of m/e 291 + m/e 293
(Fig. 6) in 151 sequential mass scans closely resembled the peak
envelope recorded from the total ion monitor. GC peaks in the mass
chromatogram determined from m/e 83 (C+HC12) eluted late in the
chromatogram while GC peaks containing m/e 117 (C+Clg) generally
eluted earlier (Fig. 7). Approximately 1-2 ug of toxaphene was
required for residue confirmation using El-mass chromatograms con-
structed from several intense ion fragments (Fig. 8) with the aid
of a large volume injection system described earlier.
Recently, mass spectrometric characterization of purified toxaphene
constituents was reported by Casida et al.4 Chemical ionization
mass spectra were obtained from Casida of two purified constituents
with empirical formulas of CioH-^QClg anc^ G10Hllc-'-7< Each CI-MS
spectrum shnwed loss of a Cl from the molecule to form (M-C1)+ ions
and (M-Cl£) . This data permitted general correlation of empirical
formula and observed CI-MS.
Examination of toxaphene by CI-MS with direct probe access resulted
in spectra characterized by multiple ion clusters. These clusters
represented isomeric series of C^gHijClg with variable numbers of
hydrogen for each degree of chlorine substitution (Fig. 9). The
total ion curve represented the volatilization of toxaphene from the
direct probe (Fig. 9). The volatilization curve based on masses in
the 400-500 range was similar to the total ion current (TIC) with
only slight fractionation of toxaphene during volatilization (Fig. 9) .
Using the intensities of the mass fragments in CI spectra, the
concentration of the various isomeric series were estimated (Table 1) .
Two series of nonachloro toxaphene constituents (C^oHyClg and
were apparent from the direct probe examination.
After determining the applicability of CI-MS to the characterization
of toxaphene, GC-CI-MS was employed to examine GC peaks. A Finnigan
quadropole GC-CI-MS with a PDF 8-m computer system was used. The
computer software generated reconstructed gas chromatograms (RGC)
which presented specified ion-fragment intensity in each sequentially
acquired MS scan as a normalized curve. This format differed from
that of the MASH GC-MS system in that the latter computer programs
present the gas chromatogram as a histogram. The GC-CI-MS spectra
of toxaphene were several orders of magnitude simpler than corre-
sponding GC-EI-MS spectra.
19
-------
8688855
Figure 5. Characteristic EI-MS spectra of four major toxaphene constituents.
20
-------
CM
Figure 6. Mass chromatogram from 151 continuous GC-EI-MS scans.
Plot of combined ion intensities of m/e 291 + m/e 293.
21
-------
fc
S § .
8 .
Figure 7. Mass chrdmatograms from 151 continuous GC-EI-MS scans.
Upper plot was of m/e 83, bottom m/e 117.
22
-------
TOXAPHENE DETECTION SOLVENT INJECTION StSTEH
30 |P CONTAINING 2
R J
§ .
U V)
R .
R .
B ,
P .
Si .
a .
3
TOTAL ION CURRENT
MASS SEARCH - 15W-161]
2 MlCKEffKS
u> S
00 0) O
Figure 8. GC-MS large volume, "SOL-VENT" injection of 2 ug of
toxaphene in 30 ul. Mass chromatogram from 101
sequential GC-EI-MS scans combining intensity of
masses 159 and 161.
23
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SFBCTRH MJfiER 18
TtMRFHB€.aB
SPECTFUI
Figure 9. Isobutane-direct probe CI-MS of toxaphene. Cl isotope
clusters at 303 represent C15; 341, C16; 374, Cly; 409,
Clg; 445, Clg. Toxaphene direct probe CI-TIC plots.
Left curve; - TIC for all masses; right curve; - TIC
for masses 400-500.
24
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Table 1. RELATIVE CONCENTRATION RATIOS OF CHLORINATED TOXAPHENE CONSTITUENTS DETERMINED FROM
THE DIRECT PROBE CI-MS
Relative concentration ratio'
Ion fragment0
Empirical formula**
Hexaa
1.5-2.0
305
C10H10C16
Hepta
5
341
C10H11C17
Octa
4
375
C10H10C18
Nona
1-2
409 411
C10H9C19 C10H11C19
Deca
1.5-2.0
445
C10H10C110
NJ
Ui
aNuiriber of chlorines substituted on camphene ring.
^Determined from ratio of intensities of each m/e cluster to sum of isotope cluster intensities for Clg-
10.
cBased on Cl isotope.
^Loss of Cl from parent ion assumed [M-C1]+
-------
Examination of CI spectra of toxaphene was best approached after
a brief review of the molecular weights of several of the possible
empirical formulas for increasing chlorination of major constituents
(Table 2) and a review of the Cl isotope abundance ratio (Fig. 10).
Selected spectra which have been background substracted were plotted
in Fig. 10, 11 and 12. The spectrum numbers designated the number
of sequential scans and the number of a corresponding spectrum used
for substraction of background. These spectra were related to toxa-
phene GC peaks presented in the GC-CI-MS TIC plot (Fig. 13). The
CI-RGC of the TIC of toxaphene (Fig. 13) was similar to that of the
EI-MS-TIC (Fig. 14).
The Cl-mass spectra of the less complicated portion of the GC curve
were characterized by intense (M-C1)+ ions which reflected the
number of chlorine atoms attached to the camphene nucleus. In these
cases the empirical formula was readily obtained from the (M-C1)+ ion.
Cl2 was also lost from the molecule and gave rise to a Cl isotopic
cluster at M-70. In many spectra unusual chlorine isotope patterns,
when compared to the expected chlorine isotope pattern (Table 2),
may only be explained by assuming that GC peaks are mixtures of
components which have molecular weights differing by 2 or 4 hydrogens.
Thus the mixtures of CiQHgClg and CiOHllcl9 suggested earlier by
direct probe Cl-mass spectra were corroborated. In addition many
other isomers which have one of several empirical formula presented
in Table 2 were also indicated.
Two atypical GC-CI-MS toxaphene constituents were noted. GC compo-
nents examined in scan numbers 76 and 84 had base ion clsuters other
than the M-C12 fragments (Fig. 15). These mass peaks in scan numbers
76 and 84 were m/e 243 and 245, respectively. M/e 243 corresponded
to CgHyCl^ and m/e 245 corresponded to CgHoCl^. However the ion
cluster at m/e 245 was a mixture of CgHyCl^ and CgHgC^"1" (m/e=247)
in the ratio of 1:3. Toxaphene RGCs were then obtained for m/e 243
and 245 (Fig. 16). These ion fragments were apparent in all of the
CI-MS scans and may reflect the substitution pattern of the toxaphene
isomers. While the relative sensitivity of these ions remains to be
determined, they are perhaps the most characteristic for toxaphene
constituents of the ions we have examined.
Comparisons of toxaphene residues in brook trout with toxaphene
standards were made using GC-CI-MS RGCs. The residue in a cleaned
up extract from a brook trout exposed to toxaphene for 141 days was
examined by GC-CI-MS. Scan numbers of RGCs from the brook trout
residue did not directly correspond with those of the toxaphene
standard since the computer software required longer scan intervals
for lower concentrations of sample. The CI-TIC for the sample extract
was plotted in Fig. 17. RGCs for the CIj toxaphene series revealed
significant alteration of the sum of toxaphene isomers C^oHgCly
(M-C1=339) + C10H1;LC17 (M-C1=341) and C10H13C17 (M-C1=343)
(Fig. 18). A major constituent in the toxaphene RGC-343 (scan
numbers 56 to 57) was greatly reduced in the corresponding RGC-343
26
-------
scan 101-107 of the brook trout extract. However, comparison of toxaphene
and toxaphene residue RGCs for the toxaphene isomer CiQEgClj (M-C1=339)
were more similar (Fig. 19). At least one RGC-339 peak, scan numbers
67-72, from the toxaphene standard was identical to that of the correspond-
ing brook trout RGC-339 peak, scan numbers 121-127.
GC-MS-Computer and in particular GC-CI-MS-Computer permitted characteriza-
tion of the major constituents of toxaphene. In addition, RGCs or mass
chromatograms generated by a GC-MS-Computer system were advantageous
tools for the identification of environmental toxaphene residues.
27
-------
Table 2. EMPIRICAL FORMULA OF TOXAPHENE ISOMERS FROM CI-MS
Cla
5
6
7
8
9
10
Formula
C10H9C15
C10H11C15
C10H13C15
C10H8C16
ClOHiQClg
C10H12C16
C10H7C17
C10H9C17
C10HHC17
C10H6C18
C10H8C18
C10H10C18
C10H5C19
C10H7C19
C10H9C19
C10H4C110
C10H6C110
G10H8C110
M+Hb
305
307
309
339
341
343
373
375
377
407
409
411
441
443
445
475
477
479
M-C1
269
271
273
303
305
307
337
339
341
371
373
375
405
407
409
439
441
443
a
35C1 isotope
b Mass
28
-------
ISOTOPE
RHTIOS
10
8.
8.
F-
r-
ta.
TB 80 90 IBB 110 M8138 1* ISO MB :
no «B «o an
Ftgure 10. Theoretical Cl Isotope patterns for substitution of 1-10 Cl.
Calculated from abundance ratio of 35ci to ^CI. This infor-
mation is used when examining chemical ionization spectra of
toxaphene to determine if isotope clusters are homogenous.
CI-MS spectra of toxaphene GC component. Spectrum, No. 35
subtracted from No. 32. Principal constituents were
C10H10C16 (M-C1=305) and C1oHllcl5 (M-C1=271).
29
-------
1
130 1« ISO Ml :
i 218 zao zao aw aa ate
CW-T£»R€HE
1 II l|l rlli| i i t i If IT All li 111 l,l|r ,
TO BO 30 100 110 120 130 I« 13 IBB 170 IBB 190 &j 218 220 230 WO IJSD SED 2W J399 IBB 3(
|
B 310 aai aaa
Figure 11. Upper CI-MS of toxaphene constituents. Spectrum, No. 47
subtracted from No. 45. Mixture of CiQHgCly (M-C1=339)
and C^oHllCU (M-C1=341). Note variation of isotope
cluster starting at mass 339 for Cly. Lower CI-MS also
toxaphene constituents. Spectrum No. 52 subtracted from
50. Primarily CiQHgCly (M-C1=339). Compare isotope
cluster at m/e 339 for Cly with corresponding mass isotope
cluster in Figure 12.
30
-------
SFEETFUt HMER E3 - 79
JIB =0 23J Z« SO JBO '
Figure 12. Upper CI-MS of toxaphene constituents. Spectrum No. 73
subtracted from 69. A mixture of C10H8C18 (M-C1=373)
and C10H10C18 (M-C1-375). Relative ratio of mixture 1:20.
Lower CI-MS also toxaphene constituents. Spectrum No. 94
minus 92. A mixture of Cl0HgCl9 (M-C1=409) and
(M-C1=411). Relative ratio of mixture 20:1.
31
-------
CIM - TQXFPHENE
RfiC - TOTAL ION CURRENT
10 20 30
SPECTRUM NUMBER
50 60 70 80 90 100 110 120 133 140 150 160 170
Figure 13. RGC-TIC for GC-CI-MS of toxaphene obtained and plotted using
Finnigan-System Industries computer system.
32
-------
TOXAPHENE SEPARATION ON OV-7
•^k"
8 .
CT
Figure 14. GC-EI-MS-TIC histogram of toxaphene obtained and plotted using
Digital Equipment Corporation's MASH computer system.
33
-------
JL
r bs Sw 250 :«! i™ a» a» 301
S So » W'«
m a> so ioi 110 OT ia» i« iso 'wo M MO iao a» M iao an
aio S bo ato 350 an S
Figure 15. Upper CI-MS of toxaphene constituents
C^oHllcl7) having an atypical mass fragment at
243 (C8H7Cl4+). Lower CI-MS also toxaphene constituents
having atypical mass fragment C8H
-------
CIM - TOXflPHENE
g
«•"•
8_
S3-
0 10 20 30 10 50 60 70
90 100 110 123 130 110 150 160 170
CIM - TOXHPHENE
E>
0 10 20 30
SPECTRUM NUTCER
-50 60 70 80 90 100 110 120 130 110 150 160 170
Figure 16. RGC's from CI-MS of toxaphene. Masses scanned were 243 and 245,
35
-------
CIM - TQX BFDCK TROUT FISH RESIDUE
£J
2_ R6C TOTAL ION CURRENT
230 210 250 zee
SPECTRM IWflER
Figure 17. RGC-CI-MS-TIC of an extract from a brook trout exposed
to toxaphene for 141 days. Large peak from spectra
numbers 165-172 is due to sample contamination by
di-2-ethylhexyl phthalate.
36
-------
CIM - TOX BROCK TROUT FISH RESIDUE
o
0 10 20 30 40 50 60 70 80 SO 1(30 118 120 13C3 140 150 163 170 180
SPECTRUM NUMBER
CIM - TOXRPHENE
10 20 30 4
SPECTRUM NUM3ER
SO 60 70 80
100 110 120 130 110 150 160 170
Figure 18. Upper RGC-CI-MS is from an extract of a brook trout exposed
to toxaphene for 141 days. Mass scanned was 343.
Lower RGC-CI-MS of toxaphene standard, mass scanned
was 343.
37
-------
CIM - TQXflPHBC
10 20 30 40 50
SPECTRUM NUMBER
70 80 90 100 110 12C
CIM - TQX BROOK TROUT FISH RESIDUE
10 20 30 4
SPECTRUM NUMBER
,!
50 60 70
90 100 110 120 130 H0 150 160 170
Figure 19. Upper RGC-CI-MS of toxaphene. Mass scanned was 339.
Lower RGC-Ct-MS of an extract from a brook trout exposed
to toxaphene for 141 days. Mass scanned was 339.
38
-------
SECTION VII
REFERENCES
1. Frear, D. E. H. Pesticide Index, Fourth Edition. State College,
Pennsylvania, College Science Publishers, p. 372, 1969.
2. Zweig, G. and J. Sherma. Analytical Methods for Pesticides
and Plant Growth Regulators. Gas Chromatographic Analysis
New York, New York, Academic Press, Inc., p. 514-518, 1972.
3. Stalling, D. L. GC-MS Analysis of Toxaphene Residues. Bureau
of Sport Fisheries and Wildlife. (Presented at 165th American
Chemical Society Meeting, Division of Pesticide Chemistry.
Dallas, Texas. April 9-13, 1973.) Abstract #77.
4. Casida, J. E., R. L. Holmstead, S. Khalifa, J. R. Knox, T.
Ohsawa, K. J. Palmer and Y. W. Rosalind. Toxaphene Insecticide:
A Complex Biodegradable Mixture. Science 183:520-521, 1973.
5. Mayer, F. L., Jr., P. M. Mehrle, Jr., and W. P. Dwyer. Toxaphene
Effects on Reproduction, Growth, and Mortality of Brook Trout.
U.S. Environmental Protection Agency, Environmental Research Laboratory,
Duluth, Minnesota, EPA-600/3-75-013, 1975.
6. Tindle, R. C. and D. L. Stalling. Apparatus for Automated Gel
Permeation Cleanup for Pesticide Residue Analysis. Analytical
Chemistry 44:1768-1772, 1972.
7. Field, F. H. Chemical lonization Mass Spectrometry. IX.
Temperature and Pressure Studies with Benzylacetate and t-
Amylacetate. J. Amer. Chem. Soc. 91:2827-2839, 1969.
8. Benville, P. E., and R. C. Tindle. Dry Ice Homogenization
Procedure for Fish Samples in Pesticide Residue Analysis.
J. Agr. Food Chem. 18(5):948-949, 1970.
9. Hesselberg, R. J., and J. L. Johnson. Column Extraction of
Pesticides From Fish, Fish Food and Mud. Bull. Environ.
Contam. & Toxicol. 7^:115-120, 1972.
10. Uthe, J. F., J. Reinke, and H. Gesser. Extraction of Organo-
chlorine Pesticides from Water by Porous Polyurethane Coated
with Selective Absorbent. Environmental Letters 3(2):117-135,
1972.
11. Stalling, D. L. and J. N. Huckins. Silicic Acid PCB-Pesticide
Separation Method. PCB Newsletter, p. 1-3, March 1972.
39
-------
12. Zumwalt, R. W., K. Kuo, and C. W. Gehrke. Application of a
Gas-Liquid Chromatographic Method for Amino Acid Analysis: A
System for Analysis of Nanogram Amounts. J. Chromatog. 55;267-
280, 1971.
40
-------
SECTION VIII
LIST OF PUBLICATIONS
Stalling, D. L. GC-MS Analysis of Toxaphene Residues. 165th
American Chemical Society Meeting, Division of Pesticide
Chemistry. Dallas, Texas. April 9-13, 1973. Abstract #77.
41
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SECTION IX
GLOSSARY OF ABBREVIATIONS
GC Gas chromatography
SCOT Support-coated open tubular
PCB Polychlorinated biphenyls
El Electron impact
CI Chemical ionization
GC-MS Combined gas chromatography and mass spectrometry
GC-EI-MS Combined gas chromatography and electron impact
mass spectrometry
GC-CI-MS Combined gas chromatography and chemical ionization
mass spectrometry
TIC Total ion current
RGC Computer reconstructed gas chromatogram
M Mass
42
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
REPORT NO.
EPA-600/3-76-076
3. RECIPIENT'S ACCESSION NO.
TITLE AND SUBTITLE
ANALYSIS AND GC-MS CHARACTERIZATION OF
TOXAPHENE IN FISH AND WATER
5. REPORT DATE
August 1976 (Issuing Date)
6. PERFORMING ORGANIZATION CODE
. AUTHOR(S)
David L. Stalling
James N. Huckins
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Fish Pesticide Research Laboratory
Fish and Wildlife Service
United States Department of Interior
Columbia, Missouri 65201
10. PROGRAM ELEMENT NO.
1BA608
11. CONTRACT/GRANT NO.
EPA-IAG-0153D
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
13. TYPE OF REPORT AND PERIOD COVERED
Final (4/72-3/74)
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Sensitive methods for the detection and identification of toxaphene in
water and fish were described. Polyurethane foam, gel permeation and silicic
acid chromatography were utilized to permit accurate quantitation of multi-
component toxaphene residues. A method for characterization of changes in
isomer composition of toxaphene residues in fish was reported. The chemical
composition of toxaphene was examined by electron impact and chemical ionization
mass spectrometry. Chemical ionization gas chromatography-mass spectrometry
was particularly applicable to the analysis and confirmation of toxaphene
in residues in environmental samples.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Chromatographic analysis
Mass spectroscopy
Methodology
Research
Trout
Pesticides
Identifying
Chemical composition
Detection
Water
Toxaphene
Brook trout
7C
7.D
3. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS (ThisReport)
unclassified
21. NO. OF PAGES
53
20. SECURITY CLASS (Thispage)
unclassified
22. PRICE
EPA Form 2220-1 (9-73)
43
m,'SGPO: 1976-657-695/5488 Region 5-11
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