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Analysis of Fish Tissue for Kepone,
Mirex, Atrazine , Linuron , and Alachlor
January 1977 ^XTTT>^
X^1 Ro^X
Final Report /> - - f'o\
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Is „ 9 1918 §'
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N^m^X
Prepared for EPA by:
V M *- ^' " "~
^ ^r.. =.T v^^'cal Biochemistry Laboratories, Inc.
1 p
. 0. Box ICG 7
™ Columbia, Missouri 65201 y ^ Envfrofnmestal Pf^scSo*
(31-) U74-8579 . (^a in lafonotiM HM
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Csntsr (SPM52)
841.&tstiUitS!rert ^
Signed: PMMMpMi,rA ^W £
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, -^ -> c^__^^f^/e. /^ ^/
ar"y Broptehart , Senior Chemist
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VlT "t/7 /Johnson , /I.Aboratory Manager
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EPA Report Cotledioi
Information Resource
US EPA Region 3
Philadelphia, PA 191
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CONTENTS
— Section I - Kepone
| Summary of Procedure
•) Table I - Kepone Results
• . Section II - Mirex
' Summary of Procedure
• Table II - Mirex Results
Section III - Atrasine and Simazine
•Summary of Procedure
Table III - Atrazine and Simazine Results
1 Section IV - Alachlor and Linuron
Summary of Procedure
Table IV - Alachlor and Linuron Results
|Cc,-iltr (2PM52)
a,l O.^taat Stoat
PA 19187
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Section I - Kepone
Procedures used in analysis of fish for Kepone
The desiccated fish samples were extracted with
Ethyl Acetate/Toluene (3+1) and brought to a volume of
I! 25ml except for samples 2, 4, 8, 11, 15, 21, 24, 25,
/ 30, and 36. These 10 samples had aliquots removed equal
to lOg of tissue before the Na-SCL tissue mixture was
•j ' extracted.
/ 0.5ml aliquots of the extracts were placed on 6mm X
_( 165mm florisil columns topped with Na2SOlt. PCB's, DDT's '
• and other pesticides which might interfere with the Ke-
• pone analysis were eluted with 4ml of 50% dischloromethane,
49.55% of Petroleum Ether and 0.35% of Acetonitrile, this
•fraction was discarded. Kepone was eluted with 15ml of
Ethyl Acetate/Toluene (3+1) and concentrated to a volume
of 1ml of Ethyl Acetate/Toluene (3+1). Four microliter
Ialdquots of these solutions were injected into a Searle
GLC equipped with a Ni electron capture detector with
the following parameters:
Column - £' X 4mm I.D., 5% SE-30 on 80/100 GC-Q
Coluir-n Temperature - 190°C
Injec-ccr Temperature - 215°C
Detec-jcr Temperature - 290°C
Kepone was quantitated by comparing peak heights of
Kepone in samples TO peak heights of Kepone standards.
Four fortified samples were analyzed with this group, the
average recovery was 72"%. All data is uncorrected for
recovery.
Samples 37, 35, and 28 have higher than normal de-
tection limits because of interfering lipid materials.
Samples 6, 28, 29, 32, 34, 35, and 37 because of
interferences or positive results were reinjected on the
column described below:
1.5% OV-17, 2.0% OV-210 on 80/100 GC-Q. 4mm I.D. X
6ft. long
Injector Temperature - 235°C
Column Temperature - 205°C
TamD^rature - 300°C
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Percent Recovery of Kepone Fortified Samples
Fortified ppm ppm Fortified Recovery Applicable t<
•afr No. Level(ppm) Control Sample ppm Percent Samples
18385-Spl 0.5 0.014 0.36 0.32 64 1 •*• 10
Ba385-Sp2 0.5 <0.02 0.37 0.37 74 -11 '-»• 20
*8385-Sp3 0.5 <0.02 0.27 0.27 57 21 -»• 30
8385-Sp'4 0.5
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Table I - Kepone Results
Lab No^ Customer I. D._ Location ppm Kepone
18385-1 # 1 Nanticoke 0.04
18385-2 ' // 2 Nanticoke 0.02
18385-3 # 3 Nanticoke 0.02
18385-4. # 4 Nanticoke <0.02
18385-5 # 5 Nanticoke 0.02 •
18385-6 # 6 Potomac <0.05
18385-7 # 7 Potomac 0.02 .
18385-8 # 8 Potomac <0.02
18385-9 __ #9 Potomac 0.03
18-3a5-'rO " " "#10 — ^ ^Potomac - --•-' - - 0.-Q3
18385-11 #11 Sassafras <0.02
18385-12 #12 Sassafras <0.02
18335-13 #13 Bohemia <0.02
18385-14 #14 Bohemia <0.02
18385-15 #15 Bohemia <0.02
18385-16 #16 Bohemia <0.02
13385-17 #17 Bohemia <0.02
18385-18 #18 Little Elk <0.02
18335-19 #19 Little Elk <0.02
18385-20 #20 Elk <0.02
18385-21 #21 Elk 0.03
18335-22 #22 Elk <0.02
18385-23 #23 Choptank <0.02
18385-2^ #24 Choptank <0.02
18385-25 #25 Choptank 0.04
18385-26 #26 Choptank <0.02
18385-27 #27 Choptank <0.02
18385-28 #28 James <0.07
18385-29 #29 James <0.05
18385-30 #30 James <0.02
18385-31 #31 James <0.02
18385-32 #32 James 0.09
18385-34 #34 Rappahannock 0.08
18385-35 #35 Rappahannock <0.07
18385-36 #36 Rappahannock 0.03
18385-37 #37 Rappahannock <0.07
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Section II - Mirex
Procedures used in analysis of fish for Mirex
»
Five milliliter aliquots of the Ethyl Acetate/Toluene
(3+1) extracts were cleaned up by gel permeation chroma-
tography with the following parameters: Column 35 X 2.5cm
SX-3, Dump 100ml, Collect 100ml, Wash 10ml, flow rate
5ml/minute. The cleaned up extracts were concentrated
to 5ml and 1ml aliquots were placed on 165 X 6mm I.D.
Silica Gel Columns. Mirex was eluted with 8ml 0..5% Benzene
in Petroleum Ether. The eluate was concentrated to a
Irnl volume and two to four microliter aliauots were in-
jected into a Searle GLC equipped with a Ni electron
capture detector at the following conditions:
Column - 6' X Umm I.D. 1.5% OV-17, 2.0% OV-210 an
100/120 GC-Q
Column Temperature - 200°C
Inlet Temperature - 220°C
Detector Temperature - 300°C
Percent Recovery of Mirex
Fortified Recovery Applicable
Lab No. Lev^l yg Blank yg _ Percent _ to Samples
385 Mirex 0.58 <0.01 0.56 97 all ten
GPCSP
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Table TI - Mirex Results
/
Lab No.
18385-4
18385-8
18385-11
18385-15
18385-21
18385-24
18385-25
18385-30
18385-36
Customer I.D.
# 4
. # 8
#24
#25
#30
#36
Location
Nanticoke-
Potomac
Sassafras
Bohemia
Elk
Choptank
Choptank
James
Rappahannock
-vindicates less than.
ppm Kepone
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01'
<0.01
<0.01 .
<0.01
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Section III - Atrazine and Simazine
Two ml aliquots of the Ethyl Acetate/Toluene ex-
tracts were concentrated to dryness under an air stream.
The residue was transferred to a separa'tory 'funnel with
10ml of pet ether,-and 10ml acetonitrile. The separatory
funnel was shaken and the lower (acetonitrile) layer was
collected. The hexane layer was extracted with two ad-
ditional portions of acetonitrile. The combined lower
layers were concentrated to just dryness on a rotary
evaporator and transferred with benzene to a tube marked
at 2ml and brought to volume. A 1ml aliquot was placed
on a 15cm X 6mm i.d. florisil column and eluted with
10ml benzene and 15ml 10% acetonitrile in benzene. The
eluate was concentrated to just dryness and brought to
1ml with benzene. Two to four microliter aliquots of the
sancle solutions were injected into a GLC equipped with
a Ni electron capture detector at the following para-
meters :
Column - 6' X Umm i.d., 1.5% OV-i?, 2% OV-210 on
ICO/120 mesh GC-Q
Column Te~cera~ure - 170°C
Inlet Temperature - 190°C
Detector Temperature - 300°C
Quantitation was performed by comparing peak heights
of standard injections of Simazine and Atrazine to the
peak heights of Simazine and Atrazine in the sample.
Percent Recovery of Atrazine and Simazine
Lab No.: 18385-11
Atrazine Simazine
Fortified Level (ppm) 5.7 ' U.9
Control (ppm) <0.6 <0.4
Recovery (ppm) "4.9 2.9
Recovery (Percent) 85 % 60 %
Vindicates less than, when present.
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1 Table III -
ILab TK..
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18"J8S-2
• 18385-4
• 1 f 3 385-8
18385-11
1- 18385-15
18385-21
/' 18385-24
1" 18385-25
13385-30
- 18385-36
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•^indicates
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Atrazine and Simazine Results
Customer I.D. . Location ppm Atrazine
# 2 • Nanticoke <0.2 •
# 4 - Nanticoke <0.2
# 8 Potomic <0.2
#11 Sassafras <0.6*
#15 ~ ; ~" Bohemia . <0.2
#21 Elk <0.2
#24 Choptank <0.2
"" #25- -Choptank - ---<0.2
#30 James <0.2
#36 - -- —-Rappahannock . <-X. 0*
limits raised because of interference.
less than, when present.
ppm Simazine
<0.4
<0.4
<0.4
. <0.4
<0.4
<0.4
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Section IV - Alachlor and Linuron
Aliquots containing IQg of tissue were taken from
the Na SOU, tissue mixture of the designated samples. ' •'
Alachlor and Linuron were hydrolyzed to diethyl and di-
chloro analine respectively. The analine hydrolysis
products were steam distilled into an acidic trap. This
acidic solution was then extracted with petroleum ether
to remove codistilled lipophylic substances. The solu-
tion was then adjusted to a pH of Ca 8 and the analines
extracted with dichloromethane. After concentration to
a small volume, the extracts were .transferred to a 6 X
•0.5 cm florisil column topped with 1cm of Na^SO^. Ana-
lines were eluted with llml of dichloromethane. The
eluate was left uncovered at ambient conditions to con-
centrate, the volume of the solutions adjusted to 1ml.
Four microliter aliquots were injected into a Gas Liquid
Chromatograph equipped with a flame ionization detector
with the following conditions:
Column - 3.8% UCW 98, U' X Umm i.'d.
Column Temperature - 150°C
Inlet Temperature - 170°C
Detector Temperature - 230°C
Quantisation T-^as performed by plotting peak heights
of four standards against amount injected for each analine.
From the resultant curve and sample peak height, the
amount of analine in the sample injection was obtained.
PPM were calculated by multiplying this number by factors
accounting for volumes, dilutions, sample weight, and
gravimetric factors to express data in terms of the parent
compound.
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Recovery wa'j vory.poor,for the fish, tissue spikes.
Faotorr; which may havr- contributed to this low recovery'"'
were the presence of Na_SCK which altered the ionic
strength of the-hydrolysis solutions. It was difficult
to dissolve the Na~SOt and may have irreversibly bound
some of the compounds of interest. A great deal of foam-
ing occured with most samples which may have prevented .
distillation of the analine.
As shown in the recovery table, plant material sam-
ples yield quite good recovery.
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lo. \IV - Alachlor and Linuron Results
Lab J-i'o^ Gus Loirier 1 . D. Luca l_ion_ ppm Alachlor ppm Linuron
!838f$-2 # 2 Nanticoke <0.1 <0.2
18385-4 i^4 .Nanticoke ..... <0.1 - ..... <0.2
183FJ5-8 # 8 Potomic- ' <0.1' "- "•--•-'<0.2
183.85-11 #11 Sassafras <0.1 - <0.2
183/85-15 #15 - Bohemia <0 . 1 <0.2
18385-21 #21 Elk <0.1 <0 . 2
16385-24 #24 Choptank <0.1 <0.2
18385-25 #24 Choptank" <0.1 <0.2
18385-30 #30 James <0.1 <0.2
18385-36 #36 Rappahannock <0.1 <0.2
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_,'Percent Recovery; of Alachlor and Linuron from Fortified Samples
I . D . :
Spike A Spike B
Alachlor Linuron Alachlor Linuron
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Fortified
^-L.e ue 1 (p p m)_.
Control (ppm)
Fortified
Samples(ppm)
Recovery (ppm)
Recovery (%)
0.07
0.07
7%
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<0.2
0.47
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0.47
47%
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