oEPA
United States
Environmental Protection
Agency
Environmental Research
Laboratory
Duluth MN 55804
EPA 600 3-90 023
March 1990
Research and Development
Analytical Procedures and
Quality Assurance
Plan for the
Determination of
Xenobiotic Chemical
Contaminants in Fish
-------
EPA/600/3-90/023
March 1990
U.S. ENVIRONMENTAL PROTECTION AGENCY
NATIONAL OIOXIN STUDY
PHASE I I
Analytical Procedures and Quality Assurance Plan for
the Determination ofXenobiotic Chemical Contaminants in Fish.
December 1989
Environmental Research Laboratory-DuIuth
6201 Congdon Blvd.
Duluth, HN 55804
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NOTICE
The information in this document has been funded wholly or in part by the
U.S. Environmental Protection Agency. It has been reviewed technically and
administratively. Mention of trade names of commercial products does not
constitute endorsement or recommendation for use.
12/89 QA/QC Xenobiotics ii
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ACKNOWLEDGEMENTS
Technical contributions to this research were made by:
Brian C. Butteruorth
Douglas U. Kuehl
*§£i_£2£E2£Sli2D
Phillip J. Marquis
Marie L. Larsen
Larry G. Holland
Christine E. Soderberg
Jennifer A. Johnson
Kevin L. Hogfeldt
Alan E . Mozol
Elizabeth A. Lundmark
Daniel M. Fr-emgen
Sandra Naumann
Murray Hackett
Kent Johnson
Harvey D. Corbin, Jr.
Df- Raymond L. Hanson
John Dargan
Dr. Thomas Tiernan
Dr. Michael Taylor
12/89 QA/QC Xenobiotics
i i i
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FOREWORD
Directed by Congressional mandate, the U.S. Environmental Protection Agency
during 1983 initiated the National Dioxin Study, a survey of environmental
contamination by 2,3',7,8- tetrachIorodibenzo-p-dioxin (TCDD) in the United
States. Results of this study are published in the National Dioxin Study:
Tiers 3,5,6, and 7, EPA 400/4-82-003. This laboratory, the Environmental
Research Laboratory - Duluth, was responsible for one part of the Study, the
analysis of fish samples. The most significant findings of these analyses was
the observation that fish contamination was more widespread than previously
thought, and that a primary source of TCDD was discharge from pulp and paper
production using chlorine.
A second more detailed characterization of anthropogenic organic chemical
contaminants in fish was conducted in subsequent analyses during what is now
called Phase II of the National Dioxin Study. This document describes the
analytical methods used for the determination of the level of contamination of
polychI orinated biphenyls, pesticides, and industrial compounds in fish. A
companion document (EPA /600/3-90/022) describes the analytical methods used
for the determination of levels of contamination of fitfteen biosignificant
polychlorinated d i benzo-p-di ox ins and dibenzofurans in those same fish.
12/89 QA/QC Xenobiotics iv
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TABLE OF CONTENTS
I . Introduction 1
II. Preparation of Sample Extract 4
A. Sample Handling Methodology 4
1. Shipment of Samples to ERL-Duluth 4
2. Sample Logging and Coding Procedures 4
3. Tissue Preparation and Storage Procedures.-.4
B . Extraction of Tissue Samples 7
1. Soxhlet Extraction 7
2 . Fortification with Surrogate Standards 9
3. Fortification with Target Analytes 9
C. Isolation of Xenobiotic Chemical, Contaminants. ..11
1. Gel Permeation Chromatography 11
2 . Silica Gel Chromatography 11
3. Fortification with Internal Standards 11
III. Standards and Reagents 12
IV. Analysis of Extracts 13
A. Gas Chromatographic Operating Parameters 13
B. Mass Spectrometric Operating Parameters 13
V. Quality Assurance/Quality Control Procedures 14
A. General Procedures of Operation 14
1. Sample Analysis Set 14
2. Sample Tracking 16
3. Data Storage 16
4. Data Review 16
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B. Procedures for Analytical Quality Assurance 16
1. Gas Chromatography-Mass Spectrometry
a. Instrument Maintenance 16
b. Gas Ch roma tog raphy ...16
1. Column Resolution 17
2. Relative Retention Time 17
c. Mass Spec t romet ry 17
1. Sensitivity 17
2. Spectral Quality 17
2. Gel Permeation Ch roma t og raphy 18
a. GPC Column Flow Rate 18
b. GPC Column Resolution 18
c. Collection Cycle 18
3. Silica Gel Chromatography 18
C. Criteria for Quantitative Analysis 18
1. Gas Chromatographic Relative Retention Time.18
2 . Analyte Identification Criteria 19
3. Signal to Noise 19
4. Relative Response Factor 19
5. Surrogate Standard Recovery 19
6. Total Analyte Recovery 19
D. Quality Control 20
1. Continual Bias Assessment 21
2. Continual Precision Assessment 21
3. Quality Control Chart 21
VI. Quantification of Target Analytes 22
A. Quantification Procedures 22
B. Determination of Minimum Level of
Quant i f icat ion 23
12/89 QA/QC Xenobiotics vi
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Tables
Table 1 -- List of Target Analytes, Internal
Standards, and Surrogate Compounds and
Their Quant i tat ion Ions 2
Table 2 -- Codes for the SCC Number and
Matrix Type 7
Table 3 -- Surrogate Standard and
Internal Standard Solutions... 6
Table 4 -- Target Analyte Fortification
Solutions 10
Table 5 -- Gas Chroroatography / Mass
Spectrometry Operating Parameters 14
Table 6 -- Composition and Approximate
Concentrations of Calibration Solutions for
Full-Range Data Acquisition 15
Table 7 --Target Analytes with low recoveries
for this method 20
Figures
Figure 1 -- Bioaccumulative Pollutant Study
Database Output 5
Figure 2 -- Schematic of Analytical Procedures...8
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I. INTRODUCTION
i
This document, developed for Phase II of the U.S. EPA National Oioxin Study,
describes the analytical procedures and quality assurance plan for the
determination of xenobiotic chemical contaminants in fish. The analytical
approach includes:
- a simple sample preparation methodology that produces a single
extract which minimizes analyte losses,
- a procedure that is cost effective in terms of man power,
chemical reagents, and instrumentation,
- a characterization and quantification of a certain set of
chemical contaminants,
- an identification of unknown contaminants by screening the data.
The set of analytes quantified was derived through considerations that included,
but were not limited to, history (data from previous monitoring efforts),
toxicology, persistence, bioavailability potential, total yearly production, and
feasibility of analyses. A list of target analytes is presented in Table 1.
Limits of quantitat ion for the Target Analytes are as follows:
Target Analytes 2.5 ppb
(except for PCBs)
Polychlorinated Biphenyls
Level of Ch lorination: 1-3 1.25 ppb
4-6 2.50 ppb
7-8 3.75 ppb
9-10 6.25 ppb
Fish were provided by the U.S. EPA Regional labs working with state
environmental agencies.
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Table 1 .
AMALYTE CAS
B i pheny.1 -d,_ (Internal Standard)
lodobenzene (Surrogate)
1,3,5-Trichlorobenzene
1 , 2,4-Trichlorobenzene
1 ,2,3-Trichlorobenzene
Hexachlorobutadiene
1 , 2,4,5-Tetrachlorobenzene
1 ,2,3,5-Tetrachlorobenzene
B i p h e n y I
1 ,2,3,4-Tetrachlorobenzene
Pentachlorobenzene
NUMBER
108703
v,120821
87616
87683
95954
634902
92524
634662
608935
Phenanth rene- d. „ ^Internal Standardl
1 - I odonaph tha I ene (Surrogate)
T r i f 1 u r a I i n
Alpha-BHC
Hexach I orobenzene
Pentachloroanisole
Gamma-BHC (Lindane)
Pentachloronitrobenzene
Diphenyl disulfide
Hept ach I or
Chlorpyrifos
Isopropal in
Octach lorostyrene
Heptachlor Epoxide
Oxych I ordane
Chlordane, Trans-
ChlordaneA Cis-
Chrysene-d.a (Internal Standard)
Nonach I or , Trans-
DDE, p,p'-
D i e I d r i n
N i t r o f e n
E n d r i n
Per thane
Nonach 1 or , Cis
4,4' -D i iodobiphenyl (Surrogate)
Hethoxych I or
Dicofol (Kelthane)
H i r e x
1582098
319846
1 18741
1825214
58899
82688
882337
76448
2921882
33820530
29082744
1024573
27304138
5103742
5103719
39765805
72559
60571
1836755
72208
72560
5103731
72435
1 15322
2385855
QUANT
ION
164
204
180
180
180
225
216
216
154
216
266
188
127
306
219
284
280
219
295
218
272
197
280
380
353
185
373
373
240
409
246
277
283
317
223
409
406
227
139
272
RRT
1 .000
0.309
0.461
0.548
0.625
0.629
0.891
0.891
1.010
1.015
1 .378
1 .000
0.763
0.855
0.890
0.912
0.924
0.979
0.994
1 .076
1 . 185
1 .308
1 .382
1 .395
1 .406
1.410
1 .477
1 .524
1 .000
0.779
0.805
0.807
0.836
0.840
0.844
0.875
0.876
1.017
1.017
' 1.079
12/89 QA/OC Xenobiotics
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Table 1. LIST OF TARGET ANALYTES, INTERNAL STANDARDS, AND
QUANT
ANALYT! ___________________ £*i_!iyMBER ___________ J.ON _____ RRT
Pol ych lor i nated Biphenyls, Cl 1-10
Monoch lorobiphenyls 27323188 - 188 0.318
DichlorobiphenyLs 25512429 222 0.452
Trichlorobiphenyls 25323686 256 0.556
Tetrachlorobiphenyls 26914330 292 0.575
Pentachlorobiphenyls 25429292 326 0.801
Hexach lorobipheny Is 26601644 360 0.818
Heptach I orobi pheny I s 28655712 394 0.881
Octachlorobiphenyls 31472830 430 1.022
Nonachlopobiphenyls 53742077 464 1.250
Oecach I orobi pheny I s 2051243 498 1.288
12/89 QA/OC Xenobioties
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I I . PREPARATION OF SAMPLE EXTRACT
A. Sample Handling Hethodology
1. Shipment of Samples to_ ERL-Duluth : The EPA Regional
Offices are responsible for the collection of the fish samples.
Frozen fish wrapped in aluminum foil are sent to the ERL-Duluth
Iaboratory.
2. Sample Logging and Coding Procedures; The Sample
Control Center (SCO or EPA Regional Offices notify ERL-Duluth
when samples have been shipped. Upon arrival, the samples are
checked to make sure they are in good condition and the Shipment
Records are complete. ERL-Duluth personnel complete the chain of
custody forms and then notifies SCC that samples arrived safely or
if there were any problems with the samples (example: a
mislabeled sampled, no species identification).
Samples are initially placed in a large walk-in freezer.
Aliquots(100-500 g) of ground fish tissue samples (sec. I . A . 3. )
are transferred to laboratory freezer A. Extracted samples are
stored in laboratory freezer B. Completed samples are taken to a
locker plant for long term storage. A locker plant log is kept
according to Episode and SCC numbers.
A computerized data base was developed for sample tracking and
data storage. The episode number, SCC number, date sample was
received, matrix type, latitude, longitude, description of
sampling site, and state from which the sample came are entered
into the data base. Figure 1 is a sample output of the data base.
The first two letters of the SCC number indicate whether the
sample is an Environmental, Method or Matrix Blank, or Duplicate
Sample. All Environmental samples begin with the letter D. The
Blank and Duplicate samples begin with the letter 0 followed by a
D or an R for duplicate or reference fish sample, respectively.
Table 2 lists the possible codes for the SCC number, and matrix '
type. Episode numbers for Blanks and Fortified Matrix samples are
entered as 0000.
Tissue preparation and storage procedures: Fish tissue is
ground frozen at ERL-Duluth in a stainless steel meat grinder.
Each sample is processed through the grinder three times which
homogenizes it thoroughly. For whole fish samples, the entire fish
including organs and fillets are ground. The ground tissue is
stored at - 2 0 ° C in solvent rinsed glass jars with aluminum lined
plastic lids.
12/89 QA/QC Xenobiotics
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NDS PHASE II: B I OACCUHUL AT I VE
Sample Tracking System
POLLUTANTS IN
ERL-0 L o c .
FISH
1234
EPISODE *: 4444
SCC *: DP022030
Sampling Information:
Sampling Office: ERL-Duluth
State & City: MN Duluth
Sampling Contact: Regional Coordinator
Date Sampled: 8/23/87
Site Location: M N Lester River 3 Lake Superior, Duluth
Latitude: N 44 24' 34'' Longitude: W 94 24' 53''
Analysis Lab: D Date Received: 8/31/87
Matrix Type: F PF Steelhead Species Code: A2
Sample Composite: 5
Analytical :
Extraction Date
GC/HS ID
LAB ID
Weight
XLipid
DPE Indication
PCDD/PCDF
O/ O/ 0
Pesticide & Industrial
11/ 3/87
DR871213
B110387JJ
20.00
3.2
Chemicals
Mass lipid on GPC:
0.68
Comments:
Xenobiotic Definitions:
QA Flags:
E - exceeds highest calibration standard
D - below limit of quantitation
Limits of Quantitation:
Pesticides- 2.5 0 p p b
PCBs: 1-3 chloro - 1.25 ppb
4-6 chloro - 2.50 ppb
7-8 chloro - 3.75 ppb
9-10 chloro - 6.25 ppb
12/89 QA/QC Xenobiotics
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JLiayiS-ii lioaccumul at i ve Poll
EPISODE #: 4444 SCC » :
Target Ana lyte
1 ,3,5-Trichlorobenzene
1, 2, 4-Trichloro benzene
1,2,3-Trichlorobenzene
Hexachlorobutadiene
1 ,2,4,5-Tetrachlorobenzene
1 ,2,3,5-Tetrachlorobenzene
B i phenyl
1 ,2,3,4-Tetrachlorobenzene
Pentachlorobenzene
T r i f I u r a I i n
Alpha-BHC
Hexach lorobenzene
Pentachloroanisole
Gamma-BHC (Lindane)
Pentachloronitrobenzene
Di phenyl disulfide
Heptachlor
Chi orpyr i f os
Isopropalin
Octach lorostyrene
Heptachlor Epoxide
Oxych I ordane
Ch I ordane , Trans -
Ch 1 ordane , C i s-
Nonachlor, Trans-
DDE, p,p'-
D i e Idr i n
Ni trof en
E n d r i n
Per thane
Nonachlor, Cis
Methoxych I or
Dicofol (Kelthane)
Mi rex
Total Monoch I orobi pheny I
Total D i ch I orob i pheny I
Total Tr i ch lorofaipheny I
Total Tet rach I orobi pheny I
Total Pentach I orobi phenyl
Total Hexachlorobi phenyl
Total Heptach I orobi pheny I
Total Octach I orobi pheny I
Total Nonach 1 orob i pheny I
Total Decach I orobi pheny I
Total Polych 1 or i na ted Biphenyls
utants In Fish Data
DP022030
CASRN QA Flag
108-70-3
120-82-1
87-61-6
87-68-3
95-95-4
634-90-2
92-52-4 D
634-66-2
608-93-5
1582-09-8 0
319-84-6
118-74-1
1825-21-4
58-89-9 D
82-68-8
882-33-7
76-44-8
2921-88-2
33820-53-0
29082-74-4
1024-57-3
26880-44-8
5103-74-2
5103-71-9
39765-80-5
72-55-9 E
60-57-1
1836-75-5
72-20-8
72-56-0
3734-49-4
72-43-5
115-32-2
2385-85-S E
27323-18-8
25512-42-9
25323-68-6
26914-33-0
25429-29-2 E
26601-64-4 E
28655-71-2 E
31472-83-0
53742-07-7
2051-24-3
base_0i
ERL-D
|tgut
Loc.: 1234
CONCN Cng/g)
ND
NO
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
NO
ND
ND
0.25
2.34
13.2
23.4
1.23
17.2
33.1
45.2
1234
21.2
18.4
118
1 1 .4
60.6
265
187
39.8
564
Mercury ( AA analysis)
SURROGATE RECOVERY;
Iodobenzene
Iodonaphthaiene
4,4' -Di iodobiphenyl
7439-97-6 0.34
12
48
93
ug/g
12/89 QA/QC Xenobiotics
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IS^iS.li £odes_for_SCC_Numbers_and_matrix_tyee^
Environmental sample QA sample
FirstLetter: D Q
Second Letter:
A
B
C
D
E
F
G
H
Y
J
- - Reg
i
- - R e g i
-- Reg
-- Reg
-- Reg
- - Reg
-- Reg
-- Reg
-- Reg
-- Reg
i
i
i
i
i
i
i
i
on
on
on
on
on
on
on
on
on
on
1
2
3
4
5
6
7
8
9
10
B -- Method blank
D -- Laboratory duplicate
R -- Reference fish or
fortified matrix
Matrix Code
F - - Fish
L -- Lab duplicate
R -- Reference fish
Y -- Method Blank
Matrix Type
UB -- Whole bottom
BF -- Bottom fillet
PF -- Predator fillet
WP -- Whole predator
B. Extraction of Tissue Samples.
Figure 2 is a schematic of the analytical procedures.
1. SoxhIet Extraction: Ground fish tissue (20 g) is blended
with anhydrous sodium sulfate (100 g) in a 250 mL beaker to
completely dry the sample. Two-thirds of the mixture is
transferred to a coarse fritted soxhlet extraction thimble and
spiked with Surrogate Standard Solution A (25 uL), Table 3. Also,
at this time the Fortified Matrix Sample and the Fortified
Duplicate Sample, if used, are spiked with 25 ul of Target Analyte
Solution (one of eight Target Analyte Fortification Solutions,
Table 4). The remaining sample is added to the thimble and the
sample is extracted for at least 12 hours with hexane/methylene
chloride (1:1, v:v). The extract is then quantitatively
transferred to a Kuderna-Danish (KD) apparatus fitted with a
3-ball Snyder column and reduced in volume to less than 5 mL on a
steam bath. The extracts are further reduced under carbon
filtered air to remove all solvent. The KD sample tubes with
lipid are weighed. Two 0.40 g aliquots are prepared for Gel
Permeation Chromatography (GPC) by weighing into 5 ml tubes. The
empty sample tube is dried and reweighed to determine the percent
lipid.
12/89 QA/OC Xenobiotics
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to
oo
v£>
Figure 2. Schematic of Analytical Procedures
o
o-
CO
FNVRQNFMTM SAMME
A) NET COLLECTION
B) SHOCK COLLECTION
AIM SURROGATE AMAIYTFS
IODOBENZENE
HOOONAPTHA1ENE
4.4--DIOOOBIPHENYL
RESIDUE STORAGE
TOTAL LESS 1 GRAM
PREP FISH
A) GRIND FILLET OR
B) GRIND WHOLE
EXTRACTION
A) BLEND 20g TISSUE
B) EXTRACT WTTH
SOLVENT REMOVAL
A) KUDERNA-DANISH
APPARATUS
C) DETERMINE TOTAL
LIPID
uv
DETECTOR
1
loo o
w
/
\
fASTE
/
21g SILICA GEL
ADD NTFRNA1 STANDARDS
DloPHENANTHRENE
D,zCHRYSENE
GEL PERMEATION CHROMATOGRAPY
COLLECT FRACTION 1.7 TIMES THE
DISTANCE FROM APEX OF DEHP
TOTHEAPEXOFPYRENE
SI1ICA GF1 CHRQMATOGRAPHY
A) ACTIVATE 130 C*. OVERNIGHT
B) DEACTIVATE 1%H
-------
2. Fortification with Surrogate Standards:
Each sample is .fortified with Surrogate Standard Solution A (25
u L) prior to soxhlet extraction. The standards in this solution
have been selected to represent various types of chemicals found
in the list of target analytes, and are used to evaluate the
recovery of target analytes in cleaned-up environmental samples.
Table 3. _Su££oga£e_S£anda£d_and_2.n£e£na^_S^andard_So^u£jions
Surrogate Standard Solution A (25 uL)
Compound
I odobenzene
1 - I odonaph tha I ene
4,4' -Di iodobiphenyl
1 25
125
125
Internal Standard Solution (10 uL)
Compound
Biphenyl-D.g
Phenanth rene-D
Ch rysene-D .
50
75
3. Fortification with Target Analytes: A blank
matrix sample is fortified with one of eight Target Analyte
Fortification Solutions (25 uL), Table 4, to evaluate the
overall accuracy of a subset of the target analytes. Two blank
matrix samples will be fortified with the same solution
once in every five (20X) sample sets to evaluate precision.
12/89 QA/QC Xenobiotics
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Solution A: Aroclor 1254 at 500 ug/ml (A-1) and 1000 ug/ml
(A-2) in toluene.
Solutions B.C and D: Each have Target Analytes at 125 ug/ml
(B-1, C-1, D-1) and 250 ug/ml (B-2, C-2, 0-2)
1,2,3-Trichlorobenzene
1,2,4,5-Tetrachlorobenzene
B i p h e n y I
Alpha-BHC
Chlordane, cis
D i c o f o I
Endr i n
Diphenyl disulfide
Hexachlorobenzene
Mi rex "
Octachlorostyrene
Pentachlorobenzene
Per thane
1 ,2,4-Trichlorobenzene
1,2,3,4-Tetrachlorobenzene
Gamma-8HC (Lindane)
Chlordane, trans-
DDE, p,p'
N i trofen
Heptachlor
IsopropaI in
Honachlor, cis
Oxychlordane
Pentachloroni trobenzene
Trifluralin
Hexachlorobutadiene
1 ,3,5-Trichlorobenzene
1,2,3,5-Tetrachlorobenzene
Hethoxychlor
Chlorpyri fos
D i e I d r i n
Heptachlor Epoxide
Nonachlor, trans-
Pentachloroanisole
12/89 QA/OC Xenobiotics
10
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C. Isolation of Xenobiotie Chemical Contaminants.
1. GeI Permeation Ch roma tog raphv: A GPC system is used to
isolate xenobiotic chemical contaminants from biological molecules
(fish lipid). The GPC column (2.5 X 50 cm) (ACE Glass Company) is
packed with previously swelled Biobead SX-3. The GPC injection
port valve is fitted with a 0.075 mm stainless steel screen filter
to remove particulates. The solvent is pumped at 5 mL/min. The
absorbance of the effluent is monitored with a 254 nm UV detector
(Varian Aerograph). Each aliquot of extract is diluted with 2 ml
of elution solvent. The supernatant is quantitatively transferred
into a sample loop of a 24 port auto-sampler with three additional
1 ml washes of the sample vial. The loops of the auto-sampler are
loaded sequentially onto the GPC column under computer control. A
GPC performance standard solution (sec. IV.B.1) is run to
determine the collection period. This sample is run prior to each
sample set. Xenobiotic chemical contaminants which elute 4
minutes after the elution apex of Di-2-ethylhexylphthalate, DEHP,
and 1.7 times the elution volume between the apex of DEHP and
Pyrene are collected in a K D . Each sample (two loops) are
collected in a single KD. Hexane (10 ml) is added to the KD and
the sample is reduced in volume (5 ml) on a steam bath using a 3 -
ball Snyder column. The sample is further reduced in volume to
0.5 ml with a stream of dry filtered air at 40 C prior to silica
gel chromatography.
2. Silica Ge I Chromatographv: A Kontes column packed with
freshly prepared, partially deactivated silica gel is used to
remove naturally occurring cholesterol and fatty acids.
The column (9 mm X 19 cm plus a 50 ml reservoir) is packed with
glass wool, anhydrous sodium sulfate (0.5 cm), silica gel (2.1 g
about 7 cm), and anhydrous sodium sulfate (0.5 cm). The column is
pre-eluted with 50 ml of hexane and the sample is quantitatively
transferred to the column with three 0.5 ml methylene
chloride/hexane (15%, v:v) washes. The column is then eluted with
an additional 58.5 mL of the same solvent. Toluene (1 ml) is
added to the collection vial as a "keeper". The sample is reduced
in volume (0.5 ml) with a stream of dry filtered air, 40° C, and
quantitatively transferred with toluene to a tapered vial (1 mL).
3. Fortification with Interna I Standards. The samples are
reduced to 90 uL and fortified with 10 uL of Internal Standard
solution (Table 3) and stored in a microvial for GC/MS analysis.
12/89 QA/QC Xenobiotics 11
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III. Standards and Reagents
A. Reagents
1. Solvents: Only pesticide grade distilled in glass
solvents are used. They are: hexane, methylene chloride,
toluene, acetone, and cylcopentane (Burdick and Jackson and
Fischer Scientific).
2. Sodium Sulfate: Sodium sulfate (Baker Chemical Company
reagent grade anhydrous) is baked at 650°C in a furnace for
24 hours, cooled, and stored in an empty hexane solvent bottle.
3. GPC Packing: Biobead SX-3 (BIORAD Corporation) are
swollen in the elution solvent, cyclopentane/methylene chloride
(1:1, v:v) .
4. Silica Gel: Silica-Gel-60 (Merck-Darmstadt) is activated
overnight at 2 2 5 ° C. It is then deactivated by adding distilled
water ( 1 X w:w) and shaken at high speed for four hours to
disperse the water. The mixture is allowed to equilibrate for
eight hours.
B. Standards
All pesticide standards are made from pure standard materials.
1. GPC Performance Check Solution: Prepare a solution of
5 mg/ml Oacthal, 4 mg/ml DEHP, and 0.2 rug/ml Pyrene.
2. MS Performance Check Solution: Prepare a 5 ng/ul solution of
decafluorotriphenylphosphine (DFTPP) in toluene.
3 . Silica-Gel Performance Check Solution: Prepare a solution
containing 2 mg/ml Dieldrin and 10 mg/ml cholesterol in an
appropriate solvent.
4. Internal Standards: Chrysene-d.,, phenanthrene-d.., and
biphenyl-d-g are used as internal standards. Table 1
indicates which internal standard the target analytes are
referenced to in quantitation. Table 6 indicates the
concentration of the internal standards in the calibration
solutions and in the solution used to add the internal
standards to the samples just prior to MS analysis.
5. Surrogate Compounds: lodobenzene, 1 - Iodonaphtha Iene, and
4,4'-diiodobiphenyl are used as surrogate compounds. Each
are present at 125 ug/ml (Table 3) in the sample spiking
solution. Table 6 indicates the concentration present in
the five calibration solutions.
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6 . Pesticides and PCB Standards: A stock solution is made
contain ing the pesticides listed in Table 1 and the PCS
congeners listed in Table 6. Five calibration solutions
are made at the concentrations listed in Table 6.
7. Fortification Solutions: The pesticides are divided into
three fortification solutions at two different concentrations
(Table 4). Aroclor 1254 is used as the PCB fortification
solution at the concentrations listed in Table 4 .
IV. Analvsis of Extracts
Samples are analyzed on a Finnigan-MAT Model 4500 GC/HS
with SUPERINCOS software and supplemental public domain software (1,2)
provided by the U.S. EPA laboratories in Cincinnati, OH. All Target
Analytes will be quantified individually and the results reported as unique
values, except for PCBs, which will be reported by total congener at each
degree of chlorination. An analysis set includes an analysis of a mass
spectrometer performance check solution (sec. III.B.2), an analytical
standard, an unfortified solvent (instrument blank), and twelve prepared
samples. The GC/HS operator reviews the MS performance solution,
analytical standard, and instrument blank data before starting the analysis
of samp Ies .
as Ch romatog rapi c Operating Parameters; A Finnigan-MAT
Model 9610 GC is fitted with a 60 m X 0.32 mm 10 08-5 fused silica
capillary column (J & W Scientific) and operated in a temperature
programmed mode. The capillary column is interfaced directly with the
ionizer. Injections are made in splitless mode. Specific operating
parameters are provided in Table 5.
Mass Spectrometric Operating Parameters: A Finnigan-MAT
Model 4500 mass spectrometer is used in the electron impact mode.
Specific operating parameters are provided in Table 5. The
positive identification of target analytes is based upon a reverse
library search threshold value and relative retention time (RRT).
Quantification of the target analytes is based on the response factors
(RF) relative to one of the three internal standards listed in Table 1.
Table 1 is formatted so that the target analytes follow the internal
standard used in quantification. RRTs and RFs are initially
determined using data from triplicate analysis of each of five
target analyte quantification solutions (Table 6).
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GC Parameters:
Injector Temp.: 250° C
Initial Temp.: 100° C held for 1 min.
First Ramp: 5° C/min to 175° C
Second Ramp: 3° C/min to 280° C hold for 20 min
MS Parameters:
Cycle time: 1.0 second
Acquisition time: 0.95 second
Scan Rate: 1.0 second
Scan Range: 95 - 550 amu
EIectron Voltage: 70 eV
Emission Current: 0.30 mA
Manifold Temp.: 95° C
Ionizer Temp.: 150° C
V. Quality Assurance/Quality Control (QA/QC^
A. General Procedures of Operation.
1. Sample Analysis Set : Analysis of samples is
done in sets of twelve consisting of:
a. Blank; A METHOD BLANK (blank extraction
apparatus) is analyzed with each set.
b. Fortified Matrix; A blank matrix
sample is fortified with one of eight different
mixtures of Target Analytes (Table 4) and analyzed
with each set.
c. Dupl icate; Each analysis set contains
one duplicate sample. In four of five (80%) of
the sample sets the duplicate is an environ-
mental sample previously chosen for
analysis in that set. In one of five (20%) of the
sample sets the duplicate is a blank matrix
sample that has been fortified with the same
target analyte subset as the Fortified Matrix
Sample. This additional type of duplicate insures
that sufficient data is available at the end
of the study to evaluate precision on all target
ana Iytes .
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Table 6. Composition and Approximate Concentrations of Calibration
Analyte/Znt. Std./
4 ___ CAL_5
PCS CaI. Congeners
C^ 2- 0.25 0.50 1.25 2.50 5.00
C12 2,3- 0.25 0.50 1.25 2.50 5.00
C13 2,4,5- 0.25 0.50 1.25 2.50 5.00
C14 2,2',4,6- 0.50 1.00 2.50 5.00 10.00
C15 2,2',3,4,5'- 0.50 1.00 2.50 5.00 10.00
C16 2,2',4,4- ,5,6'- 0.50 1.00 2.50 5.00 10.00
C17 2,2',3,4,5,6,6- 0.75 1.50 3.75 7.50 15.00
Clg 2,2',3,3',4,5,6'- 0.75 1.50 3.75 7.50 15.00
CI1Q 1.25 2.50 6.25 12.50 25.00
All Target Analytes
other than PCBs listed
in Table 1 0.50 1.00 2.50 5.00 10.00
Internal Standards
Chrysene-d12 7.50 7.50 7.50 7.50 7.50
Phenanthrene-d1Q 7.50 7.50 7.50 7.50 7.50
Biphenyl-d1Q 5.00 5.00 5.00 5.00 5.00
Surrogate Compounds
lodobenzene 0.50 1.00 2.50 5.00 10.00
1-lodonaphthalene 0.50 1.00 2.50 5.00 10.00
OiSO .KOO 2^50 5A00 10^.00,
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d . Env i ronmenta I Samp I es; Nine Environmental
Samples are analyzed with each set.
i. Samp Ie Tracking: A sample tracking and logging
system is used to assure that no samples are
lost (see sect i on I - A).
3. Data Storage: Data folders consisting of all
hard copy output is maintained for each sample.
In addition, all raw GC/HS data is stored on
magnetic tape.
4. Data Review: GC/HS data is initially reviewed
during sample set acquisition by the GC/HS operator
to assure that all instrumental QA parameters are being
met. Final review and release of the data is the
responsibility of the Project Hanager. Once the quality
assurance criteria have been met, the quantification
information is entered into the database. Quality
assured data is then transferred to 8 IOACC/STORET
for availability to the EPA Regions. Before release
to the public, all transferred data is verified for
completeness by the database manager.
B. General Procedures o f Analytical Quality Assurance;
1. Gas Chromatography-Hass S pec t romet rv Sys t em:
a. 1ns t rument Maintenance: The GC/HS system
is maintained according to the manufacturer's
suggested schedule. The maintenance schedule
is indicated on a calendar located near each
instrument. Log books will be kept for: Daily
instrument settings; Samples analyzed;
Maintenance; and Data Storage. Instrumental
problems resulting in more than two days of down
time are to be reported to the EPA Hass
Spectrometry Facility Supervisor to discuss
solutions to the problems.
b. Gas Chromatography: The performance of the
GC is evaluated by determination of the
number of theoretical plates of resolution, and by
relative retention of the Surrogate Standards.
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1. Co L umn Resolution: The number of
theoretical plates of resolution, N, is
determined at the time the calibration curve
is generated using Chrysene-d^p and monitored
with each sample set. The value of N shall not
decrease by more than 20X. The equation for N
is given as follows:
N - 16 (RT / U)2
where, RT - Retention Time of
Chrysene-d^Q in seconds
U = Peak width of
Chrysene-d.g in seconds.
2. Relative Retention Time: Relative
retention times of the internal standards
shall not deviate by more than +/- 3 % from
the values calculated at the time the
calibration curve was generated.
c. Mass Spect romet ry: The performance of the
mass spectrometer will be evaluated for both
sensitivity and spectral quality.
1. Sensitivity; The signal to noise value
must be at least 3.0 or greater for m/z 198
from an injection of 10.0 ng decafluorotri-
phenylphosphine CDFTPP).
2. Spectral Quality: The intensity of
ions in the spectrum of DFTPP must meet the
criteria listed below:
127 30-60X mass 198
197 < 1X mass 198
198 base peak
199 5-9X mass 198
442 >40X mass 198
443 17-23X mass 442
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2. Gel Permeat i on Chromatographv; The GPC is
maintained when needed as determined by visual
inspection (column discoloration, leaks, cracks, etc)
measurement of flow rate, and routine measurement of
contamination of instrument blanks.
a. GPC Co lumn FIow Rate; The flow rate of the
GPC is measured three times during an analysis:
1) before the GPC resolution solution, 2) after all
samples are loaded but before analysis and 3) after
all samples have been analyzed. Flow rate should not
vary by more than +/- 0.2 mL/min.
b. GPC Column Resolution: A 350 ul injection of a
performance solution containing Dacthal (5 mg/mL),
OEHP (4 mg/ml), and Pyrene (0.2 mg/mL) must be run
daily to evaluate column resolution, and to determine
analyte starting and ending collection volume.
c. Collection Cycle: Proper operation of the
GPC will also be evaluated by recording the time
during an analysis cycle that the collection/waste
valve is in the collect position. This is
accomplished most easily by recording the valve
position on the second pen of a dual pen recorder-
The start and end of the collect cycle must not
deviate by more than +/- 2 ml.
3. Silica Gel Ch romatographv: The silica gel
column will be evaluated by its ability to resolve
cholesterol from a select model target analyte,
Dieldrin. A solution (1.0 ml) containing Dieldrin
(2.5 mg/mL) and cholesterol (10 mg/mL) is spiked onto a
silica gel column and eluted with methylene
chloride/hexane (15X, v:v, 60 m L ) . The eluant,
analyzed by flame ionization detector/gas chromatography
(FID/GC) must not contain more than 1 0 X of
the cholesterol while at least 90% of the Dieldrin must
be recovered.
C. Criteria for Quantitative Analysis; All of the
following quality assurance criteria must be met before a
quantitative value may be reported for an analyte.
1 . Gas Chromatographie R eI a t i ve Retention Time;
Relative retention times of the target analytes shall
not deviate by more than +/- 3 X from the values
established during the generation of the calibration
curve (see Table 1 for RRT data).
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2- Analvte !dent i f i eat i on Cr i ter i a ; Reverse search
identification of an anatyte (SEAR) must have an FIT
value of 800 or greater.
3. Signal to Noise: The quantification ion must have
a signal to noise value of at least 3.0.
*• Relative Response Factor: The relative response
factor for each analyte quantification ion relative to
the appropriate internal standard quantification ion
must not deviate by more than 20X from' the value
determined on the previous day (within a 24 hour period)
and uithin SOX of the mean value from the calibration
curve. The target analytes Endrin, Dicofol, and Oeca-
chIorobiphenyI must not deviate by more than SOX from
the previous day.
A control chart is maintained on the daily response
factors for each target analyte.
Surrogate Standard R ecove ry : The percent recovery
(XR) of each surrogate standard will be determined
for all samples, as shown below:
XRs = 100[Co/Ca]
where XRs = surrogate percent recovery
Co = observed concentration of
surrogate
Ca actual concentration of
surrogate added to the sample.
The percent recovery must be within 25 and 130
percent for iodonaphtha Iene and 50 and 130 percent
for 4,4'-diiodobiphenyl. The recovery of iodobenzene
qualitatively indicates the extent of evaporative
losses that the analytes listed in Table 7 may experience,
Total Analyte Recovery; The overall accuracy of
quantification of all target analytes is evaluated
by the analysis of a subset of target analytes
fortified into a matrix blank. Recovery of the
fortified analytes must fall within the range of 50 to
130X except for those listed in Table 7. The analytes
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Table 7. Target Analytes with low recoveries for
1 ,3,5-Trichlorobenzene
1 ,2,4-Trichlorobenzene
1 ,2,3-Trichlorobenzene
1,2,4,5-Tetrachlorobenzene
1,2,3,5-Tetrachlorobenzene
1 ,2,3,4-Tetrachlorobenzene
Pentachlorobenzene
Hexachlorobutadiene
listed in Table 7 show recoveries that fall in the range
of 20 to 30X for this method. An average analyte
recovery (XAR) for all target analytes will be calculated
and must be greater than 35X but less than 130X.
A control chart for total analyte recovery and analyte
recovery is maintained for each spiking solution.
To determine total analyte recovery first calculate
the percent recovery (XR) for each fortification analyte
using,
XRa = 100((Ai-Bi)/Ti)
where XRa = analyte percent recovery
Ai = measured analyte concentration in
fortification sample after
analysis.
Si = natural analyte concentration in
sample before fortification.
Ti = known true concentration of
analyte fortification level.
Then calculate XAR by,
XAR = (Summation of XRa) /N
where N = number of fortification
analytes in spiking solution.
Quality Control ; Quality control charts displaying
quantitative bias (XB> and precision (XP) are maintained
for each analyte using LOTUS 123 software, Lotus Development
Corporation. Percent bias and percent precision will be
recorded and the control chart will be updated after each
analysis set. Complete statistics may be done for bias and
precision at the completion of the project.
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Continual Bias Assessment:
XB = (100(Ca-Cb>/T) - 100
where Ca = determined concentration after analysis
Ca = concentration present before spike added,
T known value of the spike.
Continual Precision Assessment:
Precision of quantification of ea>ch target analyte
will be assessed separately for duplicate environmental
samples and duplicate fortified matrix samples.
XP 100[
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VI. Quantification of Target Ana I vtes:
A. Quantification Procedures
Response factors are determined for each target analyte and surrogate
compound relative to one of the three internal standards. The
response factors are determined by:
RF * AxCIS/AlSCX
where A.. = peak area of quant i tat ion ion for a target analyte
or a surrogate compound,
A. s = peak area of quantitat ion ion for either
Biphenyl-d.j0, Phenanthrene-d^Q, or Chrysene-d..-,
C.g * injected quantity of the internal standard,
C.. = injected quantity of the target analyte or
surrogate compound.
Public domain software was provided by the EPA Office of
Research and Development, Environmental Monitoring and
Support Laboratory for the automated identification and
quantification of the target analytes. The data reduction
software uses the following formula to calculate target
analyte concentrations:
CONC = ((QA * NUM * QRV) * FESV) / (VIA * SIZE)
where QA = concentration as calculated using the
response factor from the daily standard,
NUM = factor to convert to number of ug/ml,
QRV = Quan Report Volume (0.100 ml),
VIA = Volume Internal Standard added to (0.100 ml),
FESV = Final Effective Sample Volume,
SIZE = sample size (g).
The FESV term accounts for the total lipid present in the
sample and the amount injected on the GPC. The FESV is
calculated by:
FESV = Final Volume (ml) * (Total Lipid (g) / Lipid on GPC (g))
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Calculations for determining surrogate spikes and fortified
amounts use the following equation:
COMC « (SA * FESV) / (FSRV * SIZE)
where SA * spike amount,
FSRV » Final Effective Surrogate Volume,
FESV, SIZE * same as above.
The FSRV term is equal to the FESV term. The concentration
of a target analyte is denoted in the final report if it
exceeds the calibration range, ('£' flag), or is below the
quant i tat i on limit, CD' flag).
B. Determination of Minimum LeveI of Quantification
The calculated method detection limits (MDLs) for the analytes, (determined
according the Federal Register 1988, Vol. 40, Appendix B, Part 136,
Definition and Procedure for the Determination of the Method Detection
Limit, Rev. 1.11), are unreaIisticaI Iy low in comparison to the analysis of
the xenobiotic calibration solutions over a two month period. Based on the
analysis of the calibration solutions a minimum level of quantification was
determined for each analyte, as given in the Introduction, which accurately
reflects the instrumental detection limits.
U.S. GOVERNMENT PRINTING OFFICE 1990/748-159/00430
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