United States
Environmental Protection Agency
Office of Water
Washington, D.C. 20460
EPA 503/6-90-002
May 1986
BIOACCUMULATION
MONITORING GUIDANCE:
4. ANALYTICAL METHODS FOR U.S. EPA
PRIORITY POLLUTANTS AND 301 (h)
PESTICIDES IN TISSUES FROM
ESTUARINE AND MARINE ORGANISMS
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EPA 503'6-9OOC2
May 1966
BIOACCUMULATION
MONITORING GUIDANCE:
ANALYTICAL METHODS FOR U.S. EPA
PRIORITY POLLUTANTS AND 301 (h)
PESTICIDES IN TISSUE FROM
ESTUARINE AND MARINE ORGANISMS
Prepared by:
Tetra Tech, Inc.
11820 Northup Way, Suite 100
Beltevue, Washington 98005
Prepared for:
Marine Operations Division: 301 (h) Program
Office of Marine and Estuarine Protection
U.S. Environmental Protection Agency
401 M Street SW
Washington, D.C. 20460
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This report is one element cf the Bioaccumulation Monitoring Guidance
Series. Tne purpose of this series is to provide guidance for monitoring
of priority pollutant residues in tissues of resident marine organisms.
Trsese guidance documents were prepared for the sewage discharge program
of Section 301(h) of the Clean Water Act unoer the U.S. EPA Office of Marine
end Estuarine Protection, Marine Operations Division. Two kinds of monitoring
guicance are provided in this series; recommendations for sampling ana
analysis assigns, and aids for interpretation of monitoring c'ata,
Some oasic assumptions were made in developing tne guidance presentee
in these documents: 1) each bioaccumulation monitoring program will be
designed to meet the requirements of the 30i(h) regulations, 2) tissue
samples i-.il! be collected from appropriate locations near the sewage discharge
and from en unpolluted reference site, 3) the initial chemicals of concern
are the U.S. EPA priority pollutants ana 301{h) pesticides, and 4) the
monitoring cata should be suitable for a meaningful evaluation of the potential
hazards to living marine resources as well as human health. It should
be recognized that the design of a monitoring program reflects tne site-
specific characteristics of the pollutant discharge and the receiving environ-
ment. Thus, site-specific considerations may lead to a moaification of
the generic recarmendations herein. Finally, although these guidance documents
were prepared specifically for monitoring of sewage discharges under the
301(h) program, their potential use extends to assessment and monitoring
cf bioaccunul ation resulting from otner kinds of pollutant discharges into
marine and estuarine environments.
11
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I ' ' ' ' v
L, J i \ . _ . 1 I J
PREFACE ii
LIST OF FIGURES
LIST OF TABLES vi
'ir*|/M-'^t'ir''-\p*"i**~»i"f'c ^ 1
H'-IVlUrt utl JbCi'iwFi t b ' ' I
INTRODUCTION ix
SECTION I. ANALYSIS OF EXTRACTABLE ORGANIC COMPOUNDS
IN ESTUARINE AND MARINE TISSUES
1.0 SCO^E AND APPLICATION 1-1
2.*0 SUMMARY OF METHOD 1-4
3.0 INTERFERENCES 1-5
4.0 SAFETY 1-6
5.0 APPARATUS AND EQUIPMENT 1-6
6.0 REAGENTS AND CONSUMABLE MATERIALS 1-10
7.0 SAMPLE COLLECTION, PREPARATION, AND STORAGE 1-14
8.0 CALIBRATION AND STANDARDIZATION 1-16
9.0 QUALITY ASSURANCE/QUALITY CONTROL 1-21
10.0 PROCEDURE 1-24
11.0 QUANTITATIVE DETERMINATION (CALCULATIONS) 1-35
12.0 PRECISION AND ACCURACY 1-42
13.0 REFERENCES 1-43
SECTION II. ANALYSIS OF VOLATILE OR6ANIC COMPOUNDS
IN ESTUARINE AND MARINE TISSUES
1.0 SCOPE AND APPLICATION 11-1
2.0 SUMMARY OF METHOD 11-2
iii
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5.0 AP3AF,ATL3 A!,3 £3^I?/!E,'iT
O , 0 ~~r-.G;.i»>5 "ti'J L ->. iS U;'l--.u L.t i'l*- i trt .. .-M_5
7.0 SAI-.PLE C;LLE:TI::;, PREPARATION, ;;;D STORAGE
s.c CALIBRATION AND STANDARDIZATION
or, ,> i i ^ v r "' "* "5""' ""_".".
j , J J J r,:_» i I L ^. i i '. _ *. - -
10. C PROCEDURE ":-2C
11.0 QUANTITATIVE DETERMINATION (CALCULATIONS) 11-26
1 "> Pi n->^.-T-i«(i,nii'i«^/>!i'Kf-v TT-?7
i 4. . U rr.l^^iu.i n!>J nowUriiOT - * ^'
SECTION III. ANALYSIS OF METALS AND METALLOIDS
IN ESTUARINE AND MARINE TISSUES
1.0 SCOPE AND APPLICATION III-i
2.0 SUMMARY OF METHOD HI-1
3.0 DEFINITION'S ^11-^
4.0 INTERFERENCES iii-ii
5.0 SAFETY III-4
6.0 APPARATUS AND EQUIPMENT III-4
7.0 REAGENTS AND CONSUMABLE MATERIALS III-6
8.0 SAMPLE COLLECTION, PREPARATION, AND STORAGE III-7
9.0 CALIBRATION AND STANDARDIZATION III-9
10.0 QUALITY CONTROL III-11
11.0 PROCEDURE II1-13
12.0 CALCULATIONS III-21
13.0 PRECISION AND ACCURACY 111-21
14.0 REFERENCES 111-21
iv
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FIGURES
_N umber Page
1-1 Relative response calibration curve 1-45
1-2 Extracted ion current profiles for chrornatographically
resolved labeled (^2/z) anc* unlabeled (mi/z) pairs 1-45
1-3 Extracted ion current profiles for (3A) unlabeled compound,
(3B) labeled compound, and (3C) equal mixture of unlaoeled
and labeled compounds 1-45
1-4 Flow chart for sample preparation 1-46
II-l Apparatus for vacuum distillation and cryogenic concen-
tration II-29
II-2 Relative response calibration curve 11-30
II-3 Extracted ion current profiles for (A) the unlabeled pol-
lutant, (B) the laPeled analog, and (C) a mixture of the
labeled and unlabeled compounds 11-30
III-l Quality control chart 111-24
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C C
_t J
1-1 Gas chromatography of extractable compounds i-47
1-2 OFTPP nass- intensi ty specifications 1-50
1-3 Precision and accuracy of method blanks 1-51
II-l Volatile organic analytes 11-31
II-2 BF3 mass-intensity specification 11-32
II-3 Percent spike recoveries for volatile priority pollutants
using vacuum distillation 11-33
Ill-i General information for each priority pol 1 utant metal 111-25
III-2 Typical data obtained on a certified reference material
(N3S oyster tissue) 111-26
v1
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Tnis document has been reviewed by the 301 (h) Task Force of tre Environ-
mental Protection Agency, which includes representatives from the Water
Management Divisions of U.S. EPA Regions I, II, HI, IV, IX, and X; tne
Office of Research and Development - Environmental Research Laboratory -
Narragansett (located in Narragansett, RI and Newport, OR), and the Marine
Operations Division in the Office of Marine and Estuarine Protection, Office
of Water.
This technical guidance document was produced for the U.S. Environmental
Protection Agency under the 301(h) post-decision technical support contract
No.- 63-01-6938, Allison J. Duryee, Project Officer. This report was prepared
by Tetra Tech, Inc., under the direction of Dr. Thcnas C. Ginn.
c r " T »m' T
O _ o I 1 J . 1 i
The primary authors were Mr. Robert C. Barrick and Mr. Harry R. Seller.
The assistance of Mr. Raleigh C. Farlow is appreciated.
Existing U.S. EPA analytical methods were incorporated into Section I
whenever possible. Specifically, many sections were adapted from the Contract
Laboratory Program for Qrganics Analysis (Section I, reference 2) and U.S. EPA
Method 1625 Revision B (Section I, reference 3), which was developed by
the Industrial Technology Division of the Office of Water Regulation and
Standards.
Validation data presented in Section I (Precision and Accuracy) were
generated by California Analytical Laboratories and Weyerhaeuser Technology
Center .
vn
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errT'H" T T
-JwU 1 lUH 1 1
Tne primary c'jtr.crs .-.ere Xr. ROD en C. Barri>, enc !!r. -erry R.
The assistance cf Mr. Raleigh C. Farlow is appreciated.
The procedure described in Section II is largely a compilation cf
methods cevelooec bv U.S. EPA. Specifically, tne rr.etnods were ceve'cc-c
by tr.e Environmental Monitoring Systems Laboratory (EMSL) in Las Vecas
{Section II, references 1 and 2) and the Industrial Technology Division
of the Office of Water Regulation anc Stancaras (Section II, refc-*-enc~ 2''.
Dr. M. Hiatt (Analytical Technologies, Inc., national City, CA, previously
at EMSL Las Vegas) was a valuable source of technical information presenter
in tnis document.
SECTION III
The primary authors were Mr. Robert C. Barries, Mr. Harry R. Seller,
and Mr. Robert V. Several 1. The assistance of Dr. Charles R. Lytle is
apprec iatec.
Validation cata presented in Section III (Precision ana Accuracy)
were generated by Analytical Service Laboratories, Ltd.
Mention of trace names or commercial products nerein coes not constitjte
endorsement for use 3y U.S. EPA or Tetra Tech, Inc.
vi n
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The tnree analytical metnocs in this document have been designed to
be consistent with probable uses of 501 (h) bioaccumulation monitoring cats.
Comparison of tissue c en tarn in ant concentrations from contaminated and relative'_,
uncontarr.inatec areas ana estimation cf the potential health effects cf
b i OcCC'jnul £tc-G Substances often require sensitive analytical tecr.nic^es
for a wide range of chemically diverse pollutants. The recommenced 301(h)
procedures allow for sensitive analyses of the target compounds *itr; a
reasonacle amount of laboratory effort. Organopnospnate 201(n) pesticices
have net yet been tested with the recommended techniques (i.e., Malathicn,
Paratnicn, jernetor, Guth ion). Analyses for 2 , 5 ,7,3-TCuD rtith appropriate
detection limits will require the dedicated U.S. EPA Contract Laboratory
Program procedure for dioxin analysis (9/15/33), which involves selected
ion monitoring (SIX) GC/MS analysis.
Tnere are currently no formally approved U.S. EPA procedures for analyzing
priority pollutants and 301(h) pesticides in biological tissue. However,
various U.S. EPA procedures were reviewed during development of tnis report
fe.g., Interim Methods for the Sampling and Analysis of Priority Pollutants
in Sediments and Fish Tissue (1977, revised 1980); Contract Laboratory
Program procedures for organics analysis and inorganics analysis]. Conse-
quently, the recommended 301 (h) procedures include portions of U.S. EPA
analytical and quality assurance procedures that were considered appropriate
for sensitive, full-scan analyses (e.g., at the low parts per billion level
for organics analysis). The 301(h) methods have been assembled accorcirg
to gjidei ines for EMSL (Environmental Monitoring and Support Laboratory,
Cincinnati) analytical methods (as specified in EPA-600/8-83-020).
ix
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SECTION I
ANALYSIS OF EXTRACTA3LE ORGANIC COMPOUNDS
AND MARINE TISSUES
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CONTENTS
1.0 SCOPE AND APPLICATION 1-1
2.0 SUMMARY OF METHOD 1-4
3.0 INTERFERENCES 1-5
4.0 SAFETY 1-6
5.0 APPARATUS AND EQUIPMENT 1-6
6.0 REAGENTS AND CONSUMABLE MATERIALS 1-10
7.0 SAMPLE COLLECTION, PREPARATION, AND STORAGE 1-14
8.0 CALIBRATION AND STANDARDIZATION 1-16
*
9.0 QUALITY ASSURANCE/QUALITY CONTROL 1-21
10.0 PROCEDURE 1-24
11.0 QUANTITATIVE DETERMINATION (CALCULATIONS) 1-35
12.0 PRECISION AND ACCURACY 1-42
13.0 REFERENCES 1-43
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ANALYSIS OF EXTRACTA3LE ORGANIC COMPOUNDS
IN ESTUARINE AND MARINE TISSUES
1.0 SCOPE AND APPLICATION
1.1 This method is designed to determine the semivolatile priority pollutants
(Tab'e 1-1) associated with the Clean Water Act Section 301(h) regulation
[40 CFR 125.5S(k) and (v)]. Additional compounds anenaole to extraction
and analysis by capillary column gas chrcmatography-mass spectrometry (GC/KS)
and/or gas chromatography-electron capture detection (GC/ECD) may be suitable
for analysis, subject to testing.
These procedures are applicaole when low part per billion analyses
are required to monitor differences between oody burdens in organisms from
relatively uncontaminated reference areas and contaminated estuarine and
marine environments. In addition, the procedures are applicable when low
detection limits are required for the estimation of potential health effects
of bioeccxiulated substances.
Two GC/MS options included in the methods are analyses by isotopa
dilution GC/MS (preferred) or by a GC/KS internal standard technique (minimum
required). In both cases, the laboratory procedures for sample extraction
and concentration of the resulting extract are identical. Compound-specific
recovery corrections used in the isotope dilution technique are designed
to increase the accuracy of the analysis and the comparability of results
among laboratories. In addition, use of the multiple recovery stencrds
in each analysis increases confidence in the validity of detection limits
reported for undetected target compounds. By forcing a search for every
recovery standard in the sample extract (over 50 are available), the technique
also increases the efficiency of detection and reporting frequency of target
compounds that otherwise may be overlooked in complex extracts.
1-1
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1.2 Tne cornpounas listed in Table I-i incline pesticides suoject to regulation
under Section 301 (h; of the Clean water Act. However, the epplic=Dility
of this Tiethcd to non-chlorinated organopnosphorous pesticides (Malatrnor,,
Parathion, Demeton, and Guthion) has not been demonstrated. Chemists at
the Food and Drug Administration have recently published a technique for
determining organophosphate pesticides of wide ranging polarity in matrices
including fatty animal tissue (J.J. Blaha and P.J. Jackson, J. Assoc. Anal.
Chem., Vol. 68, pp. 1095-1099, 1985). The technique involves 1iquid-1icuid
partitioning and gel permeation chromatography [both are included in this
recomenced 301(h) procedure] as well as N/P alkali thermionic or flame
photometric detection. Further work is required to determine the suitability
of the recommended 301(h) procedure for organophosphorous pesticides.
1.3 The detection limit of this method is usually dependent on the level
of interferences rather than instrumental limitations. The limits listed
in Table 1-1 represent the minimum quantity that can be detected with no
interferences present.
Lower limits of detection (LLD) are established by analysts basea
on their experience with the instrumentation and with interferences in
the sample matrix being analyzed. LLD are greater than the instrumental
detection limits in Table 1-1 because they take into account sample inter-
ferences. To estimate LLD, the noise level should be determined in the
retention window for the quantitation mass of representative analytes.
These determinations should be made for at least three field samples in
the sample set under analysis. The signal required to exceed the average
noise level by at least a factor of two should then be estimated. This
signal is the minimum response required to identify a potential signal
for quantification. The LLD is the concentration corresponding to tr,e
level of this signal based on calibrated response factors. Based on best
professional judgment, this LLD would then be applied to samples in the
set with comparable or lower interference. Samples with much higher inter-
ferences (e.g., at least a factor of two higher) should be assigned LLD
at a multiple of the original LLO.
1-2
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These LLO values may be less than the rigorously defined metricc
Tin-its specified in the revised "Guidelines EstaDlishing Test Procedures
for the Analysis cf Pollutants" (£0 CFR Part 136, 10/26/84). This latter
proceaure requires tr.e analysis of seven replicate samples anc a statistical
determination of the method detection limit with 99 percent confidence.
Data quantified between the LLD and the rigorous method detection limit
are valid and useful in environmental investigations of Tow-level contamination,
but have a lower statistical confidence associated witn them than data
quantified aDove the method detection limit.
LLD for the described method on a wet weight basis are 10 ug/kg for
aromatic hydrocaroons and pnthaTates (GC/MS analysis) and 10-20 ug/kg for
chlorinated hydrocarbons and halogenated ethers (GC/MS analysis). Detection
limits for GC/MS analyses of pesticides are 50 ug/kg (wet weignt); the
GC/ECD detection limits for pesticides are 0.1-5 ug/kg. A method cetect^on
limit of 20 ug/kg is attainable for GC/ECD analysis of total PCBs.
1.4 The GC/MS portions of this method are for use only by analysts experienced
with GC/MS or under the close supervision of such qualified persons. Labora-
tories unfamiliar with the analyses of environmental samples by GC/MS should
run the performance tests in reference 1 before beginning.
1.5 This procedure has been designed to analyze for a Targe number of
organic compounds with wide-ranging chemical properties (e.g., polarity,
molecular weight) while minimizing procedural complexity. The accuracy
and sensitivity that could be attained by conducting a dedicated analysis
for only one of the compounds in Table 1-1 cannot be attained in such a
comprehensive analysis.
1.6 Several analytes are particularly susceptible to decomposition during
analysis. Benzidine can be subject to oxidative Tosses during solvent
extraction. Hexachl orocyclopentadiene is subject to thermal decomposition
in the inlet of the gas chromatograph, chemical reaction in acetone solution,
and photochemical decomposition. The polycyclic aromatic hydrocarbons
are also photosensitive, especially benzo(a)pyrene.
1-3
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2.C SUM'-^RY OF METHOD
2.1 A maceratcG hcrrocemzeG 20 to 25 g tissue sar.p'e is Scxnlet extrict = c
end the extract is dried over sodium sjlfate. Biological mecromolecules are
removed from the extract by gel permeation chromatogrepny (GPC) v,-ith 3"'o
Beads S-X3 (or equivalent) (reference 2). A portion of the extract (20 percent;
is subjected to alumina cnromatogrephy to separate polar conpojncs fror,
pesticides end PCBs prior to capillary column GC/ECD analysis (reference 2).
The remaining SO percer.t of the extract is analyzes for acid, base, ana
neutral compounds by capillary column GC/MS. An isotope cilution technique
(EPA Metnod 1625 Revision B, reference 3) is recommended for all compounds
analyzed by GC/MS. This technique involves spiking tne homogenized tissue
sample with the stable isotope labeled analogs of most of the pollutants
to be en^yzec by GC/f-'S. The advantage of isotope cilution is that re" iaDie-
recovery corrections can be made for each analyte with a labeled analog
or a chemically similar analog.
2.1.1 Mjch of the text of EPA Met hoc 1625 Revision 3 has been incorporated
into this method in mocified forn. The modifications were necessary because,
in relation to Methoc 1625 Revision B, the present method involves different
sample matrices (biological tissues), different calibration requirements,
and cGditicnal analytes (pesticides and PCBs, both requiring GC/ECD analysis].
2.2 Identification cf compounds is performed by comparing the GC retention
times and background-corrected characteristic spectral masses with those
of authentic standards. Tentative identifications of low levels of pesticides
and PCEs ere maae by comparing GC retention times to standards. The identities
cf pesticides end PCBs ere confirmed by GC/ECD analysis on an alternative
column phase or by GC/MS when sufficient concentrations occur.
2.3 Quantitative analysis is performed by GC/MS using extracted ion current
profile (EICP) areas. Isotope dilution, with labeled analogs of pollutants
acting as recovery standards, is the method of quantification when labeled
compounds are available. When the isotope dilution technique is usea anc
1-4
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r'v^i C'~ « N^ C* ' i ~ C vv t, / d'l I « " T j ^ v ^> ^ u xJ I VT .. I X^ 'u * -rf i^' M s, ^ . . i» [ ^ . !,' i ^-
C'Onpcuncs Gjantifiea by GC/MS fi^e >"9porteG after correcting for metnoa
recoveries wricn t°.e isotope ai'jfion tecr.nicue is usea.
3.1 Solvents, reagents, glcss^are, ~rz ctr.er sample processing r.arc-.-.ere
nay yield a^ti^acts anc/Dr elevctec case" ir,es, causing -i s intercretat ior,
of cnrcncto:'"c~5 anc suectrj. All materials snou^c ?e cer'icrstratec tc
oe free from interferences unoer tr.e conditions of thie analysis c-y running
netn^G bla^KS initially cnc with each sample lot (Sect. 9.4). Specific
selection of reagents and purification of solvents by distillation in all-
glass systems a^e recuired. Hic^-purity , distil led- in.-gl ess solvents are
corviercTally available (e.g., Bjrdick end Jackson Laboratories, Muskegon,
MI). An effective way of cleaning laboratory glassware is covering with
cluniT'jm foil, r.eating at 450C C fcr severs; hours, and rinsing wit P. polar
ana ncr-cclar solvents oefcre use. Note tnat r.eatin; '.'.itno^t sjssecuent
sc'ven* ri.-sir.g may not eliminate laboratory resiGu-es of PCBs ana certain
otner crlorinateo rydrccarbons.
3.2 Phtralates are common laboratory contaminants that are usea widely
as plasticizers. Phthalates can derive from plastic labware, plastic tubing,
plastic cloves, plastic coated glassware clamps, and have been found as
a contaminant in Na2SQ4. Polytetraf1uoroethylene (PTFE) can be usea instead
of polypropylene or polyethylene to minimize tnis potential source of
contamination. However, use of PTFE labware will not necessarily precluce
all phthalate contamination.
3.3 Interferences coextracted from tissue samples limit the method detection
and quantitation limits. For this reason, sample extract cleanup is necessary
to yield reoroducible and reliable analyses of low level contaminants in
tissue samples.
1-5
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4.0 SAFETY
4.1 The toxicity or care inogen ic i ty of each compound or reagent used in
this method has not been precisely determined. However, each chemical
compound should be treated as a potential health hazard. Exposure to these
compounds should be reduced to the lowest possible level. The laboratory
is responsible for maintaining a current awareness file of OSHA regulations
regarding the safe handling of the chemicals specified in this method.
A reference file of data handling sheets should also be made available
to all personnel involved in these analyses. Additional information on
laboratory safety can be found in references 4-6.
4.2 The following compounds covered by this method have been tentatively
classified as known or suspected human or mammalian carcinogens: benzene,
benzo(a) anthracene, 3,3'-dichlorobenzidine , benzo( a) pyrene , d ibenzo(a ,h) anthra-
cene, N-nitrosod imethyl anrine, 4,4'-DDT, alpha-, beta-, delta-, and gamma-
hexachlorocycl ohexane, and PCBs. Primary standards of these compounds
should be prepared in a hood, and a NIOSH/MESA approved toxic gas respirator
should be worn when high concentrations are handled.
5.0 APPARATUS AND EQUIPMENT
5.1 Soxhlet Extractor - 50 ml extractor (Corning 3740-S, or equivalent),
with 250 ml flask (Corning 4320-250, or equivalent) and condenser with
34/45 joint. Cellulose thimbles of the appropriate size should be cleaned
with the extraction solvent mixture for at least 30 cycles.
5.2 Drying Column - 30 cm x 2 on borosilicate glass chromatography column
with glass wool plug. Glass wool is extracted with the appropriate solvents
and allowed to dry before use.
1-6
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5.3 Kutierna-Danish (K-D) Apparatus -
5.3.1 Concentrator tuDe - 10 ml, graduated (Kontes K-570050-1G25,
or equivalent. A ground glass stopper (19/22 joint) is used to prevent
evaporation of extracts.
5.3.2 Evaporation flask - 500 ml (Kontes K-570050-0500, or equivalent),
attached to concentrator tube with springs (Kontes K-662750-0012).
5.3.3 Snyder column - three ball macro (Kontes K-503000-0232, or
equivalent).
5.3.4 Snyder column - two ball micro (Kontes K-469002-0219, or equiva-
lent).
5.3.5 Silicon carbide boiling chips - approximately 10/40 mesh, extracted
with methylene chloride and heated at 4500 C for 1 h minimum. Note that
boiling chips can be a significant source of contamination if not properly
cleaned.
5.4 Separatory Funnel - 500 ml, borosilicate glass with PTFE stopcock.
5.5 Borosilicate Glass Beaker - 400 ml and 100 ml.
5.6 Water Bath - heated, with concentric ring cover, capable of temper-
ature control (+2° C) , installed in a fume hood.
5.7 Sample Vials - amber glass, 2-5 ml with PTFE-lined screw cap.
5.8 Analytical Balance - capable of weighing 0.1 mg.
5.9 Micro-grinder (e.g., Tekmar Tissuemizer, Tekmar Company, Cincinnati, OH).
1-7
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5.10 Nitrogen evaporation cevice equipped «itri a water Datn tnet can z±
naintained at 35-40° C. Tr.e N-Evap by Organonation Associates, Inc., ScJtn
Berlin, XA is suitable.
5.11 Balance - capable of 100 g to tne nearest 0.01 g.
5.12 Disposable Pasteur Pipets - sealed with aluminum foil and annealed
at 450° C fcr several h, and rinsed witn solvents before use.
5.13 Drying Oven.
5.14 Annealing Oven - capable of reaching 45QO C.
5.15 Dessicator.
5.16 Chromatograpny Column for Alumina - 5 ml disposable Dorosilicate
glass serological pipet with borosilicate glass wool plug. (Glass wool
must be extracted with the appropriate solvents and allowed to dry oefore
use.)
5.17 Gel Permeation Chromatography Cleanup Device -
5.17.1 Automated system: gel permeation chromatograph (GPC) , e.g.,
Analytical Biochemical Labs, Inc. GPC Autoprep 1002, including
25 mm ID x 600-700 mm glass column packed with 70 g of Bio
Beads S-X3.
Syringe, 10 ml with Luer Lok fitting.
Syringe Filter Holder and Fitters - stainless steel and
TFE, Gelman 4310 or equivalent.
5.17.2 Manual system assembled from parts (Wise, R.H., D.F. Bishop,
R.I. Williams, and B.M. Austern. Gel permeation chroniatography in tne
1-8
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/MS ana
ysis of organ -ics in sieges. U.S. EP^., K-niciscl Err, ire ".rental
Researcn Laboratory, Cincinnati, CH. ^5268) (see reference 2, p. D-25).
5.18 Gas Chronatog raph - 1) one equipped with electron capture detector
(ECO) and 2) one interfaced to trie mass spectrometer (Sect. 5.19). Both
snould have splitless injection ports for capillary column, temperature
programs with 30° C hold, and should meet all the performance specifications
in Sect. 9.9.
5.18.1 Column - 30^5 n x 0. 25 +_ 0.02 rrm i .d . 5 percent pnen yl , 9-1 percent
methyl, 1 percent vinyl silicone bonded phase (0.25 in film thickness) fused
silica capillary column (J & W D8-5, or equivalent).
5.19 Mass Spectrometer - 70 eV electron impact ionization, should repeatedly
scan fron 35 to 450 a^u in 0.95 to 1.00 second and snould produce a unit
resolution (valleys between m/z 441-442 less than 10 percent of the height
of the 441 peak) , background-corrected mass spectrum from 20 ng decaf luorotri-
phenyl phosphine (DFTPP) introduced through the GC inlet. The spectrum
shoulc meet the mass- inten si ty criteria in Table 1-2 (reference 7). The
mass spectrometer should be coupled with the GC such that the end of the
capillary column terminates within one centimeter of the ion source but
does net intercept the electron or ion beans. All portions of the column
which connect the GC to the ion source should remain at or above the column
temperature during analysis to preclude condensation of less volatile compounds.
5.20 Data System - should collect and record MS data, store mass intensity
data in spectral libraries, process GC/MS data, generate reports, and should
compute and record response factors.
5.20.1 Data acquisition - mass spectra should be collected continuously
throughout the analysis and stored on a mass storage device.
5.20.2 Mass spectral libraries - user created libraries containing
mass spectra obtained from analysis of authentic standards should be employed
to reverse search GC/MS runs for the compounds of interest (Sect. 8.2).
1-9
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5.20.3 Data processing - the data system should be used to sesrcr ,
locate, identify, and quantify the compounds of interest in each GC/'-'S
analysis. Software routines should be employed to compute retention times
and peak areas. Displays of spectra, mass chromatograms, and library compar-
isons are required to verify results.
5.20.4 Response factors and multipoint calibrations - the data system
should be used to record and maintain lists of response factors (response
ratios for isotope dilution) and multipoint calibration curves (Sect. £;.
Computations of relative standard deviation (coefficient of variation)
are useful for testing calibration linearity.
6.0 REAGENTS AND CONSUMABLE MATERIALS (partially adapted from references
2 and 3)
6.1 Reagents
6.1.1 Acetic acid, acetone, benzene, n_-hexane, isooctane, rnethanol ,
and methylene chloride (CH2Cl2) (pesticide quality, distiHed-in-glass).
6.1.2 Alumina - neutral, super Woelm or equivalent (Universal Scientific,
Atlanta, GA). Extract alumina with methylene chloride for 30-40 cycles
in a Soxhlet extractor to remove contamination. Allow solvent to evaporate.
Prepare activity III alumina by adding 7 percent (v/w) reagent water to
neutral alumina that has been activated at approximately 225° C for at
least 2 h or preferably overnight. Store in tightly sealed, clean glass
container.
6.1.3 Hydrochloric acid - concentrated, make 2N HC1 with reagent
water. Solvent clean in a separatory funnel with methylene chloride.
6.1.4 Potassium hydroxide - reagent grade, 5N in reagent water.
Solvent clean in a separatory funnel with methylene chloride.
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6.1.5 Sodium sulfete - reagent grade, granular anhydrous, rinsed
with Ch^Cl? (20 mL/g) and conditioned at 450° C for 1 h minimum.
6.1.6 Reagent weter - water in whicn the compounds of interest en.c
interfering compounds are not detected by this method.
6.2 GPC Calioration Solutions:
6.2.1 Corn oil - 200 mg/mL in CH2C12-
6.2.2 Bis(2-ethylhexyl)phthslate and pentachlorophenol - 4 mg/mL
in CH2Cl2.
6.3 Stock Standard Solutions - purchased as solutions or mixtures with
certification to their purity, concentration, and authenticity, or prepared
from materials of known purity and composition. If the compound purity
is 96 percent or greater, the weight may be used without correction to
compute the concentration of the standard. When not being used, standards
are stored in the dark at -20 to -10° C in screwcapped vials with PTFE-
linec lids. A mark is placed on the vial at the level of the solution
so that solvent evaporation loss can be detected. The vials are brought
to roan temperature prior to use. Any precipitate is redissolved and solvent
is added if solvent loss has occurred.
6.3.1 Preparation of stock solutions - prepare in methylene chloride,
benzene, isooctane, or a mixture of these solvents according to the steps
below. Observe the safety precautions given in Sect. 4. The large number
of labeled and unlabeled acid and base/neutral compounds used for combined
calibration (Sect. 8) and calibration verification (9.9.1.3) require high
concentrations (approximately 40 mg/mL) when individual stock solutions
are prepared, so that dilutions of mixtures will permit calibration with
all compounds in a single set of solutions. The working range for most
compounds is 1-50 ug/mL. Compounds with a reduced MS response may be prepared
at higher concentrations.
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Siancarcs for GC/ECD nave lower .vorking ranges (e.g., 0.04 to 2.G jc.<~i_
for single component pesticides) than GC/i-'S standards. Ho^e.-er, GC/'E-D
stock solutions sr.C'jia De preoerec ,-,itn 51 least 10 rg of tr.e pure r-.2-.er~i"
(e.g., in iO ~L of solvent) 10 reduce potential weighing er^or.
6.3.2 Dissolve an appropriate amount of assayed reference material
in a suitable solvent. For example, weigh 400 mg naphtha! ene in a 10 rrL
ground glass stoppered volumetric flask and fill to the mark with benzene.
After the naphthalene is completely dissolved, transfer the solution to
a 15 ml vial with PTFE-lined cap.
6.3.3 Stock standard solutions should be checked for signs of degradation
prior to the preparation of calibration or performance test stancarcs.
Quality control check samples that can be used to determine the accuracy
of calibration stancarcs are available from the U.S. Environmental Protection
Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio
45268.
6.3.4 Stock stancaro solutions should be replaced after 6 no, or
sooner if comparison with quality control check samples indicates a cr.ange
in concentration.
6.4 Injection Internal Standard Solutions
6.4.1 GC/MS internal standard solution - prepare 2,2'-difluoroDiDhenyi
(DFB) at a concentration of 2 mg/mL in benzene.
6.4.2 GC/ECD internal standard solution - prepare decafluorobenzo-
phenone (DF8P) at a concentration of 2.5 ug/mL in isooctane.
6.5 GC/MS Secondary Dilution Standards - using stocx solutions (Sect.
6.3), prepare a secondary standard containing each of the unlabeled priority
pollutants in Table 1-1 at a concentration of 100 ug/mL, or at a higher
concentration appropriate to the MS response of the compounds.
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b.6 Laoe'ea CornDOunG SDiKing So'uticn - prepare a spiking sc1 -tier frc~
stock star.carc solutions prepared es ir Sec*. 6.3, or from mixtures, at
a ccncar.tr a:i en of iCO ug/rrL, or at a concentration appropriate to t'r.e
MS response of each compound. The deuterium and 1->C-1 aDel eu conpourics
listed In Taole 1-1 are commercially available individually or as r.-ixtures
(e.g., :-,erck Sharp i Dohrne/Isotcpes, Montreal, Canada),
6.7 Solutions for obtaining authentic mass spectra (Sect. 8.2) - preparc-
;nixtures of labeled ana unlaoeled compounds at concentrations tnat will
assure that authentic spectra are obtained for storage in libraries.
6.8 Calibration Solutions - the concentrations of calibration solutions
suggested in the following sections are intended to bracket concentrations
that will be encountered curing sample analysis without overloading GC
columns or saturating detection systems.
6.8.1 GC/MS calibration solutions - comoine 0.1 ml of the spiking
solution (Sect. 6.6) with 10, 50, 100, 200, ana 500 uL of the secondary
dilution solution (Sect. 6.5) and bring to 1.00 ml total volume each.
This v.'ill produce calibration solutions of nominal 1, 5, 10, 20, and 50 ug/nL
of the pollutants end a constant nominal 10 ug/mL of the labele:: corcpouncs.
Spike each solution with 10 uL of the GC/MS internal standard solution,
yielding 20 ug/nL.
6.8.2 PCS calibration solutions -
6.8.2.1 Aroclor stock solution for GC/MS - prepare a solution
in hexane with 250 ng/uL of each of three PCB mixtures, Aroclor 1016, Aroclor
1254, and Aroclor 1260.
6.S.2.2 Aroclor standard solution for GC/ECO - dilute the stock
solution (Sect. 6.8.2.1) to one-tenth its original concentration. It is
essential that this solution be prepared directly from the batch used for
Sect. 6.8.2.1. Combine 20, 50, 250, 500, and 1,000 uL of the diluted standard
1-13
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the GC/ECD intGrr.a*. s: = r.c = rc s:-l-ticn arc Dr'r-g eacr so".,:::"
to a fine' volume cf 5.0 r.L.
This will proauce calibration solutions of nominel concentrations
of 100, 250, 1,253, 2,5CC, ana 5,000 ng/nL cf the i:l:l Aroclor mixture
and a constant nominal concentration of 50 ng/rr.L of internal standard.
C.S.3 Pesticide calioration solution - combine 20 uL of the GC/ECD
internal standard solution with 2, 5, 10, 50, and 100 uL of a 20 ug/r,L
stock solution cf all chlorinated pesticides listed in Table 1-1 (excect
toxapnene) and bring to a 1.0 ml total volume. This will produce calibration
solutions of 40, 100, 200, 1,000, and 2,OCO ng/mL of eacn pesticice arc
a constant internal stancara concentration of 50 ng/mL.
6.8.4 Tcxapnene calibration solution - prepare toxephene solutions
of 100, 250, 1,250, 2,500, and 5,000 ng/mL with constant internal standard
concentration of 50 ng/mL.
6.8.5 DFTPP solution - prepare at 20 ug/mL in acetone.
6.9 Stability of Solutions - all standard solutions (Sect, 6.4-6.8.4)
should be analyzed within 43 h of preparation and on a monthly basis thereafter
for signs of degradation. Standards will remain acceptable if response
factors relative to the internal standard correspond within +_! 5 percent
to those obtained in the initial analysis of the standard.
7.0 SAMPLE COLLECTION, PREPARATION, AND STORAGE
7.1 In the field, sources of contamination include sampling gear, grease
from ship winches or cables, ship engine exhaust, dust, and ice used for
cooling. Efforts should be made to minimize handling and to avoid sources
of contamination. This will usually require that resection (i.e., surgical
removal) cf tissue be performed in a controlled environment (e.g., a labora-
tory). For example, to avoid contamination from ice, the samples snould
be wrapped in aluminum foil, placed in watertight plastic bags, and immediate!y
1-14
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cooled In a covered ice cnest. Organises snculc not be frozen prior to
resection if analyses will only be conducted on selected tissues (e.g.,
internal organs), because freezing may cause internal organs to rupture
and contaminate other tissue. If organisms are eviscerated on board the
survey vessel, the remaining tissue may be wrapped as described above ana
frozen.
7.2 To avoid cross-contamination, all equipment used in sample handling
should be thoroughly cleaned before each sample is processed. All instruments
must be of a material that can be easily cleaned (e.g., stainless steel,
anodized alur.inun, or borosilicate glass). Before the next sample is processed,
instruments snould be washed with a detergent solution, rinsed with tap
water, soaked in high-purity acetone and methylene chloride, and finally
rinsed with reagent water. Work surfaces should be cleaned with 95 percent
ethanol and allowed to dry completely.
7.3 Resection should be carried out by or under the supervision of a competent
biologist. Each organism should be handled with clean stainless steel
or quartz instruments (except for external surfaces). The specimens should
come into contact with precleaned glass surfaces only. Polypropylene and
polyethylene surfaces are a potential source of contamination and should
not be used. To control contamination when resecting tissue, separate
sets of utensils should be used for removing outer tissue and for resecting
tissue for analysis. For fish samples, special care must be taken to avoid
contaminating target tissues (especial ly muscle) with slime and/or adhering
sediment from the fish exterior (skin) during resection. The incision
"troughs" are subject to such contamination; thus, they should not be included
in the sample. In the case of muscle, a "core" of tissue is taken from
within the c"ea boarded by the incision troughs, without contacting then.
Unless specifically sought as a sample, the dark muscle tissue that may
exist in the vicinity of the lateral line should not be mixed with the
light muscle tissue that constitutes the rest of the muscle tissue mass.
7.4 The resected tissue sample should be placed in a clean glass or TFE
container which has been washed with detergent, rinsed twice with tap water,
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rinsea once -with distilled water, rinsed witn acetone, end, finally, rinses
with hign-purity nethylene chloride. Firing of the glass jar at 450° C
may be substituted for the final solvent rinse, but precautions must be
taken to avoid contamination as the container is cooled after drying.
7.5 The U.S. EPA and other federal agencies (e.g., National Bureau of
Standards) have not yet provided specific guidance regarding holding tines
and temperatures for tissue samples to be analyzed for seni-volatile organic
compounds. Until U.S. EPA develops definitive guidance, the following
holding conditions should be observed. Resected tissue samples should be
maintained at -20° C and extracted as soon as possible, but witnin ID days
of sample receipt. Complete analyses should be performed within 40 days.
These holding times are based on the Contract Laboratory Program requirements
for sediments (reference 10). Liquid associated with the tissue sample
ttust De maintained as part of trie sample (the liquid will contain lipic
material).
8.0 CALIBRATION AND STANDARDIZATION (adapted from reference 2)
8.1 Estaolish the GC/MS operating conditions in Table 1-1. Analyze stancards
per the procedure in Sect. 10.2 to demonstrate that the analytical system
meets tne detection limits in Table 1-1 and the mass-intensity criteria
in Table 1-2 for 20 ng DFTPP.
8.2 Mass Spectral Libraries - detection and identification of compounds
of interest are dependent upon spectra stored in user created libraries.
8.2.1 Obtain a mass spectrum of each pollutant, labeled compound,
end the internal standard by analyzing an authentic standard either singly
or as part of a mixture in which there is not interference between closely
eluting components. Confirmation that only a single compound is present
is attained by examination of the spectrum. Fragments not attributable
to the compound under study indicate the presence of an interfering compound.
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8.2.2 Adjust the analytical conditions and scan rate (for tnis test
only) to produce an undi storied spectrum at GC peak maximum. An undis-
torted spectrum will be oDtained if five complete spectra are collected
across tr.e upper half of the GC peak. Software algorithms designer to
"enhance" the spectrum may eliminate distortion, but may also eliminate
authentic masses or introduce other distortion.
8.2.3 The authentic reference spectrum is obtained under DFTPP tuning
conditions (Sect. 8.1 and Table 1-2) to normalize it to spectra from other
i n s t r u.i en t s .
8.2.4 The spectrum is edited for entry in the library by saving the
five most intense mass spectral peaks and all other mass spectral peaks
greater than 10 percent of the base peak. This edited spectrum is stored
for reverse searcn ana for compound confirmation.
8.3 Polar Compound Detection - demonstrate that unlabeled pentachlorophenol
and benzldine are detectable at the 10 ug/mL level (per all criteria in
Sect. 10.4). The 10 ug/mL calibration standard (Sect. 6.8.1) can be used
to demonstrate this performance.
8.4 Calibration with the isotope dilution technique - isotope dilution
technique is used when labeled compounds are available and interferences
do not preclude its use. If either of these conditions precludes isotope
dilution, the internal standard method (Sect. 8.5) is used and noted as
such in the report.
8.4.1 A calibration curve encompassing the concentration range is
prepared for each compound determined. The relative response (pollutant
to labeled) vs. concentration in standard solutions is plotted or computed
using a linear regression. The example in Figure 1-1 shows a calibration
curve for an unlabeled compound and its labeled analog. Also shown are
the ^10 percent error limits (dotted lines). Relative Response (RR) is
determined according to the procedures described below. A minimum of five
data points are employed for calibration.
1-17
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3.4.2 The relative response of a pollutant to its labeled analog
is determined from isotope ratio values conputed from acquired data. Three
isotope ratios are usea in tnis process:
RX = the isotope ratio measured for the pure pollutant
Ry = the isotope ratio measured for the labeled compound
RTC = the isotope ratio of an analytical mixture of pollutant and labeled
compounds.
The m/zs are selected such that Rx>Ry. If Fh is not oetween 2 Ry and 0.5RX,
the method does not apply and the sample is analyzed by the internal standard
method (Sect. 8.5).
8.4.3 Capillary colors usually separate the pel 1 utant- labe- ed pair,
with the labeled conpound eluting first (Figure 1-2). For this case,
Rx = (area mi/z)/l
at the retention tirce of the pollutant (RT2) and
Ry = l/(area m2/z)
at the retention time of the labeled compound (RT^). Also,
Rm = [area mi/z (at RT2)]/[area m2/z (at RTi)]
as measured in the mixture of the pollutant and labeled compounds (Figure 1-2),
and RR = Rm.
8.4.4 Special precautions are taken when the pollutant and its labeled
analog are not chromatographically separated and have overlapping spectra,
or when another labeled compound with interfering spectral masses overlaps
the pollutant (a case that can occur with isomeric compounds). In such
cases, it is necessary to determine the respective contributions of the
1-18
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pollutant and labeled compounds tc the respective EICP areas. If the pea.
-------
no label 53 analog, and to measure labeled compouncs for iritral cDcra^o1' >
statistics (Sect. 9.5.1) .
8.5.1 Response factors - calioration requires the determination of
response factors (RF) ^hich are defined by the following equation:
RF = (As x Cis)/(Ais x Cs)
where:
AS = the area of the target peak in the daily stancard
ATS = the area of the internal standard peak
Cis = the concentration of the internal standard (ug/mL)
Cs = the concentration of the compound in the daily standard (jg/nL).
8.5.1.1 The response factor is determined over the range of
concentrations described in Sect. 6.8.1, 6.8.2, 6.8.3, and 6.8.4. The
amount of internal standard added to each extract is the same so that C-;s
remains constant. The RF is plotted versus concentration for each compound
{or class of compounds in the case of toxaphene) in the standard (Cs) to
produce a calibration curve.
8.5.1.2 Linearity - if the response factor (RF) for any compound
is constant (less than 35 percent coefficient of variation) over the calibration
range, an averaged response factor may be used for that compound; otherwise,
the complete calibration curve for that compound should be used over the
range.
8.6 Combined Calibration - by using calibration solutions (Sect. 6.8.1)
containing the pollutants, labeled compounds, and the internal standard,
a single set of analyses can be used to produce calibration curves for
the isotope dilution and internal standard methods. These curves are verified
each shift by analyzing the 10 ug/mL calibration standard (Sect. 9.9.1).
Pesticide and PCB calibration standards must be analyzed separately by
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r. D ' " ^fj-ar-j: cAnno* b ~ r-r
£.7 Ongoing CiV:orafion (see Stct. 9.9)
9.0 CL'ALITY ASSURANCE/QUALITY CONTROL Tor further g uicance, see Quality
Ass Jrar.c e/Qual ity Control (QA/QC) for 301 (n) Monitoring Programs: Guicancc-
on Field ana Laooratory Methods (Tetra Tech 19S6).j
9.1 Eacn laoorstcrv tr.at uses znis :r,etncc is require- to operate 2 ~cr~;".
quality assurance progra.r. The requirements of this program consist cf
an initial demonstration of laboratory capability, analyses of replicates
ar.a n.atrix spikes usea to e/aKete ana cocument aate quality, ana analysis
of stanaarcs ana blanks usea to test continued performance.
9.2 Initial Demonstration of GC/MS Capability - the analyst should make
an initial demonstration of the ability to generate acceptaole accuracy
and precision witn the GC/MS component of this method. This ability is
estaolishea as describee in reference i.
9.3 The analyst is permitted to modify this method to improve separations
or lo,-/er the costs of measurements, provided that the new met nod is aenonstratea
to perform comparably to the present method (i.e., with comparable spike
recoveries ana precision).
9.4 Blanks - method blanks snould be analyzed by GC/MS and GC/ECD to demon-
strate freedom from contamination.
9.4.1 At least one method blank must be induced with eacn batch cf sam-
ples; method blanks must constitute at least 5 percent of all samples analyzed.
9.4.2 Method blank concentrations of compounds of interest and cf
potentially interfering compounds should be less than 5 percent of the
expected values for the corresponding analytes in samples anc below trie
LLD, if possible. It is recommended that if blank concentrations of compounds
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cf interest fc-xce" "ntr;^ 1 ates; -'- creator tr.an 1C percent cf tr.e ccrreszcr.; :r,;
anslyte conccntrat icns in samples, sa^p'ie analysis snoulc ce ra'tea ^r. r-i'
lie contamination sojrce is Gnnir-£t=G.
9.5 S^ircec S3i."."";-:-s ='"£ rec-'irea to assess metnoc performance on tne sa~^'i-
matr ix .
9.5.1 For samples analyzed by isotope dilution, the percent recovery
(p) of lebelec conpouncs can be computed by the internal standard met roc.
(Sect. £.5; a°c serves as an indication of analytical accuracy (cut r:-t
necessarily of extraction efficiency). After the analysis of five samples,
compute the average percent recovery (P) and the standard Deviation of
the percent recovery ^s->) for the laoeled ccrrpouncs only. Express ir-.e
accuracy assessment as a percent recovery interval from P - 2sp to P + 2sp.
For example, if P = 90 percent and sp = 10 percent, the accuracy interval
is expressed as 70-110 percent. Upcate the accuracy assessment for each
conpojna en a regular basis (e.g., after each 5-10 new accuracy mea
9.5.2 Laboratories unaole to use isotope dilution must analyze matrix
spikes of pollutants (other than pesticides and PCBs) at a frequency cf
5 percent of all samples analyzed or once with each sample set, whicneve'-
is more frequent. Compounds should be added at concentrations one to five
times those in the sample.
9.5.3 All laboratories are recuired to spike samples with PCBs and/or
pesticiaes at a frequency of 5 percent of all samples analyzed or once
per sample set, whichever is more frequent. The spike can be a standard
pesticide mixture or an Aroclor mixture, whichever is considered to be
more representative of the sample. The mixture should be added at one
to five times the sample concentration of these compounds.
9.6 Replicates (i.e., analyses of at least two separate aliquots from
a tissue homogenate) must be analyzed by GC/MS and GC/ECD to monitor the
precision of laboratory analyses. At a minimum, 5 percent of the analyses
should be laboratory replicates. A triplicate analysis should be performed
with each sample batch of over 40 samples.
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9.7 The Icooratory snoulc maintain recoras to aefine tne quality cf csta
tnat is generates. These records induce documentation of Panics arc resorts
cf IcDel-EG compouna recovery (5e:t. 5.5.ij, if tre latter is app"ic=o"e.
9.£ Tne laooratory snoulc, on an ongoing oasis, cerncnstrate tnrougn cal iora tic-"
verification that tne analysis system is in control (Sect. 9.9.1.3).
9.9 System and Laboratory Performance
9.9.1 At tne Beginning anc enc of eacn 12-h srnft during w
are performed, GC/I-'S system performance and calibration are verifiec
all pollutants and labeled compour.cs. For these tests, analysis cf tre
10 uc/mL calibration standards (Sect. 6.8.1) should be used to verify all
performance criteria. The GC/ECD performance is checkea at the beginning
anc enc cf each shift or at least every 6 h by analyses of 250 ana 100 ug/mL
solutions of the PC3 and pesticide stancarcs (Sect. 6.8.2.Z anc 6.6.2).
9.9.1.1 Retention times - the absolute GC/MS retention time
of 2,2'-difluorobipheny 1 should be within the range of 1078 to 1248 sec.
Tne aosoiute GC/ECG retention time of 4,4'-DDT should be within the range
cf 1C-50 and 1200 sec.
9.9.1.2 GC resolution fcr GC/MS analysis - the valley neight
between anthracene and phenanthrene at m/z 178 {or the analogs at m/z 188}
should not exceed 10 percent of the taller of the two peaks.
GC resolution for GC/ECD analysis - the valley height between two
peaks should not exceed 25 percent of the taller of the two peaks for the
following pairs: beta- and delta-HCH, dieldrin and 4,4'-DDE, 4,4'-DDD
and endrin aldehyde, and endosulfan sulfate and 4,4'-DDT.
9.9.1.3 Ongoing calibration verification - conpute the concentration
of each pollutant (Table 1-1) by isotope dilution (Sect. 8.4) for those com-
pounds tnat have labeled analogs. Compute the concentration of each pollutant
1-23
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:' s s no 1 e D e "; e G & n a } c j v, i * ,-, t r, e n e a r e s ; e "i u t i P. g 1 a o e 1 c c s t a r c a
Commute :ne concentration cf the U^eleci compouncs Dy the internal star.carc
r~5t"oc. -'-.150 COT. DJte inc: i .' icje'i :e st "' c ice- concer tr=tiors c tct= 1 22E
snc i ox a 3 n. -"e concentrations oy the interne! standard ~ethoc (GC''EC3-).
Tnese cere en -ra 1 1 or-s are carpu-ec casec cr, the cal iSraticn cate je'e-'rir.s-
in Sect. S. Preparations of new calibration standards or revisions of
calioration curves ere required if observed responses cf analytes vary
from predicted responses Dy more than +_20 percent. Samples and blanks
may be run only after calibration performance meets this control li-rit.
9.9.1.4 Multiple peaks - escn ccmpcjnd injeciec shou'c give
a single, distinct GC peak.
9.9.2 DFTPP spectrum validity - inject 1 uL of the DFTPP solution
(Sect. 6.8.5 jither separately cr within a few seconcs of injection of
the standard (Sect. 9.9.1) analyzed at the beginning of eacn shift. The
criteria in Table 1-2 should be met.
10.0 PROCEDURE (see Figure 1-4)
10.1 Sample Processing and Extraction
10.1.1 Mince tissue sample with a scalpel and homogenize tne sanole
to a uniform consistency with a micro-grinder. Care must be taken to ensure
that the micro-grinder is thoroughly cleaned after each use. This usually
entails disassembly of the unit. Devices with large surface areas (e.g.,
blenders, meat grinders) should be avoided, as they are difficult to clean
ana a small sample is difficult to remove after grinding.
10.1.2 Dry weight determination - if sample size permits and dry
weight concentrations are required, dry weight determinations may be performed
as follows: transfer an aliquot of approximately 3 g (weighed to the nearest
0.1 g) to a preweighed dish. Allow the sample to dry in a 1050 c oven
overnight, and determine the solid residue weight (to the nearest 0.1 c) .
Calculate ana report the percent solids (Ts) as:
1-24
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Ts = [dry residue wt (g)]/~wet sample wt (g}[ x 100^
10.1.3 Weigh a 20 to 25-g (wet weight) tissue aliquot to the nearest
0.1 g. Add any excess liquid from the sample directly to the round bottci
flas< to be used for Soxhlet extraction; this will help to avoid saturating
the cellulose extraction thimble with tissue fluids, which can lessen extraction
efficiency. Also add approximately 50 ml methanol and 140 ml CH2C12 to
the flask.
Spike the tissue aliquot with 10 ug of each stable isotope labeled
base/neutral compound and 15 ug of each labeled acid compound. Mix the
spiked, homogenized tissue sample with approximately 30 g precleaned Na2$04
in a beaker to improve the texture of the sample. Place the mixture into
a precleaned Soxhlet thimble for extraction. Add 20 ml of methanol directly
to the thimble and stir to enhance the removal of water from the sample.
Cover the sample with a thin layer of solvent-cleaned glass wool.
10.1.3.1 An as yet unvalidated procedure may enhance extraction
efficiency for phenolic materials. This procedure entails adding 20 ml
of acetic acid directly to the thimble (rather than methanol, as specified
in 1C.1.3). The purpose of adding acetic acid is to reduce the extraction
pH and thus to increase the solvent solubility of phenols. Acetic acid,
1 ike methanol , would facilitate water removal. The acetic acid step is
recommended only if laboratory validation data is made available.
10.1.4 Soxhlet extract the ti ssue/Na2S04 mixture with
(2/1) for 24 h (60-90 cycles). Stir the sample in the thimble at least
twice (after the first cycle and after approximately 12 h) to prevent solvent
channeling (replace the glass wool cover). The Soxhlet apparatus should
be wrapped up to the condenser with aluminum foil to ensure even heating
during cycl ing.
1-25
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10.1.6 Liquid-1iquid extraction -
10.1.6.1 After Soxhlet extraction, transfer me extrac* to a
500 ml separatory funnel. Rinse the Soxhlet flask with clean extraction
solvent anc add this rinse to the extract in trie separatory funnel. Wash
the solvent extract with approximately 100 ml of pH 2, 50 percent saturated
Na2S04 organic-free water; the pH of the water should be adjusted with
solvent-cleaned HC1. Collect and store the CH2Cl2 layer. The purpose
of washing the extract with an acidic, aqueous solution is to remove water
and nethanol from the C^Cl? and :o enhance the partitioning of acidic,
organic compounds into the CH2C12 layer. Re-extract the acidic, aqueous
phase twice with 60 ml of clean CH2C12 and add both extracts to the initial
CH2Cl2 fraction.
10.1,6.2 Adjust the pH of the aqueous phase to >_12 with solvent-
cleaned, 6 N KOH. Back-extract the base compounds three times with 60 ml
CH2C12- The pH adjustment to alkaline conditions ehances the partitioning
of basic compounds into the CH2Cl2 layer. Combine all CH2C12 layers from
Sect. 10.1.6.1 and 10.1.6.2.
10.1.6.3 Formations of emulsions or precipitates during liquid-
liquid extraction should be noted and considered when reviewing results.
The addition of N32S04 may reduce emulsions. However, if the emulsion
interface between layers is more than one-third the volume of the solvent
layer, the analyst must emloy mechanical techniques to complete the phase
separation. The optimum technique depends on the sample and may include
stirring, filtration of the emulsion through pre-cleaned glass wool, centrifu-
gation, or other physical methods (reference 2).
10.1,7 Dry the total combined solvent extract by pouring it through
an anhydrous Na2S04 drying column (approximately 30 cm x 2 on). Use approxi-
mately 30 ml of CH2C12 to rinse the drying column and combine this with
the dried extract. Collect the extract in a Kuderna-Danish (K-D) concentrator
containing 1 to 2 clean boiling chips.
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10.1.8 Attacn a 3-oall nacro Snyaer column to the K-D concentrator
and concentrate the extract on an 80° C water bath. Place tr.e K-D apparatus
on the hot water oath so that the concentrator tune is partially immersed
in the hot water and the entire lower rounded surface of the flask is bathed
with hot vapor. At the proper rate of distillation, the balls of the column
will actively chatter but the chambers will not flood with condensed solvent.
When the apparent volume reaches 5 ml, remove the K-D apparatus from the
water bath and rinse the flask with 3 ml CH2C12 draining into the concentrator
tube. Reduce the contents of the concentrator tube to 3 ml using a stream
of purified N2 gas, never allowing tre extract to go to cryness.
10.1.9 Alternative methods of extract concentration may be used if
evidence of acceptaole performance [i.e., retention of more volatile compounds
(e.g., naphthalene) comparable to that of K-D concentration] is provided.
10.1.10 Accurately measure the extract volume, Vj, remove a small
aliquot ( 100 uL) and apply directly to a preweighed microbalance weighing
boat. Allow the solvent to evaporate on a hotplate at low temperature
(less than 40° C). Quickly remove the boat when it is dry and weigh the
residue and boat to the nearest 0.1 mg. Calculate the total extracted
residue, R-r, as:
*T (mg} - [Vt (ml) x W (mg) x 103]/Va (uL)
where:
Vy = the extract volume ( 3 ml) in mL
Va = the aliquot volume transferred to the weighing boat in uL
W = the residue weight in mg as determined with the analytical micro-
balance.
Report the total extracted residue as Rj (mg)/sample wet weight (g).
10.1.11 Extract cleanup - GPC cleanup is required to separate biological
macromolecules from the analytes.
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10.1.11.1 GPC set-JD and calibration (reference 2).
10.1.11.1.1 Place 70 g of Bio Beads S-X3 in a 400-mL beaker.
Cover the beads with Cr^Cl?. Allow the beads to swell overnight. Transfer
the swelled beads to the column and start pumping solvent through the column,
from bottom to top, at 5 ml/min . After 1 h, adjust the pressure on the
column to 7-10 psi and pump for an additional 4 h to remove air from the
column. Adjust the colunn pressure as required to maintain 7-10 psi.
10,1.11.1.2 Calibration of the column - Load 5 ml of tne
corn oil solution into sample loop No. 1 and 5 mL of the phthalate-PCP
solution into loop Ho. 2. Inject the corn oil solution and collect 10 rnL
fractions for 36 min. Determine the corn oil elution pattern by evaporation
of each fraction to dryness followed by a gravimetric determination of
the residue. Analyze the phthalate PCP fractions by GC/FID, a UV-spectro-
photometer at 254 nm, or a GC/MS system. Plot the concentration cf each
component in eacn fraction versus total el uant volume. Choose a "durnptirne"
that allows >_85 percent removal of the corn oil and ^_85 percent recovery
of the phthalate. Select the "collect time" to extend at least 10 min
after the elution of pentachloropienol . Wash the column at least 15 min
between samples. Typical parameters are: dumptime, 30 min (150 ml); collect
time, 36 min (ISO ml); and wash time, 15 min (75 ml). The S-X3 Bio Beads
column may be reused for several months, but should be checked by frequent
system recalibration for every 20 extracts loaded onto the GPC.
10.1.11.2 Extract cleanup - Prefilter or load all extracts via
the filter holder to avoid particulates that might cause system blockage.
Load the 3-mL extract onto the GPC column. Do not apply excessive pressure
when loading the GPC. Purge the sample loading tubing thoroughly with
solvent between extracts. Process the extracts using the dump, collect,
and wash parameters as selected from the calibration and collect the cleaned
extracts in 400-mL beakers.
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ar.c recalibrate the system ones for every 20 extracts loaaec onto tr:e GDC.
Tr.s: recoveries and eiutien profiles are reocrtec as del 1 veraol es.
10.1.12 Transfer tr.e extract to e Kucernc-Oonisr, (<-D) ccr,: entratc-r
consisting of a 10-mL concentrator tube, a 500-mL evaporative flasK, boiling
cnips, and a Snycer column. Carefully concentrate the extract to 2.5 nL
using metnocs previously cescribec and the f<2 gas Dl Givccwn technique.
Nitrogsn blowdown should be performed at approximately 353 C. A gentle
stream of clean, cry N2 (filterec through a column cf activated carocn;
should oe used. The ir.side walls cf the tube containing the extract shculc
DC- rinsed ccwn with trie appropriate solvent several tires Curing concentration.
The extract must not be allowed to go to dryness.
10.1.13 Use a 20 percent aliquot (500 uL) cf the extract for alpine
cotuirn cleanup ana subsequent GC/ECD analysis for pesticides and PC3s.
Use the remaining 80 percent (2 ml) for GC/MS analysis. Carefully reduce
the 2 ml extract to 400 uL using the N2 blowdown technique.
1C. 1.13.1 Solvent excnange of extract for al'jnina cleanup (reference
2) - transfer 0.5 ml of the extract to a separate concentrator tube. Add
5 ml of hexane and a boiling chip and mix using a vortex mixer. Attacn
a twc-Dali micro-Snyder column. Pre-wet the Snyder column by adding 0.5 ml
of hexane to the top of the column. Place the K-D apparatus on a hot water
bath (80-900 C) so that the concentrator tube is partially immersed in
tne rot water. Adjust the vertical position of tne apparatus and the water
temperature as required to complete the concentration in 5 to 10 min.
Concentrate the extract to an apparent volume of approximately 1 ml. Use
N£ bl owe own to reduce the volume to 0.5 ml. Dilute to 1 nL by adding C',5 mL
of acetone. Proceed with alumina column cleanup.
10.1.14 Alumina column setup and use - The alumina column cleanup
is required to remove polar interferences prior to GC/ECD analysis of pesticices
ano FCBs (reference 2).
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10.1.14.1 Add 3 3 of activity III neutral alumina to tns clean
5 ml dispcsaole serological pipet (with glass wool plug). Tap trie column
to settle the alumina. Do not pre-wet the alumina with solvent.
10.1.14.2 Transfer the 1.0 ml hexane/acetone extract (Sect.
10.1.13.1) to the top of the alumina column with a disposable Pasteur pipet.
Collect the eluate in a 10 rnL K-D concentrating tube. Add 1 ml of hexane
to the original extract concentrator tube to rinse it. Transfer these
rinsings to the alumina column. El ute the column with an additional 9 ml
of hexane. Do not allow the column to go dry during the addition and eluticn
of the sample.
10.1.14.3 Note that batches of alumina may differ and storage
may alter the water content of deactivated alumina. Thus, column performance
must be checked regularly and for each batch of alumina. PCB and pesticide
standards (e.g., from Sect. 6.8.2.2, 6.8.3) and a suitable model polar
compound (e.g., tribromophenol) should be used to determine the appropriate
elution volumes for these pollutants. Recovery of single PCB or pesticide
components should be greater than 85 percent and tne tribromophenol should
not oe detected.
10.1.14.4 Concentrate the eluate to 500 uL using a nicro-Snyder
column and the N2 blowdown technique {e.g., Sect. 10.1.13.1).
10.1.14.5 Care must be taken to allow the N£ gas to create only
a small dimple on the surface of the solvent and prevent blowdown to dryness.
Submit extract for GC/ECD analyses.
10.2 GC/MS Analysis
10.2.1 Establish the following operating conditions for the GC (Table
1-1): 5 min at 300 C; 30-280° C at 80 C/min; isothermal at 280° C until
benzo(g,h,i)perylene) elutes. Make certain that the concentrated extract
or standard is at room temperature and make note of any precipitate that
does not redissolve.
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1C. 2.2 ncd 5 .L c* tr.e GC/'-'S "interne', star.car-; sc'^ticn tc t~e 4'1D -._
extract to yield a 10 ug spike. Add the solution i~ned iatel y prior to
injection tc minimize tr.e possibility of less oy evaporation, acsc^Dtio^,
or reaction. Mix thoroughly.
10.2.2.1 It is advised that a late eljting internal injection
standard (e.g., 5-alpha-cholestane} oe used in audition to DF3. The ^se
of early eluting (DF3) and late elating injection stanaards «ill allow
the analyst to detect and compensate for problems in the GC injection port
related to differential loading of ana'ytes onto the GC column.
10.2.3 Inject 1.0-1.5 uL and start the GC column initial isothermal
hold. Start MS data collection after the solvent peak elutes. Stop cata
collection after the benzo(g,h ,i)perylene elutes.
10.2.3.1 Dilution and re-injection are required for samples
that exceed the upper concentration limit of the calibration standards.
Data for compounds witnin the calibration range should be acquired in the
inif'el run. Data for compounds exceeding the calibration range should
be acquired after dilution. Respike the sample with labeled compounds
and assume 100 percent recovery. This assumption is net unreasonaDle con-
sidering the high concentrations of native compounds involved when dilution
is necessary.
10.3 GC/ECD Analysis
10.3.1 The recommended GC conditions are modified from those specified
in reference 2:
Helium carrier gas: 4 mL/rr.in at 280° C and 25 psi
Septum purge: 15 mL/min
Split vent: none
Initial temperature: 60° C, initial hold - 2 min
Program at 25° C/min to 160° C
1-31
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Program at 50 c/~in from 160° C
Final temperature: 2700 C;
hold until decachlorobiphenyl elutes
Injection port temperature: 225° C
10.3.2 Add 10 uL of the GC/ECD internal standard solution to the
500 uL extract to yield a 25 ng spike. Add the solution immediately prior
to injection to minimize the possibility of loss by evaporation, adsorption,
or reaction. Mix thoroughly. Inject 1.0-1.5 uL.
10.3.3 Dilution and re-injection are required for samples that exceec
the upper concentration limit of the calibration standards. Data for compounds
within the calibration range should be retained from the initial run.
Data for compounds exceeding the calibration range should be acquired after
d ilution.
Column overloading can result in abnormal peak shape, which can reduce
the accuracy of quantification. It may also result in a marked increase
in the retention time of the peak maximum, which can displace an analyte
frcm the retention time window established with standards at lower concentra-
tions .
10.4 Qualitative Determination
10.4.1 Qualitative determination is accomplished by comparison of
data from analysis of a sample or blank with data from analysis of the
shift standard (Sect. 9.9.1) and, for GC/MS analyses, with data stored
in the spectral libraries (Sect. 8.2.4). Identification is confirmed when
spectra and retention times agree per the criteria below.
10.4.2 Labeled compounds and pollutants having no labeled analog:
10.4.2.1 The signals for all characteristic masses stored in
the spectral library (Sect. 8.2.4) should be present and should maximize
within the same two consecutive scans.
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10.4.2.2 Either l) tne background corrected extracted ion current
Defile (EICP) areas, or 2) the corrected relative intensities of tne r.iss
spectra] peaks at the GC peak maximum snould agree within a factor of two
(0.5 to 2 times) for all masses stored in the spectral library.
10.4.2.3 The retention time difference between an analyte ana
the nearest eluting internal standard during sample analysis should be
within +_5 scans or +_5 sec (whichever is greater) of this difference in
the shift standard (Sect. 9.9.1).
10.5 Pollutants Having a Labeled Analog:
10.5.1 The signals for all characteristic masses stored in the spectral
library (Sect. 8.2.4) should be present and should maximize within tne
same two consecutive scans.
10.5.2 Either 1) the background corrected EICP areas, or 2) the corrected
relative intensities of the mass spectral peaks at the GC peak maxirnur,
should agree within a factor of two for all masses stored in the spectral
1 ibrary.
10,5.3 The retention time difference between the pollutant and its
labeled analog should agree within ^2 scans or +_2 sec (whichever is greater)
of this difference in the shift standard (Sect. 9.9.1).
10.6 If an experimental mass spectrum contains masses that are not present
in the reference mass spectrum, an experienced spectrometrist is to determine
the presence or absence of the compound.
10.7 Chlorinated Pesticides and PCBs
10.7.1 Single component chlorinated pesticides are tentatively identified
by comparison of sample peak relative retention times to those of authentic
standards (Sect. 6.8.3). Three times the standard deviation of relative
1-33
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retention times established from calibration standarcs (Sect. 6.8.2; cin
be used to calculate relative retention tine window boundaries. Confirm
the identities of pesticides by comparing the relative retention t:r,es
of sample and standard peaks on another column pnase (e.g., 865 dimetnyl-
[14%]-cyanopropyl phenyl polysiloxane or J&W DB-1701). Confirmation by
GC/MS is required when concentrations are sufficient.
10.7.2 Peaks of multi-component mixtures (PCBs and toxaphene) are
tentatively identified in samples by comparison of relative retention times
to those of authentic standards (Sect. 6.8.2.2 and 6.8.4). Three times
the standard deviation of relative retention times established fron standards
can be used to calculate relative retention time window boundaries. Choose
as many peaks as possible while avoiding those with potential interferences
(e.g., PCBs co-eluting with DDT and DDE isomers). Label on all sample
chroma tog rams the peaks identified as PC8 and toxaphene congeners. All
GC/ECD chromatograms are part of the del iverables. Interpretation of chrofna-
tograms requires the attention of an experienced analyst. Confirm the
identities of all selected congeners by injection on an alternate colunn
phase (e.g., J&W 03-1701). Confirmation by GC/MS is required if concentrations
are sufficient.
10.8 Tentatively Identified Compounds (GC/MS Analysis) - The ten non-target
peaks of greatest area in the RIC (reconstructed ion chromatogram) should
be identified and quantified, if possible.
10.8.1 Guidelines for making tentative identification (reference 10):
1) Tentative identifications should be based on a forward search
of the EPA/NIH mass spectral library. Sample spectra should
be visually compared with the most similar library match.
2) Relative intensities of major ions in the reference spectrum
(ions greater than 10 percent of the most abundant ion)
should be present in the sample spectrum.
1-34
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3) The relative intensities of the major ions should d;ree
within +20 percent. (Example: For an ion with an abundance
of 50 percent in the standard spectra, trie corresponding
sample ion abundance must be between 30 and 70 percent.)
4) Molecular ions present in reference spectrum should be present
in sample spectrum.
5) Ions present in the reference spectrum but not in the sample
spectrm should be reviewed for possible subtraction frcm
the sample spectrum because of background contamination
or co-eluting compounds. Data system library reduction
programs can sometimes create these discrepancies.
10.S.1.1 If, in the opinon of the mass spectral specialist,
tentative identification can be made, the compound should be reported
as jjnjqiown. The mass spectral specialist should give additional classification
of the unknown compound if possible (e.g., unknown phthalate, unknown hydro-
carbon, unknown aromatic compound, unknown chlorinated compound). If probable
molecular weights can be distinguished, include then.
10.8.2 Tentative quantification - quantification of TIOs will be
based on the internal standard technique and an assumed response factor
of one (in the absence of data from authentic standards). The uncertain
nature of this quantification should be clearly noted in the data report.
11.0 QUANTITATIVE DETERMINATION (CALCULATIONS)
11.1 Isotope Dilution - by adding a known amount of a labeled compound
to every sample prior to extraction, correction for losses of the pollutant
during the analysis can be made because the pollutant and its labeled analog
exhibit similar behavior during extraction, concentration, and gas chroma-
togrephy. Note that pollutants and their labeled analogs are not always
retained identically by complex matrices, so their behavior during the
extraction step may differ.
1-35
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11.1.1 Relative response (RR) values for sample mixtures are use:!
in conjunction wiih calibration curves cescriced in Sect. 9.4 tc determine
concentrations directly, so long as labeled compound spiking levels are
constant.
11.1.2 Specifically, the concentration, C(in ug/kg), can be determines
as:
C( ug/kg) = CA( ug/kg) x RR x n
where:
CA = the concentration of the stable isotope-labeled compound as spiked
into the sample, wet weight
RR = relative response of unlabeled pollutant to isotope labeled surrogate
in the sa/nple
RRi = relative response of ith point in calibration
Zi = absolute amount of unlabeled compound of ith point in calibration
^Ai = absolute amount of labeled compound of i^h point in calibration
n = number of calibration points.
11.2 Internal Standard - all data reported as determined by this method
are uncorrected for method recoveries.
11.2.1 GC/MS internal standards method - Compute the concentration
in the sample, C {in ug/kg), using the response factor, RF, determined
from calibration data, and the following equation:
C(ug/kg) = (Ax x Zis x 1.25 x 103)/(S x Ais x RF)
1-36
-------
where:
Ax = the area at the characteristic mass for the compouna in the
sample
A-js = the area of the characteristic mass for the internal standard
Z-js = the absolute amount, in ug, of the GC/MS internal standard added
to the final extract prior to instrumental analysis
S = sample wet weight (g) that was extracted.
11.2.1.1 The stable isotope labeled compound recovery, X, is
determined and reported for each sample in the following manner:
X = [C(ug/kg)/CA{ug/kg)j x 100%
where:
CA = the concentration of the stable isotope labeled compound as spiked
into the sample
11.2.2 GC/ECD internal standard method -
11.2.2.1 Pesticides - compute the concentration in the sample,
C (in ug/kg), using the response factor (RF, determined from calibration
data) and the following equation:
C(ug/kg) = (Ax x Zis x 5 x 103)/(S x A1s x RF)
where:
Ax = the area of the integrated GC peak for the compound in the sample
(Ax represents the summation of areas for a group of GC peaks
if toxaphene is being quantified)
A-js = the area of the integrated GC peak for the internal standard
1-37
-------
Z^s ~ the absolute amount, In ug, of the GC/ECD Internal standard
added to the final extract prior to instrumental analysis
S = the sa.-nple wet weight (g) that was extracted.
11.2.2.2 Accurate PCB quantification is difficult to achieve
in routine full-scan analyses. It has been common practice to quantify
PCBs with packed-column GC/ECD by comparing several selected peaks in samples
to corresponding peaks in commercial Aroclor formulations that most closely
resemble the sample. Shortcomings of this technique have been described
elsewhere (e.g., references 8 and 9). The critical difficulties with this
procedure relate to two factors: (1) environmental PCB assemblages often
differ considerably from comnercial Aroclor mixtures because of the variable
properties of PCB congeners (e.g., aqueous solubility, volatility, suscepti-
bility to biodegradation) and (2) the ECD has a markedly variable response
to the 209 PCB congeners depending on the numoer and position of chlorine
atoms on the biphenyl nucleus (e.g., reference 10).
It has been suggested that "the least systematic error [in PCB quantifi-
cation] will be given by the summation of all or at least nearly all areas
of PCB peaks corrected by their individual ECD-response factor and their
biphenyl content" (reference 11). Another alternative is to use GC/MS
instead of GC/ECD. However, GC/MS analysis is relatively insensitive unless
selected ion monitoring (SIM) is used, which can involve considerable effort
and expense.
The quantification technique recommended in this 301 (h) document relies
on high resolution (capillary column) GC/ECO and a determination of response
factors for resolvable PCB peaks (as suggested in the previous paragraph).
The technique is modified from the Webb and McCall technique (reference 12),
which has been widely used for packed-column PCB quantification. Briefly,
the resolved peaks in a PCB standard (Sect. 6.8.2.1, 6.8.2.2) are quantified
by GC/MS and GC/ECD. The GC/MS results are used to correct for the variability
of ECD response. Samples are analyzed and quantified by GC/ECD. Total
PCBs are calculated as the sum of all resolved, response factor-corrected
PCB peaks.
1-38
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11.2.2.2.1 GC/MS analysis of PCB standard - Each resolvable
peak in a PCB calibration standard (Sect. 6.3.2.1) is quantified by GC/MS,
which can identify the chlorine content of biphenyls in each peak and can
quantify PCBs based on their chlorine content. This quantification does
not require that the analyst know the exact identity of the congeners consti-
tuting a peak, only the chlorine content must be determined.
Another GC/MS calibration standard is necessary to perform this quanti-
fication. An MS response factor standard consisting of representatives
of all the congener groups (mono- through decachlorobiphenyl) is necessary
to convert areas of peaks in ion plots to the appropriate masses of chloro-
biphenyls. A standard solution should be made with approximately 10 ng/uL
of each of the following congeners (see reference 13 for an explanation
of these choices):
2
2,3
2,4,5
2,2',4,6
2,2',3,4,5'
2,2', 4,4', 5,6'
2,2', 3,4',5,6,6'
2,2', 3,3',4,5',6,6'
2,2',3,3',4,4',5,5',6,6' - (used for nona- and deca-congeners).
These congeners are available from Ultra Scientific, Inc. (Hope, RI) except
the heptachloro-congener, which is available from Wellington Environmental
Consultants, Inc. (Guelph, Ontario, Canada). The primary quantification
ions used for mono- through deca-chlorobiphenyl are: 188, 222, 256, 292,
326, 360, 394, 430, 464, and 498. The spectrum for each peak should be
manually confirmed at least once for the Aroclor standard.
1-39
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Relative amounts of cc-e'iuting congeners of different chlorine conta-
in a giver peak in the stancard can oe aetermined raring GC/MS analysis.
Co-e'uticn c^n t? accojr.tsc for .-,itn appropriate response factors. Fcr
example, if a peak is composed of tetracnloro- and pentachlorc-isomers
as determined by ion plots of n/z 292 and 32G, tne 2,2',4,6 response f-ictc'
is used for tne m/z 292 area and the 2,2',3,4,5' response factor for tne
m/z 326 area. Care muse be taken to ensure that M-70 ions are not interprets;:
as M+ ions if congeners differing by two chlorine atoms co-elute.
11.2.2.2.2 GC/ECD analysis of PCB standard (Sect. 6.3.2.2) -
Each resolvable peak in the PCB calibration stances is quantifier;
by GC/ECD according to the internal standard technique (Sect. 8.5). The
GC/ECD araTysis is performed with the same GC column phas-3 and temperature
program used for GC/MS analysis of the standard. An ECO response factor
(RF) is establisnea for each peak based on the GC/MS analysis of tne PCB
standard using tne equation defined :n Section 8.5.1
where:
^s = the mean concentration of the peak in the PCB stancard as
determined by GC/MS (determined with at least three replicate
analvses).
11.2.2.2.3 GC/ECD PCB quantification in samples - Total
PCBs are calculated as the sum of all PCB peaks identified in a sample
(Sect. 10.7.2):
C (ug/kg, wet wt) =
n
£ [(Ax x Z-s x 5 x 500)/(S x Ais x RF)]i
1-40
-------
i - escn identif;ec PCS peak, ,vitn n total peaks
Ax = area c* the integrated GC Deak for tne compound in the sample
-JS = area of the integrated GC peak for the internal standard
TS = the absolute amount, in ug, ox ths GC/ECD internal stancar;
acced to the final extract prior to instrumental analysis
S = tne sample wet weight (g) extracted
RF = calibration response factor (Sect. 8.5.1).
11.2.2.2.4 This quantification method involves two nctewcrtny
imitations:
(1) Interferences cen be a significant problem in ECO analyses.
PCS peaks co-eluting with interferents may be neglected
or quantified, in either case resulting in a decrease in
accuracy. It is essential that experienced analysts evaluate
chromatograms to determine the presence of suspected inter-
ferents. Interferents suspected of overwhelming PCS peaks
should be neglectec. Trie alumina column cleanup step is
designed to preclude major interferences. High resolution
capillary columns also reduce the potential for co-eluting
interferences.
(2) When two or more congeners have identical retention times
on a given column phase, it is impossible to determine their
relative concentrations in a peak when using GC/ECD. Thus,
it is not possible to determine whether sample peaks are
composed of the same relative combination of congeners as
corresponding standard peaks. Thus, the response factor
for a peak may be different during calibration and sample
analysis. The potential error in assigning appropriate
reponse factors has been minimized in this technique by
the use of high resolution capillary columns.
1-41
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11.2.2.2.5 Alternative techniques of detection [e.g., Hall
electrolytic conductivity detector {HECD) or MS (with selected ion monitoring}]
can provide comparable or superior PCS identification and quantification
relative to ECD (e.g., references 13 and 14} and are acceptable substitutes
for ECD detection. Although ECD is widely available and is more sensitive
for PCBs than HECD or MS, HECO has a linear response to chlorine content
and is more specific to chlorinated compounds, and MS offers more definitive
compound identification than ECD.
11.2.2.3 Quantify PCBs by summing the response factor-corrected
areas of the characteristic PCB peaks identified in Sect. 10.7.2. Report
the results as total PCSs.
11.3 Report results for all pollutants and labeled compounds found in
all standards and samples, in ug/kg, to two significant figures. Note
in the report all compounds that have not been recovery corrected. Report
results for blanks as total ng/sa-nple.
12,0 PRECISION AND ACCURACY
Multiple laboratory comparison studies of the precision and accuracy
attainable with this technique are not available. Limited precision data
for environmental samples are available; they derive from replicate analyses
of English sole muscle and liver tissue (reference 15). Mean coefficients
of variation for total PCBs were 11 percent for muscle tissue (calculated
from six duplicates) and 15 percent for liver tissue (calculated from one
duplicate and one triplicate). These PCB results were generated by packed
colimn analysis and comparisons of sample chromatograms to Aroclor standards.
The accuracy of these PCB analyses was not assessed. Validation data using
the PCB quantification procedure in this 301(h) document has not yet been
generated.
A method test was recently conducted with spiked blanks. Replicate
blanks were spiked with known amounts of labeled and unlabeled compounds.
1-42
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The amounts of the unlabelec co~ipojnds were calculates jsinc tie isc-tooe
dilution technique (i.e., the calculatec amounts of the unlabeled compouncs
were adjusted for the recovery of the labeled compounds). The ratio of
the calculated amount of the unlabeleci compounds relative to tneir actual
spiked amount (expressed as percent) is given in Table 1-3. The procedure
for this method test included two additional cleanup steps (metallic mercury
and reverse phase column chromatography) that are not part of this 301 (h)
tissue protocol. Therefore results for the 301(h) tissue procedure should
be comparable or superior to those of the method test.
13.0 REFERENCES
1. "Performance Tests for the Evaluation of Computerized Gas Chronatography/-
Mass Spectrcmetry Equipment anci Laboratories," USEPA, EMSL/Cincinnati, OH
45268, EPA-600/4-80-025 (April 1980).
2. U.S. Environmental Protection Agency. 1984 (revised January, 1985).
U.S. EPA Contract Laboratory Program - statement of work for organics
analysis, multi-media, multi-concentration . IFB WA 85-J176, J177,
J178.
3. Ped. Register, Vol. 49, No. 209, October 26, 1984, pp. 43416-43429.
4. "Carcinogens - Working with Carcinogens," DREW, PHS, CDC, NIOSH, Publica-
tion 77-206 {Aug 1977).
5. "OSHA Safety and Health Standards, General Industry," OSHA 2206, 29 CFR
1910 (revised Jan 1976).
6. "Safety in Academic Chemistry Laboratories," ACS Publications, Committee
on Chemical Safety, 3rd Edition (1979).
7. Eichel berger, J.W., I.E. Harris, and W.L. Budde, "Reference compound
to calibrate ion abundance measurement in gas chromatography-mass
spectrometry," Anal. Chem. Vol. 47, 1975, pp. 995-1000.
8. Duinker, J.C., M.T.J. Hillebrand, K.H. Palmork, and S. Wilhelmsen,
"An evaluation of existing methods for quantitation of PCBs in environ-
mental samples and suggestions for an improved method based on measurement
of individual components," Bull. Environm. Contam. Toxicol. Vol. 25,
1980, pp. 956-964.
9. AT ford-Stevens, A.L., W.L. Budde, and T.A. Bellar, "Interlaboratory
study on determination of PCBs in environmentally contaminated sediments,"
Anal. Chem. Vol. 57, 1985, pp. 2452-2457.
1-43
-------
10. Mullin, M.D., C.M. PDchini, S. McCrindle, K. Ronkes, S.H. Safe, ana
L.M. Safe, "High-resolution PCB analysis: synthesis and chromatograpnic
properties of all 209 PCB congeners," Environ. Sc i . Techno!. Vol. 18,
1984, pp. 463-476.
11. Bal1schmiter , K., and M. Zell , "Analysis of PCB by glass capillary
gas chronatography," Fresenius Z. Anal. Chem. Vol. 302, 1980, pp. 20-31.
12. Webb, R.G., and A.C. McCall, "Quantitative PCB standards for electron
capture gas chromatography," J. Chromatographic Science, Vol. 11,
1973, pp. 366-373.
13. Gebhart, J.E., T.L. Hayes, A.L. Al ford-Stevens, and W.L. Budde, "Mass
spectrometric determination of polychlorinated biphenyls as isomer
groups," Anal. Chem. Vol. 57, 1985, pp. 2458-2463.
14. Sonchik, S, , D. Madeleine, P. Macek, and J. Longbottom, "Evaluation
of sample preparation techniques for the analysis of PCBs in oil,"
J. Chronatograpnic Science, Vol. 22, 1984, pp. 265-271.
15. Tetra Tech, Inc. Comm en cement Bay nearshore/tideflats remedial investi-
gation. Vol. 1. Final report prepared for the Washington State Department
of Ecology and U.S. Environmental Protection Agency. 1985.
1-44
-------
I
*»
tn
50 -
01
O
D.
at
Ut 1.0
oc
UJ
UJ
QC
0.1 -
I
10
5 10 20
CONCENTRATION (Mg/mL)
I
50
ADAPTED FROM REFERENCE 3
1.
AREA AT
AREA AT
M,IZ
ADAPT ID H«iM HI f!«( Nil 3
(3A)
M,/Z
M./Z
(30)
M,IZ
M,IZ
(3C)
M,/Z
M,/Z
AREA = 46100
AREA = 4780
AREA = 43600
AREA = 2650
AREA = 48300
AREA = 49200
ADAPT (0 FHOM BIFIKINf! 3
3.
Figure 1-1. Relative response calibration curve.
Figure 1-2. Extracted ion current profiles for chromatographically resolved labeled
(m?/z) and unlabeled (m,/z) pairs.
Figure 1-3. Extracted ion current profiles for (3A) unlabeled compound, (3tt) labeled
compound, and (3C) equal mixture of unlabeled and labeled compounds.
-------
-25 gm HOMOGENIZED
T.SSUE SAMPLE
S?IK= 10 -j.c EACH
ISOTOPE
MIX SAMP_E
WITH NiA
SOXHLET EXTRACT
I
WASH EXTRACT
ATPH<2
ORGANIC AQUEOUS
I
ADJUST pH>i2
I
SOLVENT EXTRACT
I
I
DRV EXTRACT
CONCENTRATE TO 3 ml
GPC
I
CONCENTRATE TO 2.5 ml
80V.
20%
CONCENTRATE
ADD GGVS
INTERNAL STANDARD
INJECT ON
GC/MS
RESIDUE WEIGHT
DETERM.'NATlON
EXCHANGE
AND CONCENTRATE
ALUMIMA COLUMN
CLEANUP
CONCENTRATE
ADDGC.ECD
INTERNAL STANDARD
FOR ISOTOPE DILUTION TECHNIQUE RECOMMENDED BUT NOT REQUIRED
INJECT ON
GC/ECD
Figure 1-4. Flow chart for sample preparation.
1-46
-------
GAS CHROMATOGRAPHY OF EXTRACTA3LE COMPOUNDS
Compcy-c
2,2' -cifluoroDicnenyi (DFB)
N-nitrosc5;Tetny 1 an me
pnef>o" -^5>* '
pr.enoi
ais(2-cnloroethyl letner-ag
Dis(Z-cnloroetny 1 ) ether
1 ,3-dichlorobenzene-C4
1 ,3-dichlorooenzene
l,4-dicnlorcoenzene-d4
1,4-oicnlorobenzene
1 ,2-dlcPi1. oroDenzene-o<;
1 , 2-dichlorocenzene
bis(2-ch!oroisopropyl )ether-di2
bis(2-chloroisopropylj ether
nexacnloroethar.e-i3c.(''
hexachloroetnane
N-nitrosocn-n-propylamine
nitrooenzene-Oj
nitrooenzene
isophorone- Og
isophorone
2,4-dimEtny 1 phenol-d3
2, 4 -dimethyl phenol
bis (2-cnloroethoxy) methane
1.2,4-trichlcrooenzene-d3
1 , 2,4-tr ichlorosenzene
naphtha lene-dg
naphthalene . ,
hexach' aroDutadiene- ^4
hexacn: orooutacnene
hexacnlorocycl open tad iene-^c^
hexacnl orocyc 1 open taa iene
2-ch1oronaDhtnalene-C7
2-chloronapnthaiene
biphenyl-aiQ
biphenyl
acenaprthylene-ds
acenaphtnylene
d imethy 1 phthal ate-Q4
dimethylphthalate
2,6-dinUrotoluene-d3
2,6-dinurotoiuene
acenapntnene-diQ
acenaphthene
dibenzofuran-dg
dibenzofuran
fluorene-oiQ
f luorene
4-chlorophenylphenyl ether-ds
4-chlorophenylphenyl ether
diethyl phthal ate-d4
diethyl pntnalate
2, 4-din itrotol uene-d3
2,4-dinitrotoluene
1,2-diphenylhydrazine-da
1,2-dipnenylhydrazine^ '
Re tent
Sec
1163
385
596
700
696
704
722
724
737
740
758
760
788
799
819
823
830
845
849
881
889
921
924
939
955
958
963
967
1005
1006
1147
1142
1185
1200
1205
1211
1245
1247
1269
1273
1283
1300
1298
1304
1331
1335
1395
1401
1406
1409
1409
1414
1344
1359
1433
1439
Quant i tatior
Ion Tir.e (Prirr.aryj
Sei4:''
-------
"A3LH
1437
179
C tpner-vl ar. me
N-nitrosoc * o^ery i am m e- j*
N-nurcsod iD^er.y'i a/nine '
4-brorcDieryi pneny", efer
nexacnlcroDenzene-'^CK
nexacnlorobenzene
pnenantnrene-c- G
Dr.enan^nrene
antnracene-a^g
anthracene
oioenzothiopnene-dg
dibenzothiopnene
carbazole
di-n-outyl phthalate-d4
di-n-Dutyl pntna'ate
f luoranthene-ain.
f luoranthene
pyrene-cio
pyrene
benzidine-dg
benzicme
butylbenzyl phthalate
cnrysene-d^2
cnrysene
ben20(a)ar,thracene-di2
benzo(a)anthracene
3 , 3 ' - (5 i c r, 1 or ober. 2 1 d i n e- dg
3,3' -dichlorooenzicine
bis(2-ethylhexyl)phthalate-d4
bis(2-etnylnexyl] phthalate
di-n-octyl phthalate-d4
di-n-octyl pntnalate
benzo(b) fluoranthene-di2
benzo(b)f1uoranthene
benzo(ic)fluoranthene-di2
benzo(K') f Igor an then e
benzo( a) pyrene-di2
benzo(a) pyrer.e
benzo(g,n,i)perylene-di2
benzo(g,h,i jperylene
indeno U, 2, 3-c,d) pyrene
d ibenzo{ a, h) anthracene
2-chlorophenol-d4
2-cnlorophenol
2-nitrophenol-d4
2-nitrophenol
2,4-dichlorophenol-d3
2,4-dichlorophenol
4 -chloro-3 -methyl phenol -03
4-chloro-3-methylpnenol
2,4,6-trichlorophenol-d2
2,4,6-trichlorophenol
2,4,5-tr ichloropnenol
2,3,6-trichloropnenol
2,4-dinitrophenol-d3
2,4-dinitropnenol
4-nitrophenol-d4
4-nitrophenol
2-methyl-4,6-dinitropheno1-d2
2-methyl-4,6-dinitrophenol
pehtachloropnenol-13Cg
ptntachl orophenol
1435
1438
1439
1493
1521
1522
1573
15£3
1538
1592
1559
1564
1650
1719
1723
1613
1817
1844
1852
1854
1855
2060
20Si
2083
2082
2090
2086
2088
2123
2124
2239
Z240
2281
2286
2287
2293
2350
2352
2741
2750
2650
2660
701
705
898
900
944
947
1086
1091
1162
1165
1170
1195
1323
1325
1349
1354
1433
1435
1559
1561
. £JO
.226
.CC1
.283
.3C8
.OCO
.357
.OC3
.365
.003
1.340
1.0C3
1.419
1.473
1.002
1.559
1 . 002
1.556
1.004
1.594
1.000
1.771
1.789
1.001
1.790
1.004
1.794
1.001
1.825
1.000
1.9Z5
1.000
1.961
1.002
1.966
1.003
2.021
1.001
2.357
1.003
0.967
0.970
0.603
1.006
0.772
1.002
0.812
1.003
0.934
1.005
0.999
1.003
1.007
1.028
1.138
1.002
1.160
1.004
1.232
1.001
1.340
1.001
A t 7
175
169
248
292
2S4
188
173
133
173
192
184
167
153
149
212
202
212
202
192
134
149
240
223
240
228
253
252
153
149
153
149
264
252
264
252
264
252
288
276
276
278
132
128
143
139
167
162
109
107
200
196
196
196
187
184
143
139
200
198
272
266
I
1
';
i
1
i
1
;
1
1
1
1
3
1
1
1
1
1
1
5
5
1
1
1
1
1
5
5
1
1
1
1
1
1
1
1
1
1
2
2
2
2
1
1
2
2
1
1
2
2
2
2
2
2
20
20
6
6
13
13
5
5
:?;
NN?
4-3PPE
6CLBNZ
PHENANTHRN
ANTHRACENE
DIBNZTHIO
CARBAZOLE
OINBP
FLUQRANTHN
PYRENE
3ZID
BJTBNZPHT
CHRYSE'tE
SAA
33-2CLBZID
B2ETHXPHTH
2NOCTP
BBF
BK.F
BAP
BGHIP
INDENO-PYR
OB An A
2-CLPHK
2-NPHN
24-2CLPHN
4-CL2-MPHN
246-3CLPHN
236-3CLPHN
24-2NPHN
4-NPHN
46-2NOCRES
5CLPHN
i:"-is-4
S6-20-5
1C1 -55 -5
i:3-74-i
so -C 1-3
120-12-?
132-75-0
86-74-2
S4-74-2
206-44-0
129-00-0
92 -67 -5
55-66-7
213-01-9
56-55-3
91-94-1
117-61-7
117-84-0
205-99-2
207-08-9
50-32-8
191-24-2
193-39-5
53-70-3
95-57-8
88-75-5
120-83-2
59-50-7
88-06-2
93-37-55
51-28-5
100-02-7
534-52-1
87-86-5
a Relative retention times for labeled compounds are referenced to DF8. Relative retention
for unlabeled compounds are referenced to their labeled analogs or to the most chemically similar
most closely eluting labeled compounds if labeled analogs are not listed.
1-48
-------
TA3LE
-i
{Continued;
C3T50 jrc
2ecsf'uC'=3e'>zc3"e'1cre ;DP3P>
tciis-.e'e nit^e
ircc^or 1242 (PCS flixtjre)
Arccior 1254 (PCS mixture)
Arccior 1260 (PCS mixture)
a-HCH
C -HCH
y-nCH
5-HCH
a i d r : n
'eotachlor
"estacr'ar eoox :de
y-cn!orsar.e
a-»ncosui fsn
a-cfilorcare
c-eicrin
4,4'-OOE
C -end os u If an
enar in
e<-drin aldehyde
4,4'-OOD
enoosulfan sulfate
y-Cf.lordene
4.4'-OOT
2,3,7,6-TCDD'6'
Additional 301 (h) Pesticides:
Oemeton
Gutnicn
Malatnion
Parathioi
Methcxycnlor
Mire*
Relative
Rj? ten 1 1 on
Time
to DF3
0.736
1.2-1.9
1.32
1.36
1.41
1.43
1.64
1.70
1.83
1.85
1.88
1.91
1.98
2.00
Z.02
2.02
2.07
2.10
2.13
2.13
2.17
2.01
1.19
1.19
1.51
1.52
*' Z
.
231, 233
183, 181
183. 181
183, 181
183, 161
263, 265
100, 272
353, 255
373, 375
195, 207
373, 375
241, 263
246, 24S
207, 195
263, 277
235. 165
272, 387
336, 333
235, 237
320, 322
127, 99, 174
291, 109, 139
238, 227, 274
272, 237, 274
SC/WS
.
10
:s
10
10
2
3
2
3
2
2
2
2
2
2
2
2
2
2
2
5
2
2
3
SC .' E - D
5
100
100
100
100
20
5
5
20
5
5
15
10
5
5
5
30
5
5
30
30
30
10.-
.(5)
40
50
100
O:ES
TCXAPii;SE
?C = S
PCBS
PCBS
6CI-CHX-A
6CL-CKX-3
LISDANE
6CL-CHX-D
-L3RIN
HE°TACHLDR
"ttPCb. E?CX
ENDOSULFAN
DIELOR.'N
ODE
ENDRIN
ENDRIN-iLD
ODD
ENDOSLFN-S
DOT
DIOXIN
SYSTOX
GUTHSON
MALATHION
PARATHION
METHOXYCL
MlREX
CASSS
5:O35-2
53469-21-?
11C97-69-1
11096-B2-5
319-34-6
319-34-7
319-86-3
5S-39-9
3:9-00-2
76-44-S
1C24-57-3
115-29-7
60-57-1
72-55-9
115-29-7
72-20-8
'421-93-4
72-54-8
1031 -07-8
50-29-3
1746-C1-6
3065-48-3
86-50-0
121-75-5
56-38-2
72-43-5
2385-85-5
1 Deuterium labeled recovery (surrogate) standard; isotopically labeled surrogates oo not have
ODES CDCes.
2 *JC-1abelea recovery (surrogate) standard; isocopically laoeled surrogates do not have ODES cooes.
^ Detected as azobenzene.
Detected as diphenylanine.
^ Low level amounts (<2 ng) of DDT are denydrohalogenated and converted to DOE at variable rates
en tne GC system.
6 Acceptable detection limits will be attainable with the U.S. EPA Contract Laboratory Program
Oioxin Analysis procedure [SoU/S«diment Matru, Multi-Concentration, Selectefl Ion Monitoring (SiK)
GC/MS Analysis; 9/15/83].
Column: 30 m x 0.25 mm i.d., 94S methyl, 41 phenyl, IS vinyl bonded phase fused silica capillary
(J4« 08-5, or equivalent)
Tefflperature program (GC/HS): 5 «in at 30° C; 30-280° C at 8° C per min; isothermal at 280° C until
benzoig, h,i)perylene elutes.
Carrier gas linear velocity: 30 OT/sec, helium.
1-49
-------
TABLE 1-2. DFTP? MASS-INTENSITY SPECIFICATIC
Mass Intensity Recuireo
51 30-60fo of mass 198
68 Less than 2'= of mass 69
70 Less than 2;= of mass 69
127 40-60% of mass 193
197 Less than 1°; of mass 193
193 Base peak, 100% relative abuncance
199 5-9^ of mass 198
275 10-3C;o of mass 19S
565 1:, of mass 193
441 Less than mess 443
442 Greater tr.an 40;» of mass 193
443 17-23% of mass 442
1-50
-------
TABLE 1-3. PRECISION AND ACCURACY OF METHOD BLANKS
Percent Recoverya
EPA Priority Pollutants
Phenols
phenol
2, 4-dimethyl phenol
2-chlorophenol
2,4-dichlorophenol
4-chl oro-3-methyl phenol
2,4,6-trichlorophenol
pentachlorophenol
2-nitrophenol
4-nitrophenol
2,4-dinitrophenol
4, 6-dinitro-2-methyl phenol
Aromatic Hydrocarbons
naphthalene
acenaphthene
acenaphthylene
fluorene
phenanthrene
anthracene
fluoranthene
pyrene
benz(a)anthracene
chrysene
benzo ( b) f 1 uoranthene
benzo ( k) f 1 uoranthene
benzo (a) pyrene
indeno(l, 2, 3-cd) pyrene
dibenzo( a, h) anthracene
benzo (ghi)perylene
Chlorinated Hydrocarbons
1,2-dichlorobenzene
1,3-dichlorobenzene
1,4-dichlorobenzene
1,2,4-trichlorobenzene
2-chloronaphthalene
hexachlorobenzene
hexachloroethane
hexachlorobutadiene
hexachlorocyclopentadiene
Blank
1
96
92
100
98
99
98
110
96
110
99
110
110
120
110
120
120
120
120
120
120
120
100
110
130
160
150
120
100
87
110
93
110
160
73
98
23
Blank
2
102
120
102
101
97
100
110
99
110
100
100
110
110
110
120
120
120
120
110
110
120
102
113
125
161
189
120
110
120
120
98
110
110
104
105
25
Blank
3
91
110
110
100
110
140
120
120
110
98
110
120
120
120
130
120
110
130
150
95
150
110
120
130
170
180
120
no
94
120
130
120
120
69
130
24
Blank
4
96
97
104
110
100
100
110
110
110
110
no
120
120
120
120
130
130
130
130
110
130
110
110
130
160
190
120
no
120
130
100
120
no
77
110
25
Mean
96
105
104
102
102
110
113
106
no
102
108
115
118
115
123
123
120
125
129
109
130
106
113
129
163
177
120
108
105
120
105
115
125
81
111
24
Coeff. of
Variation
4.7
5.0
4.2
5.2
5,7
18.6
4.4
10.3
0.0
5.5
4.7
5.0
4.3
5.0
4.1
4.1
6.8
4.6
13.4
9.5
10.9
5.0
4.2
1.9
3.0
10.6
0.0
4.7
16.4
6.8
15.9
5.0
19.0
19.6
12.4
3.9
1-51
-------
TABLE 1-3. (Continued)
Phthalates
bis(2-ethy1hexyl)phtha1ate 120 130 120 130 125 4.6
benzylbutylphthalateb 270 298 120 310 250 35.2
di-n-butylphthalate 120 120 130 120 123 4.1
di-n-octylphthalate 120 120 120 120 120 0.0
dlethylphthalate 120 120 130 120 123 4.1
dimethylphthalate 110 120 140 120 123 10.3
Halogenated Ethers
bis(2-ch1oroethy1)ether 91 93 91 100 94 4.6
bis(2-ch1oroisopropy1)ether c c c 61
bis(2-ch1oroethoxy)methane 200 110 190 140 160 26.5
4-chlorophenylphenylether 120 120 140 120 1Z5 8.0
4-bromophenylphenylether 150 140 120 150 140 10.1
Qrganonitrogen Compounds
nitrobenzene 37 21 19 c 19 38.4
N-nitrosodipropylamine 26 135 150 73 96 59.7
N-nitrosodimethylamine 110 120 110 120 115 5.0
N-nitrosodiphenylamine 76 89 173 77 104 44.9
2,4-dinitrotoluene 120 120 120 100 115 8.7
2,6-dinitrotoluene 130 74 130 100 109 24.9
benzidine 0000
3,3'-dichlorobenzidine 120 170 140 120 138 17.2
1,2-diphenylhydrazine 120 110 140 100 118 14.5
Miscellaneous
isophorone 130 120 72 120 111 23.6
a Metnod blanks were processed after spiking with known amounts of unlabeled
and labeled compounds. Recovery-corrected concentration of unlabeled priority
"poTlutants was calculated using the recovery of labeled analogs for each
compound. The final percent recovery for the unlabeled compounds was then
computed as the ratio of the calculated concentration to the known spike
level of each expound.
b Benzyl butyl phthalate results are anomolously high because of laboratory
contamination traced to mercury used in the sulfur-removal step of the
procedure. The contamination was subsequently eliminated.
c Spectral interferences precluded quantification.
1-52
-------
SECTION II
ANALYSIS OF VOLATILE ORGANIC COMPOUNDS
IN ESTUARINE AND MARINE TISSUES
-------
CONTENTS
Pace
1.0 SCOPE AND APPLICATION Ii-1
2.0 SUMMARY OF METHOD 11-2
3.0 INTERFERENCES II-3
4.0 SAFETY 11-5
5.0 APPARATUS AND EQUIPMENT 11-6
6.0 REAGENTS AND CONSUMABLE MATERIALS 51-9
7.0 SAMPLE COLLECTION, PREPARATION, AND STORAGE II-12
8.0 CALIBRATION AND STANDARDIZATION 11-14
9.0 QUALITY CONTROL 11-18
10.0 PROCEDURE 11-20
11.0 QUANTITATIVE DETERMINATION (CALCULATIONS) 11-26
12.0 PRECISION AND ACCURACY 11-27
13.0 REFERENCES 11-28
-------
ANALYSIS OF VOLATILE ORGANIC COMPOUNDS
IN ESTUARINE AND MARINE TISSUES
i.O SCOPE AND APPLICATION
1.1 This method is designed to determine the volatile toxic organic pollutants
associated with Clean Water Act Section 3Cl(h) regulation [40 CFR 125.53(k)
and (v)] and additional compounds amenable to purge and trap gas chronatography-
mass spectrometry (GC/MS) (Table II-l).
1.2 The chemical compounds listed in Table II-l can be determined in biological
tissue samples collected from estuarine and marine environments by this
method.
1.3 The detection limit of this method is usually dependent on the level
of interferences rather than instrumental limitations.
Lower limits of detection (LLD) are established by analysts based
on their experience with the instrumentation and with interferences in
the sample matrix being analyzed. LLD are greater than instrumental detection
limits because they take into account sample interferences. To estimate
LLD, the noise level should be determined in the retention window for the
quantitation mass of representative analytes. These determinations should
be made for at least three field samples in the sample set under analysis.
The signal required to exceed the average noise level by at least a factor
of two should then be estimated. This signal is the minimum response required
to identify a potential signal for quantification. The LLD is the concentration
corresponding to the level of this signal based on calibrated response
factors. Based on best professional judgment, this LLD would then be applied
to samples in the set with comparable or lower interference. Samples with
much higher interferences (e.g., at least a factor of two higher) should
be assigned LLD at a multiple of the original LLD.
II-l
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These LLD values may be less than the rigorously defined method detection
limits specified in the revised "Guidelines Establishing Test Procedures
for the Analysis of Pollutants" (40 CFR Part 136, 10/26/84). This latter
procedure requires the analysis of seven replicate samples and a statistical
determination of th emethod detection limit with 99 percent confidence.
Data quantified between the LLD and the rigorous method detection limit
are valid and useful in environmental investigations of low-level contamination,
but have a lower statistical confidence associated with them than data
quantified above the method detection limit.
The LLD are roughly 5-10 ppb (wet weight) with the exception of acrolein
and acrylonitrile, which have not been thoroughly tested on tissue matrices
with this method.
2.0 SUMMARY OF METHOD
2.1 Volatile organic compounds are vacuum extracted from a macerated,
5-g (wet wt) tissue sample and concentrated in a cryogenic trap cooled
with liquid nitrogen (references 1 and 2). The cryogenic trap is then
transferred to a conventional purge-and-trap device. The extract is diluted
to a 5 mL volume with water and treated as an aqueous sample. In the purge-
and-trap device, the volatile organic compounds are purged from the aqueous
phase into a gaseous phase with an inert carrier gas. The volatile compounds
are passed into a sorbent column and trapped. After purging is completed,
the trap is back'flushed and heated rapidly to desorb the compounds into
a gas chrcmatograph (GC). The compounds are separated by GC and detected
with a mass spectrometer (MS).
Analysis is carried out by GC/MS either according to the isotope dilution
technique (U.S. EPA Method 1624 Revision B; reference 3) or U.S. EPA Method
624 (reference 4). Both of these methods were developed for water/wastewater
sample matrices. The isotope dilution technique, which requires spiking
the sample with a mixture of stable isotope labeled analogs of the analytes,
is the technique of choice because it provides reliable recovery data for
11-2
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each analyte. Method 624 requires spiking samples with only three surrogate
compounds and does not allow for recovery corrections. If uniformly high
recoveries can be attained with Method 624, then addition of numerous labeled
compounds (Method 1624B) and recovery corrections are unnecessary. However,
until such performance can be demonstrated, Method 1624B provides a detailed
and valuable assessment of analytical performance.
Hiatt (reference 5) proposed another vacuum distillation procedure
that did not include a purge-and-trap device. In this technique, volatile
organic compounds are transferred directly from a cryogenically cooled
trap to a fused-silica capillary column for GC/MS analysis. This capillary
column technique allows for optimum resolution and rapid conditioning between
samples. However, the performance of the technique has not been thoroughly
tested (reference 6). A potential problem is that water can enter the
capillary column and cause chromatographic problems or freeze, effectively
plugging the column. Thus, Hiatt1 s original procedure (references 1 and 2),
which has been tested more thoroughly, is recommended here.
Vacuum distillation is recommended rather than direct purge-and-trap
extraction (i.e., without vacuum distillation) because the former technique
has been demonstrated to allow for better recoveries of spiked compounds
than the latter technique (reference 1; comparisons based on similar spiking
level s).
2.2 Laboratories may use alternative analytical procedures provided that
evidence of performance comparable to the recommended procedure is provided.
3.0 INTERFERENCES
3.1 Impurities in the purge gas, organic compounds out-gassing from the
plumbing upstream of the trap, and solvent vapors in the laboratory account
for the majority of contamination problems. The analytical system is demon-
strated to be free from interferences under conditions of the analysis
by analyzing blanks initially and with each sample lot (samples analyzed
II-3
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on the same 8 h shift), as described in Sect. 9.3. Common laboratory solvents
(e.g., methylene chloride) are often contaminants in volatiles analyses.
3.1.1 Newly packed traps should be conditioned overnight at 1700-180° C
by backflushing with an inert gas at a flow rate of 20-30 ml/min. Traps
must be conditioned daily for a minimum of 10 min before use.
3.2 There is potential for ambient contamination of samples and extracts
when using vacuum and cryogenic concentration techniques. Care must be
taken to eliminate any leaks in the vacuum extraction and concentration
device. A critical source of potential contamination is pump oil vapor
and exhaust from the vacuum pump; this should not be a problem if the system
is properly sealed. A cold trap is placed between the vacuum pump and
concentration trap to prevent condensation of pump oil vapors in the concen-
tration trap (Figure II-l). All materials in the vacuum extraction and
concentration device that contact the sample and its vapors must be made
of stainless steel and/or borosilicate glass. All connections and seals
must be free of elastomers or grease that either outgas or allow penetration
of ambient contaminant vapors.
3.3 Samples can be contaminated by diffusion of volatile organic compounds
(particularly methylene chloride) through the bottle seal during shipment
and storage. A field blank prepared from reagent water and carried through
the sampling and handling protocol serves as a check on such contamination.
3.4 Contamination by carry-over can occur when high level and low level
samples are analyzed sequentially. When an unusually contaminated sample
is analyzed, it should be followed by analysis of a reagent water blank
to check for carry-over. Because the transfer lines, trap, and other parts
of the system can retain contaminants and interferences, frequent bakeout
and purging of the entire system may be required.
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4.0 SAFETY
4.1 The toxicity or care inogen ic ity of each compound or reagent used in
this method has not been precisely determined; however, each chemical compound
should be treated as a potential health hazard. Exposure to these compounds
should be reduced to the lowest possible level. The laboratory is responsible
for maintaining a current awareness file of OSHA regulations regarding
the safe handling of the chemicals specified in this method. A reference
file of data handling sheets should also be made available to all personnel
involved in these analyses. Additional information on laboratory safety
can be found in references 7-9.
4.2 The following compounds covered by this method have been tentatively
classified as known or suspected human or mammalian carcinogens: benzene,
carbon tetracnl oride, chloroform, and vinyl chloride. Primary standards
of these toxic compounds should be prepared in a hood, and a NIQSH/MESA-
approved toxic gas respirator should be worn when high concentrations are
handled.
4.3 The following safety measures must be employed when handling cryogenic
and vacuum systems:
4.3.1 Liquid nitrogen (LN2) must not be allowed to contact flesh
since it will cause extreme frostbite and deaden (kill) tissues.
4.3.2 The concentrator and cold traps must never be closed off or
sealed after allowing any concentration of liquid air. The liquid air
will vaporize, resulting in tremendous pressure build up and explosive
damage to the vacuum system. Always vent any vessel imred iately after
removing the cryogenic or LN2 bath. Wear safety goggles when working with
cryogenic and vacuum systems.
II-5
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5.0 APPARATUS AND EQUIPMENT
5.1 Sample Handling Equipment
5.1.1 Stainless steel spatula, rinsed wi th methanol and oven-dried
at 1500 c.
5.1.2 Sample vessel - Pyrex flask with 15 mm 0-ring connector, washed
with detergent and rinsed with distilled water and oven-dried at 4500 C.
5.1.3 0-ring, Buna N, sonicated with 50 percent methanol/water then
dried by vacuum at 600 c.
5.1.4 Tissue homogenizer (e.g., Tekmar Tissuemizer, Tekmar Co., Cin-
cinnati, OH) - must be free of volatiles and solvents before use.
5.2 Apparatus for Vacuum Distillation and Cryogenic Concentration {Figure
II-l).
5.2.1 Vacuum pump, capable of achieving 10-3 Torr and 25 L/min.
5.2.2 Vaccum/pressure gauge - with a range of subatmospheric pressure
to 10 psi.
5.2.3 Concentrator trap or purge flask, 25 ml capacity (Tekmar Part
No. 14-0957-024 or equivalent) modified with 9 mm 0-ring connectors.
5.2.4 Cold trap - glass trap (easily produced by glassblowing, Figure
11-1} with 0-ring fittings (e.g., Kontes 671750-009).
5.2.5 Transfer line, 1/4 in o.d. glass-lined stainless steel tubing.
Lines should be kept as short as possible to minimize sample carryover.
5.2.6 Vacuum valves, Nupro B-4BKT or equivalent.
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5.2.7 Dewar flasks, 665 ml or 1,000 nL, for liquid nitrogen D2th.
5.2.8 Assorted compression fittings ancj graphite ferrules (Figure II-l).
5.2.9 Ultrasonic bath, Branisonic 12 or equivalent.
5.2.10 Heater tape - to wrap around stainless steel lines and valve
bodies to maintain a temperature of 60° C.
5.2.11 Pinch clasps, Thomas - to secure 0-ring connections.
5.3 Purge-ana-Trap Device - capable of meeting specifications listed in
U.S. EPA Method 1624 B (see below). Complete devices consisting of a purging
device (the concentrator trap, Sect. 5.2.3), a Tenax/silica trap, and a
desorber are commercially available (e.g., Tekmar Model LSC-2, Tekmar Co.,
Cincinnati, OH).
5.3.1 Trap - 25 to 30 cm x 2.5 mm i.d. minimum, containing the following:
5.3.1.1 Methyl silicons packing - one +_0.2 cm, 3 percent OV-1
on 60/80 mesh Chromosorb W, or equivalent.
5.3.1.2 Porous polymer - 15 +1.0 on, Tenax GC (2,6-diphenylene
oxide polymer), 60/80 mesh, chrcmatographic grade, or equivalent.
5.3.1.3 Silica gel - 8 +1.0 cm, Davison Chemical, 35/60 mesh,
grade 15, or equivalent.
5.3.2 Desorber - should heat the trap to 175 +5° C in 45 sec or less.
The polymer section of the trap should not exceed 180° C, and the remaining
sections should not exceed 220° C.
5.3.3 Commercial purge and trap devices are easily coupled to GC
systems.
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5.4 GC/KS (Gas Chromatograph-Mass Spectrometer) System.
5.4.1 GC - should be linearly temperature programmable with initial
and final temperature holds.
5.4.2 GC column - 6 ft long x 0.1 in i.d. {stainless steel or glass)
packed with 1 percent SP-1000 on Carbopak B, 60/80 mesh or equivalent.
5.4.3 MS - 70 eV electron impact ionization; capable of repeatedly
scanning from 20 to 250 amu every 2 to 3 sec.
5.4.4 GC/MS interface - GC to MS interfaces constructed of all-glass
or glass-lined materials are recommended. Glass can be deactivated by
silanizing with dichloro-dimethyl silane.
5.5 Data System - should collect and record MS data, store mass intensity
data in spectral libraries, process GC/MS data and generate reports, and
should calculate and record response factors.
5.5.1 Data acquisition - mass spectra should be collected continuously
throughout the analysis and stored on a mass storage device.
5.5.2 Mass spectral libraries - user created libraries containing
mass spectra obtained from analysis of authentic standards should be employed
to reverse search GC/MS runs for the compounds of interest.
5.5.3 Data processing - the data system should be used to search,
locate, identify, and quantify the compounds of interest in each GC/MS
analysis. Software routines should be employed to compute retention times
and extracted ion current profile (EICP) areas. Displays of spectra, mass
chromatograms, and library comparisons are required to verify results.
5.5.4 Response factors and multipoint calibrations -- the data system
should be used to record and maintain lists of response factors (response
ratios for isotope dilution) and generate multi-point calibration curves.
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Computations of relative standard deviation (coefficient of variation)
are useful for testing calibration linearity.
5.6 Other Materials
5.6.1 Syringe, 10 uL _* 1 percent of volume.
5.6.2 Syringe, 50 uL +_ 1 percent of volume.
5.6.3 Syringe, 5 ml +_ 1 percent of volume, gas-tight with shut-off.
5.6.4 Bubble flowneter.
6.0 REAGENTS AND CONSUMABLE MATERIALS
6.1 Reagent Water
6.1.1 Reagent water is defined as water free of interferences (i.e.,
interferents are not observed at the detection limits of the compounds
of interest).
6.1.2 Prepare water by boiling 1 L of freshly distilled water down
to 900 mL and transferring the water to a l-l volumetric flask that has
been modified by replacing the ground glass joint with a 15-mm i.d., Buna-N
0-ring connector.
6.1.3 Connect the flask to the distillation apparatus at the sample
chamber site and evacuate for 15 min while continuously agitating the flask
in an ultrasonic cleaner.
6.1.4 After evacuation, release an inert gas (^2 or He can be used)
into the flask until equilibrium is obtained, then seal with a cap made
from a Buna-N 0-ring connector.
6.2 Methanol - pesticide quality or equivalent.
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6.3 Stancard Solutions - purchased as solutions or mixtures with certification
as to their purity, concentration, and authenticity, or prepared from materials
of known purity and composition. If compound purity is 96 percent or greater,
the weight may be used without correction to calculate the concentration
of the standard.
6.4 Preparation of Stock Solutions - prepare in methanol using liquid
or gaseous standards per the steps below. Observe the safety precautions
given in Sect. 4.
6.4.1 Place approximately 9.8 ml of methanol in a 10 ml ground glass
stoppered volumetric flask. Allow the flask to stand unstoppered for approxi-
mately 10 min or until all methanol-wetted surfaces have dried. In each
case, weigh the flask, inmediately add the compound, then immediately reweigh
to prevent evaporation losses from affecting the measurement.
6.4.1.1 Liquids - using a 100 uL syringe, permit two drops of
liquid to fall into the methanol without contacting the neck of the flask.
Alternatively, inject a known volume of the compound into the methanol
in the flask using a micro-syringe. With the exception of 2-chloroethylvinyl
ether, stock standards of compounds that boil above room temperature are
generally stable for at least 4 wk when stored at 4° C.
6.4.1.2 Gases ( chloromethane, bromomethane, chloroethane, vinyl
chloride) - fill a valved 5 mL gas-tight syringe with the compound. Lower
the needle to approximately 5 mm above the methanol meniscus. Slowly introduce
the compound above the surface of the meniscus. The gas will dissolve
rapidly in the methanol.
6.4.2 Fill the flask to volume, stopper, then mix by inverting several
times. Calculate the concentration in mg/mL (ug/uL) from the weight gain
(or density if a known volume was injected).
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6,4.3 Transfer tne stock solution to a Teflon sealed screw-cap bottle.
Store, with minimal heaaspace, in trie dark at -10 to -20° C.
6.4.4 Prepare fresh standards weekly for the gases and 2-chloroethylvinyl
ether. All other standards are replaced after 1 mo, or sooner if comparison
with check standards indicates a change in concentration of over 10 percent.
Quality control check standards that can be used to determine the accuracy
of calibration standards are available from the U.S. Environmental Protection
Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio.
6.5 Labeled Compound Spiking Solution - from stock standard solutions
prepared as above, or from mixtures, prepare the spiking solution to contain
a concentration such that a 5-10 uL spike into each 5 tnL sample "extract",
blank, or aqueous standard analyzed will result in a concentration of 10 ng/mL
of each labeled compound. For the gases and for the water soluble compounds
(acrolein, acrylonitrile) , a concentration of 50 ng/mL may be used. Include
the internal standards (Sect. 8.1.2) in this solution so that a concentration
of 10 ng/mL in each sample, blank, or aqueous standard will be produced.
6.6 Secondary Standards - using stock solutions, prepare a secondary standard
in methanol to contain each pollutant at a concentration of 250 ug/mL.
For the gases and water soluble compounds (Sect. 6.5), a concentration
of 1.25 mg/mL may be used.
6.7 Aqueous Calibration Standards - the concentrations of calibration
solutions suggested in this section are intended to bracket concentrations
that will be encountered during sample analysis that will not overload
the analytical system. Use sufficient amounts of the secondary standard
(Sect. 6,6) and reagent water to produce concentrations of 5, 10, 20, 50,
and 100 ug/L in the aqueous calibration standards. The concentrations
of gases and water soluble compounds will be higher (i.e., 25, 50, 100,
250, and 500 ug/L). Analysts may use a wider range of standard concentrations
if linearity can be demonstrated.
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6.8 Aqueous Performance Standard - an aqueous stancard containing a" 1
pollutants, internal standards, labeled compounds, and BFB (4-bromofl uoro-
benzene) Is prepared daily, and analyzed each shift to demonstrate performance
(Sect. 11). This standard should contain either 10 or 50 ug/L of the labeled
and pollutant gases and water solub'e compounds, 5 ug/L of BFB, and 10 ug/L
of all other pollutants, labeled compounds, and internal standards. It
may be the nominal 10 ug/L aqueous calibration standard (Sect. 6.7).
6.9 A methanol ic standard containing all pollutants and internal standards
is prepared to demonstrate recovery of these compounds when syringe injection
and purge-and-trap analyses are compared. This standard should contain
either 10 ug/mL or 50 ug/mL of the gases and water soluble compounds, and
10 ug/mL of the remaining pollutants and internal standards (consistent
with the amounts in the aqueous performance standard in Sect. 6.8).
6.10 Other standards that may be needed are those for testing of BFB per-
formance (Sect. 8.2.1) and for collecting mass spectra for storage in spectral
libraries (Sect. 8.1.4).
6.11 High Purity Helium - 99.999 percent.
6.12 Liquid Nitrogen (LN£).
7.0 SAMPLE COLLECTION, PREPARATION. AND STORAGE
7.1 In the field, sources of contamination include sampling gear, grease
from ship winches or cables, ship engine exhaust, dust, and ice used for
cooling. Efforts should be made to minimize handling and to avoid sources
of contamination. This will usually require that resection (i.e., surgical
removal) of tissue be performed in a controlled environment (e.g., a labora-
tory). For example to avoid contamination from ice, the samples should
be wrapped in aluminum foil, placed in watertight plastic bags and immediately
iced in a covered ice chest. Aluminum foil should be cleaned by heating
at over 105° C before use. Solvent cleaning is unacceptable unless heating
is performed afterward. Organisms should not be frozen prior to resection
11-12
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if analyses will only be conducted on selected tissues, Because freezing
may cause internal organs to rupture and contaminate other tissue (e.g.,
muscle). If organisms are eviscerated on boara the survey vessel, the
remaining tissue may be wrapped as described above and frozen.
7.2 To avoid cross-contamination, all equipment used in sample handling
should be thoroughly cleaned before each sample is processed. All instruments
must be of a material that can be easily cleaned (e.g., stainless steel,
anodized aluminum, or borosilicate glass). Before the next sample is processed,
instruments should be washed with a detergent solution, rinsed with tap
water, soaked in high-purity acetone and methylene chloride, and finally
rinsed with reagent water.
7.3 Resection should be carried out by or under the supervision of a competent
biologist. Each organism should be handled with clean stainless steel
or quartz instruments (except for external surfaces). The specimens should
come into contact with precleaned glass surfaces only. Polypropylene and
polyethylene surfaces are a potential source of contaminatin and should
not be used. To control contamination when resecting tissue, separate
sets of utensil should be used for removing outer tissue and for dissecting
tissue for analysis. For fish samples, special care must be taken to avoid
contaminating target tissues (especial ly muscle) with slime and/or adhering
sediment from the fish exterior (skin) during resection. The incision
"troughs" are subject to such contamination; thus, they should not be included
in the sample. In the case of muscle, a "core" of tissue is taken from
within the area boarded by the incision troughs, without contacting them.
Unless specifically sought as a sample, the dark muscle tissue that may
exist in the vicinity of the lateral line should not be mixed with the
light muscle tissue that constitutes the rest of the muscle tissue mass.
7.4 The resected tissue sample should be placed in a clean glass or TFE
container which has been washed with detergent, rinsed twice with tap water,
rinsed once with distilled water, and heated at 105° C for several hours.
Jars should be heated at 1050 c and allowed to cool immediately before
use.
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7.5 The U.S. EPA and other federal agencies (e.g., National Bureau of
Standards) have not yet provided specific guidance regarding holding times
and temperatures for tissue samples to be analyzed for volatile organic
compounds. Until U.S. EPA develops definitive guidance, the following
holding conditions should be observed. Resected tissue samples should
be maintained at -200 C and analyzed as soon as possible, but within 10 days
of sample receipt. The 10 day holding time is based on the Contract Laboratory
Program regulations for sediments to be analyzed for voUtiles (reference 10).
8,0 CALIBRATION AND STANDARDIZATION
8.1 Initial Calibration
8.1.1 Calibration by the isotope dilution technique -- the isotope
diTution technique is used for the purgeable organic compounds when appropriate
labeled compounds are available and when interferences do not preclude
the analysis. If labeled compounds are not available or interferences
are present, the internal standard technique (Sect. 8.1.2) is used. A
calibration curve encompassing the concentration range of interest is prepared
for each compound determined. The relative response (RR) vs. concentration
(ug/L) is plotted or computed using a linear regression. An example of
a calibration curve for a pollutant and its labeled analog is given in
Figure II-2. Also shown are the _+10 percent error limits (dotted 1 es) .
Relative response is determined according to the procedures described below.
A minimum of five data points is required for calibration (Sect. 6.7).
8.1.1.1 The relative response (RR) of pollutant to labeled compound
is determined from isotope ratio values calculated from acquired data.
Three isotope ratios are used in this process:
Rx = the isotope ratio measured in the pure pollutant (Figure II-3A)
Ry = the isotope ratio of pure labeled compound (Figure II-3B)
Rfji = the isotope ratio measured in the analytical mixture of the pollutant
and labeled compounds (Figure II-3C).
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The correct way to calculate RR is:
RR = (Ry - Rm)(Rx + l)/(Rm - Rx)(Ry
If Rfn is not between 2Ry and 0.5RX, the method does not apply and the sample
is analyzed by the internal standard technique (Sect. 8.1.2).
8.1.1.2 In most cases, the retention times of the pollutant
and labeled compound are the sane and isotope ratios (R's) can be calculated
from the EICP areas, where:
R = (area at m^zj/farea at m2/z)
If either of the areas is zero, it is assigned a value of one in the calcu-
lations; that is, if: area of mi/2=50,721, and area of m2/z=0, then R=50721/l =
50720. The m/z's are always selected such that Rx>Ry. When there is a
difference in retention times (RT) between the pollutant and labeled compounds,
special precautions are required to determine the isotope ratios.
Rx , Ry, and R^ are defined as follows:
Rx = [area mi/z (at
Ry = l/[area m2/z (at RTi)]
Km = [area m\/z (at RT2)]/[area m2/z (at RTi)]
8.1.1.3 An example of the above calculations can be taken from
the data plotted in Figure II-3 for a pollutant and its labeled analog.
For these data, Rx=168920/l= 168920, Ry=l/60960=0. 00001640, and Rnf% 868/82 508
= 1.174. The RR for the above data is then calculated using the equation
given in Sect. 8.1.1.1. For the example, RR=1.174. Note: Not all labeled
compounds el ute before their pollutant analogs.
8.1.1.4 To calibrate the analytical system by isotope dilution,
analyze a 5 ml aliquot of each of the aqueous calibration standards (Sect. 6.7}
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spiked with an appropriate constant amount of the labeled compound spiking
solution (Sect. 6.5), using the purge and trap procedure in Sect. 10.
Compute tne RR at each concentration.
8.1.1.5 Linearity - if the ratio of relative response to concen-
tration for any compound is constant (less than 20 percent coefficient
of variation) over the five point calibration range, an averaged relative
response/concentration ratio may be used for that compound; otherwise,
the complete calibration curve for that compound should be used over the
5 point calibration range.
8.1.2 Calibration by internal standard - used when criteria for isotope
dilution (Sect. 8.1.1} cannot be met. The method is applied to pollutants
having no labeled analog and to the labeled compounds themselves. The
internal standards used for volatiles analyses are bromochloromethane,
2-bromo-l-chloropropane, and 1,4-dichlorobutane. Concentrations of the
labeled compounds and pollutants without labeled analogs are computed relative
to the nearest eluted internal standard.
8.1.2.1 Response factors - calibration requires the determination
of response factors (RF), which are defined by the following equation:
RF = (As x Cis)/{A1s x Cs)
where:
As = the EICP area at the characteristic m/z for the compound in the
daily standard
A-JS = the EICP area at the characteristic m/z for the internal standard
Cis = the concentration (ug/L) of the internal standard
C$ - the concentration of the pollutant in the daily standard.
8.1.2.2 The response factor is determined at 5, 10, 20, 50,
and 100 ug/L for the pollutants (optionally at five times these concentrations
for gases and water soluble pollutants - see Sect. 6.6 and 6.7), in a way
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analogous to that for calibration by isotope dilution (Sect, £.1.1.4}.
The RF is plotted against concentration for eacn compound in tne stancarc
(Cs) to produce a calibration curve.
8.1.2.3 Linearity - if tne response factor (RF) for any compound
is constant (less than 35 percent coefficient of variation) over the five
point calibration range, an averaged response factor may be used for that
compound; otherwise, the complete calibration curve for that compound should
be used over the five point range.
8.1,3 Combined calibration - by adding the isotopically labeled compounds
and internal standards (Sect. 6.5) to the aqueous calibration standards
(Sect. 6.7), a single set of analyses can be used to produce calibration
curves for the isotope dilution and internal standard methods.
8,1.4 Mass spectral libraries - detection and identification of the
compound of interest during calibration and sample analysis are dependent
upon the spectra stored in user created libraries.
8.1.4.1 Obtain a mass spectrum of each pollutant and labeled
compound and each internal standard by analyzing an authentic standard
either singly or as part of a mixture in which there is no interference
between closely el uted components. That only a single compound is present
is determined by examination of the spectrum. Fragments not attributable
to the compound under study indicate the presence of an interfering conpound.
Adjust the analytical conditions and scan rate (for this test only) to
produce an undistorted spectrum at the GC peak maximum. An undistorted
spectrum will usually be obtained if five complete spectra are collected
across the upper half of the GC peak. Software algorithms designed to
"enhance" the spectrum may eliminate distortion, but may also eliminate
authentic ions or introduce other distortion.
8.1.4.2 Obtain the authentic reference spectrum under BFB tuning
conditions {Table II-2) to normalize it to spectra from other instruments.
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8.1.4.3 The spectrum is eaitec by saving me five nest intense
mass spectral peaks and all other mass spectral peaks greater tran 10 percent
of the base peak. This spectrum is stereo for reverse search ana for car.pcuna
confirrnation.
8.2 Ongoing Calibration
8,2.1 The 8F3 stana5"d must be analyzed at the beginning of eacn
8-h shift. The tuning criteria in Table 11-2 must be met before blenKs
and samples may be analyzed.
8.2.2 At the beginning anc end of each 8-h shift, system calibration
should be verified by purging the aqueous performance standard (Sect. 6.8).
8.2.2.1 Calibration is tested by computing the concentration
of unlebeled compounds by the isotope dilution technique (Sect. 8.1.1)
for compounds with labeled analogs. Concentrations of unlabeled compounds
without labeled analogs are calculated according to the internal standard
technique (Sect. 8.1.2).
A complete (five-point) recalibration should be performed when results
vary from predicted concentrations by more than _+25 percent. The last
sample analyzed before failing criteria should then be reanalyzed. If
the results differ by more than +20 percent (i.e., twice the median repro-
ducibility for replicate analysis of tissue samples, Table II-3), then
it is to be assumed that the instrument was out of control during the original
analysis and the earlier data should be rejected. Reanalysis of samples
should progress in reverse order until it is determined that there is _<20
percent difference between initial and reanalysis results.
9.0 QUALITY CONTROL [For further guidance, see Quality Assurance/Quality
Control (QA/QC) for 301(h) Monitoring Programs: Guidance on Field and
Laboratory Methods (Tetra Tech 1986).]
9.1 Each laboratory that uses this method is required to operate a formal
quality assurance program. The minimum requirements of this program consist
11-18
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of an initial cemor.stration of laboratory Capability, analysis of samples
spiked with labelec conoouncs to evaluate anc document data quality, ana
analysis of stancaras ana Dianas as tests of continues performance.
9.2 initial Demonstration of Laooratory Capaoility
9.2.1 Analyze the aqueous performance stendara (Sect. 6.3) according
to the purge-anc-trap procedure in Sect. 10. Compute the area at tne primary
m/z (Taole II-l) for each compound. Compare these areas to those o&tairsec
by injecting one uL cf the methanoiic standarc (Sect. 6.9) to ceterr.ine
compound recovery. The recovery should be greater than 20 percent for
the water soluble compounds (acrolein and acrylonitrile), and 60-110 percent
for all other compounds. This recovery snculd be cemonstratec initially
for each purge-and-trap GC/MS system. The test should be repeated only
if tne purge and trap or GC/MS systems are modified in any way that might
result in a change in recovery.
9.3 Blanks
9.3.1 Reagent water blanks must be analyzed to demonstrate freeaorn
from carry-over (Sect. 3) and contamination.
9.3.1.1 The level at which the purge-and-trap system will carry
greater than 5 ug/L of a pollutant of interest into a succeeding blank
should be determined by analyzing successively larger concentrations of
these compounds. When a sample contains this concentration or more, a
blank should be analyzed immediately following this sample to demonstrate
no carry-over at the 5 ug/L level.
9.3.1.2 With each sample lot (samples analyzed on the same 8-h
shift), a blank should be analyzed immediately after analysis of the aqueous
performance standard (Sect. 8.2.2) to demonstrate freedom from contamination.
If any of the compounds of interest, except common laboratory contaminants
(e.g., methyiene chloride and toluene), or any potentially interfering
compound is found in a blank at greater than 10 ug/L (assuming a response
11-19
-------
factor of 1 relative to the nearest eluted internal stancara for canpouncs
not listed in Table II-l), analysis of samples is halted until the source
of contamination is eliminated anc a clank snows no evicence of contaT;inaticr-.
at this level. This control action also applies if rr.ethylene cnlorice
or toluene "is cetectec in a blank at greater than 50 ug/L.
9.4 Sample Spiking
9.4.1 The laboratory snould spike all samples 'with labeled cornpouncs
to assess metnod performance on the sample matrix.
9.4.2 Spike anc analyze each sample according to the method beginning
in Sect. 10.
9.4.3 Compute the percent recovery (P) of the labeled compounds using
the internal standard technique (Sect. 6.1.2).
9.5 Replicates
9.5.1 Replicate analyses (i.e., analyses of two subsamples from the
same tissue homogenate) must be performed to monitor laboratory precision.
9.5.2 At least one laboratory duplicate should be run for cases of up
to 20 samples. For cases of over 20 samples, one blind triplicate and addi-
tional duplicates must be run for a minimum of 5 percent replication overall.
10.0 PROCEDURE
10.1 Sample Processing
10.1.1 Mince tissue sample with a scalpel and homogenize the sample
to a uniform consistency with a micro-grinder. Care must be taken to ensure
that the micro-grinder is thoroughly cleaned after each use. This usually
entails disassembly of the unit. Devices with large surface areas (e.g.,
blenders, meat grinders) should not be used, as they are difficult to clean
11-20
-------
and a. small sample Is difficult to remove after grinding. Liquia associated
with the sample should be retained throughout the procedure.
10.1.2 Dry weight determination - if sample size permits and dry-
weight concentrations are required, dry weight determinations may be performed
as fellows: transfer an aliquot of approximately 3 g (weighed to the nearest
0.1 g) to a preweighed dish. Allow the sample to dry in an oven at 1050 r
overnight and determine the solid residue weight to the nearest 0.1 g.
The percent total solids is calculated as:
Ts = [dry residue wt (g)]/[wet sample wt (g)]
Dry weight determinations should not be made at the cost of having insufficient
sample for volatiles analysis. Significant decreases in the size of samples
used for extraction will decrease attainable detection limits.
10.1.3 Immediately after homogenization, use a stainless steel spatula
to transfer a 5-g aliquot to a preweighed sample vessel (Sect. 5.1.2).
Weigh the transferred portion to the nearest 0.1 g.
10.1.4 Spike 50 nanograms of each labeled compound (or 250 nanograms
of gaseous and water soluble compounds) into 2 ml of reagent water and
add to the sample matrix. Seal the sample vessel with an 0-ring connector
and clamp and sonicate for 10 min. After sonication, store the sample
contained in the sample vessel overnight in a refrigerator/freezer and
analyze the next day.
10.2 Vaccim Distillation and Concentration (Reference 2)
10.2.1 The vacuum extractor must be airtight and free of moisture
before an extraction can be started.
10.2.2 A clean 100 mL pyrex flask is connected to the vacuum distillation
apparatus at the sample vessel site (see Figure II-l), the vacuum pump
started, and V2-V4 opened to evacuate the apparatus. Line condensation
11-21
-------
is prevented by warming the transfer lines while evacuating the systerr.
Heating tape is effective in creating even transfer line temperatures and
can be used continuously during the procedure.
10.2.3 The vacuum apparatus is pressurized with helium by closing
₯3 and opening Vi. The apparatus is tested for leaks with a helium leak
detector or Snoop , and appropriate adjustments are made as necessary.
When the apparatus has been found to be airtight, close Vj, open V3 and
then heat the transfer lines and concentrator trap to 1000 C for 5 min
to eliminate any residual contamination.
10.2.4 The flask containing the sample should be immersed in liquid
nitrogen, before the flask is uncapped. To begin the distillation, close
V;? (with V3 and V4 remaining open), cool the concentrator trap with a liquid
nitrogen bath, and replace the empty sample vessel with the cooled sample
flask. Disconnect the vacuum source by closing ₯3. Open V2 to permit
vapors from the sample vessel to reach the concentrator trap. Immerse
the sample vessel in a 500 C water bath and sonicate for 5 min.
10.2.5 Connect the vacuum source to the sample vessel by opening
V3« The lower pressure hastens the transfer of volatile compounds from
the sample to the cooled concentrator trap. After 15 min of vacuum, close
V3 and open Vi to fill the system with helium to atmospheric pressure.
Close Vi and V2 to isolate the condensate. The distillation is now completed
and the condensate is ready for transfer to a purge-and-trap device. The
condensate can be held in the liquid nitrogen bath for up to 1 h prior
to analysis. Care should be taken to ensure that moisture does not freeze
in the narrow glass tubing in the concentrator trap. Careful drying of
the system prior to analysis and maintenance of an airtight system will
preclude this problem.
10.2.6 Disconnect the sample concentrator trap from the vacuum apparatus
and connect it to the purge-and-trap device. Some outgassing is observed
when the sample condensate is melted; therefore, the condensate should
be kept frozen until the concentrator trap is attached tc the purge-and-
11-22
-------
trap device. After attacr.nent, warm the concentrator trap walls to loosen
the concensate and allow the ring of ice formed during condensation to
crop to the bottom of the trap. To tnis partially melted extract add 3 ~L
of reagent water containing 50 ng of each of the internal standards (bromo-
cnloromethane, Z-bromo-l-chloropropane, and 1,4-dichlorobutane). The internal
stancards are added after vacuum extraction to allow the analyst tne assess
analytical losses of labeled compounds during the extraction/ concentration
procedure.
10.3 Purge-and-Trap Procedure
10.3.1 Because commercial purge flasks must be slightly Codified
(with 0-ring fittings) to be attached to the vacuum distillation apparatus,
a sinple 0-ring adapter is necessary to connect the purge flask to the
commercial device for which it was designed. The modified purge flask
(Sect. 5.2.3) used in this procedure can be fitted to a commercial purge-
and-trap device (e.g., a Tekmar ALS interfaced with a Tekmar LSC-2) with
9 mm 0-ring fittings fused to short sections of glass tubing. Commercial
purge-and-trap devices are almost entirely automated and are easy to operate
with manufacturer's instructions.
10.3.2 Purge the extract solution with the concentrator trap immersed
in an ice-water bath for 5 min followed by immersion in a 550 C-water bath
for an additional 7 min. This provides conditions for reproducibly melting
the frozen extracts in order to obtain reproducible purging efficiencies.
10.3.3 The GC conditions for analysis are as follows:
Injector zone temp. 2250 c
Initial GC oven temp. 60° C
Final GC temp. 1750 c
Initial hold time 3 min
Ramp rate 80 C/min
Final hold time 24 min
Jet separator oven temp. 2250 c
11-23
-------
10.4 Qualitative Determination - accompl isned by comparison of data
analysis of a sample or blank with data from analysis of the shift standard
(Sect. 8.2.2). Identification is confirmed when spectra and retention
times agree according to the criteria below.
10.4.1 Labeled compounds and pollutants having no labeled analog:
10.4.1.1 The signals for all characteristic masses stored in
the spectral library (Sect. 8.1.4.3) should be present and should maximize
within the same two consecutive scans.
10.4.1.2 Either 1) the background corrected EICP areas or 2) the
corrected relative intensities of the mass spectral peaks at the GC peak
maximum should agree within a factor of two (0.5 to 2 times) for all masses
stored in the library.
10.4.1,3 The retention time relative to the nearest eluted internal
standard should be within +7 scans or +_20 sec, whichever is greater, of
this difference in the shift standard.
10.4.2 Pollutants having a labeled analog:
10.4.2.1 The signals for all characteristic masses stored in
the spectral library should be present and should maximi ze within the same
two consecutive scans.
10.4.2.2 Either 1) the background corrected EICP areas or 2} the
corrected relative intensities of the mass spectral peaks at the GC peak
maximum should agree within a factor of two for all masses stored in the
spectral library.
10.4.2.3 The retention time difference between the pollutant
and its labeled analog should agree within +2 scans or +6 sec, whichever
is greater, of this difference in the shift standard.
11-24
-------
10.4.2.4 If the experimental mass spectrum contains masses that
are not present in the reference spectrum, an experienced spectronetri st
is to determine the presence or absence of the compound.
10.5 Tentatively Identified Compounds (GC/MS Analysis) - The ten non-target
peaks of greatest area in the RIC (reconstructed ion chromatogram) should
be identified ana quantified, if possible.
10.5.1 Guidelines for making tentative identification (reference 10):
(1) Tentative identifications should be based on a forward
search of the EPA/NIH mass spectral library. Sample spectra
should be visually compared with the most similar library match.
(2) Relative intensities of major ions in the reference spectrun
(ions greater than 10 percent of the most abundant ion) should
be present in the sample spectrum.
(3) The relative intensities of the major ions should agree
within +_20 percent. (Example: For an ion with an abundance
of 50 percent in the standard spectra, the corresponding sample
ion abundance must be between 30 and 70 percent.)
(4) Molecular ions present in reference spectrum should be
present in sample spectrum.
(5) Ions present in the reference spectrum but not in the
sample spectrum should be reviewed for possible subtraction from
the sample spectrum because of background contamination or co-eluting
compounds. Data system library reduction programs can sometimes
create these discrepancies.
10.5.1.1 If, in the opinion of the mass spectral specialist,
no valid tentative identification can be made, the compound should be reported
11-25
-------
as unknown. The mass spectral specialist should give additional classification
of the unknown compound if possible (e.g., unknown hydrocarbon, unknown
aromatic compound, unknown chlorinated compound). If probable molecular
weights can be distinguished, include them.
10.5.2 Tentative quantification - quantification of TIOs will be
based on the internal standard technique and an assumed response factor
of one (in the absence of data from authentic standards). The uncertain
nature of this quantification should be clearly noted in the data report.
11.0 QUANTITATIVE DETERMINATION (CALCULATIONS)
11.1 Isotope Dilution - by adding a known amount of a labeled compound
to every sample prior to vacuum distillation, correction for recovery of
the pollutant can be made because the pollutant and its labeled analog
exhibit similar behavior during purging, desorption, and gas chromatography.
Note that pollutants and their labeled analogs are not always retained
identically by complex matrices, so their behavior during the extraction
step may differ. Use of this technique is to enable correction for analytical
losses after extraction, not for matrix recovery.
11.1.1 Relative response (RR) values for sample mixtures are used
in conjunction with calibration curves described in Sect. 8.1.1 to determine
concentrations directly, so long as labeled compound spiking levels are
constant.
11.1.2 For the isotope dilution technique, concentration is calculated
as follows:
C {ug/kg, wet wt tissue) =
CA (ug/kg) x RR x n
RRi * ZAi
11-26
-------
where
CA = the concentration of the stable isotope labeled compound as
spiked into the sample
RR = relative response of unlabeled pollutant to isotope labeled
surrogate in the sample
Ri = relative response at itn point in calibration
Z-j = absolute amount of unlabeled compound at i^n point of calibration
= absolute amount of labeled compound at i^1 point in calibration
n = number of calibration points.
11.2 Internal Standard - calculate the concentration using the response
factor determined from calibration data (Sect. 8.1.2) and the following
equation:
Concentration = (As x CiS)/(Ais * RF) where the terms are as defined
in Sect. 8.1.2.1, except that C-js is in ug/kg (wet tissue) and As is the
EICP area at the characteristic m/z for the analyte in the sample.
11.3 If the EICP area at the quantitation mass for any compound exceeds
the calibration range of the system, a smaller sample aliquot should be
analyzed if possible. However, sample sizes of less than 0.5 g are not
recommended because such small samples may not be representative.
11.4 Report results for all pollutants and labeled compounds found in
samples, in ug/kg (wet weight, unless dry weight is required) to three
significant figures. Pollutants and labeled compounds in blanks should
be reported in ng/sample.
12.0 PRECISION AND ACCURACY
12.1 Recoveries from replicate spiked water and tissue analyses are presented
in Table II-3 (references 1 and 2). These analyses were not performed
with the isotope dilution technique and recovery results are uncorrected.
11-27
-------
13.0 REFERENCES
1. Hiatt, M.H., "Analysis of Fish and Sediment for Volatile Priority
Pollutants," Anal. Chem. Vol. 53, 1981, pp. 1541-1543.
2. Hiatt, M.H., and T.L. Jones. Isolation of Purgeable Organics from
Solid Matrices by Vacuum Distillation. U.S. Environmental Protection
Agency, Region IX, Las Vegas Laboratory, 1984.
3. Fed. Register, Volume 49, No. 209, October 26, 1984, pp. 43407-43415.
4. Fed. Register, Volume 49, No. 209, October 26, 1984, pp. 43373-43384.
5. Hiatt, M.H., "Determination of Volatile Organic Compounds in Fish
Samples by Vacuum Distillation and Fused Silica Capillary Gas Chrorna-
tography/Mass Spectrometry," Anal. Chem. Vol. 55, 1983, pp. 506-516.
6. Hiatt, M.H. 4 November 1985. Personal Communication (phone by Mr. Harry
Beller). Analytical Technologies, Incorporated, National City, CA.
7. "Working with Carcinogens," DHEW, PHS, NIOSH, Publication 77-206 (1977).
8. "OSHA Safety and Health Standards, General Industry," 29 CFR 1910,
OSHA 2206, (1976).
9. "Safety in Academic Chemistry Laboratories," American Chemical Society
Publication, Corrniittee on Chemical Safety (1979).
10. U.S. Environmental Protection Agency. 1984 (revised January, 1985).
U.S. EPA Contract Laboratory Program - statement of work for organ ics
analysis, multi-media, multi-concentration. IFB WA 85-J176, J177,
J178.
11-28
-------
VACUUM
GAUGE
V4" S.S.
TEE UNION
i
INJ
10
25mL
PURGE
FLASK
{CONCENTRATOR
TRAP)
NUPRO B-4BKT
LIQUID
NITROGEN
BATH
VACUUM
PUMP
COLD TRAP
LIQUID
NITROGEN
BATH
AMI'11 II I HUM Kill HI Nil ,'
NOTf: PURtU Aim IHAP OlVICf IS NOT IfKllinin IH I II.Illll
Figure II-l. Apparatus for vacuum distillation and cryogenic concentration.
-------
10 -
I
CJ
o
01
U>
Z
O
GL
CO
IU
DC
Ul
Ul
a:
1.0 -
01
I
to
I
20
I
SO
CONCENTRATION
ADAPTED FROM R[F{RtNCE 3
T
100
2.
(A)
AREA 168920
M./Z
M,/Z
(B)
AREA 60960
M ,/Z
M,/Z
(C)
M ,/Z 96868
M?/Z 82508
M../Z
M,/Z
AOWUD FROM RfFIRCHCt J
Figure M-2. Relative response calibration curve.
Figure 11-3. Extracted ion current profiles for (A) the unlabeled pollutant, (B) the
labeled analog, and (C) a mixture of the labeled and the unlabeled compounds.
-------
TABLE II-l. VOLATILE ORGANIC ANALYTES
I
CO
Analyte
Acrolein
Acrylonitrile
Benzene
Bromod ichl oromethane
Bromoform
Bromomethane
Carbon tetrachloride
Chlorobenzene
Chloroethane
2-chloroethylvinyl ether
Chloroform
Chi oromethane
Dibromochl oromethane
1,1-dichloroethane
1,2-dichloroethane
1,1-dichloroethene
trans-1 ,2-dichloroethene
1,2-dichl oropropane
cis-lt3-dichloropropene
trans-1 ,3-dichloropropene
Ethyl benzene
Methylene chloride
1,1,2,2-tetrachloroethane
Tetrachloroethene
Toluene
1 ,1,1-trichloroethane
1,1,2-trichloroethane
Trichloroethene
Vinyl chloride
CASRN
107-02-8
107-13-1
71-43-2
75-27-4
75-25-2
74-83-9
56-23-5
108-90-7
75-00-3
110-75-8
67-66-1
74-87-3
124-48-1
75-34-3
107-06-2
75-35-4
156-60-5
78-87-5
10061-01-5
10061-02-6
100-41-4
75-09-2
79-34-5
127-18-4
108-88-3
71-55-6
79-00-5
79-01-6
75-01-4
ODES
ACROLEIN
ACRYLNTRLE
BENZENE
2CLBRMETHA
BROMOFORM
METHYLBR
CARBON TET
CLBNZ
ETHYL CL
2-CLEVE
CHLOROFORM
METHYL CL
2BRCLMETH
11-2CLETH
12-2CLETH
11-2CLETHE
12-2CLETHE
12-2CLPRP
C13-2CLPRE
T13-2CLPRP
ETHYLBENZ
METHYLE CL
4CLETHAN
4CLETHE
TOLUENE
111-3CLETH
112-3CLE1H
3C LETHE
VINYL CL
Quantitation
Ion (m/z)
56
53
78
83
173
94
117
112
64
63
83
50
129
63
62
96
96
63
75
75
106
84
83
164
92
97
97
130
62
Secondary
Ion(s)
55
51, 52
--
85, 129
171, 175
96
119, 121
114
66
65, 106
85
52
206, 208, 127
65, 83
64, 98
61, 98
61, 98
65, 114
77
77
91
86
85, 168
129, 131, 166
91
99, 117, 119
83, 85, 99
95, 97, 132
64
-------
fASLE II-2. 3FB MASS-INTENSITY SPEC:
Xass Intensity Required
50 15-40% of mass 95
75 30-60% of mass 95
95 Base peak,
100% relative abundance
96 5-9% of mass 95
173 <2% of nass 174
174 >50S of mass 95
175 5-9% of mass 174
176 >95% but <101S of mass 174
177 5-9% of mass 176.
11-32
-------
TABLE II-3. PERCENT SPIKE RECOVERIES FOR VOLATILE
PRIORITY POLLUTANTS USING VACUUM DISTILLATIONS
Spiking Compound
Average Percent
Recovery (Water)b
Average Percent
Recovery (Tissue)0
Chloromethane
Sromomethane
Vinyl chloride
Chloroethane
Methylene chloride
1 , 1-dichloroethene
1 ,1-dichloroethane
trans-1 ,2-dichloroethene
Chi oroform
1,2-dichloroethane
1,1,1-trichloroethane
Carbon tetrachloride
Acryl oni tr il e
Bromod ic hi or om ethane
1,2-dichloropropane
J.ran_s-l,3-dichloropropene
Tnchloroethene
Benzene
Dibromochloromethane
1 , 1, 2 -tri chloroethane
cis-1 ,3-dichloropropene
Bromofom
Tetrachl oroetnene
1,1,2,2-tetrachloroetnane
Tol uene
Chlorobenzene
Ethyl benzene
2-chloroethyl vinyl ether
Acrolein
Average compound recovery
105 + 22
110 + 23
83 + 12
103 + 16
126 + 22
98+5
96 + 5
98+5
93+8
98 + 10
104 + 9
102 + 10
85 + 13
108 + 10
104 + 7
109 + 9
105 + 9
106 + 7
102 + 11
95+8
109 + 9
104 + 14
105 + 9
90+9
106 + 7
101 + 7
103 + 5
94 + 50
113 1 76
102 i 8
85 + 22
126 + 75
64 + 11
69 + 22
LCd
74+8
90+6
86+9
107 + 31
92+5
92+8
91+9
NAe
64 + 11
54+7
52 + 9
65 + 11
57 + 10
56+9
66 + 7
54 9
NDf
NO
61 + 10
NO
64 + 15
ND
NA
76 +_ 20
a From references 1 and 2.
b Reagent water was spiked with 25 ug/L of each compound except acrolein
and acrylonitrile, which were added at 100 ug/L. The recoveries are averaged
from 9 analyses and were calculated by comparing vacuum extracted determinations
to determinations for which spikes were added directly to a purge-and-trap
device.
c Ten-gram fish samples were spiked at 25 ppb. The recoveries were averaged
from 12 analyses.
d Laboratory contamination of fish prevented the generation of valid data.
e Compound was not added to this matrix.
f Not determined.
11-33
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SECTION III
ANALYSIS OF METALS AND METALLOIDS
IN ESTUARINE AND MARINE TISSUES
-------
CONTENTS
Page
1.0 SCOPE AND APPLICATION III-l
2.0 SUMMARY OF METHOD III-l
3.0 DEFINITIONS III-2
4.0 INTERFERENCES III-2
5.0 SAFETY III-4
6.0 APPARATUS AND EQUIPMENT III-4
7.0 REAGENTS AND CONSUMABLE MATERIALS III-6
8.0 SAMPLE COLLECTION, PREPARATION, AND STORAGE II1-7
9.0 CALIBRATION AND STANDARDIZATION III-9
10.0 QUALITY CONTROL III-ll
11.0 PROCEDURE 111-18
12.0 CALCULATIONS 111-21
13.0 PRECISION AND ACCURACY 111-21
14.0 REFERENCES III-Z1
-------
ANALYSIS OF METALS AND METALLOIDS IN
ESTUARINE AND MARINE TISSUES
1.0 SCOPE AND APPLICATION
1.1 This metnod is designed to determine antimony, arsenic, beryllium,
catfrnin, chromium, copper, lead, mercury, nickel, selenium, silver, thallium,
and zinc concentrations in biological tissue samples. The method may be
used for the analysis of varying tissue types such as edible muscle and
livers of estuarine and marine organisms.
1.2 A universal wet oxidation (acid digestion) procedure is recommended
that is capable of providing a clean extract suitable for analysis by atomic
absorption spectrophotmetry (AAS). This digestion has proven effective
when determining the priority pollutant metals listed above {with the possible
exception of beryllium and thallium) (e.g., Table III-2). Because of a
lack of reference materials certified for beryllium and thallium, little
is known regarding method suitability for these elements.
1.3 Limits of detection (LOD) are listed in Table III-l. These may vary
depending on the element being measured, method of detection, and instrumental
sensitivity.
2.0 SUMMARY OF METHOD
2.1 A macerated 5-g sample of tissue is homogenized wet, subsampled and
digested using a wet oxidation method. The resulting extract is analyzed
for the metals of interest using various atomic absorption (AA) techniques
such as:
III-l
-------
direct aspiration (DFAA) = for higher concentration metals
graphite furnace (GFAA) = for lower concentration metals
hydriae generation (HYDAA) = for hydride forming elements (antimony,
arsenic , selenium)
cold vapor (CVAA) = for mercury.
2.2 Al ternative methods of detection (e.g., inductively coupled plasma
emission spectrometry) may be used providing their performance and limitations
have been establ ished.
3.0 DEFINITIONS
Certified Reference Materials (CRM): A homogeneous sample that has
been analyzed a sufficient number of times by numerous qualified laboratories.
The data ere compiled and certified values are determined through statistical
analysis. A number of CRM are commercially available in a wide range of
matrices for metals analyses (e.g., NBS Oyster Tissue, SRM 1566) (Reference 1).
Control Standard: A solution, independent of the calibration standards
whose analyte concentration is known. These are often analyzed as an external
check after calibration.
Limit of Detection (LOD): The LOD is the lowest concentration level
that can be determined to be statistically different from a blank. The
recommended value for LOD is 3cr, where a is the standard deviation of the
blank in replicate analyses (reference 2).
Matrix Modifier: A reagent added to a sample that alters some aspect
of its composition (references 3-5).
4.0 INTERFERENCES
4.1 Interferences should be considered to be any chemical or physical
phenomenon that can influence the accuracy of the data during an analytical
III-2
-------
operation. These can have eitner a positive or a negative effect on the
result depending on their nature.
4.2 Contamination of the sample caT occur during any stage of collection,
handling, storage, or analysis. Potential contaminant sources must be
known and steps should be taken to minimize or eliminate them. Some of
the most commor) sources of contamination include prolonged exposure of
the tissue to metal-containing fumes and dust; insufficiently clean sample
containers, storage facilities and testing apparatus; and the use of contam-
inated reagents during analysis (reference 6).
In general, clean laboratory procedures are extremely important when
performing trace metal analysis.
4.3 Most instrumental methods are prone to matrix interferences, which
can either suppress or enhance the analyte signal. If a matrix interference
is suspected, its effect should be determined and corrective action taken.
Some common matrix interferences are listed below along with suggested
corrective measures (references 7, 8).
4.3.1 High sample viscosity - usually due to dissolved solids and
high acid content. Match the matrix of the calibration standards with
the samples where possible.
4.3.2 Non-specific absorption (light scatter) - usually due to dissolved
solids or suspended particulates, which absorb analyte radiation. Background
correction (see instrument manufacturer's instructions) should be used
whenever this occurs.
4.4 Many chemical interferences, some of which are poorly understood,
can occur during instrumental analysis of the sample extracts. A great
many of these interferences have been addressed in the literature and in
most cases a sample pretreatment or instrumental modification has been
proposed as a remedy (e.g., reference 9).
III-3
-------
5.0 SAFETY
Laboratory personnel should be well versed in standard laboratory
safety practices. It is the responsibility of all staff and management
to ensure that safety training is mandatory. The laboratory is responsible
for maintain ing a current awareness file of OSHA regulations regarding
the safe handling of the chemicals specified in this method. A reference
file of data handling sheets should also be made available to all personnel
involved in these analyses. Additional information on laboratory safety
can be found in references 10-12.
5.1 Chemicals and reagents should be properly labelled and stored in an
area appropriate to their properties. Any reagents whose composition or
properties may change with time must be dated and properly disposed of
on or before the expiration date.
5.2 Areas where strong oxidizing agents and flammable or explosive materials
are used should be well labeled and the necessary restrictions imposed.
5.3 Where laboratory apparatus and instrumentation are used, the manu-
facturer's safety precautions should be strictly followed.
6.0 APPARATUS AND EQUIPMENT
6,1 Sample Containers - wide-mouth screw cap jars made of either glass
or non-contaminating plastic (linear or high density polyethylene or equiva-
lent). All containers should be pre-rinsed with dilute acid and distilled
deionized water (DOW) as described in Sect. 10.6.
6.2 Dissection Tools - scalpels should be made of high-quality, corrosion-
resistant stainless steel, while tweezers and cutting surfaces should be
plastic or teflon. All tools should be thoroughly rinsed with DOW prior
to use and between samples.
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6.3 Tissue Gr inder/Homogen i zer - (e.g., Tekmar Ti ssuemizer, Tekrnar Co.,
Cincinnati, OH) a standard tissue homogenizer can be used with ninor modifi-
cations. If the apparatus contains stainless steel parts, they snould
be replaced with tantalum or titanium. Stainless steel blades used during
homogenization have been found to be a source of nickel and chromium contam-
ination .
6.4 Digestion Vessels - 125 ml borosilicate glass Erlenmeyer flasks equipped
with all glass reflux caps (Tuttle covers). Tuttle covers or equivalent
reflux caps are essential for preventing evaporative loss of volatile compounds
or elements during high temperature digestion. They are commercially available
(Fisher Scientific) or are easily produced from borosilicate test tubes.
6.5 Hot Plate - a thermostatically controlled plate with a range of 75 to
4000 C.
6.6 Fume Hood - a properly constructed hood capable of withstanding acid
fumes. It must be equipped with an exhaust fan having sufficient capacity
to remove all fumes.
6.7 Atomic Absorption Spectrophotometer (AAS).
6.7.1 The AAS must have sufficient sensitivity and stability to perform
within the specifications required by the method (Sect. 11). The instrument
should have automatic background correction, direct aspiration flame, as
well as flameless capabilities. The instrument must have a routine maintenance
program to ensure proper performance and trouble-free operation. All source
la/nps should be handled with care and the exit windows kept free of dust and
fingerprints. Periodic intensity and stability checks of the lamps should
be made. Replace any lamps showing signs of deterioration (reference 13).
6.7.2 A graphite furnace (also called carbon rod) attachment for
the AAS is recomierKJed when determining most elements in the low concentration
ranges. Most, if not all, AAS manufacturers offer this equipment as an
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accessory. The stability and sensitivity afforded by the furnace is typically
one to two orders of magnitude better than direct aspiration (reference 14).
6.7.3 In addition to the graphite furnace, another flameless attachment
can be used in conjunction with the AAS to determine the hydride-form ing
elements (arsenic, antimony, and selenium). Most such attachments may
also be used to analyze for mercury using the cold vapor technique. These
methods are preferable to the graphite furnace since they vaporize the
analyte from the sample matrix prior to detection.
7.0 REAGENTS AND CONSUMABLE MATERIALS
The purity of all reagents used for trace metal determinations is
extremely important. Reagents should be checked for purity prior to use
to confirm the absence of contamination (reference 6). Low level analyses
wiTI require Ultrex grade acids (J.T. Baker) or equivalent. Instra-Analyzed
grade acids {J.T. Baker) or equivalent may be suitable for less sensitive
analyses. Copper contamination may be particularly troublesome when Instra-
Analyzed acids are used.
7.1 Distilled Deionized Water (DDW) - a water purified by distillation
(or equivalent) followed by conditioning with a mixed bed ion exchanger.
Such units are commercially available and yield a water with a typical
resistivity of 18 megohms/cm.
7.2 Hydrochloric Acid - concentrated (35 percent).
7.3 Hydroxylamine Hydrochloride [20 percent (w/v)]: - dissolve 20 g of
American Chemical Society (ACS) grade NH20H-HC1 in 100 mL of ODW. Store
in a precleaned glass or plastic bottle - prepare weekly.
7.4 Nitric Acid - concentrated (70 percent).
7.5 Perchloric Acid - concentrated (70 percent).
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7.6 Sodium Borohydride, ACS Grade Granular or Powder.
7.7 Sodium Hydroxide, ACS Grade - pellets or flakes,
7.8 Stannous Chloride [20 percent (w/v}] - dissolve 20 g of ACS grade
SnCl2 in 20 ml of concentrated hydrochloric acid. Warm gently until solution
clears, cool and add DDW until the solution reaches a 100 ml volume. Store
in a precleaned glass or plastic bottle - prepare fresh daily.
7.9 Stock Standard Solutions - These standards (typically 1,000 ppm) can
be purchased as certified solutions or prepared from ACS grade metal salts
and pure compounds. Suitable procedures for preparing stock solutions
are well documented (e.g., reference 15) and include the steps below.
7.9.1 Accurately weigh 1,000 mg of pure metal or metal equivalent
of the salt and dissolve in a minimum amount (usually about 20 ml) of an
appropriate acid. Once the reagent is dissolved, dilute the solution to
1,000 ml with DDW and store in a precleaned plastic bottle. The solution
is usually stable for at least 1 year but must be checked periodically
against an in-house control standard (Sect. 10).
8.0 SAMPLE COLLECTION, PREPARATION, AND STORAGE
8.1 The major difficulty in trace metal analyses of tissue samples is
controlling contamination of the sample. In the field, sources of contamination
include sampling gear, winches or steel cables, engine exhaust, dust, or
ice used for cooling (reference 16). Care must be taken during handling
to avoid these and any other possible sources of contamination. For example,
stainless steel collection and handling devices (e.g., grab samplers or
sieves used for infaunal collection from sediments) are suitable. The
ship should be positioned such that the engine exhausts do not fall on
deck during sampling. To avoid contamination from melting ice, the samples
should be wrapped in aluminum foil and placed in watertight plastic bags.
The ojter skin of the fish or shell of the shellfish is protection against
metals contamination from the aluminum foil.
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8.2 Sample resection (i.e., surgical removal of tissue) and any subsampling
of the organisms should be carried out in a controlled environment (e.g.,
a dust-free room). In most cases, this requires that the organisms be
transported on ice to a laboratory, rather than being resected on board
the sampling vessel. It is recommended that whole organisms not be frozen
prior to resection if analyses will be conducted only on selected tissues,
because freezing may cause internal organs to rupture and contaminate other
tissue. If organisms are eviscerated on board the survey vessel, the remaining
tissue (e.g., muscle) may be wrapped as described above and frozen.
8.3 Resection is best performed under "clean room" conditions. The "clean
room" should have positive pressure and filtered air. The "clean room"
should also be entirely metal-free and isolated from all samples high in
contaminants (e.g., hazardous waste). At a minimum, care should be taken
to avoid contamination from dust, instruments, and all materials that may
contact the samples. The best equipment to use for trace metal analyses
is made of quartz, TFE, polypropylene, or polyethylene. Quartz utensils
are ideal but expensive. To control contamination when resecting tissue,
separate sets of utensils should be used for removing outer tissue and
for removing tissue for analysis. For bench liners and bottles, borosilicate
glass would be preferred over plastic if trace organic analyses are to
be performed on the same sample.
8.4 Resection should be conducted by or under the supervision of a competent
biologist. For fish samples, special care must be taken to avoid contaminating
target tissues (especially muscle) with slime and/or adhering sediment
from the fish exterior (skin) during resection. The incision "troughs"
are subject to such contamination; thus, they should not be included in
the sample. In the case of muscle, a "core" of tissue is taken from within
the area boarded by the incision troughs, without contacting them. Unless
specifically sought as a sample, the dark muscle tissue that may exist
in the vicinity of the lateral line should not be mixed with the light
muscle tissue that constitutes the rest of the muscle tissue mass.
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3.5 After the appropriate tissues are resected, samples should De store-
in suitable containers (Sect. 6.1) and frozen at -20° C until analysis.
Although specific holding times have not been recommended by U.S. EPA or
other federal agencies (e.g., National Bureau of Standards), a holding
time of 6 mo (except for mercury samples, which should be held no longer
than 28 days) would be consistent with that recommended for water samples.
9.0 CALIBRATION AND STANDARDIZATION
9.1 Calibration standards are prepared by serial dilutions of the stock
solutions. The acid matrix of the standrds should be as closely matched
to the samples as possible (i.e., approximately 1 percent (v/v) HN03 and
4 percent (v/v) HClO/i).
Mixed standards of more than one element may be prepared only after
their compatibility has been determined. Some common mixed standards are
as follows:
Cd, Cu, Pb, Ni, and Zn
As, Se, and Sb
9.1.1 Do not add an incompatible anion to a mixed or single element
standard. For example, adding chloride to a silver standard could form
a precipitate of silver chloride (AgCl).
9.1.2 Do not mix metals that are incompatible in solution. For example,
lead and chromium may form a precipitate of lead chromate (PbCr04).
9.2 Concentration ranges of the standards should bracket those for the
samples to be analyzed. At least four analyses (one blank and three standards
of increasing concentration) should be used to calibrate the instrument
at the beg in ing of each shift.
9.3 Stability of a calibration standard varies with element, acid matrix,
concentration, and presence of other elements. As a general rule, standards
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should be continuously monitored and replaced when necessary. As a matter
of protocol, the following can be used as a guideline:
less than 0.1 ppm - prepare daily
0.1 to 1 ppm - prepare weekly
1.0 to 10 ppm - prepare monthly
10 to 100 ppm - prepare quarterly
100+ ppm - prepare yearly (at a minimum)
9.4 Initial Standardization - follow manufacturer1s suggestions for standard-
izing the instrument and check sensitivity performance with specifications.
If performance is acceptable, proceed with analysis; if not, refer to manu-
facturer's troubleshooting guide.
9.5 After standardizing the instrument, analyze an independent control
standard as a check. If the result is acceptable, proceed; otherwise,
troubleshoot calibration standards, control standard, or instrument.
9.6 Ongoing Calibration (reference 17) - the instrument should be tested
with a single point calibration every 2 h during an analysis run or at
a frequency of 10 percent of the analyses, whichever is more frequent.
A calibration check must also be run after the last sample in a laboratory
shift. A standard concentration in the middle of the initial calibration
range should be used.
If the difference between the ongoing calibration result and the known
standard concentration is greater than ^10 percent (or +2Q percent for
mercury analysis), the instrument must be recalibrated and the preceding
10 samples reanalyzed for the analytes affected.
9.7 In the event that a sample is outside of the linear response of the
instrument, it must be diluted to within range or reanalyzed using a less
sensitive setup. This is commonly accomplished by calibrating the instrument
with higher concentration standards using a secondary or tertiary wavelength
having less sensitivity.
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10.0 QUA LI TY CGNTRCL ,see reference 17 ana Quality Assurance/Qua! it./
Control tQA/QC) for 301(h) Monitoring Programs: Guidance on Field arc
Lcooretcry Metnoos (Terra Teen 1936].]
A quality control program enables trie assessment of the precision
and accuracy of data. Precision is estimated by analysis of replicates.
Accuracy is estimated by the analysis of blanks, spiked samples, and/or
laboratory control samples (reference 18).
10.1 Replicates can be cnosen to reflect the precision of most stages
of the overall analytical metnod. Replicates can consist of different
subsamples of a tissue homogenate or replicate instrumental analyses of
the same digestion extract.
10.1.1 Replicate analyses of tissue subsamples are important Decause
"the greatest potential for sample deterioration and/or contamination occurs
during preanalysis steps of sample collection, handling, preservation,
and storage" (reference 19).
10.1.2 Replicate analyses of a digestate focus only on the bench
chemistry and/or instrumental variability of the method. Together with
replicate analysis of tissue subsamples, they can be used to assess the
impact of each stage on the overall precision of the analytical result.
10.1.3 At least one replicate (a subsample of a tissue homogenate)
must be analyzed from each group of samples of a similar matrix type and
concentration for each batch of samples or for each 20 samples, whichever
is more frequent. If two analytical methods are used for the same element
in a batch of samples, duplicates must be run by each method used.
The relative percent differences (RPD) for each component are calculated
as follows:
D - D
RPD =
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where
DI = first sample value.
$2 = second sample value.
10.2 As in the case of replicates, blanks can be chosen to address most
stages of the overall analytical method. They include transportation,
dissection, reagent, and calibration blanks.
10.2.1 Transportation blanks are derived from empty containers that
have been stored with samples in the field and carried with them to the
laboratory. A small amount of 5 percent (v/v) HNQ3 is used to rinse the
inside of the container. The acid rinse is then retained for analysis.
Transportation blanks serve as estimates of contamination during preanalysis
steps (Sect. 10.1.1).
*
10.2.2 Resection blanks are used to estimate concentration from resection
utensils that may carry over from one sample to the next. They are prepared
by collecting a final rinse after cleaning utensils that have been used
for resection. The final rinse should be performed with a known volume
of 5 percent (v/v) HN03. One resection blank should be analyzed for each
batcn of samples.
10.2.3 Reagent (preparation) blanks are al iquots of 5 percent (v/v)
HN03 that are processed through each sample preparation step (e.g., reagent
addition, digestion, dilution). At least one reagent blank must be prepared
for each batch of samples or for every 20 samples, whichever is more frequent.
Reagent blanks serve as estimators of contamination resulting from the
chemical analysis steps.
All samples with at least one analyte concentration that is less than
10 times the corresponding concentration in the associated reagent blank
must be redigested and reanalyzed.
10.2.4 Calibration blanks consist of 5 percent (v/v) HN03 and are
analyzed each time the instrument is calibrated, at the beginning of each
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analysis run, and at a frequency of 10 percent during a run. Calibration
blanks are used to ensure that the analytical instrument is not introducing
false positive results during analysis. (Ongoing calibration quality assurance
is discussed in Sect. 9.6.)
10.3 The results obtained from the reagent blanks can be used to calculate
the LOO (Sect. 3} for the method. This is the assigned minimum value above
which reliable data can be reported. Results for at least the reagent
blank should be reported with the final data set.
10.4 Spiked samples are samples to which small volumes of standard solutions
of the elements of interest have been added. Spiked samples provide a
means of assessing losses during digestion, distillation, or other pretreatment
steps. The spike is added before the pretreatment steps and should be
0.5 to 2.0 times the concentrations of the elements in the sample. At
least one spiked sample must be analyzed for each batch of samples of a
similar matrix type and concentration or for each 20 samples, whichever
is more frequent.
10.4.1 The percent recovery for each element is calculated as follows:
% Recovery = (spike + sample result) - (sample result) x 1QO
J (spike added)
Spike percent recoveries should not be used to determine a correction factor
to compensate for losses.
10.4.2 If graphite furnace AA is used, a single analytical spike
is required after any digestion steps to determine if the method of standard
additions (MSA) is required (reference 17 was used to develop this section).
The spike should be added at a concentration (in the sample) that
is twice the LOD. The unspiked sample aliquot must be compensated for
any volume change in the spiked samples by addition of DDW to the unspiked
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sample aliquot. The percent recovery of the spike should be calculated
as in Sect. 10.4.1
10.4.2.1 If the sample absorbance or concentration is >50 percent
of the spikel ana the spike recovery is between 85 percent and 115 percent,
the sample should be quantified directly from the calibration curve.
10.4.2.2 If the spike recovery is less than 40 percent, the
sample must be diluted and rerun with another spike. Dilute the sample
by a factor of 5 to 10 and rerun. This step must only be performed once.
If after dilution the spike recovery is still <40 percent, there are inter-
ferences associated with the instrumental technique that prevent GFAA analysis
of the sample.
10.4.2.3 If the spike recovery is >40 percent and the sample
absorbance or concentration is <50 percent of the spikel, report the analyte
as less than the LOD or less than the LOD times the dilution factor if
the sample was diluted.
10.4.2.4 If the sample absorbance or concentration is >50 percent
of the spikel and the spike recovery is <85 percent or >115 percent, the
sample must be quantified by MSA.
10.4.2.5 The following procedures should be incorporated into
MSA analyses.
a) Data from MSA calculations must be within the linear range
as determined by the calibration curve generated at the
beginning of the analytical run.
l[Note that spikel is defined throughout Sect. 10.4.2 as (absorbance or
concentration of spike sample) minus (absorbance or concentration of the
sample.]
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b) The sample and three spikes must be analyzed consecutively
for MSA quantitation (the "initial" spike run data is speci-
fically excluded from use in the MSA quantitation).
c) Spikes (post-digestion, as for the "initial" spike in Sect.
10.4.2) should be prepared such that:
Spike 1 is approximately 50 percent of the sample
absorbance.
Spike 2 is approximately 100 percent of the sample
absorbance.
Spike 3 is approximately 150 percent of the sample
aosorbance.
d) The data for each MSA analysis should be clearly identified
in the raw data documentation along with the slope, intercept
and correlation coefficient (r) for the least squares fit
of the data.
10.5 Laboratory control samples are certified reference material s (CRM)
submitted blind to the laboratory. CRM provide an estimate of the accuracy
of the overall method. A CRM must be chosen that has a similar matrix
to samples and contains all the analytes. CRM can be purchased from a
number of agencies and are available in a range of matrices (e.g., U.S. EPA
Trace Metals in Fish Tissue or NBS Oyster Tissue).
10.5.1 Unlike an analyte spike (Sect. 10.4), a CRM tests the dissolution
technique as well as instrument calibration and matrix interferences.
10.5.2 A minimum of one CRM should be analyzed for each survey or
2 percent of the total number of samples (i.e., 1 per 50 samples), whichever
is more frequent).
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10.5.3 The percent recovery for eacn element for the overall method
is calculated as follows:
% Recovery = -^ x 100
where
x = the analytical result for the element
REF = the certified result for the element.
The data obtained for each reference material should be used to troubleshoot
the method if results fall outside the acceptable range (i.e., the 95 percent
confidence interval). Percent recovery values should not be used to determine
a correction factor to compensate for apparent procedural losses,
10.6 Maintenance of Records - the data obtained from any QC work should
be recorded in an organized manner to allow for easy retrieval and reviewing.
If sufficient data have been collected, it is recommended that these be
plotted on a control chart for a quick visual assessment. A typical control
chart for CRM results is presented in Figure III-l.
10.6.1 The quality control chart can be used to determine if the
following recommended guidelines are met:
10.6.1.1 Not more than 5 percent of the results lie outside two standard
deviations (warning limit). A result outside three standard deviations
requires action.
10.6.1.2 There are no regular periodic variations.
10.7 Cleaning and preparation of labware is an integral part of a quality
assurance/quality control (QA/QC) program. Many cleaning procedures have
been proposed in the literature that are suitable for decontaminating equip-
ment. The main concerns with cleaning are removing elements of interest
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from labware while maintaining an inactive surface. Sons cleaning procedures
tend to be too harsh, producing an surface with an ion exchange capacity.
In this case a solution could partially or completely "lose" an analyte
to the container walls {references 20, 21).
10.7.1 If at all possible, labware should not be used for work where
analyte concentrations vary by more than ten times. For example, never
use glassware for tissue analysis that has also been used for sediments.
If one can use dedicated glassware, the cleaning requirements are greatly
simp! if led.
10.7.2 A good universal cleaning procedure for glass and plasticware
is out! ined below.
10.7.2.1 Wash labware with a metal-free detergent and warm water.
10.7.2.2 Rinse at least three times with tap water followed
by distilled deionized water (DDW).
10.7.2.3 Soak equipment or labware in a dilute acid (25 percent
HN03) bath for 24 h. If possible, the bath should be maintained at an
elevated temperature {70° C).
10.7.2.4 Rinse labware with large volumes of DDW and use imme-
diately. If a time lapse must exist, the apparatus should be stored under
dust-free conditions and rinsed further with DDW prior to use.
NOTES: - Change the acid batch periodically such that no significant
buildup of metals occurs.
At no time should a metal containing reagent such as chromic
acid be used.
10.8 Round Robin or Interl aboratory Check Programs - In addition to the
quality control measures discussed above, all laboratories should participate
in interlaboratory check programs.
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11.0 PROCEDURE
11.1 Homogenize samples prior to analysis to ensure that a representative
aliquot is taken. Any grinder or homogeni zer that has been found to be
free of contamination may be used (Sect. 6.3). Samples should be ground
wet to avoid losses of volatile elements (e.g., Hg, Se) during drying.
The liquid associated with a sample after thawing should be retained as
part of the sample.
11.2 Transfer the sample paste to a container suitable for storage. If
not immediately analyzed, the samples should be frozen (-200 C) until required.
Containers should be tightly sealed to prevent moisture loss or gain during
storage.
*
11.3 Dry Weight Determination - if sample size permits and dry-weight
concentrations are required, dry weight determinations may be performed
as follows: transfer an aliquot of approximately 3 g (weighed to the nearest
0.1 g) to a preweighed dish. Allow the sample to dry in an oven at 1050 c
overnight, and determine the solid residue weight to the nearest 0.1 g.
The percent total solids is calculated as:
Ts = [dry residue wt (g)]/[wet sample wt (g)]
Dry weight determinations should not be made at the cost of having insufficient
sample for metals analysis. Significant decreases in the size of samples
used for extraction will decrease attainable detection limits.
11.4 Accurately weigh representative al iquots of homogenized tissue to
the nearest 0.1 mg. If sample size permits, approximately 5 g is required
to maintain optimum detection limits. Transfer the weighed tissue to a
precleaned 125-mL Erlenmeyer flask equipped with an all-glass reflux cap.
Analyze a sufficient number of reagent blanks, sample duplicates, analyte
spikes, and certified reference materials concurrently (Sect. 10).
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11.5 Add 10.0 ml of concentrated nitric acid (ACS grade or better), replace
cap and swirl. Allow flask to stand at room temperature for about 15 hours
in a dust-free ventilated environment. Periodically swirl the contents
to help solubilize the tissue.
11.6 After 15 hours, gently heat the flask to approximately 1000 C - hold
at this temperature for 1 hour. Gradually increase the temperature in
50° C increments to a maximum of 2500 c. Continue digesting until all
tissue has been solubilized. This usually takes about 4 hours. Do not
rush the initial digestion as losses of volatile elements will likely occur.
Once digestion is complete, cool flasks to room temperature and add 4.0 ml
of perchloric acid.
CAUTION: Perchloric acid is a strong oxidizing agent. The analyst
must be fully aware of the precautions assod a ted with
Its use. This procedure (i.e., use of perchloric acid
at sub-fuming temperatures) has been safely performed
without a perchloric hood, but a perchloric hood is
strongly recommended nonetheless. Laboratories that
do not carefully monitor perchloric acid digestions
will be endangered without perchloric hoods. Safety
precautions and background information pertaining to
perchloric acid can be found in reference 22.
11.7 Return flasks to the hotplate which has been cooled to about 200° C.
Continue heating for 1 hour, then increase plate temperature to 300° C.
Hold at this temperature until all traces of nitric acid fumes have disappeared
and the solutions have become clear. Do not overheat flasks or allow perchloric
fumes (dense white) to appear. If perchloric fumes appear, reduce heat
immediately. Remove the extracts when clear and cool them to room temperature.
11.8 When the digestion is complete, rinse the caps into the flasks and
transfer the extract to a precleaned 100-mL volumetric flask. Rinse the
Erlermeyer flask three times with DDW and combine with the extract previously
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added to the volumetric flask. Adjust the volume with DDW and transfer
to a precleaned plastic bottle.
NOTE: Some elements are not as stable as others in solution and therefore
should be analyzed first. Stability can be determined by daily analysis
of the extracts, however, the following can be used as a guideline:
Sb, Pb, Hg, Se and Ag - analyze within one day
As and Cd - analyze within two days
Cr, Cu, Ni and Zn - analyze within one week
Be and Tl - to be determined.
11.9 Instrumental Analysis - The extracts will be analyzed using various
techniques of atomic absorption spectrophotometry (AAS). The method of
choice (i.e., GFAA vs HYDAA) depends on instrument availability, analyte
concentration and sample matrix. In some instances it may be useful to
use more than one AAS method to confirm a result.
11.9.1 Follow the manufacturer's instructions for initial setup and
calibrate as outlined in Sect. 9 of this method. As every instrument responds
uniquely to a given set of conditions, it is the analyst's responsibility
to develop the optimum set of parameters. Use calibration standards and
CRM to ensure that optimum conditions exist.
11.9.2 Table III-l lists some general information for each of the
priority pollutant metals.
11.9.3 It is possible to use alternate methods of detection providing
they have been validated using a sufficient number of previously analyzed
samples or CRM.
11.10 All data generated must be clearly recorded on a strip chart, printer
or manually logged in prepared tables. The order in which the extracts
are analyzed should be the same as it appears in the records. The data,
when assembled, should be reported in consistent units (i.e., mg/L) to
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avoid errors when calculating the final results (ug/g). The final report
should contain all necessary methodology, results, quality control data
(e.g., reagent blank values) and limits of detection for each element.
The report must clearly state if any data were blank-corrected.
12.0 CALCULATIONS
12.1 All results are reported as micrograms of element per wet gram of
tissue:
ug/g ELEMENT
(wet weight basis)
where:
C = concentration (may be blank corrected) of element in final extract
(ug/mL)
V = volume of final extract (mL)
W = weight of wet tissue (g)
Reagent blank corrections may be made and blank values must always be reported.
13.0 PRECISION AND ACCURACY
In order to estimate precision and accuracy (single lab, multi-operator),
a number of CRM and analyte spikes were analyzed using this method. Table
III-2 summarizes typical data obtained. No data are currently available
for beryllium or thai!inn.
14.0 REFERENCES
1. Taylor, O.K. 1985. Standard reference material s: handbook for SRM
users. National Bureau of Standards Special Publicaton 260-100.
National Bureau of Standards, Washington, DC.
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2. Keith, L.J., W. Grummet, J. Deegan, Jr., R.A. Libby, J.K. Taylor,
and G. Wentler, 1983. Principles of environmental analysis. Anal.
Chem. 55:2210-2218.
3. Manning, D.C., and W. Slav in. 1983. The determinatin of trace elements
in natural waters using the stabilized temperature platform furnace.
Applied Spectroscopy 37:1-11.
4. Hinderberger, E.J., M.L. Kaiser, and S.R. Koirtyohann. 1981. Furnace
atonic absorption analysis of biological samples using the L'vov platform
and matrix modification. Atomic Spectroscopy 2:1-7.
5. Sturgeon, R.E., S.N. Willie, and S.S. Berman. 1985. Preconcentration
of selenium and antimony from seawater for determination by graphite
furance atomic absorption spectrometry. Anal. Chem. 57:6-9.
6, Murphy, T.J. 1976. The role of the analytical blank in accurate
trace analysis, pp. 509-539. In: Accuracy in Trace Analysis: Sampling,
Sample Handling, and Analysis. National Bureau of Standards Special
Publication 422. National Bureau of Standards, Washington, DC.
7. Skoog, D.A. 1985. Principles of Instrumental Analysis. Saunders,
Philadelphia, PA. pp. 270-279.
8. Veil Ion, C. 1976. Optical atomic spectroscopic methods, pp. 123-181.
In: Trace Analysis: Spectroscopic Methods for Elements. D. Winefordner
(ed). Wiley, New York.
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