Method 200.11
Determination of Metals in Fish Tissue by
Inductively Coupled Plasma-Atomic Emission Spectrometry
Revision 1.3
April 1987
Theodore D. Martin, Eleanor R. Martin
and
Gerald 0. McKee
Inorganic Analyses Section
Physical and Chemical Methods Branch
Larry Lobring, Quentin Pickering
and
William Horning
Aquatic Biology Section
Biological Methods Branch
U. S. Environmental Protection Agency
Office of Research and Development
Environmental Monitoring and Support Laboratory
Cincinnati, Ohio 45268
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INDEX
Section
Number Subject Page
1 Scope and Application 1
2 Summary of Method 2
3 Definitions 2
4 Interferences 3
5 Safety 5
6 Apparatus and Equipment 6
7 Reagents and Consumable Materials 8
8 Sample Collection, Preservation and Storage 15
9 Calibration and Standardization 15
10 Quality Control 16
11 Procedure 19
12 Calculations 21
13 Precision and Accuracy 21
14 References 23
TABLES
1. Recommended Wavelengths with Locations for
Background Correction and Method Detection Limits
2. Inductively Coupled Plasma Instrument
Operating Conditions
3. Precision and Accuracy Data of Laboratory
Control Standards
4. Accuracy Data in Bluegill Fillet
5. Precision Data in Bluegill Fillet
6. Analyses Data of Bluegill Fillet
7. Comparative Data to Nitric Acid - Hydrogen Peroxide
Digestion
8. Analyses Data - NBS SRM 1566 Oyster Tissue
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NOTICE
This document has been peer and administratively reviewed within EPA.
Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
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ACKNOWLEDGMENT
The authors gratefully acknowledge William H. McDaniel, U.S.
Environmental Protection Agency (USEPA), Region 4, Analytical Support
Branch, and Gerald McKinney, USEPA, Region 7, Laboratory Branch,
Environmental Services Division, for the review and comments regarding this
method. The analytical support of James O'Dell is also acknowledged.
iii
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Method 200.11
Determination of Metals in Fish Tissue by
Inductively Coupled Plasma-Atomic Emission Spectrometry
1. SCOPE AND APPLICATION
1.1 This method is an inductively coupled plasma (ICP) atomic emission
spectrometric procedure for use in the determination of naturally
occurring and accumulated toxic metals in the edible tissue portion
(fillet) of the fish. The tissue must be taken from a fresh, not
previously frozen, fish to prevent loss of analyte or contamination
of the tissue due to cell lysis and resulting fluid exchange. The
method is not intended to be used for the analysis of dried fish
tissue. This method is applicable to the analyses of the following
metals:
Chemical Abstract Services
Metal Registry Numbers (CAS RN)
Aluminum (Al) 7429-90-5
Antimony (Sb) 7440-36-0
Arsenic (As) 7440-38-2
Beryllium (Be) 7440-41-7
Cadmium (Cd) 7440-43-9
Chromium (Cr) 7440-47-3
Copper (Cu) 7440-50-8
Lead (Pb) 7439-92-1
Nickel (Ni) 7440-02-0
Selenium (Se) 7782-49-2
Thallium (Tl) 7440-28-0
Zinc (Zn) 7440-66-6
1.2 This method also may be used for the spectrochemical analyses of
other elements commonly found in fish tissue. Specific analytes
included are the following:
Chemical Abstract Services
Analyte Registry Numbers (CAS RN)
Calcium (Ca) 7440-70-2
Iron (Fe) 7439-89-6
Magnesium (Mg) 7439-95-4
Phosphorus (P) 7723-14-0
Sodium (Na) 7440-23-5
1.3 Specific instrumental operating conditions are given and should be
used whenever possible. However, because of the differences
between various makes and models of spectrometers, the analyst
should follow the instrument manufacturer's instructions in
adapting the instrument's operation to approximate the recommended
conditions given in this method.
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1.4 Table 1 lists the recommended wavelengths with locations for
background correction for the metals presently Included 1n this
method. Also listed in Table 1 are the method detection limits
(MDLs) (1) for certain metals determined in fish tissue using
conventional pneumatic nebullzation for sample introduction into
the ICP.
1.5 Once the tissue samples have been collected, approximately 20 fish
fillet samples including the mandatory quality control samples can
be analyzed using this method during the 1.5 day work period
required to complete the analysis.
2. SUMMARY OF METHOD
2.1 A 1 to 2 gram sample of fish tissue is taken from a fresh (not
previously frozen) fish and transferred to a preweighed, labeled
polysulfone Oak Ridge type centrifuge tube. The tissue is
dissociated using tetramethylammonium hydroxide (2, 3), low heat
and vortex mixing. The resulting colloidal suspension is cooled in
an ice bath and then partially oxidized with the addition of
hydrogen peroxide while allowing the sample to stand overnight at
room temperature. The following day the metals are solubilized by
acidification with nitric acid and heat, and then diluted with
deionized, distilled water to a weight/volume ratio equal to 1 gram
fish tissue per 10 ml of solution. The diluted sample is vortex
mixed, centrifuged and finally the acidified aqueous solution is
analyzed by direct aspiration background corrected ICP atomic
emission spectrometry. The determined metal concentration is
reported in microgram/gram (ug/g) wet fish tissue weight.
2.2 The basis of the determination step of the method is the
measurement of atomic emission by optical spectroscopy. The sample
is nebulized and the aerosol that is produced is transported to the
plasma torch where excitation occurs. Characteristic atomic-line
emission spectra are produced by a radio-frequency ICP. The
spectra are dispersed by a grating spectrometer and the intensities
of the lines are monitored by photomultiplier tubes. The
photocurrents from the photomultiplier tubes are processed and
controlled by a computer system. Background correction is required
to compensate for the variable background contribution of fish
matrix and reagents to the analyte determination. The location
recommended for background correction for each analyte is given in
Table 1.
3. DEFINITIONS
3.1 Fish tissue - the skinless edible muscle tissue of the fish
commonly referred to as the fillet.
3.2 Calibration blank - a volume of deionized, distilled water
containing all reagents used to prepare the tissue for analyses.
(See 7.10).
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3.3 Calibration check standard - a single standard solution containing
all dissolution reagents and each analyte at known concentration
used to verify the calibration (See 7.11.1).
3.4 Quality control sample - a solution obtained from a source
different from that used to prepare the standard stock solution
(7.7) having known concentration values to be used to verify the
single metal stock solutions (See 10.2).
3.5 Laboratory control standard - a standard solution containing all
analytes of interest at known concentration, spiked into the
reagents matrix and carried through the entire analytical scheme as
a sample (See 10.5).
3.6 Method blank - a solution of the reagent matrix carried through the
entire analytical scheme as a sample (See 10.4).
4. INTERFERENCES
4.1 Occurrences of chromium contamination of biological samples from
the use of stainless steel have been reported in the literature
(4). Use of special cutting implements and dissecting board made
from materials that are not of interest is recommended. Knife
blades made of titanium with Teflon handles have been successfully
used.
4.2 Sample contamination and losses are held to a minimum because the
collected sample is preserved, processed and analyzed from the same
polysulfone centrifuge tube. However, antimony and chromium are
not stable in the fish matrix analysis solution and therefore, the
sample should be analyzed within 24 hours after completion of the
preparation procedure (See 11.2 to 11.7).
4.3 The processed sample ready for analysis will contain a precipitate
and possibly floatable solids as a surface layer partially covering
the analysis solution. Nevertheless, physical occlusion of metals
in these solids is not expected. Percent recovery of known spike
concentrations for all metals is near or exceeds 90% (See 13.2)
4.4 Since all samples are diluted to the same weight volume ratio
(1 gram/10 ml), all samples have a similar concentration of the
major constituents in the matrix. The major constituent elements
(Ca, K, Mg, Na and P) in the fish tissue matrix that are measured
do not suppress analyte signal intensities nor cause interelement
spectral interferences for the wavelengths and analytical
conditions recommended. However, these elements represent less
than 1/10 of the 2% dissolved solids aspirated. Since the
unmeasured constituents account for the majority of the matrix, it
is suspected that they cause the shifts in background intensity and
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molecular band contribution to wavelength signal near 190
nanometers (nm). Although background correction adjacent to the
wavelength will compensate for the majority of the broad band
interferences, it has been demonstrated that the use of correction
factors based on the reading molecular band signal at 386.17 nm can
be useful in providing the additional correction needed for the
thallium wavelength (190.8 nm).
4.5 It is reported that dissolved solids exceeding 1500 to 2000 mg/L
can cause a reduction in signal intensities. When spiked fish
tissue samples are additionally diluted by a factor of 4 to reduce
the dissolved solids to 0.5% or less, most of the observed signal
suppression biases of 5% to 15% are eliminated. However, with
dilution the MDLs are raised and the precision and accuracy of low
level analyses are affected. Since the suppression effect on each
element is nearly constant over the narrow concentration range of
interest, the slight negative bias experienced is considered
acceptable for the advantage of lower detection limits.
4.6 The number of interelement spectral interferences in the fish
tissue matrix is minimal. Listed below are all interelement
correction factors determined for the wavelengths and background
correction locations recommended in this method. Obviously, these
factors are only applicable to the instrument used in the
development of this method. However, they can be used as a guide
and are evidence that except for spiked samples, most fish tissue
analyses would not require interelement correction factors. It
should be noted that if a listed interferent is present at a
concentration of 10 yg/g or less, its apparent concentration on the
analyte channel is less than the analyte's determined MDL.
INTERELEMENT CORRECTION FACTORS
Analyte Interferent Factor
As Al + .0080
As Be - .0027
As Ni - .0056
Cr Cu - .0007
Cr Ni + .0006
Cr Fe - .0003
Pb Al - .0234
Pb Cu + .0008
Sb Cr + .0150
Sb Ni - .0087
Se Fe - .0205
Tl Molecular + .0036
Zn Cu + .0013
In Ni + .0039
A 1 yg/g concentration of interferent would either add to or
subtract from the analyte an apparent concentration in ug/g equal
to the value of the correction factor.
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4~.7 The following "off-the-line" background correction locations should
be avoided because of existing spectral Interference.
4.7.1 The low side (- 0.07 nm) of the 190.8 nra thallium wavelength
has a spectral interference from phosphorus.
4.7.2 Background correction on the low side of the 193.7 nm
arsenic wavelength below - 0.06 nm may result in a severe
negative bias.
4.7.3 The high side (+ 0.07 nm) of the 196.0 nm selenium
wavelength has a severe undefined spectral interference
originating from the tetramethylammonium hydroxide.
4.7.4 Background correction on the low side of the 259.9 nm iron
wavelength below - 0.06 nm may result in spectral
interference from 259.8 nm iron wavelength.
4.7.5 The low side (- 0.05 nm) of the 308.2 nm aluminum wavelength
has a spectral interference from argon.
4.7.6 The low side (- 0.04 nm) of the 213.8 nm zinc wavelength
read in the 2nd order has a weak spectral interference from
magnesium.
5. SAFETY
5.1 All personnel handling environmental samples known to contain or to
have been in contact with human waste should be immunized against
known disease causative agents.
5.2 Precautions should also be taken to minimize potential bacterial
infections from handling and dissecting fish. Basic good
housekeeping and sanitation practices and use of rubber or plastic
gloves is recommended.
5.3 Mobile and remote sampling locations should be equipped with a
communication system to summon help in case of an emergency. It is
recommended that field personnel not work alone.
5.4 Material safety data sheets for all chemical reagents should be
available to and understood by all personnel using this method.
Specifically, tetramethylammonium hydroxide (25%), hydrogen
peroxide (50%) and concentrated nitric acid are moderately toxic
and extremely irritating to skin and mucus menbranes. Use these
reagents in a hood whenever possible and if eye or skin contact
occurs, flush with large volumes of water. Always wear safety
glasses or a shield for eye protection when working with these
reagents.
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6. APPARATUS AND EQUIPMENT
6.1 Tissue dissecting equipment
6.1.1 Dissecting Board: Polyethylene or other Inert, nonmetalUc
material, any non-wetting, easy-to-clean or disposable
surface is suitable. Adhesive backed Teflon or plastic film
may be convenient to use.
6.1.2 Forceps: Plastic, Teflon or Teflon coated.
6.1.3 Surgical Blades: Disposable stainless steel with stainless
steel or plastic handle. (See 4.1)
6.1.4 Scissors: Stainless steel.
6.1.5 Plastic bags with water tight seal, metal free.
6.1.6 Label tape: Self-adhesive, vinyl coated marking tape,
solvent resistant, usable for temperatures from + 121 C to
- 23* C.
6.1.7 Polyvinyl chloride or rubber gloves, talc-free.
6.2 Labware - All glassware, polysulfone and Teflon containers must be
soaked and washed with detergent, rinsed with tap water, soaked in
(1 + 1) nitric acid (7.3.1) rinsed again with tap water followed by
deionized, distilled water (7.1). The use of chromic acid must be
avoided.
6.2.1 Glassware: Class A volumetric flasks and pipets of various
volumes.
6.2.2 Micropipets: Reusable graduated lambda micropipets with a
0.1 mL capacity.
6.2.3 Oak Ridge type centrifuge tubes: 30 mL capacity,
polysulfone tube with polypropylene screw closure (available
from most suppliers of laboratory equipment).
6.2.4 Storage bottles: Narrow-mouth bottles, Teflon FEP
(fluorinated ethylene propylene) with Tefzel ETFE (ethylene
tetrafluorethylene) screw closure, 125 mL and 250 mL
capacities.
6.2.5 Wash bottle: One-piece stem, Teflon FEP bottle with Tefzel
ETFE screw closure, 125 mL capacity.
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6.3 Sample processing equipment
6.3.1 Pi pet suction apparatus: Chrome plated octal with rubber
adapter, made for use with lambda and other reusable
micropipets. Clay Adams 4555, Curtln Hatheson Scientific
CAT 059-709 or equivalent.
6.3.2 Rinse stand and clamp to hold pipet suction apparatus.
6.3.3 Test tube rack: Polycarbonate tube size 25-30 mm, 3 x 8
array.
6.3.4 Dish pan: Pan of molded high density polyethylene, with an
interior dimension of 14" x 12".
6.3.5 Single pan balance: Balance capable of weighing to the
nearest 0.01 gram.
6.3.6 Analytical balance: Balance capable of weighing to the
nearest 0.0001 gram.
6.3.7 Vortex mixer: Vortex mixer with neoprene mixing head and
built-in rheostat control.
6.3.8 Centrifuge: Steel cabinet with guard bowl, capable of
reaching 2000 r.p.m. compatible with centrifuge tubes
(6.2.3), electric timer and brake.
6.3.9 Drying oven: Gravity convection oven, with thermostatic
control capable of maintaining 65* C * 5* C with an interior
dimension no smaller than 14" x 6" x 6".
6.4 Analytical instrumentation
6.4.1 The ICP instrument may be a simultaneous or sequential
spectrometer system that uses ionized argon gas as the
plasma. However, the system and the processing of
background corrected signals must be cooputer controlled.
The instrument must be capable of meeting and complying with
the requirements and description of the technique given in
Section 2.2 of the method. The instrument must be equipped
with a nebulizer capable of accepting 2% dissolved solids.
6.4.2 A variable speed peristaltic pump is required to deliver
both standard and sample solutions to the nebulizer.
6.4.3 The use of mass flow controllers to regulate the argon flow
rates, especially through the nebulizer, are highly
recommended. Their use will provide more exacting control
of reproducible plasma conditions.
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7. REAGENTS AND-CONSUMABLE MATERIAL
7.1 Deionlzed, distilled water: Prepare by passing distilled water
through a mixed bed of cation and anion exchange resins. Use
deionized, distilled water for the preparation of all reagents and
as dilution or rinse water. The purity of this water must be
equivalent to ASTM Type II reagent water of Specification D 1193
(5).
7.2 Hydrogen peroxide (H202)(CAS RN 7722-84-1), 50$, stabilized
purity certified.
7.3 Nitric acid (HNOa), cone, (sp.gr. 1.41) (CAS RN 7697-37-2), ACS
reagent grade or equivalent. Redistilled acid is acceptable.
7.3.1 Nitric acid, (1+1): Add 500 mL cone. HN03 (7.3) to 400 ml
deionized, distilled water (7.1) and dilute to 1 liter.
7.4 Hydrochloric acid (HC1), cone. (sp. gr. 1.19, CAS RN 7647-01-0);
ACS reagent grade or equivalent.
7.4.1 Hydrochloric acid, (1+1): Add 500 ml cone. HC1 (7.4) to 400
mL deionized, distilled water (7.1) and dilute to 1 liter.
7.5 Tetramethylammonium hydroxide [(Ch^NOH], (CAS RN 75-59-2),
TMAH 25% aqueous solution, electronic grade 99.9999% (metals basis)
ALFA #20932 or equivalent.
7.6 Sodium hydroxide (NaOH) (CAS RN 1310-73-2), ACS reagent grade or
equivalent.
7.7 Standard stock solutions' may be purchased or prepared from ultra-
high purity grade chemicals or metals. All salts must be dried for
1 h at 105 C unless specified otherwise.
(CAUTION: Wash hands thoroughly after handling.)
Typical stock solution preparation procedures follow:
7.7.1 Aluminum solution, stock (1 mL = 1000 pg Al) - Dissolve
0.100 gram aluminum metal in an acid mixture of 4 mL (1 + 1)
HC1 (7.4.1) and 1 mL cone. HN03 (7.3) in a beaker. Warm
gently to effect solution. When solution is complete,
transfer quantitatively to a 100 mL volumetric flask and
dilute to the mark with deionized, distilled water (7.1).
Store the solution in a screwcap Teflon FEP storage bottle
(6.2.4).
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7.7.2 Antimony solution, stock (1 mi » 1000 ug Sb) - Dissolve
0.2669 gram potassium antimony! tartrate
[K(SbO)C4H406] (CAS RN 11071-15-1) in deionized,
distilled water (7.1) and dilute to 100 mL in a volumetric
flask. Store the solution in a screwcap Teflon FEP storage
bottle (6.2.4).
7.7.3 Arsenic solution, stock (1 mL « 1000 ug As) - Dissolve
0.1320 gram arsenic trioxide (AS20a) (CAS RN 1327-53-3)
in 20 mL deionized, distilled water (7.1) containing 0.4 g
sodium hydroxide (NaOH) (7.6). Acidify the solution with 2
mL cone. HN03 (7.3) and dilute to 100 mL in a volumetric
flask with deionized, distilled water. Store the solution
in a screwcap Teflon FEP storage bottle (6.2.4).
7.7.4 Beryllium solution stock (1 mL = 500 ug Be) - Do not dry.
dissolve 0.9830 gram beryllium sulfate (BeS044H20) in
deionized, distilled water, (7.1), add 1.0 mL cone. HN03
(7.3) and dilute to 100 mL in a volumetric flask with
deionized, distilled water. Store the solution in a
screwcap Teflon FEP storage bottle (6.2.4).
7.7.5 Cadmium solution stock (1 mL = 1000 ug Cd) - Dissolve 0..100
gram cadmium metal in 4 mL cone. HN03 (7.3), dilute to
100 mL in a volumetric flask with deionized, distilled
water. Store the solution in a screwcap Teflon FEP storage
bottle (6.2.4).
7.7.6 Calcium solution stock (1 mL = 1000 ug Ca) - Suspend 0.2498
gram calcium carbonate (CaCOs) dried at 180° C for 1 hr
before weighing, in deionized, distilled water (7.1).
Dissolve cautiously by adding dropwise a minimum amount of
(1+1) HC1 (7.4.1). Add 10.0 mL (1+1) HC1 (7.4.1) and dilute
to 100 mL in a volumetric flask with deionized, distilled
water (7.1). Store the solution in a screwcap Teflon FEP
storage bottle (6.2.4).
7.7.7 Chromium solution, stock (1 mL = 1000 ug Cr) - Dissolve
0.1923 gram chromium trioxide (Cr03) in deionized,
distilled water (7.1). When solution is complete, acidify
with 1 mL cone. HN03 (7.3) and dilute to 100 mL in a
volumetric flask with deionized, distilled water (7.1).
Store the solution in a screwcap Teflon FEP storage bottle
(6.2.4).
7.7.8 Copper solution, stock (1 mL = 1000 ug Cu) - Dissolve
0.100 gram copper metal in 2 mL cone. HN03 (7.3). Dilute
to 100 mL in a volumetric flask with deionized, distilled
water (7.1). Store the solution in a screwcap Teflon FEP
storage bottle (6.2.4).
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7.7.9 Iron solution, stock (1 ml • 1000 yg Fe) - Dissolve
0.100 gram iron metal in 4 mL (1+1) HC1 (7.4.1). Dilute to
100 ml in a volumetric flask with deionized, distilled water
(7.1). Store the solution in a screwcap Teflon FEP storage
bottle (6.2.4).
7.7.10 Lead solution, stock (1 ml « 1000 yg Pb) - Dissolve 0.1613 .
gram lead nitrate [Pb(N03)2] in a minimum amount of
(1+1) HNOa (7.3.1). Add 5 ml cone. HNOa (7.3). Dilute
to 100 ml in a volumetric flask with deionized, distilled
water (7.1). Store the solution in screwcap Teflon FEP
storage bottle (6.2.4).
7.7.11 Magnesium solution, stock (1 ml = 1000 yg Mg) - Dissolve
0.100 gram magnesium metal in 2 ml (1+1) HC1 (7.4.1) and
dilute to 100 ml in a volumetric flask with deionized,
distilled water (7.1). Store the solution in a screwcap
Teflon FEP storage bottle (6.2.4).
7.7.12 Nickel solution, stock (1 mL = 1000 yg Ni) - Dissolve 0.100
gram nickel metal in 5 ml hot cone. HN03 (7.3). Cool and
dilute to 100 ml in a volumetric flask with deionized,
distilled water (7.1). Store the solution in a screwcap
Teflon FEP storage bottle (6.2.4).
7.7.13 Phosphorus solution, stock (1 ml = 1000 ug P) - Dissolve
0.3745 gram ammonium phosphate, monobasic [(NH4)H2P04]
(CAS RN 7722-76-1) in deionized, distilled water (7.1) and
dilute to 100 mL in a volumetric flask. Store the solution
in a screwcap Teflon FEP storage bottle (6.2.4).
7.7.14 Potassium solution, stock (1 ml = 1000 ug K) - Dissolve
0.1907 gram potassium chloride (KC1) previously dried at
110° C for 3 hrs, in deionized, distilled water (7.1) and
dilute to 100 ml in a volumetric flask. Store the solution
in a screwcap Teflon FEP storage bottle.
7.7.15 Selenium solution, stock (1 ml = 1000 wg Se) - Dissolve
0.1414 gram selenium dioxide (Se02) in deionized,
distilled water (7.1) and dilute to 100 ml in a volumetric
flask. Store the solution in a screwcap Teflon FEP storage
bottle (6.2.4).
7.7.16 Sodium solution, stock (1 ml = 1000 yg Na) - Dissolve 0.2542
gram sodium chloride (NaCl) in deionized, distilled water
(7.1). Add 1.0 ml cone. HMOs (7.3) and dilute to 100 mL .
in a volumetric flask with deionized, distilled water
(7.1). Store the solution in a screwcap Teflon FEP storage
bottle (6.2.4).
10
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7.7.17 Thallium solution, stock (1 mL = 1000 yg Tl) - Dissolve
0.1303 gram thallous nitrate (T1N03J in deionized,
distilled water (7.1). Add 1.0 ml cone. H»3 (7.3) and
dilute to 100 mL in a volumetric flask with deionized,
distilled water (7.1). Store the solution in a screwcap
Teflon FEP storage bottle (6.2.4).
7.7.18 Zinc solution, stock (1 ml = 1000 yg Zn) - Dissolve
0.100 gram zinc metal in 5 ml cone. HMh (7.3). Dilute to
100 mL with deionized, distilled water (7.1). Store the
solution in a screwcap Teflon FEP storage bottle (6.2.4).
7.8 Prepare four 100 mL mixed standard solutions by coobining aliquots
from the appropriate individual stock solutions (7.7) in volumetric
flasks and diluting to the mark with deionized, distilled water
(7.1). Prior to preparing the mixed standard solutions, each stock
solution should be analyzed to determine purity and should be
compared to a quality control check sample (10.2) to verify its
concentration. For the wavelength and background correction
positions recommended, prepare the mixed standard solution using
the following listed aliquot volumes of the individual stock
standards. Transfer the prepared mixed standard solutions in
screwcap Teflon FEP storage bottles (6.2.4).
7.8.1 Mixed standard solution I (Volume = 100.0 nL)
Analyte
Al
Ca
Cd
Cu
Mg
Sb
Se
Stock
Solution
7.7.1
7.7.6
7.7.5
7.7.8
7.7.11
7.7.2
7.7.15
Aliquot
Vol., mL
10.0
10.0
2.0
1.0
10.0
5.0
5.0
Analyte
Cone., ug/mL
100
100
20
10
100
50
50
7.8.2 Mixed standard solution II (Volume = 100.0 nL)
Analyte
As
Cr
Stock
Solution
7.7.3
7.7.7
Aliquot
Vol.. mL
10.0
5.0
Analyte
Cone., yg/mL
100
50
7.8.3 Mixed standard solution III (Volume = 100.0 mL)
Analyte
Na
Pb
Tl
Zn
Stock
Solution
7.7.16
7.7.10
7.7.17
7.7.18
Aliquot
Vol., mL
10.0
10.0
5.0
5.0
Analyte
Cone., yg/mL
100
100
50
50
11
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7.8.4 Mixed standard solution IV (Volume - 100.0 mL)
Stock Aliquot Analyte
Analyte Solution Vol., mL Cone., ug/mL
Be 7.7.4 2.0 10
Fe 7.7.9 10.0 100
K 7.7.14 20.0 200
Ni 7.7.12 2.0 20
P 7.7.13 10.0 100
7.9 Prepare four instrument calibration standards, each in 100 ml
volumetric flask by adding in the following order 10 ml TMAH (7.5)
and 5 ml H?02 (7.2) to 10 ml of each of the four mixed standard
solutions (7.8)(See Note 1). Allow the four solutions to stand
open for 30 minutes to vent released oxygen. After standing, add 5
ml of cone. HN03 (7.3) to each solution and dilute to the mark
with deionized, distilled water (7.1). Transfer the prepared
calibration standards in screwcap Teflon FEP storage bottles
(6.2.4).
Note 1: Prior to adding the TMAH, ^2 and HNOa to
calibration standard IV (7.9.4), add 1 mL of (1 + 1) HC1
(7.4.1) and mix. The addition of HC1 prevents the
formation of a precipitate. Also, when ^2 is added
to calibration standard IV, it must be added dropwise to
prevent sudden and violent effervescence.
7.9.1 Calibration Standard I (Volume 100.0 ml)
Analyte Cone., ug/mL
Al 10.0
Ca 10.0
Cd 2.0
Cu 1.0
Mg 10.0
Sb 5.0
Se 5.0
7.9.2 Calibration Standard II (Volume = 100.0 mL)
Analyte Cone., yg/ml
As 10.0
Cr 5.0
7.9.3 Calibration Standard III (Volume = 100.0 mL)
12
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Analyte Cone., ug/ml
Na 10.0
Pb 10.0
Tl 5.0
Zn 5.0
7.9.4 Calibration Standard IV (Volume = 100.0 nt) (See Note 1)
Analyte Cone., wg/mL
Be 1.0
Fe 10.0
K 20.0
Ni 2.0
P 10.0
7.10 Prepare a calibration blank by diluting the combination solution of
10 ml TMAH (7.5), 5 mL fyOz (7.2) and 5 mL cone. HNOa (7.3)
to 100 ml in a volumetric flask with deionized, distilled water
(7.1). Store the calibration blank in a screwcap Teflon FEP
storage bottle (6.2.4).
7.11 Prepare a calibration check standard stock solution in a 100 mi
volumetric flask by combining the following listed aliquot volumes
of the individual stock standards and diluting to the mark with
deionized, distilled water (7.1). Transfer the stock solution in a
screwcap Teflon FEP storage bottle (6.2.4).
Stock Aliquot Analyte
Analyte Solution Vol., mL Cone., ug/mL
Al 7.7.1 1.0 10.0
As 7.7.3 1.0 10.0
Be 7.7.4 2.0 10.0
Ca 7.7.6 2.0 20.0
Cd 7.7.5 1.0 10.0
Cr 7.7.7 1.0 10.0
Cu 7.7.8 1.0 10.0
Fe 7.7.9 1.0 10.0
K . 7.7.14 10.0 100.0
Mg 7.7.11 2.0 20.0
Na 7.7.16 2.0 20.0
Ni 7.7.12 1.0 10.0
P 7.7.13 10.0 100.0
Pb 7.7.10 1.0 10.0
Sb 7.7.2 1.0 10.0
Se 7.7.15 1.0 10.0
Tl 7.7.17 1.0 10.0
Zn 7.7.18 1.0 10.0
13
-------
7.11.1 At the time of calibration prepare the calibration
check standard (3.3) in a 100 nt volumetric flask by
adding in the following order, 10 ml TMAH (7.5), 5 ml
H202 (7.2) and 5 ml cone. HNOa (7.3) to 10 ml
of the calibration check standard stock solution
(7.11) and diluting to the mark with deionized,
distilled water (7.1). Transfer the calibration
check standard in a screwcap Teflon FEP storage
bottle (6.2.4).
Calibration Check
Analyte Std. Cone., yg/mL
Al 1.0
As 1.0
Be 1.0
Ca 2.0
Cd 1.0
Cr 1.0
Cu 1.0
Fe 1.0
K 10.0
Mg 2.0
Na 2.0
Ni 1.0
P 10.0
Pb 1.0
Sb 1.0
Se 1.0
Tl 1.0
Zn 1.0
7.12 Prepare the laboratory control standard stock solution in a 200 ml
volumetric flask by combining the following listed aliquot volumes
of the individual stock solution and diluting to the mark with
deionized, distilled water (7.1). Transfer the laboratory control
standard solution in a screwcap Teflon FEP storage bottle (6.2.4).
Stock Aliquot Analyte
Analyte Solution Vol., ml Cone,
Al 7.7.1 10.0 50
As 7.7.3 10.0 50
Be 7.7.4 1.0 2.5
Cd 7.7.5 1.0 5.0
Cr 7.7.7 2.0 10
Cu 7.7.8 5.0 25
Ni 7.7.12 5.0 25
Pb 7.7.10 5.0 25
Sb 7.7.2 5.0 25
Se 7.7.15 10.0 50
Tl 7.7.17 5.0 50
Zn 7.7.18 10.0 50
14
-------
7.13 Prepare an Instrument wash acid solution by diluting 50 ml of cone.
HN03 (7.3) to 1 liter with deionized, distilled water (7.1).
Store in a convenient manner. This solution is to be used to flush
the solution uptake system and nebulizer between standards and
samples.
7.14 Ice, crushed
8. SAMPLE COLLECTION, PRESERVATION AND STORAGE
8.1 Fish samples are collected using a variety of equipment, methods
and techniques such as trot lines, trawls, seines, dredges, nets,
ichthyocides and electrofishing. The technique used must be free
from contamination by metals. For example, permanganate may be
used to detoxify Rotenone but should not come in contact with the
fish to be analyzed (6).
8.2 Appropriate individual tissue samples should be taken soon after
collection of the fish and must be taken prior to freezing(7). If
dissection of the tissue cannot be performed immediately after
collection, each fish should be placed in a plastic bag, sealed and
placed on ice or refrigerated at approximately 4* C.
8.3 Prior to dissection, the fish should be rinsed with metal-free
water and blotted dry. Dissection should be performed within 24
hours of collection. Each individual fillet sample should also be
rinsed with metal-free water blotted dry, placed in a preweighed,
labeled polysulfone centrifuge tube (6.2.3) and frozen at - 20° C
or below (dry ice).
8.4 Skinless fillet samples of approximately 1-2 gm (1cm x 0.5 cm x 2
cm) should be cut from the fish using a special implement (See 4.1)
and handled with plastic forceps (8,9).
8.5 A maximum holding time for frozen samples has not been determined.
9. CALIBRATION AND STANDARDIZATION
9.1 Specific wavelengths and background correction locations given in
Table 1 and instrument operating conditions given in Table 2 should
be used whenever possible. However, because of the differences
among various makes and models of spectrometers, the analyst should
follow the instrument manufacturer's instructions in adapting the
instrument's operation to approximate the recommended operating
conditions. Other wavelengths and background correction locations
may be substituted if they can provide the needed sensitivity and
are corrected for spectral interference.
15
-------
9.2 Allow the instrument to become thermally stable before beginning.
This usually requires at least 30 minutes of operation prior to
calibration.
9.3 Profile the instrument and adjust the plasma to a previously
established condition by regulating the argon flow rate through the
nebulizer while monitoring the intensity ratio of selected atom/ion
wavelengths [e.g., Cu(I) 324.75 nm/Mn(II) 257.61 nm]
9.4 Calibrate the instrument according to the instrument
manufacturer's instructions using the prepared calibration blank
(7.10) and calibration standards (7.9).
9.5 The following operational steps should be used for both standards
and samples.
9.5.1 Using a peristalic pump introduce the standard or sample
nebulizer at a uniform rate (e.g., 1.2 ml min.~l).
to
9.5.2 To allow equilibrium to be reached in the plasma, aspirate
the standard or sample solution for. 30 seconds after
reaching the plasma before beginning integration of the
background corrected signal.
9.5.3 Use the average value of four, 4 seconds background
corrected integration periods as the atomic emission signal
to be correlated to analyte concentration.
9.5.4 Between each standard or sample, flush the nebulizer and
solution uptake system with the wash acid solution (7.13)
for a period of 60 seconds.
9.6 Analyze the calibrations check standard (7.11.1) and blank (7.10)
immediately following calibration, at the end of the analyses and
periodically throughout the sample run. The analyzed value of
the calibration check standard should be within an interval of
95% to 105% of the expected value. If the value is outside the
interval, the instrument should be recalibrated and all samples
following the last acceptable calibration check standard should
be reanalyzed.
10. QUALITY CONTROL
10.1 Each laboratory using this method is required to operate a formal
quality control (QC) program. The minimum requirements of this
program consist of an initial demonstration of laboratory
capability and the analysis of spiked samples as a continuing
check on .performance. The laboratory is required to maintain
performance records that define the quality of data thus
generated. Specific minimum QC requirements consist of:
16
-------
10.1.1 Verify the purity and concentration of the single metal
stock standard solutions (See 10.2).
10.1.2 Determine the method.detection limit for each analyte of
interest (See 10.3).
10.1.3 Analysis of method blanks to detect introduction of reagent
and labware contamination (See 10.4).
10.1.4 Analysis of a laboratory control standard to demonstrate
continuing laboratory performance (See 10.5).
10.1.5 Demonstration of the ability to generate acceptable data of
known accuracy and precision with this method (See 10.6).
10.2 Prepare the quality control sample (3.4) in the same acid matrix as
the diluted aliquot of the stock standard solution to be verified.
The concentration of the analyte should be the same in both
solutions and be within the range of 1 to.10 wg/mL. The
concentration and purity of each stock single metal solution must
be verified before preparation of the mixed standards (7.8). The
concentration of the mixed standard solutions (7.8, 7.11 and 7.12)
should be verified with a quality control sample (3.4) every 3
months.
10.3 The method detection limit (MDL) in wg/g must be determined for
each of the following analytes: Al, As, Be, Cd, Cr, Cu, Ni, Pb,
Sb, Se, Tl, and Zn. Except for As, Cu and Zn, the MDLs for all
analytes must be determined in the fish tissue matrix. Because of
background concentrations in fish tissue, the MDL determination of
As, Cu and Zn should be completed by spiking the method blank (3.6)
matrix. The MDL determinations should be made using seven
replicate samples prepared as described in the procedure (11.) and
with each sample analyzed from a separate and newly prepared
calibration curve. The concentration of the spike in the sample
should be approximately 3 times the estimated detection limit. The
determined MDL values tested in Table 1 can be used as a guide.
(Actual solution concentration in ug/mL are 10% the listed
values.) Appropriate dilutions of the laboratory control standard
stock solution (7.12) may be used for spiking.
10.4 A method blank (3.6) is to be analyzed with each group of samples.
Prepare the method blank by transferring 1.0 mL TMAH (7.5) to a
clean preweighed, labeled 30 mL polysulfone Oak Ridge type
centrifuge tube (6.2.3). Carry the blank through the entire
procedure (11.) as a 1.0 gram sample ending with a final solution
volume of 10 mL. The method blank values for the following
metals: Al, As, Be, Cd, Cr, Cu, Ni, Pb, Sb, Se, Tl, and Zn should
be below the metal's respective MDL. If the method blank indicates
contamination, attention should be given to the cleaning procedure
and the purity of the reagents should be verified.
10.5 A laboratory control standard (LCS) (3.5) is to be analyzed with
each group of samples. The LCS should contain the following
metals: Al, As, Be, Cd, Cr, Cu, Ni, Pb, Sb, Se, Tl and Zn each
17
-------
at a concentration of approximately 10 times its respective MDL.
To prepare the LCS, pipet 0.1 ml of the laboratory control stock
standard (7.12) into a clean preweighed, labeled 30 ml polysulfone
Oak Ridge type centrifuge tube (6.2.3). Add 1 mL of TMAH (7.5) and
carry the LSC through the entire procedure (11.) as a sample ending
with a final volume of 10 ml. The analyzed values should be within
* 2 standard deviations of an established mean value determined
from seven prior replicate analyses. Data in Table 3 may be used
as a guide until a sufficient number of replicates have been
determined. If an analyzed value is greater than * 2 standard
deviations, it is outside the warning limits. If it is greater
than * 3 standard deviations, the analysis is out of control. When
the analysis is out of control, take appropriate steps (10.2) to
verify the concentration of the LCS stock standard and calibration
standards. Also, take steps to ensure random contamination is not
operative.
10.6 To demonstrate precision and accuracy select one fish from each
group of samples (20 or less) and at the time of dissection collect
three adjacent 1.0 ± 0.1 gram fillets. Prepare and analyze two of
the fillet samples as duplicates to determine precision. Spike the
third fillet sample with a 0.1 ml aliquot of the laboratory control
stock standard to estimate the accuracy of the analysis.
10.6.1 To measure the precision of the analysis, the relative
difference (RD) between the duplicate analyses of Cu, Zn and
other measurable metals is compared to a previously
established critical relative difference (CRD) determined
from 15 prior duplicate analyses of the same type of tissue
and species of fish. The RD between sample duplicates is
determined by dividing their difference in concentration by
their mean concentration. The CRD can be calculated using
the following equation:
CRD = 3.27
Xi
where: Ri = is the calculated difference between the
duplicates in each set,
7i = is the mean value for the duplicate set, and
n = is the number (15) of duplicate sets analyzed.
18
-------
10.6.2 To estimate accuracy the mean value of the duplicate
analyses (10.6.1) is subtracted from the spiked fillet value
and percent recovery of the spike is determined. A spike
recovery outside the interval of 90% to 110% of the expected
values for the metals Al, Cd, Pb and Zn, can be used to
alert the analyst that the accuracy of the analysis is out
of control. Dilute an aliquot of the spiked sample 1*1
with the calibration blank solution and reanalyze the
sample. Increased percent recovery to within the desired
interval indicates a physical, dissolved solids interference
and that the accuracy is within the expected limits of the
method. Recovery of all spiked metals should not be less
than 85% or more than 115%.
11. PROCEDURE
11.1 At the start of sample processing, remove the cap from the
preweighed, labeled centrifuge tube (6.2.3) containing the sample
and reweigh the tube to determine the weight of the tissue by
difference. This can be done using a single pan balance; wipe the
outside of the centrifuge tube with a Kimwipe or suitable paper
tissue and place the tube upright in a tared 100 mL Griffin
beaker. The weight of the tissue should be between 1 and 2 grams
and expressed to the nearest 10 milligrams. Record the tissue
weight.
11.2 Using a 2 mL graduated pipet add a volume of 25% tetramethyl-
ammonium hydroxide (TMAH) (7.5) equal to the weight of the tissue
(1 ml TMAH = 1 gram tissue). The aliquot of TMAH should be to the
nearest tenth of a mL equal to the tissue weight (e.g., 1.6 mL of
TMAH for 1.62 grams of tissue). With the TMAH added, replace and
tighten the cap securely. (This will minimize the odor caused in
heating the sample mixture.) Place the sample in an open rack for
adequate heating and place the rack in a drying oven preheated to
65° C * 5° C and warm the sample for one hour.
11.3 After an hour of heating, remove the sample from the oven,
retighten the cap if loose, and mix the sample for a few seconds
using a vortex mixer set at median power setting. Return the
sample to the drying oven and heat for an additional hour.
11.4 After the second hour of heating, again vortex mix the sample and
place the mixed sample in an ice water bath for 30 minutes. This
can be done by placing the entire sample rack in a pan of ice water
of sufficient volume to envelop the base of the tube to just above
the level of the sample liquid. After 30 minutes, remove the
cooled sample from the bath and add 0.5 mL of cold 50% hydrogen
peroxide (7.2). Immediately recap the tube and tighten the cap
securely. DO NOT MIX THE SAMPLE. The action of the peroxide will
begin soon after its addition, indicated by foaming inside the
19
-------
tube. It is essential that the cap be securely tightened to
prevent losses. If the peroxide action is extrenely vigorous,
return the sample to the ice water bath to slow the reaction. If
more than one sample is being prepared, treat each sample
individually before proceeding to the next sample. Allow the
capped sample to stand overnight at room temperature for the
available oxygen to react and to complete the linited oxidation
process.
11.5 The following morning, vortex mix and then acidify the sample with
cone, nitric acid (7.3) to between 4% and 5% (v/v) acid. The
volume of nitric acid added to each sample is based on the final
volume of sample. The final sample volume is calculated by
multiplying the wet tissue weight by 10. Using a 1 mL graduated
pipet add the appropriate volume of nitric acid as indicated in the
following table:
Weight of Final Sample Volume of
Tissue, g Volume, mL Cone. HNOg Added, mL
0.80 - 1.04 8 to 10 0.4
1.05 - 1.24 10 to 12 0.5
1.25 - 1.44 12 to 14 0.6
1.45 - 1.64 14 to 16 0.7
1.65 - 1.84 16 to 18 0.8
1.85 - 2.04 18 to 20 0.9
2.05 - 2.24 20 to 22 1.0
After the acid addition, recap the tube and vortex mix the sample.
Return the tube to the drying oven preheated to 100° C and heat the
sample for an hour to solubilize the metals before proceeding.
Note: After the acid is added, solids will fall out of solution
and a precipitate will form. This is normal and to be expected.
11.6 After the period of solubilization, cool the tube to room
temperature. Uncap the tube and place the tube on the single pan
balance in a tared 100 mL Griffin beaker. Adjust the final volume
of the sample by adding deionized, distilled water from a "squeeze"
wash bottle (6.2.5) while weighing the tube to an appropriate
weight to maintain the contant weight/volume ratio of 1 gram/10
mL. The appropriate weight is calculated by multiplying the wet
tissue weight by 10 and adding the product to the recorded weight
of the empty tube.
11.7 After dilution is completed, recap the tube and vortex mix the
sample. After mixing, centrifuge the sample at 2000 r.p.m. for 10
minutes. After centrifuging, the sample may contain floatable
solids as a surface layer as well as the precipitate. Also, some
20
-------
particles may adhere to the wall of the tube. This condition is
normal and should not cause concern unless the analysis solution
actually contains suspended material. In this situation the sample
may require filtration through glass wool and also necessitate
filtration of the method blank (See 10.2) to verify the absence of
contamination from the glass wool. The sample is now ready for
analysis. Analyze the sample within 24 hours of preparation (See
4.2).
11.8 Aspirate the sample into the ICP using the same operating
conditions used in calibration (9.) while making certain the
precipitate is not disturbed and inadvertently aspirated. If the
surface of the analysis solution is partially covered with
floatable solids, proceed by removing the tip of the aspiration
tube from the wash solution (7.13) and allow an air bubble segment
to form in the sample uptake line. Reverse the pump flow and while
back pumping the air bubble insert the aspiration tube past the
floatable solids into the sample solution. Change the pump flow
back to uptake direction and aspirate the sample.
11.9 If a determined analyte of interest exceeds 10 vg/g dilute the
sample (1 + 3) with calibration blank solution (7.10) and reanalyze
the sample to verify the determined concentration.
12. CALCULATIONS
12.1 If dilutions are performed, the appropriate factor must be applied
to sample values.
12.2 Data read from the instrument in wg/mL should be rounded to the
thousandth place.
12.3 To express the data in concentrations of ug/g wet tissue weight
multiply the rounded ng/mL data by a factor of 10.
12.4 Report pg/g wet tissue weight data up to three significant figures.
12.5 Do not report data below the determined MDL.
13. PRECISION AND ACCURACY (Single laboratory, EMSL-Cincinnati)
13.1 The analyses data presented in Tables 4, 5 and 6 were generated
without the use of heating the sample to solubilize the metals
after the addition of nitric acid. The heating step was added
during the evaluation of the method primarily to improve the nature
and appearance of the processed sample and to facilitate ICP
analyses. Although the complete usefulness of the heating step is
not known at this time, it is anticipated that an increase in
dissolved solids will occur.
13.2 Table 4 lists accuracy data from two groups of seven bluegill
fillet samples. All fillets were taken from the same fish, and
spiked with 12 analytes (Al, As, Be, Cd, Cr, Cu, Ni, Pb, Sb, Se, Tl
and Zn) at two different concentration levels. The concentration
21
-------
of each analyte added to the first group of fillets was
approximately 40% of the spike concentration added to the second
group. The spike concentration selected for each analyte was a
convenient concentration for pipetting and for most analytes
similar multiple of the determined MDL concentration. (See Table 1
for a listing of the determined MDL concentration for each
analyte.) The spike concentration used for Cu and Zn were higher
MDL multiples because of naturally occurring concentrations in the
bluegill fillet. These data are expressed as the mean
concentration of the recovered spike and percent recovery based on
the theoretical value. Seven separate calibrations were used in
these determinations with one sample from each group being analyzed
on each calibration.
13.3 Table 5 lists the precision data that corresponds to the accuracy
data given in Table 4. These data are expressed as standard
deviation and relative standard deviation around the mean
concentration of the recovered spike.
13.4 Table 6 lists the mean, standard deviation, relative standard
deviation, median and range values from the analyses of 14 unspiked
fillet samples. These fillets were used as controls and taken from
the same bluegill fish used in the accuracy and precision study
described in 13.2 and 13.3.
13.5 Table 7 lists comparative data to a vigorous nitric acid-hydrogen
peroxide digestion. Twelve fillets were taken from the same
bluegill fish that had been exposed for four days to the following
metal concentrations:
Metal Nominal Concentration, mg/L
Al 0.5
Be 0.5
Cd 1
Cu 0.5
Ni 2
Pb 5
Sb 1
Zn 2
The first group of four fillets was processed according to
procedure given in this method.
The second group of four fillets was processed as described in
procedure of this method with the exception that the samples were
not heated during the acid solubilization step.
The third group of four fillets was processed using a vigorous
nitric acid-hydrogen peroxide digestion with the final dilution
containing 5% (v/v) hydrochloric acid.
22
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13.6 Table 8 lists the mean, standard deviation and percent recovery
data from the analysis of four 0.25 gram aliquots of NBS SRM 1566
Oyster Tissue. These analyses were completed utilizing the heating
step following the addition of nitric acid.
14. REFERENCES
1. Definition and Procedure for the Determination of the Method
Detection Limit, Appendix A, U. S. Environmental Protection Agency,
Office of Research and Development, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio.
2. Gross, S. B., and E. S. Parkinson, Analyses of Metals in Human
Tissues Using Base (TMAH) Digests and Graphite Furnace Atomic
Absorption Spectrophotometry, Atomic Absorption Newsletter, Vol.
13, No. 4, pp. 107-108, 1974.
3. Murphy, L., E. E. Menden, P. M. Eller, and H. G. Petering, Atomic
Absorption Determination of Zinc, Copper, Cadmium and Lead in
Tissues Solubilized by Aqueous Tetramethylammonium Hydroxide,
Analytical Biochemistry, Vol. 53, pp. 365-372.
4. Versieck, 0., and F. Barbier, Sample Contamination as A Source of
Error in Trace-Element Analysis of Biological Samples, Talanta,
Vol. 29, pp. 973-984, 1982.
5. Annual Book of ASTM Standards, Volume 11.01, American Society for
Testing and Materials, 1916 Race St., Philadelphia, Pennsylvania,
19103.
6. Standard Methods for the Examination of Water and Wastewater, 16th
Edition, 1985. Part 1006 Fish; Sample Collection and Preservation.
7. Ney, J. J., and M. G. Martin, Influences of Prefreezing on Heavy
Metal Concentrations in Bluegill Sunfish, Water Res., Vol. 19, No.
7, pp. 905-907, 1985.
8. The Pilot National Environmental Specimen Bank, NBS Special
Publication 656, U. S. Department of Commerce, August, 1983.
9. Koirtyohann, S. R., and H. C. Hopps, Sample Selection, Collection,
Preservation and Storage for Data Bank on Trace Elements in Human
Tissue, Federation Proceedings, Vol. 40, No. 8, June, 1981.
23
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Table 1. Recommended Wavelengths with Locations For
Background Correction and Method Detection Limits (MDL)
Analyte Wavelength, 1 nm
Al
As
Be
Ca
Cd
Cr
Cu
Fe
K
Mg
Na .
Ni .
P
Pb
Sb
Se
Tl
Zn
308.215
193.696
313.042
315.887
226.502
205.552 X 2
324.754
259.940
766.491
279.079
588.995
231.604 X 2
214.914 X 2
220.353
206.883
196.026
190.864
213.856 X 2
Location for
Bkgd. Correction
+ 0.061 nm
+ 0.061 nm
- 0.061 nm
+ 0.061 nm
+ 0.061 nm
- 0.030 nm
- 0.061 nm
+ 0.061 nm
- 0.061 nm
- 0.061 nm
+ 0.061 nm
- 0.030 nm
+ 0.030 nm
+ 0.061 nm
+ 0.061 nm
- 0.061 nm
+ 0.061 nm
+ 0.030 nm
HDL, yg/g
Wet Tissue Weight
0.3
0.4*
0.02
0.02
0.05
0.05*
0.08
0.2
0.2
0.6
0.5
0.07*
(1) Wavelength X 2 indicates wavelength is read in second order.
(*) MDL determined in method blank matrix.
-------
Table 2. Inductively Coupled Plasma Instrument Operating Conditions
Forward rf power 1100 watts
Reflected rf power < 5 watts
Viewing height above
work coil 16 mm .
Argon supply Liquid argon
Argon pressure 40 psi
Coolant argon flow rate 19 L min-1
Aerosol carrier argon
flow rate 630 cc min-1
Auxiliary (plasma)
argon flow rate 300 cc min-1
Sample uptake rate
controlled to 1.2 ml min-1
-------
Table 3. Laboratory Control Standards
Precision and Accuracy
Concentration, yg/g
ANALYTE
Al
As
Be
Cd
Cr
Cu
Ni
Pb
Sb
Se
Tl
Zn
THEO.
VALUE
2.00
2.00
0.10
0.20
0.40
0.80
1.00
1.00
1.00
1.00
2.00
4.00
LEVEL #1
ANALYSIS
MEAN
1.98
2.05
0.10
0.21
0.41
0.80
0.97
1.01
0.99
1.01
2.01
3.96
RSD(2)
7.3%
6.5%
5.4%
1.9%
4.3%
2.3%
3.0%
7.5%
5.9%
19.0%
5.5%
1.2%
THEO.
VALUE
5.00
5.00
0.25
0.50
1.00
2.00
2.50
2.50
2.50
2.50
5.00
10.0
LEVEL #2
ANALYSIS
MEAN
4.93
5.15
0.25
0.51
1.00
1.98
2.45
2.47
2.53
2.56
5.02
9.85
RSD(2)
1.7%
2.3%
1.7%
1.7%
2.4%
0.7%
1.0%
2.2%
2.8%
5.0%
2.3%
0.9%
1. The data are the result of analyzing one laboratory control standard solution
at each level on seven separate calibrations.
2. RSD is relative standard deviation.
-------
Table 4. Accuracy Data in Bluegill Fillet
Concentration, ug/g Wet Tissue Ueight
LEVEL #1
ANALYTE
Al
As
Be
Cd
Cr
Cu
Ni
Pb
Sb
Se
Tl
Zn
THEO.
SPIKE
VALUE
2.00
2.00
0.10
0.20
0.40
0.80
1.00
1.00
1.00
1.00
2.00
4.00
ANALYSIS
MEAN
1.75
1.91
0.10
0.19
0.35
0.75
0.90
0.92
0.94
1.13
2.13
3.72
PERCENT
RECOVERED
88%
96%
100%
95%
88%
94%
90%
92%
94%
113%
107%
93%
THEO.
SPIKE
VALUE
5.00
5.00
0.25
0.50
1.00
2.00
2.50
2.50
2.50
2.50
5.00
10.00
LEVEL #2
ANALYSIS
MEAN
4.42
4.72
0.25
0.46
0.89
1.85
2.21
2.21
2.27
2.60
4.85
8.70
PERCENT
RECOVERED
88%
94%
100%
92%
89%
93%
88%
88%
91%
104%
97%-
87%
-------
Table 5. Precision Data in Bluegill Fillet
Concentration, pg/g Wet Tissue Weight
LEVEL #1
LEVEL #2
ANALYTE
Al
As
Be
Cd
Cr
Cu
Ni
Pb
Sb
Se
Tl
Zn
ANALYSIS
MEANU)
1.75
1.91
0.10
0.19
0.35
0.75
0.90
0.92
0.94
1.13
2.13
3.72
STD.
DEV.
0.081
0.142
0.004
0.005
0.020
0.018
0.017
0.081
0.057
0.178
0.088
0.199
RSD(2)
4.6%
7.4%
4.0%
2.6%
5.7%
2.4%
1.9%
8.8%
6.1%
15.7%
4.1%
5.3%
ANALYSIS
MEAN(I)
4.42
4.72
0.25
0.46
0.89
1.85
2.21
2.21
2.27
2.60
4.85
8.70
STD.
DEV.
0.195
0.203
0.005
0.018
0.038
0.048
0.050
0.107
0.107
0.268
0.145
0.342
RSD(2)
4.4%
4.3%
2.0%
3.9%
4.3%
2.6%
2.3%
4.8%
4.8%
10.3%
3.0%
3.9%
1. Analysis mean of the spike value tested in Table 4.
2. RSD is relative standard deviation.
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Table 6. Analyses Data of Bluegill Fillets(l)
Concentration, vg/g Wet Tissue Weight
ANALYTE
1.
2.
As
Ca
Cu
Fe
K
Mg
Na
P
Zn
14 fil
RSD is
ANALYSIS
MEAN
0.64
192
0.16
0.98
3980
313
255
2220
4.49
lets taken
relative
STD.
DEV.
0.189
130
0.033
0.132
94
10
12
81
0.280
from one fish.
RSD(2)
29.5%
67.7%
20.6%
13.4%
2.4%
3.2%
4.7%
3.6%
6.2%
CALCULATED
MEDIAN
0.66
132
0.16
0.94
4010
313
256
2220
4.57
RANGE
0.28
113
0.10
0.76
3750
294
236
2130
3.89
- 0.97
- 511
- 0.22
- 1.17
- 4060
- 332
- 276
- 2390
- 4.87
standard deviation.
-------
Table 7* Comparative Data to Nitric Acid - Hydrogen Peroxide Digestion
Concentration, vg/g Wet Tissue Weight
TMAH PREPARATION
ANALYTE
AT
As
Be
Ca
Cd
Cr
Cu
Fe
K
Mg
Na
Ni
P
Pb
Sb
Se
Tl
Zn
HEATED
ANALYSIS
MEAN
< 0.3
0.43
0.22
170
0.07
< 0.05
0.29
1.21
. 3470
268
92.4
0.22
2120
1.44
< 0.2
< 0.6
< 0.5
3.72
STD.
DEV.
0.075
0.156
68
0.024
0.106
0.328
33
5.1
7.79
0.067
69
1.02
0.509
UNHEATED
ANALYSIS
MEAN
< 0.3
< 0.4
0.07
111
0.05
< 0.05
0.23
1.04
3540
272
96.0
0.16
2060
0.63
< 0.2
< 0.6
< 0.5
3.71
STD.
DEV.
0.026
17
0.029
0.069
0.179
63
9.3
5.90
0.045
52
0.631
0.347
ACID DIGESTION
ANALYSES
MEAN
2.88
< 0.3
0.13
131
0.06
< 0.05
0.25
1.60
3430
266
110
0.19
1900
1.00
< 0.2
< 0.6
< 0.5
3.40
STD.
DEV.
1.22
0.096
64
0.019
0.088
0.240
136
12
12.
0.070
52
0.755
0.500
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Table 8. Analyses Data - NBS SRM 1566 Oyster Tissue
Concentration, pg/g Dry Weight
ANALYTE
As
Ca
Cd
Cr
Cu
Fe
K
Mg
Na
Ni
P
Pb
Se
Zn
PUBLISHED
CERTIFIED
VALUE
13.4 * 1.9
1500 ± 200
3.5 ± 0.4
0.69 * 0.27
63.0 * 3.5
195 * 34
9690 * 50
1280 * 90
5100 * 300
1.03 * 0.19
8100*
0.48 * 0.04
2.1 * 0.5
852 * 14
ANALYSIS
MEAN
12.9
1290
3.11
< 0.05
59.0
134
8620
1120
4550
0.77
7110
0.53
2.47
743
STD.
DEV.
0.39
73
0.029
0.54
3.0
115
5
48
0.093
52
0.303
0.656
4.7
PERCENT
RECOVERED
96%
86%
89%
94%
69%
89%
88%
89%
75%
88%
110%
118%
87%
DILUTION(I)
MEAN
12.3
1360
3.14
N.D.(2)
60.0
141
9500
1220
4830
N.D.
7340
N.D.
N.D.
830
PERCENT
RECOVERED
91%
91%
90%
95%
72%
98%
95%
95%
91%
97%
1. The dilution mean is the reanalyses of the prepared aliquots combined and
diluted 1+3 with calibration blank solution. Reported concentration adjusted
for dilution.
2. N.D, - Not detected below MDL.
* Phosphorus value not certified.
-------
DATE: March 30, 1987
SUBJECT: Disposition of Comments, Deliverable 1320[A],
EMSL-C1ndnnat1 No. 733, Method 200.11 Determination of Metals In
Fish Tissue by Inductively Coupled Plasma-Atomic Emission Spectrometry
FROM: Theodore D. Martin, Research Chemist
Inorganic Analyses Section
Physical and Chemical Methods Branch
TO: Robert L. Booth, Director
Environmental Monitoring and Support
Laboratory - Cincinnati
Method 200.11 Determination of Metals In F1sh Tissue by Inductively Coupled
Plasma-Atomic Emission Spectrometry was completed on March 6, 1987, and
distributed to twelve reviewers for concurrent technical and administrative
review. Nine of the twelve reviewers returned comments by the required date of
March 20, 1987. All reviewers found the method acceptable, requiring only
minor revisions as noted with comments in the margins of the text.
The final revision (1.3) of the method, based on the comments received that
did not require speculative Interpretation, was completed March 26, 1987. The
roost notable change was the incorporation 1n the solubilizatlon procedure of a
heating step following the addition of nitric add. Although this change does
not appear to significantly improve recoveries. It did facilitate the
spectrometric analysis of the processed sample. The change is discussed 1n the
precision and accuracy section of the method.
EMSL-CI:TDMartin:lbh:STC:rm.554:x7312:0600d:3/30/87
-------
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
OPTICE OF RESEARCH AND DEVELOPMENT
ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY
CINCINNATI, OHIO 45268
DATE:
SUBJECT:
FROM:
TO:
Transmittal of Environmental Monitoring and Support Laboratory
Cincinnati (EMSL-C1ncinnat1) Deliverable No. 1320[A], EMSL-
Cincinnatl No. 733, "Chemical Methods for Extraction of
Inorganic Pollutants from Biological Tissue"
Robert L. Booth, Director
Environmental Monitoring and Support
Laboratory - Cincinnati
William A. Whittington, Director
Office of Water Regulations and Standards (WH-551)
Office of Water Programs
Attached are two copies of Method 200.11 "Determination of Metals in
Fish Tissue by Inductively Coupled Plasma-Atomic Emission Spectrometry"
which is being submitted in fulfillment of Deliverable Item No. 1320[A]
"Chemical Methods for Extraction of Inorganic Pollutants from Biological
Tissue" authored by Theodore Martin, Eleanor Martin, and Gerald McKee of the
Inorganic Analyses Section, Larry Lobring, Quentin Pickering, and William
Horning of the Aquatic Biology Section, EMSL-Cincinnati.
The purpose of this research was to develop and standardize an
analytical method for the measurement of metals in fish tissue that is less
time consuming, more precise and equally accurate as the existing
procedure. The method currently recommended for fish tissue analyses is a
difficult, whole fish analyses that requires a wet digestion with sulfuric
acid, dry ashing of 450° C, wet digestion with nitric acid and a dissolution
step followed by atomic absorption measurement. Most regional laboratories
are using their own variations of this method that eliminates some of these
steps. The attached method requires a dissociation step using
tetramethylammonium hydroxide, heat, oxidation with hydrogen peroxide,
dissolution with nitric acid and measurement by inductively coupled plasma
atomic emission spectrometry. This method is applicable for the analyses of
aluminum, antimony, arsenic, beryllium, cadmium, calcium, chromium, copper,
iron, lead, magnesium, nickel, phosphorus, selenium, sodium, thallium and
zinc. It contains single laboratory precision, accuracy and method
detection limit data we generated using the method and a section detailing
the quality assurance practices we believe are considered necessary to
produce valid data.
This product has been prepared according to Agency format and satisfies
the objective for the analysis of edible fish fillets for the elements
specified. This method is less time consuming, more precise and as accurate
as the Agency recommended method. We plan to distribute this method to the
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regional laboratories for comment prior to further method development. We
plan on continuing research on this technique for additional types of tissue
and more analytes. If your staff or others are Interested 1n discussing any
of the technical details of the report, please contact Gerald McKee at
684-7372 or Ted Martin at 684-7312.
Attachment: (2)
As Stated
cc: Larry Jensen without attachment
Vaun Newill without attachment
Donald Ehreth with attachment
Courtney Riordan with attachment
Elenora Karicher with attachment
Edmund Notzon with attachment
Frederick Leutner with attachment
Charles Plost with attachment
Cornelius Weber with attachment
Betty Thomas with attachment
James Lichtenberg with attachment 4 •
Gerald McKee with attachment ^jj |
Kathie Fieler with tfeo;copie^^%mej2prandum and attachment
* - v-* WV« ™f'f!f
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