626 JULSHAMN & BRENNA: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO.
3, 2002
FOOD CHEMICAL CONTAMINANTS
Determination of Mercury in Seafood by Flow Injection–Cold
Vapor Atomic Absorption Spectrometry after Microwave
1
Digestion: NMKL Interlaboratory Study
KAARE JULSHAMN and JAN BRENNA
Institute of Nutrition, Directorate of Fisheries, PO Box 185 Sentrum, N-5804 Bergen, Norway
Collaborators: B. Åsman; A. Ekman; A. El-Ghauoui; K.-O. Gjerstad; D. Grønningen; S.H. Hammer; A.M. Jensen;
H. Liukkonen-Lilja; J. Mikkola; T. Ruth; L.O. Sedal; B. Solli; E.-R. Venalainen; N. Wollertsen
Ten laboratories participated in an interlaboratory
method-performance (collaborative) study of a
method for the determination of mercury in foods
of marine origin by flow injection–cold vapor
atomic absorption spectrometry after wet digestion
using a microwave oven technique. The study was
preceded by a training round of samples of known
identity. The method was tested on a total of 7 sea-
food products: blue mussel (Mytilus edulis), cod
muscle (Gadus morhua), crab (Cancer pagurus),
scampi (Nephrops norwegicus), black scabbard
fish (Aphnopus carbo), longnose velvet dogfish
(Centroscymus crepidater), and Portuguese dog-
fish (Cenbroscymus coelolepis) with mercury con-
centrations of 0.14, 0.24, 0.35, 0.59, 1.42, 4.2, and
13.2 mg/g, respectively. The materials were pre-
sented to the participants in the study as blind du-
plicates, and the participants were asked to perform
single determinations on each sample. Repeatability
relative standard deviations (RSDr) for mercury
ranged from 2.4 to 14.0%. Reproducibility relative
standard deviations (RSDR) ranged from 7.7 to
16.6%. HORRAT values for all samples were <1.0.
ncreased industrial activity has resulted in higher levels of
I toxic metals such as mercury in the environment. These
metals accumulate in animal tissues and are eventually
taken up by humans through the food chain. The adverse toxic
effects of mercury on humans are well documented (1). Fish
and other seafood products usually contain significantly
higher concentrations of mercury than do other foods (2). The
authorities in the European Union and other countries that im-
port seafood products require documentation of the mercury
content from the producing country.
Received July 13, 2001. Accepted by AP September 24, 2001.
Corresponding author’s e-mail: kare.julshamn@nutr.fiskeridir.no.
1
Nordic Committee on Food Analysis (General Secretariat, c/o National
Veterinary Institute, Department of Food and Feed Hygiene, PO Box 8156,
Dep. N-0033 Oslo, Norway).
Concern about mercury pollution has intensified the search
for analytical methods that require minimal sample prepara-
tion and give contaminant-free analysis with good analytical
sensitivity.
Since the introduction of microwave digestion in 1975 (3),
there has been significant interest in using microwave heating
to replace conventional heating during acid digestion. The
major advantage of microwave digestion using a
closed-vessel container is that the digestion time is signifi-
cantly reduced, and problems caused by contamination and
losses through volatilization are also minimized. Because of
these advantages, the microwave digestion technique is be-
coming more widely used in modern analytical laboratories
(e.g., in a number of Nordic Committee on Food Analysis
[NMKL] methods).
The most commonly used method for mercury determina-
tion is cold vapor atomic absorption spectrometry (CVAAS)
because of its ease of operation, high sensitivity, and relatively
inexpensive operational cost. Since it was introduced by
Hatch and Ott (4), the method has been significantly im-
proved. More recently, several flow injection (FI) approaches
have been reported (5–7). FI has numerous advantages such as
a high sample throughput, low carryover effects, small sample
size and, thus, better tolerance of interferences, and also low
consumption of reagents (6, 7).
The NMKL decided to undertake an interlaboratory study
of the determination of total mercury in seafood products by
FI–CVAAS, using a wet digestion procedure in a microwave
oven, in order to evaluate performance characteristics (true-
ness, repeatability, and reproducibility) in the analysis of judi-
ciously chosen seafood products. The method described in
this paper has been in use and accredited by Norwegian ac-
creditation authorities for several years at the Institute of Nu-
trition, Directorate of Fisheries, Bergen, Norway (8).
Interlaboratory Study
A description of the method and an invitation to participate
in the study were sent to 16 laboratories in Denmark, Finland,
Iceland, Norway, and Sweden. Ten laboratories accepted and
agreed to follow the method procedure and the time schedule
 JULSHAMN & BRENNA: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 3, 2002 627
Table 1. Expected mercury concentrations of materials
included in the study
Expected values,
Material µg/g (dry weight)a
Blue mussel (Mytilus edulis) 0.14 ± 0.01
Cod muscle (Gadus morhua) 0.24 ± 0.02
Crab (Cancer pagurus) 0.35 ± 0.03
Scampi (Nephrops norwegicus) 0.59 ± 0.04
Black scabbard (Aphnopus carbo) 1.42 ± 0.15
Longnose dogfish (Centroscymus crepidater) 4.2 ± 0.2
Portuguese dogfish (Cenbroscymus coelolepis) 13.2 ± 0.4
a
Each value is the mean ± standard deviation of 10 independent
determinations per sample.
of the study. The design, conduct, and interpretation of the
study followed guidelines recommended by AOAC INTER-
NATIONAL (9) and NMKL (10). Participating laboratories
represented industry, commercial laboratories, universities,
and government laboratories.
Study Materials
Each participant received 7 test materials (samples 1–4
were obtained from Rieber & Søn ASA, Bergen, Norway, and
samples 5–7 were obtained from The Institute of Marine Re-
search, Bergen, Norway): blue mussel (Mytilus edulis; meat
from fresh, frozen mussel that was cooked, minced, dried, and
milled to fine powder); cod muscle (Gadus morhua; cod mus-
cle powder made of fresh, frozen muscle of cod that was
cooked, dried, and milled to fine powder); crab meat (Cancer
pagurus; meat from fresh, frozen crab that was cooked, minced,
dried, and milled to fine powder); scampi powder (Nephrops
norwegicus; tails from fresh, frozen Norwegian lobster that
were cooked, minced, dried, and milled to fine powder); black
scabbard fish (Aphnopus carbo); longnose velvet dogfish
(Centroscymus crepidater); and Portuguese dogfish
(Cenbroscymus coelolepis; meat without skin from the 3 fish
species were minced, freeze-dried, and milled to fine powder).
The expected values of mercury in the materials used in the
study were obtained from the homogeneity study (Table 1).
The homogeneity of the test materials was investigated by
estimating within- and between-container variations of the 7
study materials. The homogeneity test was performed by tak-
ing 5 subsamples of 5 g from each seafood sample. Two repli-
cates of 0.25 g (according to the method) from each
subsample were digested. In total, 10 replicates of each food
sample were analyzed for mercury. Results were analyzed by
1-way analysis of variance (ANOVA). Values of P > 0.05
were obtained for all test materials, indicating that the samples
were homogeneous.
Protocol
Before the collaborative study, participants were given the
opportunity to become familiar with the method in a pretrial
test. The materials included 2 certified reference materials
(CRMs), Dorm-2 (dogfish muscle) and Tort-2 (lobster
hepatopancreas), both purchased from the National Research
Council Canada. The dogfish muscle contained a certified
mercury concentration of 4.64 µg/g, and the lobster
hepatopancreas had a certified value of 0.27 µg/g. Eight labo-
ratories reported results for the CRMs. The results showed
that some of the laboratories had problems in analyzing the
samples with acceptable trueness and repeatability. The prob-
able reason was insufficient experience with the method.
The 7 test materials used in the collaborative study were
packed dry in small plastic containers. They were presented to
participants as blind duplicates, that is, as 14 randomly coded
materials. The fact that blind duplicates were included in the
set of materials was not disclosed to the participants. Partici-
pants were asked to perform single determinations of the mer-
cury concentration of the materials according to the method
described below and to report results in mg/kg on a dry weight
basis. Participants were also asked to give information about
the microwave oven and the temperature program used, as
well as to report name and model of the AAS instrument, type
of FI system, background corrector, and acid purity used. The
residual moisture content of the dried test samples ranged
from 2 to 8%. The participants were asked to determine dry
matter for separate portions and report the values in mg/kg,
dry weight.
METHOD
Areas of Application
The method is applicable to the determination of mercury
in various types of seafood products. The method has been
tested primarily on dry products but may be used for fresh
samples as well.
Principle
Concentrated nitric acid and hydrogen peroxide are added
to samples that are then digested in a microwave oven. Any
commercially available laboratory microwave oven may be
used. The concentrations of mercury are determined by
FI–CVAAS using external standardization.
Chemicals and Reagents
(a) Concentrated nitric acid (HNO3).—65% Suprapur
(Merck, Darmstadt, Germany).
(b) Nitric acid, 0.65% (w/v).—Dilute 10 mL concentrated
nitric acid (a) to 1000 mL with water.
(c) Hydrogen peroxide (H2O2).—30%, analytical quality.
(d) Ultrapure water.—Specific resistance, >18 MΩ/cm.
(e) Hg standard.—1000 mg/L (commercial standard
solution).
(f) Hg standard solutions.—Dilute mercury standard (e)
to appropriate volumes with 0.65% nitric acid (b). The solu-
tions are prepared fresh daily.
628 JULSHAMN & BRENNA: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 3, 2002

Table 2. Interlaboratory study results (mg/g dry matter) for the determination of mercury in seafooda
Laboratory Blue mussel Cod muscle Crab Scampi Black scabbard Longnose dogfish Portuguese dogfish

1 0.150 0.253 0.350 0.530 1.24 3.64 10.9

1 0.150 0.245 0.356 0.540 1.19 3.86 11.4
2 0.165 0.258 0.368 0.538 1.26 4.07b 11.3
b
2 0.145 0.227 0.297 0.524 1.17 2.46 11.4
3 0.152 0.255 0.337 0.598 1.42 3.61 10.5
3 0.154 0.240 0.353 0.603 1.27 3.56 10.9
4 0.133 0.227 0.373 0.576 1.45 3.81 14.2b
4 0.157 0.257 0.390 0.616 1.51 4.21 12.3b
5 0.151 0.260 0.380 0.600 1.40 4.20 14.1
5 0.150 0.270 0.380 0.620 1.50 4.46 14.0
6 0.115 0.223 0.351 0.551 1.38 4.14 12.1
6 0.113 0.209 0.328 0.544 1.37 4.01 11.8
7 0.083 0.228 0.331 0.280 1.24 4.20 13.0
7 0.102 0.190 0.295 0.510 1.32 4.10 12.9
8 0.045b 0.309 0.373 0.592 1.43 3.79 10.6
8 0.170b 0.321 0.376 0.714 1.40 3.97 11.5
9 0.151 0.318 0.429 0.647 1.49 4.32 13.9
9 0.144 0.245 0.380 0.598 1.33 4.32 13.8
10 0.118 0.228 0.334 0.541 1.30 3.40 11.2
10 0.132 0.219 0.320 0.750 1.34 3.30 11.1

a
Analyzed as blind duplicates.
b
Outlier by the Cochran test (P < 0.01).

(g) Potassium permanganate (KMnO4).—Analytical
quality.
(h) Potassium permanganate solution, 5% (w/v).—Weigh
25 g potassium permanganate (g) into 500 mL volumetric
flask, and dilute to volume with water (d). Prepare solution
daily.
(i) Concentrated hydrochloric acid (HCl).—37%, analyti-
cal grade.
(j) Hydrochloric acid, 3.7% (w/v).—Dilute 100 mL con-
centrated HCl (i) to 1 L with water (d).
(k) Sodium hydroxide (NaOH).—Analytical grade.
(l) Sodium hydroxide solution, 10% (w/v).—Weigh 100 g
NaOH (k) into 1 L volumetric flask, and dilute to volume with
water (d).
(m) Sodium borohydride (NaBH4).—Analytical grade (tin
chloride as alternative).
(n) Sodium borohydride solution, 0.3% (w/v).—Weigh
3 g sodium borohydride (m) into 1 L volumetric flask, add
5 mL NaOH solution (l), and dilute to volume with water (d).
(o) Argon.—With purity of $99.998%.
Apparatus and Equipment
(a) Freeze-drier and thermal drier.
(b) Analytical balance.
(c) Laboratory microwave oven.—Equipped with 100 mL
digestion containers and capable of withstanding a pressure of
30 bar.
(d) Dispenser.
(e) Glassware.—25–1000 mL volumetric flasks; 250 mL
beakers.
(f) Polyethylene tubes or glass tubes with screw
caps.—10–50 mL.
(g) Automatic pipets with tips.
(h) Atomic absorption spectrometer.—Equipped with
background corrector (Zeeman, deuterium lamp or
high-current HCl pulsing).
(i) FI and cold vapor equipment.
(j) Autosampler.
(k) Autosampler cups.—2 mL.
(l) Electrodeless discharge lamp, hollow cathode lamp, or
boosted hollow cathode lamp for mercury.
Procedure
Dry matter content has to be determined for separate test
portion by freeze-drying or by thermal drying at 105°C until
constant weight is obtained (e.g., 12 h). Drying does not ap-
pear to cause a loss of mercury. Weigh into the digestion con-
tainer an amount of homogeneous sample corresponding to
0.20–0.25 g dry material. Each digestion series must contain
 JULSHAMN & BRENNA: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 3, 2002 629

2 reagent blanks, that is, HNO3 (Reagents, a) and H2O2 (Re-

agents, c) without sample material. If possible, include CRMs
HORRAT

0.77

0.51

0.68

0.98
0.73

0.58

0.95
containing mercury in amounts corresponding to those found
in the samples to be analyzed in order to reveal systematic or
random errors.
Note: The following section should be regarded as an ex-
Horwitz value of RSDR, %

ample. Digestion programs and amounts of acids will vary

with different digestion systems.
21.6

15.1

12.9

10.9
19.6

18.1

17.5
This program is suggested for 6 containers. For fewer or
more containers, the applied power for each step should be al-
tered proportionally, for example, for 12 containers the power
for each step should be doubled.
Digestion
R, µg/gh

0.064

0.101

0.109

0.269

0.295

0.982

3.65

Add 2 mL concentrated HNO3 (Reagents, a) and 0.5 mL

Table 3. Statistical analysis of interlaboratory study data for the determination of mercury (mg/g dry matter) in seafooda

H2O2 (Reagents, c) to each container. Seal containers and

place them in microwave oven. Start the program: S1, 250 W,
1.00 min; S2, 0 W, 1.00 min; S3, 250 W, 5.00 min; S4, 400 W,
RSDR, %g

16.6

7.7

8.8

10.7
14.3

10.9

16.6

5.00 min; and S5, 650 W, 5.00 min.

Let containers cool sufficiently before opening, and rinse
down with water any condensed water in cap and on sides of
vessels. Quantitatively transfer sample solution to 25 mL vol-
umetric flask, add KMnO4 (Reagents, h) until coloration is
sR, mg/kgf

0.023

0.104

0.347
0.036

0.038

0.095

obtained for 2–3 s, and dilute to volume with water. Then

Each value is the number of laboratories retained after elimination of outliers; each value in parentheses is the number of laboratories removed as outliers.
1.29

transfer sample solution to polyethylene tube (Apparatus, f).

The calibration of the microwave power is described else-
where (11).
Determination
r, µg/ge

0.028

0.190

0.417

0.825
0.065

0.080

0.227

If necessary, replace burner head with quartz cell. Ensure

that sample solutions do not contain any particles. Connect Hg
lamp and switch on AAS instrument. Adjust to correct wave-
length (253.6 nm) and slit (0.7 nm). Let instrument warm up
RSDr, %d

7.28

4.98

3.74

2.43
9.20

8.03

for ca 30 min. Correct gas flow of reductant, carrier solution,

14.0

and argon to obtain maximum sensitivity and repeatability.

Prepare appropriate standard solutions (i.e., 0.5, 1, 2, and
4 µg/L). Add 1 drop KMnO4 (Reagents, h) to standard solu-
tions, and dilute to 25 mL with nitric acid (Reagents, b).
sr, mg/kgc

0.010

0.067

0.147

0.292
0.023

0.028

0.080

Calculation
Calculate calibration curves for mercury by simple linear
regression on the basis of reagent blank-corrected data ob-
Mean, µg/g

tained from FI–CVAAS measurements. Calculate concentra-

0.154

0.277

0.393

0.637

1.50

4.43

tion of Hg in sample, as follows:

12.1

C = ms/w
RSDR = reproducibility relative standard deviation.
RSDr = repeatability relative standard deviation.

where C = concentration in sample (µg/g), ms = amount of

No. of labsb

sR = reproducibility standard deviation.

9(1)
9(1)

10(0)

10(0)
10(0)

10(0)

10(0)

mercury in sample solution (µg), and w = weighed amount of

sr = repeatability standard deviation.

R = reproducibility; R = 2 2 × S R .

sample (g).
r = repeatability; r = 2 2 × S r.
Analyzed as blind duplicates.

Results and Discussion

Portuguese dogfish
Longnose dogfish

The mean result from 8 laboratories in the pretrial test for

Black scabbard

mercury in dogfish muscle was 4.38 ± 0.39 mg/kg (1 standard

Blue mussel

Cod muscle

deviation [SD]); the certified value was 4.64 ± 0.26 mg/kg

Sample

Scampi
Crab

(95% uncertainty). Agreement was not found between the re-

a

h
c

f
630 JULSHAMN & BRENNA: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 3, 2002
ported results and the certified results by using z-scores, pro-
cedure 11. The mean result from 8 laboratories for mercury in
lobster hepatopancreas was 0.26 ± 0.04 mg/kg (1 SD); the cer-
tified value was 0.27 ± 0.06 mg/kg (95% uncertainty), and an
agreement was found (12).
Ten laboratories reported results in the interlaboratory
study (Tables 2 and 3). Results were statistically evaluated ac-
cording to the protocol of the International Union of Pure and
Applied Chemistry (IUPAC) for the design, conduct, and in-
terpretation of collaborative analytical studies (13). Results
were tested for the presence of outliers by using the Cochran
and the Grubbs tests. Results judged as outliers according to
one of these tests were not included in the final estimation of
the method-performance parameters. The results for blue
mussel from Laboratory 8, longnose velvet dogfish from Lab-
oratory 2, and Portuguese dogfish from Laboratory 4 were
found to be outliers by the Cochran test at the P < 0.01 level:
the replicate values of 0.045 and 0.170, 4.07 and 2.46, and
12.3 and 14.2 µg/g, respectively, were too far apart.
None of the data represented extreme values according to
the Grubbs test. Table 3 presents the estimated performance
characteristics of the method. The repeatability relative stan-
dard deviation (RSDr) of the method for the determination of
mercury was estimated to be between 2.4 (Portuguese dog-
fish) and 14.0% (scampi). The reproducibility relative stan-
dard deviation (RSDR) was estimated to be between 7.7 (black
scabbard fish muscle) and 16.6% (scampi and blue mussel).
The last column in Table 3 shows the HORRAT values,
computed as follows:
RSDR/2C–0.1505
where C is the relative concentration of the analyte in ques-
tion. The expression in the denominator is suggested by
Horwitz et al. (14) as an empirical formula to estimate RSDR
in an “acceptable” method-performance study. The formula is
based on several thousand such studies, although no further
details of the deviation are quoted. According to Horwitz et
al. (14), a series of ratios close to or consistently smaller than
1.0 indicates acceptable precision of a method. Correspond-
ingly, HORRAT values consistently near or greater than
2 probably indicate an unacceptable method with respect to pre-
cision. The same approach has been adopted by IUPAC (13).
Table 3 shows that the present method has an acceptable
precision for mercury determination. The HORRAT values
are <1.0 for all test materials.
Collaborators’ Comments
Laboratories 2 and 5 reported that they used tin chloride as
a reducing agent instead of sodium borohydride.
Response to the comment of Laboratories 2 and 5.—The
method gives a choice between tin chloride and sodium
borohydride as proposed in the method of the European Com-
mittee for Standardization (CEN) for mercury (15).
Laboratory 4 reported that it diluted the sample solutions to
50 mL with water and stabilized the calibration solutions to a fi-
nal concentration of 1% (v/v) HNO3 and 0.5% (w/v) K2Cr2O7.
Laboratory 6 reported that the method description needs a
more precise formulation for “keeping the color for a mo-
ment.” The laboratory requested a description of the quality of
the water used for dilution. The laboratory indicated that the
determination of dry matter should have been performed with
separate samples to avoid losing mercury.
Response to the comments from Laboratory 6.—It is nec-
essary to keep the color only for 1–2 s. The text of the method
was corrected. Only high-quality water is used throughout the
analysis. There is no indication of mercury loss by
freeze-drying or thermal drying at 105°C.
Laboratory 8 reported using 0.05% (w/v) potassium per-
manganate solution instead of 5% (w/v) potassium permanga-
nate solution (Reagents, h), 3% (v/v) HCl instead of 10% (v/v)
HCl (Reagents, j), 0.08% (w/v) sodium borohydride instead
of 0.3% (w/v) sodium borohydride (Reagents, n), standard so-
lutions with concentrations of 2.5, 5.0, 10, and 20 µg/L for the
external standard curve, and the quartz cell warmed to 100°C.
Laboratory 8 questioned the stability of the potassium per-
manganate solution and the Hg standard solutions.
Response to the comments of Laboratory 8.—The potas-
sium permanganate solution should be prepared weekly, and
the Hg standard solutions should be prepared daily. This infor-
mation is included in the text of the method.
Laboratory 9 reported that a minimum of potassium per-
manganate solution was used. The laboratory recommended
keeping the sodium borohydride solution in an air-tight flask
to minimize contact with air.
Acknowledgments
We are indebted to the following collaborators and co-
workers for their skillful participation in this study:
Birgitta Åsman, AgroLab Scandinavia AB, Kristianstad,
Sverige, Sweden
Asta Ekman, Helsingfors Stads Miljøcentral,
Miljølaboratoriet, Finland
Anu El-Ghauoui, Lahtis Stads Kontroll-och
Forskningslaboratoium, Lahtis, Finland
Karl Olav Gjerstad, Næringsmiddeltilsynet for
Midt-Rogaland, Stavanger, Norway
Dag Grønningen, Veterinærinstituttet, Oslo, Norway
Svein Harald Hammer, Næringsmiddeltilsynet i Salten,
Bodø, Norway
Arne M. Jensen, Næringsmiddelkontrollen i Trondheim,
Trondheim, Norway
Helena Liukkonen-Lilja, VTT Bio-och, Livsmedelsteknik,
Esbo, Finland
Jorma Mikkola, Vanda Stads Livsmedels-och
Milølaboratorium, Finland
Tomas Ruth, SGAB, Luleå Tekniska Univeristet, Sverige,
Sweden
Laila O. Sedal, Fiskeridirektoratets Ernæringsinstitutt,
Bergen, Norway
Berit Solli, Fiskeridirektoratets Ernæringsinstitutt, Bergen,
Norway
 JULSHAMN & BRENNA: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 3, 2002 631
Eija-Riitta Venalainen, Anstalten for Veterinæmedisin och
Livsmedel, Helsingfors, Finland
Nina Wollertsen, Fiskeridirektoratets Sentrallaboratorium,
Bergen, Norway
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