Journal of the Persian Gulf

(Marine Science)/Vol. 2/No. 4/June 2011/8/11-18

Determining Total Mercury in Samples from the Persian Gulf and the
Caspian Sea: Comparison of Dry Ash and Wet Extraction Mothods
Agah, Homira 1*; Fatemi, S. Mohamad Reza 2; Mehdinia, Ali 1; Savari, Ahmad3
1-Dept. of Marine Bioseience, Iranian National Institute for Oceanography, Tehran, IR Iran, Email: agah_hom@yahoo.com
2- Dept. of Marine Biology, Science and Research Branch, Islamic Azad University, Tehran, IR Iran
3- Dept. of Marine Biology, Khorramshahr University of Marine Science and Technology,
Khorramshahr, IR Iran
Received: September 2010

Accepted: April 2011

201l Journal of the Persian Gulf. All rights reserved.

Abstract
Monitoring of mercury in environmental samples, with its proven toxicity on the food chain, requires
sensitive and accurate analytical techniques. In this study, two methods for identification and
quantification of total mercury in biological and sediment samples are compared: i) dry ash preparation
and subsequent established procedures for Combustion Atomic Absorption Spectrometry, (AMA 254) and
ii) wet extraction consisted of classical and microwave oven digestion methods combined with cold vapor
Atomic Absorption (CV AAS) analyzing method. In order to compare the accuracy of the methods for
total mercury determination, biological and sediment certified Reference Materials and environmental
samples were analyzed. The precision and accuracy of the applied analytical methods were compared on
eight Certified Reference Materials, Dorm2, Dolt 2 and Tort 2, trace metals in dog fish muscle, liver and
Lobster (provided by the national research council of Canada); NIST 1556a, NISTt-2976, IAEA 142 trace
metals and mercury in oyster, mussels and fish, respectively; IAEA 086 and IAEA405 trace elements in
hair and in polluted estuary sediment (provided by international Atomic Energy), respectively. Ten
environmental (fish and sediment) samples and one inter-comparison sample (BCR 710, Total and
methylmercury in oyster tissue) as well as certified samples were analyzed in six replications. The
detection limits (DLs) in the combustion AAS (AMA), CEM (MDS 2000) microwave oven CVAAS,
CEM (Mars 5) microwave oven CV AAS and in normal oven CV AAS for biological and sediment were
compared. The AMA 254 with lower detection and quantification limits was more sensitive analyzing
method in comparing with the other methods.
Keywords: AMA 254, CEM Microwave oven, Total mercury, Dry ash, Wet exttaction

1. Introduction
Mercury, a naturally occurring element, enters the
environment through natural and anthropogenic
sources. The toxicity of mercury and especially
methylmerecury, which shifts to human via
consumption of marine products is well known

(Oken et al., 2005; Schober et al., 2003).

Monitoring mercury in the environmental
samples with accurate and reliable analysis
techniques has been an overriding aim of any
research in recent decades. In general,
mercury analysis methods take advantage of
dry ash, wet digestion (Normal oven /
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Agah et al. / Determining Total Mercury in Samples from the Persian Gulf and the Caspian Sea: Comparison ,

in detectiing total mercury in environmental

samples.

microwave oven / distillation / solvent

extraction) and fusion techniques. Majority of
mercury determination techniques (in solid
samples) require preliminary digestion of
sample. The most frequently used instrumental
technique is Cold Vapor Atomic Absorption
Spectrometry (CV AAS) (Baeyens et al.,
1999; Nguyen et al., 1998; French et al.,
1998; Dos Santos et al., 2000; Wagemann et
al., 2000; Storelli et al., 2001). Other
instrumental techniques include Atomic
Fluorescence Spectrometry (AFS), a popular
and sensitive technique (Nguyena et al., 2005;
Jasperson, et al., 2000; US EPA, 1996; Bloom
Fitzgzrald et al., 1988; Bloom et al. 1989),
Inductive Coupled Plasma Atomic Emission
Spectrometry
(ICP AES) (Rudner et al.,
1998), Inductive Coupled plasma Mass
Spectrometry (ICP MS) (Smith, et al.,1993)
and Graphite Furnace Atomic Absorption
Spectrometry (GF AAS) (Alexiu et al., 2004;
Bulska et al., 2001; Schintu et al., 1992). In
most methods, mercury is reduced to the
elemental state and some methods require predigestion of the sample prior to reduction. All
of the afforementioned analytical techniques
are to some extent, time-consuming and
potentially succeptible to contamination
during pretreatment of samples.
Cold vapor (CV) AAS (AMA 254) is the
most widely used and is based on direct
combusting of sample (dry ash) ,preconcentration of Hg by amalgamation with
gold and Atomic Absorption Spectrometry
(IAEA, 2004).
In this study, Combustion AAS (AMA 254)
method is compared with CEM (MDS 2000 and
MARS 5, CEM Corporation) microwave ovens
and normal oven digestion methods in conjunction
with CV AAS analyzing technique in order to
assess the more sensitive and accurate technique

2. Material and Methods

All Teflon and glasswares used in extractions
and analyses in wet extraction techniques were
washed with detergent (Extrant 2%), soaked in 5%
nitric acid overnight and rinsed thoroughly with
Milli-Q water. In addition, all Teflon vessels used in
CEM microwave ovens were cleaned with 10 ml
HNO3 (65%, Merck, pa) for 10 minutes under 120
PSI pressure.
2.1. Combustion Atomic Absorption Spectrometry
Method (AMA 254)
Ten to fifty mg of sample was weighed into the
analyzing shuttle (nickel) and introduced into the
instrument. All samples were analyzed as follows:
60 seconds drying at 100C, 150 seconds
decomposing at 550C and 45 seconds cooling
down. Figure 1 demonstrates the schema of
mercury
analyzer,
AMA
254.
Thermal
decomposition was performed in a quartz
combustion tube containing cobalt oxalate and a
mixture of manganese oxide, cobalt and calcium
acetate
catalyst,
where
halogens
and
nitrogen/sulfur oxides were trapped inside it.
Mercury was selectively trapped and preconcentrated in the gold amalgamation. The
amalgamator was then rapidly heated and released
the mercury vapor to a series of two absorbance
cuvettes, then mercury was detected at 253.6 nm
(Agah et al., 2007).
2.2.Wet Extraction Methods
In this study, biological and sediment samples
were digested with classical and microwave ovens.

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Journal of the Persian Gulf (Marine Science)/Vol. 2/No. 4/June 2011/8/11-18

turntable CEM (Mars 5) microwave oven. A ramped

pressure program was applied (up to 150 PSI)
during 15 minutes, followed by a stable pressure for
15 minutes (100 % power) and 10 minutes for
cooling down. The digestion sequence was
controlled by a temperature probe placed in one of
the 14 samples. After cooling down, microwave
vessels were vented and extractions were diluted to
a final volume of 50 mL using M.Q water. Total
mercury was analyzed by CVAAS.
Normal oven CV AAS: Sediments with low
mercury levels need to optimize sample weight
(>1g) and dilution volume (<50 mL). In classical
digestion method, 1 gr. sediment was weighted in a
Teflon vessel and digested with Nitric acid and
hydrochloric acid (4:1, v: v) over night in normal
oven at 605 C (Muhaya et al., 1998). After
cooling, the resulting solution was diluted with M.Q
water.

Fig.1: Schema of the mercury analyzer system, (Cizdziel

et al., 2002)

CEM (MDS 2000) microwave oven CVAAS: In

order to analyze total mercury in fish sample, 0.1 to
0.15 g lyophilized and homogenized reference and
collected samples were digested with 6 ml HNO3
(65% nitric acid, Suprapur) in CEM (MDS 2000,
CEM Corporation) microwave oven (Nguyena, et
al., 2005) for one hour and a ramped pressure
program was applied (up to 150 PSI). Then 1 mL
H2O2 (Suprapur) was slowly added to the vessels.
The vessels were returned to the microwave oven
and a second digestion step of 30 minutes at 80 PSI
(100% power) was applied. After digestion and
cooling, microwave vessels were vented and
extractions were diluted to a final volume of 50 ml.
using M.Q water (>18 M Cm-1). Sediment sample
(0.15-0.2 g) was digested with 8 mL HCl:HNO3, 1:4
(v/v) in CEM (MDS 2000) microwave oven for one
minute at 30 % power, 4 minutes at 80 % power and
60 minutes at 100% power. All the cooling and
diluting processes were as mentioned for biological
samples (Nguyena et al., 2005).
CEM (MARS 5) Microwave oven CVAAS:
Aliquots (about 0.15g) of lyophilized and
homogenized biological samples were accurately
weighted into 50 ml. digestion vessels of CEM
(MARS 5, CEM Corporation) Microwave oven,
followed by acid digestion using 6 mL HNO3 (65 %
nitric acid, Suprapur) and 1 mL H2O2 (30 %
analytical grade) (Agah, et al., 2008a). Digestion
vessels were capped and placed into a 14-position

2.3. Analysis
Total mercury analysis was performed by CVAAS
(Hg Analyzer II, Thermo Separation Products) in all
the wet extraction techniques (Agah, et al., 2008b).
Figure 2 demonstrates the schema of Cold Vapor
Atomic Absorption Spectrometry. Prior to analysis, all
of the sediment extraction materials were filtered
through a 0.45m (Minisart, Sartorius) filter. In this
technique, 2.5 mL tin (II) chloride (SnCl2) was added
to the reaction cell and bubbled by carrier gas (pure
nitrogen) to sweep the reducing reagent free of
mercury before sample was injected into the reaction
through a sample loop. In order to clean the sample
loop about 20 mL HCI 2% (W/W) was flushed into
the reaction cell. Mercury in the resulting solution as
Hg+2 was reduced to elemental mercury by addition of
a reducing reagent such as tin(II) chloride. The
elemental mercury was then separated from solution
and introduced into the optical path of an Atomic
Absorption Spectrometer, which had a wavelength
at the spectral line of mercury (253.7 nm). In Figure
13

Agah et al. / Determining Total Mercury in Samples from the Persian Gulf and the Caspian Sea: Comparison ,

In each sample batch, procedural blanks and three

3, the process of the analyzing techniques are

compared.
The precision and accuracy of the applied
analytical methods were compared on eight Certified
Reference Materials, Dorm2, Dolt 2 and Tort 2, trace
metals in dog fish muscle, liver and Lobster (provided
by the national research council of Canada); NIST
1556a, NISTt-2976, IAEA 142 trace metals and
mercury in oyster, mussels and fish, respectively;
IAEA 086 and IAEA405 trace elements in hair and in
polluted estuary sediment (provided by International
Atomic Energy), respectively. Ten environmental
(fish and sediment) samples and one inter-comparison
sample (BCR 710, Total and methylmercury in oyster
tissue) as well as certified samples were analyzed in
six replications.

sample replicate were included. The limits of

detections were set as three times standard
deviations of the procedural blanks (Table 1).

Biological

AAS (n= 14)

Normal oven CV

AMA 254 (n=77)

Combustion AAS

CV AAS (n= 6)

CEM (MARS 5)

CV AAS (n=44)

CEM (MDS 2000)

(RM)

Reference Materials

Table1. The detection limits of the methods for biological

samples (ng.g-1d.w).

48

12

0.28

20

0.28

RM
Sediment
RM

As shown in Table 1, the detection and

quantification limits obtained by Combustion AAS
method was lower than that obtained by classical
method, CEM (MDS 2000, MARS 5) microwave
oven AAS and lower than the environmental quality
target. To achieve an easier comparison of the
results in terms of precision and accuracy, z-score
values for the reference materials were determined
as folows:
Z=(A-)/V; Where, A= obtained result, =
Certified value and V =Standard deviation of RM.
The results were considered satisfactory when Zscores ranged between 2 and +2 and questionable
when ranged between 2 and 3 and or 2 and 3. The
Z-score values greater than 3 or less than 3 were
regarded as unsatisfactory (Miller, 2000).

Fig.2: Cold Vapor Atomic Absorption Spectrometry (from

the Manual for mercury Module).

3. Results
Total mercury concentrations were calculated for all
certified and environmental samples in microgram per
gram in dry weight (g g-1d.w.) The average of total
mercury concentrations with corresponding standard
deviations (sd) are presented in Tables 2, 3, 4 and 5.

Fig.3: Companison between digestion process of different

methods

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Journal of the Persian Gulf (Marine Science)/Vol. 2/No. 4/June 2011/8/11-18

Combusti
on AAS,
AMA 254

CEM
(Mars
5) CV
AAS

CEM
MD
2000
CV

Technique
s

Table 2. Average results for certified biological samples in gg-1d.w. (n=6).

RM

Nist 2976

Nist 1566a

IAEA 142

Tort 2

Dolt 2

Dorm 2

IAEA 086

Certified Values

0.0610.004

0.0640.007

0.1260.0
10

0.270.06

2.140.28

4.640.026

0.53-0.612

0.55
1128
8
0.3030.022
0.6
1132
1

0.67
1074
4
2.3030.091
-0.26
1005
2

0.34
984
5.4
4.7370.0272
-1.8
911
2

0.4
1024
4
0.554-0.612
-

0.3060.002

2.0660.038

4.180.072

z-score
Recovery %
RSD %
The present study
z-score
Recovery %
RSD %
The present study
z-score
Recovery %
RSD %

1.25
1089
5

0.8
1082
1.5

The present study

0.0660.004

0.0690.001

Under detection limit

0.2
1022
1.6
0.1280.0
02

Table 3. Total mercury in certified sediment samples (g

g -1 d.w. n=6)
RMs

IAEA 405

Certified values

0.810.06

AMA 254

CEM Microwave oven

CV AAS

Normal oven AAS

Mean

0.810.04

Recovery

1005

Z Score

-0.017

Mean

0.800.07

Recovery

998

Z Score

-0.16

Mean

0.830.01

Recovery

1021

Z Score

0.36

Five Sea Cat fish speciemen were sampled from

Lengeh port at the Persian Gulf. Total mercury in the
fish muscle were determined by AMA 254 and CEM
2000 microwave oven CV AAS methods. Results (Table
4) demonstrated higher precision for AMA 254.
Total mercury content in sediments of the
Caspian Sea (Babolsar, Noshahr, Anzali and
Golestan Gulf) were analyzed by acid digestion and
classical method (Table 5).
Table 5. Mercury Content (g g-1 d.w.) of sediment samples
collected from the Caspian Sea

Table 4. Concentrations of mercury in fish speciemen from the

Persian Gulf
CEM MDS 2000
CVAAS (g g-1d.w)
RSD%

Means (n=6)

CEM (MDS
2000) CV

Samples

Depth
(m)

AMA 254
(n=6)

 0.0190.004

Babolsar

Coastal

0.0120.004

 0.0060.002

Noshahr

Coastal

0.013-0.006

 0.0170.008

Anzali

0.0170.007

 0.0360.004

Golestan s1

0.0400.005

0.0430.004

Golestan s2

3.5

0.0360.003

Fish AMA 254 (g g

1
Samples weight
d.w)
(gr.)
Means
RSD
(n=6)
%

Under the detection limit

4. Discussion

0.2070.007

Catfish
(n=1)

225

0.1540.003

12

0.1700.020

Catfish
(n=2)

257

0.1930.010

0.234-0.009

Catfish
(n=2)

293

0.2380.005

13

0.2060.028

Catfish
(n=2)

321

0.1560.006

14

0.1640.022

Catfish
(n=2)

541

0.1980.007

0.2880.009

BCR710

Our results demonstrated that Combustion

Atomic Absorption (AMA 254) instrument is an
affordable and accurate alternative technique with
much lower detection limit for environmental
samples (Table 1). Although the detection limits in
our study for wet extraction methods were relatively
higher than dry ash (AMA 254) technique

0.306-0.008 3

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Agah et al. / Determining Total Mercury in Samples from the Persian Gulf and the Caspian Sea: Comparison ,

comparable with literature data (Houserova et al.,

2005; Ramalhosa et al., 2001; Garcia et al., 2001;
Rodriguez Pereiro et al., 1998).
Comparing the results and Z-Score values (Table 2)
of certified reference material showed good agreement
with certified mercury values in CEM (MDS 2000 and
MARS 5) microwave ovens CV AAS techniques,
where concentration was higher than the quantification
limits of these techniques (0.15 and 0.03 g g-1 d.w,
respectively). Figure 4 shows the comparison
between our results and the certified values. AMA
254 is more accurate in low mercury levels (Figure 4).

Application of sedimental oven them 0.5gr is an

advantage of Classical digcstion method. In this study
sediment samples prepared from five stations at the
Caspian Sea were analyzed and compared using CEM
microwave oven and AMA 254 (Table 5).
Cold vapor generation is a stable technique, but
our results and literature data corresponding to the
CEM 2000 microwave oven CV AAS indicated some
problems in the detection of low concentration and /
or identified the need to optimize the sample size,
dilution volume, digestion programming or selecting
better digestion system (such as CEM, MARS 5).

Fig. 4: Comparison between the peresent results (n=6) and the

certified values.

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Agah et al. / Determining Total Mercury in Samples from the Persian Gulf and the Caspian Sea: Comparison ,
Journal of the Persian Gulf (Marine Science)/Vol. 2/No. 4/June 2011/8/11-18

Journal of the Persian Gulf

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