Analytica Chimica Acta 508 (2004) 107–117

Methylmercury determination in sediments and fish tissues from

the Nerbioi-Ibaizabal estuary (Basque Country, Spain)
Jon Sanz Landaluze∗ , Alberto de Diego, Juan Carlos Raposo, Juan Manuel Madariaga
Department of Analytical Chemistry, University of The Basque Country, P.O. Box 644, E-48080, Bilbao, Basque Country, Spain

Received 1 August 2003; received in revised form 18 November 2003; accepted 24 November 2003

Abstract

The analysis of methylmercury in extracts from environmental solid samples by gas chromatography coupled to microwave induced
plasma atomic emission spectrometry (GC–MIP/AES) after the ethylation of the extract and the preconcentration of the volatile products
in hexane has been critically investigated. In order to correct potential sources of random error along the analytical procedure affecting the
overall repeatability of the analysis, the use of the inorganic mercury naturally occurring in the sample as internal standard in the analysis of
methylmercury is proposed. A study to establish the best conditions to achieve a quantitative recovery of methylmercury without damaging
its chemical structure has also been carried out. Magnetic stirring (without heating) of the sediment or fish tissue with 2 mol dm−3 HNO3 or
10% methanolic KOH, respectively, during 90 min has been considered as the most effective procedure to release methylmercury preserving
its structure. The proposed method has been validated using certified reference materials (CRM-580, CRM-463 and DOLT-2), assessing
its quality in terms of accuracy, repeatability and detection limit. Finally, several sediment and fish samples collected in the estuary of the
Nerbioi-Ibaizabal (Bilbao, Basque Country) have been analyzed following the procedures proposed. The results obtained show the validity
of the proposed method to analyze real samples.
© 2003 Elsevier B.V. All rights reserved.

Keywords: Speciation; Mercury species; Routine analysis; GC; MIP/AES; Nerbioi-Ibaizabal estuary; Fish; Sediments

1. Introduction ysis of mercury is, therefore, mandatory in environmental

Speciation analysis of mercury in environmental samples
has been a subject of great concern during the last four
decades. Some pollution disasters [1,2] have occurred in the
past, resulting in death of thousands of people and many
other seriously affected. The origin of these disasters has
been mainly, natural methylation of inorganic mercury in
the environment [3] and direct use of methylmercury in in-
dustrial and agricultural applications [4]. Although almost
all the industrial uses of mercury have been considerably
reduced, the specific physical–chemical properties of mer-
cury species (solubility, biomethylation, bioaccumulation,
etc.) make them stable and persistent in the environment.
Toxicity and bioavailability of mercury species is highly
dependent on its chemical structure [5,6]. Speciation anal-
∗ Corresponding author. Tel.: +34-94-601-55-51;
fax: +34-94-464-85-00.
E-mail address: qabsalaj@lg.ehu.es (J. Sanz Landaluze).
studies. Numerous analytical methods have been described
in the literature to measure mercury species in environmen-
tal samples [7–12]. Analytical determination of mercury
species are usually carried out making use of home-made
hyphenations between an appropriate separation technique
(gas or liquid chromatography, electrophoresis, selective
reduction) and a sensitive and selective detector (electron
capture, atomic absorption and fluorescence spectrome-
try, mass spectrometry). Gas chromatography coupled to
microwave induced plasma atomic emission spectrometry
(GC–MIP/AES) is a promising alternative for mercury spe-
ciation [13–16]. Nowadays hyphenation between these two
techniques is commercially available, resulting in an ex-
cellent option for non-research private laboratories to make
routine mercury speciation analysis.
Analysis of methylmercury in sediments and fish tissue
deserves special attention, due to the extreme toxicity of
methylmercury and its ability to enter and biomagnify up
in the tropic chain [17]. A huge variety of methods based

0003-2670/$ – see front matter © 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2003.11.070
108 J. Sanz Landaluze et al. / Analytica Chimica Acta 508 (2004) 107–117
on the combination of different analytical techniques have
been proposed to measure methylmercury in solid samples
of environmental significance. Most of them involve several
steps (quantitative extraction, derivatisation, preconcentra-
tion, quantification) in which random errors may multiply
and give rise to low precision and lack of repeatability of
the overall analysis. Another potential source of poor re-
peatability is the drift in sensibility typically observed when
some detectors such as MIP/AES are used. The use of an-
alytical tools (internal standard, isotopic dilution) to take
into account and correct all these potential sources of er-
ror is mandatory if accurate and reproducible data is to be
obtained.
Administration start to be sensitive towards mercury pol-
lution problem, which has stimulated the development of
more accurate, precise and sensitive methods to determinate
mercury and its compounds in a wide range of matrices.
Since new legislation is being promoted in order to limit
the concentration of methylmercury in different environ-
mental samples, routine analysis of this species is increas-
ingly demanded. Laboratories devoted to routine analysis
of environmental samples requires ready-to-use analytical
methods which must fulfil, besides the accuracy, precision
and sensibility demanded to any analytical method, several
conditions: (i) simple, low cost and fast sample treatment;
(ii) commercially available equipment supported by a ro-
bust after-sale service, compatible with the requirements of
accreditation agencies; and (iii) if possible, automatic pro-
cedures resulting in high sample throughput and improved
repeatability.
In this paper, the analysis of methylmercury in extracts
from sediments and fish samples by ethylation of the ex-
tract, preconcentration of the volatile compounds in hexane
and later GC–MIP/AES separation and detection has been
investigated. The instrumental variables and conditions for
mercury speciation by GC–MIP/AES in synthetic samples
have been optimized in a previous work [16] using a chemo-
metric approach. A new method to overcome the effect
of different sources of random errors along the analytical
method, based on the use of the inorganic mercury origi-
nally present in the sample as internal standard is presented.
The aim is to propose a ready-to-use method based on this
approach to be applied in routine analysis of methylmer-
cury. The quality of the proposed method has been checked
by analysis of several certified reference materials and its
applicability to the analysis of real samples has been proved
by processing several fish and sediments collected in the
estuary of the Nerbioi-Ibaizabal (Bilbao, Basque Country).
2. Experimental work
2.1. Cleaning procedures
Rigorous cleaning procedures of all the laboratory ware
and other equipment that comes into contact with samples
must be employed in order to avoid contamination of sam-
ples. All glassware and plastic ware was washed with a
common detergent, thoroughly rinsed with Milli-Ro quality
water (Milli-Q Model 185, Millipore, Bedford, MA, USA)
and soaked into a clean dilute HNO3 (15%) bath for 24 h.
Afterwards, the material was rinsed with Milli-Q (Milli-Q
Model 185, Millipore, Bedford, MA, USA) quality water
and stored into mercury-free plastic bags. Best materi-
als for sample storage and processes are Pyrex and silica
(quartz) glass, and Teflon (PTFE or FEP). Plastics such as
polypropylene are not recommended since these materials
can contribute to either contamination or losses of mercury
via adsorption on the wall container [10,18].
2.2. Reagents
All reagents used were of analytical-reagent grade unless
otherwise stated. The solutions were prepared using Milli-Q
quality water. Acid solutions (HNO3 , HCl, H2 SO4 and
HAc) were prepared by dilution in water of, respectively,
65% HNO3 (Suprapur, Merck, Darmstadt, Germany), 37%
HCl (tracepur, Merck), 35% H2 SO4 (Suprapur, Merck) and
glacial HAc (analytical-reagent grade, Merck). Solutions
of NaB(C2 H5 )4 (Strem Chemicals, Bischheim, France)
were prepared in a nitrogen atmosphere immediately be-
fore use. 2 mol dm−3 buffer solutions were daily prepared
by dissolving appropriate amounts of glacial HAc and
sodium acetate (analytical-reagent grade, Merck) in wa-
ter. The buffer solution was further purified prior to use
by reacting with 1 ml of 0.1% NaB(C2 H5 )4 and purging
with nitrogen during 15 min. 10% (w/v) methanolic KOH
was prepared dissolving KOH (analytical-reagent grade,
Merck) in methanol (analytical-reagent grade, Merck). 25%
tetramethylammonium hydroxide (TMAH) solution was
directly obtained from Avocado Research Chemicals (Ward
Hill, MA, USA). Ultra-pure hexane was purchased from
Merck.
In GC-AED determination oxygen (99.999%) and hydro-
gen (99.999%) were used as reagent gases to enhance the
combustion of organic compounds and to improve the base-
line stability, respectively. Helium (99.9999%) was used as
both the carrier and the plasma gas. Purge of the optics of
the spectrometer was accomplished by pre-purified (N2 pu-
rifier, Hewlett-Packard) nitrogen (99.999%). All gases were
purchased from Carburos Metálicos (Barcelona, Spain).
2.3. Stock solutions and certified reference materials
Stock solutions (∼1000 ␮g ml−1 ) of CH3 Hg+ were pre-
pared by dissolving methylmercury chloride (99.8%, Strem
Chemicals) in a mixture of acetone–water (1%). The stock
solutions were stored in the refrigerator and protected
against light. More diluted solutions (∼1000 ng ml−1 ) were
prepared weekly by appropriate dilution in water of the
stock solution. These solutions were used to produce cali-
brate solutions following the standard addition method.
 J. Sanz Landaluze et al. / Analytica Chimica Acta 508 (2004) 107–117
Several certified reference materials were used to check
the accuracy of the proposed method for methylmercury
determination, e.g., CRM-580 (polluted sediment) and
CRM-463 (tuna fish muscle tissue), both from the Com-
munity Bureau of Reference (BCR, Brussels, Belgium),
and DOLT-2 (Dogfish liver) from the National Research
Council (NRC, Canada).
2.4. Safety considerations
Organomercury compounds, and specially methylmercury
are extremely toxic. It may cause neurological damage as
well as kidney malfunction. Direct contact with the skin may
lead to death. Precautions and adequate clothing are abso-
lutely necessary when manipulating the reagent. Disposal of
all mercury-containing waste from the experiment was done
in accordance with facility guidelines.
2.5. Instrumentation
Gas chromatographic separations were carried out in a HP
model 6890 GC system (Agilent Technologies, Palo Alto,
CA, USA) with a split/splitless injector. Injections were
made by means of an HP 7386 Series automatic sampler.
An HP 2350 microwave induced plasma/atomic emission
detector MIP/AED (Agilent Technologies Inc.) was cou-
pled to the GC with a commercial available transfer line.
Data acquisition and processing were achieved by means
of a 3592A ChemStation System (Agilent Technologies).
An OV-1701 (14% cyanopropyl–86% dimethylpolysilox-
ane, 15 m × 0.53 mm i.d., 3.0 ␮m film thickness, Quadrex,
Woodbridge, CT, USA) capillary column was used to
achieve a correct separation in gas chromatography.
A 50 ml round-bottomed open borosilicate vessel
(150 mm × 35 mm i.d.) with a Microdigest Model A301
(2.45 Gz, maximum power 200 W) microwave digestor
from Prolabo (Briare, France) equipped with a TX32 pro-
grammer which allows the applied energy to be selected
from 10 to 200 W in increments of 10 W was used for the
extraction from the solid samples. The time of exposure,
up to 99 min, can be set in steps of 1 min. The focused
single-mode microwave digester is an open system with re-
spect to atmospheric pressure equilibration. The microwave
energy of the magnetron can be delivered with a great re-
producibility and is focused on the sample with maximum
intensity by the waveguide. A refluxing condenser unit
on the top of the sample prevents possible losses of the
analytes by volatilization. Temperature of the sample can
be continuously measured in real time with a Megal 500
thermometer from Prolabo.
All the solutions were prepared using a Mettler-Toledo
AE2000 (Columbus, OH, USA) analytical balance
(±0.0001 g) and 20, 100, 1000 and 5000 Eppendorf mi-
cropipettes (Hamburg, Germany) with a precision of ±2%.
A laboratory freeze dryer Cryodos-50 from Telstar Instrumat
(Sant Cugat del Valles, Catalonia, Spain) and an agate mor-
109
tar Pulverisette-6, (Fritsh, Idar-Oberstein, Germany) were,
respectively, used to lyophilize and ground the samples.
2.6. Study area
The Nerbioi-Ibaizabal estuary (Bilbao, Basque Country)
was originally the largest estuary area on the Cantabrian
coast. Nowadays the estuary is 15 km long, an average of
100 m wide, and its channel depth ranges from 2 m in the
upper estuary to 9 m at the mouth. The estuary is formed
by the tidal part of the main river Nerbioi-Ibaizabal and
four minor rivers (Kadagua, Asua, Galindo, Gobelas) which
discharge into the main course (Fig. 1).
The natural features of the estuary have been dramati-
cally modified by urban, industrial and port developments.
During the last 150 years the Nerbioi-Ibaizabal estuary
has received wastes from many sources (mineral sluicing,
industrial wastes and urban effluents) which have signifi-
cantly degraded the environmental quality of the estuary.
Nowadays, its water and sediments have extremely low con-
centrations of dissolved oxygen and high content of organic
matter and heavy metals [19,20]. A significant decrease in
the flux of heavy metal contaminants has occurred over re-
cent decades, due to implementation of environmental pro-
tection policies, improvement of waste-treatment systems,
and closure of some major factories during recent periods of
economic recession. Despite these improvements the con-
taminated sediments from intertidal areas act as long-term
sources of heavy metals to the aquatic environment.
Sediment and fish samples collected in different locations
of the Nerbioi-Ibaizabal estuary (Fig. 1) have been analyzed
following the procedures proposed here.
2.7. Sample collection, storage and preservation
Sediment samples were manually collected from the top
sediments (5–10 cm depth), stored in sealed plastic bags,
transported to the laboratory in cold boxes and frozen until
lyophilisation. Lyophilisation of the sediments was carried
out at −45 ◦ C and 0.05 mbar during 24 h. The lyophilized
samples were grounded and sieved through a 63 ␮m nylon
sieve and stored in hermetically closed Pyrex vials at 4 ◦ C
until analysis.
Fish samples (thick-lip grey mullet, Chelon Labrosus)
were collected in different points of the estuary and trans-
ported to the laboratory in cold boxes inside sealed plas-
tic bags. Liver was immediately isolated and lyophilized at
−45 ◦ C and 0.05 mbar for 24 h. Dried liver samples were
grounded and sieved in a similar way than sediments and
stored in the refrigerator until analysis.
2.8. Analytical procedures
2.8.1. Methylmercury determination in sediments (Fig. 2)
In the case of non-heated extraction procedure, 0.1–0.2 g
of sample were accurately weighted in a screw-capped glass
110 J. Sanz Landaluze et al. / Analytica Chimica Acta 508 (2004) 107–117
Fig. 1. Location of the sampling points in the Nerbioi-Ibaizabal estuary.
vial, 10 ml of acid were added and the vials were capped.
The slurry was homogenated by magnetic stirring during the
time selected in each experiment (5–300 min). In the case
of microwave-assisted extraction, 0.1–0.5 g of sample and
10 ml of acid were added in a borosilicate vessel, which was
located in the microwave oven; the refluxing condenser unit
was placed on the top of the vessel and the sample was heated
at 60% of the highest power during 10 min with magnetic
stirring. In both cases and after the extraction step, the mix-
ture was centrifuged for 5 min at 4000 rpm and the super-
natant was separated from the sediment by means of a Pas-
teur pipette. The sediment was rinsed twice with water and
all the fractions were mixed and made up with water to 25 ml.
Aliquots of 4.9 ml of this solution were used to prepare a
five-point calibration curve for methylmercury by the stan-
dard addition method. 5 ml of buffer (pH, 4.8) were added
to each aliquot, followed by different amounts of CH3 Hg+ ,
1 ml of hexane and 1 ml of 0.1% NaB(C2 H5 )4 . The mix-
tures were shaken for 10 min and the organic layers were
transferred to 2 ml glass vials for GC–MIP/AES analysis.
2.8.2. Methylmercury determination in fish samples (Fig. 2)
About 0.2 g of sample were magnetically stirred in a
screw-capped glass vial together with 10 ml of alkaline so-
lution (25% TMAH or 10% methanolic KOH). In most
of the cases, the solid phase was completely dissolved but
sometimes it was necessary to centrifuge during 5 min at
4000 rpm to separate the solid residue from the liquid phase.
One milliliter of HAc was added to the liquid phase and the
sample was made up with water to 25 ml. To produce the
calibrants following the standard addition method, aliquots
of 4.9 ml of the extract were mixed with 5 ml of buffer, in-
creasing amounts of CH3 Hg+ , 1 ml of hexane and 1 ml of
1% NaB(C2 H5 )4 . After shaking the mixtures for 10 min,
the vials were uncapped, another 1 ml of 1% NaB(C2 H5 )4
was added to each one and the mixtures were again shaken
for another 10 min. The organic layers were separated and
transferred to 2 ml glass vials and stored until GC–MIP/AES
analysis.
2.8.3. GC–MIP/AES analysis of the extracts
The analysis of the extracts was performed by GC–MIP/
AES using the best conditions listed in Table 1, which were
previously optimized [16] using synthetic aqueous mixtures
of Hg2+ and CH3 Hg+ . Some other recommendations sug-
gested in the same article were also observed during the anal-
ysis of the extracts: (i) the acetic acid–acetate buffer solution
was purified by purging with nitrogen after reaction with
 J. Sanz Landaluze et al. / Analytica Chimica Acta 508 (2004) 107–117 111

0.15 g sediment 0.2 g biological tissue

10 mL HNO3 10 mL KOH 10%

Leaching

2.0 mol·dm -3 in methanol

Magnetic stirring Magnetic stirring
90 minutes 90 minutes
Derivatisation

4.9 mL extract + 4.9 mL extract +

+ 5 mL HAc/Ac- (pH=4.8) + 1 mL ác. acético
+ 1 mL NaBEt4 0.1% (m/v) + 5 mL HAc/Ac- (pH=4.8)
+ 1 mL NaBEt4 1.0% (m/v)

1 mL hexane. Mechanical stirring

Extraction

1 mL hexane
Liquid

Mechanical stirring 10 minutes.

10 minutes.
1 mL NaBEt4 1.0% (m/v)
Mechanical stirring 10 min.
Detection

GC-MIP/AES
2 µL organic phase
Analysis conditions in Table 1
Quantification

2+
Hg in the sample as internal standard
10-12 point standardaddition method

Fig. 2. Scheme of the proposed methods for methylmercury analysis in sediments and fish tissues.

0.1% NaB(C2 H5 )4 , which resulted in high quality blank sig- 3. Results and discussion
nals, improving the detection limit in methylmercury anal-
ysis; (ii) rigorous cleaning procedures were observed for all 3.1. Use of the inorganic mercury originally present
the glass and plastic ware used throughout the analysis, in in the sample as internal standard in the analysis of
order to reduce contamination sources and memory effects; methylmercury
(iii) the methylmercury solutions used to spike the aliquots
of the extracts in the standard addition procedure were pre- The procedure proposed for the analysis of methylmer-
pared every week, in an attempt to keep artefact formation cury in solid samples involves several steps: extraction,
of methylmercury to a minimum. derivatization, preconcentration, separation and detection.
The procedure is quite long and the sources of random
errors numerous. In addition, the authors have detected in
Table 1 preliminary works drifts in the sensitivity of the MIP/AES
Optimum GC–MIP/AES operating conditions for methylmercury analysis detector when analyzing standard solutions of methylmer-
[16] cury. All these factors result in poor repeatability of the
GC MIP/AES methylmercury analysis. In an attempt to take into account
Carrier gas: He Solvent vent ON: 0.1–0.55 min
and correct the effects derived from random errors and
Injection mode: splitless Transfer line temperature: 250 ◦ C shift in sensitivity, the authors propose the use of the in-
Injection port temperature: 180 ◦ C Cavity block temperature: 250 ◦ C organic mercury naturally present in the sample (ambient)
Injection volume: 2 ␮l Wavelength: 253.65 nm as internal standard in the analysis of methylmercury using
Column: OV-1701 (15 m × Helium flow at cavity vent: the standard addition method. This means just plotting the
0.53 mm, 3.08 # 61549; m) 100 ml min−1
Column head pressure: 40 psi O2 pressure: 38 psi
CH3 Hg+ to Hg2+ signals ratio rather than the CH3 Hg+
H2 pressure: 13.4 psi signal in the calibration curve. The original inorganic mer-
Temperature program: cury in the sample fulfils all the requirements of an internal
90 ◦ C (0.5 min) standard: (i) its chemical structure and physico-chemical
35 ◦ C min−1 (4 min) behavior is very similar to that of CH3 Hg+ ; (ii) it under-
210 ◦ C (1 min)
goes similar reactions than the analyte along the analytical
112 J. Sanz Landaluze et al. / Analytica Chimica Acta 508 (2004) 107–117

Area of
18
CH3HgC2H5
16

14

12

10

0
-10 -5 0 5 10 15 20 25 30 35 40

(a) Conc. of CH3Hg+ added (ng·mL-1)

Ratio of the
0.008
areas of
0.007 CH3HgC2H5 and
Hg(C2H5)2
0.006

0.005

0.004

0.003

0.002

0.001

0
-5 0 5 10 15 20 25 30 35

(b) Conc. of CH3Hg+ added (ng·mL-1)

Fig. 3. Determination of methylmercury in the CRM-580 reference sediment: calibration curve by the standard addition method: (a) without correction
by internal standard and (b) using the inorganic mercury naturally present in the sample as internal standard.

procedure; and (iii) there is absolutely no doubt that its
concentration remains constant in all the calibrant solutions.
Furthermore, it does not require further manipulation of the
sample. Its use as internal standard in CH3 Hg+ analysis by
the proposed method rends promising results, as it can be
observed in Fig. 3 in the case of the CRM-580.
3.2. Extraction of methylmercury from sediments
and fish samples
The extraction step is nowadays one of the most contro-
versial points in the scientific community working on the
analysis of methylmercury in environmental solid samples.
The formation of methylmercury as an artefact has been
recognized, especially when using the vapor distillation
technique applied to aquatic samples [21]. In May 1998,
representatives of the main laboratories devoted to mercury
speciation hold a workshop in Wiesbaden/Mainz (Germany)
to discuss about the magnitude of the problem. The conclu-
sions of this meeting were summarized in a special number
of Chemosphere [22]. The controversy, nevertheless, contin-
ues nowadays, introducing the possibility not only of artefact
methylation of inorganic mercury but also demethylation
process during extraction and preparation steps [23–26].
Keeping this in mind, different extracting agents and
extraction procedures were investigated in this work. The
aim was to select a procedure able to quantitatively extract
the analyte from the sample without damaging its chemical
structure and, as far as possible, keeping to a minimum
the possibility of artefact formation of methylmercury. The
study was performed using different certified reference
materials, e.g., CRM-580, CRM-463 and DOLT-2. 25%
TMAH and 10% methanolic KOH were used as extract-
ing agents for biological samples and 2 mol dm−3 HNO3 ,
H2 SO4 , HCl and HAc for sediments. The first two acids
were also considered in the case of the CRM-463 material.
The methylmercury extraction efficiency of these extracting
agents was investigated using two different approaches in
the case of sediments (non-heating magnetic stirring during
90 min and microwave-heating stirring during 3 min at 60%
of total power). In the case of fish samples, only the first
approach was considered.
Fig. 4 shows the recovery of methylmercury from the
CRM-580 sediment obtained in each experimental condition
 J. Sanz Landaluze et al. / Analytica Chimica Acta 508 (2004) 107–117
Conc. CH3Hg+
180 (ng·g-1)
CRM-580
-1
160 75.5 3.7 ng·g
140
120
100
80
60
40
20
0 0
MA MAE MA MAE MA MAE MA MAE
HNO3 HNO3 HCl HCl H2SO4 HAc HAc
H2SO4
Fig. 4. Methylmercury analysis of the CRM-580 reference sediment
(solid line: methylmercury certified concentration, dotted lines: ±error):
comparison among different extraction procedures. Acid concentrations:
2 mol dm−3 . MA: non-heating magnetic stirring of the sample during
90 min; MAE: microwave assisted heating of the sample during 3 min.
Errors bars, standard deviation of each experimental condition tested,
corresponding to, at least, five independent determinations.
considered. The methylmercury content in the extract is
systematically higher when microwave power is applied
to accelerate the leaching process. The opposite behavior
is only observed when H2 SO4 is used as extracting agent.
This suggests that microwave power is efficiently acting
as a promoter of methylation. This is in good agreement
with one of the main conclusions from the Mainz work-
shop, that is, some extraction methodologies apparently
result in higher methylmercury concentrations than the real
ones [27], suggesting the possibility of artifact production
of methylmercury. The most important ones are hot alka-
line digestion, supercritical fluid extraction, acid digestion
with concentrated (>6 mol dm−3 ) hydrochloric acid and
microwave assisted acid digestion (hydrochloric and nitric
acid). The huge difference between the results obtained
using HAc as extracting agent indicates that the potential
methylating characteristics of this acid (it has methyl groups
in its chemical structure which could be eventually accepted
by the inorganic mercury present in the sample) are greatly
enhanced by microwave heating. The use of dilute HCl
(2 mol dm−3 ) also rends higher methylmercury concentra-
tions than expected, whatever it is the extraction procedure.
The Mainz workshop recognized problems connected to
acid leaching when using concentrated HCl (>6 mol dm−3 ).
The results obtained here suggest that methylation may also
occur with more diluted HCl.
Recoveries in good agreement with the certified
methylmercury concentration were only obtained with non-
heating extraction using HNO3 as extracting agent. If the
precision of the analysis is taken into account, non-heating
extraction with H2 SO4 is also acceptable. Better repeata-
bility (comparable to the reproducibility associated to the
certified concentration of the reference material) was ob-
113
-1
Conc. (ng·g ) CRM-580
75.5 3.7 ng·g-1
90
80
70
60
50
40
30
20
10
0
50 100 150 200 250 300 350
Extraction time (min.)
Fig. 5. Effect of extraction time on the analysis of methylmercury in the
CRM-580 reference sediment by GC–MIP/AES after non-heating mag-
netic stirring of the sample in 2 mol dm−3 HNO3 (solid line: methylmer-
cury certified concentration, dotted lines: ±error).
tained with the HNO3 non-heating extraction than with the
rest of conditions investigated.
The extraction time was also investigated with HNO3 as
extracting agent. The results are shown in Fig. 5. As can be
seen, quantitative recovery of methylmercury was achieved
with extraction times longer than 1 h. Longer times did not
result in decomposition nor artefact formation of methylmer-
cury. This suggests that non-heating extraction of the sedi-
ment using 2 mol dm−3 HNO3 is not promoting methylation.
Even though non-heating procedure is longer, and taking
into account that extreme conditions seem to power artificial
formation of methylmercury, non-heating leaching of the
sample with 2 mol dm−3 HNO3 during 90 min was finally
adopted as standard extraction procedure in the analysis of
the sediments collected in the Nerbioi-Ibaizabal estuary.
The results obtained from the CRM-463 are shown in
Fig. 6a. Taking into account the results from the sediments,
only non-heating treatment was considered in the case of
biological samples. The efficiency of acidic (HNO3 and
H2 SO4 ) and alkaline (KOH and TMAH) extracting agents
were compared. Recoveries in good agreement with the
certified methylmercury concentration were obtained with
both 10% methanolic KOH and 25% TMAH. The repeata-
bility of the analysis is comparable to the reproducibility
claimed by the CRM producers. Acidic extracting agents
resulted in poor recoveries, quite far from the expected
ones. Althougth some older studies successfully describe
the use of HCl or H2 SO4 at different concentrations to
extract CH3 Hg+ from biological tissues [28–30], all these
extraction procedures were heat-assisted. A soft extraction
procedure (non-heating), which seems to be appropriate to
minimize the artifact formation of methylmercury, leads to
lower extraction rates. Apparently, acids are not appropriate
for biological tissues, which is in concordance with results
from other authors [31]. Similar results were obtained using
KOH or TMAH as extracting agents with the non-heating
114 J. Sanz Landaluze et al. / Analytica Chimica Acta 508 (2004) 107–117

Table 2
Conc. CH3Hg+ CRM-364 Results obtained in the methylmercury analysis of several certified refer-
4 ( g·g-1) 3.04 0.16 g·g-1
ence materials
3.5
Number of Measured value Certified value
3 analysis
2.5 CRM-580 11 75.8 ± 1.8 ng g−1 75.5 ± 3.7 ng g−1
CRM-463 8 3.08 ± 0.43 ␮g g−1 3.04 ± 0.16 ␮g g−1
2 DOLT-2 6 777.4 ± 164.7 ng g−1 744.8 ± 52.7 ng g−1
1.5

1
within-day and the between day precision are statistically
0.5 comparable (Fcalculated < Fcritical ) [32]. The relative stan-
0 dard deviation (R.S.D.) of all the determinations is 2.7%.
KOH TMAH HNO3 H2SO4
(a) An absolute detection limit of 3 pg, calculated as the con-
centration corresponding to the blank signal plus three times
Conc. CH3Hg+ DOLT-2
the standard deviation of the blank, has been estimated for
(ng·g-1) 744.8 57.2 ng·g
-1
the determination of methylmercury by GC–MIP/AES. This
means about 5 ng g−1 if 0.2 g of solid sample are processed.
1000

900 This detection limit allows the analysis of most contami-

nated sediments and fish samples and is well above the WHO
800
health standards of 0.5 mg kg−1 for fish tissue [6]. If pris-
700 tine samples are to be analyzed, a previous preconcentra-
600
tion step should be achieved. If larger amount of sample is
processed solubilisation problems may arise and foam may
500 appear, especially in the case of biological samples and also
400 in sediments with a high organic matter content.
(b) KOH TMAH
3.4. Analysis of Nerbioi-Ibaizabal samples
Fig. 6. Methylmercury analysis of the: (a) CRM-463 and (b) DOLT-2
reference biological tissues after non-heating magnetic stirring of the
sample in the extracting agent during 90 min: comparison among dif-
Sediment and fish samples were collected in different
ferent extracting agents. Concentrations: acids, 2 mol dm−3 , methanolic points (Fig. 1) of the Nerbioi-Ibaizabal estuary from July
KOH, 10% (w/v) and TMAH, 25% (w/v) aqueous solution (solid line: 2000 to March 2002 and they were analyzed for methylmer-
methylmercury certified concentration, dotted lines: ±error). Error bars: cury using the methods proposed here. Water samples
standard deviation of each experimental condition tested, corresponding were also collected in the same places. After filtering them
to, al least, five independent determinations.
through 45 ␮m nylon filters, they were processed like the
sediment extracts. In all the cases, the concentration of
treatment of the DOLT-2 reference material (Fig. 6b). CH3 Hg+ found was below the detection limit, estimated
Consequently, 10% methanolic KOH with non-heating ex- in about 100 ng dm−3 . Total mercury in sediment extracts
traction during 90 min was selected to treat the fish samples was measured by cold vapor quartz furnace atomic ab-
from the Nerbioi-Ibaizabal estuary analyzed in this work. sorption spectrometry using NaBH4 as reducing agent. In
It is important to notice that biological tissues require more this case, the dried sediments were previously subjected to
concentrated NaB(C2 H5 )4 to derivatise the analytes. In ad- microwave assisted acid leaching (10 min at 62.8% of total
dition, the derivatisation step must be repeated twice in this
case. This is probably related to the high organic content of Table 3
the sample, which consumes an important part of the reagent. Repeatibility of the proposed method for methylmercury analysis: analysis
of variance (ANOVA) of the results obtained with the CRM-580 reference
sediment
3.3. Analytical figures of merit
Sample Day 1 Day 2 Day 3
(ng g−1 ) (ng g−1 ) (ng g−1 )
The accuracy of the method was checked using three
certified reference materials, one sediment (CRM-580 and 1 75.12 78.55 78.21
2 75.17 76.16 80.29
two biological tissues (CRM-463 and DOLT-2). The results 3 73.75 74.96 76.28
(Table 2) are in good agreement with the certified values. X̄ 74.68 ± 0.80 76.15 ± 1.69 78.26 ± 2.00
The precision of the analysis was evaluated by repeat-
Results of analysis of variance
ing the methylmercury analysis of the certified reference Variance Fcalculated = 3.47 Fcritical = 6.94
material CRM-580. Three measurements were repeated per between days
day in three different consecutive days (Table 3). Analysis Variance Fcalculated = 6.57 Fcritical = 6.94
of variance (ANOVA) of the results indicates that both the within a day
 J. Sanz Landaluze et al. / Analytica Chimica Acta 508 (2004) 107–117 115

Table 4
Total mercury (Hgtot ) and methylmercury (CH3 Hg+ ) concentrations found in samples from the Nerbioi-Ibaizabal estuary
Sample site Water, CH3 Hg+ (ng l−1 )a Sediment Fish, CH3 Hg+ (ng g−1 (cm))

Hgtot (␮g g−1 ) CH3 Hg+ (ng g−1 )

1 <D.L. 20.7 ± 5.3 (41)

8.6 ± 4.8 (34)
2 <D.L. 5.73 ± 0.34 62.9± 6.3 76.2 ± 8.5 (30)
61.9 ± 7.1 (31)
3 <D.L. 1.27 ± 0.32 71.3 ± 5.1
4 <D.L.
5 0.69 ± 0.11 <D.L.
6 2.40 ± 0.62 30.1 ± 5.1
7 <D.L. 80.1 ± 7.5
8 <D.L. 2.18 ± 1.10 66.7 ± 5.9 84.2 ± 10.5 (25)
91.0 ± 8.9 (26)
9 <D.L. 1.41 ± 0.57 60.9 ± 5.7
10 0.97 ± 0.64 55.9 ± 5.6
11 <D.L. 1.39 ± 0.40 26.2 ± 4.8
In parenthesis, the fish length is mentioned.
a Estimated detection limit (D.L.): 0.1 ng ml−1 .

power and using HCl 5.15 mol dm−3 as extracting agent) for
quantitative mercury recovery. The results summarized in
Table 4 are in all the cases the average of three independent
measurements.
As described in Section 2, all the solid samples were dried
by lyophilization. Freeze drying step using lyophilization is
an almost routinely procedure to prepare sediments [33–35]
and biota matrices [36–39] prior to sample analysis. Such
treatment is used by almost all the agencies and institutions
to produce certified reference materials of such matrices.
For biota matrices (as fishes) much more discussions have
raised in the last years about possible looses and transforma-
tions between species during the lyophilization process, but
nowadays most of the certified materials for biota are dried
by lyophilization. With the aim of reproducing the prepara-
tion procedure of the certified materials used in this work
to check the accuracy of the proposed method, all the solid
samples collected from the Nerbioi-Ibaizabal estuary were
lyophilized and homogenizated.
As indicated in the Section 2.7, methylmercury was mea-
sured in the liver of the fishes (thick-lip grey mullet, Chelon
Labrosus). It was also tried to repeat the analysis in mus-
cle tissue using the same procedure. No reproducible data
was obtained in this case, due to problems in the derivati-
zation step. Using more concentrated NaBEt4 or a higher
volume of reagent did not help to overcome the problem.
Sometimes no signal for methylmercury was detected, even
if methylmercury had been previously spiked to the sample.
Methylmercury concentrations in sediments, on the con-
trary, are significantly higher than those reported for samples
of similar origin, while total mercury content is not far dif-
ferent from other equivalent environments (Table 5). These
preliminary results suggest that the Nerbioi-Ibaizabal sys-
tem may be especially active in methylating inorganic mer-
cury, provided that direct input of CH3 Hg+ into the system
is not very probable. Quite high organic matter content in
sediments from this estuary (an average of 9.3%, with points
up to 30%) [20,40], considerably higher than those reported
for other estuaries (2–7%) [41–46], may be partially respon-
sible of this methylating activity. Microbial activity should
also be taken into account. Considering the serious implica-
tions for human health that may derive from these results,

Table 5
Total mercury (Hgtot ) and methylmercury (CH3 Hg+ ) concentrations found in samples from different locations
Study location Sample type Water, CH3 Hg+ (ng l−1 ) Sediment
Hgtot (␮g g−1 ) CH3 Hg+ (ng g−1 )

Scheldt (Belgium) [41] Estuary 0.1–0.5 0.8–1.4 5–15

Wisconsin [42] River – – –
Idrija (Slovenia) [43] River 0.05–0.2 0.5–2 0.1–1
Ontario (Canada) [44] Lake 0.07–0.35 – –
Anadyr (Russia) [45] Estuary – 0.08–2.1 0.1–0.6
Pigúeña (Spain) [46] River <5 – –
Nerbión-Ibaizabal [40] Estuary – 1–5 –
Nerbión-Ibaizabal (this work) Estuary <D.L. 0.7–5.7 8–80
D.L.: detection limit.
116 J. Sanz Landaluze et al. / Analytica Chimica Acta 508 (2004) 107–117
the exceptionally high methylmercury contents found in sed-
iments from the Nerbioi-Ibaizabal estuary deserve further
and deeper investigation.
Methylmercury concentrations found in liver from
thick-lip grey mullet, Chelon Labrosus, are significantly
higher than the only value reported for the same species
(9 ng g−1 ) [47]. In that work, it is not specified which part
of the fish was analyzed, being this fact of special rele-
vance. Methylmercury levels reported in the literature [47]
for other fish species that live in similar ecosystems are
usually higher (50–200 ng g−1 ) than the values found in this
work. The length and weight of the fish use to be correlated
with the methylmercury content [48,49] but, in this case,
no significant correlation was observed.
The few sampling points considered in this study forces to
be extremely careful in the interpretation of the results ob-
tained. The total mercury content in the sediments analyzed,
as well as the methylmercury concentration in water and in
fish, is comparable to, or even lower than, those found in
samples from other similar environments. The fact that the
methylmercury content in sediments seems to be consider-
ably higher in the Nerbioi-Ibaizabal than in other estuaries
might suggest that a noticeable amount of inorganic mercury
reaching the sediment is methylated here. The methylmer-
cury produced seems to remain in the sediment rather than
re-dissolving in the aqueous phase and becoming available
to plankton and finally to fish. Further investigation is de-
served, nevertheless, to confirm or reject this observation
which, at this stage of work (only 11 sampling points), may
be accused of being merely speculative. Anyway, these pre-
liminary data demonstrate the principal aim of this part of
the work, that is, the validation of the proposed methodol-
ogy as a valid tool for the analysis of real sediment and fish
samples.
Acknowledgements
This work has been financially supported by the Spanish
Government (R.P.: MEC 171.310-0632/97). J.S.L. is grateful
to the Spanish Government for his pre-doctoral fellowship.
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