DETERMINATION OF ORGANIC MERCURY IN BIOTA, PLANTS AND
CONTAMINATED SEDIMENTS USING A THERMAL ATOMIC
ABSORPTION SPECTROMETRY TECHNIQUE
M. VÁLEGA, S. ABREU, P. PATO, L. ROCHA, A. R. GOMES, M. E. PEREIRA∗
and A. C. DUARTE
Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal
(∗ author for correspondence, e-mail: [email protected]; Tel: +351 234 370 737,
Fax: +351 234 370 084)
(Received 10 August 2005; accepted 24 January 2006)
Abstract. A simple, rapid procedure for the determination of organic mercury in sediments, plants
and fish tissues has been developed and validated. Extraction and separation of organic mercury
compounds from the sample matrix was achieved by an established procedure based on an acid
leaching of the sample (H2 SO4 /KBr/CuSO4 ), followed by extraction of the organic mercury halide
with toluene and back-extraction with an aqueous solution of thiosulphate. Detection and quantification of mercury, in the liquid extracts, was made by atomic absorption spectrometry (AAS),
following thermal decomposition of the sample. The method was evaluated using Certified Reference Material (CRM) BCR 463 (tuna fish), BCR 580 (estuarine sediment), IAEA-140TM (sea
plant homogenate) and NRCC TORT-2 (lobster hepathopancreas). The recovery factors for organic
mercury in all tested CRM were between 81–107%. The precision of the method has relative standard deviations of less than 10% for sediments and fish tissues and of less than 16% for plant
material. The method was successfully applied to natural samples of sediments, plants, macroalgae and fish tissues collected from an estuarine ecosystem and could, therefore, be used for routine
analyses.
Keywords: atomic absorption spectrometry with thermal decomposition (AAS), environmental matrixes, organic mercury compounds
1. Introduction
Mercury is toxic, bio-accumulative and persistent in the environment and its behaviour and transport are dependent on its chemical forms (Dong et al., 2004).
Determinations of total or inorganic mercury do not provide adequate information
about its impact on the environment. In particular, organic mercury compounds are
generally more toxic than inorganic mercury salts due to their higher solubility in
lipids, which increases the potential for biological uptake and bio-concentration.
Organic mercury compounds, including methylmercury, are effectively taken up
by aquatic biota with bio-concentration factors of 104 to 107 (Wiener et al., 2002).
These compounds could be a major source of mercury to humans, especially when
fish and seafood are the main components of the diet (Wiener et al., 2002). In
Water, Air, and Soil Pollution (2006) 174: 223–234
DOI: 10.1007/s11270-006-9100-7
C
Springer 2006
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aquatic biota, organic mercury, in the form of methylmercury, is the dominant form
(85 to 90% of total mercury), although it can represent a small fraction of the total
mercury in sediments (Horvat, 1996). Other organic mercury compounds, such
as dimethylmercury, have been detected rarely in sediments, since is readily lost
from the aquatic system and is not considered to be available for uptake by aquatic
organisms (Ullrich et al., 2001).
Since the early 1960s, there has been a growing need to develop more efficient methods of determining mercury and its compounds in a wide variety of
natural matrices (Costley et al., 2000). The methodologies used for organic mercury determination (usually as methylmercury) in environmental samples include
a pre-treatment performed by acid leaching, alkaline digestion, distillation procedures and volatilization (Hintelmann, 1999). Most methods are based on Westöö’s
method or modifications of it, in which the extraction of organic mercury compounds is performed by acid leaching of the sample with HX (X = Cl, Br or
I), followed by the extraction of the organic mercury halide with an organic solvent (benzene or toluene) and back-extraction with a aqueous solution of cysteine
or thiosulphate. Accurate determination of organic mercury compounds is made
more difficult by the formation of artefacts, particularly the conversion of inorganic mercury into organic mercury during sample preparation. However, careful
acid leaching (H2 SO4 /KBr/CuSO4 ) avoids the formation of artefacts (Falter et al.,
1999; Hintelmann, 1999).
The methods described in the literature for separating and quantifying organic
mercury compounds in environmental samples, are based on the combination of
an appropriate separation technique such as gas chromatography (GC) or high
performance liquid chromatography (HPLC), coupled to a sensitive and selective
detector. Examples of hyphenated techniques with GC are GC-ECD (Canário et al.,
2004), GC-MIP (Pereiro et al., 1998), GC-AFS (Bryce et al., 2004), GC-AAS (Salih
et al., 1998), GC-ICP-MS (Wasik et al., 1998), GC-GD-AES (Velado et al., 1998)
and for HPLC are HPLC-ICP-MS (Wan et al., 1997), HPLC-CV-AFS (Ramalhosa
et al., 2001), HPLC-CV-AAS (Falter and Schöler, 1994), HPLC-AES (Colon and
Barry, 1990), HPLC-API-MS (Harrington et al., 1998).
Most hyphenated systems above are not commercially available, they are time
consuming and involve complex analytical pre-treatment (quantitative extraction,
derivatisation, separation, pre-concentration, detection and quantification), which
may introduce more uncertainty and give rise to low precision and a lack of repeatability (Quevaullier, 1999; Landaluze et al., 2004).Thus, there is a need for the
development of a simple and precise analytical methodology that can be adapted
for routine analysis.
The main objective of this work was to develop an accurate and precise method
for the determination of organic mercury in environmental matrices. The procedure
was applied to several samples collected in a mercury contaminated coastal lagoon
(Pereira et al., 1998), in order to evaluate the concentrations of organic mercury in
sediments, plants, macroalgae and fish tissues.
DETERMINATION OF ORGANIC MERCURY
225
2. Experimental
2.1. REAGENTS
All the chemicals were of analytical-reagent grade and where possible “Hg-free”
reagents were used. Working solutions were prepared using ultra-pure water from a
Milli-Q model 185 system. Plastic and glassware material were cleaned by soaking
into 3% Decon (24 h), then soaking in 25% HNO3 (24 h) and finally rinsed with
Milli-Q water.
2.2. EXTRACTION
PROCEDURE
Figure 1 shows the schematic procedure for the extraction of organic mercury
compounds, where all the solutions were prepared daily. About 50–200 mg of
Figure 1. Schematic representation of organic mercury extraction procedure.
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CRM of fish, plant and sediment were accurately weighed into 50 mL polypropylene centrifuge tubes and then 5 mL of KBr (18%) in H2 SO4 (5%) and 1 mL of
CuSO4 (1 mol L−1 ) were added to the tube. The tubes were held at room temperature for 15 min and then treated with 5 mL of toluene, followed by vigorous agitation for 15 min, to extract organic mercury. The organic phase was
separated by centrifugation (1681 ×g for 15 minutes) and then 3 mL of the organic extract was decanted to glass vessels and stored. The extraction process
was repeated twice more and each time the organic extract was retained. In
the initial experiments, the toluene extraction was optimised to three extractions because further extractions did not significantly increase ( p≤ 0.05) the
organic mercury concentrations. The organic mercury compounds retained in
the toluene were back-extracted into an aqueous sodium thiosulphate solution
0.002 mol L−1 (5 mL). Procedural blanks were carried out for quality assurance
purposes.
2.3. DETERMINATION
OF TOTAL AND ORGANIC MERCURY IN
CRMS
MATERIALS
Extracted solutions from each of the CRMs were analysed directly by atomic absorption spectrometry (AAS) with thermal decomposition, using an Advanced Mercury Analyser (AMA, LECO 254). This methodology is simple and based on a
thermal decomposition of the sample and collection of the mercury vapour on a
gold amalgamator. The liquid sample (1 mL maximum) is placed into a nickel boat
and located in a quartz combustion tube, containing a catalyst. The sample is firstly
dried at 120 ◦ C prior the combustion at 680–700 ◦ C in an oxygen atmosphere. The
mercury vapour is collected in a gold amalgamator and after a pre-defined time
(120–150 sec) the gold amalgamator is heated at 900 ◦ C. The released mercury is
transported to a heated cuvette (120 ◦ C) and then analysed by atomic absorption
spectrometry (AAS) using a silicon UV diode detector. Operational conditions used
included a drying time: 350–700 sec; decomposition time: 150 seconds; waiting
time: 40 seconds. The total time for each analysis varied between 9 min and 15 min
when 0.5 and 1 mL of sample is used, respectively. The major advantage of using the
thermal decomposition technique for mercury determinations is that it is not require
complex manipulation of the sample, such as derivatisation processes. Therefore,
an improvement of the sample throughput is obtained which is especially important
in environmental analysis whenever a large number of samples are involved. For
total mercury determinations the CRMs materials were directly analysed by atomic
absorption spectrometry (AAS) with thermal decomposition, using the equipment
refereed above. Since CRMs are solid samples the operational conditions used are
different, regarding the drying time. For total mercury determinations the operation
conditions were: drying time: 10 sec; decomposition time: 150 sec; waiting time:
45 sec. The amount of samples used varied between 50 and 500 mg.
DETERMINATION OF ORGANIC MERCURY
227
3. Results and Discussion
The accuracy of the method developed for organic mercury was carried out using the reference materials: BCR 463 (tuna fish), BCR 580 (estuarine sediment)
from the Community Bureau of Reference, IAEA-140TM (sea plant homogenate)
from the International Atomic Energy Agency and NRCC-TORT-2 (lobster hepathopancreas) from the National Research Council Canada. The AAS instrument
has internal calibration (with inorganic mercury standards) between 0 and 500 ng
of Hg and the calibration of the instrument was carried out periodically. Based on a
study by Hall and Pelchat (1997), and on our own experience, the drift in the slope
of the calibration curve over a period of 30 days was <2%. The limit of detection,
10.5 ± 3.0 pg (n = 15; 95% confidence level), was defined as three times the
standard deviation of the blank concentrations. The concentration of the organic
mercury in CRMs, expressed in μg g−1 , were calculated using the concentration
of the metal in the aqueous phase, the volume of aqueous phase (usually 5 mL),
a dilution factor accounting for extraction volumes used and the mass of sample
weighed. The results were blank corrected.
The measured concentrations of organic mercury in the CRMs were compared to
the certified values using an F test (two-tail test) for a comparison of the standard
deviations and a t test for a comparison of the means (Miller and Miller, 2000)
(Table I). For the standard deviations, the null hypothesis was that the variances of
the certified value and the measured value did not differ significantly. For the means
of the certified values and the measured values the null hypothesis adopted was that
there was no difference between the two values. The values of F and t were lower
than the respective critical value ( p ≤ 0.05) for each CRM and we conclude that
there are no significant differences between the variances and the means of certified
and measured value, at the 5% level for each CRM. The standard deviations for
the certified values were calculated for n = ∞ (t = 1.96), using the value of the
confidence interval given by the institutions responsible for certifying the reference
materials.
Recoveries of organic mercury were 88–106% for BCR-580 (sediments),
81–103% for IAEA-140 TM (plants) and 97–105% for BCR-463 and 91–107% for
NRCC-TORT-2 (fish matrices). These values confirm the accuracy of the methodology, even though they differ depending on the matrix used. The reproducibility
of the methodology was evaluated by conducting several independent analyses.
The results indicate a relative standard deviation (RSD) always lower than 10%
for certified materials of fish and sediments; however, for plant certified material
(IAEA-140TM) RSD was 16%. The high RSD for the IAEA-140TM material was
probably due to the low concentration of organic mercury on this material. The low
RSD values demonstrate the good precision of the methodology.
The method developed for determination of organic mercury compounds was
applied to sediment and plant samples collected in a salt marsh of a contaminated coastal lagoon (Pereira et al., 1998). In salt marshes, plants can interact
Cert. value
±
±
±
±
a
±
±
±
±
28
132
7
3
Recoverya (%) n
0.03
92
0.04 100
4
100
0.001 95
Exp. value
Total mercury ± s ( ± g g−1 )
0.08 2.61 ±
0.03 0.27 ±
1.5 132.5 ±
0.03 0.036 ±
Cert. value
12 2.85
22 0.27
19
132
7 0.038
Recoverya (%) n
0.08
2.82 ± 0.04
93
6.5 × 10−3 1.52 × 10−1 ± 4.05 × 10−3 100
1.85 × 10−3 7.38 × 10−2 ± 3.04 × 10−3 97.7
5.35 × 10−5 6.21 × 10−4 ± 8.34 × 10−5 98.6
Exp. value
Recovery factor = (Exp. Value/Cert. Value) × 100
n,= number of replicates.
BCR 463
3.04
NRCC Tort-2 1.52 × 10−1
BCR 580
7.55 × 10−2
IAEA- 140TM 6.26 × 10−4
CRM
Organic mercury ± s (μg g−1 )
TABLE I
Total and organic mercury concentrations (μg g−1 ) determined in the certified reference materials
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M. VÁLEGA ET AL.
DETERMINATION OF ORGANIC MERCURY
229
with the surrounding sediment exuding oxygen and organic compounds and the intense microbial activity in the interfacial zone between the roots and the sediment
can influence the distribution and the availability of trace metals (Alloway, 1995;
Mendelssohn et al., 1995). Due to difficulties associated with organic mercury determinations, there are only few works presenting levels of these compounds in this
type of ecosystems (Heller and Weber, 1998). In order to evaluate the vertical distribution of total and organic compounds in a mercury contaminated salt marsh of the
Portuguese coast, sediment cores were collected at two sites, Stations A and B, in
the most contaminated area of the lagoon. Sediment cores were sliced in 5 cm layers
and root biomass separated from each sediment layer. Total and organic mercury
compounds were analysed in belowground biomass (Figure 2) and in sediments
(Figure 3). Obtained results showed that total mercury concentrations in sediments
and in belowground biomass have an increase in the sub-surface layers and that
Figure 2. Vertical profiles of total mercury (μg g−1 ) and organic mercury species (μg g−1 ) concentrations in root biomass at Station A and Station B.
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M. VÁLEGA ET AL.
Figure 3. Vertical profiles of total mercury (μg g−1 ) and organic mercury species concentrations
(μg g−1 ) in sediments at Station A and Station B.
Figure 4. Total mercury (A) and organic mercury (B) concentrations (μg g−1 dwt) in Enteromorpha
intestinalis, Gracilaria verrucosa and Fucus vesiculosus in the Ria de Aveiro (error bars represent
standard deviation).
DETERMINATION OF ORGANIC MERCURY
231
Figure 5. Total mercury in muscle of sea bass (μg g−1 fwt) in different stations (D1 to D4).
total mercury concentrations in belowground biomass (Figure 2) of the two sites
were quite different. Also the percentage values of organic mercury compounds in
belowground biomass samples were quite different, with maximum values of 3.5%
at 7.5 cm depth in one site and 3.3% at 22.5 cm depth in the other site.
The method was also applied to macroalgae samples collected in the same coastal
lagoon in Portugal (Coelho et al., 2005). Primary producers represent an important
pathway for mercury incorporation in aquatic food webs. Macroalgae may represent a substantial pool of mercury, as a result of its high growth rate and capacity
to bind trace metals. Total and organic mercury levels of the dominant species
(Enteromorpha, Fucus and Gracilaria) were determined (Figure 4). Total mercury
in the macroalgae tissues ranged from 0.02 to 2.1 μg g −1 dwt. Fucus was the most
contaminated algae, followed by Gracilaria and Enteromorpha. As a whole, organic mercury never exceeded 15% of total mercury content, but tended to increase
with distance to metal source on all macroalgae indicating complex physiological
responses from these primary producers in areas of high and low mercury concentrations. The developed method was also applied to the fish sea bass D. labrax, a
specie with a high commercial value that enter the contaminated the Portuguese
coastal lagoon every year (Rebelo, 1994). Samples were captured at four sites (D1
to D4) at different distances to the most contaminated area and the fish specimens
were dissected and muscle tissue separated. Total mercury in muscle of sea bass,
are shown in Figure 5 (Abreu et al., 2000). Total mercury concentrations in muscle
ranged from 0.03 to 1.7 μg g−1 fwt, the highest values being found in individuals
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M. VÁLEGA ET AL.
captured in the most contaminated area. In general, smaller individuals showed
lower mercury concentrations. Ingestion of contaminated food and exposure to a
contaminated environment, may contribute to the higher mercury accumulation in
fish captured in the contaminated area. Organic mercury determinations gave results for the muscle of the fish D. labrax that are always higher than 95% of the total
metal amount. Total and organic mercury compounds were analysed in triplicate in
all samples. Reference materials and procedure blanks were always run in parallel
with the samples, and added to each batch of samples for analysis.
The intention of this work was not to discuss the obtained results, but to show
important applications of the developed methodology.
4. Conclusions
The proposed method to determine organic mercury compounds involves a well
known extraction procedure of these compounds from different matrices and an
analysis of the extracted organic mercury by thermal atomic absorption spectroscopy. The method is relatively rapid (9 to 15 min, for analysis after extraction)
and showed good agreement with the certified values for two fish tissues, one estuarine sediment and one sea plant. The obtained results revealed that the extraction
method can be applied with success to the tested matrices, allowing several samples
to be analysed in a single day, making possible its application in routine analyses,
since the used equipment (LECO AMA-254) is a simple and a commercial available one, normally used for the determination of total mercury concentrations in
routine laboratories.
Acknowledgements
The authors would like to acknowledge to Portuguese Science and Technology
Foundation (FCT) for financial support under the project SFRH/BD/18682/2004.
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