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Analysis of mercury and selenium during subcritical water treatment of fish tissue by various atomic spectrometric methods
Microchemical Journal 106 (2013) 357–362
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Analysis of mercury and selenium during subcritical water treatment of fish tissue by various atomic spectrometric methods Akira Ohki ⁎, Kentaro Hayashi, Joji Ohsako, Tsunenori Nakajima, Hirokazu Takanashi Department of Chemistry, Biotechnology, and Chemical Engineering, Graduate School of Science and Engineering, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890-0065, Japan
a r t i c l e
i n f o
Article history: Received 28 August 2012 Received in revised form 18 September 2012 Accepted 18 September 2012 Available online 26 September 2012 Keywords: Subcritical water Fish tissue Dissolution Methylmercury Selenium
a b s t r a c t Subcritical water treatment of biomass and food waste (especially marine product) is now very attractive in terms of the production of useful chemicals. Big marine fish, such as tuna, usually contain a high level of Hg, which is considered safe because of the sequestration by Se species. In this study, such fish tissues were treated with subcritical water, and the resulting aqueous phase was analyzed for Hg and Se to estimate the dissolution behavior of these toxic elements. The determination of total Hg in the aqueous phase was carried out by heat vaporization atomic absorption spectrometry (HVAAS), while that of Se was done by inductively coupled plasma mass spectrometry (ICP-MS). The dissolution behavior of those elements was favorably monitored. The speciation of Hg in the aqueous phase was performed by HPLC with cold vapor atomic fluorescence spectrometry aided by UV irradiation–oxidation (HPLC-UV-CVAFS), and the Hg species detected was mainly methylated Hg. For the speciation of Se, size-exclusion chromatography (SEC) with ICP-MS was attempted to use, and it was found that high molecular and insoluble Se species, which were originally contained in the tissue, was gradually decomposed into lower molecular hydrophilic species, and dissolved in the aqueous phase. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Subcritical water (SW) is formed from pure water under high temperature and high pressure, and it gives a low dielectric constant and a high ionic product. The former leads to an increase in the solubility of organic compounds, while the latter produces the promotion of chemical reactions, such as hydrolysis. The SW treatment is used for the production of useful chemicals, such as organic acids and amino acids, from biomass and food waste [1,2]. For such raw materials, various samples including marine product have been attempted [3–9]. Mercury (Hg) is well-known as a toxic element, and especially its organometallic form is very toxic. Therefore, according to the Japanese effluent standards, alkyl compounds of Hg are regulated as “not detectable”. It is also known that fish (especially large marine fish such as tuna) often contain high levels of Hg and methylated Hg. However, these Hg species have been considered not so toxic because the prevention of toxicity works in a certain mechanism. Parizek and Ostadalova firstly reported the importance of selenium (Se) in the prevention of Hg toxicity in 1967 [10], and then many researchers have shown such a toxicity prevention and tried to elucidate the mechanism in various living organisms [11]. It has been said that the toxicity prevention is caused by the sequestration of Hg (and/or methylated Hg) by Se. The detoxification of Hg caused by the formation of a complex ⁎ Corresponding author. Tel.: +81 99 285 8335; fax: +81 99 285 8339. E-mail address: [email protected] (A. Ohki). 0026-265X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.microc.2012.09.008
between Se-containing proteins (selenoproteins) and Hg in organisms has been studied by the use of size-exclusion chromatography (SEC) with inductively coupled plasma mass spectrometry (ICP-MS) [12,13], and a similar complex has been found [14,15]. Tavakoli and Yoshida reported that when the SW treatment of squid wastes was performed, copper, zinc, and cadmium contained in the organisms were dissolved into the aqueous phase [16]. There have been no studies on the analysis of Hg and Se in the aqueous phase during the SW treatment of marine organisms, although many studies have been done about the determination and speciation of those elements in aqueous media and biological samples [17–21]. It is possible that toxic free Hg species (especially methylated Hg species) contained in the organisms are dissolved into the aqueous phase, and thus the contamination of those species in the product and/or effluent may occur when the SW treatment of marine organisms is carried out. In this study, the SW treatment of fish tissue was carried out in an autoclave, and the Hg and Se species in the resulting aqueous solution was analyzed. The determination of total Hg in the aqueous phase was carried out by heat vaporization atomic absorption spectrometry (HVAAS), while that of total Se was done by ICP-MS. The speciation of Hg was performed by high performance liquid chromatography (HPLC) with cold vapor atomic fluorescence spectrometry (CVAFS) aided by UV irradiation–oxidation (HPLC-UV-CVAFS), while that of Se was carried out by SEC-ICP-MS. The dissolution mechanism of Hg and Se in the SW treatment of fish tissue was estimated in terms of the dissolution behavior of Hg and Se species observed by those analytical methods. Further, the
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use of SW treatment as a novel method for the extraction of specific elements from biological samples was discussed. 2. Experimental 2.1. Samples and reagents Two fish tissue (muscle) samples, southern bluefin tuna (Thunnus maccoyii) and mackerel (Scomber japonicas), which had been purchased at a local supermarket, were tested. Certified reference materials, DORM-2 (dogfish muscle) and DOLT-3 (dogfish liver), were obtained from the National Research Council, Canada. Analytical-grade reagents were used for the preparation of all solutions. Ultrapure water was prepared by Purelab Ultra Ionic (Organo Corporation, Tokyo, Japan) and used throughout the experiments. 2.2. Analysis of Hg and Se in fish tissue The determination of total Hg in the fish tissue was performed by the use of HVAAS (MA-2000; Nippon Instruments Corporation, Tokyo, Japan). For the speciation of Hg, i.e., the fractional determination of mercuric Hg (Hg 2 +) and methylated mercuric Hg (CH3Hg +) in the fish tissue, firstly the extraction of Hg species in the sample was carried out. An aqueous solution (10 mL) containing 0.1% (v/v) HCl, 0.15 (m/v) KCl, and 0.1% (v/v) 2-mercaptoethanol was shaken with 0.1 g of powdery dried sample in a stoppered centrifuge tube for 12 h. After centrifugation, the solution was analyzed by HPLC-UV-CVAFS. The HPLC system consisted of a PU-2080 pump (JASCO Corporation, Tokyo, Japan) with a Rheodyne injector. The separation of Hg species occurred in a 150 mm × 4.6 mm Wakosil-II 5C18 HG column (Wako Pure Chemical Industries, Osaka, Japan), and the flow rate of the mobile phase (CH3OH : H2O=1:9 solution containing 0.01% (v/v) 2-mercaptoethanol) was adjusted to 0.85 mL/min. According to the manufacturer's protocol, the photooxidation of CH3Hg+ to Hg2+ was performed in a PSA 10.570 UV-oxidation system (PS Analytical; Orpington, UK) using an oxidant solution, while the CVAFS measurement was carried out using a PSA 10.025 Millennium Merlin instrument (PS Analytical). The determination of total Se in the fish tissue sample was performed by microwave-assisted acid digestion followed by ICP-MS equipped with a collision cell (H2 mode) (Agilent 7500cx, Agilent Technologies, Tokyo, Japan). The sample was digested with a mixture of HNO3 and H2O2 using a microwave processor (ETHOS 1; Milestone Inc., Shelton, CT, USA) in a similar manner described in our previous papers [22–24]. The measurements of Hg and Se were done in triplicate and the mean value and standard deviation were obtained. 2.3. SW treatment of fish tissue Powdery dried fish tissue (0.5 g) was mixed with 10 mL deionized water and the mixture was placed in a 25 mL autoclave (SAN-AI science Co. Ltd., Nagoya, Japan). The atmosphere in the autoclave was purged by argon, and it was heated to a desired temperature (100–250 °C) in an electric oven (FP-41; Yamato Science Co., Ltd., Tokyo, Japan). After cooling and centrifugation to remove the majority of insoluble solid, the aqueous phase was filtrated with a membrane filter (0.2 μm; ADVANTEC, Tokyo, Japan) to remove oil and remaining insoluble solid, and analyzed for Hg and Se. In order to measure the mass balance of Hg, the insoluble solid removed and the oil trapped on the membrane filter were analyzed for Hg by HVAAS in some cases. Total organic carbon (TOC) in the aqueous phase obtained was determined by an automatic TOC analyzer (TOC-V CSH; Shimadzu Corporation, Kyoto, Japan). The determination of total Hg in the aqueous phase was carried out by HVAAS, while that of total Se was done by ICP-MS (H2 mode). The speciation of Hg in the aqueous phase was carried out by HPLC-UV-CVAFS in a similar manner described above. The Se species in the aqueous phase was
analyzed by SEC-ICP-MS. The SEC system consisted of a PU-2080 pump (JASCO corporation) with a Rheodine injector and Superdex Peptide 10/300 GL column (GE Helthcare Japan, Tokyo, Japan), and the flow rate of the mobile phase (100 mM ammonium acetate, pH 8.0) was adjusted to 0.7 mL/min. The column was calibrated with standard polyethyleneglycols using a refractive index detector (RI-2031, JASCO corporation). Also, the aqueous phase was analyzed by SEC with UV detector (UVD) at 220 nm using a UV-2025 instrument (JASCO corporation). 3. Results and discussion 3.1. Analysis of Hg and Se in fish tissue Firstly, the analysis of Hg and Se present in the fish tissues used (southern bluefin tuna and mackerel) was carried out. The determination of total Hg in the fish tissue was conducted by HVAAS, while that of Se was done by microwave-assisted acid digestion followed by ICP-MS. Also, the fractional determination of Hg 2+ and CH3Hg + was carried out by the extraction with a 2-mercaptoethanol solution followed by HPLC-UV-CVAFS. The results are listed in Table 1. To confirm the accuracy of the results, two certified reference materials, DORM-2 and DOLT-3, were analyzed by the same method. The concentrations of total Hg and Se measured were about the same as the certified values both for DORM-2 and DOLT-3. Also, the concentrations of CH3Hg + obtained were very close to the certified values. It is proved that the extraction and HPLC-UV-CVAFS method effectively works for the determination of CH3Hg + present in the biological samples. For southern bluefin tuna (tuna) and mackerel, the total Hg concentrations were 4.01 and 1.09 μg-Hg/g, respectively, in which 84% and 63% of Hg were methylated species. The molar ratios of total Hg and total Se for tuna and mackerel were 0.41 and 0.16, respectively, which were similar to those reported in literatures [25,26]. 3.2. SW treatment of fish tissue A powdery dried fish tissue (tuna) was subjected to the SW treatment in an autoclave at 220 °C in the liquid/solid (dry) ratio of 20. To investigate the progress of SW reaction, the dissolution of organic matter from the tissue into the aqueous phase was examined, and thus TOC in the aqueous phase was measured after the filtration. In Fig. 1, plots of the TOC against the treatment time are presented. The zero point of time in Fig. 1 and subsequent related figures was set when the oven temperature reached the desired temperature. Therefore, the interior of autoclave may not reach the set temperature at the zero point, and thus the increase in the TOC seems slow in early time. The degree of TOC increased as the time elapsed, and reached ca. 18,000 mg/L at 120 min. Considering the carbon content of the fish tissue (53% in dry base), the TOC yield is ca. 60%, which is similar to that reported in a previous study about the SW treatment of fish tissue [3]. It is evident that the biopolymers, such as proteins and sugars, in the tissue are hydrolyzed, and solubilized into the aqueous phase when the fish tissue is treated under the SW conditions. In fact, an appreciable amount of peptide species was observed in the aqueous phase after the SW treatment (see Fig. 5). This will be described later. 3.3. Determination of total Hg in the aqueous phase during SW treatment It is proposed that the Hg and Se species present in the fish tissue are dissolved into the aqueous phase when the SW reaction proceeds. Firstly, the determination of total Hg in the aqueous phase was attempted by the use of HVAAS when the 220 °C-SW treatment for tuna above mentioned was carried out. As mentioned before, the interior of autoclave may not reach the set temperature at the zero point of time. Therefore, as shown in Fig. 2, the dissolution of Hg scarcely occurred at 30 min.
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Table 1 Analysis of Hg and Se in fish tissue. Fish tissue
Hg species (μg-Hg/g, dry weight) CH3Hg+
Total Hg
DORM-2 DOLT-3 Tuna Mackerel a
Total Se (μg-Se/g, dry weight)
Hg2+
Certified
Measured
Certified
Measured
Measured
Certified
Measured
4.64 ± 0.26 3.37 ± 0.14 _ _
4.50 ± 0.09 3.35 ± 0.04 4.01 ± 0.01 1.09 ± 0.02
4.47 ± 0.32 1.59 ± 0.12 _ _
4.39 ± 0.44 1.55 ± 0.08 3.35 ± 0.10 0.69 ± 0.02
nda 1.64 ± 0.03 0.39 ± 0.04 0.35 ± 0.04
1.40 ± 0.09 7.06 ± 0.48 _ _
1.44 ± 0.05 6.83 ± 0.33 3.87 ± 0.24 2.66 ± 0.03
Not detected.
Afterward, the total Hg concentration increased and reached 101 μg-Hg/L at 90 min and then somewhat decreased. It is apparent that the dissolution of Hg from the fish tissue into the aqueous phase takes place due to the SW treatment, and the dissolution behavior can be effectively monitored by HVAAS. The Hg concentration (101 μg-Hg/L) corresponds to ca. 50% of Hg which is originally present in the fish tissue. The mass balance study showed that only 15% of Hg originally present occurred in the insoluble solid and oil phase. Therefore, the fate of the rest of Hg (ca. 35%) is not clear. Probably, the rest of Hg is released to gas phase to form volatile species, such as Hg 0, and the release of Hg is promoted as the reaction time elapsed, so that the Hg concentration in the aqueous phase was decreased. However, the capture of the volatile species was not successful. When the SW treatment was done at a higher temperature (250 °C), the dissolution of Hg into the aqueous phase was more promoted and reached 107 μg-Hg/L at 60 min and then greatly decreased as the time elapsed. When the SW temperature is higher, the release of Hg into the gas phase may be enhanced, and thus the decrease in the Hg concentration in the aqueous phase is more remarkable. As shown in Table 2, the dissolution of Hg from the fish tissue into the aqueous phase was quite small when the treatment temperature was at 180 °C or below. It appears that the hydrolysis of proteins is not effective when the temperature is 180 °C or below, and thus the sequestration of Hg by Se (selenoproteins) in the fish tissue is still efficiently working. However, when the temperature becomes 220 °C, the hydrolysis of proteins including selenoproteins considerably occurs, resulting in the dissolution of Hg into the aqueous phase. When other fish tissue (mackerel) was used in the SW treatment at 220 °C, the dissolution of Hg into the aqueous phase similarly occurred (Table 2). The concentrations of Hg in the aqueous phase was gradually increased when the time elapsed, and reached 33.7 μg-Hg/L at 120 min, which corresponded to ca. 60% of Hg originally present in the fish tissue.
TOC (mg/L)
15000
10000
5000
0
The speciation of Hg in the aqueous phase was conducted by the use of HPLC-UV-CVAFS when the SW treatment of fish tissue was carried out. As shown in Table 3, the majority of Hg species found in the aqueous phase was CH3Hg +, and the sum of the concentrations of Hg 2+ and CH3Hg + in the aqueous phase was about the same as the total Hg concentrations measured by HVAAS shown in Table 2. This result suggests that the analysis by HPLC-UV-CVAFS favorably works for the speciation of Hg during the SW treatment, and other Hg species do not occur in the aqueous phase. It is found that the methylated Hg species, which is originally present in the fish tissue, is dissolved into the aqueous phase, i.e., the free form of methylated species, which should be very toxic, occurs in the aqueous phase when the SW treatment of fish tissue is carried out. For mackerel, ca. 30% of Hg originally present in the tissue was Hg 2+ (see Table 1). However, the Hg species found in the aqueous phase after the SW treatment was mainly CH3Hg + (Table 3). It is presumed that the Hg 2+ released from the fish tissue is reduced to volatile Hg 0. 3.5. Determination of total Se in the aqueous phase during the SW treatment When the SW treatment for tuna was carried out, the total Se concentration in the aqueous phase was measured by ICP-MS. As shown in Fig. 3, the total Se concentration increased and reached 172 μg-Se/L at 120 min when the 220 °C-SW treatment was performed. The Se concentration (172 μg-Se/L) corresponds to ca. 95% of Se which is originally contained in the fish tissue. It is found that the dissolution of Se from the fish tissue into the aqueous phase is effectively monitored, and almost all of Se present in the fish tissue is dissolved into the aqueous phase during the SW treatment, which is different from the result for Hg above mentioned. When the SW treatment was done at a high temperature (250 °C), the dissolution of Se was considerably enhanced and ca.
Concentration of total Hg (µg-Hg/L)
20000
3.4. Speciation of Hg in the aqueous phase during SW treatment
0
30
60
90
120
Treatment time (min) Fig. 1. TOC in the aqueous phase in SW treatment (tuna).
150
120 100
220 ºC
80 60 250 ºC
40 20 0
0
30
60
90
120
150
Treatment time (min) Fig. 2. Concentration of total Hg in the aqueous phase in SW treatment (tuna).
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200
Fish tissue
Tuna
Mackerel
a
Temperature (°C)
Time (min)
100 140 180 220 250 220 250 220 220 220
90 90 90 90 90 120 120 60 90 120
Concentration of total Se (µg-Se/L)
Table 2 Concentration of total Hg and Se in the aqueous phase in SW treatment. Concentration in aqueous phase Total Hg (μg-Hg/L)
Total Se (μg-Se/L)
1.1 6.8 nda 101 75.5 90.4 39.3 14.5 28.8 33.7
50.6 52.0 82.5 138 180 172 180 74.1 110 126
250 ºC
150 220 ºC
100
50
0
0
30
60
90
120
150
Treatment time (min)
Not detected. Fig. 3. Concentration of total Se in the aqueous phase in SW treatment (tuna).
100% of Se was released into the aqueous phase at 90 min. Although the dissolution of Hg from the tissue was quite small when the treatment temperature was 180 °C or below, that of Se considerably occurred (Table 2). It is proposed that some portion of Se species present in the fish tissue are very hydrophilic, whereas the majority of Hg species are strongly sequestrated in the tissue. For mackerel, the total Se concentration in the aqueous phase was 126 μg-Se/L when the 220 °C-SW treatment (120 min) was performed (Table 2). The Se concentration corresponds to ca. 100% of Se which is originally present in the fish tissue. 3.6. Speciation of Se in the aqueous phase during SW treatment When the 220 °C-SW treatment for tuna was carried out, the speciation of Se in the aqueous phase was conducted by SEC-ICP-MS to clarify the fate of selenoproteins originally present in the fish tissue. The results are shown in Fig. 4. In the chromatogram for the 30 min-treatment, only two peaks were observed in low molecular weight region (Peaks 1 and 2). Commercially available Se compounds, selenomethionine and selenocystine, gave their peaks in the Peak 1 region and the Peak 2 region, respectively, while SeO42 − and SeO32 − provided their peaks in the Peak 1 region and the Peak 2 region, respectively. Therefore, it is proposed that Peaks 1 and 2 in Fig. 4 are assigned to some kinds of selenoamino acids and inorganic Se species, such as SeO42 − and SeO32 −. Consequently, it is found that when the treatment time is 30 min, only hydrophilic Se species are dissolved into the aqueous phase from the fish tissue. When the treatment time was 60 min, a broad peak at 1–6 kDa region in the chromatogram appeared. The broad peak shifted to lower molecular weight region while the size of Peak 1 increased, as the reaction proceeded. When the treatment time was 120 min, the broad peak merged to Peak 1 to give the left shoulder of Peak 1. It is evident that the selenoproteins present in the fish tissue are hydrolyzed by the SW treatment, and the hydrolyzed proteins (selenopeptides), which should be hydrophilic, are dissolved into the aqueous phase. The selenopeptides undergo more hydrolysis
to finally give monomeric (and/or short-oligomeric) selenoamino acids. The size of Peak 2 gradually decreased as the reaction proceeded. The phenomenon cannot be clearly explained, but it is presumed that the oxidation of SeO32− into SeO42− occurs. When the same SW treatment mentioned above was performed, the aqueous phase was also analyzed by SEC-UVD at 220 nm. The results are shown in Fig. 5. In the chromatogram for the 30 min-treatment, peaks are observed only in low molecular weight region. These peaks are presumably attributed to amino acids (monomeric and/or short-oligomeric) and organic acids, which are hydrophilic and thus are dissolved into the aqueous phase in early time. When the treatment time was 60 min, a broad peak from 1–6 kDa in the chromatogram appeared, and shifted to lower molecular region as the time elapsed. The broad peak should be assigned to protein decomposition products (peptides), and this phenomenon is quite similar to that seen in Fig. 4 (SEC-ICP-MS chromatogram). When the 220 °C-SW treatment for mackerel was carried out, the Se species in the aqueous phase was analyzed by SEC-ICP-MS. The results are shown in Fig. 6. The chromatograms are very similar to those for tuna shown in Fig. 4. When the 30 min-SW treatment was conducted, the chromatogram gave only two peaks in low molecular weight region. As the treatment time elapsed, a broad peak at 1–6 kDa region appeared, and shifted to lower molecular region. As a result, it is found that the hydrolysis of selenoprotein originally present in the fish tissue occurs in a similar manner, which is independent of the kind of fish.
Treatment time
Tuna Mackerel a
From Table 2.
Time (min)
90 120 90 120
Total Hga (μg-Hg/L)
101 90.4 28.8 33.7
2 kDa
1 kDa
Peak 2 Peak 1
500 cps
30 min
60 min
Table 3 Speciation of Hg in the aqueous phase in SW treatment (220 °C). Fish tissue
6 kDa
90 min
Speciation of Hg Hg2+ (μg-Hg/L)
CH3Hg+ (μg-Hg/L)
5.0 4.8 4.9 1.4
93.8 74.4 23.7 31.4
120 min 0
5
10
15
20
25
30
35
Retention time (min) Fig. 4. SEC-ICP-MS chromatogram of Se species in the aqueous phase in SW treatment (tuna, 220 °C).
A. Ohki et al. / Microchemical Journal 106 (2013) 357–362
Treatment time
6 kDa
2 kDa
1 kDa 600 mV
30 min
60 min
90 min
120 min 0
5
10
15
20
25
30
35
Retention time (min) Fig. 5. SEC-UVD chromatogram for the aqueous phase in SW treatment (tuna, 220 °C).
3.7. Dissolution mechanism of Hg and Se from fish tissue It has been proved that the monitoring and speciation of Hg and Se in the aqueous phase during the SW treatment are efficiently conducted by the use of various atomic spectrometric methods. Based on the results obtained, the dissolution mechanism of Hg and Se from fish tissue into the aqueous phase is estimated. Fish tissues sometimes contain a considerable amount of methylated Hg species, which may be strongly bound to the Se atom in high molecular and insoluble Se species, such as selenoproteins. Therefore, the methylated Hg species do not provide appreciable toxicity. As previously mentioned, the hydrolysis of proteins in the fish tissue is not effective when the SW treatment temperature is 180 °C or below, and the temperature of 220 °C is needed to attain the efficient decomposition of proteins including selenoproteins. As seen in the SEC-ICP-MS chromatogram (Fig. 4), when the 220 °C-SW treatment was performed, only low molecular Se species were detected in early time (30 min). As described before, when the treatment time is 30 min, the interior temperature of autoclave does not reach the set temperature. It is evident that high molecular Se species (selenoproteins) are not decomposed and are still present in the fish tissue after the 30 min-treatment because the interior temperature is not insubstantial. Also, the 30 min-treatment hardly gave the dissolution of Hg into the aqueous phase (Fig. 2). Consequently, it is proved that the Hg species are strongly sequestrated by the selenoproteins in the fish tissue, and the decomposition of the proteins is necessary for the dissolution of Hg into the aqueous phase. Contrary to the Hg species originally present in the fish tissue, a certain portion of Se species, such as
Treatment time
6 kDa
2 kDa
1 kDa 800 cps
30 min
361
selenoamino acids and inorganic Se species, are quite hydrophilic. Even for the 100 °C-treatment, almost half of Se contained in the fish tissue was dissolved into the aqueous phase (Table 2). Eventually, the mechanism of Hg dissolution in the SW treatment will be depicted as follows. When the SW treatment of fish tissue is carried out under sufficient conditions, the insoluble proteins including selenoproteins in the tissue are hydrolyzed to form hydrophilic peptides. The lower molecular weight Se species (selenopeptides) tends to be dissolved into the aqueous phase, and then the Hg species including CH3Hg + sequestrated by the insoluble selenoproteins are also dissolved into the aqueous phase. Since the sequestration of Hg by Se species does not work in the aqueous phase, it is evident that highly toxic free CH3Hg + really occurs in the aqueous phase when the SW treatment of fish tissue is performed. It is possible that such a toxic Hg species is contaminated in the effluent produced by the SW treatment process and/or some final products, such as organic acids and amino acids. Although the Hg species contained in the fish tissue was not completely dissolved into the aqueous phase when the SW treatment was carried out, almost all of Se species in the tissue was eluted into the aqueous phase. Therefore, on the other hand, the SW treatment can be used as a novel method for the extraction of Se from biological samples. A great advantage of this method is that the extraction solvent is only pure water, and thus there should be no contamination from the solvent components, such as acids, bases, surfactants, and protein-decomposition agents. For the speciation of Se, the selenoproteins originally present in fish tissue is decomposed into hydrophilic selenopeptides, which can be detected by the use of SEC-ICP-MS. Therefore, the detection of selenopeptides in the aqueous phase when the SW treatment of biological sample is performed will be the proof of the presence of selenoproteins in the sample. 4. Conclusions The SW treatment of fish tissue (tuna and mackerel) was carried out in an autoclave, and the Hg and Se species in the resulting aqueous solution were analyzed. The monitoring of total Hg in the aqueous phase was successfully carried out by HVAAS, while that of total Se was done by ICP-MS. The speciation of Hg in the aqueous phase was favorably attained by HPLC-UV-CVAFS, and the Hg species detected was mainly CH3Hg+. For the speciation of Se, SEC-ICP-MS was successfully used, and the dissolution behavior of Se species from fish tissue into the aqueous phase was clearly depicted. Thus, it is proposed that high molecular and insoluble Se species, such as selenoproteins, are hydrolyzed to form lower molecular and soluble species (selenopeptides) during the SW treatment. Consequently, according to the analysis of Hg and Se in the aqueous phase during the SW treatment of fish tissue, it is proved that the highly toxic methylated Hg species in free form occurs in the aqueous phase. Therefore, when the SW treatment of fish tissue is performed, we need to pay attention to the contamination of toxic Hg species in the effluent as well as in the products. On the viewpoint of analytical technique, it can be stated that the SW treatment is a novel method for the extraction of Se species from biological samples. Acknowledgments
60 min
This work was supported by a Grant-in-Aid for Scientific Research (B) (No. 22350069) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.
90 min References
0
5
10
15
20
25
30
35
Retention time (min) Fig. 6. SEC-ICP-MS chromatogram of Se species in the aqueous phase in SW treatment (mackerel, 220 °C).
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[16] O. Tavakoli, H. Yoshida, Effective recovery of harmful metal ions from squid wastes using subcritical and supercritical water treatments, Environ. Sci. Technol. 39 (2005) 2357–2363. [17] Y. Gao, Z. Shi, Z. Long, P. Wu, C. Zheng, X. Hou, Determination and speciation of mercury in environmental and biological samples by analytical atomic spectrometry, Microchem. J. 103 (2012) 1–14. [18] W. Maher, F. Krikowa, M. Ellwood, S. Foster, R. Jagtap, G. Raber, Overview of hyphenated techniques using an ICP-MS detector with as emphasis on extraction techniques for measurement of metalloids by HPLC-ICPMS, Microchem. J. 105 (2012) 15–31. [19] Q. Liu, Determination of mercury and methylmercury in seafood by ion chromatography using photo-induced chemical vapor generation atomic fluorescence spectrometric detection, Microchem. J. 95 (2010) 255–258. [20] E. Kapolna, P. Fodor, Speciation analysis of selenium enriched green onions (Allium fistulosum) by HPLC-ICP-MS, Microchem. J. 84 (2006) 56–62. [21] T. Ferri, F. Coccioli, C. De Luca, C.V. Callegari, R. Morabito, Distribution and speciation of selenium in Lecythis ollaria plant, Microchem. J. 78 (2004) 195–203. [22] Y.-H. Xu, A. Iwashita, T. Nakajima, H. Yamashita, H. Takanashi, A. Ohki, Effect of HF addition on the microwave-assisted acid-digestion for the determination of metals in coal by inductively coupled plasma-atomic emission spectrometry, Talanta 66 (2005) 58–64. [23] A. Iwashita, T. Nakajima, H. Takanashi, A. Ohki, Y. Fujita, T. Yamashita, Effect of pretreatment conditions on the determination of major and trace elements in coal fly ash using ICP-AES, Fuel 85 (2006) 257–263. [24] A. Ohki, K. Yamada, T. Furuzono, T. Nakajima, H. Takanashi, Analysis of trace elements in flue gas desulfurization water in coal combustion system and the removal of boron and mercury from the water, Energy Fuel 25 (2011) 3568–3573. [25] A.I. Cabanero, Y. Madrid, C. Camara, Selenium and mercury bioaccessibility in fish samples: an in vitro digestion method, Anal. Chim. Acta 526 (2004) 51–61. [26] M. Plessi, D. Bertelli, A. Monzani, Mercury and selenium content in selected seafood, J. Food Compos. Anal. 14 (2001) 461–467.
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Microchemical Journal journal homepage: www.elsevier.com/locate/microc
Analysis of mercury and selenium during subcritical water treatment of fish tissue by various atomic spectrometric methods Akira Ohki ⁎, Kentaro Hayashi, Joji Ohsako, Tsunenori Nakajima, Hirokazu Takanashi Department of Chemistry, Biotechnology, and Chemical Engineering, Graduate School of Science and Engineering, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890-0065, Japan
a r t i c l e
i n f o
Article history: Received 28 August 2012 Received in revised form 18 September 2012 Accepted 18 September 2012 Available online 26 September 2012 Keywords: Subcritical water Fish tissue Dissolution Methylmercury Selenium
a b s t r a c t Subcritical water treatment of biomass and food waste (especially marine product) is now very attractive in terms of the production of useful chemicals. Big marine fish, such as tuna, usually contain a high level of Hg, which is considered safe because of the sequestration by Se species. In this study, such fish tissues were treated with subcritical water, and the resulting aqueous phase was analyzed for Hg and Se to estimate the dissolution behavior of these toxic elements. The determination of total Hg in the aqueous phase was carried out by heat vaporization atomic absorption spectrometry (HVAAS), while that of Se was done by inductively coupled plasma mass spectrometry (ICP-MS). The dissolution behavior of those elements was favorably monitored. The speciation of Hg in the aqueous phase was performed by HPLC with cold vapor atomic fluorescence spectrometry aided by UV irradiation–oxidation (HPLC-UV-CVAFS), and the Hg species detected was mainly methylated Hg. For the speciation of Se, size-exclusion chromatography (SEC) with ICP-MS was attempted to use, and it was found that high molecular and insoluble Se species, which were originally contained in the tissue, was gradually decomposed into lower molecular hydrophilic species, and dissolved in the aqueous phase. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Subcritical water (SW) is formed from pure water under high temperature and high pressure, and it gives a low dielectric constant and a high ionic product. The former leads to an increase in the solubility of organic compounds, while the latter produces the promotion of chemical reactions, such as hydrolysis. The SW treatment is used for the production of useful chemicals, such as organic acids and amino acids, from biomass and food waste [1,2]. For such raw materials, various samples including marine product have been attempted [3–9]. Mercury (Hg) is well-known as a toxic element, and especially its organometallic form is very toxic. Therefore, according to the Japanese effluent standards, alkyl compounds of Hg are regulated as “not detectable”. It is also known that fish (especially large marine fish such as tuna) often contain high levels of Hg and methylated Hg. However, these Hg species have been considered not so toxic because the prevention of toxicity works in a certain mechanism. Parizek and Ostadalova firstly reported the importance of selenium (Se) in the prevention of Hg toxicity in 1967 [10], and then many researchers have shown such a toxicity prevention and tried to elucidate the mechanism in various living organisms [11]. It has been said that the toxicity prevention is caused by the sequestration of Hg (and/or methylated Hg) by Se. The detoxification of Hg caused by the formation of a complex ⁎ Corresponding author. Tel.: +81 99 285 8335; fax: +81 99 285 8339. E-mail address: [email protected] (A. Ohki). 0026-265X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.microc.2012.09.008
between Se-containing proteins (selenoproteins) and Hg in organisms has been studied by the use of size-exclusion chromatography (SEC) with inductively coupled plasma mass spectrometry (ICP-MS) [12,13], and a similar complex has been found [14,15]. Tavakoli and Yoshida reported that when the SW treatment of squid wastes was performed, copper, zinc, and cadmium contained in the organisms were dissolved into the aqueous phase [16]. There have been no studies on the analysis of Hg and Se in the aqueous phase during the SW treatment of marine organisms, although many studies have been done about the determination and speciation of those elements in aqueous media and biological samples [17–21]. It is possible that toxic free Hg species (especially methylated Hg species) contained in the organisms are dissolved into the aqueous phase, and thus the contamination of those species in the product and/or effluent may occur when the SW treatment of marine organisms is carried out. In this study, the SW treatment of fish tissue was carried out in an autoclave, and the Hg and Se species in the resulting aqueous solution was analyzed. The determination of total Hg in the aqueous phase was carried out by heat vaporization atomic absorption spectrometry (HVAAS), while that of total Se was done by ICP-MS. The speciation of Hg was performed by high performance liquid chromatography (HPLC) with cold vapor atomic fluorescence spectrometry (CVAFS) aided by UV irradiation–oxidation (HPLC-UV-CVAFS), while that of Se was carried out by SEC-ICP-MS. The dissolution mechanism of Hg and Se in the SW treatment of fish tissue was estimated in terms of the dissolution behavior of Hg and Se species observed by those analytical methods. Further, the
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use of SW treatment as a novel method for the extraction of specific elements from biological samples was discussed. 2. Experimental 2.1. Samples and reagents Two fish tissue (muscle) samples, southern bluefin tuna (Thunnus maccoyii) and mackerel (Scomber japonicas), which had been purchased at a local supermarket, were tested. Certified reference materials, DORM-2 (dogfish muscle) and DOLT-3 (dogfish liver), were obtained from the National Research Council, Canada. Analytical-grade reagents were used for the preparation of all solutions. Ultrapure water was prepared by Purelab Ultra Ionic (Organo Corporation, Tokyo, Japan) and used throughout the experiments. 2.2. Analysis of Hg and Se in fish tissue The determination of total Hg in the fish tissue was performed by the use of HVAAS (MA-2000; Nippon Instruments Corporation, Tokyo, Japan). For the speciation of Hg, i.e., the fractional determination of mercuric Hg (Hg 2 +) and methylated mercuric Hg (CH3Hg +) in the fish tissue, firstly the extraction of Hg species in the sample was carried out. An aqueous solution (10 mL) containing 0.1% (v/v) HCl, 0.15 (m/v) KCl, and 0.1% (v/v) 2-mercaptoethanol was shaken with 0.1 g of powdery dried sample in a stoppered centrifuge tube for 12 h. After centrifugation, the solution was analyzed by HPLC-UV-CVAFS. The HPLC system consisted of a PU-2080 pump (JASCO Corporation, Tokyo, Japan) with a Rheodyne injector. The separation of Hg species occurred in a 150 mm × 4.6 mm Wakosil-II 5C18 HG column (Wako Pure Chemical Industries, Osaka, Japan), and the flow rate of the mobile phase (CH3OH : H2O=1:9 solution containing 0.01% (v/v) 2-mercaptoethanol) was adjusted to 0.85 mL/min. According to the manufacturer's protocol, the photooxidation of CH3Hg+ to Hg2+ was performed in a PSA 10.570 UV-oxidation system (PS Analytical; Orpington, UK) using an oxidant solution, while the CVAFS measurement was carried out using a PSA 10.025 Millennium Merlin instrument (PS Analytical). The determination of total Se in the fish tissue sample was performed by microwave-assisted acid digestion followed by ICP-MS equipped with a collision cell (H2 mode) (Agilent 7500cx, Agilent Technologies, Tokyo, Japan). The sample was digested with a mixture of HNO3 and H2O2 using a microwave processor (ETHOS 1; Milestone Inc., Shelton, CT, USA) in a similar manner described in our previous papers [22–24]. The measurements of Hg and Se were done in triplicate and the mean value and standard deviation were obtained. 2.3. SW treatment of fish tissue Powdery dried fish tissue (0.5 g) was mixed with 10 mL deionized water and the mixture was placed in a 25 mL autoclave (SAN-AI science Co. Ltd., Nagoya, Japan). The atmosphere in the autoclave was purged by argon, and it was heated to a desired temperature (100–250 °C) in an electric oven (FP-41; Yamato Science Co., Ltd., Tokyo, Japan). After cooling and centrifugation to remove the majority of insoluble solid, the aqueous phase was filtrated with a membrane filter (0.2 μm; ADVANTEC, Tokyo, Japan) to remove oil and remaining insoluble solid, and analyzed for Hg and Se. In order to measure the mass balance of Hg, the insoluble solid removed and the oil trapped on the membrane filter were analyzed for Hg by HVAAS in some cases. Total organic carbon (TOC) in the aqueous phase obtained was determined by an automatic TOC analyzer (TOC-V CSH; Shimadzu Corporation, Kyoto, Japan). The determination of total Hg in the aqueous phase was carried out by HVAAS, while that of total Se was done by ICP-MS (H2 mode). The speciation of Hg in the aqueous phase was carried out by HPLC-UV-CVAFS in a similar manner described above. The Se species in the aqueous phase was
analyzed by SEC-ICP-MS. The SEC system consisted of a PU-2080 pump (JASCO corporation) with a Rheodine injector and Superdex Peptide 10/300 GL column (GE Helthcare Japan, Tokyo, Japan), and the flow rate of the mobile phase (100 mM ammonium acetate, pH 8.0) was adjusted to 0.7 mL/min. The column was calibrated with standard polyethyleneglycols using a refractive index detector (RI-2031, JASCO corporation). Also, the aqueous phase was analyzed by SEC with UV detector (UVD) at 220 nm using a UV-2025 instrument (JASCO corporation). 3. Results and discussion 3.1. Analysis of Hg and Se in fish tissue Firstly, the analysis of Hg and Se present in the fish tissues used (southern bluefin tuna and mackerel) was carried out. The determination of total Hg in the fish tissue was conducted by HVAAS, while that of Se was done by microwave-assisted acid digestion followed by ICP-MS. Also, the fractional determination of Hg 2+ and CH3Hg + was carried out by the extraction with a 2-mercaptoethanol solution followed by HPLC-UV-CVAFS. The results are listed in Table 1. To confirm the accuracy of the results, two certified reference materials, DORM-2 and DOLT-3, were analyzed by the same method. The concentrations of total Hg and Se measured were about the same as the certified values both for DORM-2 and DOLT-3. Also, the concentrations of CH3Hg + obtained were very close to the certified values. It is proved that the extraction and HPLC-UV-CVAFS method effectively works for the determination of CH3Hg + present in the biological samples. For southern bluefin tuna (tuna) and mackerel, the total Hg concentrations were 4.01 and 1.09 μg-Hg/g, respectively, in which 84% and 63% of Hg were methylated species. The molar ratios of total Hg and total Se for tuna and mackerel were 0.41 and 0.16, respectively, which were similar to those reported in literatures [25,26]. 3.2. SW treatment of fish tissue A powdery dried fish tissue (tuna) was subjected to the SW treatment in an autoclave at 220 °C in the liquid/solid (dry) ratio of 20. To investigate the progress of SW reaction, the dissolution of organic matter from the tissue into the aqueous phase was examined, and thus TOC in the aqueous phase was measured after the filtration. In Fig. 1, plots of the TOC against the treatment time are presented. The zero point of time in Fig. 1 and subsequent related figures was set when the oven temperature reached the desired temperature. Therefore, the interior of autoclave may not reach the set temperature at the zero point, and thus the increase in the TOC seems slow in early time. The degree of TOC increased as the time elapsed, and reached ca. 18,000 mg/L at 120 min. Considering the carbon content of the fish tissue (53% in dry base), the TOC yield is ca. 60%, which is similar to that reported in a previous study about the SW treatment of fish tissue [3]. It is evident that the biopolymers, such as proteins and sugars, in the tissue are hydrolyzed, and solubilized into the aqueous phase when the fish tissue is treated under the SW conditions. In fact, an appreciable amount of peptide species was observed in the aqueous phase after the SW treatment (see Fig. 5). This will be described later. 3.3. Determination of total Hg in the aqueous phase during SW treatment It is proposed that the Hg and Se species present in the fish tissue are dissolved into the aqueous phase when the SW reaction proceeds. Firstly, the determination of total Hg in the aqueous phase was attempted by the use of HVAAS when the 220 °C-SW treatment for tuna above mentioned was carried out. As mentioned before, the interior of autoclave may not reach the set temperature at the zero point of time. Therefore, as shown in Fig. 2, the dissolution of Hg scarcely occurred at 30 min.
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Table 1 Analysis of Hg and Se in fish tissue. Fish tissue
Hg species (μg-Hg/g, dry weight) CH3Hg+
Total Hg
DORM-2 DOLT-3 Tuna Mackerel a
Total Se (μg-Se/g, dry weight)
Hg2+
Certified
Measured
Certified
Measured
Measured
Certified
Measured
4.64 ± 0.26 3.37 ± 0.14 _ _
4.50 ± 0.09 3.35 ± 0.04 4.01 ± 0.01 1.09 ± 0.02
4.47 ± 0.32 1.59 ± 0.12 _ _
4.39 ± 0.44 1.55 ± 0.08 3.35 ± 0.10 0.69 ± 0.02
nda 1.64 ± 0.03 0.39 ± 0.04 0.35 ± 0.04
1.40 ± 0.09 7.06 ± 0.48 _ _
1.44 ± 0.05 6.83 ± 0.33 3.87 ± 0.24 2.66 ± 0.03
Not detected.
Afterward, the total Hg concentration increased and reached 101 μg-Hg/L at 90 min and then somewhat decreased. It is apparent that the dissolution of Hg from the fish tissue into the aqueous phase takes place due to the SW treatment, and the dissolution behavior can be effectively monitored by HVAAS. The Hg concentration (101 μg-Hg/L) corresponds to ca. 50% of Hg which is originally present in the fish tissue. The mass balance study showed that only 15% of Hg originally present occurred in the insoluble solid and oil phase. Therefore, the fate of the rest of Hg (ca. 35%) is not clear. Probably, the rest of Hg is released to gas phase to form volatile species, such as Hg 0, and the release of Hg is promoted as the reaction time elapsed, so that the Hg concentration in the aqueous phase was decreased. However, the capture of the volatile species was not successful. When the SW treatment was done at a higher temperature (250 °C), the dissolution of Hg into the aqueous phase was more promoted and reached 107 μg-Hg/L at 60 min and then greatly decreased as the time elapsed. When the SW temperature is higher, the release of Hg into the gas phase may be enhanced, and thus the decrease in the Hg concentration in the aqueous phase is more remarkable. As shown in Table 2, the dissolution of Hg from the fish tissue into the aqueous phase was quite small when the treatment temperature was at 180 °C or below. It appears that the hydrolysis of proteins is not effective when the temperature is 180 °C or below, and thus the sequestration of Hg by Se (selenoproteins) in the fish tissue is still efficiently working. However, when the temperature becomes 220 °C, the hydrolysis of proteins including selenoproteins considerably occurs, resulting in the dissolution of Hg into the aqueous phase. When other fish tissue (mackerel) was used in the SW treatment at 220 °C, the dissolution of Hg into the aqueous phase similarly occurred (Table 2). The concentrations of Hg in the aqueous phase was gradually increased when the time elapsed, and reached 33.7 μg-Hg/L at 120 min, which corresponded to ca. 60% of Hg originally present in the fish tissue.
TOC (mg/L)
15000
10000
5000
0
The speciation of Hg in the aqueous phase was conducted by the use of HPLC-UV-CVAFS when the SW treatment of fish tissue was carried out. As shown in Table 3, the majority of Hg species found in the aqueous phase was CH3Hg +, and the sum of the concentrations of Hg 2+ and CH3Hg + in the aqueous phase was about the same as the total Hg concentrations measured by HVAAS shown in Table 2. This result suggests that the analysis by HPLC-UV-CVAFS favorably works for the speciation of Hg during the SW treatment, and other Hg species do not occur in the aqueous phase. It is found that the methylated Hg species, which is originally present in the fish tissue, is dissolved into the aqueous phase, i.e., the free form of methylated species, which should be very toxic, occurs in the aqueous phase when the SW treatment of fish tissue is carried out. For mackerel, ca. 30% of Hg originally present in the tissue was Hg 2+ (see Table 1). However, the Hg species found in the aqueous phase after the SW treatment was mainly CH3Hg + (Table 3). It is presumed that the Hg 2+ released from the fish tissue is reduced to volatile Hg 0. 3.5. Determination of total Se in the aqueous phase during the SW treatment When the SW treatment for tuna was carried out, the total Se concentration in the aqueous phase was measured by ICP-MS. As shown in Fig. 3, the total Se concentration increased and reached 172 μg-Se/L at 120 min when the 220 °C-SW treatment was performed. The Se concentration (172 μg-Se/L) corresponds to ca. 95% of Se which is originally contained in the fish tissue. It is found that the dissolution of Se from the fish tissue into the aqueous phase is effectively monitored, and almost all of Se present in the fish tissue is dissolved into the aqueous phase during the SW treatment, which is different from the result for Hg above mentioned. When the SW treatment was done at a high temperature (250 °C), the dissolution of Se was considerably enhanced and ca.
Concentration of total Hg (µg-Hg/L)
20000
3.4. Speciation of Hg in the aqueous phase during SW treatment
0
30
60
90
120
Treatment time (min) Fig. 1. TOC in the aqueous phase in SW treatment (tuna).
150
120 100
220 ºC
80 60 250 ºC
40 20 0
0
30
60
90
120
150
Treatment time (min) Fig. 2. Concentration of total Hg in the aqueous phase in SW treatment (tuna).
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200
Fish tissue
Tuna
Mackerel
a
Temperature (°C)
Time (min)
100 140 180 220 250 220 250 220 220 220
90 90 90 90 90 120 120 60 90 120
Concentration of total Se (µg-Se/L)
Table 2 Concentration of total Hg and Se in the aqueous phase in SW treatment. Concentration in aqueous phase Total Hg (μg-Hg/L)
Total Se (μg-Se/L)
1.1 6.8 nda 101 75.5 90.4 39.3 14.5 28.8 33.7
50.6 52.0 82.5 138 180 172 180 74.1 110 126
250 ºC
150 220 ºC
100
50
0
0
30
60
90
120
150
Treatment time (min)
Not detected. Fig. 3. Concentration of total Se in the aqueous phase in SW treatment (tuna).
100% of Se was released into the aqueous phase at 90 min. Although the dissolution of Hg from the tissue was quite small when the treatment temperature was 180 °C or below, that of Se considerably occurred (Table 2). It is proposed that some portion of Se species present in the fish tissue are very hydrophilic, whereas the majority of Hg species are strongly sequestrated in the tissue. For mackerel, the total Se concentration in the aqueous phase was 126 μg-Se/L when the 220 °C-SW treatment (120 min) was performed (Table 2). The Se concentration corresponds to ca. 100% of Se which is originally present in the fish tissue. 3.6. Speciation of Se in the aqueous phase during SW treatment When the 220 °C-SW treatment for tuna was carried out, the speciation of Se in the aqueous phase was conducted by SEC-ICP-MS to clarify the fate of selenoproteins originally present in the fish tissue. The results are shown in Fig. 4. In the chromatogram for the 30 min-treatment, only two peaks were observed in low molecular weight region (Peaks 1 and 2). Commercially available Se compounds, selenomethionine and selenocystine, gave their peaks in the Peak 1 region and the Peak 2 region, respectively, while SeO42 − and SeO32 − provided their peaks in the Peak 1 region and the Peak 2 region, respectively. Therefore, it is proposed that Peaks 1 and 2 in Fig. 4 are assigned to some kinds of selenoamino acids and inorganic Se species, such as SeO42 − and SeO32 −. Consequently, it is found that when the treatment time is 30 min, only hydrophilic Se species are dissolved into the aqueous phase from the fish tissue. When the treatment time was 60 min, a broad peak at 1–6 kDa region in the chromatogram appeared. The broad peak shifted to lower molecular weight region while the size of Peak 1 increased, as the reaction proceeded. When the treatment time was 120 min, the broad peak merged to Peak 1 to give the left shoulder of Peak 1. It is evident that the selenoproteins present in the fish tissue are hydrolyzed by the SW treatment, and the hydrolyzed proteins (selenopeptides), which should be hydrophilic, are dissolved into the aqueous phase. The selenopeptides undergo more hydrolysis
to finally give monomeric (and/or short-oligomeric) selenoamino acids. The size of Peak 2 gradually decreased as the reaction proceeded. The phenomenon cannot be clearly explained, but it is presumed that the oxidation of SeO32− into SeO42− occurs. When the same SW treatment mentioned above was performed, the aqueous phase was also analyzed by SEC-UVD at 220 nm. The results are shown in Fig. 5. In the chromatogram for the 30 min-treatment, peaks are observed only in low molecular weight region. These peaks are presumably attributed to amino acids (monomeric and/or short-oligomeric) and organic acids, which are hydrophilic and thus are dissolved into the aqueous phase in early time. When the treatment time was 60 min, a broad peak from 1–6 kDa in the chromatogram appeared, and shifted to lower molecular region as the time elapsed. The broad peak should be assigned to protein decomposition products (peptides), and this phenomenon is quite similar to that seen in Fig. 4 (SEC-ICP-MS chromatogram). When the 220 °C-SW treatment for mackerel was carried out, the Se species in the aqueous phase was analyzed by SEC-ICP-MS. The results are shown in Fig. 6. The chromatograms are very similar to those for tuna shown in Fig. 4. When the 30 min-SW treatment was conducted, the chromatogram gave only two peaks in low molecular weight region. As the treatment time elapsed, a broad peak at 1–6 kDa region appeared, and shifted to lower molecular region. As a result, it is found that the hydrolysis of selenoprotein originally present in the fish tissue occurs in a similar manner, which is independent of the kind of fish.
Treatment time
Tuna Mackerel a
From Table 2.
Time (min)
90 120 90 120
Total Hga (μg-Hg/L)
101 90.4 28.8 33.7
2 kDa
1 kDa
Peak 2 Peak 1
500 cps
30 min
60 min
Table 3 Speciation of Hg in the aqueous phase in SW treatment (220 °C). Fish tissue
6 kDa
90 min
Speciation of Hg Hg2+ (μg-Hg/L)
CH3Hg+ (μg-Hg/L)
5.0 4.8 4.9 1.4
93.8 74.4 23.7 31.4
120 min 0
5
10
15
20
25
30
35
Retention time (min) Fig. 4. SEC-ICP-MS chromatogram of Se species in the aqueous phase in SW treatment (tuna, 220 °C).
A. Ohki et al. / Microchemical Journal 106 (2013) 357–362
Treatment time
6 kDa
2 kDa
1 kDa 600 mV
30 min
60 min
90 min
120 min 0
5
10
15
20
25
30
35
Retention time (min) Fig. 5. SEC-UVD chromatogram for the aqueous phase in SW treatment (tuna, 220 °C).
3.7. Dissolution mechanism of Hg and Se from fish tissue It has been proved that the monitoring and speciation of Hg and Se in the aqueous phase during the SW treatment are efficiently conducted by the use of various atomic spectrometric methods. Based on the results obtained, the dissolution mechanism of Hg and Se from fish tissue into the aqueous phase is estimated. Fish tissues sometimes contain a considerable amount of methylated Hg species, which may be strongly bound to the Se atom in high molecular and insoluble Se species, such as selenoproteins. Therefore, the methylated Hg species do not provide appreciable toxicity. As previously mentioned, the hydrolysis of proteins in the fish tissue is not effective when the SW treatment temperature is 180 °C or below, and the temperature of 220 °C is needed to attain the efficient decomposition of proteins including selenoproteins. As seen in the SEC-ICP-MS chromatogram (Fig. 4), when the 220 °C-SW treatment was performed, only low molecular Se species were detected in early time (30 min). As described before, when the treatment time is 30 min, the interior temperature of autoclave does not reach the set temperature. It is evident that high molecular Se species (selenoproteins) are not decomposed and are still present in the fish tissue after the 30 min-treatment because the interior temperature is not insubstantial. Also, the 30 min-treatment hardly gave the dissolution of Hg into the aqueous phase (Fig. 2). Consequently, it is proved that the Hg species are strongly sequestrated by the selenoproteins in the fish tissue, and the decomposition of the proteins is necessary for the dissolution of Hg into the aqueous phase. Contrary to the Hg species originally present in the fish tissue, a certain portion of Se species, such as
Treatment time
6 kDa
2 kDa
1 kDa 800 cps
30 min
361
selenoamino acids and inorganic Se species, are quite hydrophilic. Even for the 100 °C-treatment, almost half of Se contained in the fish tissue was dissolved into the aqueous phase (Table 2). Eventually, the mechanism of Hg dissolution in the SW treatment will be depicted as follows. When the SW treatment of fish tissue is carried out under sufficient conditions, the insoluble proteins including selenoproteins in the tissue are hydrolyzed to form hydrophilic peptides. The lower molecular weight Se species (selenopeptides) tends to be dissolved into the aqueous phase, and then the Hg species including CH3Hg + sequestrated by the insoluble selenoproteins are also dissolved into the aqueous phase. Since the sequestration of Hg by Se species does not work in the aqueous phase, it is evident that highly toxic free CH3Hg + really occurs in the aqueous phase when the SW treatment of fish tissue is performed. It is possible that such a toxic Hg species is contaminated in the effluent produced by the SW treatment process and/or some final products, such as organic acids and amino acids. Although the Hg species contained in the fish tissue was not completely dissolved into the aqueous phase when the SW treatment was carried out, almost all of Se species in the tissue was eluted into the aqueous phase. Therefore, on the other hand, the SW treatment can be used as a novel method for the extraction of Se from biological samples. A great advantage of this method is that the extraction solvent is only pure water, and thus there should be no contamination from the solvent components, such as acids, bases, surfactants, and protein-decomposition agents. For the speciation of Se, the selenoproteins originally present in fish tissue is decomposed into hydrophilic selenopeptides, which can be detected by the use of SEC-ICP-MS. Therefore, the detection of selenopeptides in the aqueous phase when the SW treatment of biological sample is performed will be the proof of the presence of selenoproteins in the sample. 4. Conclusions The SW treatment of fish tissue (tuna and mackerel) was carried out in an autoclave, and the Hg and Se species in the resulting aqueous solution were analyzed. The monitoring of total Hg in the aqueous phase was successfully carried out by HVAAS, while that of total Se was done by ICP-MS. The speciation of Hg in the aqueous phase was favorably attained by HPLC-UV-CVAFS, and the Hg species detected was mainly CH3Hg+. For the speciation of Se, SEC-ICP-MS was successfully used, and the dissolution behavior of Se species from fish tissue into the aqueous phase was clearly depicted. Thus, it is proposed that high molecular and insoluble Se species, such as selenoproteins, are hydrolyzed to form lower molecular and soluble species (selenopeptides) during the SW treatment. Consequently, according to the analysis of Hg and Se in the aqueous phase during the SW treatment of fish tissue, it is proved that the highly toxic methylated Hg species in free form occurs in the aqueous phase. Therefore, when the SW treatment of fish tissue is performed, we need to pay attention to the contamination of toxic Hg species in the effluent as well as in the products. On the viewpoint of analytical technique, it can be stated that the SW treatment is a novel method for the extraction of Se species from biological samples. Acknowledgments
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This work was supported by a Grant-in-Aid for Scientific Research (B) (No. 22350069) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.
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Retention time (min) Fig. 6. SEC-ICP-MS chromatogram of Se species in the aqueous phase in SW treatment (mackerel, 220 °C).
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