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Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Determination of toxic heavy metals in Echinodermata and Chordata species from South Korea a
a
a
a
a
b
Ji Yeon Choi , Girum Habte , Naeem Khan , Eun Yeong Nho , Joon Ho Hong , Hoon Choi , c
a
Kyung Su Park & Kyong Su Kim a
Department of Food and Nutrition, Chosun University, Gwangju, Republic of Korea
b
Food Contaminants Divisions, Food Safety Evaluation Department, Ministry of Food and Drug Safety, Cheongwon, Republic of Korea c
Korea Institute of Science and Technology, Seoul, Republic of Korea Accepted author version posted online: 11 Jun 2014.Published online: 30 Jun 2014.
To cite this article: Ji Yeon Choi, Girum Habte, Naeem Khan, Eun Yeong Nho, Joon Ho Hong, Hoon Choi, Kyung Su Park & Kyong Su Kim (2014): Determination of toxic heavy metals in Echinodermata and Chordata species from South Korea, Food Additives & Contaminants: Part B: Surveillance, DOI: 10.1080/19393210.2014.932311 To link to this article: http://dx.doi.org/10.1080/19393210.2014.932311
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Food Additives & Contaminants: Part B, 2014 http://dx.doi.org/10.1080/19393210.2014.932311
Determination of toxic heavy metals in Echinodermata and Chordata species from South Korea Ji Yeon Choia, Girum Habtea, Naeem Khana, Eun Yeong Nhoa, Joon Ho Honga, Hoon Choib, Kyung Su Parkc and Kyong Su Kima* Department of Food and Nutrition, Chosun University, Gwangju, Republic of Korea; bFood Contaminants Divisions, Food Safety Evaluation Department, Ministry of Food and Drug Safety, Cheongwon, Republic of Korea; cKorea Institute of Science and Technology, Seoul, Republic of Korea
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a
(Received 30 March 2014; accepted 4 June 2014) This study aimed at analysing concentrations of heavy metals including arsenic, lead, cadmium, aluminium and mercury in commonly consumed seafood species belonging to Echinodermata (Anthocidaris crassispina and Stichopus japonicus) and Chordata (Halocynthia roretzi and Styela plicata). The samples were digested by a microwave system and analysed for As, Cd and Pb by inductively coupled plasma mass spectrometer, for Al by inductively coupled plasma-optical emission spectrometer and Hg by Direct Mercury Analyser. The analytical method was validated by determining sensitivity, linearity, precision, spiking recoveries and analysis of the Standard Reference Material (SRM) NIST 1566-b, an Oyster Tissue. Results showed considerably higher accumulation of Al and As in analysed samples, compared to Pb and Cd, while Hg had the lowest contamination. On comparison, the obtained results with the recommended standards by the Food and Agriculture Organization, European Commission and Ministry of Food and Drug Safety of Korea, it was concluded that the analysed seafoods were safe and thus would not pose a threat to consumers. Keywords: heavy metal; Echinodermata; Chordata; ICP-MS; ICP-OES; DMA
Introduction The sea and more particularly the aquatic system are the ultimate repository of all types of industrial, agricultural, municipal, domestic and nuclear wastes (Falandysz et al. 2005; Bhattacharyya et al. 2013). These pollutants cause undesirable changes in the physicochemical (total nitrogen, inorganic phosphorus, electrical conductivity, etc.) and biological factors (macrophytes) of the ecosystem (Zhang et al. 2010). Among these innumerable contaminants, pollution by heavy metals in aquatic environment has become a global phenomenon because of its toxicity and persistence for several decades in the aquatic environment (Cheung & Wang 2008). Many of the heavy metals, which are discharged as a waste, can bioaccumulate in the tissues of resident organisms like fishes, oysters, crabs, shrimps, seaweeds, shellfishes, etc. (Hollis et al. 2001; Rainbow 2007). The contamination extent has increased markedly in the last 50 years due to technological developments and increased consumer use of materials containing these metals (Reddy et al. 2007). From an environmental point of view, coastal zones can be considered as the geographic space of interaction between terrestrial and marine ecosystems that are of great importance for the survival of a large variety of plants, animals and marine species (Castro et al. 1999). Pollution of the aquatic environment by inorganic chemicals has *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
been considered a major threat to the aquatic organisms. The agricultural drainage water containing pesticides and fertilisers and effluents of industrial activities and run-offs in addition to sewage effluents supply the water bodies and sediment with huge quantities of inorganic anions and heavy metals (EC 2002). The aquatic organisms are sensitive to heavy metals when the concentrations of the metals reach a significant level in the water and sediment. This is especially so in the case of invertebrate marine products like Echinodermata and non-vertebrate Chordata. Invertebrates tend to accumulate more heavy metals than fish as a result of differences in the evolutionary strategies adopted by various phyla (Batvari et al. 2013). Increased levels of As, Cd and Pb in food may constitute a food safety risk. Ingestion of As may induce peripheral vascular diseases and skin disease such as hyperkeratosis (WHO 2001). Intake of Pb can cause damage to the central and peripheral nervous system and memory deterioration (Carpenter 2001). Both Cd and Pb can cause kidney damage (Diamond & Zalups 1998), and long-term exposure to Cd may cause skeletal damage, which may result in fractures (Alfvén et al. 2000). Hg, especially organic mercury, is believed to cause neurobehavioural effects after exposure even at lower doses (David 2001). Aluminium compounds have the potential to affect the reproductive system (JECFA 2006).
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J.Y. Choi et al.
South Korea occupies the southern portion of the Korean Peninsula, an area of 98,480 km2, but the rural area is only 20% of the total and thus the population is concentrated around the coast. The country is surrounded by the East, West and South Seas, a coastline that extends for about 2413 km. Endowed with an abundance of fisheries resources, Koreans have developed a distinct seafood culture with annual per capita seafood consumption of 48.1 kg in 2005 (Yoon 2008). However, not much has been done about metal pollution in food like Echinodermata and Chordata of the coastal regions. Hence, it is important to investigate the levels of heavy metals in these organisms to assess whether the concentration is within the permissible level and will not pose any hazard to the consumers. In this study, we determined Pb, Cd, As, Hg and Al levels in the edible muscle of four commonly available Echinodermata and Chordata species, which have great economic as well as ecological importance in the region. Samples were prepared by microwave-assisted digestion and analyses were done by inductively coupled plasma mass spectrometer (ICP-MS) for Pb, Cd and As (López-Alonso et al. 2007; Damerau et al. 2012), by inductively coupled plasma-optical emission spectrometer (ICP-OES) for Al (Khan et al. 2013) and by Direct Mercury Analyser (DMA) for Hg (Chen & Chen 2006). Materials and methods Instrumentation The analytical determination of Pb, Cd and As was carried out with ICP-MS, and Al was analysed using ICP-OES. The quadrupole ICP-MS used was an Elan DRC II model (PerkinElmer SCIEX, Norwalk, CT, USA). Metal concentrations were calculated based on the isotopes 208Pb, 111Cd and 75As as presented in Table 1, which also includes the ICP-MS operating conditions. A Varian Model 730-ES
simultaneous CCD, ICP-OES (Wyndmoor, PA, USA) was used for Al determination, and the instrumental conditions are listed in Table 2. Before each experiment, the instrument was checked for daily performance using Elan 6100 DRC Sensitivity Detection Limit Solution (N8125034, PerkinElmer Pure, Norwalk, CT, USA). A microwave reaction system, model Anton Paar Multiwave 3000 (Graz, Austria) programmable for time and power between 600 and 1400 W and equipped with 20 high pressure polytetrafluoroethylene-tetrafluoromethylene vessels (MF 100), was used to digest the samples. Mercury analysis was done using a DMA (MA-2000, NIC, Tokyo, Japan). The conditions used during analysis were wavelength 253.65 nm, interface filter 254 nm, Silicon UV photodetector and platinum as a catalyst, purge 60 sec, amalgam 12 sec and record 30 sec. Sample collection and preparation Commonly consumed food species of Echinodermata (Anthocidaris crassispina and Stichopus japonicus) and Chordata (Halocynthia roretzi and Styela plicata) were collected from all over South Korea, making a total of 222 samples, as mentioned in Table 3. The samples were placed into sterile bags, separated by species, kept in ice to maintain a temperature of approximately 4–5°C and brought to the food analysis laboratory (Chosun University) on the same day. Both Echinodermata and Chordata samples were dissected using high-quality stainless steel scalpels to separate the edible muscle tissue parts. These filets were rinsed with tap water followed by deionised distilled water and dried between filter paper layers. For determination of moisture, each sample was dried at 105°C in an oven (HB-502M, Han Back, Korea), until constant weight was achieved. These were powdered in a blender (MR 350CA, Braun, Spain), properly labelled and stored in polyethylene bags in refrigerator (MICOM CFD-0622, Samsung, Korea) at –20°C until analysis. Several blanks were processed in the same way
Table 1. ICP-MS operating parameters. Nebulizer Spray chamber RF power (kW) Ar gas flow rates (L min−1) Plasma Auxiliary Nebulizer Lens voltage (V) Torch horizontal alignment (mm) Torch vertical alignment (mm) Scanning mode Dwell time (m s−1) Sweeps/reading Sampling depth (mm) Sample uptake rate (mL min−1) Isotopes
Meinhard Cyclonic 1.35 16 1.0–1.3 1.0–1.07 6.25 0.5–1.0 0.2–0.5 Peak hopping 50 20 6.0–8.0 0.24 208 Pb, 112Cd, 75As
Table 2.
ICP-OES operating parameters.
RF power (kW) Nebulizer RF generator (MHz) Argon gas flow (L min−1) Plasma Auxiliary Nebulizer Spray chamber Plasma viewing Processing mode Read delay (sec) Rinse (sec) Replicates Element wavelengths (nm)
1.3 Sea spray 27.12 16 1.5 0.94 Cyclonic Axial Area 30 30 3 27 Al (396.153)
Food Additives & Contaminants: Part B Table 3.
Details of 222 samples analysed in this study.
Family
Common name
Local name
Scientific name
N
Sea squirts Warty sea squirts Sea urchin Sea cucumber
Meongge Mideoduck Sungge Haesam
Halocynthia roretzi Styela plicata Anthocidaris crassispina Stichopus japonicus
67 66 26 63
Chordata
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Echinodermata
to check for possible contamination. In order to avoid cross-contamination, all glassware used for metal analyses were washed with detergent, rinsed in distilled water, presoaked in 5% HNO3 for more than 24 h, rinsed with deionised water and allowed to air-dry at room temperature before use. Determination of heavy metals A slightly modified version of the method used by Khan et al. (2013) was employed in the present study. In brief, homogenised samples (0.5 g of wet tissue), 7 mL of HNO3 and 1 ml of H2O2, both analytical grade (Dongwoo Fine Chem, Iksan, Korea), were put into a microwave digestion vessel. These were digested using a microwave combustion procedure as follows: (1) 1000 W at 80°C for 5 min, (2) 1000 W at 50°C for 5 min, (3) 1000 W at 190°C for 20 min and (4) 0 W for 30 min for cooling. After cooling, the filtered extracts of the digested samples were transferred into a 25-ml flask and brought to volume with Milli-Q purified water (18.2 MΩ cm, Millipore, Bedford, MA, USA). Reagent blanks were processed simultaneously. Samples and blanks were analysed in triplicate for Pb, Cd and As using ICP-MS and for Al using ICP-OES. For Hg analysis, the method used by Chen and Chen (2006) was employed. In brief, approximately 50 mg of powdered sample was taken into the sample boat loaded with cover layers of catalyst B (Al2O3) and catalyst M (Ca (OH)2 + Na2CO3), above and below the sample. These were preheated for 4 min at 350°C and then for 4 min at 850°C to vaporise mercury, which was collected in a gold amalgamation tube. The tube was heated at 155°C to release mercury, which was measured by an UV photodetector at 253.65 nm wavelength. All analytical values are expressed as mg kg−1 wet weight of tissue. The instruments were calibrated with standard solutions prepared from commercially available chemicals. Standard solutions of Pb, Cd, As and Al were prepared by diluting a 10 mg L−1 multi-element stock solution (AnApure KRIAT Co., Ltd., Daejeon, Korea) with the same acid mixture used for sample solutions. Mercury standard solutions were prepared by diluting 10 μg mL−1 of mercury stock solution (Kanto Chemical Co., Inc., Tokyo, Japan) with 1% L-cysteine. These
3
working solutions were prepared immediately prior to use. The analytical quality of the work was checked by analysing the Oyster Tissue SRM 1566-b (NIST, Gaithersburg, MD, USA). Analytical method validation To validate the analytical methods used, linearity, precision, accuracy and spike recovery were examined. Correlation coefficients (R2) were used to establish linearity for the calibration curves of analytes using linear regression analysis (Pacquette et al. 2012; Khan et al. 2014). Sensitivity of the analytical methods, limit of detection (LOD) and limit of quantification (LOQ) were related to the slope of the analytical curve and the ratio of 3 and 10 times the standard deviation of the blank, respectively (Khan et al. 2014). From the relative standard deviation of 10 repeated measurements of one sample, precision was determined via the coefficient of variation (CV%) (Jeong et al. 2014). To check the accuracy of the analytical method, NIST (National Institute of Standards and Technology, Gaithersburg, MD, USA) SRM 1566b (Oyster Tissue) was analysed to determine the percentage recovery of Cd, Pd, Al, As and Hg. For all elements, the analytical quality was further verified by spiking experiments followed by determining recovery percentages (Association of Official Analytical Chemists 2012; Khan et al. 2013). Statistical analysis Results were subject to analysis of variance using Statistical Package for Social Sciences (SPSS) software to examine whether heavy metal concentrations varied significantly between the seafoods studied and between species of those seafoods. Statistical significance was assessed using Duncan’s multiple range test at α = 0.05. Possibilities less than 0.05 (p < 0.05) were considered statistically significant. All statistical calculations were performed with SPSS 9.0 for Windows. Results and discussion All results obtained from the procedures performed to check the analytical quality showed good results. Mean
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recoveries based on the standard addition of known amounts of pure reference standard solutions to the samples were ≥90.0% with CV values less than 5%. LOD and LOQ of the instruments, spiking recovery results and R2 are presented in Table 4. Recovery of the Oyster Tissue SRM (NIST SRM 1566b, MD, USA) results indicated good agreement between certified and analytical values (Table 5). Knowledge of heavy metal concentration in marine products is extremely important with respect to both human consumption and management of the ecosystem. In this study, the levels of Pb, Cd, As, Hg and Al accumulation in edible muscle of A. crassispina, S. japonicus, H. roretzi and S. plicata were determined and are summarised in Table 6. In all species, the mean concentration of Al and As appeared to be considerably higher when compared with other metals. Hg was the lowest heavy metal accumulated in all samples, with results even below the LOD for all H. roretzi and S. plicata samples. The mean concentrations of Pb, Cd, As, Hg and Al were in the range 0.03–0.24, 0.01–0.23, 0.74–6.22, Pb > Cd > Hg. In H. roretzi and S. plicata, the mean metal concentration decreased in the same order as those of Echinodermata samples. Several possible explanations could account for the variation in metal accumulation rate. Metabolic rate of the organisms, exposure route, Table 4.
Method validation results (means of five determinations). Sensitivity
Metal Pb Cd As Hg Al
Table 5.
Enchinodermata
Correlation coefficient
Chordata
LOD (mg kg−1)
LOQ (mg kg−1)
Spiking Recovery (%)
Precision CV%
Spiking recovery (%)
Precision CV%
R2
0.002 0.001 0.002 0.002 0.004
0.007 0.003 0.008 0.006 0.012
101.1 96.0 98.4 98.8 99.1
1.98 3.02 1.72 3.21 4.60
101.1 97.2 97.0 95.6 98.5
2.35 2.11 1.58 5.03 2.12
0.9997 0.9994 0.9998 0.9992 0.9994
Average results of five determinations of standard reference material NIST 1566-b, Oyster Tissue.
Analyser
Metal
ICP-MS
Pb Cd As Hg Al
DMA ICP-OES
metal mobility, bioavailability and species of the chelator present in water and sediment of the coastal areas and time spent in the contaminated water are among the major factors (Gokoglu et al. 2008; Offem & Ayotunde 2008; Vinodhini & Narayanan 2008; Gundogdu et al. 2011). In addition, factors such as pH, temperature, salinity, nutrients, organic matter, organic carbon and environmental conditions of the ecosystem influence the bioavailability and bioaccumulation rate of metals (Kamala-Kannan & Krishnamoorthy 2006). In general, there was no significant difference (p > 0.05) in metal accumulation rate in the edible muscle between Echinodermata samples, except for Pb where the accumulation rate was statistically different (p < 0.05) between the two species. There was a significant difference (p < 0.05) in metal accumulation rate between Chordata samples in their edible muscle, except for Pb where the accumulation rate was statistically similar (p > 0.05) between the two species. Also, there was a significant difference (p < 0.05) between Echinodermata and Chordata in metal accumulation rate in their edible muscle, except for Cd and As where there exists statistical similarity (p > 0.05) in metal accumulation rate between these metals. So far, there are no international (Codex Alimentarius 1995; EC 2006) as well as local standards (Ministry of Food and Drug Safety 2013) set as limits of Pb, Cd, As, Hg and Al specifically both for Echinodermata and for Chordata. But taking into consideration the maximum limit of 0.3 mg kg−1 for Pb in other marine products like fish, molluscs, shellfish and crustaceans (Codex
Certified (mg kg−1) 0.31 2.48 7.65 0.037 197.2
± ± ± ± ±
0.01 0.08 0.65 0.001 6.0
Measured (mg kg−1) 0.32 2.39 7.75 0.036 190.6
± ± ± ± ±
0.01 0.04 0.10 0.001 4.7
Recovery (%)
CV (%)
101.6 96.4 101.3 97.3 96.7
2.23 2.51 1.56 2.82 2.48
Food Additives & Contaminants: Part B Table 6.
Metals concentrations (mean ± SD mg kg−1) in different species of Echinodermata and Chordata.
Species Echinodermata A. crassispina S. japonicus Chordata H. roretzi
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S. plicata Note:
ab
5
Pb
Cd
As
Hg
Al
0.10a ± 0.05 (0.04–0.21) 0.06 ± 0.05 (0.01–0.27)
0.04a ± 0.02 (0.01–0.10) 0.05a ± 0.07 (0.01–0.36)
2.22ab ± 0.67 (1.21–3.53) 2.24ab ± 1.82 (0.27–8.91)
0.009a ± 0.002 (
Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Determination of toxic heavy metals in Echinodermata and Chordata species from South Korea a
a
a
a
a
b
Ji Yeon Choi , Girum Habte , Naeem Khan , Eun Yeong Nho , Joon Ho Hong , Hoon Choi , c
a
Kyung Su Park & Kyong Su Kim a
Department of Food and Nutrition, Chosun University, Gwangju, Republic of Korea
b
Food Contaminants Divisions, Food Safety Evaluation Department, Ministry of Food and Drug Safety, Cheongwon, Republic of Korea c
Korea Institute of Science and Technology, Seoul, Republic of Korea Accepted author version posted online: 11 Jun 2014.Published online: 30 Jun 2014.
To cite this article: Ji Yeon Choi, Girum Habte, Naeem Khan, Eun Yeong Nho, Joon Ho Hong, Hoon Choi, Kyung Su Park & Kyong Su Kim (2014): Determination of toxic heavy metals in Echinodermata and Chordata species from South Korea, Food Additives & Contaminants: Part B: Surveillance, DOI: 10.1080/19393210.2014.932311 To link to this article: http://dx.doi.org/10.1080/19393210.2014.932311
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Food Additives & Contaminants: Part B, 2014 http://dx.doi.org/10.1080/19393210.2014.932311
Determination of toxic heavy metals in Echinodermata and Chordata species from South Korea Ji Yeon Choia, Girum Habtea, Naeem Khana, Eun Yeong Nhoa, Joon Ho Honga, Hoon Choib, Kyung Su Parkc and Kyong Su Kima* Department of Food and Nutrition, Chosun University, Gwangju, Republic of Korea; bFood Contaminants Divisions, Food Safety Evaluation Department, Ministry of Food and Drug Safety, Cheongwon, Republic of Korea; cKorea Institute of Science and Technology, Seoul, Republic of Korea
Downloaded by [UZH Hauptbibliothek / Zentralbibliothek Zürich] at 15:01 01 July 2014
a
(Received 30 March 2014; accepted 4 June 2014) This study aimed at analysing concentrations of heavy metals including arsenic, lead, cadmium, aluminium and mercury in commonly consumed seafood species belonging to Echinodermata (Anthocidaris crassispina and Stichopus japonicus) and Chordata (Halocynthia roretzi and Styela plicata). The samples were digested by a microwave system and analysed for As, Cd and Pb by inductively coupled plasma mass spectrometer, for Al by inductively coupled plasma-optical emission spectrometer and Hg by Direct Mercury Analyser. The analytical method was validated by determining sensitivity, linearity, precision, spiking recoveries and analysis of the Standard Reference Material (SRM) NIST 1566-b, an Oyster Tissue. Results showed considerably higher accumulation of Al and As in analysed samples, compared to Pb and Cd, while Hg had the lowest contamination. On comparison, the obtained results with the recommended standards by the Food and Agriculture Organization, European Commission and Ministry of Food and Drug Safety of Korea, it was concluded that the analysed seafoods were safe and thus would not pose a threat to consumers. Keywords: heavy metal; Echinodermata; Chordata; ICP-MS; ICP-OES; DMA
Introduction The sea and more particularly the aquatic system are the ultimate repository of all types of industrial, agricultural, municipal, domestic and nuclear wastes (Falandysz et al. 2005; Bhattacharyya et al. 2013). These pollutants cause undesirable changes in the physicochemical (total nitrogen, inorganic phosphorus, electrical conductivity, etc.) and biological factors (macrophytes) of the ecosystem (Zhang et al. 2010). Among these innumerable contaminants, pollution by heavy metals in aquatic environment has become a global phenomenon because of its toxicity and persistence for several decades in the aquatic environment (Cheung & Wang 2008). Many of the heavy metals, which are discharged as a waste, can bioaccumulate in the tissues of resident organisms like fishes, oysters, crabs, shrimps, seaweeds, shellfishes, etc. (Hollis et al. 2001; Rainbow 2007). The contamination extent has increased markedly in the last 50 years due to technological developments and increased consumer use of materials containing these metals (Reddy et al. 2007). From an environmental point of view, coastal zones can be considered as the geographic space of interaction between terrestrial and marine ecosystems that are of great importance for the survival of a large variety of plants, animals and marine species (Castro et al. 1999). Pollution of the aquatic environment by inorganic chemicals has *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
been considered a major threat to the aquatic organisms. The agricultural drainage water containing pesticides and fertilisers and effluents of industrial activities and run-offs in addition to sewage effluents supply the water bodies and sediment with huge quantities of inorganic anions and heavy metals (EC 2002). The aquatic organisms are sensitive to heavy metals when the concentrations of the metals reach a significant level in the water and sediment. This is especially so in the case of invertebrate marine products like Echinodermata and non-vertebrate Chordata. Invertebrates tend to accumulate more heavy metals than fish as a result of differences in the evolutionary strategies adopted by various phyla (Batvari et al. 2013). Increased levels of As, Cd and Pb in food may constitute a food safety risk. Ingestion of As may induce peripheral vascular diseases and skin disease such as hyperkeratosis (WHO 2001). Intake of Pb can cause damage to the central and peripheral nervous system and memory deterioration (Carpenter 2001). Both Cd and Pb can cause kidney damage (Diamond & Zalups 1998), and long-term exposure to Cd may cause skeletal damage, which may result in fractures (Alfvén et al. 2000). Hg, especially organic mercury, is believed to cause neurobehavioural effects after exposure even at lower doses (David 2001). Aluminium compounds have the potential to affect the reproductive system (JECFA 2006).
Downloaded by [UZH Hauptbibliothek / Zentralbibliothek Zürich] at 15:01 01 July 2014
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J.Y. Choi et al.
South Korea occupies the southern portion of the Korean Peninsula, an area of 98,480 km2, but the rural area is only 20% of the total and thus the population is concentrated around the coast. The country is surrounded by the East, West and South Seas, a coastline that extends for about 2413 km. Endowed with an abundance of fisheries resources, Koreans have developed a distinct seafood culture with annual per capita seafood consumption of 48.1 kg in 2005 (Yoon 2008). However, not much has been done about metal pollution in food like Echinodermata and Chordata of the coastal regions. Hence, it is important to investigate the levels of heavy metals in these organisms to assess whether the concentration is within the permissible level and will not pose any hazard to the consumers. In this study, we determined Pb, Cd, As, Hg and Al levels in the edible muscle of four commonly available Echinodermata and Chordata species, which have great economic as well as ecological importance in the region. Samples were prepared by microwave-assisted digestion and analyses were done by inductively coupled plasma mass spectrometer (ICP-MS) for Pb, Cd and As (López-Alonso et al. 2007; Damerau et al. 2012), by inductively coupled plasma-optical emission spectrometer (ICP-OES) for Al (Khan et al. 2013) and by Direct Mercury Analyser (DMA) for Hg (Chen & Chen 2006). Materials and methods Instrumentation The analytical determination of Pb, Cd and As was carried out with ICP-MS, and Al was analysed using ICP-OES. The quadrupole ICP-MS used was an Elan DRC II model (PerkinElmer SCIEX, Norwalk, CT, USA). Metal concentrations were calculated based on the isotopes 208Pb, 111Cd and 75As as presented in Table 1, which also includes the ICP-MS operating conditions. A Varian Model 730-ES
simultaneous CCD, ICP-OES (Wyndmoor, PA, USA) was used for Al determination, and the instrumental conditions are listed in Table 2. Before each experiment, the instrument was checked for daily performance using Elan 6100 DRC Sensitivity Detection Limit Solution (N8125034, PerkinElmer Pure, Norwalk, CT, USA). A microwave reaction system, model Anton Paar Multiwave 3000 (Graz, Austria) programmable for time and power between 600 and 1400 W and equipped with 20 high pressure polytetrafluoroethylene-tetrafluoromethylene vessels (MF 100), was used to digest the samples. Mercury analysis was done using a DMA (MA-2000, NIC, Tokyo, Japan). The conditions used during analysis were wavelength 253.65 nm, interface filter 254 nm, Silicon UV photodetector and platinum as a catalyst, purge 60 sec, amalgam 12 sec and record 30 sec. Sample collection and preparation Commonly consumed food species of Echinodermata (Anthocidaris crassispina and Stichopus japonicus) and Chordata (Halocynthia roretzi and Styela plicata) were collected from all over South Korea, making a total of 222 samples, as mentioned in Table 3. The samples were placed into sterile bags, separated by species, kept in ice to maintain a temperature of approximately 4–5°C and brought to the food analysis laboratory (Chosun University) on the same day. Both Echinodermata and Chordata samples were dissected using high-quality stainless steel scalpels to separate the edible muscle tissue parts. These filets were rinsed with tap water followed by deionised distilled water and dried between filter paper layers. For determination of moisture, each sample was dried at 105°C in an oven (HB-502M, Han Back, Korea), until constant weight was achieved. These were powdered in a blender (MR 350CA, Braun, Spain), properly labelled and stored in polyethylene bags in refrigerator (MICOM CFD-0622, Samsung, Korea) at –20°C until analysis. Several blanks were processed in the same way
Table 1. ICP-MS operating parameters. Nebulizer Spray chamber RF power (kW) Ar gas flow rates (L min−1) Plasma Auxiliary Nebulizer Lens voltage (V) Torch horizontal alignment (mm) Torch vertical alignment (mm) Scanning mode Dwell time (m s−1) Sweeps/reading Sampling depth (mm) Sample uptake rate (mL min−1) Isotopes
Meinhard Cyclonic 1.35 16 1.0–1.3 1.0–1.07 6.25 0.5–1.0 0.2–0.5 Peak hopping 50 20 6.0–8.0 0.24 208 Pb, 112Cd, 75As
Table 2.
ICP-OES operating parameters.
RF power (kW) Nebulizer RF generator (MHz) Argon gas flow (L min−1) Plasma Auxiliary Nebulizer Spray chamber Plasma viewing Processing mode Read delay (sec) Rinse (sec) Replicates Element wavelengths (nm)
1.3 Sea spray 27.12 16 1.5 0.94 Cyclonic Axial Area 30 30 3 27 Al (396.153)
Food Additives & Contaminants: Part B Table 3.
Details of 222 samples analysed in this study.
Family
Common name
Local name
Scientific name
N
Sea squirts Warty sea squirts Sea urchin Sea cucumber
Meongge Mideoduck Sungge Haesam
Halocynthia roretzi Styela plicata Anthocidaris crassispina Stichopus japonicus
67 66 26 63
Chordata
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Echinodermata
to check for possible contamination. In order to avoid cross-contamination, all glassware used for metal analyses were washed with detergent, rinsed in distilled water, presoaked in 5% HNO3 for more than 24 h, rinsed with deionised water and allowed to air-dry at room temperature before use. Determination of heavy metals A slightly modified version of the method used by Khan et al. (2013) was employed in the present study. In brief, homogenised samples (0.5 g of wet tissue), 7 mL of HNO3 and 1 ml of H2O2, both analytical grade (Dongwoo Fine Chem, Iksan, Korea), were put into a microwave digestion vessel. These were digested using a microwave combustion procedure as follows: (1) 1000 W at 80°C for 5 min, (2) 1000 W at 50°C for 5 min, (3) 1000 W at 190°C for 20 min and (4) 0 W for 30 min for cooling. After cooling, the filtered extracts of the digested samples were transferred into a 25-ml flask and brought to volume with Milli-Q purified water (18.2 MΩ cm, Millipore, Bedford, MA, USA). Reagent blanks were processed simultaneously. Samples and blanks were analysed in triplicate for Pb, Cd and As using ICP-MS and for Al using ICP-OES. For Hg analysis, the method used by Chen and Chen (2006) was employed. In brief, approximately 50 mg of powdered sample was taken into the sample boat loaded with cover layers of catalyst B (Al2O3) and catalyst M (Ca (OH)2 + Na2CO3), above and below the sample. These were preheated for 4 min at 350°C and then for 4 min at 850°C to vaporise mercury, which was collected in a gold amalgamation tube. The tube was heated at 155°C to release mercury, which was measured by an UV photodetector at 253.65 nm wavelength. All analytical values are expressed as mg kg−1 wet weight of tissue. The instruments were calibrated with standard solutions prepared from commercially available chemicals. Standard solutions of Pb, Cd, As and Al were prepared by diluting a 10 mg L−1 multi-element stock solution (AnApure KRIAT Co., Ltd., Daejeon, Korea) with the same acid mixture used for sample solutions. Mercury standard solutions were prepared by diluting 10 μg mL−1 of mercury stock solution (Kanto Chemical Co., Inc., Tokyo, Japan) with 1% L-cysteine. These
3
working solutions were prepared immediately prior to use. The analytical quality of the work was checked by analysing the Oyster Tissue SRM 1566-b (NIST, Gaithersburg, MD, USA). Analytical method validation To validate the analytical methods used, linearity, precision, accuracy and spike recovery were examined. Correlation coefficients (R2) were used to establish linearity for the calibration curves of analytes using linear regression analysis (Pacquette et al. 2012; Khan et al. 2014). Sensitivity of the analytical methods, limit of detection (LOD) and limit of quantification (LOQ) were related to the slope of the analytical curve and the ratio of 3 and 10 times the standard deviation of the blank, respectively (Khan et al. 2014). From the relative standard deviation of 10 repeated measurements of one sample, precision was determined via the coefficient of variation (CV%) (Jeong et al. 2014). To check the accuracy of the analytical method, NIST (National Institute of Standards and Technology, Gaithersburg, MD, USA) SRM 1566b (Oyster Tissue) was analysed to determine the percentage recovery of Cd, Pd, Al, As and Hg. For all elements, the analytical quality was further verified by spiking experiments followed by determining recovery percentages (Association of Official Analytical Chemists 2012; Khan et al. 2013). Statistical analysis Results were subject to analysis of variance using Statistical Package for Social Sciences (SPSS) software to examine whether heavy metal concentrations varied significantly between the seafoods studied and between species of those seafoods. Statistical significance was assessed using Duncan’s multiple range test at α = 0.05. Possibilities less than 0.05 (p < 0.05) were considered statistically significant. All statistical calculations were performed with SPSS 9.0 for Windows. Results and discussion All results obtained from the procedures performed to check the analytical quality showed good results. Mean
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4
J.Y. Choi et al.
recoveries based on the standard addition of known amounts of pure reference standard solutions to the samples were ≥90.0% with CV values less than 5%. LOD and LOQ of the instruments, spiking recovery results and R2 are presented in Table 4. Recovery of the Oyster Tissue SRM (NIST SRM 1566b, MD, USA) results indicated good agreement between certified and analytical values (Table 5). Knowledge of heavy metal concentration in marine products is extremely important with respect to both human consumption and management of the ecosystem. In this study, the levels of Pb, Cd, As, Hg and Al accumulation in edible muscle of A. crassispina, S. japonicus, H. roretzi and S. plicata were determined and are summarised in Table 6. In all species, the mean concentration of Al and As appeared to be considerably higher when compared with other metals. Hg was the lowest heavy metal accumulated in all samples, with results even below the LOD for all H. roretzi and S. plicata samples. The mean concentrations of Pb, Cd, As, Hg and Al were in the range 0.03–0.24, 0.01–0.23, 0.74–6.22, Pb > Cd > Hg. In H. roretzi and S. plicata, the mean metal concentration decreased in the same order as those of Echinodermata samples. Several possible explanations could account for the variation in metal accumulation rate. Metabolic rate of the organisms, exposure route, Table 4.
Method validation results (means of five determinations). Sensitivity
Metal Pb Cd As Hg Al
Table 5.
Enchinodermata
Correlation coefficient
Chordata
LOD (mg kg−1)
LOQ (mg kg−1)
Spiking Recovery (%)
Precision CV%
Spiking recovery (%)
Precision CV%
R2
0.002 0.001 0.002 0.002 0.004
0.007 0.003 0.008 0.006 0.012
101.1 96.0 98.4 98.8 99.1
1.98 3.02 1.72 3.21 4.60
101.1 97.2 97.0 95.6 98.5
2.35 2.11 1.58 5.03 2.12
0.9997 0.9994 0.9998 0.9992 0.9994
Average results of five determinations of standard reference material NIST 1566-b, Oyster Tissue.
Analyser
Metal
ICP-MS
Pb Cd As Hg Al
DMA ICP-OES
metal mobility, bioavailability and species of the chelator present in water and sediment of the coastal areas and time spent in the contaminated water are among the major factors (Gokoglu et al. 2008; Offem & Ayotunde 2008; Vinodhini & Narayanan 2008; Gundogdu et al. 2011). In addition, factors such as pH, temperature, salinity, nutrients, organic matter, organic carbon and environmental conditions of the ecosystem influence the bioavailability and bioaccumulation rate of metals (Kamala-Kannan & Krishnamoorthy 2006). In general, there was no significant difference (p > 0.05) in metal accumulation rate in the edible muscle between Echinodermata samples, except for Pb where the accumulation rate was statistically different (p < 0.05) between the two species. There was a significant difference (p < 0.05) in metal accumulation rate between Chordata samples in their edible muscle, except for Pb where the accumulation rate was statistically similar (p > 0.05) between the two species. Also, there was a significant difference (p < 0.05) between Echinodermata and Chordata in metal accumulation rate in their edible muscle, except for Cd and As where there exists statistical similarity (p > 0.05) in metal accumulation rate between these metals. So far, there are no international (Codex Alimentarius 1995; EC 2006) as well as local standards (Ministry of Food and Drug Safety 2013) set as limits of Pb, Cd, As, Hg and Al specifically both for Echinodermata and for Chordata. But taking into consideration the maximum limit of 0.3 mg kg−1 for Pb in other marine products like fish, molluscs, shellfish and crustaceans (Codex
Certified (mg kg−1) 0.31 2.48 7.65 0.037 197.2
± ± ± ± ±
0.01 0.08 0.65 0.001 6.0
Measured (mg kg−1) 0.32 2.39 7.75 0.036 190.6
± ± ± ± ±
0.01 0.04 0.10 0.001 4.7
Recovery (%)
CV (%)
101.6 96.4 101.3 97.3 96.7
2.23 2.51 1.56 2.82 2.48
Food Additives & Contaminants: Part B Table 6.
Metals concentrations (mean ± SD mg kg−1) in different species of Echinodermata and Chordata.
Species Echinodermata A. crassispina S. japonicus Chordata H. roretzi
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S. plicata Note:
ab
5
Pb
Cd
As
Hg
Al
0.10a ± 0.05 (0.04–0.21) 0.06 ± 0.05 (0.01–0.27)
0.04a ± 0.02 (0.01–0.10) 0.05a ± 0.07 (0.01–0.36)
2.22ab ± 0.67 (1.21–3.53) 2.24ab ± 1.82 (0.27–8.91)
0.009a ± 0.002 (
