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Toxic metals (Al, Cd, Pb and Hg) in the most consumed edible seaweeds in Europe
Chemosphere 218 (2019) 879e884
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Toxic metals (Al, Cd, Pb and Hg) in the most consumed edible seaweeds in Europe rrez a, *, Soraya Paz a, Carmen Rubio a, Inmaculada Frías a, Angel J. Gutie nica Martín c, Consuelo Revert d, Arturo Hardisson a lez-Weller b, Vero Dailos Gonza a
Department of Toxicology, University of La Laguna, La Laguna, Tenerife, Canary Islands, 38071, Spain Health Inspection and Laboratory Service, Canary Health Service, S/C de Tenerife, Canary Islands, 38006, Spain Canary Health Service, Las Palmas de Gran Canaria, 35004, Spain d Department of Physical Medicine and Pharmacology, University of La Laguna, Tenerife, Canary Islands, 38071, Spain b c
h i g h l i g h t s The consumption of seaweeds are increasing due to its health benefits. Seaweeds have a high absorption capacity and they accumulate toxic metals. Asian seaweeds showed the highest Cd, Pb and Al levels. European seaweeds have the highest Hg content. Asian wakame algae contributes greatly to Cd intake.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 July 2018 Received in revised form 23 November 2018 Accepted 25 November 2018 Available online 26 November 2018
Algae are becoming increasingly common because of their importance in vegan and vegetarian diets. Although they are a source of essential minerals, vitamins and antioxidants, these marine organisms have a high absorption capacity that can lead to the accumulation of toxic metals which are dangerous in humans. The objective of this study is to determine the content of toxic metals (Al, Cd, Pb and Hg) in edible seaweed samples marketed in Spain (Europe) to assess the toxicological risk from the intake of these metals. A total of 73 European and Asian algae samples marketed in Tenerife (Canary Islands, Spain) were analyzed by ICP - OES (Inductively Coupled Plasma e Optical Emission Spectrometry) and by CVAAS (Cold Vapor e Atomic Absorption Spectrophotometry). The major toxic metal was Al, whose highest level was recorded in seaweed salad (57.5 mg Al/kg dry weight). Regarding the origin, the highest concentrations of Al (38.9 mg/kg dw), Cd (0.59 mg/kg dw) and Pb (0.40 mg/kg dw) were found in Asian algae, which may be due to the high levels of industry in the Asian countries. However, the highest concentration of Hg (0.017 mg/kg dw) was found in European algae. The consumption of 5 g of dehydrated seaweed per day represents a percentage of contribution to the tolerable weekly intake of Cd of 22.7%, this percentage may entail a risk when considering total Cd intake. However, the consumption of 5 g a day of dehydrated seaweed would not, pose a risk to the health of adults. © 2018 Elsevier Ltd. All rights reserved.
Handling Editor: Martine Leermakers Keywords: Seaweeds Toxic metals Risk assessment ICP-OES CV-AAS
1. Introduction Algae are becoming more common in Europe due to new trends in food. They are a staple food in vegan and vegetarian diets, where you can find a large number of products made with algae.
* Corresponding author. Gutie rrez). E-mail address: [email protected] (A.J. https://doi.org/10.1016/j.chemosphere.2018.11.165 0045-6535/© 2018 Elsevier Ltd. All rights reserved.
Although algae are a source of essential minerals, vitamins and antioxidants, they have a high absorption capacity that can lead to a high accumulation of toxic metals (Sangiuliano et al., 2017; Paz et al., 2018). Different studies have shown correlations between the content of metals in sediments, water and algae (Akcali and Kucuksezgin, 2011; Rybak et al., 2012). Pollution of the marine environment has increased considerably and the study of the toxic metal content in algae can serve as a pollution indicator. Algae are one of the best marine pollution bioindicators because
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they are stable, efficient and the concentration of pollutants is directly related to the environment (Zbikowski et al., 2007; Shams El-Din et al., 2014). These organisms, moreover, are capable of absorbing and accumulating metals without suffering damage (Shams El-Din et al., 2014). Toxic metals such as aluminum (Al), cadmium (Cd), lead (Pb) and mercury (Hg) are environmental pollutants originating from anthropogenic activities such as mining or uncontrolled use of pesticides (Rubio et al., 2017a, 2017b; Paz et al., 2018). Al is a neurotoxic metal without a function in the human body that tends to accumulate in the brain, bones, liver and kidneys. Prolonged exposure to high levels of Al has been linked to neurodegenerative diseases such as Alzheimer's disease (Arvand and Kermanian, 2012). Cd has toxic effects because of its high half-life and its bioaccumulation (Barbier et al., 2005). The divalent nature of Cd gives it the ability to form a large number of stable coordination complexes with biomolecules, thereby altering their functioning. This metal mainly affects the renal system, causing irreversible damage to the renal tubules, which are involved in the mechanisms of nutrient reabsorption (Rubio et al., 2018a). Pb, another neurotoxic metal, tends to accumulate in the body causing serious damage to the central nervous system (CNS), especially in developing children and fetuses (Rubio et al., 2005, 2018b), and can cause nephropathies, alterations of the gastrointestinal tract and Alzheimer's disease (Rubio et al., 2005; Nordberg et al., 2007). Hg has a high toxicity and a marked tendency to accumulate and biomagnify along the trophic chain (Paz et al., 2017; Rodríguez et al., 2018). The toxic effects of this element are related to the chemical form in which it is found, as well as the way of entry into the body, for example, when entry is through the digestive tract methylmercury is the mercury compound with the greatest impact and this accumulates in nervous tissues. Bearing in mind the growing consumption of algae in Europe, marine pollution and the high capacity of absorption and accumulation of toxic metals of these marine organisms, the determination of the content of toxic metals (Al, Cd, Pb and Hg) that may pose a risk to health is necessary. An analytical technique normally used for the determination of the metal content in seaweeds is ICP-OES (Inductively Coupled Plasma e Optical Emission Spectrometry) which is a technique based on an optical method (Tsui et al., 2006; Intawongse et al., 2018). The ICP-OES technique offers relatively low detection limits, wide concentration ranges, high sensitivity and accuracy (Skoog et al., 2008; Luis et al., 2015). The determination of the Hg content in seaweeds is generally performed by the CV-AAS (Cold Vapor e Atomic Absorption Spectrophotometry) technique, where the samples are exposed to lower rez, 2002; Rodríguez temperatures to prevent the loss of Hg (Rupe
et al., 2018). 2. Material and methods Analytical grade chemical reagents and high purity distilled water obtained from the Milli-Q water purification system (Millipore, MA, United States) have been used. Furthermore, in order to avoid possible contamination plastic (polyethylene and highdensity polypropylene) and glass material, previously washed with laboratory detergent Acationox (Merck, Germany), distilled water and 65% nitric acid (HNO3) have been used. 2.1. Samples A total of 73 samples of edible algae marketed on the island of Tenerife (Canary Islands, Spain) from Asian and European coastal waters (Table 1) were analyzed. The algae were purchased in shopping centers and health food shops and stored at room temperature until treatment. 2.2. Determination of Al, Cd and Pb by inductively coupled plasma optical emission spectroscopy (ICP - OES) Three grams of each sample, previously homogenized, were weighed in porcelain capsules (Staalich, Germany) using an analytical balance (Metler Toledo, OH, United States) and subjected to drying in an oven (Nabertherm, Germany) at 70e75 C for 24 h. The hot acid digestion of the samples was then performed with 65% HNO3 (Merck, Germany) and subjected to incineration in a muffle furnace (Nabertherm, Germany) with a temperature-time program of 420 C for 24 h, until obtaining white ash (Hardisson et al., 2001; rrez et al., 2008; Rubio et al., 2017a). The resulting ashes were Gutie dissolved in 1.5% nitric acid (HNO3) up to a volume of 25 mL in a volumetric flask for further analysis. The metals were determined by an inductively coupled plasma atomic emission spectrometer (ICP-OES) model ICAP 6300 Duo Thermo Scientific (Waltham, MA, United States) with an Auto Sampler autosampler (CETAX model ASX-520). The instrumental conditions were as follows: approximate radiofrequency power of 1150 W; gas flow (nebulizer gas flow, auxiliary gas flow) of 0.5 L/ min; injection of the sample to the 50 rpm flow pump; stabilization time of 0 s (Rubio et al., 2018a). The instrumental wavelengths (nm) for each metal were: Al (167), Cd (226.5) and Pb (220.3). As for the limits of quantification for each metal, they were calculated as three and ten times the standard deviation (SD) resulting from the analysis of 15 targets (IUPAC, 1995), and were the following: Al (0.012 mg/L), Cd (0.001 mg/L) and Pb (0.001 mg/L). The quality control of the method, to ensure the accuracy of the analytical procedure, was performed by studying the recovery
Table 1 Descriptive data of the analyzed algae samples. Common name
Species
No. of samples
Origin
Growing method
Packaging
Wakame
Undaria pinnatifida
15
Organic
Plastic
Himanthalia elongata Laminaria ochroleuca Undaria pinnatifida
8 2 15 15 10
Bay of Biscay and Portuguese waters. Galicia (Spain) China Thailand Bay of Biscay and Portuguese waters. Galicia (Spain) Taiwan
Sea spaghetti Kombu Wakame algae salad Seaweed salad
Undaria pinnatifida, Laminaria ochroleuca, Himanthalia elongata
8
Bay of Biscay and Portuguese waters. Galicia (Spain).
Conventional Organic Conventional Organic
Tinned
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Table 2 Certified concentration in mg/kg (mean ± SD, n ¼ 3) of the reference materials and recovery percentages (%) for the studied metals. Material
Metal
Certified Concentration (mg/kg)
Obtained Concentration (mg/kg)
Recovery (%)
SRM 1515 Apple leaves BCR 279 Sea lettuce
Al Cd Pb
286 ± 9 0.27 ± 0.02 13.1 ± 0.4
284 ± 0.50 0.26 ± 0.01 12.8 ± 0.20
99.3 96.3 97.7
percentage obtained with the reference material under reproducible conditions. The reference materials used were SRM 1515 Apple leaves from the “National Institute of Standards and Technology” (NIST) for the determination of Al and BCR 279 Sea Lettuce from the “British Certified Reference” (BCR) for the determination of Cd and Pb. Table 2 shows the certified and obtained concentrations, as well as the recovery percentages.
EDI ðmg=dayÞ ¼Concentration of each metal ðmg=kg fresh weightÞ$Mean consumption ðkg=dayÞ Having calculated the estimated daily intake for each metal, the percentage contribution to tolerable daily or weekly intake was calculated. The contribution percentage is calculated as follows:
Contribution percentage ð%Þ ¼ ½ðEDI ðmg=dayÞÞ=
2.3. Determination of mercury by cold vapor atomic absorption spectrophotometry (CV-AAS)
ðGuideline valueÞ$100
0.2 mg of each sample were weighed in model 4744 acid digestion pumps with a Parr Instrument Teflon sample vessel, adding 5 mL of 1:1 sulfonitric solution, previously prepared. It was then digested in a microwave oven with a temperature program which took 3 min to reach 85 C, 9 min to reach 145 C, 4 min to reach 180 C, 15 min at 185 C and 25 min to reach 30 C. After which, the samples were put into 10 mL volumetric flasks with 1.5% HNO3. The mercury determination was performed by cold vapor atomic absorption spectrophotometry (CV-AAS). Table 3 shows the recovery percentages obtained for each oxidizing agent used, with the 1: 1 sulfonitric solution used in the digestion treatment of the samples having the highest percentage of recovery (Hardisson et al., 1999). Table 4 shows the results of the mercury concentrations obtained with the reference materials used (NIST SRM 1577 BL or bovine liver and BRC-278R MT or mussel tissue) for the digestion by microwave and 1:1 sulfonitric solution (Hardisson et al., 1998). Finally, BCR-278 R MT was used as the reference material due to its higher percentage of recovery.
2.5. Statistical analysis A statistical analysis was conducted using the IBM Statistics SPSS 22.0 for Windows ™ program. Firstly, the normality of the samples was checked by the Kolmogorov-Smirnov and Saphiro Wilk test and Levene's equality of variances test. In the absence of normality of the data, nonparametric tests were applied with the Kruskalrrez et al., 2008). This statistical analysis was Wallis test (Gutie carried out to confirm the existence or not of significant differences (p < 0.05) between the different samples according to their origin (Asia and Europe) and according to their species. 3. Results and discussion 3.1. Toxic metal content in the analyzed algae species Table 5 shows the average concentrations (mg/kg dry weight) of each metal analyzed and the standard deviations (SD) for each sample. Al was the major toxic metal found in the analyzed commercial algae and the highest average concentration of Al (57.7 mg/kg dw) was in the seaweed salad from Asia. No significant differences (p ¼ 0) were found in the aluminum content between the studied species. As for the Cd, it is worth mentioning the concentration recorded in the wakame seaweed from Asia, whose average level was 1.11 mg/kg dw. The highest average level of Pb (0.49 mg/kg dw) was found in the Asian seaweed salad. Finally, the highest average concentration of Hg (0.024 mg/kg dw) was recorded in European kombu (L. ochroleuca) algae. No significant differences were detected for any of the studied toxic metals, except for the Hg content with significant differences between the Asian seaweed salad, where Hg was non-detected, and the rest of the studied seaweeds. In general, the Asian seaweed salad had the highest concentrations of Al and Pb. European Regulation (EC) No 1881/2006 of the
2.4. Calculation of intake The evaluation of the dietary intake requires the previous calculation of the estimated daily intake (EDI) which is obtained as follows:
Table 3 Statistical study of the recovery of different mineralization procedures to determine Hg. Oxidizing Agent/mL
H2SO4/HNO3/10 (1:1) HNO3/10 H2SO4/HNO3/10 (1:1) H2SO4/HCl/10 (1:1) Lumatom/5
45/15 100/1 100/1 100/1 45/24
C/h
Reactor
Recovery (%)
No Yes Yes Yes No
96.7 86.2 83.8 82.8 93.7
± ± ± ± ±
5.0 3.8 3.4 4.8 5.0
P2 >0.05 <0.01 <0.01 <0.01 <0.01
Table 4 Accuracy of Hg concentrations measured in a reference material (ng/g of fresh weight). Reference material
Samples
Procedure
Obtained concentration
Certified concentration (95%)
Recovery (%)
NIST SRM 1577 BL BCR-278 R MT
11 11
Microwave þ H2SO4/HNO3 Microwave þ H2SO4/HNO3
3.5 ± 0.8 0.193 ± 0.011
4±2 0.195 ± 0.010
87.7 98.9
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Table 5 Mean concentrations (mg/kg) of the analyzed metals and standard deviations (SD) of the algae samples. Metal
Al Cd Pb Hg a
Wakame (Undaria pinnatifida)
Sea spaghetti (Himanthalia elongata)
Kombu (Laminaria ochroleuca)
Seaweed salad
Europe
Asia
Europe
Europe
Europe
Asia
31.5 ± 17 0.04 ± 0.03 0.30 ± 0.1 0.012 ± 0.002
20.0 ± 4.4 1.11 ± 0.3 0.31 ± 0.06 0.011 ± 0.001
19.1 ± 8.6 0.11 ± 0.12 0.23 ± 0.07 0.015 ± 0.003
34.7 ± 22 0.08 ± 0.1 0.38 ± 0.2 0.024 ± 0.001
15.5 ± 4.2 0.12 ± 0.05 0.27 ± 0.07 0.016 ± 0.002
57.7 ± 35 0.06 ± 0.06 0.49 ± 0.2 < LOQa
Concentration less than the limit of quantification.
Commission of December 19, 2006, which sets the maximum content of certain contaminants in food products, did not set maximum limits for toxic metals in these types of products (EC, 2006). However, French legislation sets maximum values of Cd, Pb and Hg in edible algae (Burtin, 2003). Bearing in mind the limit of Cd established at 0.5 mg/kg of dry weight by the French directive, the wakame seaweed coming from Asia would exceed this limit and would not be suitable for the human consumption. Table 6 shows the comparison of the metal content of seaweeds found by other authors. Studies carried out by Khan et al. (2015) on Asian wakame algae show concentrations of Cd (0.072 mg/kg dw) which are higher than European wakame algae, and of Pb (0.05 mg/ kg dw) which are lower than the levels found in the European and Asian wakame algae analyzed here (Khan et al., 2015). A more recent study by Rubio et al. (2017a) in which wakame seaweed was analyzed, reports average levels of Al (11.7 mg/kg dw) which are considerably lower than those recorded here in both European and Asian wakame algae, Cd levels (0.06 mg/kg dw) lower than the Asian algae but greater than the European algae studied here, and Pb (0.07 mg/kg dw) lower than the Asian and European wakame algae analyzed here (Rubio et al., 2017a). Likewise, the same study reports average concentrations in kombu algae of Al (7.97 mg/kg dw), Cd (0.07 mg/kg dw) and Pb (0.07 mg/kg dw) which are lower than those found in the kombu algae analyzed in the present study. Whereas the average concentrations found by Rubio et al. (2017a) in sea spaghetti seaweed of Al (7.04 mg/kg dw) and Pb (0.02 mg/kg dw) are lower than sea spaghetti algae analyzed, but the average level of Cd (0.82 mg/kg dw) is greater than that found here. 3.2. Toxic metal content of the analyzed algae according to zone Fig. 1 shows the average concentration according to the area which the algae samples come from (Asia and Europe). Regarding the content of Al, the highest average level was recorded in the algae from Asia (38.9 mg/kg dw). In addition, significant differences (p < 0.05) in Al content between Asian and European algae were detected. Besides which, the highest average concentrations of Cd (0.59 mg/kg dw) and Pb (0.40 mg/kg dw) were found in Asian algae, confirming the existence of significant differences in the content of
Table 6 Comparison of the metal content of edible seaweeds found by other authors. Author
Origin
Type
Khan et al. (2015) Rubio et al. (2017a)
Asia Europe
The present study, 2018
Asia Europe
Wakame Wakame Kombu Sea spaghetti Wakame Kombu Sea spaghetti
Concentration (mg/L) Al
Cd
Pb
e 11.7 7.97 7.04 20.0 31.5 34.7 19.1
0.072 0.06 0.07 0.82 1.11 0.04 0.08 0.11
0.05 0.07 0.07 0.02 0.31 0.30 0.38 0.23
Fig. 1. Toxic metal content (mg/kg dw) according to the origin of the analyzed seaweeds.
Cd and Pb between algae from Asia and those from Europe. However, the highest average content of Hg (0.017 mg/kg dw) was recorded in algae from Europe. The analyzed European algae came from the coasts of Galicia, north of Spain. The Galician coasts are exposed to large scale human activity, which has been increasing over the years. This human activity, as well as the environmental disasters that have affected the Galician coasts, such as the sinking of the Prestige oil boat, have damaged this natural environment rezwith the consequent increase in pollutants, such as Hg (Pe pez et al., 2006). Lo The highest concentrations found in Asian algae may be due to the high levels of industry in Asian countries such as China or Thailand. This industrialization leads to greater environmental pollution (Rodríguez et al., 2018). Industrial effluents can be a source of toxic metals entering the sea with the consequent absorption and accumulation of these by organisms such as algae (Khan et al., 2015; Paz et al., 2018). 3.3. Evaluation of dietary intake Table 7 shows the estimated daily intake (EDI) values and percentages of contribution to intake taking into account the maximum values set by different institutions. The consumption of algae should be limited to about 5 g of dehydrated algae per day (Rubio et al., 2017a), taking into account the manufacturers recommendations on the labeling of these products and where they recommend not to exceed 5 g of dehydrated seaweed per day. According to this average consumption,
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Table 7 Estimated daily intake values (mg/day) and contribution percentages (%) of the edible analyzed seaweeds. Species
Wakame (Undaria pinnatifida) Sea spaghetti (Himanthalia elongata) Kombu (Laminaria ochroleuca) Seaweed salad a b c
Origin
Asia Europe
Asia Europe
EDI (mg/day)
Contribution percentages (%)
Al
Cd
Pb
Hg
Ala,c
Cda,c
Pbb,c
Hga,c
100 158 95.5 174 289 77.5
5.55 0.20 0.55 0.40 0.30 0.60
1.55 1.50 1.15 1.90 2.45 1.35
0.05 0.06 0.08 0.12 0.00 0.09
1.02 1.61 0.98 1.77 2.95 0.79
22.7 0.82 2.25 1.64 1.23 2.45
4.53 4.38 3.36 5.55 7.16 3.94
0.13 0.16 0.19 0.31 0.00 0.22
Tolerable weekly intake of: 1 mg/kg body weight/week for Al (EFSA, 2011a), 2.5 mg/kg bw/week for Cd (EFSA, 2011b) and 4 mg/kg bw/week for Hg (EFSA, 2012). Tolerable daily intake of 0.5 mg/k bw/day for Pb (AECOSAN, 2012). Mean weight of an adult taken as 68.48 kg (AECOSAN, 2006).
the consumption of Asian wakame algae would account for 22.7% of the tolerable weekly intake (TWI) of Cd, this contribution percentage is high since the intake of total Cd may be a health risk. The highest percentage of contribution to the TDI of Pb (7.16% for adults) comes from the consumption of the seaweed salad from Asia, likewise this seaweed salad is the one that contributes the highest percentage to the TWI of Al, which is 2.95% of the established value. Regarding the percentage of contribution of Hg, the consumption of 5 g daily of any of these algae does not represent an important percentage of intake of this metal. 4. Conclusions Al is the most noteworthy toxic metal in all the analyzed samples, with the Asian seaweed salad having the highest average Al concentration (57.7 mg/kg dw). Regarding the metal content by zones, Asian algae have the highest concentrations of Al, Cd and Pb while the algae from Europe have the highest Hg content. The consumption of 5 g per day of dehydrated Asian wakame algae would mean a Cd contribution percentage of 22.7% of the TWI set at 2.5 mg/kg bw/week. In conclusion, the intake of toxic metals from the consumption of 5 g/day of the algae analyzed would not pose a health risk. Conflicts of interest The authors declare that they have no conflict of interests. Funding sources This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References ~ ola de Consumo, Seguridad Alimentaria y Nutricio n), AECOSAN (Agencia Espan Científico de la AESAN en relacio n a criterios de 2012. Informe del Comite n de concentraciones para la discusio n de propuestas de límites de estimacio n de determinados metales pesados y otros elementos de objetos de migracio mica destinados a entrar en cont. Rev. Com. Cient. 16, 11e20. cera ~ ola de Consumo, Seguridad Alimentaria y Nutricio n), AECOSAN (Agencia Espan 2006. Spanish Model Diet for the Determination of Consumer Exposure to Chemicals. Ministry of Health, Social Services and Equality, Madrid, Spain. Akcali, I., Kucuksezgin, F., 2011. A biomonitoring study: heavy metals in macroalgae from eastern Aegean coastal areas. Mar. Pollut. Bull. 62 (3), 637e645. Arvand, M., Kermanian, M., 2012. Potentiometric determination of aluminum in foods, pharmaceuticals, and alloys by AlMCM-41-modified carbon paste electrode. Food Anal. Meth. 6, 578e586. Barbier, O., Jacquillet, G., Tauc, M., Cougnon, M., Poujeol, P., 2005. Effect of heavy metals on, and handling by, the kidney. Nephron. Physiol. 99, 105e110. Burtin, A., 2003. Nutritional value of seaweeds. Electron. J. Environ. Agric. Food Chem. 498e503. EC (European Regulation), 2006. Reglamento Europeo (CE) No 1881/2006 de la n de 19 de diciembre de 2006 por el que se fija el contenido ma ximo de Comisio determinados contaminantes en los productos alimenticios. Off. J. Eur. Union
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bioindicator of heavy metals off a hot spot area on the Egyptian Mediterranean Coast during 2008-2010. Environ. Monit. Assess. 186 (9), 5865e5881. Skoog, D.A., West, D.M., Holler, J., Crouch, S.R., 2008. Fundamentos de Química Analítica, eighth ed. Cengage Learning. Tsui, M.T.K., Cheung, K.C., Tam, N.F.Y., Wong, M.H., 2006. A comparative study on metal sorption by brown seaweed. Chemosphere 65, 51e57. Zbikowski, R., Szefer, P., Latala, A., 2007. Comparison of green algae Cladophora sp. and Enteromorpha sp. as potential biomonitors of chemical elements in the southern Baltic. Sci. Total Environ. 387 (1e3), 320e332.
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Toxic metals (Al, Cd, Pb and Hg) in the most consumed edible seaweeds in Europe rrez a, *, Soraya Paz a, Carmen Rubio a, Inmaculada Frías a, Angel J. Gutie nica Martín c, Consuelo Revert d, Arturo Hardisson a lez-Weller b, Vero Dailos Gonza a
Department of Toxicology, University of La Laguna, La Laguna, Tenerife, Canary Islands, 38071, Spain Health Inspection and Laboratory Service, Canary Health Service, S/C de Tenerife, Canary Islands, 38006, Spain Canary Health Service, Las Palmas de Gran Canaria, 35004, Spain d Department of Physical Medicine and Pharmacology, University of La Laguna, Tenerife, Canary Islands, 38071, Spain b c
h i g h l i g h t s The consumption of seaweeds are increasing due to its health benefits. Seaweeds have a high absorption capacity and they accumulate toxic metals. Asian seaweeds showed the highest Cd, Pb and Al levels. European seaweeds have the highest Hg content. Asian wakame algae contributes greatly to Cd intake.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 July 2018 Received in revised form 23 November 2018 Accepted 25 November 2018 Available online 26 November 2018
Algae are becoming increasingly common because of their importance in vegan and vegetarian diets. Although they are a source of essential minerals, vitamins and antioxidants, these marine organisms have a high absorption capacity that can lead to the accumulation of toxic metals which are dangerous in humans. The objective of this study is to determine the content of toxic metals (Al, Cd, Pb and Hg) in edible seaweed samples marketed in Spain (Europe) to assess the toxicological risk from the intake of these metals. A total of 73 European and Asian algae samples marketed in Tenerife (Canary Islands, Spain) were analyzed by ICP - OES (Inductively Coupled Plasma e Optical Emission Spectrometry) and by CVAAS (Cold Vapor e Atomic Absorption Spectrophotometry). The major toxic metal was Al, whose highest level was recorded in seaweed salad (57.5 mg Al/kg dry weight). Regarding the origin, the highest concentrations of Al (38.9 mg/kg dw), Cd (0.59 mg/kg dw) and Pb (0.40 mg/kg dw) were found in Asian algae, which may be due to the high levels of industry in the Asian countries. However, the highest concentration of Hg (0.017 mg/kg dw) was found in European algae. The consumption of 5 g of dehydrated seaweed per day represents a percentage of contribution to the tolerable weekly intake of Cd of 22.7%, this percentage may entail a risk when considering total Cd intake. However, the consumption of 5 g a day of dehydrated seaweed would not, pose a risk to the health of adults. © 2018 Elsevier Ltd. All rights reserved.
Handling Editor: Martine Leermakers Keywords: Seaweeds Toxic metals Risk assessment ICP-OES CV-AAS
1. Introduction Algae are becoming more common in Europe due to new trends in food. They are a staple food in vegan and vegetarian diets, where you can find a large number of products made with algae.
* Corresponding author. Gutie rrez). E-mail address: [email protected] (A.J. https://doi.org/10.1016/j.chemosphere.2018.11.165 0045-6535/© 2018 Elsevier Ltd. All rights reserved.
Although algae are a source of essential minerals, vitamins and antioxidants, they have a high absorption capacity that can lead to a high accumulation of toxic metals (Sangiuliano et al., 2017; Paz et al., 2018). Different studies have shown correlations between the content of metals in sediments, water and algae (Akcali and Kucuksezgin, 2011; Rybak et al., 2012). Pollution of the marine environment has increased considerably and the study of the toxic metal content in algae can serve as a pollution indicator. Algae are one of the best marine pollution bioindicators because
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they are stable, efficient and the concentration of pollutants is directly related to the environment (Zbikowski et al., 2007; Shams El-Din et al., 2014). These organisms, moreover, are capable of absorbing and accumulating metals without suffering damage (Shams El-Din et al., 2014). Toxic metals such as aluminum (Al), cadmium (Cd), lead (Pb) and mercury (Hg) are environmental pollutants originating from anthropogenic activities such as mining or uncontrolled use of pesticides (Rubio et al., 2017a, 2017b; Paz et al., 2018). Al is a neurotoxic metal without a function in the human body that tends to accumulate in the brain, bones, liver and kidneys. Prolonged exposure to high levels of Al has been linked to neurodegenerative diseases such as Alzheimer's disease (Arvand and Kermanian, 2012). Cd has toxic effects because of its high half-life and its bioaccumulation (Barbier et al., 2005). The divalent nature of Cd gives it the ability to form a large number of stable coordination complexes with biomolecules, thereby altering their functioning. This metal mainly affects the renal system, causing irreversible damage to the renal tubules, which are involved in the mechanisms of nutrient reabsorption (Rubio et al., 2018a). Pb, another neurotoxic metal, tends to accumulate in the body causing serious damage to the central nervous system (CNS), especially in developing children and fetuses (Rubio et al., 2005, 2018b), and can cause nephropathies, alterations of the gastrointestinal tract and Alzheimer's disease (Rubio et al., 2005; Nordberg et al., 2007). Hg has a high toxicity and a marked tendency to accumulate and biomagnify along the trophic chain (Paz et al., 2017; Rodríguez et al., 2018). The toxic effects of this element are related to the chemical form in which it is found, as well as the way of entry into the body, for example, when entry is through the digestive tract methylmercury is the mercury compound with the greatest impact and this accumulates in nervous tissues. Bearing in mind the growing consumption of algae in Europe, marine pollution and the high capacity of absorption and accumulation of toxic metals of these marine organisms, the determination of the content of toxic metals (Al, Cd, Pb and Hg) that may pose a risk to health is necessary. An analytical technique normally used for the determination of the metal content in seaweeds is ICP-OES (Inductively Coupled Plasma e Optical Emission Spectrometry) which is a technique based on an optical method (Tsui et al., 2006; Intawongse et al., 2018). The ICP-OES technique offers relatively low detection limits, wide concentration ranges, high sensitivity and accuracy (Skoog et al., 2008; Luis et al., 2015). The determination of the Hg content in seaweeds is generally performed by the CV-AAS (Cold Vapor e Atomic Absorption Spectrophotometry) technique, where the samples are exposed to lower rez, 2002; Rodríguez temperatures to prevent the loss of Hg (Rupe
et al., 2018). 2. Material and methods Analytical grade chemical reagents and high purity distilled water obtained from the Milli-Q water purification system (Millipore, MA, United States) have been used. Furthermore, in order to avoid possible contamination plastic (polyethylene and highdensity polypropylene) and glass material, previously washed with laboratory detergent Acationox (Merck, Germany), distilled water and 65% nitric acid (HNO3) have been used. 2.1. Samples A total of 73 samples of edible algae marketed on the island of Tenerife (Canary Islands, Spain) from Asian and European coastal waters (Table 1) were analyzed. The algae were purchased in shopping centers and health food shops and stored at room temperature until treatment. 2.2. Determination of Al, Cd and Pb by inductively coupled plasma optical emission spectroscopy (ICP - OES) Three grams of each sample, previously homogenized, were weighed in porcelain capsules (Staalich, Germany) using an analytical balance (Metler Toledo, OH, United States) and subjected to drying in an oven (Nabertherm, Germany) at 70e75 C for 24 h. The hot acid digestion of the samples was then performed with 65% HNO3 (Merck, Germany) and subjected to incineration in a muffle furnace (Nabertherm, Germany) with a temperature-time program of 420 C for 24 h, until obtaining white ash (Hardisson et al., 2001; rrez et al., 2008; Rubio et al., 2017a). The resulting ashes were Gutie dissolved in 1.5% nitric acid (HNO3) up to a volume of 25 mL in a volumetric flask for further analysis. The metals were determined by an inductively coupled plasma atomic emission spectrometer (ICP-OES) model ICAP 6300 Duo Thermo Scientific (Waltham, MA, United States) with an Auto Sampler autosampler (CETAX model ASX-520). The instrumental conditions were as follows: approximate radiofrequency power of 1150 W; gas flow (nebulizer gas flow, auxiliary gas flow) of 0.5 L/ min; injection of the sample to the 50 rpm flow pump; stabilization time of 0 s (Rubio et al., 2018a). The instrumental wavelengths (nm) for each metal were: Al (167), Cd (226.5) and Pb (220.3). As for the limits of quantification for each metal, they were calculated as three and ten times the standard deviation (SD) resulting from the analysis of 15 targets (IUPAC, 1995), and were the following: Al (0.012 mg/L), Cd (0.001 mg/L) and Pb (0.001 mg/L). The quality control of the method, to ensure the accuracy of the analytical procedure, was performed by studying the recovery
Table 1 Descriptive data of the analyzed algae samples. Common name
Species
No. of samples
Origin
Growing method
Packaging
Wakame
Undaria pinnatifida
15
Organic
Plastic
Himanthalia elongata Laminaria ochroleuca Undaria pinnatifida
8 2 15 15 10
Bay of Biscay and Portuguese waters. Galicia (Spain) China Thailand Bay of Biscay and Portuguese waters. Galicia (Spain) Taiwan
Sea spaghetti Kombu Wakame algae salad Seaweed salad
Undaria pinnatifida, Laminaria ochroleuca, Himanthalia elongata
8
Bay of Biscay and Portuguese waters. Galicia (Spain).
Conventional Organic Conventional Organic
Tinned
S. Paz et al. / Chemosphere 218 (2019) 879e884
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Table 2 Certified concentration in mg/kg (mean ± SD, n ¼ 3) of the reference materials and recovery percentages (%) for the studied metals. Material
Metal
Certified Concentration (mg/kg)
Obtained Concentration (mg/kg)
Recovery (%)
SRM 1515 Apple leaves BCR 279 Sea lettuce
Al Cd Pb
286 ± 9 0.27 ± 0.02 13.1 ± 0.4
284 ± 0.50 0.26 ± 0.01 12.8 ± 0.20
99.3 96.3 97.7
percentage obtained with the reference material under reproducible conditions. The reference materials used were SRM 1515 Apple leaves from the “National Institute of Standards and Technology” (NIST) for the determination of Al and BCR 279 Sea Lettuce from the “British Certified Reference” (BCR) for the determination of Cd and Pb. Table 2 shows the certified and obtained concentrations, as well as the recovery percentages.
EDI ðmg=dayÞ ¼Concentration of each metal ðmg=kg fresh weightÞ$Mean consumption ðkg=dayÞ Having calculated the estimated daily intake for each metal, the percentage contribution to tolerable daily or weekly intake was calculated. The contribution percentage is calculated as follows:
Contribution percentage ð%Þ ¼ ½ðEDI ðmg=dayÞÞ=
2.3. Determination of mercury by cold vapor atomic absorption spectrophotometry (CV-AAS)
ðGuideline valueÞ$100
0.2 mg of each sample were weighed in model 4744 acid digestion pumps with a Parr Instrument Teflon sample vessel, adding 5 mL of 1:1 sulfonitric solution, previously prepared. It was then digested in a microwave oven with a temperature program which took 3 min to reach 85 C, 9 min to reach 145 C, 4 min to reach 180 C, 15 min at 185 C and 25 min to reach 30 C. After which, the samples were put into 10 mL volumetric flasks with 1.5% HNO3. The mercury determination was performed by cold vapor atomic absorption spectrophotometry (CV-AAS). Table 3 shows the recovery percentages obtained for each oxidizing agent used, with the 1: 1 sulfonitric solution used in the digestion treatment of the samples having the highest percentage of recovery (Hardisson et al., 1999). Table 4 shows the results of the mercury concentrations obtained with the reference materials used (NIST SRM 1577 BL or bovine liver and BRC-278R MT or mussel tissue) for the digestion by microwave and 1:1 sulfonitric solution (Hardisson et al., 1998). Finally, BCR-278 R MT was used as the reference material due to its higher percentage of recovery.
2.5. Statistical analysis A statistical analysis was conducted using the IBM Statistics SPSS 22.0 for Windows ™ program. Firstly, the normality of the samples was checked by the Kolmogorov-Smirnov and Saphiro Wilk test and Levene's equality of variances test. In the absence of normality of the data, nonparametric tests were applied with the Kruskalrrez et al., 2008). This statistical analysis was Wallis test (Gutie carried out to confirm the existence or not of significant differences (p < 0.05) between the different samples according to their origin (Asia and Europe) and according to their species. 3. Results and discussion 3.1. Toxic metal content in the analyzed algae species Table 5 shows the average concentrations (mg/kg dry weight) of each metal analyzed and the standard deviations (SD) for each sample. Al was the major toxic metal found in the analyzed commercial algae and the highest average concentration of Al (57.7 mg/kg dw) was in the seaweed salad from Asia. No significant differences (p ¼ 0) were found in the aluminum content between the studied species. As for the Cd, it is worth mentioning the concentration recorded in the wakame seaweed from Asia, whose average level was 1.11 mg/kg dw. The highest average level of Pb (0.49 mg/kg dw) was found in the Asian seaweed salad. Finally, the highest average concentration of Hg (0.024 mg/kg dw) was recorded in European kombu (L. ochroleuca) algae. No significant differences were detected for any of the studied toxic metals, except for the Hg content with significant differences between the Asian seaweed salad, where Hg was non-detected, and the rest of the studied seaweeds. In general, the Asian seaweed salad had the highest concentrations of Al and Pb. European Regulation (EC) No 1881/2006 of the
2.4. Calculation of intake The evaluation of the dietary intake requires the previous calculation of the estimated daily intake (EDI) which is obtained as follows:
Table 3 Statistical study of the recovery of different mineralization procedures to determine Hg. Oxidizing Agent/mL
H2SO4/HNO3/10 (1:1) HNO3/10 H2SO4/HNO3/10 (1:1) H2SO4/HCl/10 (1:1) Lumatom/5
45/15 100/1 100/1 100/1 45/24
C/h
Reactor
Recovery (%)
No Yes Yes Yes No
96.7 86.2 83.8 82.8 93.7
± ± ± ± ±
5.0 3.8 3.4 4.8 5.0
P2 >0.05 <0.01 <0.01 <0.01 <0.01
Table 4 Accuracy of Hg concentrations measured in a reference material (ng/g of fresh weight). Reference material
Samples
Procedure
Obtained concentration
Certified concentration (95%)
Recovery (%)
NIST SRM 1577 BL BCR-278 R MT
11 11
Microwave þ H2SO4/HNO3 Microwave þ H2SO4/HNO3
3.5 ± 0.8 0.193 ± 0.011
4±2 0.195 ± 0.010
87.7 98.9
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Table 5 Mean concentrations (mg/kg) of the analyzed metals and standard deviations (SD) of the algae samples. Metal
Al Cd Pb Hg a
Wakame (Undaria pinnatifida)
Sea spaghetti (Himanthalia elongata)
Kombu (Laminaria ochroleuca)
Seaweed salad
Europe
Asia
Europe
Europe
Europe
Asia
31.5 ± 17 0.04 ± 0.03 0.30 ± 0.1 0.012 ± 0.002
20.0 ± 4.4 1.11 ± 0.3 0.31 ± 0.06 0.011 ± 0.001
19.1 ± 8.6 0.11 ± 0.12 0.23 ± 0.07 0.015 ± 0.003
34.7 ± 22 0.08 ± 0.1 0.38 ± 0.2 0.024 ± 0.001
15.5 ± 4.2 0.12 ± 0.05 0.27 ± 0.07 0.016 ± 0.002
57.7 ± 35 0.06 ± 0.06 0.49 ± 0.2 < LOQa
Concentration less than the limit of quantification.
Commission of December 19, 2006, which sets the maximum content of certain contaminants in food products, did not set maximum limits for toxic metals in these types of products (EC, 2006). However, French legislation sets maximum values of Cd, Pb and Hg in edible algae (Burtin, 2003). Bearing in mind the limit of Cd established at 0.5 mg/kg of dry weight by the French directive, the wakame seaweed coming from Asia would exceed this limit and would not be suitable for the human consumption. Table 6 shows the comparison of the metal content of seaweeds found by other authors. Studies carried out by Khan et al. (2015) on Asian wakame algae show concentrations of Cd (0.072 mg/kg dw) which are higher than European wakame algae, and of Pb (0.05 mg/ kg dw) which are lower than the levels found in the European and Asian wakame algae analyzed here (Khan et al., 2015). A more recent study by Rubio et al. (2017a) in which wakame seaweed was analyzed, reports average levels of Al (11.7 mg/kg dw) which are considerably lower than those recorded here in both European and Asian wakame algae, Cd levels (0.06 mg/kg dw) lower than the Asian algae but greater than the European algae studied here, and Pb (0.07 mg/kg dw) lower than the Asian and European wakame algae analyzed here (Rubio et al., 2017a). Likewise, the same study reports average concentrations in kombu algae of Al (7.97 mg/kg dw), Cd (0.07 mg/kg dw) and Pb (0.07 mg/kg dw) which are lower than those found in the kombu algae analyzed in the present study. Whereas the average concentrations found by Rubio et al. (2017a) in sea spaghetti seaweed of Al (7.04 mg/kg dw) and Pb (0.02 mg/kg dw) are lower than sea spaghetti algae analyzed, but the average level of Cd (0.82 mg/kg dw) is greater than that found here. 3.2. Toxic metal content of the analyzed algae according to zone Fig. 1 shows the average concentration according to the area which the algae samples come from (Asia and Europe). Regarding the content of Al, the highest average level was recorded in the algae from Asia (38.9 mg/kg dw). In addition, significant differences (p < 0.05) in Al content between Asian and European algae were detected. Besides which, the highest average concentrations of Cd (0.59 mg/kg dw) and Pb (0.40 mg/kg dw) were found in Asian algae, confirming the existence of significant differences in the content of
Table 6 Comparison of the metal content of edible seaweeds found by other authors. Author
Origin
Type
Khan et al. (2015) Rubio et al. (2017a)
Asia Europe
The present study, 2018
Asia Europe
Wakame Wakame Kombu Sea spaghetti Wakame Kombu Sea spaghetti
Concentration (mg/L) Al
Cd
Pb
e 11.7 7.97 7.04 20.0 31.5 34.7 19.1
0.072 0.06 0.07 0.82 1.11 0.04 0.08 0.11
0.05 0.07 0.07 0.02 0.31 0.30 0.38 0.23
Fig. 1. Toxic metal content (mg/kg dw) according to the origin of the analyzed seaweeds.
Cd and Pb between algae from Asia and those from Europe. However, the highest average content of Hg (0.017 mg/kg dw) was recorded in algae from Europe. The analyzed European algae came from the coasts of Galicia, north of Spain. The Galician coasts are exposed to large scale human activity, which has been increasing over the years. This human activity, as well as the environmental disasters that have affected the Galician coasts, such as the sinking of the Prestige oil boat, have damaged this natural environment rezwith the consequent increase in pollutants, such as Hg (Pe pez et al., 2006). Lo The highest concentrations found in Asian algae may be due to the high levels of industry in Asian countries such as China or Thailand. This industrialization leads to greater environmental pollution (Rodríguez et al., 2018). Industrial effluents can be a source of toxic metals entering the sea with the consequent absorption and accumulation of these by organisms such as algae (Khan et al., 2015; Paz et al., 2018). 3.3. Evaluation of dietary intake Table 7 shows the estimated daily intake (EDI) values and percentages of contribution to intake taking into account the maximum values set by different institutions. The consumption of algae should be limited to about 5 g of dehydrated algae per day (Rubio et al., 2017a), taking into account the manufacturers recommendations on the labeling of these products and where they recommend not to exceed 5 g of dehydrated seaweed per day. According to this average consumption,
S. Paz et al. / Chemosphere 218 (2019) 879e884
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Table 7 Estimated daily intake values (mg/day) and contribution percentages (%) of the edible analyzed seaweeds. Species
Wakame (Undaria pinnatifida) Sea spaghetti (Himanthalia elongata) Kombu (Laminaria ochroleuca) Seaweed salad a b c
Origin
Asia Europe
Asia Europe
EDI (mg/day)
Contribution percentages (%)
Al
Cd
Pb
Hg
Ala,c
Cda,c
Pbb,c
Hga,c
100 158 95.5 174 289 77.5
5.55 0.20 0.55 0.40 0.30 0.60
1.55 1.50 1.15 1.90 2.45 1.35
0.05 0.06 0.08 0.12 0.00 0.09
1.02 1.61 0.98 1.77 2.95 0.79
22.7 0.82 2.25 1.64 1.23 2.45
4.53 4.38 3.36 5.55 7.16 3.94
0.13 0.16 0.19 0.31 0.00 0.22
Tolerable weekly intake of: 1 mg/kg body weight/week for Al (EFSA, 2011a), 2.5 mg/kg bw/week for Cd (EFSA, 2011b) and 4 mg/kg bw/week for Hg (EFSA, 2012). Tolerable daily intake of 0.5 mg/k bw/day for Pb (AECOSAN, 2012). Mean weight of an adult taken as 68.48 kg (AECOSAN, 2006).
the consumption of Asian wakame algae would account for 22.7% of the tolerable weekly intake (TWI) of Cd, this contribution percentage is high since the intake of total Cd may be a health risk. The highest percentage of contribution to the TDI of Pb (7.16% for adults) comes from the consumption of the seaweed salad from Asia, likewise this seaweed salad is the one that contributes the highest percentage to the TWI of Al, which is 2.95% of the established value. Regarding the percentage of contribution of Hg, the consumption of 5 g daily of any of these algae does not represent an important percentage of intake of this metal. 4. Conclusions Al is the most noteworthy toxic metal in all the analyzed samples, with the Asian seaweed salad having the highest average Al concentration (57.7 mg/kg dw). Regarding the metal content by zones, Asian algae have the highest concentrations of Al, Cd and Pb while the algae from Europe have the highest Hg content. The consumption of 5 g per day of dehydrated Asian wakame algae would mean a Cd contribution percentage of 22.7% of the TWI set at 2.5 mg/kg bw/week. In conclusion, the intake of toxic metals from the consumption of 5 g/day of the algae analyzed would not pose a health risk. Conflicts of interest The authors declare that they have no conflict of interests. Funding sources This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References ~ ola de Consumo, Seguridad Alimentaria y Nutricio n), AECOSAN (Agencia Espan Científico de la AESAN en relacio n a criterios de 2012. Informe del Comite n de concentraciones para la discusio n de propuestas de límites de estimacio n de determinados metales pesados y otros elementos de objetos de migracio mica destinados a entrar en cont. Rev. Com. Cient. 16, 11e20. cera ~ ola de Consumo, Seguridad Alimentaria y Nutricio n), AECOSAN (Agencia Espan 2006. Spanish Model Diet for the Determination of Consumer Exposure to Chemicals. Ministry of Health, Social Services and Equality, Madrid, Spain. Akcali, I., Kucuksezgin, F., 2011. A biomonitoring study: heavy metals in macroalgae from eastern Aegean coastal areas. Mar. Pollut. Bull. 62 (3), 637e645. Arvand, M., Kermanian, M., 2012. Potentiometric determination of aluminum in foods, pharmaceuticals, and alloys by AlMCM-41-modified carbon paste electrode. Food Anal. Meth. 6, 578e586. Barbier, O., Jacquillet, G., Tauc, M., Cougnon, M., Poujeol, P., 2005. Effect of heavy metals on, and handling by, the kidney. Nephron. Physiol. 99, 105e110. Burtin, A., 2003. Nutritional value of seaweeds. Electron. J. Environ. Agric. Food Chem. 498e503. EC (European Regulation), 2006. Reglamento Europeo (CE) No 1881/2006 de la n de 19 de diciembre de 2006 por el que se fija el contenido ma ximo de Comisio determinados contaminantes en los productos alimenticios. Off. J. Eur. Union
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