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Journal of Marine Systems
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m a r s y s
Heavy metals in edible seaweeds commercialised for human consumption
Victoria Besada a,⁎, José Manuel Andrade b,1, Fernando Schultze a,2, Juan José González a,2
Centro Oceanográﬁco de Vigo, Instituto Español de Oceanografía, Apdo. 1552, 36200 Vigo, Spain Departamento de Química Analítica, Universidad de A Coruña, Campus da Zapateira s/n 15071, A Coruña, Spain
a r t i c l e
i n f o
a b s t r a c t
Though seaweed consumption is growing steadily across Europe, relatively few studies have reported on the quantities of heavy metals they contain and/or their potential effects on the population's health. This study focuses on the ﬁrst topic and analyses the concentrations of six typical heavy metals (Cd, Pb, Hg, Cu, Zn, total As and inorganic As) in 52 samples from 11 algaebased products commercialised in Spain for direct human consumption (Gelidium spp.; Eisenia bicyclis; Himanthalia elongata; Hizikia fusiforme; Laminaria spp.; Ulva rigida; Chondrus crispus; Porphyra umbilicales and Undaria pinnatiﬁda). Samples were ground, homogenised and quantiﬁed by atomic absorption spectrometry (Cu and Zn by ﬂame AAS; Cd, Pb and total As by electrothermal AAS; total mercury by the cold vapour technique; and inorganic As by ﬂame-hydride generation). Accuracy was assessed by participation in periodic QUASIMEME (Quality Assurance of Information in Marine Environmental Monitoring in Europe) and IAEA (International Atomic Energy Agency) intercalibration exercises. To detect any objective differences existing between the seaweeds' metal concentrations, univariate and multivariate studies (principal component analysis, cluster analysis and linear discriminant analysis) were performed. It is concluded that the Hizikia fusiforme samples contained the highest values of total and inorganic As and that most Cd concentrations exceeded the French Legislation. The two harvesting areas (Atlantic and Paciﬁc oceans) were differentiated using both univariate studies (for Cu, total As, Hg and Zn) and a multivariate discriminant function (which includes Zn, Cu and Pb). © 2008 Elsevier B.V. All rights reserved.
Article history: Received 28 September 2007 Received in revised form 14 October 2008 Accepted 21 October 2008 Available online 29 October 2008 Keywords: Marine algae Edible seaweed Heavy metals Spain Pattern recognition
1. Introduction Asian cultures have traditionally employed seaweeds (macroalgae) as a natural source of food and medicines, and Japan, Korea, China, Vietnam, Indonesia and Taiwan are by far the largest consumers (FAO, 2002). Six million tons of fresh algae are now cultivated per year worldwide, amounting to around 90% of the commercial demand (FAO, 2002). In addition to the general use of algae extracts as additives and intermediate food products, direct consumption has been
⁎ Corresponding author. Tel.: +34 986492111; fax: +34 986498626. E-mail addresses: email@example.com (V. Besada), firstname.lastname@example.org (J.M. Andrade), email@example.com (F. Schultze), firstname.lastname@example.org (J.J. González). 1 Tel.: +34 981167000; fax: +34 981167065. 2 Tel.: +34 986492111; fax: +34 986498626. 0924-7963/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jmarsys.2008.10.010
steadily growing since the early 1980s (Hou and Yan, 1998), particularly in western countries, which had formerly been reluctant to eat seaweed. Consumers in most developed countries are turning to more natural and nutritional products such as seaweed-based products, many of which have unrivalled healthy properties (Netten et al., 2000). It is now possible to ﬁnd out a large variety of products derived from macro and microalgae throughout Spain and a huge increase in consumption is expected in the next few years (Ortega-Calvo et al., 1993). Seaweeds are harvested and cultivated in western countries mostly to extract agar, carrageenans and alginates (known as hydrocolloids or phycocolloids) (Radmer, 1996). Hydrocolloids have attained commercial signiﬁcance because they are used in various industries, particularly food production. However, many seaweeds are consumed directly after only minor preprocessing (mostly drying). Red algae (mainly, Porphyra, commercial name ‘Nori’) are consumed frequently since they
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are rich in proteins and vitamins B and C. Among brown algae, Laminaria (generic commercial name, ‘Kombu’, with more than 12 species), Undaria (generic name ‘Wakame’) and Hizikia fusiforme (generic name ‘Hiziki’ ) are the most consumed. Unfortunately, some seaweeds exhibit a high afﬁnity for heavy metals (Bryan and Hummerstone,1973) and, indeed, they have been used as biomonitors for metal pollution in estuarine and coastal waters worldwide and to evaluate the quality of their surrounding environment (Chmielewská and Medved, 2001; Topcuoglu et al., 2001; Villares et al., 2005; Melville and Pulkownik, 2006). However, heavy metal concentrations in algae are strongly dependent on the environmental parameters of the sampling sites (salinity, temperature, pH, light, nutrient concentrations, oxygen, etc) (Zbikowski et al., 2006) and on the structural differences among the algae (Favero et al., 1996). We have found few studies of the life-cycle of the seaweeds considered in this study, their physiological behaviour, their element accumulation patterns, and how the geochemical processes involved in the uptake of different elements affect the accumulation of particular metals. Without doubt, there are a great deal of research opportunities in these ﬁelds (Brown et al., 1999). Furthermore, these parameters are strongly dependent on the particular seaweed, so extrapolations cannot be made from other species. Nevertheless, some general ideas can be given in this introductory section and more speciﬁc comments are included in the Results and discussion section. The capacity of algae to accumulate metals depends on a variety of factors, the two most relevant ones being the bioavailability of metals in the surrounding water and the uptake capacity of the algae (Sanchez-Rodriguez et al., 2001). Uptake is currently considered to take place in two ways. The ﬁrst is a surface reaction in which metals are absorbed by algal surfaces through electrostatic attraction to negatives sites. This is independent of factors inﬂuencing metabolism such as temperature, light, pH or age of the plant, but it is inﬂuenced by the relative abundance of elements in the surrounding water. This seems to be the main uptake mechanism for Zn. The second is a slower active uptake in which metal ions are transported across the cell membrane into the cytoplasm. This form of uptake is more dependent upon metabolic processes (it seems to be the relevant one for Cu, Mn, Se and Ni), and it is subject to variations due to changes in temperature, light, or age of the plant (Sanchez-Rodriguez et al., 2001). It is also worth noting that physiological changes and growth can affect concentrations of metals in the macroalgal tissue (Huerta-Diaz et al., 2007). The concentrations are generally low in summer when growth rates are high and the accumulated metals are diluted, and high in winter when the metabolic processes slow down (Brix and Lyngby, 1983; Hou and Yan, 1998; Villares et al., 2002, cited in Zbikowski et al., 2006). The concentrations of Mn, Cu, Zn and Ni may be controlled by enzyme cofactors, and concentrations of Zn may also be controlled by activators of dehydrogenases and protein-synthesis enzymes. Fe, Cu and Zn may also accumulate in algal tissue (Lobban and Harrison, 1994, cited in Huerta-Diaz et al., 2007). Furthermore, the mobilisation of metals from the sediment in areas covered by large amounts of macroalgae could contribute to their accumulation. Some species are able to absorb elements directly from sediment (through the roots, in which metal concentrations are much higher than in the water column) (Zbikowski et al., 2006).
One of the seaweeds considered in the present study, Ulva, is an opportunistic alga that grows quickly under favourable conditions, and this may cause rapid dilution of elements in its tissues (Villares et al., 2005). However, seasonal ﬂuctuations in metal concentrations in Ulva rigida have been attributed to growth, age of tissue and abiotic factors (salinity and temperature), as well as to variations in metal concentrations in the environment (Villares et al., 2002). Also, because of the laminar structure of Ulva lactuca (with a relatively high surface area), it mainly takes up solubilised metals (Villares et al., 2001). In general, green algae have a lower metal-binding capacity than brown algae (Schiewer and Wong, 1999) and the latter are apparently unable to regulate the uptake of trace elements due to the presence in their cell walls of a large number of compounds (e.g. alginic acid and proteins). Surprisingly, there is little legislation on seaweeds in the European Union, so little control is exercised on potentially harmful metals. France was the ﬁrst European country to set up regulations on the use of seaweeds for human consumption as non-traditional foods. Currently 12 macroalgae and 2 microalgae are authorised as vegetables and dressings/ﬂavourings (Burtin, 2003). French limits for edible seaweeds are: Pbb 5 mg/ kg dry weight (mg/kg d.w.); Cdb 0.5 mg/kg d.w.; Hgb 0.1 mg/kg d.w.; and inorganic Asb 3 mg/kg d.w. At present, neither Spain nor the European Union (EU) has speciﬁc legislation on this subject. The EU Regulation for Pb, Cd and Hg in foodstuffs (European Commission, 2006) does not consider algae, and efforts are currently being made by a Committee of Experts within the European Commission, the “Industrial Contaminants” working group, to decide whether general advice or a special regulation should be issued. The only Spanish legislation concerns seaweeds for animal consumption and states that inorganic As should be less than 2 mg/kg d.w. and that special care should be devoted to Hizikia fusiforme (BOE, 2004). A very recent paper (Nakajima et al., 2006) also demonstrated that Hiziki ingestion can be considered equivalent to As(III) intake from polluted water as the urinary arsenic level of volunteers was close to that of individuals with hyperkeratosis and hyperpigmentation in regions endemic for arsenic poisoning; accordingly, long-term ingestion of Hiziki might cause arsenic poising. To the best of our knowledge only a very small number of papers analysing heavy metals in edible algae have been published (they will be referred to hereinafter), and this is currently an emerging ﬁeld. There is also a need to compare the metal content in algae from different production areas. The aim of this work is therefore to provide information on the concentrations of several harmful heavy metals present in commercialised seaweed samples. This may contribute to current discussions on issuing joint quality controls on seaweed quality within the European Union and help to determine whether seaweeds can be differentiated objectively by employing chemical measurements. 2. Materials and methods 2.1. Samples Samples were obtained from specialist shops throughout Spain. Most of them contained only one type of seaweed though some were mixtures of different algae (termed ‘salads’). Several
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samples of a unique seaweed were provided by a Spanish ﬁrm specialised in growing/harvesting and marketing seaweed. Efforts were made to acquire as many different samples (‘species’) as possible from both the Atlantic and Paciﬁc Oceans and cover most commercially available options. In addition, some agar samples were included in this study because of their widespread use in foods (e.g. as thickening agents in meat products and desserts). In total, 52 seaweed samples were analysed in this study: agar (Gelidium spp.); arame (Eisenia bicyclis); seaweed spaghetti (Himanthalia elongata); hiziki (Hizikia fusiforme); kombu (Laminaria spp); sea lettuce (Ulva rigida); Irish moss (Chondrus crispus); nori (Porphyra umbilicales) and wakame (Undaria pinnatiﬁda). In addition, two seaweed ‘salads’ were found, one composed of a mixture of wakame, ogonori, kombu, agar and akamodoki (algae salad number 10) and the other of wakame, nori and sea lettuce (seaweed salad number 11). All samples were dried, ground and homogenised in the laboratory prior to analysis. 2.2. Analytical measurements The treated samples of around 1 g were weighed and placed in a Teﬂon reactor ﬁtted to a stainless steel container (measurement of inorganic As required only 0.5 g). After addition of 1 mL of ultrapure water (Milli-Q, Millipore) and 6 mL of concentrated nitric acid (Merck, Suprapur), the reactor was set to 90 °C for 2 h and then to 130 °C for 4 h. The digested sample was transferred to a volumetric ﬂask and made up to 25 mL with Milli-Q water. Quantiﬁcation was performed using a Perkin-Elmer AAnalyst 800 spectrophotometer, equipped with a Zeeman background correction device (Cu and Zn by ﬂame AAS; Cd, Pb and total As by electrothermal AAS). The operational parameters and matrix modiﬁers were those recommended by the manufacturer. Total Hg was determined by the cold vapour technique, employing a Perkin-Elmer FIMS-400 system (SnCl2 as reducing agent). Determination of inorganic As included solubilisation with hydrochloric acid, reduction and subsequent extraction with chloroform, back-extraction into diluted hydrochloric acid, dry-ashing, and quantiﬁcation by hydride generation AAS employing a Perkin-Elmer FIAS 200 device (Muñoz et al., 1999a). The detection limits expressed in mg/kg dry weight were Pb: 0.008; Cd: 0.005; Hg: 0.001; Cu: 0.2; Zn: 1; total As: 0.050; and inorganic As: 0.020. All analytical protocols were included in a quality control system, which included analysis of certiﬁed reference materials, duplicated samples, procedural blanks and international interlaboratory exercises such as those from QUASIMEME and IAEA on a regular basis (Wyse et al., 2003; Quasimeme, 2004). Table 1 shows the analytical results (mean± SD) for eight independent analyses of two certiﬁed reference materials: a sea lettuce, Ulva lactuca (CRM 279) and an aquatic plant, Lagarosiphon major (CRM 60), both from the EU Community Bureau of Reference. It is seen that the results agreed with the certiﬁed ones. Due to the absence of certiﬁed (or guide) values for inorganic As in the CRMs available for different organic matrices, a sea organism (the dogﬁsh muscle, Squalus acanthias, National Research Council of Canada, DORM-2 CRM) was selected to “validate” the protocol for measuring inorganic As. This CRM had previously been analysed by Muñoz et al. (1999a,b), also for
Table 1 Comparison between experimental results and certiﬁed values for two CRMs (mg/kg dry weight) Element CRM 279 Certiﬁed Cadmium Lead Mercury Copper Zinc Total Arsenic 0.274 ± 0.022 13.48 ± 0.36 0.041–0.054⁎ 13.14 ± 0.37 51.3 ± 1.2 3.09 ± 0.20 Measured 0.271 ± 0.017 13.43 ± 2.15 0.049 ± 0.016 13.46 ± 0.78 50.4 ± 2.5 3.22 ± 0.81 CRM 60 Certiﬁed 2.20 ± 0.10 63.8 ± 3.2 0.34 ± 0.04 51.2 ± 1.9 313 ± 8 8⁎ Measured 2.29 ± 0.17 64.7 ± 4.6 0.37 ± 0.04 49.9 ± 2.9 333 ± 7 7.77 ± 2.08
inorganic As and with the same methodology as that employed here. These authors worked with samples whose inorganic As contents were in the same range as the seaweeds studied here. These were therefore the only published data we could ﬁnd to evaluate the trueness of the analytical methodology. Our results, 0.136 ± 0.007 mg/kg d.w. (mean ± SD for six independent analyses), agreed with those of Muñoz et al. (1999b), i.e. 0.145± 0.011 mg/kg d.w., and this supported a satisfactory performance of the method. In addition, in recovery studies carried out by spiking aliquots of the reference materials, the average recoveries were: 98.9% for Cd , 99.6% for Pb, 105.1% for Hg, 102.4% for Cu, 98.2% for Zn and 104.2% for total As (using the CRM 279). For CRM 60, the values were: 104.1%, 101.4%, 108.8%, 97.5%, 106.4% and 97.1% for Cd, Pb, Hg, Cu, Zn and total As, respectively. Recovery for inorganic As was 93.8%. 2.3. Statistical analysis All studies were performed using Statgraphics v5Plus and SPSS v11; all tests were performed at a 95% conﬁdence level, unless otherwise stated. 3. Results and discussion The experimental concentrations are summarised in Table 2. Although the determination of inorganic elements in various algae in different countries has been summarised elsewhere (Hou and Yan, 1998), it is difﬁcult to compare results from various authors because of differences in sample treatments and analytical procedures. Furthermore, several studies attributed seasonal variations in the metal concentrations to the algal growth rate. For instance, concentrations decreased in macroalgae during growing periods and increased during the winter dormant period (Phillips, 1994), the possible causes for which included allometric parameters (inﬂuencing metal uptake and accumulation), environmental factors (affecting metal accumulation, Brown et al., 1999), and local variation in salinity (affecting biological uptake, Netten et al., 2000). Here, a preliminary univariate study was performed considering all samples and elements. Then, multivariate studies addressed whether the heavy metals studied differentiate seaweeds according to their type and/or geographical origin. 3.1. Univariate studies 3.1.1. Cadmium Table 2 shows that Cd concentrations for different seaweeds are highly variable. The relationship between heavy metals and
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Table 2 Concentrations found for several edible seaweeds (mg/kg dry weight) Species Gelidium spp. Eisenia bicyclis Himanthalia elongate Hizikia fusiforme Laminaria spp Ulva rigida Chondrus crispus Porphyra umbilicales Undaria pinnatiﬁda Wakame, ogonori, kombu, agar and akamodoki Wakame, nori and sea lettuce Commercial name Agar Arame Seaweed spaghetti Hiziki Kombu Sea lettuce Irish moss Nori Wakame Algae salad Seaweed salad Graphical code 1 2 3 4 5 6 7 8 9 10 11 Cd 0.025–0.046 0.585–0.827 0.310–0.326 0.988–2.50 0.085–1.83 0.031–0.033 0.718–0.742 0.253–3.10 0.267–4.82 1.69–1.80 0.683–0.709 Pb⁎ 0.381–0.861 0.029–0.096 0.203–0.259 b0.008–0.531 b0.008–0.460 1.00–1.05 0.403–0.726 b0.008–0.270 b0.008–1.28 0.189–0.420 1.31–1.35 Hg 0.005–0.009 0.023–0.047 0.008–0.016 0.015–0.050 0.001–0.005 0.018–0.019 0.006–0.007 0.008–0.032 0.010–0.057 0.024–0.035 0.006–0.017 Cu 0.410–1.55 3.06–4.54 1.14–1.25 1.78–7.70 0.91–2.50 3.05–3.15 1.55–2.21 5.50–14.1 1.07–1.70 1.47–1.56 7.00–7.08 Zn 1.30–6.29 21.4–23.5 48.5–48.7 4.72–19.5 10.3–23.2 5.61–6.14 51.8–53.3 39.5–73.8 8.25–26.6 42.8–43.4 26.2–27.00 As⁎ b0.05–0.21 27.9–34.1 32.9–36.7 103–147 51.7–68.3 6.41–7.06 23.2–25.5 28.9–49.5 42.1–76.9 18.6–19.1 19.7–23.0 Inorganic As 0.025–0.135 0.041–0.170 0.166–0.245 32.1–69.5 0.052–0.443 0.151–0.177 0.217–0.225 0.132–0.338 0.045–0.346 0.240–0.283 0.117–0.223
⁎The symbol ‘b’ means lower than the limit of detection (stated value). The graphical codes correspond to those in the plots.
algae ‘classiﬁed’ by their colour (Chlorophyceae, green; Phaeophyceae, brown; and Rhodophyceae, red) has been discussed by different authors. Topcuoglu et al. (2001) found that Cd concentrations were higher in red algae (Pterocladia capillacea, mean 1.40 mg/kg d.w.) than in brown (Cystoseira barbata, mean 0.55 mg/kg d.w.) and green algae samples (Ulva lactuca, 0.50 mg/kg d.w.). On the other hand, in the present work the highest Cd concentration (4.8 mg/kg d.w.) corresponded to brown algae (wakame) (since none of Topcuoglu's seaweeds were available in the Spanish samples, comparison is not straightforward). A wakame batch from the Atlantic Ocean had a much higher concentration (4.8 mg/kg d.w.) than any other value reported previously: 1.9 and 0.71 mg/kg d.w. from Almela et al. (2002) and Netten et al. (2000), respectively. Nevertheless, two other wakame batches, also from the Atlantic Ocean, had very low values (ca. 0.3 mg/kg d.w.). It is noteworthy that the widest concentration ranges corresponded to Porphyra umbilicales and Undaria pinnatiﬁda (nori and wakame, respectively). A two-way Anova was carried out considering ‘seaweed‘ and ‘ocean’ as main factors. Table 3 shows that the average values for both the Atlantic and Paciﬁc Oceans were comparable. Results for the Atlantic region were more disperse than those for the Paciﬁc Ocean. The ‘species’ factor was not statistically signiﬁcant. Considering the French Legislation as a working reference (Almela et al., 2002) , arame (Eisenia bicyclis), hiziki (Hizikia fusiforme), Irish moss (Chondrus crispus), wakame (Undaria
pinnatiﬁda from the Paciﬁc region), algae salad and seaweed salad exceeded the 0.5 mg/kg d.w. limit. Up to 50% of the samples of kombu and nori (Laminaria spp., from the Paciﬁc region, and Porphyra umbilicalis, from the Atlantic region) exceeded the French limit. The same occurred for one third of the Undaria pinnatiﬁda (wakame) samples from the Atlantic region. 3.1.2. Lead Several samples of different algae (Table 2) had concentrations below the method limit of detection. The highest values (1.35 mg/kg d.w.) corresponded to the seaweed salad from the Atlantic region. This was not surprising because the two components were analysed separately and yielded high concentrations: 1.05 mg/kg d.w. for sea lettuce, and 1.28 mg/ kg d.w for wakame. Levels for Pb were similar to those found by other authors (Almela et al., 2002; Topcuoglu et al., 2003) although lower than those from the Black Sea (Topcuoglu et al., 2001). It is worth noting that batches of the same seaweed from different locations had different Pb levels. An Anova study showed comparability among the average values of the two main harvesting regions (Paciﬁc and Atlantic Oceans, Table 3). Nevertheless, the highest ﬁgures corresponded to the Atlantic region, and particularly to the seaweed salad and a wakame sample. Note that the conﬁdence interval for Pb in seaweed salad overlapped only with that for sea lettuce and that both species were from the Atlantic region (Fig. 1). The average value for all wakame samples was comparable to those
Table 3 Summary of main statistics and two-way ANOVA results (95% conﬁdence) Cd Mean SD Max Min 1.07 1.19 4.82 0.025 Pb 0.30 0.38 1.35 b0.008 Hg 0.018 0.013 0.057 0.001 Cu 3.79 3.43 14.1 0.41 Zn 29.37 21.48 73.8 1.30 As 46.95 33.97 147 b 0.05 inorg As 6.02 16.85 69.5 0.025
2-way ANOVA (p-values) Seaweeds 0.2044 Region 0.5500
V. Besada et al. / Journal of Marine Systems 75 (2009) 305–313
Fig. 1. Graphical effect of ‘seaweeds’ on the average results (two-way ANOVA) of trace metals (average values and conﬁdence intervals calculated using the least signiﬁcant difference, 95% conﬁdence). Codes: agar = 1, arame = 2, seaweed spaghetti = 3, hiziki = 4, kombu = 5, sea lettuce = 6, Irish moss = 7, nori = 8, wakame = 9, algae salad = 10, seaweed salad = 11.
from other algae (but not sea lettuce and seaweed salad). All experimental values were well below the French limit (5 mg/kg d.w.). 3.1.3. Mercury In general, values for total Hg (0.001–0.057 mg/kg d.w.) were highly similar to those reported previously (Netten et al., 2000; Almela et al., 2002) and clearly lower than the French limit of 0.1 mg/kg d.w. (Almela et al., 2002). The two-way ANOVA showed that the average values for the two regions were different (Atlantic, 0.021 mg/kg d.w. vs. Paciﬁc, 0.012 mg/kg d.w.), see Table 3. For the seaweed factor, arame (Eisenia bicyclis), hiziki (Hizikia fusiforme), wakame (Undaria pinnatiﬁda) and the algae salad showed higher average values than other seaweeds (Fig.1). 3.1.4. Copper Cu concentrations were similar to those reported elsewhere (Netten et al., 2000; Topcluogu et al., 2003). The Atlantic and Paciﬁc regions had different average values (2.14 mg/kg d.w. and 4.45 mg/kg d.w., respectively). Four nori samples out of 12 had huge values (14.1 mg/kg d.w.), thus affecting the comparison. After discarding them, the average values for the two regions overlapped. The Anova study (see Table 3 and Fig. 1)
revealed signiﬁcant differences between nori plus seaweed salad and all other algae. 3.1.5. Zinc As for the above metals, the experimental values for Zn were similar to those from other studies (Hou and Yan, 1998; Netten et al., 2000), and very disperse. The two-way ANOVA showed that there were differences between harvesting regions and between seaweeds (Table 3). The Atlantic average (30.9 mg/kg d.w.) was greater than the Paciﬁc average (ca. 24.5 mg/kg d.w.). Seaweeds showed three concentration levels (Fig. 1). One corresponded to agar and sea lettuce (lowest values, ca. 3 mg/kg d.w.), the second to arame, hiziki, kombu and the seaweed salad (intermediate values), and the third to seaweed spaghetti, Irish moss, nori and algae salad (highest levels, N45 mg/kg d.w.). Within the last group, seaweed spaghetti and Irish moss came from the Atlantic Ocean, while the algae salad came from the Paciﬁc region. Porphyra umbilicales (nori) was present in both Oceans and the corresponding samples showed no statistically signiﬁcant differences (though the Atlantic samples showed somewhat higher values). Finally, it is worth noting that Zn concentrations for Ulva lactuca were reported to vary seasonally (Brown et al., 1999),
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with higher ﬁgures in winter than in spring or summer. The authors concluded that these results highlight the complexity of the inter-relationships between metabolism, growth rate and metal uptake, for which there has been no adequate explanation for most seaweed species and metals. Furthermore, it was also reported that some algae, such as wakame, appeared to uptake some elements (including Zn) according to their growing stage (Netten et al., 2000). 3.1.6. Total and inorganic arsenic In general, As concentrations are higher in marine organisms than in terrestrial ones (Phillips, 1990) and this explains why seafood has higher As contents than other foods. Accordingly, the total amount of As ingested by humans depends greatly on the amount of seafood included in their diet. It is well-known that organic and inorganic species of As differ widely in their toxicity (Oygard et al., 1999), inorganic forms being in general more toxic than organic ones (López et al., 1994). Algae accumulate As, and hence play an important role in its cycle. Macroalgae accumulate moderate amounts of total As, and the concentrations are higher in brown algae than in green and red ones (Phillips, 1990). This suggests that the accumulation ratios are more heavily dependent on the particular seaweed than on the environmental level of As. The ‘Mixed Commission of the Codex Alimentarius' (Food and Agriculture Organisation-World Health Organisation, FAOWHO) recommended measuring not only the total concentration of As present in food, but also the concentrations of inorganic As (Tsuda et al., 1992), as it could happen that seafood exhibiting very high concentrations of total As have no toxicity because most of the As is in its organic forms. Our experimental results revealed that total As for wakame, arame and nori were comparable to other reports, although values for hiziki and kombu were slightly higher (Netten et al., 2000). Furthermore, our results strongly agreed with those of Almela et al. (2002). The average values for the different seaweeds were not comparable. The largest difference was caused by the hiziki samples (Hizikia fusiforme showed the highest ﬁgures, at 103–147 mg/kg d.w.). They also caused the average of the Paciﬁc Ocean to be signiﬁcantly higher than that of the Atlantic region. A two-way ANOVA (hiziki samples excluded) revealed that the Atlantic average was larger than the Paciﬁc one. Kombu (Laminaria) and wakame (Undaria) had total As concentrations of around 60 mg/kg d.w., and agar and sea lettuce had the lowest values (b7 mg/kg d.w.). For inorganic As, the values were similar to those found elsewhere (Almela et al., 2002; Agency, 2004; Nakajima et al., 2006). Nevertheless, as the high ﬁbre content in algae may affect inorganic As bioavailability (Vélez and Montoro, 2001), no direct conclusions can be drawn from the analytical data alone. Remarkably, all Hiziki samples exceeded the French limit (=3.0 mg/kg d.w.), and in fact Hizikia fusiforme is not included within the 12 macroalgae authorised for human consumption (Burtin, 2003). Here, we found levels of inorganic As of 70 mg/ kg d.w. for Hizikia fusiforme. In July 2004 the British Food Standard Agency advised people not to eat this brown algae because of its levels of inorganic As (Agency, 2004). More recently, Nakajima et al. (2006) demonstrated that Hiziki ingestion might lead to undesired consequences because of its As contents. After discarding the Hiziki samples no statistical differences were found for the remaining seaweeds (see Fig. 1).
3.2. Multivariate analysis 3.2.1. Principal component analysis A Varimax principal components analysis (PCA) yielded three PCs, which explained ca 73.3% of the variance. PC1 (33.53% of the variance) was clearly associated with total As and inorganic As, which characterised the Hizikia fusiforme samples (Fig. 2). PC2 (22.54% of the variance) was mostly associated with Cu and Zn, which differentiated Porphyra umbilicales (nori) from other seaweeds (PC2-scores N0.9 denote Porphyra samples). Since both Cu and Zn dominated this factor, they should have a common origin, which has usually been related to urban efﬂuents (Villares et al., 2002). Two other algae were discriminated further: agar and sea lettuce (Fig. 2). The former had the lowest As (total and inorganic), Cu and Zn concentrations among all the samples. The latter also had low values but higher than those for agar. PC3 (17.24% of the information) was associated mainly with Cd and Pb (which had an inverse relationship). This PC was not useful for differentiating among seaweeds. It is noteworthy that the PCA was not sensitive to the geographical regions from which the samples came. 3.2.2. Cluster analysis The best results for cluster analysis were found by employing autoscaled data, the Ward grouping method and the city block (or Manhattan) distance (suited to handling large differences in the concentrations of the metals in the samples). As expected, the dendrogram (Fig. 3) conﬁrmed the main results obtained from both the PCA and the ANOVAS. Five sample groups were observed: Group A, associated only with hiziki samples (with high total and inorganic As); Group B, associated with the nori samples (with high Cu and Zn values); Group C, clustering duplicated samples for seaweed spaghetti, Irish moss and algae salad; Group E, clustering agar, sea lettuce and seaweed salad (all with the lowest values in most metals), and also including a duplicated sample of wakame, showing very low values compared to its counterpart; and Group D, which was a mixture of different samples without a clear explanation except for their metal content (the subgroups did not correspond to speciﬁc algae).
Fig. 2. Varimax PC1-PC2 scores subspace considering autoscaled data and all the species. Codes: agar = 1, hiziki = 4, sea lettuce = 6, nori = 8.
V. Besada et al. / Journal of Marine Systems 75 (2009) 305–313
Table 4 Contingency table for the LDA discrimination models (harvesting areas, Atlantic = 1, Paciﬁc = 2) Predicted (⁎) 1 Real 1 2 18 (18) 3 (3) 2 8 (8) 23(23) Predicted (⁎⁎) 1 13 (18) 5 (4) 2 13 (8) 21 (22) Theoretical 1 26 – 2 – 26
⁎Considering all metals. ⁎⁎Considering only metals that showed discrimination capabilities after individual ANOVAS—see text for details. The values between brackets correspond to forward inclusion of variables.
Fig. 3. Dendrogram obtained using autoscaled data, the Ward grouping method and the city block distance. Codes: agar = 1, arame = 2, seaweed spaghetti = 3, hiziki = 4, kombu = 5, sea lettuce = 6, Irish moss = 7, nori = 8, wakame = 9, algae salad = 10, seaweed salad = 11.
3.2.3. Linear discriminant analysis The fact that neither PCA nor hierchical clustering differentiated among the two harvesting areas indicated that the overall variance was dominated by the inherent differences between the seaweeds. Nevertheless, since some of the metals (total As, Cu, Hg and Zn) revealed different averages for the harvesting areas when two-way ANOVAS were performed, a multivariate discrimination technique, linear discriminant analysis (LDA), was applied. LDA searches for discriminant functions (linear combinations of the experimental variables) that discriminate as much as possible among the groups deﬁned by the scientists, in this case the two geographical origins. Note that PCA and cluster analysis extract patterns which explain most of the variance of the dataset, although they do not necessarily differentiate well between the groups. On the other hand, LDA looks for discriminant functions capable of differentiating the groups as much as possible, even if these factors fail to explain most of the variance. Therefore, they are complementary techniques and are not redundant. Studies were carried out considering ﬁrst all metals and then only those that showed some discrimination capabilities in the ANOVA studies (total As, Cu, Hg and Zn). Furthermore, the forward approach was also applied as a way to consider only those metals that statistically improved the overall discrimination capability of the model. Consideration of total As, Cu, Hg and Zn yielded a 75% classiﬁcation success when all samples were used for training, but only a 65% success when leave-one-out cross-validation (LOOCV) was used. The forward approach included only Cu and Zn in the model, leading to approximately 81 and 77% success ratios for all samples in the model and LOOCV, respectively (see Table 4). Slightly better results were obtained considering all metals. The discriminant function (standardised coefﬁcients) was 1.17⁎Zn + 0.94⁎Pb + 0.48⁎As + 0.094⁎Hg + 0.073⁎Cd − 0.59⁎As (inorg) − 0.86⁎Cu, which allowed for 86 and 78.8% success ratios (all samples in the model and LOOCV, respectively; see Table 4). The forward approach yielded the 1.29⁎Zn + 0.85⁎Pb − 1.00⁎Cu function (standardised coefﬁcients), with 78.8% success in both cases. This latter option was preferred because it used fewer metals (i.e. it included less random variability) and because the
classiﬁcation ratios considering all samples and cross-validation (which simulate classiﬁcation of truly unknowns) were almost the same. It is noteworthy that Pb increased the success ratios. Though Pb itself did not differentiate the two regions, it correlated positively with Zn and Cu and accordingly the information it added to the discriminant function improved its classiﬁcation ability. The classiﬁcation functions were: Atlantic Ocean= −4.30+ 6.09⁎Pb+ 0.16⁎Zn − 0.35⁎Cu, and Paciﬁc Ocean= −1.64+ 1.75⁎Pb + 0.17⁎Cu + 0.04⁎Zn (note that Zn was not critical for the Paciﬁc region and that Pb played an important role in the Atlantic area). Fig. 4 shows the classiﬁcation scores. It is worth noting that two agar samples were always misclassiﬁed, regardless of the model. As explained above for PCA, they had very low values in most variables (but medium Zn levels) and although they were from the Atlantic ocean, they were classiﬁed as being from the Paciﬁc area. One nori sample was also misclassiﬁed as being from the Paciﬁc, probably because of its high total and inorganic As concentrations. Two algae salads (#49 and #50 in the ﬁgure) were wrongly considered as being from the Atlantic (probably because they contained wakame, which had very high concentrations of several metals).
Fig. 4. Box-Whisker representation of the classiﬁcation functions obtained by LDA (forward selection) obtained for each harvesting area: 1 characterises the Atlantic ocean (= − 4.30 + 6.09⁎Pb + 0.16⁎Zn −0.35⁎Cu), whereas 2 characterises the Paciﬁc Ocean (= −1.64 + 1.75⁎Pb + 0.17⁎Cu + 0.04⁎Zn). See text for more details.
V. Besada et al. / Journal of Marine Systems 75 (2009) 305–313 Burtin, P., 2003. Nutritional value of seaweeds. Electronic Journal of Environmental, Agricultural and Food Chemistry 2, 1–9. Chmielewská, E., Medved, J., 2001. Bioaccumulation of heavy metals by green algae Cladophora glomerata in a reﬁnery sewage lagoon. Croatica Chemica Acta 74, 135–154. European Commission, 2006. Regulation (CE) N° 1881/2006 Commission, December/19/2006, L(364), pp. 5–24. FAO, 2002. Perspectivas para la producción de algas marinas en los países en desarrollo. Report No. 968 FIIU/C968(Es). Favero, N., Cattalini, F., Bertaggia, D., Albergoni, V., 1996. Metal accumulation in a biological indicator (Ulva rigida) from lagoon of Venice (Italy). Archives of Environmental Contamination and Toxicology 31, 9–18. Hou, X., Yan, X., 1998. Study on the concentration and seasonal variation of inorganic elements in 35 species of marine algae. The Science of the Total Environment 222, 141–156. Huerta-Diaz, M.A., de Leon-Chavira, F., Lares, M.L., Chee-Barragan, A., SiqueirosValencia, A., 2007. Iron, manganese and trace metal concentrations in seaweeds from the central west coast of the Gulf of California. Applied Geochemistry 22, 1380–1392. Lobban, C.S., Harrison, P.J., 1994. Seaweed and Physiology. Cambrigde University Press. López, J.C., Reija, C., Montoro, R., Cervera, M.L., De-la-Guardia, M., 1994. Determination of inorganic arsenic in seafood products by microwaveassisted distillation and atomic absorption spectrometry. Journal of Analytical Atomic Spectrometry 9, 651–656. Melville, F., Pulkownik, A., 2006. Investigation of mangrove macroalgae as bioindicators of estuarine contamination. Marine Pollution Bulletin 52, 1260–1269. Muñoz, O., Vélez, D., Montoro, R., 1999a. Optimization of solubilization, extraction and determination of inorganic arsenic As (III) + As (IV) in seafood products by acid digestion, solvent extraction and hydride generation atomic absorption spectrometry. The Analyst 124, 601–607. Muñoz, O., Vélez, D., Cervera, M.L., Montoro, R., 1999b. Rapid and quantitative release, separation and determination of inorganic arsenic (As (III) + As (IV) in seafood products by microwave assisted distillation and hydride generation atomic absorption spectrometry. Journal of Analytical Atomic Spectrometry 14, 1607–1613. Nakajima, Y., Endo, Y., Ionue, Y., Yamanaka, K., Kato, K., 2006. Ingestion of Hijiki seaweed and risk of arsenic poisoning. Applied Organometallic Chemistry 20 (9), 557–564. Netten, C.V., Cann, S.A.H., Morley, D.R., Netten, J.P.V., 2000. Elemental and radioactive analysis of commercially available seaweed. The Science of the Total Environment 255, 169–175. Ortega-Calvo, J.J., Mazuelos, C., Hermosín, B., Sáiz-Jimenez, C., 1993. Chemical composition of Spirulina and eukaryotic algae food products marketed in Spain. Journal of Applied Phycology 5, 425–435. Oygard, J.K., Lundebye, A.K., Julshamn, K., 1999. Determination of inorganic arsenic in marine food samples by hydrochloric acid distillation and ﬂow-injection hydride-generation atomic absorption spectrometry. Journal of the Association of Ofﬁcial Analytical Chemists International 82 (5), 1217–1223. Phillips, D.J.H., 1990. Arsenic in aquatic organisms: a review, emphasizing chemical speciation. Aquatic Toxicology 16, 151–186. Phillips, D.J.H., 1994. Macrophytes as biomonitors of trace metals. In: Kramer, K.J.M. (Ed.), Biomonitornig of Coastal Waters and Estuarines. CRC Press, Boca-Ratón. QUASIMEME, 2004. QUASIMEME Laboratory Performance studies. Round 24. Exercise 472. Trace metals in biota, QUASIMEME Project Ofﬁce, FRS Marine Laboratory, Aberdeen. Radmer, R.J., 1996. Algal diversity and commercial algal products. Bioscience 46, 263–271. Sanchez-Rodriguez, I., Huerta-Diaz, M.A., Choumiline, E., Holguin-Quinones, O., Zertuche-Gonzalez, J.A., 2001. Elemental concentrations in different species of seaweeds from Loreto Bay, Baja California Sur, Mexico: implications for the geochemical control of metals in algal tissue. Environmental Pollution 114 (2), 145–160. Schiewer, S., Wong, M.H., 1999. Metal binding stoichiometry and isotherm choice in biosorption. Environmental Science & Technology 33 (21), 3821–3828. Topcuoglu, S., Güven, K.C., Kirbasoglu, C., Güngör, N., Ünlü, S., Yilmaz, Y.Z., 2001. Heavy metals in marine algae from Sile in the Black Sea, 1994– 1997. Bulletin of Environmental Contamination and Toxicology 67, 288–294. Topcuoglu, S., Güven, K.C., Balkis, N., Kirbasoglu, C., 2003. Heavy metal monitoring of marine algae from the Turkish Coast of the Black Sea, 1998–2000. Chemosphere 52, 1683–1688. Tsuda, T., Babazono, A., Ogawa, T., Hamada, H., Mino, Y., Aoyama, H., Kurumanati, N., Nagira, T., Hotta, N., Harada, M., Inomata, S., 1992. Inorganic arsenic: a dangerous enigma for mankind. Applied Organometallic Chemistry 6, 309–322.
4. Conclusions This study showed that there is a need for a national (Spanish)—or preferably European—regulation on the maximum amount of pollutants in edible seaweeds commercialised for human consumption. Here several heavy metals were measured in different commercial products. According to the limits laid down by the recent French legislation, the algae studied scored as follows: (i) Most samples exceeded the limits set for cadmium. (ii) Hizikia fusiforme showed the largest total and inorganic As concentrations, in all cases exceeding the inorganic As limit (which would preclude its consumption by humans). (iii) The results for Hg and Pd were well below the limits. (iv) Four nori (Porphyra) samples and the salads showed the highest Zn and Cu concentrations (around 8–14 and N45 mg/kg d.w., respectively). The content of these metals is not covered by the French legislation. The average metal concentrations for seaweeds from the two production areas (Atlantic and Paciﬁc oceans) were similar and could not be differentiated by either PCA or cluster analysis. Nevertheless, two-way ANOVA studies showed that total As, Cu, Hg and Zn yielded statistical differences (95% conﬁdence). Accordingly, multivariate LDA studies were made, which satisfactorily classiﬁed the samples as a function of their geographical origin, with ca. 80% classiﬁcation success. Despite this, more studies seem necessary in order to clearly differentiate between the inherent variability of the different types of seaweed and the variability caused by the region in which they are grown. In our opinion, future studies should focus not only on increasing the data for different metals and different algae but on investigating the ecology and life-history of the different seaweeds, as well as on the processes governing metal accumulation. Acknowledgements This work is a part of a broader study led by the Instituto Español de Oceanografía (IEO, Vigo), and the Secretaría General de Pesca Marítima (SGPM), Spanish Government. The authors would like to thank Juan Manuel Salinas (IEO, Santander) and Rosa María Mirás. Thanks are also due to the technicians of the IEO's Pollution Laboratory in Vigo. References
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