Journal of Marine Systems 75 (2009) 305–313

Contents lists available at ScienceDirect

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
a
Centro Oceanográfico de Vigo, Instituto Español de Oceanografía, Apdo. 1552, 36200 Vigo, Spain
b
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

Article history: Though seaweed consumption is growing steadily across Europe, relatively few studies have
Received 28 September 2007 reported on the quantities of heavy metals they contain and/or their potential effects on the
Received in revised form 14 October 2008 population's health. This study focuses on the first topic and analyses the concentrations of six
Accepted 21 October 2008
typical heavy metals (Cd, Pb, Hg, Cu, Zn, total As and inorganic As) in 52 samples from 11 algae-
Available online 29 October 2008
based products commercialised in Spain for direct human consumption (Gelidium spp.; Eisenia
bicyclis; Himanthalia elongata; Hizikia fusiforme; Laminaria spp.; Ulva rigida; Chondrus crispus;
Keywords:
Porphyra umbilicales and Undaria pinnatifida).
Marine algae
Edible seaweed Samples were ground, homogenised and quantified by atomic absorption spectrometry (Cu and
Heavy metals Zn by flame AAS; Cd, Pb and total As by electrothermal AAS; total mercury by the cold vapour
Spain technique; and inorganic As by flame-hydride generation). Accuracy was assessed by
Pattern recognition 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.

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: victoria.besada@vi.ieo.es (V. Besada), andrade@udc.es
(J.M. Andrade), fernando.schultze@vi.ieo.es (F. Schultze),
jjose.gonzalez@vi.ieo.es (J.J. González).
1
Tel.: +34 981167000; fax: +34 981167065.
2
Tel.: +34 986492111; fax: +34 986498626.
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 unri-
valled 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 consump-
tion 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 pre-
processing (mostly drying). Red algae (mainly, Porphyra,
commercial name ‘Nori’) are consumed frequently since they

0924-7963/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jmarsys.2008.10.010
306 V. Besada et al. / Journal of Marine Systems 75 (2009) 305–313
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-synth-
esis 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 tempera-
ture), 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 micro-
algae 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 equiva-
lent 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 employ-
ing 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
 V. Besada et al. / Journal of Marine Systems 75 (2009) 305–313 307

samples of a unique seaweed were provided by a Spanish firm Table 1

specialised in growing/harvesting and marketing seaweed. Comparison between experimental results and certified values for two CRMs
(mg/kg dry weight)
Efforts were made to acquire as many different samples
(‘species’) as possible from both the Atlantic and Pacific Oceans Element CRM 279 CRM 60
and cover most commercially available options. In addition, Certified Measured Certified Measured
some agar samples were included in this study because of their Cadmium 0.274 ± 0.022 0.271 ± 0.017 2.20 ± 0.10 2.29 ± 0.17
widespread use in foods (e.g. as thickening agents in meat Lead 13.48 ± 0.36 13.43 ± 2.15 63.8 ± 3.2 64.7 ± 4.6
products and desserts). Mercury 0.041–0.054⁎ 0.049 ± 0.016 0.34 ± 0.04 0.37 ± 0.04
In total, 52 seaweed samples were analysed in this study: Copper 13.14 ± 0.37 13.46 ± 0.78 51.2 ± 1.9 49.9 ± 2.9
Zinc 51.3 ± 1.2 50.4 ± 2.5 313 ± 8 333 ± 7
agar (Gelidium spp.); arame (Eisenia bicyclis); seaweed Total Arsenic 3.09 ± 0.20 3.22 ± 0.81 8⁎ 7.77 ± 2.08
spaghetti (Himanthalia elongata); hiziki (Hizikia fusiforme);
⁎Indicative value.
kombu (Laminaria spp); sea lettuce (Ulva rigida); Irish moss
(Chondrus crispus); nori (Porphyra umbilicales) and wakame
(Undaria pinnatifida). In addition, two seaweed ‘salads’ were inorganic As and with the same methodology as that employed
found, one composed of a mixture of wakame, ogonori, here. These authors worked with samples whose inorganic As
kombu, agar and akamodoki (algae salad number 10) and contents were in the same range as the seaweeds studied here.
the other of wakame, nori and sea lettuce (seaweed salad These were therefore the only published data we could find to
number 11). All samples were dried, ground and homogenised evaluate the trueness of the analytical methodology. Our
in the laboratory prior to analysis. 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±
2.2. Analytical measurements 0.011 mg/kg d.w., and this supported a satisfactory performance
of the method. In addition, in recovery studies carried out by
The treated samples of around 1 g were weighed and placed spiking aliquots of the reference materials, the average
in a Teflon reactor fitted to a stainless steel container (measure- recoveries were: 98.9% for Cd , 99.6% for Pb, 105.1% for Hg,
ment of inorganic As required only 0.5 g). After addition of 1 mL 102.4% for Cu, 98.2% for Zn and 104.2% for total As (using the
of ultrapure water (Milli-Q, Millipore) and 6 mL of concentrated CRM 279). For CRM 60, the values were: 104.1%, 101.4%, 108.8%,
nitric acid (Merck, Suprapur), the reactor was set to 90 °C for 2 h 97.5%, 106.4% and 97.1% for Cd, Pb, Hg, Cu, Zn and total As,
and then to 130 °C for 4 h. The digested sample was transferred respectively. Recovery for inorganic As was 93.8%.
to a volumetric flask and made up to 25 mL with Milli-Q water.
Quantification was performed using a Perkin-Elmer AAna- 2.3. Statistical analysis
lyst 800 spectrophotometer, equipped with a Zeeman back-
ground correction device (Cu and Zn by flame AAS; Cd, Pb and All studies were performed using Statgraphics v5Plus and
total As by electrothermal AAS). The operational parameters SPSS v11; all tests were performed at a 95% confidence level,
and matrix modifiers were those recommended by the unless otherwise stated.
manufacturer. Total Hg was determined by the cold vapour
technique, employing a Perkin-Elmer FIMS-400 system (SnCl2 3. Results and discussion
as reducing agent). Determination of inorganic As included
solubilisation with hydrochloric acid, reduction and subsequent The experimental concentrations are summarised in Table 2.
extraction with chloroform, back-extraction into diluted hydro- Although the determination of inorganic elements in various
chloric acid, dry-ashing, and quantification by hydride genera- algae in different countries has been summarised elsewhere
tion AAS employing a Perkin-Elmer FIAS 200 device (Muñoz (Hou and Yan, 1998), it is difficult to compare results from
et al., 1999a). The detection limits expressed in mg/kg dry various authors because of differences in sample treatments
weight were Pb: 0.008; Cd: 0.005; Hg: 0.001; Cu: 0.2; Zn: 1; and analytical procedures. Furthermore, several studies attrib-
total As: 0.050; and inorganic As: 0.020. uted seasonal variations in the metal concentrations to the algal
All analytical protocols were included in a quality control growth rate. For instance, concentrations decreased in macro-
system, which included analysis of certified reference materials, algae during growing periods and increased during the winter
duplicated samples, procedural blanks and international inter- dormant period (Phillips, 1994), the possible causes for which
laboratory exercises such as those from QUASIMEME and IAEA included allometric parameters (influencing metal uptake and
on a regular basis (Wyse et al., 2003; Quasimeme, 2004). accumulation), environmental factors (affecting metal accu-
Table 1 shows the analytical results (mean± SD) for eight mulation, Brown et al., 1999), and local variation in salinity
independent analyses of two certified reference materials: a (affecting biological uptake, Netten et al., 2000).
sea lettuce, Ulva lactuca (CRM 279) and an aquatic plant, La- Here, a preliminary univariate study was performed con-
garosiphon major (CRM 60), both from the EU Community sidering all samples and elements. Then, multivariate studies
Bureau of Reference. It is seen that the results agreed with the addressed whether the heavy metals studied differentiate
certified ones. seaweeds according to their type and/or geographical origin.
Due to the absence of certified (or guide) values for inorganic
As in the CRMs available for different organic matrices, a sea 3.1. Univariate studies
organism (the dogfish muscle, Squalus acanthias, National
Research Council of Canada, DORM-2 CRM) was selected to 3.1.1. Cadmium
“validate” the protocol for measuring inorganic As. This CRM Table 2 shows that Cd concentrations for different seaweeds
had previously been analysed by Muñoz et al. (1999a,b), also for are highly variable. The relationship between heavy metals and
308 V. Besada et al. / Journal of Marine Systems 75 (2009) 305–313

Table 2
Concentrations found for several edible seaweeds (mg/kg dry weight)

Species Commercial name Graphical Cd Pb⁎ Hg Cu Zn As⁎ Inorganic As

code
Gelidium spp. Agar 1 0.025–0.046 0.381–0.861 0.005–0.009 0.410–1.55 1.30–6.29 b0.05–0.21 0.025–0.135
Eisenia bicyclis Arame 2 0.585–0.827 0.029–0.096 0.023–0.047 3.06–4.54 21.4–23.5 27.9–34.1 0.041–0.170
Himanthalia elongate Seaweed spaghetti 3 0.310–0.326 0.203–0.259 0.008–0.016 1.14–1.25 48.5–48.7 32.9–36.7 0.166–0.245
Hizikia fusiforme Hiziki 4 0.988–2.50 b0.008–0.531 0.015–0.050 1.78–7.70 4.72–19.5 103–147 32.1–69.5
Laminaria spp Kombu 5 0.085–1.83 b0.008–0.460 0.001–0.005 0.91–2.50 10.3–23.2 51.7–68.3 0.052–0.443
Ulva rigida Sea lettuce 6 0.031–0.033 1.00–1.05 0.018–0.019 3.05–3.15 5.61–6.14 6.41–7.06 0.151–0.177
Chondrus crispus Irish moss 7 0.718–0.742 0.403–0.726 0.006–0.007 1.55–2.21 51.8–53.3 23.2–25.5 0.217–0.225
Porphyra umbilicales Nori 8 0.253–3.10 b0.008–0.270 0.008–0.032 5.50–14.1 39.5–73.8 28.9–49.5 0.132–0.338
Undaria pinnatifida Wakame 9 0.267–4.82 b0.008–1.28 0.010–0.057 1.07–1.70 8.25–26.6 42.1–76.9 0.045–0.346
Wakame, ogonori, kombu, Algae salad 10 1.69–1.80 0.189–0.420 0.024–0.035 1.47–1.56 42.8–43.4 18.6–19.1 0.240–0.283
agar and akamodoki
Wakame, nori and Seaweed salad 11 0.683–0.709 1.31–1.35 0.006–0.017 7.00–7.08 26.2–27.00 19.7–23.0 0.117–0.223
sea lettuce

⁎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; Phaeo-
phyceae, 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 concentra-
tions 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 corre-
sponded 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 Pb Hg Cu Zn As inorg As
Mean 1.07 0.30 0.018 3.79 29.37 46.95 6.02
SD 1.19 0.38 0.013 3.43 21.48 33.97 16.85
Max 4.82 1.35 0.057 14.1 73.8 147 69.5
Min 0.025 b0.008 0.001 0.41 1.30 b 0.05 0.025

2-way ANOVA (p-values)

Seaweeds 0.2044 0.0003 0.0000 0.0000 0.0000 0.0000 0.0000
Region 0.5500 0.0850 0.0136 0.0010 0.0120 0.0146 0.9813
 V. Besada et al. / Journal of Marine Systems 75 (2009) 305–313 309

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 compar-
ison. 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),
310 V. Besada et al. / Journal of Marine Systems 75 (2009) 305–313
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. Further-
more, 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 organ-
isms 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, FAO-
WHO) recommended measuring not only the total concentra-
tion 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 employ-
ing 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) 305–313 311

Table 4
Contingency table for the LDA discrimination models (harvesting areas,
Atlantic = 1, Pacific = 2)

Predicted (⁎) Predicted (⁎⁎) Theoretical

1 2 1 2 1 2
Real 1 18 (18) 8 (8) 13 (18) 13 (8) 26 –
2 3 (3) 23(23) 5 (4) 21 (22) – 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.

classification ratios considering all samples and cross-valida-

Fig. 3. Dendrogram obtained using autoscaled data, the Ward grouping tion (which simulate classification of truly unknowns) were
method and the city block distance. Codes: agar = 1, arame = 2, seaweed
almost the same. It is noteworthy that Pb increased the success
spaghetti = 3, hiziki = 4, kombu = 5, sea lettuce = 6, Irish moss = 7, nori = 8,
wakame = 9, algae salad = 10, seaweed salad = 11. 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
3.2.3. Linear discriminant analysis classification ability. The classification functions were: Atlantic
The fact that neither PCA nor hierchical clustering differ- Ocean= −4.30+ 6.09⁎Pb+ 0.16⁎Zn − 0.35⁎Cu, and Pacific Ocean=
entiated among the two harvesting areas indicated that the −1.64+ 1.75⁎Pb + 0.17⁎Cu + 0.04⁎Zn (note that Zn was not critical
overall variance was dominated by the inherent differences for the Pacific region and that Pb played an important role in the
between the seaweeds. Nevertheless, since some of the metals Atlantic area).
(total As, Cu, Hg and Zn) revealed different averages for the Fig. 4 shows the classification scores. It is worth noting that
harvesting areas when two-way ANOVAS were performed, a two agar samples were always misclassified, regardless of the
multivariate discrimination technique, linear discriminant model. As explained above for PCA, they had very low values in
analysis (LDA), was applied. LDA searches for discriminant most variables (but medium Zn levels) and although they were
functions (linear combinations of the experimental variables) from the Atlantic ocean, they were classified as being from the
that discriminate as much as possible among the groups Pacific area. One nori sample was also misclassified as being
defined by the scientists, in this case the two geographical from the Pacific, probably because of its high total and inorganic
origins. Note that PCA and cluster analysis extract patterns As concentrations. Two algae salads (#49 and #50 in the figure)
which explain most of the variance of the dataset, although they were wrongly considered as being from the Atlantic (probably
do not necessarily differentiate well between the groups. On the because they contained wakame, which had very high
other hand, LDA looks for discriminant functions capable of concentrations of several metals).
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 discrimina-
tion 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 Fig. 4. Box-Whisker representation of the classification functions obtained by
LDA (forward selection) obtained for each harvesting area: 1 characterises
function (standardised coefficients), with 78.8% success in both the Atlantic ocean (= − 4.30 + 6.09⁎Pb + 0.16⁎Zn −0.35⁎Cu), whereas 2
cases. This latter option was preferred because it used fewer characterises the Pacific Ocean (= −1.64 + 1.75⁎Pb + 0.17⁎Cu + 0.04⁎Zn). See
metals (i.e. it included less random variability) and because the text for more details.
312 V. Besada et al. / Journal of Marine Systems 75 (2009) 305–313
4. Conclusions
This study showed that there is a need for a national
(Spanish)—or preferably European—regulation on the max-
imum amount of pollutants in edible seaweeds commercia-
lised 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 differ-
entiate 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.
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