Environmental Pollution 215 (2016) 77e83

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Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol

Presence of trace metals in aquaculture marine ecosystems of the

northwestern Mediterranean Sea (Italy)
S. Squadrone*, P. Brizio, C. Stella, M. Prearo, P. Pastorino, L. Serracca, C. Ercolini, M.C. Abete
Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Turin, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Information regarding chemical pollutant levels in farmed fish and shellfish, along with the risks asso-
Received 15 March 2016 ciated with their consumption is still scarce. This study was designed to assess levels of exposure to 21
Received in revised form trace elements in fish (Dicentrarchus labrax), mussels (Mytilus galloprovincialis) and oysters (Crassostrea
26 April 2016
gigas) collected from aquaculture marine ecosystems of the northwestern Mediterranean Sea. Metal
Accepted 28 April 2016
concentrations showed great variability in the three species; the highest values of the nonessential el-
Available online 11 May 2016
ements As and Cd were found in oysters while the highest levels of Al, Pb and V were found in mussels.
The essential elements Cu, Mn and Zn were highest in oysters, but Fe, Cr, Ni, Se, Co and Mo levels were
Keywords:
Trace elements
highest in mussels. Fish had the lowest concentrations for all trace elements, which were at least one
Marine aquaculture order of magnitude lower than in bivalves. The rare earth elements cerium and lanthanum were found at
Mediterranean Sea higher levels in mussels than in oysters, but undetectable in fish. The maximum values set by European
Bivalves regulations for Hg, Cd and Pb were never exceeded in the examined samples. However, comparing the
Fish estimated human daily intakes (EHDIs) with the suggested tolerable copper and zinc intakes suggested a
potential risk for frequent consumers of oysters. Similarly, people who consume high quantities of
mussels could be exposed to concentrations of Al that exceed the proposed TWI (tolerable weekly
intake).
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction cadmium and lead accumulate in fish and shellfish, they pose a
potential risk to human health. Therefore, European Regulations
Marine ecosystems can receive chemical pollutants, with po- (1881/2006 and amendments) set maximum limits of these three
tential ecological and public health hazards such as metals, through contaminants in marine products.
rivers, direct discharges, or atmospheric deposition. Metals can, Mercury (Hg) is one of the most toxic heavy metals in our
therefore, be present in marine food items through accumulation environment. Natural, anthropogenic and re-emitted sources are
and for some elements-such as mercury-by means of bio- the three major origins of Hg emissions, with the most important
magnification through the marine food chain. anthropogenic sources of Hg pollution in the environment being
Some trace elements have nutritional functions and are essen- urban discharges, agricultural materials, mining, combustion and
tial to life (e.g. selenium, copper, cobalt, molybdenum, manganese industrial discharges (Zhang and Wong, 2007). The contamination
and zinc), and contribute to maintaining a good health status in chain of Hg closely follows a cyclic order, namely industry, atmo-
humans and animals (Vandermeersch et al., 2015). Other elements, sphere, soil, water, phytoplankton, zooplankton, fish and humans
(e.g. lead, cadmium, mercury, arsenic, tin, vanadium and (Kadar et al., 2006). Hg highly bio-accumulates and bio-magnifies
aluminum), have no biological functions and their intake can lead throughout the aquatic food chain (Carrasco et al., 2011). The
to adverse health effects. The World Health Organization (WHO) general population is most commonly exposed to mercury pri-
recently indicated arsenic, lead, mercury and cadmium on their marily by eating fish and shellfish that may contain methyl mercury
prioritized list of the top ten chemicals of major public health in their tissues. Methyl-mercury (CH3Hg) is the most toxic form of
concern (WHO, 2010). In particular, if toxic metals such as mercury, mercury, and affects the immune system, alters genetic and
enzyme systems, and damages the nervous system.
Fish and shellfish are an important source of cadmium (Cd)
* Corresponding author. intake in the general population. Most of the cadmium content in
E-mail address: [email protected] (S. Squadrone).

http://dx.doi.org/10.1016/j.envpol.2016.04.096
0269-7491/© 2016 Elsevier Ltd. All rights reserved.
78 S. Squadrone et al. / Environmental Pollution 215 (2016) 77e83

fish or other seafood is highly absorbable in CdCl2 form; Cd is elements in fish and shellfish from Northwestern Mediterranean
particularly accumulated in the kidneys, while Cd concentrations mariculture.
are low in muscles (ATSDR, 2012). In humans and animals, Cd af- The main objectives of the present study were:
fects a number of organs, namely kidneys, lungs, bones, placenta,
brain and the central nervous system. Cd also causes reproductive i) to analyze the concentrations of aluminum (Al), antimony
toxicity, adverse effects on the hepatic hematological and immune (Sb), arsenic (As), beryllium (Be), cadmium (Cd), cerium (Ce),
systems, (ATSDR, 2012). cobalt (Co), chromium (Cr), iron (Fe), manganese (Mn),
Lead (Pb) is an established contaminant that is mainly exposed mercury (Hg), molybdenum (Mo), lanthanum (La), nickel
to the general population via food and air. Once absorbed, Pb ac- (Ni), lead (Pb), copper (Cu), selenium (Se), tin (Sn), thallium
cumulates in high concentrations in bones, teeth, liver, lungs, kid- (Tl), vanadium (V) and zinc (Zn) in European sea bass, Eu-
neys, brain and spleen, and it can penetrate the blood-brain barrier ropean mussels and Pacific oysters from Northwestern
and the placenta (Gwaltney-Brant and Rumbeiha, 2002). Much of Mediterranean aquaculture (Ligurian Sea)
the Pb in the marine environment is strongly adsorbed onto sedi- ii) to verify the compliance with the maximum levels estab-
ments and suspended particles, reducing its availability to organ- lished by the European Commission Regulation (1881/2006
isms (Falco et al., 2006). In shellfish, Pb concentrations are higher in and amendments)
the calcium-rich shell than in the soft tissue, while in marine fish, iii) to increase the data about the potential risks to human
Pb levels are higher in the gills and skin than in other tissues (Eu- health associated with these contaminants by consuming
ropean Food Safety Authority, EFSA, 2012). aquaculture products.
In the marine environment, inorganic arsenic (As) predominates
in seawater and sediments, while bioaccumulation of the element
by organisms generally occurs as organic non-toxic compounds 2. Materials and methods
such as arsenobetaine and arsenocholine, which have been sug-
gested to be final products of detoxification processes (Francesconi 2.1. Study area and collection of samples
and Edmonds, 1996). Anthropogenic contamination can modify the
environmental bioavailability of arsenic, and influence the relative The sampling areas were located in a northwestern Mediterra-
presence of toxic forms in tissues of exposed organisms such as fish nean, Ligurian Sea (Fig. 1).
and shellfish (Fattorini et al., 2004). Samples of mussels (Mytilus galloprovincialis) (n. 25) and oysters
The Mediterranean is the largest semi-enclosed European sea. It (Crassostrea gigas) (n. 25) were collected in February from aqua-
has a narrow shelf and, in the north, is mostly bordered by culture facilities in the Gulf of La Spezia.
mountain chains sloping steeply into the sea, resulting in a narrow Mussels and oysters are bred, within and outside the east dam,
littoral zone. This semi-closed basin is characterized by several in tanks of varying widths of 500e2000 m2, consisting of floats
unique features such as low trophic potential, high seawater tem- attached together by nylon strings, stretched below the level of the
peratures, phosphorus limit primary production, low sedimenta- wave motion, to which so-called “arbors” or “reste” are knotted, on
tion rate and high maritime traffic (CIESM, 2007). which the bivalves grow. The morphology and favorable environ-
Mediterranean aquaculture has experienced a rapid growth in mental conditions of the area (especially the lack of storms), the
the last three decades. To cope with the continuous increase in depth of the seabed and freshwater undercurrents create optimal
production, Mediterranean mariculture has moved from small conditions for the growth and development of mussels.
land-based operations to large enterprises located along the European sea bass (Dicentrarchus labrax) samples (n. 25) were
coastline and more recently, to off-shore sites. Several fish marine collected in aquaculture facilities from Porto Venere (in the prov-
species are cultured in the Mediterranean, the most commercially- ince of La Spezia). Fish lengths ranged from 30 to 36 cm, and
important being the gilthead sea bream (Sparus aurata) and Euro- weights ranged from 350 to 450 g. The plant, equipped with
pean sea bass (Dicentrarchus labrax), and regarding shellfish, the floating cages in the sea is an in-shore installation for farming sea
European mussel (Mytilus galloprovincialis), the European flat oys- bass. Currently, it consists of 76 tanks and occupies an area of
ter (Ostrea edulis) and the Pacific oyster (Crassostrea gigas) (FAO, 19,000 m2.
2014). Immediately after collection, fish and bivalve samples were
Adequate information on trace elements present in seafood placed into a clean polythene bag, preserved on ice and transported
from aquaculture is often lacking, thus creating potential risks. to the laboratory. Each sample of mussels or oysters was prepared
Where cage farming exists, in addition to the natural presence of by pooling 10 or more individual samples, while at least 10 g of the
metals in the aquatic environment from geochemical and anthro- dorsal muscle from the fish was dissected for subsequent chemical
pogenic processes, another source of trace metals is from the use of analysis.
metal (e.g. copper)-based anti-foulants for protecting the nets from
fouling (Arechavala-Lopez et al., 2013). Moreover, fish diets are also 2.2. Detection of trace elements
enriched with various essential metals, such as copper, iron, man-
ganese, cobalt and chromium (CIESM, 2007), which could Samples were divided into two sub-samples, one for Hg quan-
contribute to the metal burden in fish flesh. As a result, differences tification with a Direct Mercury Analyzer (DMA-80 Analyzer from
between farmed and wild sea bream and sea bass have been found Milestone, Shelton, CT, USA) and the other for detecting all the
through trace element analysis (Arechavala-Lopez et al., 2013). other metals by Inductively Coupled Plasma-Mass Spectrometry
Our investigation took place in aquaculture systems of the Lig- (ICP-MS Xseries II, Thermo Scientific, Bremen, Germany). The Direct
urian Sea, part of the Mediterranean Sea positioned between the Mercury Analyzer performs thermal decomposition, catalytic
northwestern coast of Italy, the southeastern coast of France, and reduction, amalgamation, desorption and atomic absorption spec-
north of the islands of Corsica and Elba. Previous reports of this area troscopy without requiring pre-treatment. Between 0.05 g and 0.1 g
(Giorgi et al., 2009) revealed that wild fish and shellfish are of samples were directly weighed on graphite shuttles and pro-
potentially harmful for consumers, as some samples exceeded the cessed for Hg content.
maximum residue limits (MRL) set by the regulations for Hg and Pb. Determination of Al, As, Be, Cd, Ce, Co, Cr, Cu, Fe, La, Mn, Mo, Pb,
To our knowledge, no data are available on the levels of trace Ni, Sb, Se, Sn, Tl, V and Zn, was performed after wet digestion using
 S. Squadrone et al. / Environmental Pollution 215 (2016) 77e83 79

Fig. 1. Study area and sampling sites (Northwestern Mediterranean Sea, Gulf of La Spezia, Italy). Red and white asterisks represent the sampling sites. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)

acids and oxidants (HNO3 and H2O2) of the highest quality grade and seafood consumption for the Italian population, as reported by
(Suprapure). For this purpose, 1.5e2.0 g of samples were subjected the National Research Institute for Food and Nutrition (Leclercq
to microwave digestion (microwave oven ETHOS 1 from Milestone, et al., 2009), and BW is the human body weight (70 kg).
Shelton, CT, USA) with 7 mL of HNO3 (70% v/v) and 1.5 mL of H2O2 The consumption figures used were the 50th and 95th percen-
(30% v/v). Ultrapure water was added to the samples to reach a final tile intakes for the total population in consumers of all ages. The
weight of 50 g (Arium611VF system from Sartorius Stedim Italy obtained values were compared with the tolerable daily intake
S.p.A., Antella e Bagno a Ripoli, FI, Italy). Multi-elemental deter- (TDI) or the provisional tolerable weekly intake (PTWI) proposed
mination was performed by ICP-MS after daily optimization of by the EFSA.
instrumental parameters. Using an external standard calibration
curve; rhodium and germanium were used as internal standards. 3. Results and discussion
Analytical performances were verified by processing Certified
Reference Materials (Oyster Tissue- SRM 1566b from the National The concentrations of the investigated metals are shown in
Institute of Standard and Technology), along with blank reagents in Table 1 and Fig. 2 (essential trace elements) and Fig. 3 (nonessential
each analytical session. The limit of quantification (LOQ) for each trace elements).
element, the reference material values and the percentages of re- The metal concentrations in mussels decreased in the following
covery obtained are shown in Table S1. The analytical methods order: Al (141.616) > Fe (71.032) > Zn (24.651) > Mn (3.627) > Cu
were validated according to UNI CEI EN ISO/IEC 17025 (General (1.002) > As (0.929) > V (0.864) > Cr (0.535) > Pb (0.489)> Ni
requirements for the competence of testing and calibration (0.388) > Se (0.299) > Mo (0.155) > Co (0.123) > Ce (0.065) > Cd
laboratories). (0.049) > La (0.037) > Sn (0.022) > Hg (0.011) mg kg1 wet weight,
median values. In oysters, metal levels were as follows: Zn
2.3. Statistical analysis (382.930) > Cu (84.346) > Al (43.127) > Fe (30.418) > Mn
(4.440) > As (1.508) > V (0.864) > Se (0.250) > Pb (0.190) > Cr
In analyzing metal concentrations, the differences between the (0.180) > Ni (0.131)> Cd (0.120) > Mo (0.050) > Co (0.030) > Sn
three groups (mussels, oysters and fish) were studied using the (0.028) > Ce (0.020) > Hg (0.014) > La (0.012) mg kg1 wet weight,
Kruskal-Wallis test or, in the case of two groups (some elements median values.
were < LOQ in fish), using the Kolmogorov-Smirnov test (Table S2). Metal concentrations in fish muscle were measured in the
The range, the mean concentration and the median concentration following order: Zn (4.956) > Fe (2.829) > Cu (0.889) > As
of each quantifiable element are shown, with the relative standard (0.388) > Mn (0.199) > Se (0.173) > Al (0.153) > Ni (0.041) > Mo
deviation (Table 1). Results were considered statistically significant (0.040) > Hg (0.037) > Cr (0.024) mg kg1 wet weight, median
at p values of <0.01. Statistical calculations were performed using values. Be and Tl were below the LOQ in all samples, while Cd, Ce,
Graph Pad Statistics Software Version 6.0 (GraphPad Software, Inc., Co, La, Pb, Sn and V were below the LOQ in fish, Dicentrarchus
USA). labrax.

2.4. Estimation of daily or weekly intake 3.1. Essential trace elements

We estimated human exposure from bivalve and fish con- 3.1.1. Copper, iron, manganese and zinc
sumption by calculating the Estimated Human Daily Intake (EHDI), Copper is an essential nutrient that is incorporated into a
as follows: number of metalloenzymes involved in hemoglobin formation,
xenobiotic metabolism, carbohydrate metabolism, and in the
EHDI ¼ (C  DC)/BW antioxidant defense mechanism (ATSDR, 2004). Although copper
homeostasis plays an important role in preventing copper toxicity,
where C is the mean contaminant concentration, DC is the daily fish exposure to excessive levels of Cu can result in a number of adverse
80 S. Squadrone et al. / Environmental Pollution 215 (2016) 77e83

Table 1
Trace elements concentrations (mg kg1) in mussels, oysters and fish from Mediterranean aquaculture.

Trace element Mytilus galloprovincialis (n.25a) Crassostrea gigas (n.25a) Dicentrarchus labrax (n.25)

Min Max Median Mean SD Min Max Median Mean SD Min Max Median Mean SD

Al 100.388 263.530 141.616 162.466 59.010 22.806 72.382 43.127 42.368 20.07 0.072 0.742 0.153 0.224 0.234
As 0.838 1.155 0.929 0.942 0.100 0.883 1.695 1.508 1.368 0.360 0.072 0.563 0.388 0.414 0.069
Cd 0.045 0.060 0.049 0.050 0.000 0.104 0.126 0.120 0.117 0.010 e e <0.010 <0.010 e
Ce 0.044 0.090 0.065 0.069 0.020 0.009 0.039 0.020 0.021 0.010 e e <0.010 <0.010 e
Co 0.087 0.161 0.123 0.124 0.020 0.024 0.045 0.030 0.032 0.010 e e <0.010 <0.010 e
Cr 0.136 0.651 0.535 0.513 0.110 0.104 0.289 0.180 0.187 0.070 0.018 0.091 0.024 0.033 0.026
Cu 0.571 1.316 1.002 1.035 0.150 73.752 134.021 85.343 91.615 24.220 0.017 1.938 0.889 0.992 0.437
Fe 1.024 116.595 71.032 79.169 25.640 17.119 53.794 30.418 31.826 14.28 0.637 3.301 2.829 2.841 0.260
Hg 0.010 0.014 0.011 0.011 0.000 0.010 0.033 0.014 0.019 0.010 0.032 0.040 0.037 0.036 0.003
La 0.010 0.055 0.037 0.041 0.010 0.006 0.020 0.012 0.012 0.010 0.000 0.000 <0.010 <0.010 e
Mn 0.052 4.384 3.627 3.407 0.770 2.821 4.594 4.440 4.046 0.760 0.169 0.400 0.199 0.248 0.104
Mo 0.133 0.193 0.155 0.155 0.030 0.039 0.081 0.050 0.056 0.020 0.035 0.065 0.040 0.045 0.011
Pb 0.123 1.048 0.489 0.513 0.290 0.084 0.194 0.131 0.130 0.040 e e <0.010 <0.010 e
Ni 0.347 0.589 0.388 0.425 8.500 0.132 0.276 0.190 0.194 0.050 0.035 0.046 0.041 0.039 0.006
Se 0.245 0.361 0.299 0.291 0.000 0.171 0.300 0.250 0.251 0.050 0.030 0.211 0.173 0.179 0.016
Sn 0.017 0.030 0.022 0.023 0.290 0.019 0.036 0.028 0.028 0.010 e e <0.010 <0.010 e
V 0.024 1.429 0.864 0.868 0.090 0.046 0.162 0.087 0.092 0.040 e e <0.010 <0.010 e
Zn 0.549 42.802 24.651 28.868 0.040 318.479 546.832 382.930 411.433 97.290 4.818 5.278 4.956 4.920 0.234
a
Each sample was prepared by pooling 10 or more individuals.

health effects including liver and kidney damage, anemia, immu- including pregnant and lactating women, based on studies of zinc
notoxicity, and developmental toxicity (ATSDR, 2004). A Tolerable supplementation for up to 14 weeks (EFSA, 2014).
Upper Intake of 5 mg day1 has been established for adults (EFSA, Copper and zinc concentrations were found to be higher in
2015a,b). oysters (82.343 and 411.433 mg kg1, respectively) than in mussels
Zinc has a wide array of vital physiological functions, but chronic and fish (Table 1, Fig. 2a), in agreement with previous findings,
high zinc intake can result in severe neurological diseases (Hedera which showed higher concentration of these elements in oysters
et al., 2009). The Scientific Committee for Food (SCF , 2002) set a compared to other marine species (Pan and Wang, 2012). These
Tolerable Upper Intake Level (UL) of 25 mg day1 for adults, data are similar to those recently obtained in Crassostrea gigas and

Fig. 2. a Copper, iron, manganese and zinc concentrations (mg kg1 wet weight) in Fig. 3. a. Arsenic, cadmium, lead and mercury concentrations (mg kg1 wet weight) in
fish, oysters and mussels (logarithmic scale). b Cobalt, chromium, molybdenum, nickel fish, oysters and mussels. b. Aluminum, cerium, lanthanum, tin and vanadium
and selenium concentrations (mg kg1 wet weight) in fish, oysters and mussels. (mg kg1 wet weight) in fish, oysters and mussels (logarithmic scale).
 S. Squadrone et al. / Environmental Pollution 215 (2016) 77e83 81

Mytilus galloprovincialis by Biland
zic et al. (2016) along the Croatian still scarce. However, nickel can be genotoxic, immunotoxic and
coast of the Adriatic Sea, although the Cu levels we found in oysters hepatotoxic; it is also known to increase ROS formation, enhancing
were higher. Compared to levels found by Ochoa et al. (2013) in the risk of oxidative stress, which could be important in Ni-induced
oysters cultured in the Ebro Delta in Catalonia, Spain, we found carcinogenesis (Das, 2008). We found low levels of nickel (Table 1),
higher Cu values and lower Zn concentrations. The levels of Cu and which significantly differed in the three marine species, with
Zn differed significantly in the three species, as shown in Table S2. mussels having the highest values, followed by oysters and fish
Copper values in sea bass were comparable to those registered by (Fig. 2b).
Iamiceli et al. (2015) in the Mediterranean Sea. Selenium is a metalloid trace element required by wildlife in
Iron is an essential element for almost every organism, but is small amounts for biological functions (Ohlendorf and Heinz,
toxic in high amounts. Epidemiological studies have reported as- 2009), and is increasingly considered to be a nutraceutical
sociations of high Fe intake and/or Fe stores with an increased risk component (Ralston and Raymond, 2010). Selenium can enter
of chronic diseases such as cardiovascular disease, type II diabetes aquatic ecosystems, and can bioaccumulate and bioamplify up the
and cancer of the gastrointestinal tract (EFSA, 2004). The recom- food chain (Burger et al., 2013). Selenium values had comparable
mended daily dietary intake for iron is 40 mg in children and 45 mg values in the analyzed species, with only significant differences in
in adults (Institute of Medicine (2001)), while a tolerable upper mussels versus fish (Table S2, Table 1).
level has still not been set. In our study, the highest Fe level was
found in mussels (71.032 mg kg1), while oysters and fish had 3.2. Nonessential trace elements
lower values (Table 1, Fig. 2a). These values were higher than those
found by other authors in farmed mussels from the Mediterranean 3.2.1. Arsenic, cadmium, lead and mercury
Sea (Gue rin et al., 2011; Biland

zi
c et al., 2016) and Fe content was Arsenic is a metalloid, which can be toxic in its inorganic form,
shown to be significantly different in the three analyzed species and innocuous in some organic forms, such as arsenobetaine. The
(Table S2). uptake of As by organisms is highly dependent on element
Although low levels of manganese intake are necessary for hu- bioavailability, which varies significantly in the environment
man health, the exposure to high levels of Mn are toxic, and several (Rahman et al., 2012). It is well known that shellfish significantly
studies have suggested that ingestion of water and/or foodstuffs contribute to the dietary intake of As. We found comparable values
containing increased concentrations of this trace element may in mussels and oysters (Table 1, Table 2S and Fig. 3a), similar to
result in adverse neurological effects (ATSDR, 2012). The Tolerable those obtained by Belivermiş et al. (2016) in mussels, but lower that
Upper Intake Level (TDI) is 11 mg manganese/day for adults of 70 kg those recorded by Biland
zi
c in oysters (2016) from the Mediterra-
(ATSDR, 2012). Manganese values in the analyzed mussels and nean Sea. In fish, As content was comparable to the levels found in
oysters were similar (3.407 and 4.440 mg kg1, respectively, p European seabass from the Mediterranean Sea by Iamiceli et al.
value > 0.01 as shown in Table S2) and comparable to previous (2015).
findings in the Mediterranean Sea (Gue rin et al., 2011; Jovic and Cadmium exposure has been associated with nephrotoxicity,
Stankovi c, 2014). osteoporosis, neurotoxicity, carcinogenicity, genotoxicity, and
teratogenicity, and has been shown to have adverse effects on the
3.1.2. Cobalt, chromium, molybdenum, nickel and selenium endocrine and reproductive systems (EFSA, 2009). A provisional
Cobalt is an essential trace element present in vitamin B12, and tolerable weekly intake (PTWI) of 7 mg kg1 b.w. was set by the SCF
plays an important role in human and animal nutrition. Cobalt has (EFSA, 2009), corresponding to 70 mg of Cd for an adult of 70 kg b.w.
also been identified to play a role in regulating blood pressure and Cd was < LOQ in European sea bass (Table 1, Fig. 3a, Table S2), in
found to be necessary for proper thyroid function (Calabrese et al., agreement with previous findings in Italian fish farms (Trocino
1985). A daily oral intake of 0.6 mg cobalt (based on a lowest- et al., 2012), while a median concentration of 0.120 mg kg1 were
observed-adverse-effect level (LOAEL) of 1 mg kg1 for poly- found in oysters, which was in the range of levels previously
cythemia) appears to be the minimum risk level for humans that registered in the Mediterranean Sea (Ochoa et al., 2013; Jovi c and
can protect from the known threshold-related adverse effects Stankovic, 2014). In mussels, Cd levels were lower than those
(EFSA, 2009). detected in other Mediterranean-farmed M. galloprovincialis
Cobalt was detected in all the mussel (0.123 mg kg1 median (Kljakovic-Gaspic et al., 2010; Spada et al., 2013; Jovic and
value) and oyster samples (0.030 mg kg1 median value), while in Stankovic, 2014). Cd values were well below the maximum limits
fish; this element was undetectable (Table 1, Table S2 and Fig. 2b). set by European regulation, Reg. EU 488/2014 for Cd content in fish
Values were one order of magnitude lower than those found by (0.050 mg kg1 w.w.) and bivalves (1.00 mg kg1).
Joksimovic et al. (2011) in Mediterranean mussels, while to our There is no current PTWI set for Pb, which is an environmental
knowledge, no data are available regarding Co levels in oysters from contaminant mainly resulting from anthropogenic activities, such
the Mediterranean Sea. as mining and smelting. Due to its long half-life in the body, chronic
Chromium is an essential element and is ubiquitous in the diet Pb toxicity is of utmost concern when considering the potential risk
(EFSA, 2014). The biologically usable form of Cr plays an essential to human health (EFSA, 2010). Mussels and oysters differed
role in glucose metabolism and it is estimated that the average significantly in Pb content (Table S2); the highest median value of
human requires nearly 1 mg day1. Chromium deficiency results in Pb was found in mussels 0.489 mg kg1 w.w., with values ranging
disturbances of the metabolism of glucose, lipids and proteins from 0.123 to 1.043 mg kg1 w.w; while in oysters, the Pb median
(Mertz, 1992). The EFSA Panel on Contaminants in the Food Chain level was 0.131 mg kg1 w.w. (Fig. 3a). In sea bass fish samples, Pb
(CONTAM Panel) recently proposed a Tolerable Daily Intake (TDI) of was undetectable, although other studies have shown a range of
300 mg Cr (III) kg1 b. w. (EFSA, 2014). Chromium was detected in all 0.278e1.030 mg kg1 w.w. in farmed Mediterranean fish (Orban
investigated samples (Table 1, Table S2 and Fig. 2b); the highest Cr et al., 2002; Dugo et al., 2006). Current maximum limits for Pb
concentrations were found in mussels, lower than those found in (EU Regulation 1005/2015) are 0.30 mg kg1 w.w. and
samples from the Croatian Adriatic coast (Joksimovic et al., 2011) 1.500 mg kg1 w.w in fish and bivalves, respectively, and were
and in the Turkish coast (Belivermiş et al., 2016). never exceeded in the analyzed samples.
Nickel is ubiquitous in the environment and is essential for The general population is most commonly exposed to mercury
animal nutrition. Like other trace elements, data on Ni levels are by eating fish that may contain methyl mercury (the most toxic
82 S. Squadrone et al. / Environmental Pollution 215 (2016) 77e83

form of mercury) in their tissues. Hg bioaccumulates and bio- (Monti et al., 2005), which demonstrated a lower proportion of
magnifies to a significant extent through the aquatic food chain toxic metals, such as Hg and Pb, in farmed sea bass compared to
(Carrasco et al., 2011). The limit for total Hg in fish and seafood for wild sea bass from the Mediterranean Sea.
human consumption is 0.500 mg kg1 w.w. (Reg. EU, 1881/2006). Compared to previous data registered in Dicentrarchus labrax
Mercury content in the analyzed samples was negligible (Table 1, from Italian fish farms (Trocino et al., 2012) we found lower values
Fig. 3a), ranging from 0.011 (mussels) to 0.037 mg kg1 w.w (sea of As, Cd, Cr, Cu, Pb and Zn and higher values of Se.
bass). Oysters and mussels showed similar levels of As, Hg, Mn, Mo, Se
and Sn but differed significantly in Al, Cd, Ce, Co, Cr, Cu, Fe, La, Pb,
3.2.2. Aluminum, cerium, lanthanum, tin and vanadium Ni, V and Zn contents (Table 1, Table S2). Consequently, the con-
Little is known about the toxicity of aluminum, but the neuro- sumer is exposed to different substances (trace elements)
toxicity of Al in humans was demonstrated in patients undergoing depending on the consumed species. The EHDIs were calculated for
dialysis where insufficiently purified water was used, and the pa- all the analyzed elements and compared to the proposed TDI or
tients were therefore parenterally exposed to high concentrations TWI (EFSA) of each metal. The proposed TDIs of 5 mg day1 for
of aluminum (EFSA, 2008). A TWI (tolerable weekly intake) of 1 mg copper (EFSA, 2015a,b) and of 25 mg day1 for Zn (EFSA, 2014)
Al kg1 b.w. was recommended by the EFSA (2008), corresponding would be exceeded in frequent consumers of oysters, considering
to 10 mg of Al per week for an adult. the consumption for the Italian population, as reported by the
Mussels had the highest Al levels (Table 1, Fig. 3b), ranging from National Research Institute for Food and Nutrition (Leclercq et al.,
100. 388 to 263.530 mg kg1 w.w., while in oysters the median 2009). Similarly, the TWI of 1 mg A kg1 b.w. recommended by
value was 43.127 mg kg1 w.w. and in fish the median value was the EFSA (2008) would be exceeded in frequent consumers of
0.153 mg kg1 w.w. To our knowledge, there are no data regarding mussels.
aluminum content in Mediterranean mariculture products, and this
is therefore the first report about the presence of this ubiquitous 4. Conclusions
nonessential element. However, in a previous study (Battuello et al.,
2016), we found that Al levels in Mediterranean zooplankton from In this study we assessed the levels of 21 trace elements in fish
superficial waters ranged from 62.30 to 174.14 mg kg1; (Dicentrarchus labrax), mussels (Mytilus galloprovincialis) and oys-
zooplankton contributes to the transfer of trace metals to higher ters (Crassostrea gigas) collected from aquaculture marine ecosys-
trophic levels such as bivalves. tems of the northwestern Mediterranean Sea. Toxic metals such as
Cerium and lanthanum belong to the group of rare earth ele- cadmium, lead and mercury were found at levels below the current
ments (REE). In the case of marine environments, REE may be maximum limits set by the European Community for fish and bi-
released from industrial wastewater effluents and they affect both valves. However, consumers should be aware that maximum limits
coastal waters and sediments, hence sediment welling biota (Oral varied greatly between seafood categories and that higher levels of
et al., 2010). Dissolved REEs are reported to be present in very cadmium and lead are allowed in bivalves (1 mg kg1 of cadmium;
low concentrations in natural open seawater, typically in the order 1.5 mg kg1 of lead) compared to fish (0.05 mg kg1 of Cd,
of pg L1 (Wang and Yamada, 2007), but they can be bio- 0.30 mg kg1 of Pb). Our study confirmed that shellfish contribute
accumulated by marine invertebrates such as zooplankton and to the recommended daily element intake of several essential ele-
enter the food chain (Palmer et al., 2006). We investigated Ce and ments, although some of them may contribute to excessive intake.
La levels in marine zooplankton of the Mediterranean Sear The copper and zinc concentrations that were higher than the
(Battuello et al., 2016) and they were below the LOQ recommended reference values suggest a potential risk for con-
(0.010 mg kg1). Accordingly, these two REE were not detected in sumers if large quantities of oysters are consumed frequently.
fish, and had low levels in bivalves, but both Ce and La were higher Similarly, the nonessential but ubiquitous element aluminum could
in mussels than in oysters (Table 1, Fig. 3b). constitute a potential risk for frequent consumers of mussels.
Tin and its compounds are significant and controversial chem- Further studies are necessary for a better understanding of human
icals in the environment. They are generally regarded as being exposure to metals through the consumption of mussels and oys-
relatively immobile in food chains. Coastal regions are hot spots for ters. Pregnant and lactating women are suggested to pay particular
environmental contamination by organotin compounds due to attention to the amount of bivalves introduced into their diets, also
their proximity to harbor areas, as well as to industrial and do- considering that the estimated intake does not include the contri-
mestic points of effluent discharge (WHO, 2005).Sn concentrations bution of other contamination sources to which the population
in the analyzed samples were low and of no concern for human could be also exposed. European sea bass farmed in the study area
health in bivalves (Table 1; Table S2); Sn was undetected in fish. always had levels of trace metals below those that could pose a risk
Vanadium enters the aquatic system through multiple routes, for human health and were thus considered absolutely safe for
including release of flying ash and through run-off from naturally human consumption.
V-rich soils, irrigated areas and industrial plants (Mackey et al.,
1996). This element was one order of magnitude higher in mus-
sels than in oysters (Fig. 3b; Table 1) and undetected in fish. Acknowledgements

3.3. Human exposure to metals by farmed seafood The research was funded through an Italian Health Ministry
Research (Project n. IZS PLV 01/13 RC).
Although the three marine species were sampled from mari- The authors particularly thank the editor and the reviewers for
culture facilities of the same area, namely the Gulf of La Spezia, their suggestions, which have improved the quality of this
Ligurian Sea, metal concentrations were highly variable and manuscript.
species-dependent. European sea bass samples from the examined
area consistently had metal concentrations that were below the Appendix A. Supplementary data
values that would pose a risk for human health (regarding
maximum limits, TDI, TWI), and can therefore be considered to be Supplementary data related to this article can be found at http://
safe for human consumption. This confirms previous findings dx.doi.org/10.1016/j.envpol.2016.04.096.
 S. Squadrone et al. / Environmental Pollution 215 (2016) 77e83 83

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