Accepted Manuscript
Lead, mercury and cadmium levels in edible marine molluscs and echinoderms from the Veneto Region (north-western Adriatic Sea – Italy)
Laura Bille, Giovanni Binato, Veronica Cappa, Marica Toson, Manuela Dalla Pozza, Giuseppe Arcangeli, Antonia Ricci, Roberto Angeletti, Roberto Piro
PII: S0956-7135(14)00524-6 DOI: 10.1016/j.foodcont.2014.09.018 Reference: JFCO 4067
To appear in: Food Control
Received Date: 14 March 2014 Revised Date: 12 September 2014 Accepted Date: 15 September 2014
Please cite this article as: Bille L., Binato G., Cappa V., Toson M., Pozza M.D., Arcangeli G., Ricci A., Angeletti R. & Piro R., Lead, mercury and cadmium levels in edible marine molluscs and echinoderms from the Veneto Region (north-western Adriatic Sea – Italy), Food Control (2014), doi: 10.1016/ j.foodcont.2014.09.018.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT 1 Lead, mercury and cadmium levels in edible marine molluscs and echinoderms
2 from the Veneto Region (north-western Adriatic Sea – Italy)
3 Laura Bille a * , Giovanni Binato a, Veronica Cappa a, Marica Toson a, Manuela Dalla
4 Pozza a, Giuseppe Arcangeli a, Antonia Riccia, Roberto Angeletti a, Roberto Piro a.
5 a Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell'Università 10, Legnaro
6 (PD), 35020, Italy.
7 * Corresponding author. Tel.: +39 0498084332; fax: 049 8830268. E-mail address:
8 [email protected] (Bille Laura)
9
10 ABSTRACT
11 In this study, heavy metal levels detected in molluscs produced in the Veneto Region
12 (north-western Adriatic Sea) are presented and discussed. A descriptive analysis was
13 conducted on data collected in the framework of official monitoring activities in 14 shellfish production areas between 2007 and 2012. MANUSCRIPT Concentrations of lead (Pb), mercury 15 (Hg) and cadmium (Cd) in about 2,000 samples of 17 different species of marine
16 bivalve molluscs, echinoderms and gastropods were checked by means of atomic
17 absorption spectrometry. Hg and Pb concentrations were always below the maximum
18 levels set by the European legislation; 2.2% of the analysed samples exceeded the
19 threshold level for Cd, 86.7 % of witch belonging to purple dye murex ( Bolinus
20 brandaris ). The highest mean levels of Hg and Cd were found in purple dye murex,
-1 -1 21 with 0.10 mgACCEPTED kg and 0.88 mg kg respectively, while rayed trough shell ( Mactra 22 stultorum ) and truncate donax ( Donax trunculus ) were the species with the highest
23 mean value of Pb (0.25 mg kg -1). Species of major consumption showed mean values
24 almost ten times lower than the limits set by the European legislation (Manila clam: Pb:
25 0.20 mg kg -1; Hg: 0.04 mg kg -1; Cd: 0.07 mg kg -1; Mediterranean mussel: Pb: 0.22 mg
1 ACCEPTED MANUSCRIPT 26 kg -1; Hg: 0.03 mg kg -1; Cd: 0.13 mg kg -1). A preliminary evaluation of the impact on
27 human health of shellfish according to the detected levels indicates that the species of
28 major consumption are safe for the average consumers, although a potential risk cannot
29 be ruled out for vulnerable groups, such as women of childbearing potential and
30 children, as well as for regular or high-level shellfish consumers, particularly those who
31 frequently eat species such as murex.
32 Keywords : Heavy metals, Cadmium, Mercury, Lead, Molluscs, Gastropods.
33 1. Introduction
34 Italy is the third European producer of marine molluscs (~150.000 t), after Spain
35 (~270.000 t) and France (~250.000 t) (FAO 2011). About 70% of the Italian shellfish
36 production is consumed domestically, while the remaining 30% is exported to European
37 countries, first among them Spain (Sladonja et al., 2011). MANUSCRIPT 38 The national shellfish production relies mainly on two species, the Manila clam 39 (Venerupis philippinarum ) and the Mediterranean mussel ( Mytilus galloprovincialis ),
40 which are also the most largely consumed. Italy is the leading European producer of
41 Manila clams and the third worldwide producer of Mediterranean mussels, after China
42 and Spain (Robert et al., 2013).
43 The Italian mollusc production is mostly concentrated in the north Adriatic regions. In
44 particular, the Veneto Region is a leader in the national shellfish industry, being the first 45 producer of ManilaACCEPTED clams and the third producer of mussels in the country (Robert et 46 al., 2013). Other minor species are also produced in this area.
47 The northern Adriatic Sea is not particularly deep and is fed by three large waterways:
48 the Po, the Adige and the Brenta rivers. They flow across the Po valley, where there are
49 numerous urban settlements, as well as industrial and zootechnical activities. Moreover,
2 ACCEPTED MANUSCRIPT 50 the Venice Lagoon is classified as being a sensitive area exposed to anthropogenic
51 pollutants sources, the main being the Industries of Porto Marghera and the discharge of
52 urban waste water sewage and urban runoff from the municipality of Venice (Giusti L.
53 and Zhang H., 2002).
54 As mentioned above, this area is well known for its shellfish harvesting activity, whose
55 extensive production can be an easy target for chemical contamination. Lead (Pb),
56 cadmium (Cd) and mercury (Hg) are chemical elements of great concern from a public
57 health point of view, being environmental pollutants which can occur naturally in the
58 environment and also derive from either industrial (e.g. mining, metallurgical,
59 incineration, pesticide and varnish production activities) or agricultural sources (e.g.
60 pesticide and fertilizers use). These contaminants are highly toxic and may accumulate
61 in seafood, whose consumption can represent an important route of human exposure to
62 these harmful substances (Storelli, 2008). 63 Shellfish have an excellent ability to filter large MANUSCRIPT amounts of water, and they can 64 accumulate high concentrations of heavy metals (Soto, 1996; Roméo and Gnassia-
65 Barelli, 1995; Claisse, 1992; Fisher, 1983). The capacity of accumulating metals may
66 vary among molluscs depending on their filtering activity and their position in the water
67 column. Oysters can accumulate concentrations of Cd three times higher than mussels,
68 if placed in the same environment (Claisse, 1992). McConchie and Lawrance (1991)
69 also reported the capacity of bottom dwelling oysters to accumulate a quantity of Cd 70 which can beACCEPTED up to 10 times higher than that of oysters grown in the same site but in 71 baskets placed on the surface of the water. Metal concentration in molluscs can be
72 influenced by many physiological factors like individual size, age or breeding (Claisse,
73 1992). Regarding the latter, the levels detected in mussels or oysters at the time of
74 sexual maturity remain the same, although the concentration of metals seems to be
3 ACCEPTED MANUSCRIPT 75 lower due to the increase of the body size. This pattern is called ‘biological dilution’
76 and has been observed in these species for cadmium, copper, lead and zinc (Guéguen et
77 al., 2011).
78 The current European Food Hygiene Legislation makes the control and monitoring of
79 all classified relaying and production areas of bivalve molluscs, gastropods,
80 echinoderms and tunicates (EU Council, 2004b) mandatory, in order to ensure the
81 compliance of the product with microbiological, biotoxicological and chemical
82 parameters and its suitability for human consumption (EU Commission, 2005; EU
83 Council, 2004a; EU Commission, 2006). In order to properly perform the above
84 mentioned monitoring activities, Italy implemented specific guidelines for the
85 Veterinarian Competent Authority and for Food Business Operators (Presidenza del
86 Consiglio dei Ministri, 2010).
87 As reported in a survey on the perception of food-related risks in the EU, European 88 citizens seem to be more concerned about the chemicMANUSCRIPTal contamination of food rather 89 than bacterial contamination itself or health and nutrition issues (Eurobarometer, 2010).
90 The availability to access data on chemical contamination issues is becoming of
91 increasing significance allowing better health impact assessments of food consumption
92 and helping provide the consumer with more reliable and complete information on the
93 health risks that food contamination may cause.
94 Little information is available in literature on the heavy metals level of shellfish 95 detected in theACCEPTED framework of official monitoring programmes in Italy. 96 In this paper, levels of Pb, Hg and Cd found in the period 2007-2012 in edible molluscs
97 and echinoderms from lagoons and marine areas of the Veneto Region in the context of
98 official control activities are presented and discussed together with a preliminary
99 evaluation of the impact on human health of locally produced shellfish.
4 ACCEPTED MANUSCRIPT 100 2. Materials and methods
101 2.1 Sampling
102 Sampling was performed in the framework of the classification and monitoring activity
103 on mollusc production areas by the local competent veterinary authority. Molluscs and
104 echinoderms were collected from the lagoons and coastal areas within 12 nautical miles,
105 attributable to the Veneto Region, North eastern Italy (Fig. 1).
106 Since 2006, lagoons (Venice and the Po River Delta lagoons) and seashore of the
107 sampling area have been divided into production areas defined as “sea”, “estuarine” or
108 “lagoon” areas, containing either natural beds of bivalve molluscs or sites used for the
109 cultivation of bivalve molluscs, and from which live bivalve molluscs are taken (EU
110 Council, 2004a) and classified according to EU Regulation 854/04 (EU Council,
111 2004b). In each area, all the species for which harvesting activities are allowed are
112 sampled at least twice a year to check the level of heavy metals; the period of the year 113 as well as the exact sampling site are established MANUSCRIPT each time by the official veterinarian. 114 Since the overall sampling activity for each species covered all the year, with a slight
115 decrease during the winter months probably due to weather conditions, we can
116 reasonably consider that samples analysed are representative of the average year
117 condition.
118 The environment from which the sampled species were collected (lagoon or sea) and
119 their type of production site (farm or natural banks) are reported in Tables 2, 3 and 4. 120 About 2000 samplesACCEPTED of 17 different species representative of the Veneto Region 121 production, including bivalves, gastropods and echinoderms, were collected between
122 January 2007 and December 2012 in order to check the levels for Pb, Hg and Cd; more
123 specifically, 2038 samples were analysed for all the three metals and 54 samples of
5 ACCEPTED MANUSCRIPT 124 purple dye murex were analysed only for Cd, this being a species particularly at risk for
125 Cd accumulation.
126 The sample size, reported in brackets, is distributed as follow : Manila clam - Venerupis
127 philippinarum (871), Mediterranean mussel – Mytilus galloprovincialis (378 for Pb and
128 Hg; 377 for Cd), striped venus - Chamelea gallina (147), grooved carpet shell -
129 Ruditapes decussatus (109), smooth callista - Callista chione (90), common edible
130 cockle – Cerastoderma edule (71), minor jackknife – Ensis minor (46), variegated
131 scallop - Mimachlamys varia (41), Pacific cupped oyster - Crassostrea gigas (24),
132 European flat oyster - Ostrea edulis (17), rayed trough shell - M. stultorum (13 for Pb
133 and Hg; 14 for Cd), great Mediterranean scallop - Pecten jacobaeus (13), warty venus -
134 Venus verrucosa (9) and truncate donax - Donax trunculus (8), purple dye murex –
135 Bolinus brandaris (109 for Pb and Hg; 163 for Cd), changeable nassa – Nassarius
136 mutabilis (51) and purple sea-urchin - Paracentrotus lividus (41) (see 137 http://www.sealifebase.org consulted on 06 June MANUSCRIPT 2014). 138 In general, the most frequently sampled species were Manila clams (42.7%) and
139 Mediterranean mussels (18.5%), followed by striped venus (7.2%), grooved carpet shell
140 (5.3%) and purple dye murex (4.4%). Other minor species accounted for the remaining
141 21.9 % of samples.
142 The overall yearly distribution of samples was uniform, with a mean of 340 samples per
143 year in the case of Pb and Hg (range: 292-375) and 349 samples for Cd (range: 292 – 144 396). ACCEPTED 145 Each sample was composed of one aliquot of at least 1.5 kg of adult individuals
146 (commercial size) belonging to the same species, collected directly from the sea or
147 lagoon and placed in a sealed plastic bag. Once collected, samples where transported
148 within the day to the laboratory at refrigerated temperature, accompanied by an
6 ACCEPTED MANUSCRIPT 149 identification label and a dedicated sampling form. Once they reached the laboratory,
150 the samples were stored at -20°C pending analysis.
151
152 2.2 Experimental
153 2.2.1 Atomic Absorption Instrumentation
154 For Pb and Cd determination, a Thermo Electron (Waltham, Massachusetts, USA) M6
155 mkII Atomic Absorption Spectrometer with D2 and Zeeman background correction,
156 equipped with a GF95 graphite furnace atomiser and an FS95 autosampler, was used.
157 For Hg determination, a TDA AAS (Thermal Decomposition Amalgamation and
158 Atomic Absorption Spectrophotometry) direct analyser, FKV AMA254 (Altec Ltd,
159 Prague, CZ), was employed.
160 2.2.2 Digestion equipment
161 The digestion of mollusc samples was performed by means of a 1,600 W CEM 162 (Matthews, North Carolina, USA) Mars Xpress MANUSCRIPT Microwave sample preparation system, 163 using CEM Xpress 55 ml PFA digestion vessels.
164 2.2.3 Reagents
165 Ultrapure analytical grade concentrated nitric acid 69% TMA Hiperpur was purchased
166 from Panreac (Barcelona, ES). Ammonium dihydrogen phosphate 99.99% matrix
167 modifier and dihydrogen peroxide solution (30%) were of analytical grade and obtained
168 from Merck (Darmstadt, Germany). Water used for preparing solutions was distilled 169 and deionisedACCEPTED using a Millipore (Milford, MA, USA) MilliQ Water Purification System. -1 . -1 170 Cd (1000 mg L as Cd(NO 3)2 4H 2O in HNO 3 2%), Pb (1000 mg L as Pb(NO 3)2 in
-1 . TM 171 HNO 3 2%) and Hg (1000 mg L as Hg(NO 3)2 H2O in HNO 3 2%) ULTRAGrade
172 stock solutions were purchased from ULTRAScientific (North Kingstown, RI, USA).
173 Calibration standards were prepared daily. Certified Reference Materials BCR185R (for
7 ACCEPTED MANUSCRIPT 174 general quality control purposes), DORM4 and SRM2976 (for specific quality control
175 purposes in fish products analyses) were obtained by the EC Joint Research Centre
176 IRMM (Geel, Belgium), the National Research Council Canada INMS (Ottawa,
177 Ontario, Canada), and the National Institute of Standard and Technology (Gaithersburg,
178 MD, USA), respectively. Certified Reference Materials recovery values are reported in
179 Table 1.
180 2.2.4 Sample preparation and digestion procedure for GFAAS
181 After thawing, the shells of all the individuals included in the sample were opened and
182 the soft tissue (edible part) removed, pooled, homogenised and weighed for analyses.
183 1 g of the homogenised whole mollusc sample was mixed with HNO 3 (8ml) and H 2O2
184 (2 ml) in a PFA digestion vessel, and digested in the microwave oven. After cooling at
185 room temperature, the digests were quantitatively transferred into 25 ml class A
186 volumetric flasks and adjusted to volume with distilled deionised water. 187 2.2.5 Analytical methods MANUSCRIPT 188 Instrumental parameters for GFAAS determination were set as follows: Wavelength,
189 283.3 nm for Pb and 228.8 nm for Cd; Slit, 0.5 nm; Measurement time, 3.0 sec;
190 Background correction, D2; Atomisation temperature, 1,500 °C for Pb and 1,300 °C for
191 Cd .
192 Hg determination: a 0.5 g homogenised whole mollusc sample (edible part) was
193 analysed by means of an automatic solid/liquid mercury analyser at the following 194 analytical conditions:ACCEPTED wavelength 253.6 nm, accumulation time 200 sec, drying time 195 150 sec, decomposition time 45 sec.
196 The employed analytical methods are accredited to ISO/IEC 17025:2005 Standard and
197 in compliance with the Regulation 333/2007/EC (EU Commission 2007), laying down
198 the methods of sampling and analysis for the official control of the levels of Pb, Cd and
8 ACCEPTED MANUSCRIPT 199 Hg in foodstuffs. Detection limits (LOD) on wet weight (3 σ) were: Pb 0.016 mg kg -1,
200 Cd 0.005 mg kg -1, Hg 0.0009 mg kg -1. Limit of quantification (LOQ) on wet weight
201 (6 σ) were: Pb 0.031 mg kg -1; Cd 0.011 mg kg -1; Hg 0.002 mg kg -1. Recoveries were
202 95.3% for Pb, 100.0% for Cd and 99.0% for Hg. RSD r (n=6) were 8.9% for Pb, 7.0%
203 for Cd and 5.7% for Hg. Measurement uncertainty (k=2, p=95%) were: Pb 15% Cd
204 13% Hg 13%. Trueness of analytical data was verified by means of Certified Reference
205 Materials (BCR185R, NRCCDORM3, NISTSRM2976) analysed concurrently with
206 samples in each analytical batch. The mean recovery values found are listed in Table 1.
207
208 2.3 Calculation and statistical methods
209 Data were analysed using the statistical software Stata 12.1 (Copyright 1985-2011
210 StataCorp LP). Mean, standard deviation (sd), minimum and maximum were calculated
211 and expressed in milligrams of element per kg (mg kg -1), wet weight (ww). Values 212 below the limits of quantification (LOQ) were consiMANUSCRIPTdered equal to LOQ (upper bound 213 values). Results obtained in this work were compared with those found by other authors
214 in the same species and expressed in mg kg -1 ww. Values in ww can approximately be
215 converted to dry weight (dw) using a conversion index of 1:5 (Guéguen, 2011;
216 Giarratano, 2010; Yap, 2004).
217 The annual or seasonal trend of heavy metal levels was not considered in this work.
218 The estimation of dietary metal exposure for the most commonly produced and 219 consumed speciesACCEPTED at local level, i.e. Manila clams and Mediterranean mussels, was 220 calculated using the respective mean concentrations found in this work and assuming
221 for each of them a consumption rate equal to that reported for the general Italian
222 population by the former-Italian Research Institute for Food and Nutrition (INRAN),
223 now CRA-NUT, for the food category “bivalves”, which included the species mussels,
9 ACCEPTED MANUSCRIPT 224 clams, oysters and scallops; the mean consumption is reported to be 0.03 g kg -1 bw per
225 day , while the value of the 95 th percentile is 0.19 g kg -1 bw per day (INRAN, 2010),
226 which we assume as being attributable to high consumers.
227 The estimated daily intake for Pb (EDI), expressed in g kg -1 bw per day, was
228 compared to the available BMDL 10, i.e. the lower limit of the confidence interval of the
229 Benchmark Dose associated with a 10% incidence of toxic effects from lead for
230 developmental neurotoxicity (0.50 µg kg -1 bw per day), effects on prevalence of chronic
231 kidney disease (0.63 µg kg -1 bw per day) and effects on systolic blood pressure (1.50 µg
232 kg -1 bw per day) respectively, proposed by EFSA (EFSA, 2010). The estimated weekly
233 intakes (EWI) for MeHg and Cd expressed in g kg -1 per week were compared to the
234 available tolerable weekly intake (TWI) for methylmercury (MeHg) and Cd,
235 corresponding respectively to 1.3 µg kg -1 bw per week (EFSA, 2012a) and to 2.5 µg kg -
236 1 bw per week (EFSA, 2011). To convert the values of total mercury found in this paper MANUSCRIPT 237 to MeHg, the conversion factor of 0.8, proposed for molluscs, was applied (EFSA, 238 2012a).
239
240 3. Results and discussion
241 3.1 Lead
242 The mean concentrations of Pb detected in the 17 mollusc species are reported in Table
243 2. All mean values observed in each species were well below the 1.5 mg kg -1 limit set 244 by European ACCEPTEDlegislation (EU Commission, 2006) and none of the samples analysed 245 exceeded the threshold. Among bivalves, the highest mean value was found in rayed
246 trough shell and truncate donax (0.25 mg kg -1), while the lowest in striped venus and
247 minor jackknife (0.10 mg kg -1). The absolute maximum value (1.18 mg kg -1) was
248 detected in Manila clam and in smooth callista; the detection of such high values
10 ACCEPTED MANUSCRIPT 249 represented an occasional event, as evidenced by the p75 values (0.26 mg kg -1 and 0.22
250 mg kg -1 respectively)
251 Marine gastropods and echinoderms showed the highest percentages of values below
252 the LOQ (9% for purple dye murex and 15% for changeable nassa) and lower mean
253 values than those found in bivalves; in particular, changeable nassa had the lowest one
254 among all the considered species (0.09 mg kg -1).
255 Table 5 reports results of monitoring activities conducted by other authors, showing
256 data comparable to those found in this study even if the sample sizes, in some cases,
257 were significantly smaller than the one considered in this work.
258 Pb levels similar to those found in the present study are reported for bivalves species
259 collected in Italy along the northern and central Adriatic coast (Majori et al., 1991b;
260 Cubadda et al. 2006; Desideri et al. 2010), with the exception of higher concentrations
261 found in Mediterranean mussels by Majori et al. in 1991 in the Gulf of Trieste. Values 262 found in the Venice Lagoon in 2006 (Cubadda MANUSCRIPTet al. 2006) are very close to those found 263 in this work, while those registered in 1991 (Majori et al., 1991b) were a little higher.
264 This could indicate a decreasing temporal trend in Pb concentration, or may be due to
265 the fact that in 1991 many samples were collected in highly polluted areas near Venice
266 and Porto Marghera, which are nowadays banned to shellfish harvesting activities; on
267 the other hand, the study of 2006 included only samples collected in areas specifically
268 designated to mollusc harvesting activities, as most of the samples in the present work. 269 Species sampledACCEPTED along the Tyrrhenian coast generally showed slightly higher values 270 (Papetti and Rossi, 2009).
271 Even Pb levels found in bivalves in other countries are essentially comparable to the
272 ones we found (Guéguen et al., 2011; Usero et al., 1997; Arik Colakoglu et al., 2011;
273 Gavrilovic et al., 2001), excepting little higher values in mussels collected along the
11 ACCEPTED MANUSCRIPT 274 French Mediterranean coast (Guéguen et al., 2011) and in some areas of the south
275 Atlantic coast of Spain (Usero et al, 1997). Significantly higher mean values were
276 registered in striped venus from one of the five sampling sites of the south coast of the
277 Marmara Sea (Turkey), probably due to the presence of many industrialized areas and
278 to the contributions of different rivers (Arik Colakoglu et al., 2011). Quite lower values
279 have been reported for Pacific cupped oyster sampled from the French market (Guéguen
280 et al., 2011) and for purple sea-urchin collected in France (Noël et al., 2011).
281 Values similar to the mean concentration found in this work were reported for purple
282 dye murex, both in the products collected in Italy (Mar Ligure) (Giorgi et al., 2009) and
283 in France (Noël et al., 2011).
284
285 3.2 Mercury
286 As shown in Table 3, all samples analysed for Hg showed concentrations below the 287 European legislation limit of 0.5 mg kg -1 (EU Commission,MANUSCRIPT 2006). The mean Hg values 288 ranged from 0.01 mg kg -1 in great Mediterranean scallop, the species with the highest
289 percentage (23%) of values lower than LOQ, to 0.1 mg kg -1 in purple dye murex, for
290 which the highest value (0.31 mg kg -1) was detected.
291 Table 5 reports comparable mean values detected by other authors in some of the
292 species considered in this work.
293 Generally, similar values to those found in this work are reported for bivalves and 294 gastropods collectedACCEPTED along the Italian coast (Majori et al., 1991a; Ghidini et al., 2001; 295 Cubadda et al. 2006; Giorgi et al., 2009), with the exception of Mediterranean mussel
296 sampled in the Grado and Marano Lagoon (Majori et al., 1991a); in 2003 this area had
297 been declared a polluted site of national interest – SIN – because of the widespread Hg
298 contamination. As for Pb, Hg values registered in the Venice Lagoon in 2006 (Cubadda
12 ACCEPTED MANUSCRIPT 299 et al. 2006) were similar to ours, while those of 1991 (Majori et al., 1991 a) were
300 slightly higher; the considerations reported in chapter 3.1 are valid also in this case.
301 Even recently published national data on Hg content in several seafood species referred
302 to period 2009-2011 (Brambilla et al., 2013), are essentially similar to those detected in
303 this work for all the considered species (data not reported in Table 5).
304 Differently to Pb findings, Hg values registered on the Tyrrhenian coast (Papetti and
305 Rossi, 2009) tend to be slightly lower than those found on the Adriatic side.
306 Concerning Hg levels found in other countries in bivalves, gastropod and echinoderms
307 (Usero et al., 1997; Guéguen et al., 2011; Noël et al., 2011), they are essentially
308 comparable to ours, except for the higher values detected in Manila clams from some
309 areas of the south Atlantic coast of Spain (Usero et al., 1997) and for the lower
310 concentrations detected in Pacific cupped oyster (Guéguen et al., 2011) and murex
311 collected in France (Noël et al., 2011). 312 MANUSCRIPT 313 3.3 Cadmium
314 As shown in Table 4, concentrations of Cd above the European legislation limit of 1 mg
315 kg -1 (EU Commission, 2006) were found in 45 of the 2,092 samples analysed (2.2%);
316 86.7% of them were purple dye murexes and 7% European flat oysters, while in other
317 species (variegated scallop and stony sea-urchin) values exceeding the threshold were
318 detected occasionally. The lowest mean value of 0.02 mg kg -1 was found in rayed trough
-1 319 shell, while theACCEPTED highest one in murex (0.88 mg kg ); in the latter the highest value 320 among all considered species (5.12 mg kg -1) was also detected and 24% of the samples
321 analysed showed concentrations of Cd above the European legislation limit.
13 ACCEPTED MANUSCRIPT 322 Also in European flat oyster the calculated mean value was close to the European
323 legislation limit; however, the number of samples analysed was very low, reflecting the
324 negligible importance of its production at a local level.
325 Several comparable values for Cd concentration available in the literature are reported
326 in Table 5.
327 Values found in bivalves by other authors along the Adriatic coast are similar (Cubadda
328 et al., 2006; Gavrilovich et al., 2007; Desideri et al., 2010; Guéguen et al., 2011) or
329 slightly higher (Majori et al.,1991b; Ghidini et al., 2001) than those reported in this
330 paper; however, Hg concentrations found along the Tyrrhenian coast turned out to be
331 quite lower (Papetti and Rossi, 2009).
332 As for the Cd values reported by other countries, concentrations detected in France in
333 Mediterranean mussel and Pacific cupped oyster are similar to ours (Guéguen et al.,
334 2011). Significantly higher mean values were registered in striped Venus in some 335 sampling stations of the south coast of Spain (User MANUSCRIPTo et al., 1997) and in Turkey (Arik 336 Colakoglu et al., 2011), probably due to the presence of industrial or mining activities
337 and rivers discharge nearby the sampling point.
338 Much higher mean values of Cd, exceeding 2 mg kg -1, were reported for Pacific cupped
339 oyster in Canada (Schallie, 200; Kruzynski, 2001; Lekhi et al., 2008). Such high levels
340 of Cd were primarily due to natural oceanographic conditions (Cd concentrations in the
341 north Pacific deep waters are elevated and cadmium-rich water is brought to the surface 342 during intensiveACCEPTED upwelling events) and to the contribution of watershed and coastal 343 geology (Kruzynski, 2001; Crispo, 2001).
344 Concerning murex, concentrations reported by other Italian authors are slightly lower
345 (Ghidini et al., 2001; Giorgi et al., 2009) while the mean value found in the French
346 product is very similar (Noël et al., 2011). Many papers reported the capacity of marine
14 ACCEPTED MANUSCRIPT 347 gastropods to accumulate high levels of Cd (Roméo et al., 2006; Dallinger et al., 1989;
348 Bouquegneau et al., 1988; Bouquegneau and Martoja, 1987), which could be an
349 essential element for these species and may be involved in the process of shell
350 formation (Bouquegneau and Martoja, 1987). Higher Cd concentrations reported in this
351 study for this species were particularly detected along the south coast of the Veneto
352 region (sea areas in front of the Po River Delta). For this reason, from the end of 2011,
353 purple dye murex harvesting has been progressively banned from production areas of
354 this coastal zone and the restriction is still in force.
355
356 3.4 Estimation of metal intake and health threat related to mollusc consumption
357 Table 6 extensively reports the estimated daily intake (EDI) for Pb and the estimated
358 weekly intake (EWI) for MeHg and Cd, expressed respectively in g kg -1 per day and
359 per week, as well as the relative percentage contribution to BMDL 10 (%BMDL 10 ) or 360 TWI (%TWI) for average and high consumers ofMANUSCRIPT locally produced Mediterranean 361 mussels or Manila clams.
362 Several Italian studies had already assessed the intake of heavy metals through seafood
363 products (Storelli and Marcotrigiano, 2001; Marcotrigiano and Storelli, 2003; Cirillo et
364 al., 2010; Parstorelli et al., 2012) or had focused on the Hg and MeHg intake at a
365 national (Brambilla et al., 2013, Dellatte et al., 2014) or local level (Di Leo et al., 2010,
366 Spada et al., 2012), but a comparison with the values reported in this work can be 367 misleading consideringACCEPTED that different consumption data and conversion indexes for the 368 estimation of the fraction of MeHg/Hg have been used.
369 Taking into account the maximum percentage contribution to the TWI of MeHg (3.3 %
370 for high consumers of Manila clams) and considering fish and shellfish as being the
371 major contributors to MeHg exposure from the diet (EFSA, 2012a), the consumption of
15 ACCEPTED MANUSCRIPT 372 these locally produced species can be considered essentially safe. Moreover, two recent
373 Italian studies on Hg and MeHg intake from seafood confirm that the contribution to
374 MeHg intake by bivalves is low (Brambilla et al. 2013; Dellatte et al., 2014).
375 As for Pb, the maximum percentage contribution to BMDL 10 s found in the present work
376 for high consumers of mussels was 8.4 % for developmental neurotoxicity, 6.6% for the
377 effects on prevalence of chronic kidney disease and 2.8% for the effects on systolic
378 blood pressure; we can therefore state that the average consumer has a low possibility of
379 suffering from any toxic effects through the consumption of these locally produced
380 species. On the other hand, EFSA has expressed its concern on the neurodevelopment
381 effects that current exposure levels may have on European infants, children and
382 pregnant women and has established that widely consumed products such as cereals,
383 cereal products, vegetables and tap water are the major contributors to Pb exposure
384 (EFSA, 2010). Therefore, considering the high levels of Pb that characterize bivalves 385 compared to other food items, we can state that MANUSCRIPT in these vulnerable groups their 386 consumption should be discouraged.
387 The maximum calculated percentage contribution to Cd TWI is of 6.9% for high
388 consumers of locally produced mussels. According to EFSA, the margin between the
389 average weekly intake of Cd from food by the general population and the health-based
390 guidance values is narrow. Considering that in European adults the mean fraction of the
391 total Cd dietary intake represented by shellfish consumption is estimated to be 3.9% 392 (EFSA, 2012b),ACCEPTED a health risk for high consumers of mussels cannot be ruled out; 393 otherwise, there seems to be no realistic threat to mean consumers of both considered
394 species.
395 According to Italian statistics, only 9.4% of the general population consumes molluscs
396 (INRAN, 2010), but there are considerable differences in daily diets which vary
16 ACCEPTED MANUSCRIPT 397 according to the geographical and cultural context. For example, the mean consumption
398 of Manila clams in the Venice Lagoon area is estimated to be of 30 g day -1 per person
399 (Boscolo et al., 2006), which is more than double the amount eaten by the average
400 Italian consumer; moreover, in this same area the consumption of species such as purple
401 dye murex can be high, since this is considered a delicacy. In such an instance, health
402 risks should be considered as being concrete.
403 In this framework, more detailed investigations on the eating habits at a local level,
404 especially regarding minor species as the ones described in this paper, are necessary to
405 properly asses the health impact on humans deriving from the consumption of molluscs.
406
407 4. Conclusions
408 The results of monitoring activities performed between 2007 and 2012 on edible marine
409 molluscs collected in the north-western part of the Adriatic Sea (Veneto Region) show 410 that concentrations of Pb, Hg and Cd are generally MANUSCRIPT well under the thresholds set by 411 European legislation. Some differences were found in purple dye murex, where Cd
412 values exceeding 1 mg kg -1 were quite frequently detected.
413 Considering the estimated percentage contribution made by the consumption of locally
414 produced species like Manila clam and Mediterranean mussel to the existing health-
415 based guidance values set by EFSA for metals taken into account in our study, we can
416 conclude that there appears to be no health threat to average consumers. Nevertheless, it 417 should not beACCEPTED dismissed that high-level consumers of shellfish, such as those from 418 coastal areas, or vulnerable groups (women of childbearing age, pregnant or children)
419 might exceed the recommended intakes especially for Pb and Cd.
420 The issue of Cd content in purple dye murex will be further evaluated with dedicated
421 monitoring plans and more detailed investigations of consumption habits, especially at a
17 ACCEPTED MANUSCRIPT 422 local level. Moreover, a proper risk assessment should be performed in order to provide
423 consumers with clear information on the concerns related to the consumption of this
424 product.
MANUSCRIPT
ACCEPTED
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618
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Table 1
CRM recovery values
Pb Cd Hg CRM Certified value Mean found value Certified value Mean found value Certified value Mean found value (mg kg -1) (mg kg -1) (mg kg -1) (mg kg -1) (mg kg -1) (mg kg -1) BCR185R 0.172 ± 0.009 0.164 ± 0.014 0.544 ± 0.017 0.543 ± 0.015 - - NRCCDORM4 - - - - 0.410 ± 0.055 0.403 ± 0.010 NISTSRM2976 1.19 ± 0.18 1.15 ± 0.16 0.82 ± 0.16 0.84 ± 0.10 0.061 ± 0.004 0.059 ± 0.003
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Table 2
Lead levels (mg kg -1 ww) Production Species Environment n %a n > 1.5 mg kg -1 mean ± sd min max techniques Bivalves Manila clam ( V. philippinarum ) L C 871 42.7 - 0.20 ± 0.11 0.03 1.18 Mediterranean mussel ( M. galloprovincialis ) L/S C 378 18.5 - 0.22 ± 0.12 0.03 0.80 Striped Venus ( C. gallina ) S N 147 7.2 - 0.10 ± 0.06 0.03 0.47 Grooved carpet shell ( R. decussatus ) L C 109 5.3 - 0.20 ± 0.09 0.05 0.53 Smooth callista ( C. chione ) S N 90 4.4 - 0.17 ± 0.14 0.03 1.18 Common edible cockle (C. edule) L/S N 71 3.5 - 0.21 ± 0.11 0.03 0.55 Minor jakknife (E. minor) S N 46 2.3 - 0.10 ± 0.06 0.03 0.25 Variegated scallop ( M. varia ) S N 41 2.0 - 0.21 ± 0.15 0.03 0.59 Pacific cupped oyster ( C. gigas ) S C 24 1.2 - 0.20 ± 0.12 0.05 0.47 European flat oyster ( O. edulis ) L C 17 0.8 - 0.12 ± 0.08 0.04 0.33 Great Mediterranean scallop ( Pecten jacobaeus ) S N 13 0.6 - 0.19 ± 0.11 0.06 0.40 Rayed trough shell ( M. stultorum ) S N 13 0.6 - 0.25 ± 0.12 0.03 0.48 Warty venus ( V. verrucosa ) S N MANUSCRIPT 9 0.4 - 0.14 ± 0.05 0.08 0.21 Truncate donax ( D. trunculus ) S N 8 0.4 - 0.25 ± 0.08 0.13 0.35 Marine Gastropods Purple dye murex (B. brandaris) S N 109 5.3 - 0.12 ± 0.10 0.03 0.63 Changeable Nassa (N. mutabilis) S N 51 2.5 - 0.09 ± 0.08 0.03 0.46 Echinoderms Purple sea-urchin (P. lividus) L/S N 41 2.0 - 0.16 ± 0.09 0.03 0.50 Total 2038 100.0 0 L=lagoon; S=sea; C=cultured; N=natural banks a The percentage is calculated as the ratio between the number of samples tested for each species and the total number of samples.
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Table 3
Mercury levels (mg kg -1ww) Production Species Environment n % a n > 0.5 mg kg -1 mean ± sd min max techniques Bivalves Manila clam ( V. philippinarum ) L C 871 42.7 - 0.04 ± 0.03 0.002 0.19 Mediterranean mussel ( M. galloprovincialis ) L/S C 378 18.5 - 0.03 ± 0.02 0.002 0.18 Striped Venus ( C. gallina ) S N 147 7.2 - 0.03 ± 0.03 0.002 0.25 Grooved carpet shell ( R. decussatus ) L C 109 5.3 - 0.07 ± 0.03 0.020 0.19 Smooth callista ( C. chione ) S N 90 4.4 - 0.03 ± 0.01 0.002 0.06 Common edible cockle (C. edule) L/S N 71 3.5 - 0.06 ± 0.05 0.002 0.23 Minor jakknife (E. minor) S N 46 2.3 - 0.06 ± 0.03 0.010 0.13 Variegated scallop ( M. varia ) S N 41 2.0 - 0.02 ± 0.02 0.002 0.12 Pacific cupped oyster ( C. gigas ) S C 24 1.2 - 0.06 ± 0.03 0.002 0.12 European flat oyster ( O. edulis ) L C 17 0.8 - 0.04 ± 0.01 0.030 0.06 Great Mediterranean scallop ( Pecten jacobaeus ) S N 13 0.6 - 0.01 ± 0.01 0.002 0.03 Rayed trough shell ( M. stultorum ) S N 13 0.6 - 0.03 ± 0.01 0.010 0.06 Warty venus ( V. verrucosa ) S N 9 0.4 - 0.06 ± 0.01 0.040 0.08 Truncate donax ( D. trunculus ) S N MANUSCRIPT 8 0.4 - 0.03 ± 0.03 0.002 0.08 Marine Gastropods Purple dye murex (B. brandaris) S N 109 5.3 - 0.10 ± 0.06 0.002 0.31 Changeable Nassa (N. mutabilis) S N 51 2.5 - 0.09 ± 0.05 0.020 0.23 Echinoderms Purple sea-urchin (P. lividus) L/S N 41 2.0 - 0.04 ± 0.02 0.002 0.10 Total 2038 100.0 0 L=lagoon; S=sea; C=cultured; N=natural banks a The percentage is calculated as the ratio between the number of samples tested for each species and the total number of samples.
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Table 4
Cadmium levels (mg kg -1 ww) Production Species Environment techniques n % a n > 1.0 mg kg -1 mean ± sd min max Bivalves Manila clam ( V. philippinarum ) L C 871 41.6 - 0.07 ± 0.05 0.01 0.34 Mediterranean mussel ( M. galloprovincialis ) L/S C 377 18.0 - 0.13 ± 0.06 0.02 0.58 Striped Venus ( C. gallina ) S N 147 7.0 - 0.06 ± 0.05 0.01 0.49 Grooved carpet shell ( R. decussatus ) L C 109 5.2 - 0.08 ± 0.05 0.01 0.25 Smooth callista ( C. chione ) S N 90 4.3 - 0.05 ± 0.05 0.01 0.52 Common edible cockle (C. edule) L/S N 71 3.4 - 0.03 ± 0.03 0.01 0.18 Minor jakknife (E. minor) S N 46 2.2 - 0.04 ± 0.02 0.01 0.08 Variegated scallop ( M. varia ) S N 41 2.0 2 0.26 ± 0.31 0.05 1.40 Pacific cupped oyster ( C. gigas ) S C 24 1.2 - 0.29 ± 0.25 0.06 1.00 European flat oyster ( O. edulis ) L C 17 0.8 3 0.78 ± 0.36 0.11 1.37 Great Mediterranean scallop ( Pecten jacobaeus ) S N 14 0.7 - 0.02 ± 0.01 0.01 0.03 Rayed trough shell ( M. stultorum ) S N 13 0.6 - 0.21 ± 0.22 0.07 0.87 Warty venus ( V. verrucosa ) S N MANUSCRIPT 9 0.4 - 0.16 ± 0.07 0.09 0.29 Truncate donax (D. trunculus ) S N 8 0.4 - 0.03 ± 0.03 0.01 0.09 Marine Gastropods Purple dye murex (B. brandaris) S N 163 7.8 39 0.88 ± 1.02 0.01 5.12 Changeable Nassa (N. mutabilis) S N 51 2.4 - 0.11 ± 0.06 0.03 0.33 Echinoderms Purple sea-urchin (P. lividus) L/S N 41 2.0 1 0.11 ± 0.35 0.01 2.26 Total 2092 100.0 45 L=lagoon; S=sea; C=cultured; N=natural banks a The percentage is calculated as the ratio between the number of samples tested for each species and the total number of samples.
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Table 5
Lead, Mercury and Cadmium levels (mg kg -1 ww) reported by other authors in some species of molluscs and echinoderms investigated in this paper. Species Sampling area n Pb (mg kg -1) Hg (mg kg -1) Cd (mg kg -1) Reference Mediterranean Italy, northern Adriatic coast Mussel Venice Lagoon 30 0.15− 0.22 a 0.02− 0.05 a 0.13 − 0.15 a Cubadda et al. 2006 Italy, northern Adriatic coast: Trieste gulf 22 (44 for Hg) 1.17 (mean) 0.03 ± 0.01 0.23 (mean) Majori et al., 1991a-b Grado and Marano Lagoon 36 (72 for Hg) 0.17 (mean) 0.22 ± 0.06 0.22 (mean) Venice Lagoon 36 (72 for Hg) 0.32 (mean) 0.10 ± 0.05 0.31 (mean) Italy, northern Adriatic coast 3 0.03 ± 0.03 0.17 ± 0.02 Ghidini et al. 2001 3 0.03 ± 0.02 0.23 ± 0.04 Italy, central Adriatic coast 4 0.21 ± 0.10 0.14 ± 0.001 Desideri et al. 2010 Italy, Tyrrhenian coast 5 0.35 ± 0.03 0.01 ± 0.01 0.02 ± 0.001 Papetti and Rossi 2009 France, market 6 0.14 −0.26 0.003 − 0.02 0.06 −0.18 Guéguen et al. 2011 France, Atlantic coast 84 0.25 − 0.31 a 0.14 − 0.18 a France, Mediterranean coast 48 0.49 ± 0.26 0.04 ± 0.02 0.23 ± 0.09 France, shellfish farming areas 374 0.03 ± 0.02 0.15 ± 0.09 Manila clam Italy, northern Adriatic coast 2 0.05 ± 0.01 0.09 ± 0.003 Ghidini et al. 2001 Italy, northern Adriatic coast 4 0.02 ± 0.002 0.13 ± 0.02 2 0.03 ± 0.004 0.15 ± 0.03 Spain, southern Atlantic coast 4 in 7 locations 0.07 ± 0.02 − 0.33 ± 0.13 a 0.02 ± 0.01 − 0.39 ± 0.10 a 0.08 ± 0.02 − 0.46 ± 0.05 a Usero et al. 1997 Grooved carpet Italy, central Adriatic coast 3 0.21 ± 0.05 MANUSCRIPT 0.08 ± 0.01 Desideri et al. 2010 shell Italy, Tyrrhenian coast 5 0.44 ± 0.09 5 ACCEPTED MANUSCRIPT Table 6 Percentage contribution to Pb BMDL10 (%BMDL10 ), MeHg and Cd TWI (%TWI) in relation to mean or high consumption of Mediterranean mussels and Manila clams. Pb MeHg Cd Per-capita Per-capita daily weekly EDI a BMDL b EWI a TWI EWI a TWI Species 10 %BMDL %TWI %TWI consumption consumption (µg kg -1 bw) (µg kg -1 bw) 10 (µg kg -1 bw) (µg kg -1 bw) (µg kg -1 bw) (µg kg -1 bw) (g kg -1 bw) (g kg -1 bw) Mediterranean 0.03 0.21 0.007 NT 0.5 1.3 0.005 1.3 0.4 0.027 2.5 1.1 mussel KD 0.63 1.0 BSP 1.5 0.4 0.19 1.33 0.042 NT 0.5 8,4 0.032 1.3 2.5 0.173 2.5 6.9 KD 0.63 6.6 BSP 1.5 2.8 Manila clam 0.03 0.21 0.006 NT 0.5 1.2 0.007 1.3 0.5 0.015 2.5 0.6 KD 0.63 1.0 BSP 1.5 0.4 MANUSCRIPT 0.19 1.33 0.038 NT 0.5 7.6 0.043 1.3 3.3 0.093 2.5 3.7 KD 0.63 6.0 BSP 1.5 2.5 EDI=Estimated daily intake; EWI=Estimated weekly intake a EDI and EWIs were calculated using the mean concentration of metals found in this work for Manila clam (Pb: 0.20 mg kg -1; Hg: 0.04 mg kg -1; Cd: 0.07 mg kg - 1) and Mediterranean mussel (Pb: 0.22 mg kg -1; Hg: 0.03 mg kg -1; Cd: 0.13 mg kg -1).To convert the mean values of total mercury to MeHg, the conversion factor of 0.8 was applied b NT= BMDL10 for developmental neurotoxicity; KD=BMDL10 for effects on chronic kidney disease; BSP=BMDL10 for effects on systolic blood pressure ACCEPTED 6 ACCEPTED MANUSCRIPT MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT Highlights • Pb, Hg and Cd levels were tested in 17 molluscs species between 2007-2012 • Hg and Pb concentrations proved to be within the levels set by EU legislation • 2.2% of samples had Cd concentrations exceeding the EU legislation threshold value • 86.7 % of the contaminated samples exceeding the EU limit for Cd were purple dye murex • Most consumed species showed concentrations ten times lower than legislation limit MANUSCRIPT ACCEPTED