Multivariate data analysis of trace elements in bivalve molluscs: Characterization and food safety evaluation

Accepted Manuscript Multivariate data analysis of trace elements in bivalve molluscs: characterization and food safety evaluation Isa dos S. Barbosa, Geysa B. Brito, Gabriel L. dos Santos, Luana N. Santos, Leonardo S.G. Teixeira, Rennan G.O. Araujo, Maria Graças A. Korn PII: DOI: Reference:

S0308-8146(18)30299-1 https://doi.org/10.1016/j.foodchem.2018.02.063 FOCH 22447

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

11 August 2017 9 February 2018 12 February 2018

Please cite this article as: Barbosa, I.d.S., Brito, G.B., dos Santos, G.L., Santos, L.N., Teixeira, L.S.G., Araujo, R.G.O., Korn, M.G.A., Multivariate data analysis of trace elements in bivalve molluscs: characterization and food safety evaluation, Food Chemistry (2018), doi: https://doi.org/10.1016/j.foodchem.2018.02.063

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Multivariate data analysis of trace elements in bivalve molluscs: characterization and food safety evaluation

Isa dos S. Barbosaa, Geysa B. Britoa, Gabriel L. dos Santosa, Luana N. Santosa,b, Leonardo S. G. Teixeiraa, Rennan G. O. Araujoa, Maria Graças A. Korna*

a

Instituto de Química, Universidade Federal da Bahia, Campus Universitário de

Ondina, Salvador, 40170-280, Bahia, Brazil. b

Departamento de Ciências Exatas e Tecnológicas, Universidade Estadual de Santa

Cruz, Ilhéus, 45662-900, Bahia, Brazil.

*Corresponding author: Tel/Fax: +55 71 3283 6830 E-mail address: [email protected]

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Multivariate data analysis of trace elements in bivalve molluscs: characterization and food safety evaluation

Abstract Four species of bivalve molluscs (Anomalocardia brasiliana, Iphigenia brasiliana, Lucina pectinata and Trachycardium muricatum) were collected in the Todos os Santos Bay (TSB), Bahia, Brazil, in order to evaluate As, Cd, Co, Cu, Cr, Fe, Mn, Ni, Pb, Se, V and Zn levels and, consequently, the risk of bivalve mollusc consumption in humans. The samples were analyzed by inductively coupled plasma optical emission spectrometry (ICP OES) and inductively coupled plasma mass spectrometry (ICP-MS) after closed-vessel microwave digestion. The accuracy was confirmed using the certified reference materials of oyster tissue (NIST 1566b) and mussel tissue (NIST 2977), and the results were statistically equivalent to the certified values. Application of principal component analysis (PCA) and hierarchical cluster analysis (HCA) showed a tendency to form two groups between samples of Lucina pectinata and Trachycardium muricatum. All species showed As and Cr concentrations higher than the maximum tolerable limit specified in Brazilian legislation.

Keywords: Bivalve molluscs; Trace elements; Microwave-assisted digestion; Principal component analysis; Hierarchical cluster analysis

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1. Introduction The bioaccumulation of essential and toxic trace elements in marine animals of natural or anthropogenic origin is a food safety issue. Trophic transfer has increasingly been recognized as an important pathway for trace element accumulation in marine invertebrates. Marine organisms, especially gastropods, oysters, sponges, sea cucumbers and bivalves, have the ability to assimilate metals from their living environment (Roméo et al., 2005; Cervantes, Jiménez-Cárceles, & Álvarez-Rogel, 2009). Bivalve molluscs, which include mussels, oysters and clams, have high nutritional value. Together with their culinary value, these molluscs are regarded as a good source for proteins, lipids, carbohydrates and minerals, and have recently been studied for their beneficial effects on human health (Fernández et al., 2015; Rittenschober, Nowak, & Charrondiere, 2013). However, given their relative position in the food chain, the health risk to humans from high exposure to trace elements posed by consumption of bivalve molluscs is important, particularly in coastal areas (Hatje & Andrade, 2009; Li & Gao, 2014; Besada, Andrade, Schultze, & Gonzalez, 2011; Karnjanapratum, Benjakul, Kishimura, & Tsai, 2013; Rittenschober, Nowak, & Charrondiere, 2013; Liu, Cao, & Dou, 2017). Although many metals are essential biological elements, they are also potentially toxic to organisms above a certain concentration threshold. Thus, the concentration of trace elements in aquatic organisms and the potential risk of human consumption of these marine organisms has been the subject of several studies (Alves et al., 2017; Araújo et al., 2016; Costa, Korn, Brito, Ferlin, & Fostier, 2016; Conte et al., 2015; Santos et al., 2013a; Souza, Windmöller, & Hatje, 2011). In Brazil, bivalve molluscs are widely consumed in several regions, mainly in the Northeast, where several species are part of the typical regional cuisine. Therefore, characterizing these 3

molluscs and performing a food safety evaluation is important (Costa, Korn, Brito, Ferlin, & Fostier, 2016; Souza, Windmöller, & Hatje, 2011; Santos, Trigueiro, Lemos, Furtunato, & Cardoso, 2013b; Araújo et al., 2016). Advances in instrumentation have led to significant improvements in their accuracy, precision, limits of detection and processing of samples, thereby expanding the capacity for analysis of different foods and applications. Furthermore, the use of multivariate data analyses such as principal component analysis (PCA) and hierarchical cluster analysis (HCA) can assist by facilitating additional interpretation of results. Both PCA and HCA are pattern recognition techniques that are commonly employed in the evaluation of food data, allowing the establishment of similarities and trends in data sets (Canizo, Escudero, Pérez, Pellerano, & Wuilloud, 2018; Rodríguez-Bermúdez, LópezAlonso, Miranda, Fouz, Orjales, Herrero-Latorre, 2018; Chung et al., 2018; Dos Santos et al., 2017; Karabagias, Louppis, Karabournioti, Kontakos, Papastephanou, & Kontominas, 2017; Moncayo, Manzoor, Rosales, Anzano, & Caceres, 2017; Ferreira et al., 2017; Ferreira, 2015; Panero, Vieira, Cruz, & Moura, 2009;). In this study, the concentrations of As, Cd, Co, Cu, Cr, Fe, Mn, Ni, Pb, Se, V and Zn were evaluated in different bivalve mollusc species consumed in Todos os Santos Bay (TSB), Bahia, Brazil. To the best of our knowledge, the trace element concentrations in Lucina Pectinata, Iphigenia brasiliana and Trachycardium muricatum species have not yet been described. Principal component analysis and HCA were applied for multivariate data analysis of the trace element concentrations of the shellfish samples for characterization and food safety evaluation.

2. Experimental 4

2.1 Sample collection and preservation Four bivalve mollusc species (Anomalocardia brasiliana, Iphigenia brasiliana, Lucina pectinata and Trachycardium muricatum) were collected at seven sites along the coast of Todos os Santos Bay (TSB): L1, Acupe/Itapema; L2, Bom Jesus dos Pobres; L3, Maré Island; L4, Madre de Deus/Suape; L5, Mutá; L6, Salinas da Margarida; and L7, Tainheiros beach. Two samplings were performed, the first in September and October 2010 (dry period, P1), during which 37 samples were collected, and the second in April and May of 2011 (rainy season, P2), with 29 samples collected. It should be noted that the analyses were performed using composite samples because bivalve molluscs are small animals. For this, a certain quantity of bivalves was required to compose a sample. The bivalve molluscs were collected during low tide when the mollusc population was not covered by the tide. Afterwards, the organisms were cleaned with a brush to remove any residue and algae attached to the shells, then they were packed into boxes with ice and transported. When in the laboratory, the external surface of the bivalve molluscs was washed with distilled water while the shells were opened with the aid of a scalpel, and the liquid inside the shells was discarded. The soft tissues were removed from the shells, placed into glass containers that had been decontaminated, then stored in a freezer at -20 ºC. The biological tissues were freeze-dried (Alfa 1-4 LD Plus; Martin Christ, Germany). Dried samples were ground using a ball mill with a tungsten carbide vial set (Model 8000M; Spex Sample Prep, USA) and stored in clean polyethylene vials inside a desiccator at room temperature.

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2.2 Reagents and standards All closed vessels, polyethylene flasks and plastic containers were cleaned with 10% (v v-1) HNO3 for 24 h then rinsed with deionized water. Subsequently, all material was dried at 50 °C and stored under clean-air conditions. The plastic containers, polyethylene flasks, pipette tips, perfluoroalcoxy polymer (PFA) Teflon digestion vessels (Milestone SRL, Sorisole, Italy) and reagents that came into contact with the samples or standards were checked for contamination. All solvents and reagents were of the highest commercially available purity grade Deionized water with a resistivity of ≥18 MΩ cm was obtained using a Milli-Q Plus pure water generating system from Millipore (Molsheim, France) and was employed to prepare all standard and sample solutions. Analytical grade nitric acid (Merck, Darmstadt, Germany) was double-distilled using a model duoPUR 2.01E subboiling system (Milestone, Bergamo, Italy). Monoelemental, high-purity grade stock solutions (1 g L-1) of As, Cd, Co, Cu, Cr, Fe, Mn, Ni, Pb, Se, V and Zn and a multielemental solution (100 mg L-1) of Bi, Ge, In, Tl, Rh and Sc were purchased from Merck (Darmstadt, Germany).

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2.3 Instrumental analysis A quadrupole ICP-MS (model Xseries II, Thermo, Germany) equipped with a hexapole collision cell tecnology (CCT) was used for As, Cd, Co, Cu, Cr, Fe, Mn, Ni, Pb, Se, V and Zn determinations, and the results were expressed as micrograms per gram of dry mass. Internal standards (Ge, Rh, Sc and Tl) were added to compensate for any acid effects and instrument drift. The instrument software allows rapid switching between the standard mode (no gas, cell vented to mass analyzer chamber) and the CCT mode while continuously aspirating the sample. The premixed gases H2 (7%) in He (H2O and other impurities <5 ppm) were admitted into the CCT under flow control through stainless steel lines. The measurements were made with a nickel sampler and skimmer cones (1.0 and 0.7 mm diameter orifices) and a standard concentric nebulizer. A glass impact bead spray chamber cooled to 4°C by a Peltier cooler and a shielded Fassel torch was used to minimize the plasma potential, thereby obtaining a low and narrow initial ion energy distribution. The following isotopes were chosen for each element: 75As, 111Cd, 59Co, 63

Cu, 52Cr, 55Mn, 60Ni, 82Se, 51V, 208Pb, and 66Zn. The determinations were performed

under conditions recommended by the manufacturer, with 1350 W power and plasma, nebulizer and auxiliary argon flow rates of 13.0, 0.87 and 0.70 L min-1, respectively. For the determination of Fe and C concentrations, an inductively coupled plasma optical emission spectrometer with axially viewed configuration (VISTA PRO; Varian, Mulgrave, Australia) was used. The sample introduction system consisted of SturmanMasters spray chamber PTFE and a V-Groove nebulizer. The determination of Fe and C was performed at 238.204 and 193.025 nm, respectively. Argon (with a minimum purity of 99.996%) was used for plasma generation.

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2.4 Analytical methods 2.4.1 Microwave-assisted digestion procedure Acid digestion of the mollusc samples was performed using a commercial highpressure laboratory microwave oven (Milestone Ethos 1600 Microwave Labstation; Sorisole, Italy) operating at a frequency of 2450 Hz with an energy output of 900 W. This microwave digestion system was equipped with 10 vessels made with PFA with a volume of 100 mL. The maximum operating temperature and pressure were 300 °C and 100 bar, respectively. The freeze-dried soft tissue samples (approximately 250 mg) were digested with 7.0 mL of 2.0 mol L-1 nitric acid solution and 1.0 mL of 30% m m-1 hydrogen peroxide (H2O2, Merck, Darmstadt, Germany). The microwave oven heating program was performed in four successive steps. In the first step, the temperature was increased linearly from 25 °C to 120 °C over 5 min. In the second step, the temperature was held at 120 °C for 2 min. In the third step, the temperature was increased linearly to 180 °C over 6 min, then in the last step, the temperature was held at 180 °C for 20 min. Finally, ventilation was performed for 10 min before removing the microwave digestion rotor from the microwave oven. After the digestion procedure and subsequent cooling, the digested samples and blank solutions were diluted to a final volume of 25.0 mL with water. Blanks solutions were prepared for each lot of samples, with all experiments performed in triplicate. The extracts were stored at -4 °C in PET-HD flasks that had been previously cleaned with nitric acid (Eça, Brito, Barbosa, Korn, & Hatje, 2014). 2.4.2 Determination of the acidity and residual carbon content of digests 8

To determine the final acidity, acid-base titration of the digests was performed. The titration was carried out with a standard solution of sodium hydroxide (0.0997 mol L-1; Merck, Darmstadt, Germany) employing phenolphthalein solution used as indicator (1.0% m v-1 in ethanol). The residual carbon concentration (RCC) was determined by ICP-OES, using carbon line emission at 193.025 nm. Carbon reference solutions (25–500 mg L−1) were prepared by dissolution of citric acid (Merck, Darmstadt, Germany) in water (Bizzi, Flores, Picoloto, Barin, & Nóbrega, 2010; Gouveia et al., 2001). 2.5 Quality assurance and quality control Data were reported as the mean and standard deviation of triplicate measurements. The concentrations of each element were calculated and expressed as micrograms per gram of dry mass. The accuracy and precision of the procedure were evaluated using certified reference materials (CRM) of oyster tissue (NIST 1566b) and mussel tissue (NIST 2977) acquired from the National Institute of Standards and Technology (NIST; Gaithersburg, MD, USA). In addition, results were evaluated by addition and recovery tests. Samples for each bivalve mollusc species were diluted to obtain concentrations of 0.3, 1.0 and 3.0 µg L−1 of each analyte. 2.5 Multivariate data analysis

The multivariate data analysis was conducted by PCA and HCA to evaluate the relationship among the trace element composition and the samples of molusc species in TSB, achieved using a correlation matrix. Statistical analyses were performed using 9

STATISTICA® 6.0 for Windows (StatSoft, Inc. 1984-2004, Tusla, USA). The results obtained for element concentrations of the different species of bivalves collected during dry and rainy seasons were compared by pattern recognition of the data. To achieve this, HCA and PCA were applied to the generated data matrix (66 × 12) with the samples organized in rows and element concentrations (variables) in columns. The data had been previously autoscaled as large variations were observed between the element concentrations.

3. Results and discussion

3.1 Validation of the analytical method The results obtained for the analysis of the CRM of oyster tissue (NIST 1566b) and mussel tissue (NIST 2977) presented good agreement with the certified values (Table 1). Statistical analysis showed no significant difference between the obtained results and the certified values within a 95% confidence level. The standard deviation of experimental values were low and close to those found for the certified values, demonstrating good repeatability of the analytical method. The precision was expressed as the relative standard deviation (RSD, n=3) for each reference sample, with all values lower than 10%, showing that the analytical procedure was suitable. Additionally, the recoveries of spiked additions, which were added to the bivalve mollusc samples prior to digestion, ranged from 82% to 115% with a RSD lower than 6% in all cases (n = 3). According to these results, the analytical method was accurate and precise for trace element determination in biological tissues. Furthermore, the final acidity of digested samples ranged from 0.4 to 0.7 mol L-1 and the obtained RCC values were lower than 15%, confirming the efficiency of the microwave-assisted digestion 10

protocol (Araújo, Gonzalez, Ferreira, Nogueira, & Nóbrega, 2002). The limits of detection (LOD) and limit of quantification (LOQ) were determined from the parameters of the analytical curves. For the LOD and LOQ, 15 blank solutions were prepared and analyzed. The LOQ values obtained for ICP-MS were 0.07 µg g-1 for As, 0.06 µg g-1 for Cd, 0.2 µg g-1 for Co, 0.3 µg g-1 for Cu, 0.4 µg g-1 for Cr, 0.03 µg g-1 for Mn, 0.08 µg g-1 for Ni, 0.4 µg g-1 for Pb, 0.7 µg g-1 for Se, 0.06 µg g-1 for V and 0.6 µg g-1 for Zn. When ICP OES was employed, the LOQ for Fe determination was 6.5 μg g−1. The results obtained showed that the limits of detection and quantification were suitable for the range of concentrations of all trace elements evaluated in the mollusc samples.

3.2 Trace element concentrations in bivalve mollusc samples The concentrations of Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Se, V and Zn in the edible parts of the four species of bivalve molluscs (Anomalocardia brasiliana, Iphigenia brasiliana, Lucina pectinata and Trachycardium muricatum) collected in TSB, Bahia, expressed as milligrams per kilogram of dry mass, are summarized in Table 2 and Table S1 (see Supplementary Material). According to Table 2, samples of the Lucina pectinata species presented the highest concentrations of Cd, Cu and Pb, indicating that this species may respond differently to the concentration of these elements in the environment when compared to the other species of bivalve molluscs assessed in this study. Lucina pectinata is consumed and commercialized in the coastal regions of Brazil where it is of great socioeconomic importance. In contrast, the highest concentration values for Cr and Mn were found in samples of the species Trachycardium muricatum. As shown in Table 2, the lowest and highest concentrations of Cu were obtained 11

for Trachycardium muricatum (2.34 mg kg-1) and Lucina pectinate (569 mg kg-1), respectively. These samples were collected in area influenced by a number of Cu-based industrial activities and extensive port facilities. Concentrations of Cu, Mn, Se, V and Zn in Anomalocardia brasiliana found in the present study were similar to values reported for TSB in previous studies (Rocha et al., 2012; Souza et al., 2011; CRA, 2004), while the Fe concentration found for this species was lower than that previously reported for TSB (Souza et al., 2011). In Brazil, according to the National Health Surveillance Agency (Agência Nacional de Vigilância Sanitária - ANVISA), the maximum limits of As, Cd and Pb in bivalve molluscs suitable for human consumption are 1.00, 2.00 and 1.50 mg kg-1 (RDC 42/2013-ANVISA) and, more generally, 30.0 mg kg-1 Cu, 0.1 mg kg-1 Cr and 50.0 mg kg-1 Zn in food products (ANVISA, 1965a; ANVISA 1998b). Table 3 shows a comparison of the limit values recommended by ANVISA and the concentrations of As, Cd, Cr, Cu, Pb and Zn, expressed as milligrams per kilogram of wet mass, determined in the current study. The Cd and Zn levels in all edible bivalve species were below the legal limits established by ANVISA (2.0 and 50.0 mg kg-1, respectively) and World Health Organization (WHO) (2.0 and 100 mg kg-1, respectively) (ANVISA, 1965a; WHO, 2006). The concentrations of As obtained in samples were above the legal limit recommended by ANVISA (1.00 mg kg-1) and WHO (inorganic As 1.0 mg kg-1 wet mass) (ANVISA, 2013c; WHO, 2006). According to the literature, marine organisms accumulate substantial amounts of arsenic more efficiently than terrestrial organisms. When assessing arsenic species, it is important to consider both the toxicological implications and the biogeochemical cycle of this element in the marine environment. Marine organisms are thought to acquire arsenic through the food chain, then convert

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inorganic arsenic to arsenobetaine via monomethylarsonic (MMA) and dimethylarsinic (DMA) ions by biomethylation (Bajger, Konieczka, & Namieśnik, 2011; Santos et al., 2013a; Jia, Wang, Ma, & Yang, 2018). Comparing the results for Pb concentrations with the Brazilian legislation, three Lucina pectinata samples collected from Acupe/Itapema, Maré Island and Madre de Deus/Suape were found to exceeded the legal limit of 1.50 mg kg-1 established by ANVISA and EC (European Commission, 2006) with levels of 1.94, 7.09 and 2.21 mg kg-1, respectively. These areas are close to a number of Cu-based industrial activities and extensive port facilities, which may explain these high values. With respect to Cr, most of the samples were found to be present at a concentration above the maximum limit value (0.1 mg kg-1), except for Anomalocardia brasiliana samples collected in Madre de Deus (L4) and Tainheiros (L7). All Cu levels obtained in Lucina pectinata samples were above the maximum values established by ANVISA (30.0 mg kg-1 wet mass). These results demonstrate the need for continued monitoring of Cu in this region, mainly in bivalve molluscs that showed levels exceeding the limits of Brazilian recommendations, and therefore considered inadequate for human consumption (ANVISA, 1965a). In addition to maximum limits, ANVISA has also established recommended daily intake (RDI) values for Cu, Fe, Mn and Zn of 3, 14, 5 and 15 mg day-1, respectively. Using the data obtained for Cu, Fe, Mn and Zn in the current study, we calculated the RDI for daily consumption in milligrams per 100 g of sample. These results showed that samples of the Anomalocardia species significantly contribute to the recommended daily intake of Fe and Mn, providing 14.7% to 17.9% of the RDI, respectively. The Lucina pectinata samples showed a higher contribution to daily Cu and Zn

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intake, with the RDI for Cu exceeding the recommended daily value. This may be related to samples of this species having been collected in Ilha de Maré (L3), which presents Cu enrichment, with values higher than those recommended by ANVISA. For Zn, a percentage of 20% of the RDI was found for this species. Considering the concentrations of As, Cu and Pb obtained for some species, a monitoring program should be implemented through a validated questionnaire in order to obtain information on the actual consumption rates of bivalve molluscs in the local population. This information is important to understand the actual risk of developing carcinogenic or chronic systemic effects among mussel consumers.

3.3 Multivariate data analysis

Multivariate data analysis of the results obtained for concentrations of the tested elements in the samples of different species of bivalve molluscs was carried out with HCA and PCA. In the HCA, Euclidean distances are calculated between each sample, making it possible to visualize groupings and, consequently, samples with similar characteristics (Ferreira, 2015). On the other hand, the PCA aims to present the samples and variables in a reduced number of dimensions called principal components (PC), allowing better visualization and interpretation of the obtained data (Panero, Vieira, Cruz, & Moura, 2009; Campanha et al., 2010; Ferreira, 2015). The pre-processing used was autoscaling, in which data were centered according to their mean value then each was divided by the standard deviation such that all variables were given the same importance (Sousa, Borges Neto, Poppi, Baccan, & Cadore, 2006). This type of pre-processing was chosen because of the variation in the magnitude of element concentration values.

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3.3.1. Principal component analysis

In the principal component analysis (PCA), data are projected into smaller matrices from the original data matrix by scores and loadings. The scores matrix shows the distribution of the samples while the loadings matrix shows the importance of the variables. Thus, PCA results in two- or three-dimensional graphs of scores or loadings (Vandeginste, Massart, Buydens, De Jong, S., Lewi, P.J., & Smeyers-Verbeke., 1998; Sousa, Borges Neto, Poppi, Baccan, & Cadore, 2006). The first principal component (PC1) accounted 38.44% of the total variance. The second principal component (PC2) and third principal component (PC3) accumulated 22.97% and 9.33%, respectively, together explaining 70.74% of the total data variance, which was sufficient to explain the data. The loading values of the variables for the three PCs are presented in Table S2 (see Supplementary Material), with the most relevant loading values identified in bold. In this analysis, PC1 was mainly related to the concentrations of Cr (0.729), Fe (0.726), Ni (0.686), Mn (0.625) and Se (0.822), all of which have positive loading values, followed by the concentrations of Cd (-0.641), Cu (-0.633), Pb (-0.690) and Zn (-0.611) with negative loading values, indicating an inverse correlation between the set of elements evaluated. The PC2 was related to the concentrations of Cr (-0.626), Pb (-0.601) and V (0.787), all variables with negative loading values. On the other hand, in PC3 the Co concentrations showed a positive loading value which was inverse to the negative loading value of the As concentration, also demonstrating an inverse relationship between the presence of these elements in the molluscs. Figure 1 shows the score plot of PC2 versus PC1, in which there is a tendency

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for clustering among the mollusc species, with PC1 contributing the main information to explain this trend. The Lucina pectinata samples with negative scores in PC1 are characterized by higher concentrations of Cd, Cu, Pb and Zn, and Trachycardium muricatum samples located in the positive PC1 are characterized by higher concentrations of Cr, Fe, Mn, Ni and Se. Anomalocardia brasiliana and Iphigenia brasiliana were centered in the PC1 axis, indicating a low concentration of these elements. Lucina pectinata, Trachycardium muricatum and Iphigenia brasiliana species were dispersed along the PC2 axis due to the As, Cr and V concentrations showing directly proportional absorption of these elements, most likely related to the locations where these mollusc species were collected. All samples of Anomalocardia brasiliana had positive PC2 scores with low concentrations of the elements mentioned above. In addition, it is possible to observe outliers on these graphs as, besides presenting useful information on trends and characteristics of the data, such as clustering of objects or variables, they can also be used to identify samples that present atypical behavior, that is, they do not fit the model (Sena, Poppi, Frighetto, & Valarini, 2000). Figure 2 shows separation of the most distinct species, Lucina pectinate (negative score) and Trachycardium muricatum (positive score) in the PC1 axis through dispersing the inverse As and Co concentrations in the PC3 axis, which had negative and positive loading values, respectively. Only one sample of the Lucina pectinata species could be characterized as an outlier as it presented the highest concentrations of Ni and V when compared to the other species studied, as well as a higher Co concentration among the Trachycardium muricatum species. Anomalocardia brasiliana and Iphigenia brasiliana were centered on the PC1 axis showing an equilibrium between the concentrations of Cd, Cr, Cu, Fe, Mn, Ni, Pb,

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Se and Zn when compared to the two other mollusc species.

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3.2.2. Hierarchical cluster analysis

Hierarchical cluster analysis was applied, also using autoscaled data, employing the Ward’s linkage method with Euclidean distances to calculate the interpoint distances and similarities between samples. The HCA is represented in the form of a twodimensional chart called a Dendogram. Figure 3 shows the dendogram obtained from a data matrix generated using element concentrations found in the different mollusc species. It corroborates the results of the PCA, and is another way of observing trends and similarities in the concentration of elements among the different species of bivalve molluscs. By analyzing the dendogram it is possible to observe a trend towards group formation, mainly by Lucina pectinate and Trachycardium muricatum.

4. Conclusions This study provides information related to the concentration of trace elements in four bivalve molluscs species that are commonly consumed in TSB, Bahia, Brazil. Based on the results obtained, all species showed As and Cr concentrations above permitted levels specified in Brazilian legislation. In addition, the levels of the Cu and Pb measured in most Lucina pectinata samples were higher were than the permitted for human consumption established by ANVISA. The PCA showed a tendency toward separation between Lucina pectinata and Trachycardium muricatum species. The samples of Lucina pectinata were characterized by higher concentrations of Cd, Cu, Pb and Zn, whereas Trachycardium muricatum samples had higher concentrations of Cr, Fe, Mn, Ni and Se. Considering that most coastal residents regularly consume large

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quantities of bivalves, further investigations are needed to better elucidate human health risks via contaminated organisms in the studied region. Additionally a study of speciation of arsenic is important since the different chemical forms of this element present different toxicity levels.

Acknowledgements The authors are grateful for the financial support provided by Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB, Brazil), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil), Petróleo Brasileiro S. A. (PETROBRAS, Brazil), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at

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São Francisco do Conde, Bahia, Brazil. Food Control, 33, 193-199. Sena, M.M., Poppi, R.J., Frighetto, R.T.S., & Valarini, P.J. (2000). Avaliação do uso de métodos quimiométricos em análise de solos. Química Nova, 23, 547-556. Sousa, R.A., Borges Neto, W., Poppi, R.J., Baccan N., & Cadore S. (2006). Classificação de água de coco processada e natural por meio de HCA, PCA e teores de íons metálicos determinados por ICP OES. Química Nova, 29, 654-656. Souza, M.M., Windmöller, C.C., & Hatje, V. (2011). Shellfish from Todos os Santos Bay, Bahia, Brazil: Treat or threat? Marine Pollution Bulletin, 62 2254–2263. Panero, F. dos S., Vieira, M. de F. P., Cruz, Â. M.F., & Moura, M. F. V. (2009). Aplicação da análise exploratória de dados na discriminação geográfica do quiabo do Rio Grande do Norte e Pernambuco. Orbital The Eletronic Journal of Chemistry, 1, 228-237. Vandeginste, B.G.M., Massart, D.L., Buydens, L.M.C., De Jong, S., Lewi, P.J., & Smeyers-Verbeke, J. (1998). Handbook of Chemometrics and Qualimetrics: Part b, Elsevier: Amsterdam. WHO (World Health Organization) (2006). Summary and Conclusions of the Sixty Seventh Meeting of the Joint FAO/WHO Export Committee on Food Additives, Rome, 20–29 June 2006. World Health Organization, Geneva . Accessed 20 December 17).

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Figures Captions Figure 1. Score plot of PC2 versus PC1 for mollusc species: Anomalocardia brasiliana, Iphigenia brasiliana, Lucina pectinata and Trachycardium muricatum.

Figure 2. Score plot of PC3 versus PC1 for mollusc species: Anomalocardia brasiliana, Iphigenia brasiliana, Lucina pectinata and Trachycardium muricatum. Figure 3. Dendogram for bivalve mollucs species: Ward’s linkage with Euclidean distances.

26

Figure 1

27

Figure 2

28

Figure 3

29

Elements

As / mg kg-1 Cd / mg kg-1 Co / mg kg-1 Cu / mg kg-1 Cr / mg kg-1 Fe / mg kg-1 Mn / mg kg-1 Ni / mg kg-1 Pb / mg kg-1 Se / mg kg-1 V / mg kg-1 Zn/ mg kg-1

Table 1. Certified values of reference materials of mussel Tissue (NIST 2976) and oyster tissue (CRM 1566b) and values experimentally obtained for trace elements.

NIST 1566b

NIST 2977

Certified value

Found Value

Certified values

Found Value

7.65 ± 0.65 2.48 ± 0.08 0.371 ± 0.009 71.6 ± 1.6 n.d 205.8 ± 6.8 18.5 ± 0.2 1.04 ± 0.09 0.308 ± 0.009 2.06 ± 0.15 0.577 ± 0.023 1424 ± 46

7.53 ± 0.48 2.24 ± 0.13 0.391 ± 0.013 67.9 ± 1.3 n.d. 212.5 ± 8.0 17.9 ± 1.1 1.11 ± 0.05 0.304 ± 0.017 2.32 ± 0.12 0.525 ± 0.067 1564 ± 86

8.83 ± 0.91 0.179 ± 0.003 0.48 ± 0.13 9.42 ± 0.52 3.91 ± 0.47 274 ± 18 23.93 ± 0.29 6.06 ± 0.24 2.27 ± 0.13 1.78 ± 0.16 (1.1) 135 ± 5

9.64 ± 0.07 0.171 ± 0.029 0.43 ± 0.04 9.56 ± 0.95 3.76 ± 0.35 212 ± 13 23.58 ± 2.70 5.38 ± 0.71 1.98 ± 0.30 2.20 ± 0.21 1.26 ± 0.15 130 ± 13

Results expressed mean ± interval confidence at 95% (n= 3) Table 2. Concentration of the analytes (mg kg -1, dry mass) for each bivalve molluscs 30

species collected at four Todos os Santos Bay locations, during dry (D) and rainy period (R).

Element

Period

Anomalocardia brasiliana

Iphigenia brasiliana

Lucina pectinata Trachycardium muricatum

D

12.8 ± 2.2

17.3 ± 1.3

14.5 ± 3.4

12.4 ± 2.1

R

12.6 ± 3.4

15.6 ± 1.5

14.7 ± 4.4

10.5 ± 1.6

D

0.63 ± 0.39

0.098 ± 0.032

3.58 ± 2.22

0.24 ± 0.11

R

0.84 ± 0.96

0.133 ± 0.026

4.2 ± 2.2

0.23 ± 0.13

D

11.2 ± 7.1

8.6 ± 6.2

13.8 ± 5.6

8.4 ± 3.8

R

8.4 ± 2.9

7.2 ± 2.1

8.8 ± 1.8

12 ± 17

D

0.66 ± 0.21

1.93 ± 0.39

1.21 ± 0.22

3.4 ± 2.2

R

0.77 ± 0.17

1.04 ± 0.22

1.7 ± 1.6

1.8 ± 1.3

D

10.9 ± 9.3

10.9 ± 5.7

503 ± 683

2.34 ± 0.81

R

13 ± 14

22.6 ± 8.1

569 ± 663

4.1 ± 1.5

D

134 ± 69

691 ± 291

340 ± 61

1154 ± 823

R

186 ± 88

371 ± 110

362 ± 118

760 ± 535

D

21 ± 11

5.9 ± 2.9

2.5 ± 1.6

34 ± 17

R

32 ± 16

16.9 ± 11.2

4.9 ± 1.7

49 ± 25

D

1.83 ± 0.71

2.91 ± 0.56

1.54 ± 0.45

3.0 ± 1.5

R

1.79 ± 0.54

2.17 ± 0.79

2.4 ± 3.4

3.1 ± 1.0

D

0.61 ± 0.18

0.60 ± 0.19

13.2 ± 10.1

0.94 ± 0.42

R

0.65 ± 0.28

0.663 ± 0.058

15.1 ± 8.4

0.92 ± 0.53

D

4.4 ± 1.3

8.50 ± 0.34

2.17 ± 0.25

9.4 ± 2.7

R

4.7 ± 1.6

6.33 ± 0.80

2.01 ± 0.16

9.4 ± 2.4

As

Cd

Co

Cr

Cu

Fe

Mn

Ni

Pb

Se

31

D

1.19 ± 0.36

2.79 ± 0.63

3.03 ± 0.88

4.2 ± 2.0

R

1.56 ± 0.20

1.87 ± 0.50

3.8 ± 3.2

2.7 ± 1.2

D

46.2 ± 4.7

132 ± 39

120 ± 48

46.5 ± 2.9

R

54.4 ± 4.3

112 ± 24

205 ± 162

44.5 ± 3.3

V

Zn

Results expressed as mean ± deviation standard.

32

Locality Bivalve molluscs

As

Cd

Cu

Cr

Pb

Zn

Table 3. Results found in bivalve molluscs species (in mg kg-1, wet mass) compared to ANVISA values.

33

Anomalocardia b.

3.52

0.340

2.06

0.128

0.0612 10.5

Lucina p.

4.41

1.69

51.3

0.270

1.94

Anomalocardia b.

3.11

0.148

1.36

0.248

0.0629 10.3

Trachycardium m.

2.90

0.0622 0.715

1.55

0.337

11.5

Anomalocardia b.

2.48

0.0781 7.09

0.148

0.114

10.1

Lucina p.

2.92

0.798

376

0.283

7.09

46.4

Anomalocardia b.

3.11

0.157

1.52

0.0877 0.184

12.7

Iphigenia b.

4.45

0.0292 3.03

0.516

0.175

29.6

Lucina p.

2.67

0.583

32.0

0.286

2.21

25.5

Trachycardium m.

2.92

0.08

0.368

0.699

0.140

11.9

Anomalocardia b.

3.76

0.162

1.69

0.136

0.187

12.7

Iphigenia b.

4.40

0.0180 0.888

0.376

0.111

20.2

Lucina p.

4.01

0.386

0.326

1.45

20.1

Trachycardium m.

3.72

0.0586 0.0675 0.762

0.190

10.7

Anomalocardia b.

3.30

0.127

4.12

0.141

0.104

10.4

Iphigenia b.

3.84

0.0203 3.55

0.493

0.107

40.8

Anomalocardia b.

2.25

0.0509 0.636

0.0904 0.0776 11.4

Trachycardium m.

2.68

0.0333 0.609

0.279

0.0802 10.5

ANVISA VALUES

1.00

2.00

0.10

1.50

L1 23.9

L2

L3

L4

L5 25.9

L6

L7

30.0

50.0

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L1: Acupe/Itapema; L2: Bom Jesus dos Pobres; L3: Maré Island; L4: Madre de Deus/Suape; L5: Mutá; L6: Salinas da Margarida; L7: Tainheiros beach.

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Highlights

Bivalve molluscs species were analyzed by ICP OES and ICP-MS.

Most Lucina pectinata samples had higher concentrations of Cu, Cr and Pb.

Concentrations of As obtained in most samples were above the legal limit recommended by ANVISA

PCA and HCA showed separation between Lucina pectinata and Trachycardium muricatum species.

36