Accepted Manuscript

Title: Risk characterization for polycyclic aromatic hydrocarbons and toxic metals associated with sh consumption Author: Maria Maddalena Storelli Grazia Barone Veronica Giuliana Perrone Arianna Storelli PII: DOI: Reference: To appear in: Received date: Revised date: Accepted date: 30-10-2012 25-3-2013 26-3-2013 S0889-1575(13)00064-1 http://dx.doi.org/doi:10.1016/j.jfca.2013.03.008 YJFCA 2311

Please cite this article as: Storelli, M. M., Barone, G., Perrone, V. G., & Storelli, A., Risk characterization for polycyclic aromatic hydrocarbons and toxic metals associated with sh consumption, Journal of Food Composition and Analysis (2013), http://dx.doi.org/10.1016/j.jfca.2013.03.008 This is a PDF le 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 nal 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.

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Original research article

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Risk characterization for polycyclic aromatic hydrocarbons and toxic metals associated with fish consumption
Maria Maddalena Storelli*, Grazia Barone, Veronica Giuliana Perrone, Arianna Storelli Biosciences, Biotechnologies and Pharmaceutical Sciences Department, Veterinary

Medicine Faculty, University of Bari - Strada Prov. le per Casamassima Km 3, 70010 Valenzano (Ba), Italy

* Corresponding author. Tel.: +39 80 5443365; fax: +39 80 5443363. E-mail address: m.m.storelli@veterinaria.uniba.it (M.M. Storelli)

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Abstract

Concentrations of 11 polycyclic aromatic hydrocarbons (PAHs) and toxic metals (Hg, Cd and Pb) were measured in three fish species [(Raja miraletus (brown ray), Lepidorhombus whiffiagonis (megrim) and Lophius piscatorius (angler)] from Mediterranean Sea (Adriatic Sea). No significant difference in PAHs and metal concentrations was encountered among the fish species examined. Total PAH concentrations ranged from 209.9 ng g-1 wet weight to 227.2 ng g-1 wet weight. Lowmolecular weight (two and three rings) PAHs were observed dominating over the high molecular weight (from four to six rings) PAHs. With regard to metals, Hg exhibited the highest concentrations (0.68-0.98 g g-1 weight wet), followed by Pb (0.08-0.12 g g-1 weight wet) and Cd (0.05-0.10 g g-1 weight wet). The health risks by consumption

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of these species were assessed and did not present threat to public health concerning PAH, Pb and Cd intakes. However, the estimated exposure Hg illustrates the importance of limiting the dietary consumption of larger-sized fish. Keywords: Fish; Polycyclic aromatic hydrocarbons; PAHs; Toxic metals; Mediterranean Sea; Dietary exposure; Provisional tolerable weekly intakes; PTWI; Food safety; Food analysis; Toxic equivalency factors; TEFs

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1 Introduction

pollutants in the marine environment (Ramalhosa, 2012). They enter the aquatic

combustion of fuels, and industrial and urban effluents (Ramalhosa, 2012). Owing to their low aqueous solubility and strong hydrophobic nature, these contaminants tend to associate with suspended material with the underlying sediments as their ultimate sink (Patrolecco et al., 2010). Also toxic metals, such as Hg, Cd, and Pb, once released in the marine environment, can be rapidly adsorbed on particulate material and precipitate on the bottom (Ney and Van Hassel, 1983). Consequently fish living in the bottom waters with recurrent contact with the sediments are more exposed to sediment-associated contamination than fish living in the upper zone of the water column (Yilmaz et al., 2010). Raja miraletus (brown ray), Lepidorhombus whiffiagonis (megrim) and Lophius piscatorius (angler), important commercial species of the Mediterranean Sea, besides depending on the bottom to feed, have carnivorous feeding habits in higher trophic levels (Stergiou & Karpouzi, 2002), a property that favours accumulation of toxic compounds. Information about PAHs and toxic metal concentrations in these edible fish

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environment primarily from oil spills, atmospheric pollution resulting from incomplete

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Polycyclic aromatic hydrocarbons (PAHs) are among the most ubiquitous organic

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are, therefore, essential for evaluating possibly risks for human health. In fact, these substances express toxicity, so several of them are classified as probable or possible carcinogens by the International Agency for Research on Cancer (IARC). In addition, it is a well-known fact that the general population is most commonly exposed to metals,

examines the extent of accumulation of PAHs and some toxic metals, namely Hg, Pb and Cd in three different fish species above mentioned commonly consumed in Italy,

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2 Materials and methods

2.1 Sample collection

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Specimens of Raja miraletus (brown ray: n 35), Lepidorhombus whiffiagonis (megrim: n 28) and Lophius piscatorius (angler: n 30) were collected in the South Adriatic Sea (Mediterranean Sea) from May to July 2011. The fish lengths (brown ray: 35.0-85.7 cm, average: 66.91.8 cm; megrim: 18.2-32.3 cm, average: 23.51.9 cm; angler: 40.0-120.0 cm, average: 101.72.2 cm) and weights (brown ray: 241.5-1057.6 g, average: 915.611.3 g; megrim: 32.0-275.3 g, average: 150.28.5 g; angler: 250.9-3069.0 g, average: 1015.7232.0 g) were individually measured before they are pooled (brown ray: 11 pools; megrim: 8 pools; angler: 12 pools) for analysis. From fish of each pool, muscle tissue was taken, homogenized and kept in a deep freeze at -20 C until chemical analysis. Length has been chosen as basic measure to reflect fish age, because it is less subject to fluctuation than body weight (Diaz et al., 1994).

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and evaluates whether such contamination levels may pose risks to human health.

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especially to Hg, through the consumption of fish. In this context the present study

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2.2

Sample preparation

For PAH analyses [naphthalene (NA), fluorene (FL), phenanthrene (PHE), anthracene (AN), fluoranthene (FA), pyrene (PY), chrysene (CHR), benz[a]anthracene (BaA), benzo[a]pyrene (BaP), dibenzo[a,h]anthracene (DBahA), benzo[g,h,i]perylene

mixture (100 mL ethanol, 1 M KOH) for 2 h by mechanical shaking extracted three

times by water n-hexane (15 mL). Then, n-hexane combined extracts were dried over

on a column with a 15 mL hexane dichloromethane (3:1, v/v) eluent. The column was

sodium sulphate and was washed with 10 mL of hexane before use. The extract collected was concentrated under a gentle nitrogen stream (Hwang et al., 2012) and dissolved in dichloromethane before the analysis.

The analysis was carried out using a GC Thermo Trace Ultra coupled with a Thermo

used. Helium was used as the carrier gas and the column head pressure was maintained at 10psi to give an approximate flow rate of 1 mL/min. The injector was maintained at

L in the splitless mode. The column temperature was initially held at 80 C for 1 min, ramped to 300 C at a rate of 5 C/min, and then the temperature was held at 300 C for 20 min. The mass spectrometer was used in electron ionisation (70eV) mode and operated in the selected ion monitoring (SIM) mode using the typical PAH molecular ions (M+) m/z: 128, 166, 178, 202, 228, 252, 276 and 278.

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280 C, while the transfer line was maintained at 250 C. All injection volumes were 1

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PolarisQ mass spectrometer. A 30 m long column model DB 5-MS of 0.25 m I.D. was

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packed with 5 g of Florisil (mesh 60/100, Supelco, USA) and a top layer of anhydrous

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anhydrous sodium sulphate and concentrated by evaporation. The extract was purified

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(BghiP)], 20 g of the sample homogenates were weighed and digested in an alkaline

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The most abundant ion was used for quantification and two other ions were additionally used for confirmation. The quantification and confirmation ion abundances were determined by injection of standards under the same chromatographic conditions using full-scan (mass/charge ratio ranging m/z 50-600). PAHs were confirmed by their

determination of ion ratios. The extractive analytical procedures and the instrumental

conditions to determine metal concentrations have been described elsewhere (Storelli,

In detail, for Pb and Cd, aliquots (1-2 g) of the homogenised samples were digested in a quartz Erlenmeyer flask with 11 mL of a mixture of HNO3-HClO4 (8:3) using a hotplate heated to 150 0C. Additional aliquots of nitric acid (maximum of 0.2 mL) were added until a completely colourless solution was obtained. After evaporation the residue was

deionised water. For Hg, the samples were weighted into a conical flask and digested in

completely colourless solution was obtained. After cooling, the resultant solution was diluted to a known volume (100 mL) with deionised water according to the method

metals was determined by atomic absorption spectrophotometer (AAS) (Perkin Elmer Analyst 800). Cd and Pb was analyzed by graphite furnace technique (THGA-800 P.E.) and Hg by a hydride system (FIMS 100) after reduction by SnCl2.

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recommended by the Official Italian Agencies (G.U.R.I., 1994). The content of toxic

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10 mL of H2SO4-HNO3 (1:1). The flask was heated under reflux conditions until a

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dissolved in 2 mL of water, and finally, the volume was made up to 25 mL with

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2008).

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retention times, the identification of quantification and confirmation ions and the

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2.3

Quality control and assurance

Prior to the sample analysis, PAH standards (98-99% purity) (Absolute Standards, Analytical Tecnology S.r.l., Milano) were used to make multipoint calibration curves (1, 10, 100 and 1000 ng g-1) whose correlation coefficients ranging from 0.993 to 0.999.

PAHs ranged from 0.10 to 2.0 ng g-1 and 0.30 to 4.0 ng g-1, respectively. For metals,

reference tissue (Tort-2 Lobster Hepatopancreas, National Research Council of Canada,

Results (Hg: 0.28 0.03; Cd: 26.2 2.4; Pb: 0.32 0.18 g g-1 dry weight) were in

0.13 g g-1 dry weight) and the standard deviation were low, proving good repeatability of the methods.

from 91% to 104% (n = 3). The limit of detection (LOD) (Hg: 5; Cd: 0.10; Pb: 10 ng g-1

deviation of blanks and the standard of quantification (LOQ) are the following: Hg: 13; Cd: 0.38; Pb: 40 ng g-1 wet weight. Two empty samples blank were analysed together

and or analysis. Metal and PAH concentrations in blanks were below the detection limits in all the analyses. Blanks and calibration standard solutions were similarly analysed as the samples and calibration curves constructed. Analyses were duplicated to check the reproducibility of the results. Relative standard deviations among replicates were always less than 10%. Recovery tests were performed for the investigated metals and PAHs in selected samples by spiking analysed samples

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wet weight) is defined as the concentration corresponding to three times the standard

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The results for standard reference material displayed recoveries of the elements ranging

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good agreement with the certified values (Hg: 0.27 0.06; Cd: 26.7 0.60; Pb: 0.35

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Ottawa, Ontario, Canada) was treated and analysed in the same way as the samples.

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The limit of detection (LOD) and the limit of quantification (LOQ) for the individual

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with aliquots of the metal and PAH standards (50 ng g-1). The recovery percentages ranged from 96 to 99% for metals and from 85 to 110% for PAHs, and the concentrations were not corrected for percent recovery. Metal and PAH concentrations were determined on a g g-1 wet weight basis and ng g-1 wet weight basis, respectively.

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3 Results and discussion

3.1

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Content of PAHs and heavy metals (Hg, Cd and Pb) in fish muscle tissue

The mean values, standard deviations and range of concentrations of PAHs and Hg, Cd and Pb in studied fish species are given in Table 1. The mean total concentrations of PAHs were 227.2, 209.9 and 214.1 ng g-1 wet weight for brown ray, megrim and angler,

significantly different (p>0.05) among fish species investigated. Also PAHs

(Fl, PHE, AN) (59.8-65.4%), followed from those with 4-rings (FA, PY,CHR, BaA) (30.3-35.5%) and 2-rings (NA) (3.8-4.7%), while BaP, DBahA, and BghiP showed

This common pattern with a larger amount of lower molecular weight contaminants, could probably depend from the metabolic transformations of heavier PAHs occurring in fish liver (Meador et al., 1995). In addition, fish converting up to 99% of the PAHs to metabolites within 24 h of uptake, change the pattern and the concentration in the tissues (Varanasi et al., 1989). Data from the open literatures show that accumulation of

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levels below the limit of quantification (LOQ) in all samples.

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composition pattern was similar, being dominated by the presence of PAHs with 3 rings

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respectively. Statistical comparison revealed that total PAH concentrations were not

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Figure 1 shows the chromatograms of a PAH standard (a) and of a fish sample (b).

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these pollutants in fish is rather scarce (Perugini et al., 2007; Patrolecco et al., 2010). The lack of significant correlation between total PAHs concentrations and length of fish examined, which concurs with the results of other authors (Perugini et al., 2007), validates this assertion. Also, the concentrations and distribution pattern encountered

studies carried out either on fish from Mediterranean Sea (Perugini et al., 2007; Oliveri Conti et al., 2012) or other marine areas (Llobet et al., 2006; Ramalhosa et al., 2012). With regard to metals, the element present in the highest amounts in the muscle tissues of the species examined was Hg, while Cd and Pb remained lower (Table 1). The data analysis revealed also that there were no consistent differences in metal concentrations among the fish species (p>0.05). In particular, the mean concentrations of Hg varied from a minimum value of 0.68 in megrim to a maximum of 0.98 g g-1 weight wet in

between 0.05-0.10 g g-1 wet weight and 0.08-0.12 g g-1 wet weight, respectively. The

species are all benthic-feeding fish and with similar dietary preferences (Stergiou & Karpouzi, 2002).

Another common point to all three characterized species was that Hg concentration increased with fish length, as described by results of linear regression analysis (brown ray: R= 0.75, p<0.01; megrim: R= 0.83, p<0.001; angler: R= 0.82, p<0.001). The dependency between Hg enrichment and length of fish is an expected finding which agrees with the literature data (Polak-Juszczak, 2009). Also, the Cd and Pb results are

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lack of differences in metal concentrations might be mainly due to the fact that tested

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brown ray, while a narrow range was encountered for Cd and Pb whose levels were

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here, with prevalence of lower molecular weight PAHs, is in line with several previous

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close to those reported in literature for benthic fish from Mediterranean Sea (Tepe, 2009; Yilmaz et al., 2010; Copat et al., 2012). On the contrary, the Hg concentrations encountered here were high when compared to those in benthic fish from Mediterranean Sea (Storelli, 2008). This is probably the result of differences in fish size analyzed, because as above reported the Hg concentrations are strongly influenced by this parameter. However, the pattern of metal content followed the order published by other authors for fish from Mediterranean Sea (Falc et al., 2006), confirming a decline of Pb concentrations in this basin (Tian & Ruiz-Pino, 1995; Storelli, 2008) and a low capacity of Cd accumulation in fish muscle tissue (Vieira et al., 2011).

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3.2

Estimation of potential public health risks

and potential human health risks. The European Union legislation has established in the

including PAHs and heavy metals. Concerning PAHs, only benzo[a]pyrene (BaP) had a limit (2 ng g-1 wet weight) set by European Regulation No 1881/2006 (Official Journal

quickly metabolised in fresh fish and do not accumulate in the muscle meat the limit has been abrogated (Official Journal of the European Union, 2011). For metals, the threshold values adopted by the European Commission for Cd and Pb are 0.05 and 0.30 g g-1 wet weight, respectively, while the range of standards for Hg is 0.5-1.0 g g-1 wet weight depending on the types of fish (Official Journal of the European Union, 2006). None of the fish samples analyzed presented Pb concentrations exceeding the proposed

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of the European Union, 2006), but recently because it has been shown that PAHs are

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limit, while 20% of the brown ray and angler and 10% of megrim samples had Cd levels slightly exceeding the guideline above mentioned. With respect to Hg, the standard of 1.0 g g-1 wet weight was exceeded by 35% of brown ray, 27% of angler and 26% of megrim samples. The relevance of these data can be evaluated in terms of potential human health risks comparing the metal

concentrations found with the Provisional tolerable weekly intakes (PTWIs) established

by the Joint FAO/WHO Expert Committee on Food Additives (JECFA, 2004) for Hg (5 g kg-1 bw), Cd (7 g kg-1 bw) and Pb (25 g kg-1 bw). As illustrated in Table 2, the average exposure estimates, considering a per capita demersal fish weekly consumption of 125 g (FAO, 2007) and a body weight of 70 kg, were below the toxicological reference values for all studied elements, suggesting that consumption of these species is not a problem for human health.

However, the estimation of exposure to Hg merits special attention due to some important assumptions. In fish the highest Hg concentration is commonly found in those occupying the top trophic levels, as well as in larger and older fish (Storelli et al., 2002) and the dominant form of this toxin is methylmercury (MeHg) (70-100%) (Joiris et al., 1999; Storelli et al., 2005). Taking all this into account, the estimations of exposure based on a worst-case scenario drafted using the maximum Hg concentrations encountered in largest fish, were not to underestimate, constituting from 55.0% to 62.8% of value of total Hg PTWI. In addition, assuming that 100% of Hg found is methylmercury (EFSA, 2004), the average weekly intakes were close (see megrim and angler) or higher (see brown ray) than MeHg PTWI of 1.6 g kg-1 bw established by the Joint FAO/WHO Expert Committee on Food Additives (JECFA, 2003).

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Concerning PAHs, their contribution to dietary exposure would be rather limited since, as above reported, accumulation of these chemicals in fish muscle is negligible. However, in consideration of the fact that they have been shown to have a carcinogenic activity it would be of interest to calculate the toxicity equivalent quotient (TEQ) of

LaGoy (1992). In this way the sum of concentrations of individual PAHs

(TEQ=BaPEq), expressed as equivalents of benzo[a]pyrene (BaPEq), was compared

consumption rate of 18 g day-1 for a 70 kg adult (USEPA, 2000). The calculated toxicity

value (SV) of 5.3 ng g-1 indicating that these fish were safe for human consumption in terms of the amounts of PAHs found in their muscles. Nevertheless, it is worth highlighting that the risk may be underestimated because other PAHs such as benzo(b)fluoranthene, benzo(k)fluoranthene and indeno(1,2,3-cd)pyrene were not taken into account in this study.

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4 Conclusion

health threat resulting from the consumption of these fish from the Mediterranean, except for Hg. The Hg estimated exposure, in fact, illustrates the importance of limiting the dietary consumption of larger-sized fish. In addition, strategies to address the dietary choices of consumers could help to minimize the possible health risks associated with eating seafood.

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As a final conclusion, the results of the study show that there should not be any human

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equivalent concentrations (TEQs=2.02-2.35 ng g-1) (Table 3) were below the screening

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with the USEPA screening value (SV) for BaP, calculated considering a fish

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total PAHs, through the toxic equivalency factors (TEFs) proposed by Nisbet and

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Varanasi, U., Stein, J.E., Nishimato, M. (1989). Biotrasformation and disposition of

Vieira, C., Morais, S., Ramos, S., Delerue-Matos, C., Oliveira, M.B.P.P. (2011). Mercury, cadmium, lead and arsenic levels in three pelagic fish species from the

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PAH in Fish. In: Varanasi, U. (Ed.). Metabolism of Polycyclic Aromatic Hydrocarbons in the Environment (pp. 93-150). CRC Press, Inc., Boca Raton, Florida.

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Atlantic Ocean: Intra- and inter-specific variability and human health risks for consumption. Food and Chemical Toxicology, 49, 923-932. Yilmaz, A.B., Sangn, M.K., Yaholu, D., Turan, C. (2010). Metals (major, essential to non-essential) composition of the different tissues of three demersal fish species from skenderun Bay, Turkey. Food Chemistry, 123, 410-415.

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Figure caption

Figure 1. GC/MS Chromatograms of a PAH Standard (a) and of a fish sample (b). [naphthalene (NA), fluorene (FL), phenanthrene (PHE), anthracene (AN), fluoranthene (FA), pyrene (PY), chrysene (CHR), benz[a]anthracene (BaA), benzo[a]pyrene (BaP), dibenzo[a,h]anthracene (DBahA), benzo[g,h,i]perylene (BghiP)],

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Table 1 Concentrations of individual and total PAH (ng g-1 wet weight) and heavy metals (g g-1 wet weight) in the three fish species analyzed. Brown raya Naphthalene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Chrysene Benz[a]anthracene Total (PAHs) Hg 4.6-12.6 8.62.4 5.5-22.1 19.24.6 4.7-10.2 8.22.0 76.5-152.3 116.325.1 4.8-7.1 5.30.7 35.3-69.6 53.712.4 4.9-10.3 6.92.1 6.0-13.0 9.02.2 125.4-231.5 227.2 Megrimb 5.6-14.4 8.92.5 10.0-20.1 18.83.6 5.0-21.2 9.86.2 59.8-136.9 108.729.1 6.0-14.0 7.22.8 15.5-78.1 44.324.9 4.2-9.9 4.92.0 5.3-14.2 7.33.0 108.8-213.7 209.9 0.15-1.60 0.680.21 0.03-0.11 0.050.02 0.03-0.18 0.080.04 Anglerc

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0.28-1.54 0.980.33 Cd 0.03-0.12 0.070.01 Pb 0.02-0.17 0.120.03 a n=11 pools , 3 replicates analysis for each pools b n=8 pools , 3 replicates analysis for each pools c n=12 pools , 3 replicates analysis for each pools

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6.1-18.6 10.14.3 4.5-18.7 14.54.4 5.2-17.3 10.24.2 49.7-130.2 103.39.03 7.0-14.5 8.22.2 25.4-96.2 47.521.9 5.3-11.3 9.72.0 7.0-15.5 10.63.1 103.5-228.2 214.1 0.23-1.76 0.850.30 0.07-0.12 0.100.03 0.06-0.14 0.100.04
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Table 2 Estimated weekly intake (ng g-1 body weight) for Hg, Cd and Pb. Estimated Weekly Intake (EWI) Hg Cd Pb 0.50-2.75 0.05-0.21 0.04-0.30 1.750.59 0.130.02 0.210.05 0.27-2.86 0.05-0.20 0.05-0.32 1.210.38 0.090.04 0.140.07 0.41-3.14 0.13-0.21 0.11-0.25 1.520.54 0.180.05 0.180.07

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Table 3 Average BaPEq (ng g-1) and their sum (TEQ=BaPEq) in fish samples. PAHs Naphthalene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Chrysene Benz[a]anthracene TEQ=BaPEq a Nisbet & LaGoy, 1992 TEFsa 0.001 0.001 0.001 0.01 0.001 0.001 0.01 0.1 Brown ray 0.009 0.02 0.008 1.16 0.05 0.05 0.07 0.90 2.27 Megrim BaPEq 0.009 0.02 0.01 1.09 0.07 0.04 0.05 0.73 2.02 Angler 0.01 0.01 0.01 1.03 0.08 0.05 0.10 1.06 2.35

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Highlights

1. PAHs and Hg, Cd and Pb were measured in important commercial fish species. 2. The study was carried out in the Adriatic Sea (Mediterranean). 3. PAHs, Cd and Pb intakes not present threat to public health. 4. Hg estimated exposure shows importance of limiting larger size fish consumption.

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