Organochlorine and organobromine compounds in a benthic fish (Solea solea) from Bizerte Lagoon (northern Tunisia): Implications for human exposure

Ecotoxicology and Environmental Safety 88 (2013) 55–64

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Organochlorine and organobromine compounds in a benthic fish (Solea solea) from Bizerte Lagoon (northern Tunisia): Implications for human exposure Walid Ben Ameur a,n, Yassine El Megdiche a, Ethel Eljarrat b, Sihem Ben Hassine a, Barhoumi Badreddine a, Trabelsi Souad a, Hammami Be chir a, Damia Barcelo´ b, Mohamed Ridha Driss a a b

Laboratory of Environmental Analytical Chemistry (05/UR/12-03), University of Carthage, Faculty of Sciences, Bizerte 7021 Zarzouna, Tunisia Department of Environmental Chemistry, IDAEA-CSIC, Jordi Girona 18-26, Barcelona 08034, Spain

a r t i c l e i n f o

abstract

Article history: Received 28 March 2012 Received in revised form 25 October 2012 Accepted 26 October 2012 Available online 6 December 2012

Information on the occurrence of persistent organic pollutants (POPs) in fish from Tunisia is scarce. In this study, thirty one persistent organic pollutants including organochlorine pesticides (OCPs) (dichlorodihenyltrichloroethane and its metabolites (DDTs), hexachlorocyclohexanes (HCHs) and hexachlorobenzene (HCB)), polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs) and methoxylated polybrominated diphenyl ethers (MeO-PBDEs) were determined in solea solea muscle, from Bizerte Lagoon (northern Tunisia) and from the Mediterranean Sea (reference area) (northern Mediterranean). In the Bizerte Lagoon, contaminant concentrations generally followed this order: PCBs 4DDTs 4 PBDEs 4 MeO-PBDEs 4 HCB 4 HCHs; while in the Mediterranean Sea, pollutant concentration followed this order: MeO-PBDEs 4PCBs 4DDTs 4PBDEs 4 HCB 4HCHs. Mean levels of organochlorine compounds were 1018 and 380 ng g  1 lipid weight (lw) in fish from Bizerte Lagoon and the Mediterranean Sea, respectively. Mean concentrations of organobromine compounds were 279 and 301 ng g  1 lw in sole from Bizerte Lagoon and the Mediterranean Sea, respectively. Organohalogen concentrations in fish from Bizerte Lagoon were similar or slightly lower than those reported for other marine fish species from other locations around the world. PCB, HCH, HCB and PBDE levels were negatively correlated with lipid content, while no such correlation was seen for DDTs. Assessment based on several available guidelines suggested an insignificant human health risk for dietary intake of HCB, lindane and PBDEs associated with consumption of sole. However, the estimated lifetime cancer risk from dietary exposure to DDTs and PCBs is a potential concern. & 2012 Elsevier Inc. All rights reserved.

Keywords: Bizerte Lagoon Dietary intake Organohalogen compounds Risk assessment Sole.

1. Introduction Increasing global population and technological development has resulted in increased production of chemicals and their subsequent release into the environment. The presence of organohalogenated contaminants (OHCs), such as polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs) and polybrominated diphenyl ethers (PBDEs), in the environment has been a cause of concern because of their persistent character, bioaccumulative potential and their association with adverse effects in both humans and wildlife (Vos et al., 2000; Birnbaum and Staskal, 2004; de Witt et al., 2006; UNEP, 2009; Wan et al., 2010). These persistent, bioaccumulating organohalogen compounds, (Tanabe et al., 1994; Yogui and Sericano, 2009), have been produced and released into the natural environments, including aquatic ecosystems (Hites, 2004; Hale et al., 2006; Law et al., 2006, 2008;

n

Corresponding author. Fax: þ216 72 590 566. E-mail address: [email protected] (W. Ben Ameur).

0147-6513/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2012.10.021

Tanabe et al., 2008; Shaw and Kannan, 2009). Historically, organochlorine compounds (OCs) had widespread applications. Sources of OCPs are mainly derived from agricultural applications (Wurll and Obbard, 2005). PCBs had a wide range of industrial applications as dielectric and hydraulic fluids, prior to their ban in many countries (Tanabe, 1988). Both industrial and agricultural sources have contributed significant amounts to the environment through leakage, disposal and evaporation (Tolosa et al., 2010). PBDEs are brominated flame retardants (BFRs) used in electronics, plastics, paints, textiles, foam furniture and building materials (Alaee et al., 2003; Hale et al., 2003; Watanabe and Sakai, 2003). PBDEs are released into the environment in various of forms: associated with particles, by leaching, and by volatilization from flame-resistant products during their use and waste disposal such during incineration of municipal waste (Watanabe and Sakai, 2003; Hites, 2004; Hale et al., 2003, 2006). PBDEs have been shown to exert neurodevelopmental and endocrinedisrupting effects in laboratory animals (Costa and Giordano, 2007). In 2009, the Stockholm Convention included Penta- and Octa-BDE mixtures in the list of persistent organic pollutants

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W. Ben Ameur et al. / Ecotoxicology and Environmental Safety 88 (2013) 55–64

(POPs) (Birnbaum and Staskal, 2004; Shaw and Kannan, 2009; Stockholm Convention on POPs, 2009). Recently, attention has been drawn towards the presence of naturally-produced compounds, such as methoxylated PBDEs (MeO-PBDEs) in aquatic ecosystems. There is recent evidence of MeO-PBDEs in fish and marine mammals and measured at high concentrations in marine organisms, including top-predators (Vetter et al., 2001; Weijs et al., 2009, Shaw and Kannan, 2009). There are no known anthropogenic sources of these compounds. The origin has been suggested to be biogenic production via metabolism of PBDEs or natural production via a biobromination pathway. MeO-PBDEs are naturally-produced in the marine ¨ environment by sponges or algae (Vetter et al., 2002; Malmvarn et al., 2005). There is, at present, exceedingly little information about the biological effects and thus the toxicological potential of environmentally relevant MeO-PBDEs in laboratory animals and wildlife. A previous study showed the cytotoxic effect of 6-MeOBDE-47 in human hepatoma cell line HepG2 (An et al., 2010). Anti-bacterial and anti-inflammatory activity was observed in bacteria exposed to 20 MeO-BDE-68 (Kuniyoshi et al., 1985). The exposure of POPs to humans can take place through skin absorption, respiration and especially ingestion of contaminated food (Safe, 1998), although unfortunately, some of them can take place through accidental contamination (Chen et al., 1982; Kannan et al., 1992) and at workplace (Nordin et al., 2002). Skin absorption and respiration are not the main route of exposure to POPs. Despite the prohibition of use of these compounds in Tunisia, they have also been found in sediments (Cheikh et al., 2002; Ameur et al., 2011), in fish (Masmoudi et al., 2001; Masmoudi et al., 2007), in human breast milk and serum (Ennaceur and Driss, 2010; Hassine et al., 2012). Bizerte Lagoon (Northern Tunisia) is an area of vital environmental importance. Many resident marine species live and feed in this area, and many of the pelagic species reproduce there. However, this lagoon is subjected to many anthropogenic pressures including urbanization, industrial activities (cement works, metallurgical industry, boatyards), as well as naval and commercial shipping harbors. Lagoon shores have also been used as open-air waste-dumping sites. The direct and indirect discharges of urban and industrial wastes and runoff have resulted in the chemical contamination of the lagoon by various toxic compounds such as OCPs (Cheikh et al., 2002), halogenated aromatics compounds like PCBs (Derouiche et al., 2004), polycyclic aromatic hydrocarbons (PAHs) (Trabelsi and Driss, 2005), PBDEs and their methoxylated analogs (Ben Ameur et al., 2011) and heavy metals (Ben Garali et al., 2010). Based on the results of these previous works, and given the existence of a positive correlation between fish consumption and Tunisian breast milk levels of polychlorinated biphenyls (Ennaceur and Driss, 2010), it is necessary to clarify the status of their contamination in such Tunisian foodstuffs. Despite the presence of all these possible contaminants, there are no studies about the accumulation of persistent halogenated pollutants in the benthic marine species from Bizerte Lagoon. To acquire further data on the state of contamination of Bizerte Lagoon and to assess potential risks for fish consumers, this study investigated the residue levels of persistent organohalogen pollutants in a benthic fish species among its edible marine species. 2. Materials and methods 2.1. Sample collection Bizerte Lagoon is located in northern Tunisia (Fig. 1). It extends for about 150 km2, between latitude 37108 and 37114N and longitude 9148 and 9156E and is connected to the Mediterranean Sea and Lake Ichkeul by straight channels.

Fig. 1. Map showing sampling areas. Thirty fish samples from the Bizerte Lagoon and ten fish samples from the Mediterranean Sea (reference area) were sampled using a gill net, in December 2010 (Fig. 1). The fish were immediately sacrificed, weighed, measured, dissected and kept frozen (  20 1C) until required for chemical analyses. Each individual fish sample was lyophilized, homogenized separately and used for chemical analysis. The fish species collected in the present study was the sole (Solea solea).

2.2. Chemicals The solvents used in this study (n-hexane, acetone and dichloromethane) were pesticide quality and were obtained from Fluka (Buchs, Switzerland). Sulphuric acid was obtained from Biotechnica. Florisil (60–100 mesh) was obtained from Fluka, activated at 650 1C for 8 h and re-heated at 130 1C for 5 h before use. Anhydrous sodium sulfate suitable for use in pesticide analysis was purchased from Fluka, heated at 300 1C and stored in a 130 1C oven. OCPs standards (HCB, b-HCH, lindane, p,p0 -DDE, p,p0 -DDD, o,p0 -DDD, and p,p0 -DDT) with purities ranging from 97 to 99%, were obtained from Polyscience Corporation Analytical Standards (Niles, IL, USA) and individual standard solution of each pesticide were prepared in hexane at 1000 mg mL  1 except b-HCH which is dissolved in acetone. A standard mixture of twelve PCB congeners (PCB-18, 28, 31, 52, 44, 101, 149, 118, 153, 138, 180 and 194) at 10 mg mL  1 in heptane was purchased from Supelco (CIL, USA). Individual standard solutions of PBDEs (BDE28, BDE-47, BDE-66, BDE-85, BDE-99, BDE-119, BDE-138, BDE-153, BDE-154, and BDE-183) at 50 mg mL  1 in isooctane were purchased from Supelco (CIL, USA). The MeO-PBDE analytical mixture standard solution was purchased from Wellington Laboratories (Guelph, ON, Canada) containing 6-MeO-BDE-47, 20 -MeO-BDE-68, 5-MeO-BDE-47, 4-MeO-BDE-49, 5-MeO-BDE-100, 4-MeO-BDE-103, 5-MeO-BDE99, and 4-MeO-BDE-101 at 50 mg mL  1 in nonane. These standard solutions were further diluted by n-hexane to obtain mixed fortifying and GC calibration standard solutions for all compounds.

2.3. Sample preparation and extraction Organohalogen compounds were analyzed following the method described by Guo et al., 2008a with slight modifications. Freeze dried muscle tissue (10 g) was Soxhlet extracted with n-hexane:acetone (4:1; v/v) for 16 h at a rate of five cycles per hour. The extract was concentrated with a rotary evaporator. An aliquot of 1 mL was used for gravimetric determination of the extractable lipid content. The remaining lipids, after adding BDE-77 as internal standard were removed by treatment with concentrated sulfuric acid (4  10 mL). Further, cleanup was done on a column (40  0.5 cm ID) packed with 5 g of activated Florisil and topped with 1 g of anhydrous sodium sulfate. The extract was eluted with 50 mL of dichloromethane

W. Ben Ameur et al. / Ecotoxicology and Environmental Safety 88 (2013) 55–64 and n-hexane (1:9; v/v). The eluate was finally concentrated in a Kuderna-Danish to 0.5 mL and was ready for instrumental analysis. 2.4. Instrumental analysis The analysis of OCPs and PCBs was performed on an Agilent Model 6890 Gas Chromatograph equipped with a 63Ni electron capture detector (GC-ECD). In total, 2 mL of extract was injected in splitless mode into a PTE-5 (30 m  0.32 mm i.d., 0.32 mm film thickness) capillary column, using hydrogen (H2) carrier gas with a 1 mL min  1 flow rate. The selected chromatographic conditions were similar to those described by Ennaceur and Driss (2010). Quantitative and qualitative analysis of OCPs and PCBs were done by comparison with an external standard. Compounds analyzed by GC-ECD were confirmed in each extract sample by GC–MS using a Finningan Trace MS instrument, working at electron impact (EI) ionization mode, operated by Xcalibur Software, equipped with a DB-5 ms capillary column (30 m  0.25 mm i.d., 0.25 mm film thickness). PBDEs and MeO-PBDEs were analyzed by by GC coupled with electron capture negative ionization (ECNI)-MS. GC-ECNI-MS analyses were performed on a gas chromatograph Agilent 6890 connected to a mass spectrometer Agilent 5973 ˇ Madrid, Spain). An HP-5 ms (30 m  0.25 mm Network (Agilent Technologies Espana, i.d., 0.25 mm film thickness) containing 5% phenyl methyl siloxane (model HP 19091S-433) capillary column was used. The temperature program was from 110 1C (held for 1 min) to 180 1C (held for 1 min) at 8 1C min  1, then from 180 to 240 1C (held for 5 min) at 2 1C min  1, and then from 240 to 265 1C (held for 6 min) at 2 1C min  1, using the splitless injection mode during 1 min, and injection volume of 2 mL. The operating conditions were as follows: ion source temperature¼ 250 1C, ammonia as chemical ionization moderating gas at an ion source pressure of 1.9  10  4 Torr. Experiments were carried out by monitoring the two most abundant isotope peaks from the mass spectra corresponding to m/z¼ 79 and 81 ([Br]-). 2.5. Quality control Procedural blanks were analyzed simultaneously with every batch of five samples to check for interferences or contamination from solvent and glassware. No analytes of interest were detected. Using the described methodology, recoveries ranged from 69 to 90% for OCs and from 46 to 90% for organobromine compounds. All samples were recovery corrected. Relative standard deviations of the method (n¼5) were below the 10%, indicating acceptable repeatability of the method. Multi-level calibration curves in the linear response interval of the detector were created for the quantification and good correlation (r2 40.995) was achieved. The limit of detection (LOD) calculated as three times the signal to noise ratio, ranged from 500 to 1000 pg g  1 lipid weight for organochlorines and from 35 to 695 pg g  1 lipid weight for organobromines. Confirmation criteria for the detection and quantification of PBDEs and MeOPBDEs included the following: (a) all m/z monitored for a given analyte should maximize simultaneously 7 1 s, with signal to noise ratio Z 3 for each; and (b) the ratio between the two monitored ions should be within 15% of the theoretical value. Quantification of the target compounds was carried out by internal standard procedure with the BDE-77 as internal standard. 2.6. Statistical analysis Statistical treatment of the obtained results was performed with SPSS software (SPSS 10.0 for Windows, SPSS Inc.). Our data were not normally distributed. In the first step we tried to log-transform the data, but even then most of them were still not normally distributed. Therefore we further used non-parametric tests for these data. For general comparisons, we used k-independent sample and if difference was significant (p o 0.05), subsequent multiple comparisons between fish groups and sites were tested using Mann–Whitney U-test. Spearman’s rank correlation was used to examine the strength of associations between parameters. Statistical significance was accepted at p o0.05.

3. Results and discussion After the collect of fish, we measured length, weight and lipidcontent of each sole sample. The average length of sole collected from Bizerte Lagoon and the Mediterranean Sea is 24.472.7 cm and 23.071.9 cm, respectively. The average weight is 144751 g and 117715 g, respectively in sole from Bizerte Lagoon and the Mediterranean Sea. The lipid-content average was equal to 2.1471.2% and 4.3670.9%, respectively in sole from Bizerte Lagoon and the Mediterranean Sea. There is no significant difference between the morphometric data of fish samples in the two investigated sites (P40.05). However, there is a statistically significant difference

57

between lipid percent values of sole samples in the two studied sites (Po0.05). Organohalogen compounds were detected in sole muscle samples from both investigated areas, this could indicate their widespread contamination in the investigated aquatic environment (Table 1). OCPs, PCBs, PBDEs and MeO-PBDEs measured in all samples accounted for 20.9, 57.6, 13.1 and 8.3% of total organohalogen compounds, respectively in Bizerte Lagoon. In the Mediterranean Sea, OCPs, PCBs, PBDEs and MeO-PBDEs accounted for 11.0, 39.2, 7.00 and 42.8% of total organohalogen compounds, respectively. Our results revealed that PCBs constituted the most abundant OCs in the fish samples in both investigated areas, with mean sum concentrations of 747 and 299 ng g  1 lw in Bizerte Lagoon and the Mediterranean Sea, respectively. In the Bizerte Lagoon PBDEs represented the most abundant organobromine compounds, with mean sum concentration of 170 ng g  1 lw. In contrast, MeO-PBDEs represented the most abundant organobromine compounds, with mean sum concentration of 325 ng g  1 lw, in the Mediterranean Sea. The sum of PBDE and Me-BPBDE concentrations were lower compared to the sum of OCPs and sum of PCBs in fish samples collected from Bizerte Lagoon (Mann–Whitney, Po0.05). The organochlorine levels were significantly higher in Bizerte Lagoon than in the reference area (the Mediterranean Sea) (Table 1, Mann–Whitney, Po0.05). The relatively high organochlorine levels in Bizerte Lagoon are attributable to the many sources of agricultural, municipal, and industrial contamination in Bizerte region. In particular, these chemicals mainly arrive in the studied ecosystem as a consequence of evaporation, atmospheric fallout, surface run-off, and wastewater discharges from the intensively cultivated areas, the densely populated urban centers, the large industrial complexes, and the many waste dumps clustered along the coast (Derouiche et al., 2004; Trabelsi and Driss, 2005). The high PBDE levels in Bizerte Lagoon compared to the Mediterranean Sea may be caused by the industrial development and population growth. The most obvious sources are effluents from factories producing textile and plastic products and from local wastewater discharges (Derouiche et al., 2004; Trabelsi and Driss, 2005). In general, the concentrations of OCs in fish samples detected in this study followed this order: PCBs4DDT4HCB. Our results were in accordance with that reported in fish samples from Italy (Naso et al., 2005). The distributions of organohalogen compounds were different in fish from the two investigated areas. This result was in accordance with that reported in fish samples from the Yangtze River Delta, China (Su et al., 2010). For PCBs, OCPs as well as PBDEs, concentrations were significantly lower in Mediterranean Sea fishes compared to the Bizerte Lagoon fishes (Mann–Whitney, Po0.05). Concentrations of MeO-PBDEs in the studied species were significantly higher in the Mediterranean Sea than in Bizerte Lagoon (Mann–Whitney, p o0.05). Similar findings were obtained in a study conducted in Brazil showing MeO-PBDE levels higher in dolphin samples collected from oceanic waters than those collected from coastal waters (Dorneles et al., 2010) and in Tunisian fish samples (Ameur et al., 2011). 3.1. Organochlorine pesticides Among the organochlorine pesticides analyzed HCB, p,p0 -DDE, o,p -DDD, were most commonly detected and were dominant in almost all samples (Table 1). In our study, DDT and its derivatives followed this general order: DDE4DDD 4DDT (Fig. 2A). Our results are in accordance with reported order of DDT metabolites in fish from other countries (Perugini et al., 2004, Naso et al., 2005, Covaci et al., 0

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W. Ben Ameur et al. / Ecotoxicology and Environmental Safety 88 (2013) 55–64

Table 1 Organohalogen compound concentrations (ng g  1 lw) in Solea solea muscle from Bizerte Lagoon and the Mediterranean Sea. Bizerte Lagoon

Mediterranean Sea

Mean

SD

Range

Mean

SD

Range

HCB b-HCH g-HCH p,p0 -DDE o,p0 -DDD p,p0 -DDD p,p0 -DDT P HCHs P DDTs P OCPs

58.1 3.50 10.8 105 56.3 29.0 8.20 14.1 199 271

54.4 7.8 11.6 69.4 37.6 23.6 24.4 16.9 126 190

1.70–18.0 nd-26.5 nd-30.3 21.8–281 14.8–145 9.2–85.8 nd-87.4 nd-58.0 54.2–512 65–752

15.4 3.00 3.40 38.1 15.1 9.00 nd 6.30 62.1 84.0

1 4.9 3.5 8.4 7.3 4.80

14.5–16.5 nd-8.50 nd-7.00 30.2–46.9 10.8–23.5 4.50–14.0

2.6 11.1 13.2

3.4–08.50 55.2–74.9 73.2–98.6

PCB-18 PCB-28þ 31 PCB-52 PCB-44 PCB-101 PCB-118 PCB-149 PCB-138 PCB-153 PCB-180 PCB-194 P PCBs P OCPs þPCBs

4.30 nq 14.4 10.0 95.0 87.2 37.1 98.0 211 170 20.0 747 1018

7.7

nd-18.5

18.2 11.6 66.6 62.8 38.7 63.8 57 91.3 32.6 319 497

nd-51.9 nd-31.7 27.2–207 35.3–221 nd-111 24.5–235 132–317 49.6–285 nd-99.2 315–1417 384–2169

nd nq 3.50 3.00 25.2 33.3 5.00 28.5 120 80.5 nd 299 383

3.60 1.00 1.90 3.6 5.9 0.80 30.6 22.5

nd-7.30 1.50–3.50 23.6–27.2 29.4–36.4 nd-11.4 28–29.5 94.4–153.8 64.3–106

35.2 28.3

257–319 348–398

BDE-28 BDE-47 BDE-99 BDE-100 BDE-153 BDE-154 BDE-183 P PBDEs

17.3 96.2 3.0 2.00 12.0 13.1 27.3 171

22.4 67.1 4.80 4.4 13.3 14.7 19.5 104

nd-78.2 20.6–205 nd-15.2 nd-15.4 nd-45.6 nd-43.6 nd-77.2 51.2–354

3.60 40.5 0.41 0.85 3.00 2.91 6.00 57.2

3.43 26.1 0.71 1.5 4.48 2.58 5.24 26.1

nd-6.84 15.7–67.7 nd-1.20 nd-2.60 nd-7.76 nd-4.90 nd-9.66 38–86.7

6-MeO-BDE-47 20 -MeO-BDE-68 RMeO-PBDEs P PBDEsþ MeOPBDEs

65.5 42.4 108 279

75.4 34.9 105 112

nd-176 nd-96.7 nd-248 163–499

224 101 325 381

48 9.82 46.6 115

175–225 89.8–109 284–375 360–413

nd¼ not detected. nq¼ not quantifiable. SD¼ standard deviation.

2006, Sudaryanto et al., 2007, Hosseini et al., 2008). These results are not surprising considering the high chemical stability and hydrophobicity of p,p0 -DDE (log Kow value of 6.36) and its long half-life and, hence, persistence in both abiotic and biotic components of the aquatic ecosystems (Naso et al., 2005). The DDE/DDT ratio is commonly used to assess the chronology of DDT input into the ecosystems. This ratio ranged between 1.3 and 2.1 in fish from Bizerte Lagoon. It was remarkably higher than the ratio found in other studies (between 0.2 and 0.9) (Szlinder-Richert et al., 2008 and Li et al., 2008). High concentrations of DDE and low DDT levels in fish suggest that DDTs have not been recently used in agriculture after their ban in 1984 (Perugini et al., 2004; Tunisia Country Situation Report APEK, 2005). Another metabolite of DDT, p,p0 -DDD, was also found, but in lower amounts than p,p0 -DDE. Higher proportions of p,p0 -DDE to p,p0 -DDD suggested that DDT underwent aerobic transformation in organisms (Zhou et al., 2008). DDT mean concentrations detected in fish from Bizerte Lagoon are reviewed and compared with those found in similar studies from other Mediterranean and non-Mediterranean regions. It can be seen that the mean levels of DDTs detected in specimens from Bizerte Lagoon are generally comparable to that reported for specimens from the Marmara Sea in Turkey (231 ng g  1 lw; Coelhan et al., 2006), from Thailand (120 ng g  1 lw; Kannan et al., 1995) and Indonesia (120 ng g  1 lw; Sudaryanto et al., 2007). Our concentrations are

lower than those detected in fish from other Mediterranean aquatic ecosystems such as the Gulf of Naples in Italy (917 ng g  1 lw; Naso et al., 2005), Australia (650 ng g  1 lw; Kannan et al., 1995), Vietnam (1400 ng g  1 lw; Kannan et al., 1995) and the Ebro Delta in Spain (481 ng g  1 lw; Pastor et al., 1996). Otherwise, the DDT levels found in this study are generally higher than those reported from the Orbetello lagoon in Italy (60.7 ng g  1 lw; Corsi et al., 2005) and from marine waters of Cambodia (73 ng g  1 lw; Monirith et al., 1999). The HCH compounds were also detected in fish samples at mean levels of 14.1 and 6.30 ng g  1 lipid wt, respectively in Bizerte Lagoon and the Mediterranean Sea (Table 1). g-HCH was the predominant isomer in the analyzed samples (Fig. 2B) and coincided with results from other studies (Masmoudi et al., 2001). This suggests a preferential usage of lindane (pure g HCH, the most toxicological active HCH isomer) than the technical mixture of HCHs on Bizerte Lagoon shores farms and its usages as a human scabicide. HCH mean concentrations in the studied fish species from Bizerte Lagoon were lower than that of fish from Mediterranean regions such as Italy (142 ng g  1 lw; Corsi et al., 2005) and Turkey (77 ng g  1 lw; Coelhan et al., 2006) and similar to those of fish from the Adriatic Sea (17.3 ng g  1 lw; Stefanelli et al., 2004). Although the usage of HCB has been banned in Tunisia since 1984 (Tunisia Country Situation Report APEK, 2005), all analyzed fish samples contained HCB residues, with mean levels of 58.1 and 15.4 ng g  1 lipid wt, respectively in Bizerte Lagoon and the

W. Ben Ameur et al. / Ecotoxicology and Environmental Safety 88 (2013) 55–64

100%

100%

90%

90%

80%

80% 70%

60%

4,4' DDT

50%

4,4' DDD

40%

2,4' DDD

30%

4,4' DDE

30%

20%

2,4' DDE

20%

10% 0%

Percentage

Percentage

70%

50%

γ-HCH

40%

0%

BL MS Sampling areas

β-HCH

BL MS Sampling areas

100%

90%

CB194

80%

CB180

70%

CB153

60%

CB138

50%

CB149

40%

CB118

30%

CB101

20%

CB44

10%

CB52 Sole BL Sole MS Sampling areas

CB18

90% Percentage of total PBDEs

Percentage

60%

10%

100%

0%

59

80%

BDE 183

70%

BDE 154

60%

BDE 153

50%

BDE 100

40%

BDE 99

30%

BDE 47

20%

BDE 28

10% 0%

BL MS Sampling areas

Fig. 2. Composition of (A) DDTs; (B) HCHs; (C) PCBs and (D) PBDEs in fish muscle from the Bizerte Lagoon (BL) and the Mediterranean Sea (MS).

Mediterranean Sea (Table 1). The present finding of HCB in marine organisms from Bizerte Lagoon may be ascribed not only to its previous use as a fungicide in treatment for seeds but also to the fact that it can be released from high temperature industrial processes. It may also be present as impurity in other OCPs (Erdogrul et al., 2005). Incineration may also contribute to HCB pollution (Sudaryanto et al., 2007). The comparison with worldwide studies shows that HCB mean levels in Bizerte Lagoon fish were lower than those from Spain (284 ng g  1 lw; Pastor et al., 1996), Australia (120 ng g  1 lw; Kannan et al., 1995) and Iran (170 ng g  1 lw; Davodi et al., 2011), and higher than those from Italy (17.6 ng g  1 lw; Corsi et al., 2005), Turkey (4.92 ng g  1 lw; Coelhan et al., 2006), Thailand (4.5 ng g  1 lw; Kannan et al., 1995), Indonesia (1.6 ng g  1 lw; Sudaryanto et al., 2007), Gambodia (1.6 ng g  1 lw; Monirith et al., 1999), Vietnam (2.6 ng g  1 lw; Kannan et al., 1995) and USA (2.94 ng g  1 lw; Sajwan et al., 2008). 3.2. Polychlorinated biphenyls PCBs were found in all the samples (100% of the samples). Individual PCB congener’s distribution patterns were shown in Fig. 2C. PCB-118, -153, -138 and -180 were dominant contaminants in fishes collected from both areas, accounting, respectively for 11.7, 28.3 13.1 and 22.8% of total PCBs detected in the biota collected from Bizerte Lagoon, while in the fishes caught from the Mediterranean Sea, these congeners accounted for 11.1, 40.2 9.57 and 27.0%, respectively. Similar results have been reported for PCB-153, -180, 118 and -138 as the predominant congeners in fishes from other regions (Naso et al., 2005, Nie et al., 2005). The 180, 153, 138, and 118

PCB congeners turned out to be the most abundant due both to their high lipophilicity, stability, and persistence that facilitate the adsorption to sediments and the accumulation in the aquatic ecosystem, and to their molecular structure (Naso et al., 2005). The PCB congener distribution in the fish species from both locations (Fig. 3) showed a predominance of mid- chlorinated PCB congeners (mainly composed of tetra-, penta-, and hepta-CB congeners). The observed trends in PCB congener compositions were in accordance with those observed in Bizerte Lagoon sediments in Tunisia (Derouiche et al., 2004) and in other studies on fishes (Naso et al., 2005; Storelli et al., 2010). Concentrations of the target PCB congeners in Bizerte Lagoon fish are lower than those reported in fish from Mediterranean aquatic ecosystems such as Tunis Bay in Tunisia (185–5566 ng g  1 lw; Masmoudi et al., 2007), Adriatic sea in Italy (411–1697 ng g  1 lw; Perugini et al., 2004) and in the Orbetello lagoon in Italy (nd-16,179 ng g  1 lw; Corsi et al., 2005). These levels are higher than those found in fish, from the Ria Aveiro in Portugal (275 ng g  1 lw; Antunes et al., 2004), the Atlantic coast in USA (12–496 ng g  1 lw; Sajwan et al., 2008), Thailand (30 ng g  1 lw; Kannan et al., 1995), Gambodia (18 ng g  1 lw; Monirith et al., 1999), Indonesia (360 ng g  1 lw; Sudaryanto et al., 2007) and from the Marmara Sea in Turkey (6.95 ng g  1 lw; Coelhan et al., 2006). Comparable levels were reported in other fish species from the Gulf of Naples in Italy (2387–31,274 ng g  1 lw; Naso et al., 2005). 3.3. Polybrominated diphenyl ethers The PBDE profiles measured in Bizerte Lagoon fishes and Mediterranean Sea fishes are presented in Fig. 2D. Very similar

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patterns were obtained for sole samples in both sites. In all the samples, BDE-47 was the predominant among the seven detected P congeners. For instance, its contribution to PBDEs ranged from 33.1 to 79.7% and from 41.3 to 83.2%, respectively in sole from Bizerte Lagoon and from the Mediterranean Sea. The dominance of BDE-47 in the present study was consistent with the general pattern found in biota samples in other studies (Eljarrat et al., 2005; Labandeira et al., 2007). In this study, the aquatic biota from the Bizerte Lagoon seemed to accumulate higher proportions of tri-BDEs (BDE-28) than penta-BDEs (BDE-100, BDE-99). This result was in line with results in the Yangtze River Delta (Xian et al., 2008) and Pearl River Delta (Guo et al., 2008b). In these studies, PBDE congener profiles were markedly different from the patterns observed in marine fishes from other regions of the world, which are generally dominated by tetra- to hexa-BDEs (Eljarrat et al., 2005; Labandeira et al., 2007). In these studies, fishes accumulated higher proportion of PBDEs from di- to tetra-BDEs such as BDE15, BDE-28 and BDE-47. The debromination of higher PBDE congeners in the environment is a plausible reason for the prevalence of BDE-28. Some studies have shown that the potential decomposition of higher brominated congeners under sunlight (Bezares-Cruz et al., 2004), through microbial debromination (He et al., 2006; Lee and He, 2010) and reductive debromination catalyzed by iron and iron sulfides (Keum and Li, 2005) can lead to the formation of BDE-28. Another possible explanation of higher proportion of BDE 28 congener in fish muscle from the two investigated areas, is may be due to a different foodwebs compared to the aquatic ecosystems around the world and consequently variable fish food sources (Shaw and Kannan, 2009). The congener pattern observed in this study was similar to those described in previous studies conducted for fish samples (Xian et al., 2008; Gao et al., 2009). PBDE mean concentrations observed in Bizerte Lagoon samples were comparable to those obtained in fish from Spain (160 ng g  1 lw; Labandeira et al., 2007), Sweden (130 ng g  1 lw; Kierkegaard et al., 1999), Baltic sea (86 ng g  1 lw; Burreau et al., 1999) and China (140 ng g  1 lw; Xian et al., 2008). However, concentrations in the present study were relatively lower than those from Hardley Lake, USA (1600 ng g  1 lw; Dodder et al., 2002), Belguim (1550 ng g  1 lw; Roosens et al., 2008), Sweden ¨ et al., 1996), Canada (1612 ng g  1 lw; (2140 ng g  1 lw; Sellstrom Ikonomou et al., 2006) and Yakima River, USA (960 ng g  1 lw; Johnson and Olson, 2001). The concentrations observed in Bizerte Lagoon were relatively higher than those from Georgia coast, USA (77.5 ng g  1 lw; Sajwan et al., 2008), Japan (46 ng g  1 lw; Ohta et al., 2000), northwest Atlantic (62 ng g  1 lw; Shaw et al., 2009) and much higher than those from Eastern China coastline (3.04 ng g  1 lw; Xia et al., 2011), Sweden (17 ng g  1 lw; 100% 90% 80% Percentage

70%

Octa Cl

60%

Hepta Cl

50% 40%

Hexta Cl

30%

Penta Cl

20%

Tetra Cl

10%

Tri Cl

0%

BL

MS Sampling areas

Fig. 3. Percentage contribution of different Cl-substituted PCBs in sole muscle from the Bizerte Lagoon (BL) and the Mediterranean Sea (MS).

¨ Sellstrom et al., 1996), Vietnam (12 ng g  1 lw; Hien et al., 2012), Chile (16 ng g  1 lw; Montory and Barra, 2006), Australia (13 ng g  1 lw; Ueno et al., 2004) and Storfjorden (3.55 ng g  1 lw; Wolkers et al., 2004). 3.4. Methoxylated polybrominated diphenyl ethers Only two out of the 8 targeted MeO-PBDE congeners were found at measurable levels in the studied fish samples (Table 1): 20 -MeO-BDE-68 and 6-MeO-BDE-47. These MeO-PBDEs have also been detected in previous studies (Pena-Abaurrea et al., 2009; Su et al., 2010). Concentrations of SMeO-PBDEs ranged from nd to 248 ng g  1 lw and from 284 to 375 ng g  1 lw in sole from Bizerte Lagoon and Mediterranean Sea, respectively (Table 1). 20 MeOBDE-68 and 6-MeO-BDE-47 accounted for 39.4 and 60.6% of P the MeO-PBDE concentration in sole from Bizerte Lagoon and P 31.1 and 68.9% of the MeO-PBDE concentration in sole from the Mediterranean Sea, respectively. As suggested by Vetter (2006), a higher contribution of 20 -MeO-BDE-68 would indicate sponges as the dominant source of MeO-PBDEs, while a higher proportion of 6-MeO-BDE-47 would point to algae as the principal source of MeO-PBDEs. Therefore, the ratio between these two naturally-produced organobrominated compounds (20 -MeOBDE-68/6-MeO-BDE-47) was calculated for each individual in the present study. For fish from Bizerte Lagoon this ratio ranged from 0.30 to 0.73. For fish from the Mediterranean Sea, the above mentioned ratio ranged from 0.40 to 0.62. Thus, and according to Vetter (2006), the fish samples from both areas would be receiving MeO-PBDEs predominantly from algae. Our result showed a significant difference in MeO-PBDE concentrations in sole muscle between Bizerte Lagoon and the Mediterranean Sea. Because there is no anthropogenic source of these MeO-PBDEs, the difference in the concentrations may possibly be due to the difference in the distribution of marine sponge, algae, and other aquatic organisms that are known to synthesize these compounds. In our study there was no significant correlation between the levels of BDE-47 and 6-MeO-BDE-47 in sole (rs¼0.23, p40.05) from Bizerte Lagoon, suggesting that 6-MeO-BDE-47 is not only a possible metabolic product of BDE-47 in fish but could also come from other marine sources. This fact is also supported by Vetter et al. (2001) who did not detect significant amounts of parent compounds (PBDEs) in marine mammals from northeastern Australia despite the high levels of 60 -MeO-BDE47. Since the levels of 6-MeO-BDE 47 were highly correlated with the levels of 20 -MeO-BDE 68 in fish from Bizerte Lagoon (rs¼0.794, po0.05), it is highly plausible that these compounds have both accumulated from similar sources. The mean MeO-PBDEs concentration found in Bizerte Lagoon fish, 108 ng g  1 lw, is similar to that found in wild bluefin tuna (Thunnus thynnus) from the Mediterranean Sea, 150 ng g  1 lw (Pena-Abaurrea et al., 2009) and it was higher than that found in anchovy (coila sp) from the Yangtzer River Delta, 9.10 ng g  1 lw (Su et al., 2010) and to that found in marine species from the Sydney Harbor, 26.7 ng g  1 lw (Losada et al., 2009). 3.5. Correlation of contaminant levels with fish characteristics Since it has been reported that POP accumulation can be affected by biological factors, we tested for correlation between PCB, HCB, HCH, DDT, PBDE and MeO-PBDE concentrations and some specific characteristics of fish samples. With respect to PCBs, P we revealed statistically significant correlation between PCB concentrations, lipid content (rs¼ 0.557, po0.05), length (rs¼0.563, po0.05) and weight (rs¼0.723, po0.05). With respect P to OCPs, HCB (rs¼ 0.632, po0.05) and HCHs (rs¼  0.603,

W. Ben Ameur et al. / Ecotoxicology and Environmental Safety 88 (2013) 55–64

po0.05) were correlated with lipid content. A close significant correlation existed between DDT concentrations and lipid content (rs¼  0.672, po0.05), but no correlation was found with fish length and weight. These results were similar to those found by Deshpande et al. (2002), Chan et al. (1999) and by Ferrante et al. (2010). Similarly, levels of PBDEs were correlated with lipid content (rs¼0.726, po0.05), while these were not correlated with fish weight and length. These results were in accordance with those found by Luo et al. (2007), Mariottini et al. (2008), Borghesi et al. (2009) and by Gao et al. (2009). Finally, MeO-PBDE levels were not correlated with lipid percentage or length or weight. These results were similar to those found by Strid et al. (2010). Note that the inverse relationship between fat content and pollutant levels is consistent with the results of other authors (Larsson et al., 1991; Ferrante et al., 2010) and provides support to the hypothesis that the pollutants are diluted in the increasing fat contents (Larsson et al., 1991). 3.6. Correlation between organohalogens Pearson correlation analysis showed that the concentrations of HCB, HCHsDDTs, PCBs and PBDEs were significantly correlated to each other (Table 2), indicating a similar exposure pathway of these compounds to the studied fish species. MeO-PBDEs showed no correlation with other organohalogen compounds, suggesting that these pollutants have a different source such as sponges or algae with the POPs in the marine environment.

61

3.7. Risk assessment of human exposure To understand the magnitude of exposure to the different pollutants by fish consumption, we calculated the estimated daily intake (EDI) of HCB, lindane, DDTs, PCBs, PBDEs and MeO-PBDEs through the studied fish species consumption for the general population in Bizerte (Northern of Tunisia) (Table 3). The estimated daily intake (EDI; ng/kg body weight/day) of all target compounds by the following equation: EDI ¼

FDC X C BW

where FDC is fish daily consumption, C is the concentration of detected compounds mean, and BW is body weight. The daily fish consumption value (25 g) was obtained from the Institut National de Nutrition et de Technologie Alimentaire and we adopted 60.0 kg as the mean Tunisian body weight. EDI of DDTs and g-HCH were far below the acceptable daily intake (ADI) recommended by the Food and Agriculture Organization of the United Nations/World Health Organization (FAO/ WHO) indicating that this intake would not pose a human health risk. The literature about the dietary intakes of the selected pollutants in this study is very scarce. Our dietary intakes for HCB and PCBs, with respective values of 21.9 and 282 ng/day, were higher than those obtained in China (HCB: 11.4 ng/day; PCBs: 110 ng/day) (Yang et al., 2006) and similar to those in Sweden (HCB: 36 ng/day;

Table 2 Spearman’s rank correlation coefficients between concentrations of major POPs in sole muscle collected from Bizerte Lagoon and the Mediterranean Sea. P HCHs

HCB

HCB P HCHs P DDTs P PCBs P PBDEs P MeO-PBDEs

P DDTs

P PCBs

P PBDEs

P MeO-PBDEs

BL

MS

BL

MS

BL

MS

BL

MS

BL

MS

0.60** 0.91* 0.88*  0.73** 0.06

0.72** 0.82* 0.90*  0.84** 0.08

0.90* -0.73** 0.89** 0.06

0.88* 0.66** 0.79** 0.1

0.91*  0.74** 0.19

0.85*  0.72** 0.22

 0.77** 0.17

 0.80** 0.09

 0.42

 0.30

BL

MS

p o0.05. p o 0.001.

nn n

Table 3 National average exposures and benchmark concentrations for contaminants in fish and the estimated daily intakes of HCB, g-HCH, DDTs, PCBs, PBDEs and MeO-PBDEs through the studied fish species by human (average body wt. 60 kg) in Bizerte (Northern Tunisia). Compounds Oral RFD (mg/ CSF (mg/ kg/day) kg/day)

HCB g-HCH DDTs PCBs PBDEs Meo-PBDEs BDE 47 BDE 99 BDE 153

8.0E  04 3.0E  04 5.0E  04 2.0E  05

1.0E  04 1.0E  04 2.0E  04

1.6 1.30 0.34 2.0

Cancer benchmark concentration (ng/kg/day)

1.50 1.85 7.06 1.20

50th HR (95th (50th MEC) (ng/g MEC) (ng/g wet wt.) wet wt.)

95th HR

EDI (ng/ kg/day)

ADI (ng/ kg/day)

So BL So MS

So BL So MS

So BL

So MS

So BL

So MS

So BL

So MS

0.82 0.10 2.55 11.30 2.47 0.79

2.16 0.47 5.92 18.01 5.01 3.67

0.23 0.01 0.58 3.92

0.06 0.00 0.19 1.67

0.60 0.03 1.34 6.25

0.07 0.01 0.25 1.67

0.37 0.04 0.75 2.82 0.64 0.41

0.10 8.000 0.02 20.000 0.39 1.88 0.36 2.04

0.23 0.05 0.85 4.80 0.69 4.75

0.25 0.10 1.10 4.81 1.25 5.58

6.0E  03 2.5E  03 6.0E  03 2.5E  03 9.8E  05 2.6E  05 9.8E  05 2.6E  05 7.5E  04 1.6E  04 7.5E  04 1.6E  04

So BL¼ Sole collected from Bizerte Lagoon, So MS: Sole collected from the Mediterranean Sea. CSF¼ cancer slope factor. 50th MEC¼ 50th percentile measured concentrations; 95th MEC: 90th percentile measured concentrations HR: Hazard ratio. EDI ¼Estimated daily intake. ADI ¼Acceptable daily intake.

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PCBs: 349 ng/day) (Darnerud et al., 2006) and in South Korea (PCBs: 240 ng/day) (Moon et al., 2009). With regard to DDTs, our dietary intake of 75.1 ng/day was lower than those calculated in China (882 ng/day) (Yang et al., 2006) and in South Korea (270 ng/ day) (Moon et al., 2009). Finally, the dietary intake of PBDEs from our study with a value of 64.3 ng/day and a range from 19.3 to 133 ng/day was higher than those in USA (8.94–15.7 ng/day) (Schecter et al., 2006) and lower than those from Hong Kong (311–1677 ng/day) (Cheung et al., 2008). There is no published data about the dietary intakes of the MeO-PBDEs for comparison of the results obtained in this study. To screen the potential health significance of the estimated dietary exposure to pollutants, a method based on the benchmark concentration was also used for assessment. The detailed method has been described elsewhere (Jiang et al., 2005). In brief, hazard ratios (HRs) were estimated by dividing the average exposure by the benchmark concentrations (the benchmark concentration is derived by setting the cancer risk to 1000,000 for lifetime exposure). HRs exceeding 1 indicated that there was potential risk to human health. Two HRs were estimated to assess the potential risk, in which one was on the 50th percentile exposure and the other was on the 95th percentile exposure (Jiang et al., 2005). The oral reference dose (RfD) and cancer benchmark concentrations for contaminants are shown in Table 3. All of the concentrations (for HCB, lindane, DDTs and PCBs) were less than their oral RfD. Similar results were found in the Chinese coastal population (Jiang et al., 2005). Calculated HRs of noncancer risk for HCB and lindane were less than one based on their 50th and 95th percentile concentrations in both investigated areas. However, the HRs of cancer risk for DDTs (in Bizerte Lagoon) and PCBs (in the two studied sites) were greater than one based on their 50th and 95th percentile concentration, suggesting that daily exposure to these contaminants due to fish consumption had a lifetime cancer risk of greater than one in one million. These results indicate that certain contaminants, namely DDTs and PCBs, may be of particular concern. For the brominated compounds, there are no RfD available for the MeO-PBDEs and all PBDE congeners. To assess the potential health risk caused by PBDEs, the U.S. EPA proposed oral RfD values for BDEs 47, 99, and 153 as part of the IRIS Toxicological Review for each PBDE congener (U.S. EPA, 2008). Hazard ratios were calculated as the quotient of the 50th or 95th centile of the DI divided by the oral RfD (Table 2). With respect to PBDEs, although several authors have published toxicity data for PBDEs (Eriksson et al., 2001; Meerts et al., 2001; Zhou et al., 2002; Viberg et al., 2002), applicable risk assessment guidelines and regulations for PBDEs are thus far quite limited. In the United States, the Agency for Toxic Substances and Disease Registry (ATSDR) has derived a minimal risk level (MRL) of 3.0  104 ng kg  1 bw/day for acute-duration oral exposure and 7000 ng/kg bw/day for intermediate-duration oral exposure to lower brominated phenyl ethers, respectively. The ATSDR has also derived a minimal risk level (MRL) of 1.0  107 ng kg  1 bw/day for intermediate-duration oral exposure to BDE-209 based on a no observable adverse effect level of 1.0  109 ng kg  1 bw/d for developmental toxicity in rats exposed to BDE-209 for 19 days during gestation (http://www.atsdr.cdc.gov/mrls.html). Darnerud et al. (2001); Bocio et al. (2003) have recommended a lowest observed adverse effect level (LOAEL) of 1 mg kg  1/day, based on the most sensitive endpoints of toxic effects of PBDEs. The mean values of the HR for PBDE based on the 50th and 95th percentile concentrations of PBDEs are less than 1.0, and the estimated daily intake of PBDEs compiled herein was much lower than the limits described above, including minimal risk level for acute-duration oral exposure and intermediate-duration oral exposure to lower brominated phenyl ethers, developed by the

ATSDR, and the LOAEL recommended by Darnerud et al. (2001) and by Bocio et al. (2003). In all, health risk is minimal for PBDEs intake via sole consumption based on the current regulations and guidelines. In conclusion, based on the EDI and the HR values of the analyzed POPs, sole can safely be consumed by people.

4. Conclusion The Bizerte Lagoon, is located in a zone with heavy pollution stemming mainly from urban, agricultural and industrial activities. Fish are among the most accessible source of animal protein for Tunisian coastal inhabitants. However, there is a growing amount of evidence that Bizerte Lagoon fish species could be potentially contaminated by POPs and harmful to human health. In order to assess the potential health risks associated with these contaminants due to fish consumption, sole samples were collected from Bizerte Lagoon and the Mediterranean Sea. The results of this study supply information about some POP contents in muscle tissue of the examined species from Bizerte Lagoon and indirectly indicate the environmental contamination of the lagoon. Moreover, these results can also be used to understand the chemical quality of fish and to evaluate the possible risk associated with their consumption. This study accounted for only a limited number of contaminant. Additional surveys on other toxic substances such as heavy metals, polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/ Fs) compounds are needed, to determine their bioaccumulation in the food web and the associated risks to the ecosystems and human health.

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