Marine Environmental Research xxx (2014) 1e5

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Pilot study on levels of chemical contaminants and porphyrins in Caretta caretta from the Mediterranean Sea Cristiana Guerranti a, b, *, Matteo Baini a, Silvia Casini a, Silvano Ettore Focardi a, Matteo Giannetti a, c, Cecilia Mancusi d, Letizia Marsili a, Guido Perra a, Maria Cristina Fossi a a

Department of Physical, Earth and Environmental Sciences, University of Siena, Via Mattioli 4, 53100 Siena, Italy Institute of Clinical Physiology, National Research Council (CNR), Via Moruzzi, 1, 56100 Pisa, Italy Department of Life Sciences, University of Siena, Via A. Moro 2, 53100 Siena, Italy d ARPAT, Environmental Protection Agency of Tuscany Region, Livorno, Italy b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 September 2013 Received in revised form 20 January 2014 Accepted 25 January 2014

Perfluorinated compounds (PFCs), synthetic musks compounds (SMCs), bisphenol A (BPA), para-nonylphenol (p-NP) and polybrominated diphenyl ethers (PBDEs) are known for their toxicity and ability to interfere with the endocrine system. The aim of this study was to determine levels and distribution of the above mentioned compounds in liver samples of Caretta caretta and levels of porphyrins that have been proposed as sensitive biomarkers of exposure to contaminants. This paper reports the results for 9 specimens yet analysed. Musk ketone was never detected, PFOA was found in one sample, while PFOS was the prevalent contaminant. For PFCs the levels are lower than the results of studies of comparison. The porphyrins profile showed a predominance of protoporphyrins on coproporphyrins and uroporphyrins, with a positive statistical correlation between levels of PFOS and uroporphyrins. These data represent, for several parameters, the first evidence of contaminant levels and biomarker responses in loggerhead turtles. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Perfluorinated compounds Synthetic musk compounds Nonylphenol Polybrominated diphenyl ethers Porphyrins Bisphenol A Loggerhead turtle Biomarkers Bioindicator Endocrine disruptors

1. Introduction The loggerhead turtle, Caretta caretta (Linnaeus, 1758), is presently classified as globally endangered by the World Conservation Union (D’Ilio et al., 2011). The causes are attributed to diverse factors: one of the most important anthropogenic threats is pollution (D’Ilio et al., 2011). Sea turtles can bioaccumulate contaminants from food, sediment, water and marine debris, defined as any manufactured or processed solid waste imported into the marine environment (Coe and Rogers, 1997; Lazar and Gra can, 2011). In this regard, sea turtles are considered of increasing interest as potential bioindicators for pollution in marine ecosystems (Jerez et al., 2010; D’Ilio et al., 2011; Guerranti et al., 2013a).

* Corresponding author. Department of Physical, Earth and Environmental Sciences, University of Siena, Via Mattioli 4, 53100 Siena, Italy. Tel.: þ39 0577232879; fax: þ39 0577232806. E-mail address: [email protected] (C. Guerranti).

In recent times marine turtles have been proposed as indicators for Good Environmental Status (GES) for improving effectiveness of conservation strategies (e.g. Marine Strategy Framework Directive, MSFD, 2008/56/EC) (Fossi et al., 2012). The aim of this study is to determine levels and distribution of several chemical compounds in liver samples of stranded marine turtle C. caretta, from the Mediterranean, and levels of porphyrins. Given the delicate status of this species, it is evident the importance of using non invasive methods of sampling, or using tissues from stranded animals. Porphyrins, intermediate metabolites of heme biosynthesis or oxidative by-products of metabolites, are produced and accumulated in trace amounts in erythropoietic tissues, the liver and the kidneys and are excreted via urine or feces. Heme biosynthesis may be altered by several environmental contaminants (e.g. pesticides, polychlorinated biphenyls, trace elements) leading to changes in accumulation or excretion profiles; for this reason porphyrins have been proposed as sensitive biomarkers of exposure to contaminants (Fossi et al., 1996; Casini et al., 2002, 2006).

0141-1136/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.marenvres.2014.01.004

Please cite this article in press as: Guerranti, C., et al., Pilot study on levels of chemical contaminants and porphyrins in Caretta caretta from the Mediterranean Sea, Marine Environmental Research (2014), http://dx.doi.org/10.1016/j.marenvres.2014.01.004

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C. Guerranti et al. / Marine Environmental Research xxx (2014) 1e5

This pilot study reports the results for 9 stranded specimens of C. caretta analysed until now for their hepatic levels of perfluorinated compounds (PFCs), polybrominated diphenyl ethers (PBDEs), synthetic musk compounds (SMCs), bisphenol A (BPA) and para-nonylphenol (p-NP), all compounds known for their toxicity and ability to interfere with the endocrine system, (Chou and Dietrich, 1999; Barucca et al., 2006; Porte et al., 2006; Governini et al., 2009; Della Torre et al., 2011; Bechi et al., 2013; La Rocca et al., 2013; Mantovani et al., in press) together with porphyrins. Although numerous studies have demonstrated their presence in the environment and in many organisms, very little information is available on the levels of the above mentioned compounds and of porphyrins in turtles and in C. caretta in particular. 2. Materials and methods 2.1. Sampling The 9 liver samples of C. caretta analysed in this study were collected from dead turtles that were washed ashore along the coasts of Tuscany. For each specimen the curved carapace length (CCL) and the curved carapace width (CCW) were measured to the nearest cm. Table 1 gives an overview of biometric parameters of the specimens of C. caretta collected. Turtles were classified as 8 juveniles (CCL < 64 cm) and 1 adult (CCL > 64 cm), according to Broderick and Godley (1996). Sex was not determined except for 2 specimens (a male and a female).

BDE156) were identified and quantified using a GC/MS (ion trap mass spectrometer). Detection limits, calculated as the mean blank þ3SD, were 4 pg/g tissue. 2.2.3. p-NP and SMCs For p-NP and SMCs analysis, homogenated samples (about 5 g) were extracted by accelerated solvent extractor (ASE), according to US-EPA (1996) method 3545A. The extract was purified on a chromatographic column packed with 5 g of Florisil PR activated at 130  C for 16 h. The column was conditioned with 10 ml of hexane and the sample eluted with a mixture of diethyl ether/hexane (100 ml, 1:10), then evaporated under a stream of nitrogen and brought to final volume (50 ml) with nonane. For the quantitative analysis a GC/MS ion trap Polaris coupled to a gas chromatograph GC TraceTM 2000 (provided with AS3000 autosampler) (ThermoFinnigan) was used. The capillary column used was RTX-5MS (30 m  0.25 mm in inner diameter, 0.25 mm) provided by Restek. 2 ml of sample were injected in splitless mode, at 250  C. The temperature program applied was as follows: oven at 90  C for 2 min, then at 180  C with an increase of 20  C/min up to 225  C (for 10 min) with an increase of 4  C/min, up to a maximum of 300  C (for 50 ) with an increase of 30  C/min. The energy of the filament was set to 70 eV. The mass spectrometer has functioned with EI þ source (200  C), with a transfer line temperature of 300  C. SMCs and p-NP were quantified using a standard mix containing 4 SMCs (xylene, ketone, tonalide and galaxolide) and p-NP at 4 different concentrations as external standard. LOD, calculated as the mean blank þ3SD was 1 ng/g for each compound.

2.2. Chemical analysis Levels of contaminants were determined on lyophilized samples by chromatography-mass spectrometry, after various steps of extraction and purification. Analytical standards for chemicals were obtained by Sigma Aldrich Co. 2.2.1. PFCs PFOS and PFOA were extracted using an ion-pairing extraction procedure and measured using high performance liquid chromatography (HPLC) with electrospray ionization (ESI) tandem mass spectrometry. The analytical procedure for the extraction of liver samples was made following a method widely tested (Corsolini et al., 2008; Perra et al., 2010; Guerranti et al., 2011; Corsolini et al., 2012; Caserta et al., 2013; Guerranti et al., 2013b; Perra et al., 2013). LOD, determined as three times the signal-to-noise (S/N) ratio, was 0.4 ng/g. 2.2.2. PBDEs For PBDEs analysis, according to a method described by Corsolini et al. (2008) and Guerranti and Focardi (2011), samples were homogenized with anhydrous sodium sulphate salt and Soxhlet extracted with hexane:dichloromethane (40:60). Interferences were removed by fractionation by a multilayer silica gel column. Twenty BDE congeners (IUPAC numbers BDE3, BDE7, BDE15, BDE17, BDE23, BDE28, BDE47, BDE49, BDE66, BDE71, BDE77, BDE85, BDE99, BDE100, BDE119, BDE126, BDE138, BDE153, BDE154,

Table 1 Biometric parameters of the specimens of Caretta caretta sampled. CCL: curved carapace length; CCL: CCW: curved carapace width.

Mean Standard deviation Median Range

Weight (kg)

CCL (cm)

CCW (cm)

22 14 18 8e53

54 9 53 42e70

52 9 50 37e68

2.2.4. BPA For BPA analysis the homogenized sample (about 0.5 g) were added with 3 ml of ethyl ether, shaken for 30 min and subsequently centrifuged at 4000 rpm for 5 min. The supernatant was collected into polyester tube. This procedure was repeated for three times, once reconstituted volume of sample, the latter was evaporated under a stream of nitrogen and reconstituted in acetonitrile. Finally, it was filtered through a nylon filter (0.2 mm pore size) and brought to a final volume of 0.5 ml of acetonitrile in vials for auto-sampler. For the analytical determination the ESI-MS system operated in negative mode and every run had duration of 20 min. A volume of 20 ml was injected in a C18 column Betasil 50  2.1 mm at a flow rate of 250 ml/min. The mobile phases used were acetonitrile and 2 mM ammonium acetate. A standard solution of BPA at 4 different concentrations was used as a calibration curve for analysis. LOD: 0.5 ng/g. 2.2.5. QA/QC For each kind of analysis, data quality assurance and quality control protocols included matrix spikes, laboratory blanks, and continuing calibration verification. Blanks were analysed with each set of five tissue samples as a check for possible laboratory contamination and interferences. 2.2.6. Porphyrins Porphyrins determination was conducted by a spectrofluorimetric method. About 300 mg of lyophilized tissue was homogenised in a Turrax homogeniser; homogenate was then transferred to glass tubes to which methanol/lN perchloric acid mixture was added. After vortex-mixing, the samples were kept in the dark for 10 min and then centrifuged for 5 min at a low speed (De Matteis and Lim, 1994). The porphyrins extract in the upper layer was then used for spectrofluorimetric determination, done following the method described by Grandchamp et al. (1980). The procedure is based on three different excitation/ emission wavelengths matched to each of the porphyrins

Please cite this article in press as: Guerranti, C., et al., Pilot study on levels of chemical contaminants and porphyrins in Caretta caretta from the Mediterranean Sea, Marine Environmental Research (2014), http://dx.doi.org/10.1016/j.marenvres.2014.01.004

C. Guerranti et al. / Marine Environmental Research xxx (2014) 1e5

(uroporphyrins 405e595 nm; coproporphyrins 400e595 nm; protoporphyrins 410e605 nm). Porphyrin standards were obtained from Porphyrin Product Inc. (Logan, Utah).

Table 3 Summary of the results of porphyrins analyses. Values are in nmol/g. P CoproUroProtoporphyrins Mean Standard deviation Median

2.3. Statistics Relationships between variables were examined with the Spearman rank correlation test. Values below the LOD were considered equal to half the LOD value (half bound). 3. Results and discussion A synthesis of the results achieved is shown in Tables 2 and 3. Results are expressed in dry weight (d.w.). Musk ketone was never detected, PFOA was found in just one sample, while PFOS was the prevalent contaminant, in terms of concentration and PBDEs were found in all samples. Many of the contaminants which are considered in this study were studied for the first time in samples of turtles. PFCs, for example, have never been studied in liver of turtles and levels of PFCs found in liver samples of C. caretta from the Mediterranean are lower than these reported for loggerhead turtles from other areas and for other species of turtles (Table 4), even considering that most of the articles relates about PFCs in plasma and serum and expressed in wet weight (w.w.). All the studies found in the scientific literature confirms the prevalence of PFOS on PFOA in turtles, with the latter rarely found; probably this discrepancy is related to the fact that PFOA has a lower bioconcentration factor than PFOS (Morikawa et al., 2006). Among the PBDE congeners considered, ten out of twenty were detected and BDE47 was the predominant one (Fig. 1): its concentration in liver samples of C. caretta was on average 32.03 pg/g d. w. and it was found in all the samples analysed. The abundance followed the pattern BDE47 > BDE49 > BDE71: the percentage contribution of these congeners to the total BDE residue was 32%, 17%, 13%, respectively. PBDE47 is the congener most used in pentabrominated commercial mixtures (EU, 2001), that were banned in Europe since year 2004 (European Commission, 2003); hence this prevalence in C. caretta tissue could be due to previous uses or longrange transport from areas where penta-brominated mixtures are still used (Wania and Dugani, 2003). Moreover BDEs 47 prevailed in a wide variety of matrices analysed worldwide, such as fish tissues, birds, human milk and food (Corsolini et al., 2008; Guerranti et al., 2008; Guerranti and Focardi, 2011; De Sanctis et al., 2013). PBDEs have never been studied in liver of C. caretta prior this study. Scientific literature reports the PBDE mean levels in Green turtle’s (Chelonia mydas) liver at 1600 pg/g lipid basis (l.b.) (Hermanussen et al., 2008). For C. caretta the matrices analysed were plasma and eggs with mean values of 86 and 5.5 ng/g l.b. of PBDEs respectively (Keller et al., 2005). From the comparison with these data, even if made on different matrices/species and expressed on a lipid basis, the samples analysed in this study appear to be less contaminated; both cited papers confirm the prevalence of BDE49 in samples of turtle.

3

1726 560 1540

1145 267 1135

6591 1094 6865

9462 1647 9793

Musk xylene, between SMCs detected, resulted to be the less present, both in terms of concentration and number of samples with detectable values (1 out of 9), while the prevailing was tonalide. To the author’s knowledge SMCs have never been studied in turtles; moreover the majority of data that can be found in the scientific literature, concern exposure experiments and does not report values of concentration in the tissues of wildlife. Hu et al. (2011), for example, report higher levels of SMCs in fish: tonalide prevailed (2.9e6.8 ng/g d.w.) on galaxolide (3.3e5.3 ng/g d.w.) and both these two SMCs were detected in all samples analysed. Musk ketone, never detected in the liver samples of C. caretta analysed in this study, was instead found in fish tissues in levels ranging from 2.7 to 7.9, even if in a very limited fraction of samples, while musk xylene was never found in the work of Hu et al. (2011). The SMCs accumulation pattern observed in C. caretta liver samples was different from the pattern observed in fish by Hu et al. (2011) and the reason could include different sources of exposure between species, different elimination rates and/or different biotransformation rates. Also p-NP and BPA, found in a limited fraction of samples and in levels only slightly higher than the LODs, seem to have never been studied before in turtles. Tsuda et al. (2000), that studied alkylphenols in various molluscs and fish, found BPA (8 ng/g wet weight, w.w.) just in Japanese Smelt, while p-NP was found in almost all species in levels ranging from 2 to 19 ng/g w.w. The SMCs and alkylphenols accumulation pattern observed in C. caretta liver samples were different from the patterns observed in fish by Hu et al. (2011) and in fish and molluscs by Tsuda et al. (2000) and the reason could include different sources of exposure between species, different elimination rates and/or different biotransformation rates. Considering biometric data (CCL and weight), no correlations were found between pollutants concentration and body parameters. For what concerns porphyrins, the profile observed was Proto>Copro->Uro-; in particular Proto- was 69.66% of the total porphyrins, Copro- 18.24% and Uro- 12.10%. To the authors knowledge the present paper reports the first data on porphyrins levels in liver of C. caretta, but the same profile was observed in excreta from the same species Proto- 63.98%, Copro- 25.04% and Uro- 10.99% (Casini et al., 2012). Statistical analysis was carried out to highlight possible correlations between porphyrins and contaminants and the existence of a positive statistical correlation (Spearman rank test, r ¼ 0.76 p < 0.05) between levels of PFOS and uroporphyrins was verified. A positive correlation between contaminants and a specific porphyrin was also found in Japanese quail (Fossi et al., 1996) where it was shown that accumulation of hepatic protoporphyrin

Table 2 Summary of the results obtained as regards the levels of contaminants. All values are in ng/g d.w. with the exception of PBDEs that are expressed in pg/g d.w.. The values < LOD were considered equal to half of the LOD. P PFOS PFOA PBDE BPA Musk xylene Tonalide Galaxolide Musk ketone p-NP Mean Standard deviation Median Range of values > LOD % values > LOD

1.21 0.75 1.50 1.29e2.06 67

0.28 0.09 0.25 0.53 11

100.60 55.67 88.92 52.94e233.80 100

0.36 0.24 0.25 0.58e0.93 22

0.76 0.79 0.5 2.87 11

0.87 0.65 0.5 1.09e2.37 33

1.01 0.72 0.5 1.23e2.58 44

0.50 e 0.5 e

1.09 1.04 0.5 1.02e3.08 33

Please cite this article in press as: Guerranti, C., et al., Pilot study on levels of chemical contaminants and porphyrins in Caretta caretta from the Mediterranean Sea, Marine Environmental Research (2014), http://dx.doi.org/10.1016/j.marenvres.2014.01.004

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C. Guerranti et al. / Marine Environmental Research xxx (2014) 1e5

Table 4 Levels of PFOS and PFOA in turtles reported in the scientific literature. Species

Matrix

PFOS

PFOA

Reference

Caretta caretta

Blood