Co-occurrence of musk fragrances and UV-filters in seafood and macroalgae collected in European hotspots

Environmental Research 143 (2015) 65–71

Contents lists available at ScienceDirect

Environmental Research journal homepage: www.elsevier.com/locate/envres

Co-occurrence of musk fragrances and UV-filters in seafood and macroalgae collected in European hotspots S.C. Cunha a,n, J.O. Fernandes a, L. Vallecillos b, G. Cano-Sancho c, J.L. Domingo c, E. Pocurull b, F. Borrull b, A.L. Maulvault d, F. Ferrari e, M. Fernandez-Tejedor f, F. Van den Heuvel g, M. Kotterman h a LAQV-REQUIMTE, Laboratory of Bromatology and Hydrology, Faculty of Pharmacy, University of Porto, Rua Jorge de Viterbo Ferreira 228, 4050-313 Porto, Portugal b Departament de Química Analítica i Química Orgànica, Universitat Rovira i Virgili, Campus Sescelades, Marcel
lí Domingo, s/n, 43007 Tarragona, Catalonia, Spain c Laboratory of Toxicology and Environmental Health, School of Medicine, IISPV,Universitat Rovira i Virgili, Sant Llorenç 21, 43201 Reus, Catalonia, Spain d Division of Aquaculture and Upgrading (DivAV), Portuguese Institute for the Sea and Atmosphere (IPMA), Avenida de Brasilia, 1449-006 Lisboa, Portugal e Aeiforia srl, Loc. Faggiola 12-16, 29027 Gariga di Podenzano, Piacenza, Italy f IRTA, Ctra. de Poble Nou, E-43540 Sant Carles de la Ràpita, Tarragona, Spain g Hortimare Projects & Consultancy BV, The Netherlands h IMARES, Stichting Dienst Landbouwkundig Onderzoek, Haringkade 1, 1976CP IJmuiden, The Netherlands

art ic l e i nf o

a b s t r a c t

Article history: Received 16 January 2015 Received in revised form 3 May 2015 Accepted 5 May 2015 Available online 16 May 2015

In the last decades, awareness regarding personal care products (PCP), i.e. synthetic organic chemicals frequently used in cosmetic and hygienic products, has become a forward-looking issue, due to their persistency in the environment and their potential multi-organ toxicity in both human and wildlife. Seafood is one of the most significant food commodities in the world and, certainly, one of the most prone to bioaccumulation of PCP, what can consequently lead to human exposure, especially for coastal population, where its consumption is more marked. The aim of this work was to evaluate the co-occurrence of musk fragrances and UV-filters in both seafood and macroalgae collected in different European hotspots (areas with high levels of pollution, highly populated and near wastewater treatment plants). Despite the fact that UV-filters were detected in three different kind of samples (mussel, mullet, and clam), in all cases they were below the limit of quantification. Galaxolide (HHCB) and tonalide (AHTN) were the musk fragrances most frequently detected and quantified in samples from the European hotspots. Cashmeran (DPMI) was also detected in most samples but only quantified in two of them (flounder/herring and mullet). The highest levels of HHCB and AHTN were found in mussels from Po estuary. & 2015 Elsevier Inc. All rights reserved.

Keywords: Contaminants Fish Gas chromatography–mass spectrometry Macroalgae Musk fragrances UV-filters

1. Introduction Personal care products (PCPs) constitute a diverse group of compounds, used in soaps, lotions, toothpaste, fragrances, and sunscreens. The main classes of PCPs include ultraviolet filters (UV-filters), insect repellents, musk fragrances, disinfectants and preservatives. Due to their wide use, PCPs enter the environment both direct (disposal and wastage from external application) and indirect (excretion, washing, and swimming) (Daughton, 2007). PCPs are generally not subjected to structural alterations, resulting in the release of large quantities of unaltered compounds

n

Correspoding author. E-mail address: [email protected] (S.C. Cunha).

http://dx.doi.org/10.1016/j.envres.2015.05.003 0013-9351/& 2015 Elsevier Inc. All rights reserved.

into the environment (Brausch and Rand, 2011; Ternes et al., 2004). The incidence of PCPs in the environment may have a negative impact on human and wildlife individuals either by their accumulation or by long-term chronic exposure of aquatic organisms to these compounds (Brausch and Rand, 2011; Hao et al., 2007). Furthermore, there is some evidence that even low doses of PCPs may account to synergic toxicity effects and cumulative stress in exposed organisms (Daughton, 2004; Grung et al., 2007). For some PCPs (e.g. some synthetic musk fragrances and UV-filters) hormonal activity (oestrogenic, antiestrogenic, androgenic, antiandrogenic) have been documented in vitro as well as in vivo assays (Kunz and Fent, 2006; Zenker et al., 2008). Despite these facts, limited efforts have been made so far to regulate environmental contamination by PCPs residues. Recently, in EU the components of PCPs have been covered by new REACH regulation for chemicals

66

S.C. Cunha et al. / Environmental Research 143 (2015) 65–71

which includes hazard and environment risk assessment (EC, 2008; Treadglod et al., 2012). However, at present USA laws does not require the disclosure of chemicals in PCPs (Treadglod et al., 2012). PCPs with high lipophilicity, such as synthetic musk fragrances and organic UV filters, have been detected in surface waters, wastewater, soil and sediments as summarized in recent reviews (Kim and Carlson, 2005; Hao et al., 2007; Brausch and Rand, 2011; Balmer et al., 2005). In contrast, relatively few studies have documented the occurrence of PCPs in aquatic organisms (Buser et al., 2006; Fent et al., 2010; Nakata et al., 2007). Nakata et al. (2007) reported that two polycyclic musk fragrances, galaxolide (HHCB) and tonalide (AHTN), were the dominant compounds found in most marine organisms (lugworm, clam, crustacean, fish marine mammal and bird) collected from tidal flat and shallow water areas of the Ariake Sea, Japan. The same compounds have been also detected in mussels collected from Cambodia, China, Hong-Kong, India, Indonesia, Japan, Korea, Malaysia and Philippines, suggesting their ubiquitous contamination and widespread distribution (Nakata et al., 2012). 4-MBC (4-methylbenzylidene camphor) and OC (octocrylene), two widely used UV-filters, were found in the muscle tissue of brown trout (Salmo trutta fario) from several small Swiss rivers, all receiving inputs from wastewater treatment plants (WWTPs) at levels up to 1800 (4-MBC) and 2400 ng/g (OC) expressed in lipid weight based concentrations (Buser et al., 2006). Ethylhexyl-methoxycinnamate (EHMC or OMC) was found by Fent et al. (2010) in crustaceans (Gammarus sp.), mollusks (Dreissena polymorpha) and several fish species at levels of 133, 150 and 337 ng/g lipids respectively, from samples collected above and below WWTPs in the river Glatt, Swiss. More recently, benzophenone 3 (BP3), EHMC and OC were detected above 290 ng/g dw (dry weight) in samples of the fish species Luciobarbus sclateri, endemic of the Iberian peninsula (Gago-Ferrero et al., 2013). Most of these studies were held on river fish samples, thus there is a lack of information regarding UV-filters levels in seafood. Such data are necessary to promote ecological and human health risk assessments documenting potential consequences of environmental PCP exposures. The aim of this work was to evaluate the co-occurrence of musk fragrances and UV-filters in both fish and macroalgae collected in different European polluted areas-hotspots by using gas chromatography–mass spectrometry (GC–MS) and gas chromatography– tandem mass spectrometry (GC–MS/MS) as analytical techniques.

2. Experimental 2.1. Standards and reagents for UV-filters 2-Hydroxy-4-methoxybenzophenone (BP3; 98% purity), 2,3,4trihydroxybenzophenone (THB; 98% purity) and 2-ethylhexyl 4(dimethylamino)benzoate (EPABA; 98% purity) were purchased from Alfa Aesar (Heysham, Lancashire, UK). 3,3,5-trimethylcyclohexyl salicylate (HMS; 98% purity) 2,2′-dihydroxy-4,4′-dimethoxybenzophenone (DHMB, 99% purity) and isoamyl-4 methoxycinnamate (IMC, 95% purity) were purchased from TCI (Haven, Zwijndrecht, Belgium). Octocrylene (OC, 98% purity), 2-ethylhexyl 4-methoxycinnamate (OMC, 100% purity), 2-ethylhexyl salicylate (EHS, 99% purity), hexyl 2-[4-(diethylamino)-2-hydroxybenzoyl] benzoate (DBENZO, 99% purity), 2,4-dihydroxybenzophenone (BP1, 99% purity) and 3-(4-methylbenzylidene)camphor (4-MBC, 98.5% purity), were purchased from Sigma-Aldrich (Steinheim, Germany). The internal standard (IS) Benzophenone-d10 (BPd10-IS, 99 at% D) was also purchased from Sigma-Aldrich. Individual standard solutions of the UV-filters were prepared in methanol (HPLC grade from Sigma-Aldrich) at concentrations of

2000 mg/mL. Working mixture solutions of 100 mg/mL was prepared in acetonitrile, solvent used in the extraction. Acetonitrile (MeCN) HPLC grade was obtained from Sigma-Aldrich. Trichloroethylene was high purity grade for GC analysis and was obtained from Fluka (Buchs, Switzerland). Derivatization reagent N,O-bis(trimethylsilyl)trifluoroacetamide with 1% TMCS (BSTFA þ1%TMCS, 99% purity grade) was obtained from Fluka. Hydrochloric acid and pH test strips (0–14 pH resolution: 1.0 pH unit) were purchased from Sigma-Aldrich. Water was prepared by purifying demineralized water in a “Seradest LFM 20” system (Seral, Ransbach-Baumbach, Germany). Ultrahigh purity Helium (99.999%) for GC–MS was purchased from Gasin (Maia, Portugal). 2.2. Standards and reagents for musk fragrances The six polycyclic musk fragrances: 6,7-dihydro-1,1,2,3,3-pentamethyl-4(5H)-indanone (DPMI, cashmeran), 4-acetyl-1,1-dimethyl-6-tert-butylindane (ADBI, celestolide), 6-acetyl-1,1,2,3,3,5hexamethylindane (AHMI, phantolide), 5-acetyl-1,1,2,6-tetramethyl-3-isopropylindane (ATII, traseolide), 1,3,4,6,7,8-hexahydro4,6,6,7,8,8-hexamethylcyclopenta-(g)-2-benzopyran (HHCB, galaxolide) and 7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphthalene (AHTN, tonalide) were supplied by Promochem Iberia (Barcelona, Spain). 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-(g)-2-benzopyran-1-one (HHCB-lactone, galaxolidone) was provided by International Flavors & Fragrances Inc. (Barcelona, Spain). The nitro musk fragrances 2,4,6-trinitro-1,3dimethyl-5-tert-butylbenzene (MX, musk xylene) and 1,1,3,3,5pentamethyl-4,6-dinitroindane (MM, musk moskene) were purchased as 100 mg mL  1 individual solutions in acetonitrile from Sigma-Aldrich and Riedel de Haën (Seelze, Germany), respectively. The standard 4-aceto-3,5-dimethyl-2,6-dinitro-tertbutylbenzene (MK, musk ketone) was provided by Fluka while the internal standard (IS) d15-musk xylene (d15-MX) came as a 100 mg/mL solution in acetone from Symta (Madrid, Spain). Individual standard solutions of the synthetic musk fragrances were prepared in acetone at concentrations of 4000 mg/mL for polycyclic musks and 1000 mg/mL for musk ketone and HHCBlactone. A working mixture solution of 100 mg/mL was prepared in ethyl acetate except for MX, MM and d15-MX which were supplied directly at a concentration of 100 mg/mL in acetonitrile and used as received. Acetone and ethyl acetate were GC grade with purity 499.9% from Prolabo (VWR, Llinars del Vallès, Barcelona, Spain). The extraction solvent dichloromethane was GC grade (of 499.9% purity) from Prolabo. Ultrapure water was obtained using an ultrapure water purification system from Veolia Water (Sant Cugat del Vallés, Barcelona, Spain). Helium gas with a purity of 99.999% was used for the chromatographic analysis (Carburos Metálicos, Tarragona, Spain). 2.3. Sampling Mussels (Mytilus edulis and Mytilus galloprovincialis) were sampled at three European hotspot locations (Table 1 and Fig. 1). European flounder (Platichthys fesus), mullet (Liza aurata) and clam (Chamelea gallina) were collected from Western Scheldt, Tagus Estuary and Ebro Delta, respectively. Seaweeds (Laminaria digitata and Saccharina Latissima) were collected from Norwegian fish farm. All the samples were collected between September and December of 2013. Fish: a representative sampling, at least 100–150 g of pooled tissue (fillet fish discarded from heads, scales, tails, fins, guts, bones, and skin) per specie was homogenized in a grinder and freeze‐dried before analysis.

S.C. Cunha et al. / Environmental Research 143 (2015) 65–71

67

Table 1 Description of the samples collected in hotspot locations. Specie

Localization

No. individual per pool

Range individual length and mean

Range individual weight and mean

Mussels

Ebro Delta Po estuary Tagus estuary Western Scheldt Tagus estuary Tagus estuary Ebro Delta Solund

50 50 50 25 25 25 100 10–15

43–58 mm; 49.64 mm 40–72 mm; 48.6 mm 37–64; 47.4 mm 25–40; 30.1 cm 32–43;36.3 mm 27–47;34.4 mm 27–36 mm; 31.38 mm L. digitata 0.1–0.5 m S. Latissima 10–15 m

 2.1–6.5; 4.0 g 1.6–8.3; 3.4 g 264–708; 394 385–1030; 627.1 g 232–1435; 532.4 g  4–5 kg 4–5 kg

Flounder Mullet I Mullet II Clams Macroalgae

Mussels and clams: a representative sampling, at least 50 individuals were collected in each hotspot. Then, after rinsed with fresh water the shells were opened with stainless steel knives and the meat and intervalvular fluid were pooled together, homogenized in a grinder and freeze‐dried before analysis. Macroalgae: a total of 4–5 kg fresh weight was obtained per species by diving/snorkling. Exposure to sunlight above water while collecting was kept to a minimum to prevent degradation of contaminants due to UV radiation. Samples were drying either by sun (first set of samples) or low temperature drying in a dry room (remainder of samples). After initial drying the samples were freeze dried. 2.4. Sample extraction 2.4.1. Extraction of UV-filters The sample preparation was based on a previously described methodology (Cunha et al., 2012) with some modifications: (i) weight 3.5 g of freeze-dried sample into a 40 mL glass vial tube; (ii) add 5 mL of deionized water and 10 mL of acetonitrile (MeCN)

seal the tube, vortex and place it on a wrist action shaker for 10 min (iii) add 4 g of anhydrous MgSO4 and 1 g of NaCl; (iv) seal the tube and shake vigorously by hand for 5 min (v) centrifuge the tube at 5000g for 3 min. After QuEChERS a dispersive liquid–liquid microextraction (DLLME) procedure was performed: (vi) transfer 1 mL of the MeCN extract to a 4 mL vial tube and add 50 mL of BPd10 (IS, 1000 mg/mL) (vii) add 75 mL of trichloroethylene (viii) transfer rapidly the mixture to a 25 mL glass tube with conical bottom containing 4 mL of deionized water acidified at pH 3 (ix) seal the tube and shake gently by hand for 30 s (x) centrifuge the tube at 5000g for 1 min (xi) transfer 60 mL of the settled volume into a vial and dried using a gentle nitrogen stream at room temperature. Finally, the analytes were silylated (xii) add 50 μL of BSTFA and derivatize during 5 min in a household microwave (600 W) and inject 1 mL of the extract in the GC–MS system. 2.4.2. Extraction of musk fragrances The different matrices, including fish, mussels, clams and macroalgaes were analysed for the target fragrances using the following procedure, which was described in more detail by

Fig. 1. Location of four hotspot sampling points.

68

S.C. Cunha et al. / Environmental Research 143 (2015) 65–71

Vallecillos et al. (2015). In summary (i) 0.5 g (d.w.) of freeze-dried sample was blended with 1 g of diatomaceous earth and homogenized. (ii) The homogenate was transferred to a 11 mL stainless steel extraction cell that contained 1 g of florisil (in-cell clean-up sorbent) previously conditioned at 400 °C overnight at the bottom and was compacted with 1 g of diatomaceous earth. (iii) Then, the extraction was carried out using an accelerated solvent extraction system (ASE 200, Dionex, Sunyvale, CA, USA) with dichloromethane at 60 °C and 1500 psi for 5 min (Vallecillos et al., 2015). (iv) The sample extract was concentrated to 1 mL on a rotary evaporator (R-114, Büchi, Switzerland) at 30 °C and the IS (50 ng/g) was added to the residue before it was reconstituted to 2 mL with ethyl acetate and filtered with a 0.22 mm PTFE syringe filter. The extract was finally analysed by gas chromatography ion trap tandem mass spectrometry (GC–IT-MS/MS). 2.5. GC–MS conditions 2.5.1. Determination of UV-filters by GC–MS The gas chromatograph 6890 (Agilent, Little Falls, DE, USA) equipped with a Combi-PAL autosampler (CTC Analytics, Zwingen, Switzerland) and an electronically controlled split/splitless injection port was interfaced to a single quadrupole inert mass selective detector (5975B, Agilent) with electron ionization (EI) chamber. GC separation was performed on a DB-5MS column (30 m  0.25 mm i.d.  0.25 μm film thickness; J&W Scientific, Folsom, CA, USA). Helium was the carrier gas with a constant flow of 1 mL/min. The injection was made in splitless mode (purge-off time 60 s) at 250 °C. The oven temperature programme was as follows: 95 °C held for 1 min, ramped to 180 °C at 40 °C/min, ramped to 230 °C at 5 °C/min and then ramped 290 °C at 25 °C/min held for 6.47 min. Total run time was 22 min. The MS transfer line was held at 280 °C. Mass spectrometric parameters were set as follows: electron ionization with 70 eV energy; ion source temperature, 230 °C and MS quadrupole temperature, 150 °C. The MS system was routinely set in selective ion monitoring (SIM) mode and each analyte was quantified based on peak area using one target and two qualifier ion(s). Complete SIM parameters and retention times of the analytes are shown in Table 2. Agilent Chemstation was used for data collection/processing and GC–MS control. 2.5.2. Determination of musk fragrances by GC–MS/MS The GC-IT-MS/MS analyses were performed using a Varian ion trap GC–MS system (Varian, Walnut Creek, CA, USA), equipped with a 3800 gas chromatograph, a 4000 ion trap mass detector, a Table 2 Retention times and MS conditions for the GC–MS analysis of UV-filters. Compounds

tR (min)

Time windows

SIM ions m/za

BP10 (IS)

5.37

5.0

110, 82, 192, 54, 160

EHS HMS IMC 4-MBC BP3

7.96 8.82 8.93 9.24 9.55

7.2

195, 135, 57, 307, 195, 69, 135, 210 178, 161, 134, 248, 89, 118 254, 128, 211, 183, 155, 55 285, 242, 77, 223, 105

BP1 DHMB EPABA THB OMC OC DBENZO a

10.47 11.75 11.81 12.00 12.47 16.16 18.54

9.0

10.0 11.5

12.1 16.0 18.0

343, 73, 164, 105, 271, 373, 73, 299, 223 165, 277, 77, 145, 431, 73, 343, 105 178, 163, 134, 290, 57 249, 204, 232, 360, 70, 112, 178 454, 340, 370, 73, 280, 149, 469

Quantification ions are shown in bold type.

1079 programmable vaporising temperature injector and a CombiPal autosampler (CTC Analytics, Zwigen, Switzerland). A fused silica capillary column (3 m  0.25 mm i.d.) from Micron Phenomenex (Torrance, California, USA) was used as a guard column. The chromatographic separation was carried out on a ZB-50 analytical column (30 m  0.25 mm i.d.  0.25 mm film thickness) from Micron Phenomenex. The oven temperature programme was as follows: 70 °C hold for 3.5 min, 50 °C/min to 200 °C, then 5 °C/min to 240 °C and finally 20 °C/min to 290 °C (hold 3.4 min). Helium with a purity of 99.999% was used as carrier and collision gas at a constant flow rate of 1 mL/min. During the injection of the 10 mL, the 1079 injector operated in large volume injection (LVI) mode and a 2 mm i.d. insert liner packed with glass wool (Varian) was used. During injection in split mode at a rate of 50 mL/min the 1079 injector temperature was set at 70 °C. The ethyl acetate was purged out with a vent flow of 100 mL/min within 0.5 min (vent time). The splitless mode was then programed for 2.5 min while the temperature was increased at 100 °C/min to 300 °C for 5 min. Transfer line, manifold and trap temperatures were 280 °C, 50 °C and 200 °C respectively. The mass spectrometer was operated in the electron ionization (EI) mode (70 eV) with a filament-multiplier delay of 3 min to prevent instrument damage. For quantitative analysis of the target fragrances, MS/MS mode was applied. Retention times as well as optimal MS parameters of the target fragrances are summarized at Table 3.

3. Results and discussion 3.1. UV-filters performance Initially, slopes of the calibration curves obtained from solvents and from matrix (standards added to fillet mackerel samples free of compounds of interest) were compared, being observed a matrix suppression effect. Therefore, the use of matrix-matched calibration was required for a reliable UV-filters quantification. Linearity concentration ranged from 5–1000 ng/g (d.w.) for HS, BP3, OMC, from 20 to 1000 ng/g (d.w.) for EHS, IMC, 4-MBC, BP1, DHMB, OC and EPABA and from 100–1000 ng/g (d.w.) for THB and DBENZO. Calibration curves were constructed with 6 levels by plotting the UV-filters/I.S. area ratio against the concentration of UV-filters in the matrix-matched solutions. The analysis was performed in duplicate for each concentration. The results obtained demonstrated a good linearity within the tested interval, with correlation coefficients (r) always higher than 0.9970 for all analytes. Recovery and intra-day repeatability were determined on blank samples (fillet mackerel samples free of compounds of interest) spiked with UV-filters at two concentration levels (100 and 500 ng/g), each test being performed six times (see Table 4). Eighth compounds (EHS, HMS, IMC, 4-MBC, BP3, BP1, EPABA, and OC) had recoveries between 70% and 110%, three compounds (THB, DHMB, and DBENZO) had recoveries between 50% and 70%, and the recovery of OMC was above 115%. Good results were obtained for intra-day repeatability; more than 70% of compounds tested show a value of RSDo15% and less than 20% of the compounds show a value of RSD4 20%. Method detection limits (MLD) were determined by successive analysis of diluted extracts until a 3:1 signal-to-noise ratio was reached. The lowest assigned value obtained was 2 ng/g (d.w.) for HS, BP3, and OMC (see Table 5). Method quantification limits (MQL) were established as the lowest concentration assayed quantified with acceptable accuracy (70–110% of recovery) and precision (RSD of r20%), which were the lowest level of the calibration curve. Both, the MLD and MQL obtained were similar to those reported by Bachelot et al. (2012) and Gago-Ferrero et al.

S.C. Cunha et al. / Environmental Research 143 (2015) 65–71

69

Table 3 Retention times and MS conditions for the GC–MS/MS analysis of musk fragrances. Compounds

tR (min)

Parent ion (m/z)

Product ionsa (m/z)

CID Amplitude (V)

CID Storage level (m/z)

m/z Range

Scan time (s/scans)

DPMI ADBI AHMI ATII HHCBb AHTNb MXb MMb MK HHCB-lactone d15-MX (IS)b

7.77 8.98 9.46 10.17 10.42 10.44 11.07 11.15 12.97 15.50 10.89

191 229 229 215 243 243 282 263 279 257 294

107, 135, 173 131, 173, 187 131, 145, 187 131,171, 173 171, 213 145, 159, 187 265, 266, 281 187, 201, 211 191, 247, 280 183, 201, 239 170, 276, 295

0.82 0.92 0.92 0.88 0.96 0.96 1.08 1.02 1.07 1.00 1.11

84.1 100 100 94.7 122 103 124.2 115.9 122.9 113.2 129.5

94–201 110–239 110–239 104–225 132–253 113–253 134–292 125–273 132–289 123–267 139–304

1.08 1.01 1.01 1.01 0.53 0.53 0.59 0.59 1.05 1.03 1.04

a b

Compounds were separated using the multiple reaction monitoring. Quantification ions (m/z) are shown in bold type.

from the corresponding peak areas of each spiked sample. The linear range started at the method quantification limit (MQL), defined as the lowest calibration point, and went up to 100 ng/g (d.w.) or 250 ng/g (d.w.) depending on the target fragrance, with good linearity for all of the target fragrances (r2 40.994) provided by the presence of the IS d15-MX (50 ng/g (d. w.)). As can be seen in Table 6, the lowest MQLs were obtained working with macroalgae with values between 1 ng/g (d.w.) for the polycyclic musk fragrances DPMI, ADBI, HHCB and AHTN and up to 5 ng/g (d.w.) for the studied nitro musk fragrances. The MDLs, which were calculated as explained by Vallecillos et al. (2015), ranged between 0.25 and 5 ng/g (d.w.) for macroalgae and fish samples and from 0.5 ng/g (d.w.) to 7.5 ng/g (d.w.) for clams and mussels. As happened with the MQLs the highest MDLs were obtained with the nitro musk fragrances, 5 ng/g (d.w.) for macroalgae and fish and 7.5 ng/g (d.w.) for clams and mussels. Intra-day and inter-day repeatabilities were obtained with five replicates of each kind of sample spiked at 50 ng/g (d.w.). The presence of the IS improved the method repeatabilities obtaining intra-day repeatability values r10% for fish and macroalgae and up to 14% for more complex matrices as clams and mussels. Interday repeatability values (n¼ 5, 50 ng/g (d.w.)) were below 20% independently of the matrix analysed.

Table 4 Average of recovery (%) and repeatability (%RSD), for the UV-filters in study, obtained in fillet mackerel spiked samples using QuEChERS followed by DLLME and GC–MS analysis (n ¼6). Compounds

EHS HMS IMC 4-MBC BP3 BP1 DHMB EPABA OMC OC THB DBENZO

100 ng/g spiking level

500 ng/g spiking level

%Recovery

Intra-day %RSD

%Recovery

Intra-day %RSD

83 76 95 79 72 77 61 79 115 76 68 59

12 15 17 10 12 16 17 10 21 8 22 18

91 82 89 96 83 68 60 69 93 75 70 62

15 13 10 3 3 2 5 5 10 4 8 14

(2013). 3.2. Musk fragrances performance Due to the matrix effect values obtained by Vallecillos et al. (2015), the quantification of the target fragrances was done with one calibration curve for each kind of matrix (fish, mussels, clams and macroalgaes). The samples used to validate the method were analysed (n ¼5) to determine if any musk fragrance was present, and the results revealed peaks of HHCB and AHTN in the chromatogram for all of the samples, while HHCB-lactone was only present in the sample used for the validation of macroalgae. The average peak area of each compound detected was subtracted

3.3. Occurrence of UV-filters in seafood and macroalgae samples from hotspot locations Four (BP3, DHMB, OMC, OC) of the 12 UV-filters studied were detected in three samples (mussel, mullet, and clam), but all below the method limit of quantification (see Table 5). These data are clearly distinct to those recently reported by Picot Groz et al. (2014) and by Bachelot et al. (2012) for mussels collected from

Table 5 Performance parameters (MDL and MQL, ng/g (d.w.)) and concentrations (ng/g, dw) of target UV-filter found in each hotspot sample analysed, expressed in ng/g (d.w.). Sample type

Hotspot location

%Dry of Weight

EHS

HMS

IMC

4-MBC

BP3

BP1

DHMB

EPABA

OMC

OC

THB

DBENZO

Mussels Mussels Mussels Mullet I Mullet II

Ebro delta Tagus estuary Po estuary Tagus Tagus

13.9 21.6 17 23.6 24.3

n.d. n.d. n.d. n.d. n.d.

n.d. n.d. n.d. n.d. n.d.

n.d. n.d. n.d. n.d. n.d.

n.d. n.d. n.d. n.d. n.d.

n.d. n.d. n.d. n.d. o MQL

n.d. n.d. n.d. n.d. n.d.

n.d. n.d. n.d. n.d. oMQL

n.d. n.d. n.d. n.d. n.d.

o LOQ n.d. n.d. n.d. n.d.

o LOQ n.d. n.d. n.d. n.d.

n.d. n.d. n.d. n.d. n.d.

n.d. n.d. n.d. n.d. n.d.

Flounder Clams Performance parameters

Western Scheldt Ebro delta MDL MQL

22.6 19.4 6 20

n.d. n.d. 2 5

n.d. n.d. 6 20

n.d. n.d. 6 20

n.d. n.d. 2 5

n.d. n.d. 3 20

n.d. n.d. 6 20

n.d. n.d. 6 20

n.d. n.d. 2 5

n.d. o MQL 3 20

n.d. o MQL 23 100

n.d. n.d. 30 100

n.d. n.d.

n.d. ¼ non detected. o MQL ¼below the method detection limit.

70

S.C. Cunha et al. / Environmental Research 143 (2015) 65–71

Table 6 Performance parameters (MDL and MQL, ng/g (d.w.)) and concentrations of the target musk fragrances found in each hotspot sample analysed, expressed in ng/g (d.w.). Sample type

Hotspot location

Polycyclic musk

Nitro musk

DPMI

ADBI

AHMI

ATII

HHCB

AHTN

HHCB-lactone

MX

MM

MK

Mussels Mussels Mussels Clams Flounder L. Digitata S. Latissima. Mullet I Mullet II

Po estuary Tagus estuary Ebro delta Ebro delta Western Scheldt Solund Solund Tagus estuary Tagus estuary

o MQL o MQL o MQL o MQL 9.11 n.d. n.d. 7.72 6.10

n.d. o MQL n.d n.d n.d n.d n.d o MQL n.d

n.d. o MQL n.d n.d n.d n.d n.d n.d n.d

n.d. n.d n.d n.d n.d n.d n.d n.d n.d

34.52 13.32 8.68 33.10 5.61 o MQL 3.12 10.93 11.73

12.99 8.42 6.98 o MQL 3.55 o MQL o MQL 5.75 3.62

63.51 n.d n.d n.d n.d o MQL o MQL n.d. n.d.

n.d n.d n.d n.d n.d n.d n.d n.d n.d

n.d n.d n.d n.d n.d n.d n.d n.d n.d

n.d n.d n.d n.d n.d n.d n.d n.d n.d

Mussel

MDL MQL MDL MQL MDL MQL MDL MQL

1 2.5 0.5 2.5 1 2.5 0.25 1

2.5 5 0.5 1 1 2.5 0.25 1

2.5 5 0.25 2.5 1 2.5 0.25 2.5

2.5 5 1 2.5 1 2.5 0.5 2.5

0.5 2.5 0.25 1 0.5 2.5 0.25 1

0.5 2.5 0.25 1 0.5 2.5 0.25 1

5 7.5 0.5 2.5 2.5 5 0.25 2.5

7.5 10 5 10 7.5 10 5 5

7.5 10 5 10 7.5 10 5 5

7.5 10 5 10 7.5 10 5 5

Mullet Clams Macroalgae

n.d. ¼non detected. o MQL ¼below the method detection limit.

Portuguese and French Coasts, respectively, where were found OC, EHMC and octyl dimethyl p-aminobenzoic acid with levels up to 256 ng/g (d.w.). These differences could be explained either by the collection season in September–December of the samples (Gomez et al., 2012). Picot Groz et al. (2014) and Bachelot et al. (2012) observed seasonal trends for UV-filters, with the highest concentrations detected in the summer period, before the swimming period. Similarly, Fent et al. (2010) showed that freshwater mussels collected in a Swiss lake where bathing was practiced had higher concentrations after summer than before. The highest frequency of detection of the compounds EHMC and OC in the samples analysed, about 30%, is comparable to the previous studies mentioned. EHMC and OC are extensively used in several PCPs like sunscreens and show high lipophilicity (log Kow close to 6), contributing for their bioaccumulation in the environment. 3.4. Occurrence of musk fragrances in seafood and macroalgae samples from hotspot locations The results of musk fragrances determination, summarized in the Table 6, confirmed the forbidden nitro musks were not detected in any sample. The polycyclic musks HHCB and AHTN were quantified in most of seafood species collected from the hotspots. The higher levels of HHCB were achieved in mussels from Po estuary and clams from Ebro Delta with respective values of 34.52 and 33.10 ng/g (d.w.). Lower values of HHCB in mussels were quantified in those samples from Tagus estuary and Ebro delta. AHTN was similarly distributed in the same species but at lower concentrations, with the exception of clams, where it was not quantified. A high correlation was observed between HHCB and AHTN with Pearson's coefficient of 0.89. HHCB-lactone, the main degradation metabolite of HHCB, was only quantified in mussels from Po estuary. One sample of algae (Laminaria spp.) showed levels of HHCB up to 3.12 ng/g (d.w.). DPMI was detected in all the samples with the exception of macroalgaes, while, in case of mussel and clams DPMI was found below the MQL of 2.5 ng/g (d. w.) in flounder and mullet was found at concentrations ranged between 6.10 and 9.11 ng/g (d.w.). These concentrations are higher than those reported by Kannan et al. (2005) in Atlantic salmon

fillets and Atlantic sharpnose shark or smallmouth bass livers collected from United States with maximum concentration values ranging between 3.2 and 5.4 ng/g wet weight (w.w.) for HHCB, and between 1.6 and 1.9 ng/g (w.w.) for AHTN. While, the levels of HHCB found in mussels from Tagus estuary (13.32 ng/g (d.w.)) and Ebro estuary (8.68 ng/g (d.w.)) were comparable with the HHCB concentrations found in mussels from south of Portugal (values around 12 ng/g (d.w.)) by Picot Groz et al. (2014). Otherwise, higher concentration of HHCB, 34.52 ng/g (d.w.), was reached in mussel samples from Po estuary.

4. Conclusions To sum up, the present study reports the application of two GC–MS methodologies to explore the levels of 12 UV-filters and 10 musk fragrances in highly consumed seafood species and algae from four European hotspot locations. BP3, DHMB, OMC, OC were the UV-filters detected in mussels, clams and mullet, but all at levels below the MQL. Galaxolide (HHCB) and tonalide (AHTN) were the musk fragrances most frequently quantified in samples from the European hotspots. Cashmeran (DPMI) was also detected in most of samples but only quantified in flounder and mullet. The highest levels of HHCB and AHTN were found in mussels from Po estuary. Co-occurrence of UV-filters and musk fragrances contaminants was verified in Ebro-delta as well as in Tagus delta.

Acknowledgements The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007– 2013) under the ECsafeSEAFOOD project (Grant Agreement no. 311820).

References Bachelot, M., Li, Z., Munaron, D., Le Gall, P., Casellas, C., Fenet, H., Gomez, E., 2012. Organic UV filter concentrations in marine mussels from French coastal regions. Sci. Total Environ. 420, 273–279.

S.C. Cunha et al. / Environmental Research 143 (2015) 65–71

Balmer, M.E., Buser, H.R., Müller, M.D., Poiger, T., 2005. Occurrence of some organic UV filters in wastewater, in surface waters, and in fish from Swiss Lakes. Environ. Sci. Technol. 39, 953–962. Brausch, J.M., Rand, G.M., 2011. A review of personal care products in the aquatic environment: environmental concentrations and toxicity. Chemosphere 82, 1518–1532. Buser, H.R., Balmer, M.E., Schmid, P., Kohler, M., 2006. Ocurrence of UV-filters 4-methylbenzylidene camphor and octocrylene in fish from various Swiss rivers with imputs from wastewater treatment plants. Environ. Sci. Technol. 40, 1427–1431. Cunha, S.C., Cunha, C., Ferreira, A.R., Fernandes, J.O., 2012. Determination of bisphenol A and bisphenol B in canned seafood combining QuEChERS extraction with dispersive liquid–liquid microextraction followed by gas chromatography–mass spectrometry. Anal. Bioanal. Chem. 404, 2453–2463. Daughton, C.G., 2004. Non-regulated water contaminants: emerging research. Environ. Impact Asses. 24, 711–732. Daughton, C.G., 2007. Pharmaceuticals in the Environment: Sources and Their Management, Chapter 1, 1–58. In: Analysis, Fate and Removal of Pharmaceuticals in the Water Cycle (M. Petrovic and D. Barcelo, Eds.). Wilson & Wilson's comprehensive Analytical Chemistry series (D. Barcelo, Ed.), vol. 50, Elsevier Science. 564 pp. Fent, K., Zenker, A., Rapp, M., 2010. Widespread occurrence of estrogenic UV filters in aquatic ecosystems in Switzerland. Environ. Pollut. 158, 1817–1824. Gago-Ferrero, P., Diaz-Cruz, S., Barcelo, M., 2013. Multi residue method for trace level determination of UV-filter in fish based on pressurized liquid extraction and liquid chromatography–quadrupole-linear ion trap-mass spectrometry. J. Chromatogr. A 1286, 93–101. Gomez, E., Bachelot, M., Moillot, C., Munaron, D., Chiron, S., Casellas, C., Fenet, H., 2012. Bioconcentration of two pharmaceuticals (benzodiazepines) and two personal care products (UV filters) in marine mussels (Mytilus galloprovincialis) under controlled laboratory conditions. Environ. Sci. Technol. 19, 2561–2569. Grung, M., Lichtenhaler, R., Ahel, M., Tollefsen, K.E., Langford, K., Thomas, K.V., 2007. Effects-directed analysis of organic toxicants in wastewater effluent from zagreb, Croatia. Chemosphere 67, 108–120. Hao, C., Zhao, X., Yang, P., 2007. GC–MS and HPLC–MS analysis of boactive pharmaceuticals and personal-care products in environmental matrices. Trends Anal. Chem. 26, 569–580. Kannan, K., Reiner, J.L., Yu, S.H., Perrotta, E.E., Tao, L., Johnson-Restrepo, B., Rodan, B.

71

D., 2005. Polycyclic musk compounds in higher trophic level aquatic organisms and humans from the United States. Chemosphere 61, 693–700. Kim, S.-C., Carlson, K., 2005. LC–MS2 for quantifying trace amounts of pharmaceutical compounds in soil and sediment matrices. Trends Anal. Chem. 24, 635–644. Kunz, P.Y., Fent, K., 2006. Multiple hormonal activities of UV filters and comparison of in vivo and in vitro estrogenic activity of ethyl-4-aminobenzoate in fish. Aquat. Toxicol. 79, 305–324. Nakata, H., Sasaki, H., Takemura, A., Yoshioka, M., Tanabe, S., Kannan, K., 2007. Bioaccumulation, temporal trend and geographical distribution of synthetic musks in marine environment. Environ. Sci. Technol. 41, 2216–2222. Nakata, H., Shinohara, R.-Y., Nakazawa, Y., Isobe, T., Sudaryanto, A., Subramanian, A., Tanabe, S., Pauzi Zakaria, M., Zheng, G.J., Lam, P.K.S., Kim, E.Y., Min, B.-Y., We, S.U., Viet, P.H., Tana, T.S., Prudente, M., Frank, D., Lauenstein, G., Kannan, K., 2012. Asia-Pacific mussel watch for emerging pollutants: Distribution of synthetic musks and benzotriazole UV stabilizers in Asian and US coastal waters. Mar. Poll. Bull. 64, 2211–2218. Picot Groz, M., Martinez Bueno, M.J., Rosain, D., Fenet, H., Casellas, C., Pereira, C., Maria, V., Bebianno, M.J., Gomez, E., 2014. Detection of emerging contaminants (UV filters, UV stabilizers and musks) in marine mussels from Porutguese cosat by QuEChERS extraction and GC–MS/MS. Sci. Total Environ. 493, 162–169. Regulation (EC) No. 1272/2008 of the European parliament and of the council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing directive 67/548/EEC and 1999/45/EC and amending regulation (EC) No. 1907/2006. Ternes, T.A., Joss, A., Siegrist, H., 2004. Scrutinizing pharmaceuticals and personal care products in wastewater treatment. Environ. Sci. Technol. 38, 392A–399A. Treadglod, J., Liu, Q.T., Plant, J.A., Voulvoulis, N., 2012. Pharmaceuticals and Personal-care Products in Pollutants, Human Health and the Environment: A Risk Based Approach. vol. 8. John Wiley & Sons, pp. 207–229. Vallecillos, L., Pocurull, E., Borrull, F., 2015. Influence of pre-treatment process on matrix effect for the determination of musk fragrances in fish and mussel. Talanta 134, 690–698. Zenker, A., Schmutz, H., Fent, K., 2008. Simultaneous trace determination of nine organic UV-absorbing compounds (UV filters) in environmental samples. J. Chromatogr. A 1202, 64–74.