Marine Pollution Bulletin 62 (2011) 948–955

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Marine Pollution Bulletin

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Occurrence of perfluorinated compounds in water, sediment and mussels from theCantabrian Sea (North Spain)

Cristian Gómez a, Joana Vicente a, Beatriz Echavarri-Erasun b, Cinta Porte a, Silvia Lacorte a,⇑a Department of Environmental Chemistry, IDAEA-CSIC, Jordi Girona 18-26, 08034 Barcelona, Catalonia, Spainb Instituto de Hidráulica Ambiental ‘‘IH Cantabria’’, Grupo de Emisarios Submarinos e Hidráulica Ambiental, Universidad de Cantabria, E.T.S.I. de Caminos Canales y Puertos,Avda de los Castros s/n, 39005 Santander, Spain

a r t i c l e i n f o

Keywords:Perfluorinated compoundsWaterSedimentMusselsLC–MS

0025-326X/$ - see front matter � 2011 Elsevier Ltd.doi:10.1016/j.marpolbul.2011.02.049

⇑ Corresponding author. Tel.: +34 934006100; fax:E-mail address: slbqam@idaea.csic.es (S. Lacorte).

a b s t r a c t

Perfluorinated compounds (PFCs) have emerged as significant global environmental pollutants with per-sistent, bioaccumulative and toxic properties. The aim of this study was to determine the occurrence ofPFCs in water (wastewater, submarine emissaries and port-waters), sediment and transplanted musselsin estuarine areas of high urban and industrial impact from Northern Spain. Five PFCs of industrial usewere studied: perfluorooctanesulfonate, perfluorohexanesulfonate, perfluorobutanesulfonate, perflu-orooctanoate acid and perfluorononanoate acid. After selective extraction, samples were analyzed byUltra Performance Liquid Chromatography coupled to tandem mass spectrometry. RPFCs ranged from0.06 to 10.9 ng/L in water, with higher levels in wastewater treatment plants effluents and port watersthan in submarine emissaries. Little accumulation was observed in sediments and mussels with RPFCsranging from 0.01–0.13 ng/g dw and 0.01–0.06 ng/g ww, respectively. Most ubiquitous compounds werePFOS and PFOA. Mass fluxes of PFCs to the Cantabrian Sea are estimated and the impact to the coastalecosystem is discussed.

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1. Introduction

Perfluorinated compounds (PFCs) are organofluorine com-pounds where all the C–H bonds have been replaced by C–F bonds.They contain a hydrophobic fluorinated carbon tail and a polar,hydrophilic head attached to one end of the carbon chain (Ericsonet al., 2007). PFCs have surface-active properties and are excellentsurfactants and surface protectors due to their unique properties ofrepelling both water and oil. PFCs have been widely used for50 years in the production of fluoropolymers (for example polytet-rafluoroethylene-PTFE or polyvinylidene fluoride-PVDF) (Claraet al., 2009). They are also used in a variety of products such as tex-tile protectors, coatings, plastics, fire fighting foams, photolithogra-phy, electronic chemicals, shampoos, stain repellents for furniture,carpets and pesticides (Guo et al., 2008; Yu et al., 2009). PFCs arereleased to the environment after production, use or disposal andpersist although some may undergo chemical degradation or bio-degradation (Dinglasan et al., 2004; Naile et al., 2010). The twoPFCs which are found at the highest concentrations in the environ-ment are the perfluorosulfonates and the perfluorocarboxylic acids(Clara et al., 2009).

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As other types of contaminants, PFCs are discharged to the seaand oceans via treated or untreated wastewater effluents or riverflows (Sánchez-Avila et al., 2010a). Once released, PFCs are dilutedin open seawaters but because of their continuous input, stabilityand persistance, PFCs have been detected in coastal waters in To-kyo Bay (Yamashita et al., 2004) and at 100–1000 times lower con-centration in open waters from the Pacific and North and MidAtlantic Oceans (Yamashita et al., 2005, 2008). In addition, sedi-ment and deep ocean waters have been identified as sinks and res-ervoirs of PFCs (Higgins and Luthy, 2006; Prevedouros et al., 2006;Yamashita et al., 2008). However, data on the sources and massbalance of PFCs in the coastal area are scarce. In 2007, PFOA wasdetected close to the river mouth in Ferrara at 60 and 174 ng/Land contributed to a mass load of 0.3 kg PFOA per hour or 2.6 tonsper year to the Adriatic Sea (Loos et al., 2008). Another study car-ried out in the United States stated PFCs discharges between 0.52and 7.62 g/day in Georgia and 2.22 and 3.11 g/day in Kentucky(Loganathan et al., 2007). Recently it has been estimated that thedaily PFCs discharge to the NW Mediterranean sea were of 190 g/day, considering both river and wastewater treatment plant(WWTP) effluents (Sánchez-Avila et al., 2010a). The continuousexposure of trace PFCs levels in open seawaters and oceans leadsto their accumulation in wildlife (Guo et al., 2008), and may havesignificant negative consequences on aquatic biota (Bossi et al.,2008). For such reason, in 2006, the SCHER (Scientific Committeeon Health and Environmental Risks) classified PFCs as persistent,

C. Gómez et al. / Marine Pollution Bulletin 62 (2011) 948–955 949

bioaccumulative and toxic pollutants for the global environmentand in 2009 PFOS and its salts were included in the Stockholm-Convention-Secretariat (2009).

The aim of the present study was to determine the distributionand compartmentalization of PFCs in sea water, sediments andmussels (Mytilus galloprovincialis) samples collected in NorthernSpain. This area has an important industrial activity and hosts largecities. We have focused on perfluorooctanesulfonate (PFOS), per-fluorohexanesulfonate (PFHxS), perfluorobutanesulfonate (PFBuS),perfluorooctanoate acid (PFOA) and perfluorononanoate acid(PFNA), the most used and widespread PFCs (Ahrens et al., 2009).

2. Materials and methods

2.1. Chemicals and reagents

Native compounds of PFOS, PFOA, PFNA, PFHS and PFBS weresupplied by Wellington Laboratories (Ontario, Canada) at a concen-tration of 100 ng/mL in acetonitrile. Stock standard solutions wereprepared in acetonitrile at a concentration of 5 ng/ml for all nativecompounds and were stored at �18 �C. Perfluoro-n-(1,2,3,4-13C4)octanoate acid (m-PFOA) and sodium perfluoro-1-(1,2,3,4-13C4)octanesulfonate (m-PFOS), also from Wellington Laboratories,were used as surrogate standards. HPLC grade water, methanol(>99.9%) and acetonitrile (>99.8%) were purchased from Merck(Darmstadt, Germany) and glacial acetic acid from Panreac (Barce-lona, Spain). Activated carbon (Envicarb) was purchased as a pow-der (Supelco, USA). The molecular formulas, structures and theuses of the five studied PFCs are shown in Table SI1.

2.2. Sampling

Waters, sediments and transplanted mussels were sampled inestuarine areas from three regions in Northern Spain: Asturias,Cantabria and Basque Country (Fig. 1). These areas receive thedischarges of treated or untreated urban wastewaters via emis-saries, run-off of industrial wastes and harbour activities. Study

Fig. 1. Geographical distribution of the sampling area, comprisin

areas comprised three sea emissaries, five ports, three sewagetreatment plant effluents and one industrial wastewater effluentfrom a metallurgical industry (Table S2). Sewage treatment efflu-ents were collected at the exit of the wastewater treatmentplant (WWTP). WWTP sediments corresponded to the sedimentsof the WWTP effluent receiving river. In sea emissaries, watersamples were collected at different depths (surface, 1 m deepand at the bottom of the sea in the emissary outflow) and sed-iments at the sea bed. Port waters were collected inside the portat 1–3 m depth and sediments were sampled at the sameemplacement. After collection, samples were stored at 4 �C untiltransport to the main laboratory and waters were processedwithin 1 week while sediments were frozen, freeze dried andsieved through 120 lm.

Transplanted mussels were placed in WWTP effluents’ receivingrivers, in ports and emissaries for a period of 3 weeks. First, 40–50 mm length mussels were collected from mussel crops fromthe Galician Rías (NW, Spain), cleaned from epiphytes and intro-duced in polyethylene experimental units. Experimental unitswere acclimated in an open circuit aquarium for 7 days. At t = 0(before placing the mussels in each study site), 10 individuals fromfour experimental units were collected to quantitate the contami-nation levels before the exposure. In emissaries, the experimentalunits were anchored onto fixed structures at 30 m depth while inthe other stations, those were held between 1 and 4 m of depth.After 21 days of exposure, 10 additional mussels were collected,except from Peñarrubia emissary where the experimental unitwas lost because of a storm. Once collected, 10 mussels wereground to a homogenized mix and kept in a glass jar at �20 �C untilextraction.

Sampling was carried out in October 2009 (time zero) and re-peated in November 2009 (time 21 days). Sea sampling was per-formed by professional scuba-divers and precleaned amberglassware was used for all matrices. During the sampling andextraction process, samples were never in contact with Teflon con-tainers in order to avoid contamination and adsorption of theanalytes.

g Asturias, Cantabria and Basque country (Northern Spain).

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2.3. Sample extraction

2.3.1. Water extractionOne litre of unfiltered water was spiked with 20 lL of a 20 pg/

lL surrogate standard solution (13C-PFOS and 13C-PFOA) and wasincubated for 12–18 h at 4 �C. PFCs were extracted using WatersOasis� 6 cc of 200 mg SPE cartridge according to Sánchez-Avilaet al. (2010b). Briefly, cartridges were conditioned with 10 mL ofmethanol and 15 mL of HPLC water. After pre-concentration, ana-lytes were eluted with 15 mL of methanol; the resulting extractwas evaporated to dryness with a TurboVap and reconstituted with200 lL of a 70:30 mixture of HPLC water: acetonitrile.

2.3.2. Sediment extractionOne gram of <120 lm particle dried sediment was weighted in

polypropylene (PP) centrifuge tubes. After the addition of 20 lL ofa 5 ng/lL solution of surrogate standard solution (13C-PFOS and13C-PFOA), samples were incubated for 18 h at 4 �C. Nine millilitreof methanol and 10 mL of a 1% glacial acetic acid solution werethen added and the sample was thoroughly mixed using a vortexmixer. Samples were then extracted in an ultrasonic bath for10 min at room temperature (three times). Between each periodof 10 min, samples were thoroughly mixed. Finally they were cen-trifuged at 2500 rpm for 5 min. The supernatant was transferred toa new vial and evaporated with a TurboVap to dryness. Then, 1 mLof acetonitrile was added to the dried samples and incubated for10 min in the ultrasonic bath. The samples were purified by adding25 mg of activated carbon and 50 lL of glacial acetic acid, mixing itvigorously during 1 min. Afterward, the samples were centrifugedfor 10 min at 10000 rpm. One millilitre was transferred to a cleanmicro vial. Finally, 150 lL of a filtered extract (0.2 lm GHP Accro-disc 13 mm syringe filter, PALL Life Sciences, USA) were reconsti-tuted with 350 lL of HPLC water.

2.3.3. Mussels extractionOne gram of wet mussel sample was weighted in polypropylene

(PP) centrifuge tubes. After the addition of 1 mL of acetonitrile and20 lL of a 5 ng/lL surrogate standard solution (13C-PFOS and 13C-PFOA), samples were incubated for 18 h at 4 �C. Extraction condi-tions were similar to a previous study (Meyer et al., 2009). Eightmillilitre of acetonitrile were added and then the samples wereshaken with a vortex mixer. Samples were then extracted in anultrasonic bath for 10 min at room temperature (three times). Be-tween each period of 10 min, samples were thoroughly mixed. Fi-nally they were centrifuged at 2500 rpm for 5 min. Thesupernatant was transferred to a new vial and evaporated with aTurboVap to dryness. Then, 1 mL of acetonitrile was added to thedried samples and incubated for 10 min in the ultrasonic bath.The samples were purified by adding 25 mg of activated carbonand 50 lL of glacial acetic acid, mixing it vigorously during1 min. Afterward, the samples were centrifuged for 10 min at10000 rpm. One millilitre of the supernatant was transferred to aclean micro vial and reconstituted as for sediments.

2.4. Instrumental analysis

PFCs were measured using Ultra Performance Liquid Chroma-tography coupled to tandem mass spectrometry (UPLC–MS/MS).The system consists on an Acquity UPLC system (Waters, USA) con-nected to a Triple Quadruple Detector. An Acquity UPLC BEH C18

column (1.7 lm particle size, 50 � 2.1 mm, Waters, USA) was usedas mobile phase residue trap to remove any PFCs traces from themobile phases and tubing. The analysis was performed on a LiChro-CART HPLC RP-18e column (125 � 2 � 5 lm particle size, Merck,Germany) connected to a C18 pre-column at a flow rate of0.4 mL/min. The mobile phase was 2 mM NH4OAc (A)/acetonitrile

(B). Gradient elution started at 70% A and 30% B, increased to90% B in 5 min and increased to 100% B in 6 s, held for 54 s. Initialconditions were attained in 1 min and the system was stabilizedfor 3 min. Ten microlitres were injected. PFCs were measured un-der negative electrospray ionization and acquisition was per-formed in Selected Reaction Monitoring (SRM) using one or twotransitions from parent to product ion to identify each compound.The transitions used as well as the dwell time, cone voltage andcollision energy are given in a previous study (Fernández-Sanjuanet al., 2010). Internal standard quantitation was performed using13C-PFOS to quantitate PFOS, PFHxS, PFBuS and 13C-PFOA to quan-titate PFOA and PFNA.

2.5. Quality control parameters

Samples were extracted and analyzed in batches together witha procedural blank in order to control any external contaminationduring the whole analytical process. Seven point calibration curveswere used at a range of 2.5–100 ng/mL, covering the concentrationrange where target compounds are likely to be found in the sam-ples, with the surrogate standards at 5 ng/ml. Recovery studieswere performed in duplicate using HPLC water spiked at 0.4 ng/L.In the case of sediments and mussels, the performance of theextraction method was evaluated using the surrogate standards.

Limit of detection (LOD) was calculated as the concentrationthat yielded an S/N ratio higher than or equal to three. Instrumen-tal detection limit (LODinstr) was calculated using the lowest con-centration standard solution at 2.5 ng/mL and method detectionlimit (LODmethod) was calculated in the same way, using watersamples spiked at 0.4 ng/L.

3. Results and discussion

3.1. Method performance

The trace analysis of PFCs in water still poses some difficultiesas regards to sensitivity and blank contamination. Therefore, qual-ity control and quality assurance is of outmost importance to pro-vide accurate results. Fig. SI1 shows the LC–MS/MS chromatogramsof the three environmental matrices analyzed and a standard solu-tion at 25 ng/mL (Fig. SI1). Labeled 13C-PFOA and 13C-PFOS elutingat 3.26 and 4.24 min, respectively, were used to control any reten-tion time shifts and the instrumental response. In all cases, thesame chromatographic behavior was found for the standard solu-tion and the environmental extracts, regarding both retention timeand analytical response. These chromatograms also indicate thatthe extraction and analytical method were highly efficient sincein general no matrix interferences were observed except for portseawater (Urdaibai-sample 5) which presented some interferenceat 4 and 4.11 min. Target compounds were not detected in any ofthe blank samples, indicating no external contamination from nei-ther the extraction nor the analytical steps. The selectivity of themethod was also good. With a chromatographic run time of6 min, PFNA and PFHxS coeluted but were selectively separatedat their specific transitions.

Table 1 shows the quality parameters of the method, such asslope of the calibration curve, limits of detection, values of repro-ducibility and finally, the recoveries of target compounds. Goodcorrelations (r2 > 0.997) were obtained for all compounds over aconcentration range of 2.5–100 ng/mL using internal standardquantitation with 13C-PFOS and 13C-PFOA. The reproducibility ofthe method at 10 ng/mL was of 3–11%. In water, recovery valueswere from 60% to 122% with RSD ranging from 2% to 12% indicatingacceptable extraction efficiency. For sediment and mussel samples,recoveries were calculated using the surrogate standards from the

Table 1Quality parameters of all target compounds studied.

Quality parameters PFOS PFHxS PFBuS PFOA PFNA

Slope 1.81 5.16 2.56 3.84 0.95LODinstrumental waters (pg) 0.12 0.05 0.08 0.08 0.27LODmethod waters (pg/L) 3 1 1.8 0.6 2.2Reproducibility (% RSD, n = 3) 6 3 11 7 7Recoveries in water (% RSD) 104 ± 12 93 ± 2 104 ± 3 60 ± 10 122 ± 3Recoveries in sediments (% RSD) 100 ± 26* n.a. n.a. 108 ± 18* n.a.Recoveries in mussels (% RSD) 70 ± 7* n.a. n.a. 130 ± 16* n.a.

n.a.: not analyzed.* Recovery of surrogate standards.

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unspiked sediment and mussel samples and the method yieldedclose to 100% recovery, except for 13C-PFOA in mussels wherethe recoveries increased to 130%.

3.2. PFCs in wastewaters effluents

All wastewaters contained PFCs and were identified as a sourceof PFCs to the Cantabrian coastal waters, through discharges ofWWTP effluents or submarine emissaries (Table 2). PFCs in portswere from 0.01 to 6.57 ng/L, in WWTP effluents from 0.04 to

Table 2Levels of PFCs in waters (ng/L) at two sampling periods (at t = 0 and after 21 days), in sed

Sample location PFOS (ng/L) PFHxS (ng/L) P

Water t0 t21 t0 t21 t

Emissaries 1a 0.08 0.02 BDL BDL 01b 0.11 0.01 BDL BDL 01c 0.04 BDL BDL BDL 02a 0.03 0.03 BDL BDL B2b 0.02 0.02 BDL BDL B2c 0.02 0.05 BDL BDL 03a 0.02 0.02 BDL BDL B3b 0.03 0.02 BDL 0.01 B3c 0.02 0.02 BDL BDL 0

Port seawater 4 0.30 0.40 0.03 0.04 05 0.61 6.57 0.01 BDL 06 0.06 0.46 0.09 BDL B7 0.02 BDL BDL BDL B8 0.51 0.01 0.19 BDL B

WWTP effluents 9 0.09 0.26 0.05 0.08 110 0.71 1.80 0.06 0.08 011a 1.45 2.19 0.12 0.14 B11b 2.16 1.42 0.26 BDL 112 0.59 5.11 0.06 0.31 0

Sediment samples PFOS (ng/g) PFHxS (ng/g) P

Emissaries 1 BDL BDL B2 BDL BDL B3 BDL BDL B

Ports 4 BDL BDL B5 0.02 BDL B7 BDL BDL 08 BDL BDL B

Sediments from the river 9 BDL BDL B11 0.03 BDL B12 0.13 BDL B

Mussels samples PFOS (ng/g) PFHxS (ng/g) P

Emissaries 1 0.02 BDL B3 BDL BDL B

Ports 4 BDL BDL B5 0.06 BDL B6 BDL BDL B7 BDL BDL B8 BDL BDL B

Mussels from the river 9 BDL BDL B11 BDL BDL B12 BDL BDL B

a: at the bottom of the sea; b: one meter of deep; c: at the surface (emissary waters). S

5.11 ng/L and in emissaries from 0.01 to 0.12 ng/L (Fig. 2). Usinga one way ANOVA test with a statistical significance level of 0.05,a p-value of 0.0188 and a F value of 5.63 indicate statistical differ-ences in sample types (wastewater, port and emissary). Whilethere were no differences among port and wastewater effluents(p = 0.0918 and F = 3.67) and port and emissaries (p = 0.1761,F = 2.2), differences were observed among wastewater effluentsand emissaries (p = 0.0155, F = 9.38). PFOA, PFOS and PFNA weredetected in almost 100% of the samples while PFHxS and PFBuSwere detected sporadically. These results are in accordance with

iments (ng/g dw) and in mussels (ng/g ww).

FBuS (ng/L) PFOA (ng/L) PFNA (ng/L) RPFCs

0 t21 t0 t21 t0 t21 t0 t21

.05 BDL 0.08 0.12 0.06 0.05 0.26 0.20

.05 0.02 0.05 0.02 0.02 0.02 0.24 0.06

.02 BDL 0.07 0.05 0.04 0.06 0.18 0.11DL 0.01 0.04 0.02 0.06 0.01 0.12 0.07DL BDL 0.03 0.07 0.03 0.04 0.08 0.12.01 BDL 0.04 0.04 0.07 0.03 0.15 0.11DL BDL 0.06 0.04 0.03 0.04 0.10 0.10DL BDL 0.04 0.03 0.02 0.01 0.09 0.06.03 BDL 0.07 0.03 0.06 0.03 0.18 0.08.22 0.13 0.16 0.18 0.06 0.08 0.77 0.83.18 1.25 0.07 0.11 BDL 0.07 0.87 8.00DL BDL 0.31 0.20 0.10 0.20 0.56 0.86DL BDL 0.06 0.05 0.04 0.07 0.11 0.11DL BDL 0.06 0.05 0.04 0.07 0.80 0.12.17 1.51 0.43 0.99 0.06 0.10 1.81 2.94.89 BDL 0.17 0.47 0.04 0.04 1.88 2.39DL 0.85 0.67 0.72 0.27 0.22 2.51 4.11.19 5.08 3.38 3.53 1.40 0.85 8.39 10.89.18 0.86 0.16 0.71 0.11 0.44 1.10 7.43FBuS (ng/g) PFOA (ng/g) PFNA (ng/g) RPFCs

DL BDL BDL BDLDL BDL BDL BDLDL 0.06 BDL 0.06DL 0.01 BDL 0.01DL 0.03 0.08 0.13.01 BDL BDL 0.01DL BDL BDL BDLDL 0.02 BDL 0.02DL BDL BDL 0.03DL BDL BDL 0.13FBuS (ng/g) PFOA (ng/g) PFNA (ng/g) RPFCs

DL BDL BDL 0.02DL 0.01 BDL 0.01DL BDL BDL BDLDL BDL BDL 0.06DL 0.01 BDL 0.01DL BDL BDL BDLDL BDL BDL BDLDL 0.01 BDL 0.01DL BDL BDL BDLDL BDL BDL BDL

ample 11 a and b: effluents were sampled two times.

Fig. 2. Boxplot in logarithmic scale of PFC levels in emissaries (samples 1–3), Port waters (samples 4–8) and WWTP effluents (samples 9–12).

952 C. Gómez et al. / Marine Pollution Bulletin 62 (2011) 948–955

a previous study where PFOA and PFOS were identified as the ma-jor PFCs in European Rivers (detected in the 97% of samples) (Looset al., 2009) and in wastewaters (Sánchez-Avila et al., 2010a). Thiscompound distribution is similar to that reported by Naile et al.who analyzed wastewater and found that the concentration of[PFOS] (octa) > [PFDS] (deca) and [PFOA] (octa) > [PFNA] (non-a) > [PFDA] (deca) (Naile et al., 2010). Ahrens et al. also analyzingwastewater, found that [PFOA] > [PFDA]. As for perfluorocarboxilicacids (PFCA) with C > 9, they should be accumulated in sedimentand mussels, but former studies do not demonstrate this tendency(Naile et al., 2010).

All target compounds were detected in all WWTP effluent sam-ples (Table 2). RPFCs values varied from 1.10 to 10.9 ng/L. At t0,PFOS (0.09–2.16 ng/L), PFOA (0.16–3.38 ng/L) and PFBuS (0.18–1.19 ng/L) were the compounds detected at the highest levels, fol-lowed by PFNA (0.04–1.40 ng/L) and PFHxS (0.05–0.26 ng/L). Witha one way ANOVA test, a p-value of 0.2725 and a F value of 1.24 isobtained. Therefore, there are no significant differences withinsamples collected at t0 and t21 (Table 2). The similar concentrationsdetected among two sampling periods provide validation of themethodology used and give evidence that PFCs undergo physico-chemical and biological degradation of WWTP (Ahrens et al.,2009; Higgins et al., 2005) and therefore, are continuously releasedto receiving rivers through WWTP effluents.

The three WWTP included in this study treat mixed waterscoming both from urban and industrial areas. Galindo’s WWTP(sample 11) effluent (1500,000 hab-equivalents) contained thehighest PFCs levels, with RPFCs = 6.47 ± 3.8 ng/L, followed by Plen-tzia (sample 9) (44,000 hab-equivalent) which contained2.37 ± 0.79 ng/L and San Román (sample 10) (530,000 hab-equiva-lent) with 2.13 ± 0.36 ng/L. All three studied WWTP operate usingsecondary treatment and the PFCs distinct levels is attributed tothe different treatment processes and the wastewater origin (Sin-clair and Kannan, 2006). The habitant equivalents play an impor-tant role not only in the load of contaminants to the WWTP butalso in the volume of water treated. With a daily flow of 4.05,2.52 and 0.152 m3/s in Galindo (sample 11), San Román (sample10) and Plentzia (sample 9), respectively, these plants contributeto a mean RPFCs discharge of 2.26, 0.46 and 0.03 g/day (consider-ing both t0 and t21 sampling periods) to the receiving rivers. On theother hand, effluent from an important metallurgical plant (Ferro-atlántica, sample 12) contained RPFCs from 1.10 to 7.43 ng/L, con-sidering the two sampling periods. This water effluent isdischarged to the estuarine waters of Santander, in the CantabrianSea.

Other studies report the presence of PFCs in wastewaters. Ah-rens et al. report the presence of 21 PFCs in nine WWTP effluents.

RPFCs were between 30.5 and 266.3 ng/L, with PFOA, PFBuS, per-fluorohexanoic acid (PFHxA) and PFOS the most abundant com-pounds detected. These WWTP contributed to a discharge of PFCsbetween 0.05 and 1.6 g/day (similar to the present study) exceptfor two WWTP with high industrial inputs which contributed to11.6 and 55.61 g/day (Ahrens et al., 2009). Another study carriedout in Hong Kong, out of 14 PFCs analyzed, only PFOS, PFBuS, Per-fluoropentanoate acid (FPeA), PFOA, PFNA, PFHxA were detected atconcentrations of 0.6–49.9 ng/L in wastewaters from a WWTP of3.5 M hab-equivalents (Ma and Shih, 2010). Another study reportsthe occurrence of eight PFCs in a Kentucky WWTP, where PFCswere detected in wastewaters influents between <0.5 and184 ng/L and in effluents at levels between <0.5 and 183 ng/L, indi-cating the poor efficiency of the plant for removing these com-pounds. A similar situation was found in Georgia WWTP(Loganathan et al., 2007). In Singapore, PFOS was detected from7.9 to 374.5 ng/L in influents and from 7.3 to 461.7 ng/L in efflu-ents, while PFOA was detected from 14.1 to 638.2 ng/L in influentsand from 15.8 to 1057.1 ng/L in effluents (Yu et al., 2009). These re-sults corroborate those of Loganathan and confirm that these com-pounds are not eliminated within a WWTP operating withsecondary treatment and thus, WWTP effluents become a sourceof PFCs to receiving waters and to the sea. This same panoramawas observed for the three Spanish WWTP effluents.

3.3. PFCs in submarine emissaries

Other WWTP release treated effluents directly to the sea viasubmarine emissaries which discharge 1–3 miles from the coastline and in the deep sea bed. RPFCs were detected in the three sub-marine emissaries at levels from 0.06 to 0.26 ng/L, considering thethree depths (Table 2). The most ubiquitous compound was PFOS(0.01–0.11 ng/L), followed by PFOA (0.02–0.12 ng/L) and PFNA(0.01–0.07 ng/L). PFHxS and PFBuS were seldom detected and atvery low concentration (0.01–0.05 ng/L). With a two way ANOVAtest a p-value of 0.916 and a F value of 0.09 indicates that thereare no significant differences among sampling periods (time t0

and t21) and depths (surface, middle, bottom). PFCs levels werevery similar (p = 0.0691, F = 4.31), which indicates again an overalllow concentration but continuous inputs. Of the three emissariesstudied, slightly highest levels were found in Virgen del Mar (sam-ple 1), with mean RPFCs of 0.175 ± 0.07 ng/L (Table 2). This emis-sary has an outflow rate of 1.6 m3/s and it discharges 0.024 g/day.In Peñarrubia emissary (sample 2), the mean RPFCs was0.108 ± 0.03 ng/L, with PFOS, PFOA and PFNA concentrations rang-ing from 0.01 to 0.07 ng/L. With an effluent water outflow of0.7 m3/s, this emissary contributed to RPFCs of 0.007 g/day. Berria

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emissary (sample 3), in Santoña town (14,000 inhabitants) is con-sidered a clean area although the RPFCs were from 0.10 ± 0.04 ng/L, with PFOS, PFOA and PFNA ranging 0.01–0.07 ng/L. With an out-flow of 5.12 m3/s, this emissary contributes to 0.044 g/day. In gen-eral, the mass flow of PFCs through these three submarineemissaries is low even in the mouth of the emissary if comparedwith WWTP effluents collected directly from the plant outflow.Submarine emissaries gather water from treated or untreatedwaters and also from run-off. Therefore, contaminants present inWWTP effluents can be diluted during its pipe-transport time. Inaddition, the outflow of the emissaries create a high turbid areaproducing a rapid dispersion of the effluent in the surrounding sea-water, aided by the marine currents and rough waves of the Canta-brian Sea. Fig. 3 shows that there was not a vertical profile in thePFCs concentrations collected at three depths from the emissarymouth (One way ANOVA test with a statistical significance levelof 0.05, p = 0.0718, F = 4.22).

3.4. PFCs in port waters

In Northern Spain, port activity is very high, with merchandisetrade, fishing and sportive activities and thus ports receive the di-rect pollution from boat maintenance and repairing. In addition,the five ports studied are settled within a estuarine areas and re-ceive urban run-off and wastewater effluent discharges. Becauseof the semi-enclosed situation of ports and estuaries and the littlecirculation and refreshing of waters, port waters have been identi-fied as a source of PFCs to coastal seawaters. The incidence ofRPFCs were Urdaibai (sample 5) > Arriluze (sample 4) > Pasaia(sample 6) > Santander (sample 8) > San Vicente (sample 7) (Ta-ble 2), with levels ranging from 0.11 to 8 ng/L. PFOS was detectedbetween 0.01 and 6.57 ng/L, followed by PFOA (0.05–0.31 ng/L)and PFNA (0.04–0.20 ng/L). PFBuS was detected only in two outof five samples analyzed at 0.13–1.25 ng/L and PFHxS in four of fivesamples at 0.01–0.19 ng/L. Similar results were obtained by Willeet al. who detected PFOS at 2.3–3.6 ng/L in the harbour of Nieuw-poort, PFOS and PFHxS at 4.3–12.8 ng/L and 2.4–6.7 ng/L, respec-tively, in the harbour of Ostend and only PFOS was detected at3.3–4.8 ng/L in the harbour of Zeebrugge. Wille et al. conclude thatadverse effects towards aquatic organisms are not expected con-sidering the Predicted Non-Effect Concentration of PFOS and PFOA(Wille et al., 2010).

As demonstrated in previous studies, coastal waters receive theimpact of wastewater discharges. In Fig. 4, the differences betweenthe levels of PFCs obtained in the Cantabrian Sea are compared toother seawater areas. Catalonian coastal waters contained 0.087–8.38 ng/L of PFOS and 0.38–2.25 ng/L of PFOA (Sánchez-Avilaet al., 2010a). Maximum PFOS values were found in TomakomaiBay with an amount of 2880 ng/L (Taniyasu et al., 2005). However,

Fig. 3. Vertical profile of RPFCs in submarine e

for PFOA, the maximum values were found in Western Korea andTomakonai Bay with levels of 68.6 ng/L (Naile et al., 2010) and62.4 ng/L (Taniyasu et al., 2005), respectively. In contrast, the min-imum values for both PFOS and PFOA were found in the North Seawith a concentration of 0.07 ng/L for both compounds (Ahrenset al., 2010). In general, industrialized areas have a high pollutionimpact by PFCs.

3.5. Occurrence of PFCs in sediment

PFCs have an amphoteric character and a long fluorinated car-boxylic chain responsible for their persistence and accumulationin sediments (Senthilkumar et al., 2007) and biota (Frommeet al., 2007). The organic carbon partitioning coefficient (log Koc)of PFCs varies from 2.17 to 3.71 (Labadie and Chevreuil, 2011)and we have found that partitioning to sediments with Total Or-ganic Carbon (TOC) values from <0.1 to 4.92 herein analyzed isnot favoured.

In river sediments receiving WWTP effluents, emissaries andport sediments, the RPFCs varied from 0.01 to 0.13 ng/g dw andwere detected in 7 of the 10 samples, with PFOS and PFOA thedominant contaminants detected. Overall, it was found thatadsorption of PFCs onto sediments was very low. Sediments col-lected in the receiving river from Plentzia (sample 9) and Galindo(sample 11) WWTP effluents contained 0.02 ng/g dw of PFOA and0.03 ng/g dw of PFOS and the same situation was observed for sed-iments collected close to Ferroatlántica effluent (sample 12)(0.13 ng/g dw). Sediments collected at the bottom of submarineemissaries did not contain traces of PFCs and only PFOA was de-tected in Berria-Santoña (sample 3) at 0.06 ng/g dw, as shown inTable 2. As studied submarine emissaries discharge in open coastalwaters, high dispersion of PFCs limit the accumulation of PFCs tosediments. A similar situation was found in port sediments, witha very low concentration or detection frequency. Only Urdaibaiport (sample 5) sediments contained PFOS, PFOA and PFNA (RPFCsof 0.13 ng/g) higher than in other areas. Comparing to other sites,the low concentration of PFOS and PFOA in sediments from theCantabrian Sea were similar to sediments from Kyushu, Japanwhich contained 0.09–0.14 ng/g dw and 0.84–1.1 ng/g dw, respec-tively (Nakata et al., 2006). In sediments, Ma et al. found 0.1–30.7 ng/g dw and [PFOA] > [PFDA] > [PFUnDA] (undeca) (Ma andShih, 2010). In contrast, in San Francisco Bay, levels up to 3.1 ng/g dw for PFOS and 0.63 ng/g dw for PFOA were detected (Higginset al., 2005) and in the Tokyo Bay, 1.7 ng/g for PFOS and 0.45 ng/g dw for PFOA were identified (Zushi et al., 1950–2004). Baoet al. detected eight PFCs in sediments from Daliao River and trib-utaries (NE China) with RPFCs between 0.29 and 1.03 ng/g dw and[PFOA] > [PFNA] > [PFDA] (Bao et al., 2009). In the western coast ofKorea, Naile et al. detected 12 PFCs in estuarine water samples

missaries (surface, 1 m deep and seabed).

Fig. 4. Comparison between levels of PFCs in coastal waters (ng/L) in the Cantabrian Sea with other coastal areas, in logarithmic scale. Maximum values have been used.

954 C. Gómez et al. / Marine Pollution Bulletin 62 (2011) 948–955

with RPFCs from 0.23 to 450 ng/L and in mussels the levels werefrom 0.21 to 4.97 ng/g dw while they were not detected in sedi-ments (Naile et al., 2010). In a recent study, Labadie et al. find verylow concentration of PFCs in river sediments (from <0.7 to 4.26 ng/g) and indicate that PFOS is the major compound (4.26 ng/g) whilethe rest are found at 0.03–1.72 ng/g. Compound distribution is[PFOS] > [PFDS], but in the case of acids, [PFDoDA] (dode-cane) > [PFUnDA] > [PFDA] > [PFNA] > [PFOA]. In this study the car-bon chain is correlated with log Koc (Labadie and Chevreuil, 2011).

3.6. Occurrence of PFCs in transplanted mussels

In transplanted mussels, PFCs accumulation was very low andno specific distribution was observed. The octanol–water partition-ing coefficient of PFOS is 6.3 and thus, has a high accumulation po-tential (Table SI1). However, only 5 out of 10 samples analyzedcontained PFOS or PFOA at a concentration range between 0.01and 0.06 ng/g ww (Table 2). The reason for this low accumulationrate in mussels is attributed to (i) short exposure time and/or (ii)low water concentration of PFCs. However, some studies state thatthe low accumulation of PFCs in mussels is explained by the in-creased efflux transport activity of proteins belonging to the aden-osine triphosphate (ATP)-binding cassette (ABC) superfamily,which act as a first line of defense of the organism, preventing toxicchemicals from entering the cell (Epel et al., 2008). As an example,gill tissue fragments of Mytilus californianus exposed to 100 nMPFNA for 60 min produced an uptake and a rapid loss of PFNA fromthe tissue (Stevenson et al., 2006). Other studies have evidencedthe low accumulation potential of PFCs in mussels. In China, PFCsin Perna viridis mussels were between 0.11 and 0.58 ng/g dw forPFOS and 0.20–0.66 ng/g dw for PFOA (So et al., 2006). In a recentstudy, PFCs were not detected in oysters (Crassostrea gigas) fromthe Ebro Delta (NW Mediterranean) or in zebra mussels (Dreissenapolymorpha) from the Ebro River (Fernandez-Sanjuan et al., 2010).Thus, the non accumulation of PFCs in mussels may limit the use ofthese organisms as bioindicators of PFCs pollution, unlike othertypes of contaminants.

4. Conclusions

The Cantabrian Sea receives the impact of large municipalities(>1500,000 habitants) and of very intense industrial and harbour-ing activities. WWTP effluents and submarine emissaries havebeen identified as a source of PFCs to coastal waters, with total dai-ly inputs of 2.83 g/day. In addition, port waters are impacted by thepresence of PFCs by their direct use in boating activities or due to

the discharge of treated or untreated urban waters, industrialwastes or run-off. Wastewaters and port waters contained thehighest PFCs concentration and in emissaries, PFCs were rapidlydispersed. In all cases, the PFCs concentration in water was atthe low ng/L range and accumulation in sediments and musselswas not favoured. PFOS was the dominant compound present inall samples, followed by PFOA. Since these compounds have a highbioaccumulation and biomagnification potential, concern has aris-en because the continuous release of these compounds to the seamay cause acute or long term effects, still unravelled.

Acknowledgements

This study has been financed by the Ministry of the Environ-ment of Spain, with the project Vertitox [018/RN08/02.1]. JuanSánchez and María Fernández-Sanjuan are acknowledged for aid-ing in the sampling and in the method set up. Dr. R. Chaler, D. Fan-jul and M. Comesaña are acknowledged for MS support.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.marpolbul.2011.02.049.

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