Screening of perfluorinated chemicals (PFCs) in various aquatic organisms

ORIGINAL PAPER

Screening of perfluorinated chemicals (PFCs) in variousaquatic organisms

María Fernández-Sanjuan & Johan Meyer &

Joana Damásio & Melissa Faria & Carlos Barata &

Silvia Lacorte

Received: 22 April 2010 /Revised: 7 July 2010 /Accepted: 12 July 2010 /Published online: 31 July 2010# Springer-Verlag 2010

Abstract The aim of this study was to evaluate theoccurrence of five perfluorinated chemicals (perfluorooc-tane sulfonic acid (PFOS), perfluorooctanoic acid (PFOA),perfluorononanoic acid (PFNA), perfluorohexane sulfonicacid (PFHxS), and perfluorobutane sulfonic acid) in aquaticorganisms dwelling in either freshwater or marine ecosys-tems. Organisms selected were insect larvae, oysters, zebramussels, sardines, and crabs, which are widespread in theenvironment and may represent potential bioindicators ofexposure to PFCs. The study comprises the optimization ofa solid–liquid extraction method and determination byhigh-performance liquid chromatography coupled to tan-dem mass spectrometry. Using spiked zebra mussels at 10and 100 ng/g level, the method developed providedrecoveries of 96% and 122%, and 82% to 116%, respec-tively, and a limit of detection between 0.07 and 0.22 ng/gww. The method was highly sensitivity and robust todetermine PFC compounds in a wide array of biologicalmatrices, and no matrix interferents nor blank contamina-tion was observed. Among organisms studied, none of thebivalves accumulated PFCs, and contrarily, insect larvae,followed by fish and crabs contained levels ranging from0.23 to 144 ng/gww of PFOS, from 0.14 to 4.3 ng/gww ofPFOA, and traces of PFNA and PFHxS. Assessment of thepotential use of aquatic organisms for biomonitoring studiesis further discussed.

Keywords Perfluorinated compounds . LC-MS/MS .

Aquatic organisms . Biomonitoring

Introduction

Perfluorinated chemicals (PFCs) such a perfluorooctanesulfonic acid compound (PFOS), perfluorooctanoic acid(PFOA), or perfluorononanoic acid (PFNA) have been usedfor many years in numerous industrial products, knownwidely for their use in Teflon products, although they arealso used as additives in detergents, soaps, as surfactants,explosives, and as flame retardants [1]. They are considereda new generation of contaminants that have arouse concernbecause they are globally distributed in the environment,especially in the aquatic environment [2], have a highbioaccumulation potential, and can have an impact onaquatic organisms. PFCs are moderately water soluble, non-volatile, and thermally stable [3], and due to a strongcarbon fluorine (C–F) covalent bond, PFCs are resistant tohydrolysis, photolysis biodegradation, and metabolism [4,5]. These characteristics explain the environmental persis-tence and bioaccumulative potential of PFCs [6].

PFCs have been identified in top predators such as polarbears [7], fish, and birds [8, 9], but they have also beenidentified in oysters [10], crabs [11], and mussels [12] andedible fish from the Mediterranean Sea [13]. In thesestudies, it has been shown that PFOS and PFOA are themost concurrent and abundant compounds. Most of thesestudies are based in marine organisms, which provideevidence on the accumulation potential of PFCs even whenthey are detected at the low ng/g level in seawater. Inmarine ecosystems, oysters and mussels have been for yearsthe ideal matrix used in biomonitoring studies and havebeen used to assess the levels of several persistent organicpollutants throughout the Mediterranean [14]. However,little information is known on the accumulation of PFCs infreshwater organisms. Because PFCs are detected inEuropean surface waters at levels between 32 and

M. Fernández-Sanjuan : J. Meyer : J. Damásio :M. Faria :C. Barata : S. Lacorte (*)Department of Environmental Chemistry, IDAEA-CSIC,Jordi Girona, 18-26,08034 Barcelona, Spaine-mail: [email protected]

Anal Bioanal Chem (2010) 398:1447–1456DOI 10.1007/s00216-010-4024-x

1,371 ng/L [15], accumulation of PFCs in aquatic organ-isms is expected. The analysis of aquatic organisms ofdifferent trophic levels can be used as bioindicator ofexposure to PFCs and can provide a wider vision on thedistribution of these contaminants in the freshwater eco-system. Among others, zebra mussel (Dreissena polymor-pha) has been selected for its widespread and pestilencedistribution in rivers and lakes and for its long life cycle (2–3 years). This organism has been used to assess the levelsof several toxicants such as metals, polycyclic aromatichydrocarbons, and polychlorinated biphenyls, and it wasconcluded that accumulation of these contaminants in zebramussel tissue represents a potentially realistic hazard toorganisms (i.e., fish and birds) that feed on them [16]. Onthe other side, the American crab (Procambarus clarkii) isalso a pest organism dwelling in warm freshwaters, such asrivers, marshes, reservoirs, canals, and rice paddies, andactively colonizes new territory at the expense of the nativecrayfish. The American crab is a source of food to manyorganisms, but it is also eaten by humans all around theworld, usually known as crawfish boil or barbecued.Finally, the insect Hydropsyche exocellata is widelydistributed in freshwater ecosystems, both in pristine andwith anthropogenic pressure. Part of its life cycle is in formof sedentary larvae dwelling in the aquatic environment. Thisorganism was considered as a bioindicator of heavy metalspollution [17], but no information exists on the accumulationpotential of organic contaminants as PFCs [18].

From an analytical point of view, the extraction of PFCsfrom biological matrices is based on liquid–solid extraction[19], pressurized liquid extraction, or solid-phase extraction[20] followed by liquid chromatography coupled to tandemmass spectrometry [21]. However, one of the main draw-backs in the analysis of these compounds is the co-extractionof matrix interferents, which may affect the detection ofthese analytes and produce low recoveries due to inefficientionization. Another problem is the presence of PFCs in blanksamples due to contamination during the extraction andanalytical steps. This issue is not properly addressed in manystudies and thus may produce overestimation of the detectedPFCs. Finally, limits of detection might be compromised byblank contamination and inefficient recovery. Therefore,quality control analysis needs to be properly addressed,especially when proposing a robust method to be used in awide array of biological matrices.

The first aim of our study was to develop a robustanalytical method to determine five main PFCs in biota andprovide quality control data. The second objective was toevaluate the occurrence of five PFCs in various aquaticorganisms that are widespread in Catalan river basins and incoastal areas. Organisms included are insect larvae(Hydropsyche exocellata), zebra mussels (Dreissena poly-morpha), oysters (Crassostrea gigas), anchovies (Engraulis

encrasicholus), sardines (Sardina pilchardus), and crabs(Procambarus clarkii). The final objective is to define asuitable biomonitoring matrix that can be used to assess thepresence and potential effects of PFCs in both seawater andfreshwater organisms.

Materials and methods

Chemicals and reagents

Native compounds of PFOS, PFOA, PFNA, perfluorohex-ane sulfonic acid (PFHxS), and perfluorobutane sulfonicacid (PFBS) were supplied by Wellington Laboratories(Ontario, Canada). Stock standard solutions were preparedin acetonitrile at a concentration of 5 ng/μL for all nativecompounds and were stored at −18 °C. Perfluoro-n-(1,2,3,4-13C4) octanoic acid (m-PFOA) and sodiumperfluoro-1-(1,2,3,4-13C4) octane sulfonic acid (m-PFOS),also from Wellington Laboratories, were used as surrogatestandards. High-performance liquid chromatography(HPLC) grade water and acetonitrile were supplied byMerck (Darmstadt, Germany) and glacial acetic acid fromPanreac (Barcelona, Spain).

Sampling

Several aquatic communities in poor and good ecologicalstate were sampled throughout Catalonia (NE Spain) duringthe period 2006–2009 to evaluate the presence of PFCs.Organisms studied were selected according to their avail-ability in each ecosystem. Trichoptera larvae (H. exocel-lata) were sampled along the Besós River (NE Spain),zebra mussels (D. polymorpha) were taken at eight sitesalong the low Ebro river (NE Spain), oysters (C. gigas)were taken from four barges placed in the bays of Alfacsand Fangar in the Ebro delta, and the American crabs (P.clarkii) was also collected from the Ebro Delta. Anchovies(E. encrasicholus) and sardines (S. pilchardus) were boughtfrom the local markets and were from the Catalan coast inthe NW Mediterranean Sea. Samples were collected usingpolypropylene (PP) falcons. During sampling and storage,samples were never in contact with Teflon containers toavoid contamination of the samples.

Sample extraction and cleanup

PFCs were solid–liquid extracted from grinded and ho-mogenized wet samples using acetonitrile, based on themethod of Meyer et al. [22]. About 0.1–1 g was weightedin PP centrifuge tubes, and the surrogate standards (m-PFOS and m-PFOA) were added at a concentration of100 ng/g and incubated for 18 h at 4 °C. Acetonitrile (4.5–

1448 M. Fernández-Sanjuan et al.

9 mL), depending on the initial quantity weighed, was thenadded, 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 eachperiod of 10 min, the samples were thoroughly mixed.Afterwards, the samples were centrifuged at 2,500 rpm for5 min. The supernatant was transferred to a new vial andevaporated to dryness. Then, 1 mL of acetonitrile wasadded to the dried samples and incubated for 10 min in theultrasonic bath. The samples were purified by adding 25 mgof activated carbon and 50 μL of glacial acetic acid andwere vigorously mixed for 1 min. Afterwards, the sampleswere centrifuged for 10 min at 10,000 rpm. One milliliter ofthe supernatant was transferred to a clean micro-vial andstored until further analysis. Before injection, 150 μL ofthis extract was reconstituted in 350 μL of HPLC water,similar to the initial conditions of the mobile phase.

Instrumental analysis

PFCs were measured using combined HPLC coupled totandem mass spectrometry (MS/MS). The analytical systemconsisted of an Acquity Ultra performance LC systemconnected to a Triple Quadruple Detector (TQ Detector)(Waters, USA). An Acquity UPLC BEH C18 column(1.7 μm particle size, 50 mm×2.1 mm, Waters, USA) wasused as mobile phase residue trap to remove any contam-ination from the mobile phase. The analysis was performedon a LiChroCART HPLC RP-18e column (125 mm×2 mm×5 μm particle size, Merck, Germany), and the flowrate was set at 0.4 mL/min. Injection volume was of 10 μL.The mobile phase was 2 mM NH4OAc (A)/acetonitrile (B).Gradient elution started from 70% A and 30% B, increasedto 90% B in 5 min and to 100% B in 0.10 min and held for1 min. Initial conditions were reached in 1 min, and thesystem was stabilized for 4 min. The various PFCs weremeasured under negative electrospray ionization using two

transitions from parent to product ion to identify eachcompound. The two transitions from parent to product ionas well as the dwell time, cone voltage, collision energy,and retention times are summarized in Table 1. To identifythe target compounds, the retention time and these twotransitions were used. Internal standard quantification wasperformed using m-PFOS to quantify PFOS, PFHxS,PFBS, and m-PFOA to quantify PFOA and PFNA.

Quality control parameters

Samples were extracted and analyzed in batches togetherwith a procedural blank to control any external contamina-tion during the whole analytical process. Seven pointcalibration curves were constructed over a range of 1.25to 50 ng/mL, covering the concentration range where targetcompounds are likely to be found in the biota samples.Recovery studies were performed in duplicate using zebramussel spiked with native compounds at concentrations of10 and 100 ng/g. The reproducibility of the method wascalculated from the relative standard deviation of duplicateanalyses of spiked zebra mussel samples. Instrumentdetection limits (LODinst) were calculated using the lowestconcentration standard solution at 1.25 ng/mL for eachcompound and were calculated using three times the valueof the signal-to-noise ratio (the ratio between the peakintensity and the noise measured 1 min after the peaksignal). Method detection limits (LODmethod) were calcu-lated in the same way, using spiked zebra mussel.

Results and discussion

Method performance

LC-MS/MS analysis permitted the resolution of 5 PFCs in6 min, with PFNA and PFHxS coeluting at 3.55 min,

Table 1 Compounds analyzed ordered by elution times (min), parent ion, and MRM transitions monitored and optimized cone voltages andcollision energies for each PFCs

PFCs Retention time (min) Parent ion Transitions (m/z) Dwell time (s) Cone voltage Collision energy

PFBS 2.70 299 299>80 0.20 45 29299>99

PFHxS 3.54 399 399>80 0.05 45 35399>99

PFOA 3.26 413 413>369 0.05 17 10

m-PFOA 3.26 417 417>372 0.05 17 10

PFNA 3.54 463 463>419 0.05 16 11

PFOS 4.24 499 499>80 0.05 45 40499>99

m-PFOS 4.24 503 503>80 0.05 45 40503>99

Screening of PFCs in various aquatic organisms 1449

although they could be resolved at their specific transitions(Table 1). This short analysis time enhances samplethroughput, and no carry over effects were observed,provided a solvent was analyzed after five samples.

One of the main drawbacks in the analysis of PFCs is thepresence of trace concentration of target compounds in theblank sample. In a first step, solvent blanks were analyzedto control any contribution from the mobile phase. Tracesof PFOS, PFOA, and PFNA were observed in solventblanks. Therefore, the UPLC BEH C18 column was used asmobile phase residue trap to sorb possible PFCs present inthe solvent or in the UPLC tubing and valves. Nocontributions were further observed. In a second step,procedural blank samples were analyzed to evaluate anycontribution from the preconcentration step. Target com-pounds were not detected in any of the blank samples sincenone of the material used during extract ion (vials, tubing,

and septa) contained Teflon. Figure 1 shows a chromato-gram of blank samples (no matrix) in which only thesurrogate standards m-PFOS and m-PFOA are detected at4.26 and 3.25 min, respectively.

The analysis of PFCs in biota samples requires a highselectivity and sensitivity. Selectivity was obtained by usingactive carbon and glacial acetic acid as a clean-up step,which eliminated any lipid or matrix interference coex-tracted from the biota samples. The selectivity of the MS/MS detector and the MRM transitions used resulted inchromatograms void of interferents, even with shortanalysis time. Figure 2a shows a chromatogram of anoyster sample, in which no interferents were present and inwhich surrogate standards are perfectly resolved despite thefact that target compounds were not detected. Figure 2bshows a trichoptera larvae sample in which four of the fivetarget compounds were detected, and both retention time

Time (min)

m-PFOS

m-PFOA

Fig. 1 LC-MS/MS chromato-gram of a blank sample

1450 M. Fernández-Sanjuan et al.

and analytical response of the surrogate standards aremaintained. This selectivity was necessary to obtainoptimal sensitivity. Table 2 shows the quality parametersof the method, such as slope of the calibration curve, limitsof detection, repeatability and reproducibility, and finally,the recoveries of the target compounds. Internal standardquantification provided a response factor between 0.9 and4.9, indicating a similar behavior between labeled surrogatestandards and native compounds. Good correlations (r2>0.995) were obtained for all compounds. The response ofm-PFOS and m-PFOA throughout the analytical sequencewas used as control standards to evaluate the extractionefficiency and the ion suppression for the LC-MS/MSanalysis. To prove the suitability and lack of ion suppres-sion or enhancement, the response of the surrogate stand-ards in the standard solutions and in the samples wascompared and it was constant for all the analyzed samples.This means that there is no matrix effect, and thus, thesurrogates can be used for internal standard quantificationand no further cleanup or MS optimization is needed.

LODinstrumental was within the 0.05–0.11 ng range andgives information on the minimum amount of eachcompound detected using the developed LC-MS/MS con-ditions and without taking into consideration the extractionprocedure. LODmethod was between 0.07 and 0.22 ng/g, byextracting 1 g of sample, thus indicating that the overallsensitivity of the method (extraction and analysis) permitsthe identification of PFCs at environmental relevantconcentrations. The instrumental limits of detectionobtained were very similar to those reported in the literatureusing similar methods [23]. The repeatability of the methodprovided good relative standard deviation (RSD) values(7.1–13.9%) for the target compounds, calculated from 18measures in 5 days.

For the extraction performance, Table 2 shows that therecoveries of the target compounds in spiked zebra musselat 100 ng/g were from 82% to 116%, with RSD rangingfrom 3% to 5%, indicating acceptable extraction and clean-up efficiency. At a spiking concentration of 10 ng/g, moreclose to environmental relevant concentrations, the recov-

aFig. 2 Chromatogramsobtained for a oyster sampleand b a trichoptera larvaesample

Screening of PFCs in various aquatic organisms 1451

eries were between 96% and 122%, with RSD between 7%and 15%.

Presence of PFCs in seawater organisms

PFCs are widely distributed in water [24] and sediments[25], although little is known on their presence in aquaticbiota of different trophic level. Organisms accumulate PFCsdue to direct contact of the organisms with a contaminated

water body, due to ingestion of contaminated food or wateror due atmospheric uptake [26]. Uptake depends also on thetype of organism, showing more or less susceptibility depend-ing on their biology, physiology, body composition, anddetoxification mechanism. Therefore, the levels of PFCs canvary largely depending on both environmental and intrinsicallybiological factors. Table 3 shows the range of concentrationsof PFCs in the different organisms studied. Among organismsevaluated, insect larvae had the highest ΣPFCs concentration,

Quality parameter PFOS PFOA PFNA PFBS PFHxS

Response factor 0.9 2.1 1.1 4.9 2.3

LODinstrumental (ng) 0.08 0.10 0.05 0.10 0.11

Repeatability (% RSD n=18) 7.3 7.1 10.5 10.9 13.9

Recoveries±RSD (%, n=2) (100 ng/g) 104±3 108±5 116±4 82±3 93±4

Recoveries±RSD (%, n=2) (10 ng/g) 100±15 96±13 111±11 112±7 122±9

LODmethod (ng/g) 0.22 0.11 0.07 0.13 0.17

Table 2 Quality parameters forthe analytical method used

Presence of PFCs in seawaterorganisms

bFig. 2 (continued)

1452 M. Fernández-Sanjuan et al.

reaching a ΣPFC of 22.1 to 156.7 ng/gww, followed bysardines (0.73 to 1.32 ng/gww), anchovies (0.54 to 1.14 ng/gww), and crabs (0.44 to 1.14 ng/gww).

Oysters have the ability to filter approximately 1 L ofwater per day and live long enough to accumulate eventraces of contaminants from water samples. However, noneof the target compounds were detected in oysters collectedfrom barges located in the Ebro delta, a highly bivalveproduction area. Our results differ from those reported byNakata et al. where PFOA and PFNA were detected atconcentrations of 9.5 and 1.6 ng/gww in mussels from atidal flat area of Ariake Sea in Japan [27]. Other studiesreport concentrations of individual PFCs in mussels andoysters from south China and Japan from 0.113 to0.586 ng/gww for PFOS, 0.063 to 0.511 ng/gww forPFHxS, and 0.093 to 0.030 ng/gww for PFBS, although thesamples were collected from impacted areas [23]. In ourstudy, oysters were caged only for 2 months in Delta delEbro, which might not be enough to accumulate PFCsconsidering the low ng/L concentration of PFCs in seawater[28]. Assuming a total concentration of PFCs in water of1 ng/L and an assimilation efficiency of 10%, the amount ofcompound accumulated in the organism over the exposureperiod (60 days) should theoretically be 6 ng. The lack ofaccumulation in oysters cannot be fully explained byexposure time and the low PFC concentration in water.Biological detoxification mechanisms may play animportant role in the bioaccumulation process in mussels[29]. Bossi et al. [30] report that PFCs were notaccumulated in blue mussels (Mytilus edulis) collected inseveral areas from Denmark and attribute this phenomenato an internal detoxification mechanism of oysters whichis activated by the presence of xenobiotics. Oysters arealso known to have high activities of transmembranemultidrug transport proteins that act effluxing a variety ofpotential xenobiotics including PFCs out of the cell [31].Thus, it is plausible to hypothesize that the combination oflow levels of PFCs in water with an active transport ofthese compounds out of the organisms by multidrugtransmembrane transporters may explain the undetectedaccumulation levels.

PFCs were also determined in sardines and anchovies.PFCs were first analyzed in fish muscle and no traceswere observed. Following the analysis of whole bodyprovided detectable PFOS levels of 0.73 to 1.32 ng/gwwin sardines and from 0.54 to 1.14 ng/gww in anchovies,thus indicating that PFOS are accumulated in fish organsor skin rather than in muscle. The levels encountered arewithin the range of those reported by Llorca et al. inMediterranean fish [20], although in that study PFOA andPFBS were the main compounds detected in fish muscle.Similar to our study, Ye et al. report PFOS as thepredominant compound identified in freshwater wholefish with median concentrations of 24.4, 31.8, and53.9 ng/gww in the Missouri, Ohio, and MississippiRivers, respectively [32]. Martin et al. report thatsulfonates bioaccumulate to a greater extent than carbox-ylates of equivalent perfluoroalkyl chain length and thatdietary exposure of perfluorinated acids do not result inbiomagnification in juvenile trout [33]. In our study,perfluorinated acids were not detected in fish, and onlythe low levels of PFOS may produce biomagnification inthe marine food web.

Presence of PFCs in freshwater organisms

PFCs in freshwater are, in general, 10 to 100 times higherthan in coastal or open sea waters [24, 28]. Therefore, thebioaccumulation potential increases and thus freshwaterspecies become highly vulnerable organisms. In a firststep, and taking advantage of the widespread distribu-tion of invasive zebra mussels in Ebro river reservoirsand the high theoretical accumulation potential, theoccurrence of PFCs was assessed. Contrary to ourpresumption, zebra mussel did not contain any tracesof PFCs. This study is in accordance to Kannan et al.where PFCs were neither detected in zebra musselsfrom two locations in the Great Lakes, and only PFOSwas accumulated at 3.1 ng/gww in an area with a waterconcentration of 3.5 ng/L [34]. Contrarily to oysters thatwere exposed for 2 months, zebra mussels were dwellingin the study area for 2 years, and thus, the lack of

Samples PFOS PFOA PFNA PFHxS PFBS

Sea water organisms

Oyster (n=18) n.d. n.d n.d n.d n.d

Sardines (n=5) (0.73–1.32) n.d n.d n.d n.d

Anchovies (n=5) (0.54–1.14) n.d n.d n.d n.d

Freshwater organisms

Zebra mussel (n=8) n.d n.d n.d n.d n.d

Crabs (n=5) (0.23–0.63) (0.14–0.40) (0.07–0.11) n.d n.d

Insect larvae (n=8) (20–144) (1.3–4.3) (0.6–7.5) (0.2–0.8) n.d

Table 3 The PFCsconcentration range inwhole body aquatic organismsin ng/gww

n.d. not detected

Screening of PFCs in various aquatic organisms 1453

accumulation should be explained by other mechanismsrather than exposure time. Stevenson et al. suggest that atlow levels of PFCs may be effluxed out from cells by thecell membrane P-glycoprotein (p-gp) transporter activity.The efflux acts as a first line of defense against toxicantsand is specially expressed in mussels [29]. This mecha-nism might be responsible for the low overall accumula-tion potential for PFCs in bivalves.

Crabs (P. clarkii) is another invasive species whichcauses damage in dikes and canals. This species is widelyspread in Catalonia and serves as a source of food for manyorganisms, such as birds or fish. Crabs may live for 5 yearsand are totally adapted to dwell in waters of poor chemicalquality. The presence of PFCs was evaluated by analyzingthese compounds first in muscle and then in whole bodyincluding head. Similar to fish, the muscle did not containany traces of PFCs, but the whole body contained PFOS,PFOA, and to a lesser extent PFNA at 0.07–0.63 ng/gwwlevel. Therefore, crabs may become a source of PFCs tohigher trophic organisms, including men [35].

Finally, the occurrence of PFCs in insect larvae wasassessed by analyzing larvae collected from eight freshwa-ter bodies (Fig. 3). Each sample contains one pool of 100individuals. PFOS, due to their more lipophylic properties,was accumulated at the highest concentration (20–144 ng/gww), followed by PFOA (1.3–4.3 ng/gww), PFNA (0.6–7.5 ng/gww), and PFHxS (0.2–0.8 ng/gww). This insectbenthic larvae species can live for up to 12 months in waterattached to stones at the bottom of the river, and filteringand eating suspended edible particles. This period isenough to produce accumulation of PFCs, given the highspecific surface area of these organisms, their foraging filterfeeding behavior, and the high presence of PFCs infreshwaters compared to seawater. Highest levels are foundin samples 3 and 4, which correspond to areas nearindustries and samples 5 and 6 located near urban centers.

Toxicological implications

PFCs are present in aquatic organisms, with levels infreshwater organisms higher than in seawater organismsand represent an emerging risk for both freshwater andmarine ecosystems.

Legal initiatives have been undertaken by the NationalWater Quality Criteria from United States EnvironmentalProtection Agency (NWQC, US EPA) in 1985 [36] and bythe Stockholm Convention [37] regarding their environ-mental distribution. In addition, its use and marketing havebeen legalized, and preparations, semi-finished products orarticles, or parts thereof may not be placed on the market ifthe concentration of PFOS is equal to or higher than 0.1%by mass and for textiles or other coated materials, if theamount of PFOS is equal to or higher than 1 μg/m2 of thecoated material [38]. These actions may limit the release ofPFCs to the environment in the near future. However, manyscientific studies prove that they are widespread inbiological matrices. From a toxicological point of view,the presence of PFCs in aquatic organisms can pose severeconsequences due to their bioaccumulation capabilities, andvery little information is available on the potential toxiceffects they may cause on individuals or at population level.To derive numeric water quality values for those PFCs thathave sufficient and appropriate toxicity data, we have reliedon the Great Lakes Initiative US EPA (GLI; US EPA) in1995 [39]. The guidance provided by the GLI is intended toprovide both acute and chronic criteria for the protection offish, invertebrates, and other aquatic organisms. Forexample, the mean acute toxicity values for aquaticorganisms exposed to PFOS range from 0.089 to 134 mg/L in midge (Chironomus tentans) and water flea (Daphniapulicaria) [40]. In addition, the acute toxicity of PFOS wasevaluated for various organisms, and Giesy et al. compileno observed effect concentration data at the mg/L level and

10,0

0,1

1,0

10,0

100,0

1000,0

2 3 4 5 6 7 8

ng/g

ww

PFOS PFOA PFNA PFHxS

Fig. 3 PFCs scale logconcentrations (ng/gww) foundit in trichoptera larvae fromeight samples collected alongthe Besos river (NE Spain)

1454 M. Fernández-Sanjuan et al.

lethal concentrations (LC50) from 3.6 to 169 mg/L formicroalgae, invertebrates, amphibians, and fish [40]. Fromthese values, it can be stated that toxic effects are triggeredat relatively high water concentrations. However, thecontinuous release of these chemicals and their accumula-tion in biota might induce unknown effects which should befurther studied to protect environment and indirectly, thehuman health.

Conclusions

The monitoring of PFCs in aquatic organism, includingboth seawater and freshwater, brings new perspectives inthe assessment of the occurrence and impact of this newgeneration of environmental contaminants. While PFCswere not accumulated in bivalve species likely due tospecific detoxification mechanisms, American crabs, insectlarvae, or seawater fish were suitable bioindicators. Thepresence of PFCs in these organisms suggests that PFCs arewidespread and can have serious long-term impacts to theaquatic ecosystems, either freshwater or seawater. Manyaquatic organisms are in the base of the tropic chain andthus may be a source of PFCs to higher trophic levels,although the underlying mechanisms and biomagnificationfactors still need to be studied. It is still early to evaluate thepotential effects they may cause both at individual and atthe ecosystem level. Thus, it is worthwhile to evaluate thetoxicological endpoints in aquatic organisms to furthercontribute to an appropriate environmental managementregarding the release of these toxic, bioaccumulative andpriority compounds.

Acknowledgments This study was funded by the Ministry ofEnvironment of Spain [042/RN08/03.4] and [038-2009] projects.The authors are grateful to Dr. Albert Bertolero for facilitating the crabsamples from the Ebro Delta. Dr. Roser Charler, Dori Fanjul, andMaría Comesaña are acknowledged for invaluable MS assistance.

References

1. Kissa E (2001) Fluorinated surfactants and repellents, 2nd edn.Marcel Dekker, New York

2. Giesy JP, Kannan K (2001) Environ Sci Technol 35:1339–13423. 3M company (2001) Environmental Laboratory Project Number

EOO-1716. Water solubility in natural seawater and 3.5% sodiumchloride solution-shake flask method. 3M Company, St Paul

4. Hatfield T (2001) Screening studies on the aqueous photolyticdegradation of potassium perfluorooctane sulfonate (PFOS). 3MCompany, St Paul

5. Hatfield T (2001) Hydrolysis reactions of perfluorooctanesulfonate (PFOS). 3M Company, St Paul

6. Giesy JP, Kannan K (2002) Environ Sci Technol 36:146A–152A7. Dietz R, Bossi R, Riget FR, Sonne C, Born EW (2008) Environ

Sci Technol 42:2701–2707

8. Bossi R, Riget F, Dietz R, Sonne C, Fauser P, Dam M, Vorkamp K(2005) Environ Pollut 136:323–329

9. Li X, Wai Yin Yeung L, Xu M, Taniyasu S, Lam PKS, YamashitaN, Dai J (2008) Environ Pollut 156:1298–1303

10. Kannan K, Hansen KJ, Wade TL, Giesy JP (2002) EnvironContam Toxicol 42:313–318

11. Van de Viejver KI, Hoff PT, Van Dongen W, Esmans EL, Blust R,De Coen WM (2003) Environ Toxicol Chem 22:2037–2041

12. Cunha I, Hoff P, Van de Vijever K, Guilhermino L, Esmans E, DeCoen W (2005) Mar Pollut Bull 50:1121–1145

13. Nania V, Pellegrini GE, Fabrizi L, Sesta G, De Sanctis P,Lucchetti D, Di Pasquale M, Coni E (2009) Food Chem115:951–957

14. Porte C, Albaiges J (1994) Arch Envir Contam Toxicol 26(3):273–281

15. Loos R, Gawlik BM, Locoro G, Rimaviciute E, Contini S,Bidoglio G (2009) Environ Pollut 157(2):561–568

16. Roper JM, Cherry DS, Simmers JW, Tatem HE (1996) EnvironPollut 94(2):117–129

17. Barata C, Lekumberri I, Vila-Escalé M, Prat N, Porte C (2005)Aquat Toxicol 74:3–19

18. Van Gossum H, Bots J, Snijkers T, Meyer J, Van Wassenbergh S,De Coen W, De Bruyn L (2009) Environ Pollut 157:1332–1336

19. Szostek B, Prickett KB (2004) J Chromatogr B 813:131–13620. Llorca M, Farré M, Picó Y, Barceló D (2009) J Chromatogr A

1216:7195–720421. Jahnke A, Berger U (2009) J Chromatogr A 1216:410–42122. Meyer J, Jaspers VLB, Eens M, De Coen W (2009) Sci Tot

Environ 407:5894–590023. So MK, Taniyasu S, Lam PKS, Zheng GJ, Giesy JP, Yamashita N

(2006) Arch Environ Contam Toxicol 50:240–24824. Loos R, Locoro G, Huber T, Wollgast J, Christoph EH, de Jager

A, Gawlik BM, Hanke G, Umlauf G, Zaldívar JM (2008)Chemosphere 71:306–313

25. Higgins CP, Field J, Criddle CS, Luthy RG (2005) Environ SciTechnol 39:3946–3956

26. De Vos MG, Huijbregts MAJ, Van den Heuvel-Greve MJ, VethaakDA, Van de Vijver KI, Leonards PEG, van Leeuwen SPJ, deVoogt P, Hendriks A (2008) Chemosphere 70:1766–1773

27. Nakata H, Kannan K, Nasu T, Cho H-S, Sinclair E, Takemura A(2006) Environ Sci Technol 40:4916–4921

28. Sánchez-Avila J, Meyer J, Lacorte S (2009) Environ Pollut.doi:10.1016/j.envpol.2010.06.022

29. Stevenson CN, Macmanus-Spencer LA, Luckenbach T, LuthyRG, Epel D (2006) Environ Sci Technol 40:5580–5585

30. Bossi R, Strand J, Sortkjaer O, Larsen MM (2008) Environ Int34:443–450

31. Minier C, Lelong C, Djemel N, Rodet F, Tutundjian R, Favrel P,Mathieu M, Leboulenger F (2002) Mar Environ Res 54:455–459

32. Ye X, Strynar MJ, Nakayama SF, Varns J, Helfant L, Lazorchak J,Lindstrom AB (2008) Environ Pollut 156(3):1227–1232

33. Martin JW, Mabury SA, Solomon KR, Muir DCG (2003) EnvironToxicol Chem 22:189–195

34. Kannan K, Tao L, Sinclair E, Pastva SD, Jude DJ, Giesy JP(2005) Arch Environ Contam Toxicol 48(4):559–566

35. Clarke DB, Bailey VA, Routledge A, Lloyd AS, Hird S, MortimerDN, Gem M (2010) Food Addit Contam 27(4):530–545

36. US EPA (1985) Guidelines for deriving numerical national waterquality for the protection of aquatic organisms and their uses. PB-85-227049. US Environmental Protection Agency, NTIS, Springfield

37. Council Decision 2006/507/EC of 14 October 2004 concerningthe conclusion, on behalf of the European Community, of theStockholm Convention on Persistent Organic Pollutants. OfficialJournal L 209, 31.7.2006, p.1

38. Directive 2006/122/ECOF. The European Parliament and ofthe Council of 12 December 2006. 30th time Council

Screening of PFCs in various aquatic organisms 1455

Directive 76/769/EEC annex 1 on the approximation of laws,regulations and administrative provisions of the MemberStates relating to restrictions on the marketing and use ofcertain dangerous substances and preparations (perfluorooc-tane sulfonates). Official Journal of the European Union L372/32

39. US EPA (1995) Final water quality guidance for the Great LakesSystem: final rule. Federal Register 60:15366–5425. Washington,DC, USA

40. Giesy JP, Naile JE, Khim JS, Jones PD, Newsted JL (2010)Reviews Environ Contam Toxicol. doi:10.1007/978-1-4419-1157-5_1

1456 M. Fernández-Sanjuan et al.