Article pubs.acs.org/JAFC

Perfluoroalkyl Acid Contamination and Polyunsaturated Fatty Acid Composition of French Freshwater and Marine Fishes Ami Yamada,*,† Nawel Bemrah,† Bruno Veyrand,§ Charles Pollono,§ Mathilde Merlo,† Virginie Desvignes,† Véronique Sirot,† Marine Oseredczuk,† Philippe Marchand,§ Ronan Cariou,§ Jean-Phillippe Antignac,§ Bruno Le Bizec,§ and Jean-Charles Leblanc† †

Risk Assessment Directorate − French Agency for Food, Environmental and Occupational Health and Safety (Anses), 27-31 avenue du Général Leclerc, Maisons-Alfort 94701, France § LUNAM Université, Oniris, Laboratoire d’Etude des Résidus et Contaminants dans les Aliments (LABERCA), USC INRA 1329, Nantes 44307, France ABSTRACT: In this study, French marine and freshwater fish perfluoroalkyl acid (PFAA) contamination are presented along with their fatty acid (FA) composition to provide further elements for a risk/benefit balance of fish consumption to be assessed. The 29 most consumed marine fish species were collected in four metropolitan French coastal areas in 2004 to constitute composite samples. Geographical differences in terms of consumed species and contamination level were taken into account. Three hundred and eighty-seven composite samples corresponding to 16 freshwater fish species collected between 2008 and 2010 in the six major French rivers or their tributaries were selected among the French national agency for water and aquatic environments freshwater fish sample library. The raw edible parts were analyzed for FA composition and PFAA contamination. Results show that freshwater fishes are more contaminated by PFAAs than marine fishes and do not share the same contamination profile. Freshwater fish contamination is mostly driven by perfluorooctane sulfonate (PFOS) (75%), whereas marine fish contamination is split between perfluorooctanoic acid (PFOA) (24%), PFOS (20%), perfluorohexanoic acid (PFHxA) (15%), perfluoropentanoic acid (PFHpA) (11%), and perfluorobutanoic acid (PFBA) (11%). Common carp, pikeperch, European perch, thicklip grey mullet, and common roach presented the most unfavorable balance profile due to their high level of PFAAs and low level of n-3 long-chain polyunsaturated fatty acids (LC-PUFAs). These data could be used, if needed, in an updated opinion on fish consumption that takes into account PFAA contamination. KEYWORDS: perfluoroalkyl acid, PFAA, PFAS, n-3 LC-PUFA, fatty acid, freshwater fish, marine fish

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INTRODUCTION Fishes are an important source of high-quality proteins, iodine, selenium, and phosphorus and, above all, are considered as an important vector of fatty acids (FA) such as essential n-3 longchain polyunsaturated FA (n-3 LC-PUFA) in food consumption.1 n-3 LC-PUFA are involved in many biological functions: cell membrane formation, inflammation, platelet aggregation, blood pressure regulation, and the functioning of several organs (brain, retina, etc.). The protective role of n-3 LC-PUFA has been documented for cardiovascular disease,2 although questioned by some recent studies,3 autoimmune and neurodegenerative diseases, some cancers, infant development, asthma, mental disorders,4 and prevention of posttraumatic stress disorder.5 However, fish consumption, and especially that of oily fish, is also associated with environmental contaminant and pollutant exposure. Although several studies confronted n-3 LC-PUFA content and methylmercury, inorganic arsenic, dioxins and dioxin-like compounds, or polychlorinated biphenyls (PCB)6−8 to adopt a risk−benefit analysis approach, nothing had been found to date on per- and polyfluorinated compounds, which include perfluoroalkyl acids (PFAAs), amphiphibic substances of raising concern. Their remarkable surface tension lowering properties as well as their dust-, water-, and oil-repellent capacities make them suitable for use in various applications such as firefighting foams, food-contact paper impregnation, textile, leather © 2014 American Chemical Society

and carpet protection, cookware nonstick coating, cleaning products, ink, ski waxes, and pesticides and also make them essential in aviation hydraulic fluid.9 Their widespread use and their high thermal and chemical stabilities have led to a global environmental contamination,10,11 and they are even found in the serum of the general population as well as in umbilical cord blood12,13 at nanogram per milliliter levels. A number of epidemiological studies have been conducted, and some of them pointed out an association between an elevated PFAA serum or blood concentration and hepatotoxicity,14,15 change in thyroid profile,16−18 cardiovascular effects,19−21 birth outcome such as a lower birth weight and increased number of preterm births,22−24 obesogenic effects,25,26 effects on reproductive functions such as an earlier menopause,27 an increased time to pregnancy28 or a decrease of semen quality,29 and breast and prostate cancer.30,31 However, these associations are still controversial.32−37 The aim of this paper is to present marine and freshwater fish PFAA muscle contamination along with their FA composition for the first time, to our knowledge, to obtain further elements for a risk−benefit balance to be conducted related to fish consumption. Received: Revised: Accepted: Published: 7593

March July 8, July 8, July 8,

31, 2014 2014 2014 2014

dx.doi.org/10.1021/jf501113j | J. Agric. Food Chem. 2014, 62, 7593−7603

Journal of Agricultural and Food Chemistry

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Article

32 freshwater fish groups sampled, the 11 most consumed were selected with at least 10 individual fishes per sample, resulting in 28 composite samples. Analysis of Lipid and Fatty Acid Composition and PFAA Contamination. Analysis of PFAAs. The used methodology targeted 15 PFAAs: perfluorobutanoic acid (PFBA), perfluoropentanoic acid (PFPA), perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoroundecanoic acid (PFUnA), perfluorotridecanoic acid (PFTrDA), perfluorotetradecanoic acid (PFTeDA), perfluorobutane sulfonate (PFBS), perfluorohexanane sulfonate (PFHxS), perfluoroheptane sulfonate (PFHpS), perfluorooctane sulfonate (PFOS), and perfluorodecane sulfonate (PFDS). The analytical procedure used for PFAA determination was described elsewhere.43 Briefly, samples were pretreated by freeze-drying and liquid/solid extraction with methanol. A dispersive solid phase extraction (SPE) based on charcoal particles was then applied. Final purified extracts were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) with negative electrospray ionization on a triple-quadrupole system. Two diagnostic signals (MRM transitions) were recorded for each targeted analyte. Quantification was performed according to the isotope dilution principles: each sample was supplemented by 11 13C-labeled internal standards (13C4-PFBA, 13 C5-PFHxA, 13C4-PFHpA, 13C4-PFOA, 13C5-PFNA, 13C2-PFDA, 13C7PFUnA, 13C2-PFDoA, 18O2-PFHxS, 13C4-PFOS, and 13C4-PFOSi). Limits of detection (LOD), depending on the considered fish species and target compound, were determined on the basis of the signal-tonoise ratio (S/N > 3) and ranged from 0.005 to 0.3 ng g−1 for PFOA and PFOS and from 0.007 to 0.95 ng g−1 for other PFAA substances. Repeatability of the signal was also dependent on the matrix but was globally estimated below 18.6% for all compounds. The generated exposure data were produced under the Quality Assurance system (ISO.IEC 17025:2005 and ISO 9001:2008 standards) and using validated analytical methods, including interlaboratory comparison tests, that have proven the fitness-for-purpose of this method. Blank and quality control samples were analyzed and monitored in parallel with the samples to be characterized: the signal of each compound in the blanks was checked to ensure the absence of contamination throughout the analytical procedure, and concentrations of analytes in quality control were monitored to check the method repeatability. Analysis of Lipids and Fatty Acids. The total lipids and 48 free FAs were analyzed for the 40 marine fish composite samples. The protocol was detailed elsewhere.44 Briefly, the total lipids were analyzed by two different laboratories with two different methods for the same samples. One of the laboratories freeze-dried the samples before powdering and transfer into accelerated solvent extraction with a 70:30 (v/v) mixture of toluene/acetone. Then the samples were evaporated and the total lipids were determined by gravimetry. The second laboratory followed the AFNOR NF V04-403 standard for meat, meat products, and fish products for the extraction, purification, and esterification of the free fat.45 Then the extracts were dried, extracted with a mixture of hexane/ isopropanol 3:2 (v/v) before filtration on a Na2SO4 and Celite column, and weighed. For the free FA analysis, the FAs were extracted with hexane, filtered, esterified with a mixture of methanol/sulfuric acid 13:2 (v/v), and analyzed using a gas phase chromatograph equipped with a flame ionization detector. Their identification was based on retention time obtained for standard esters. The part of each free FA was based on the area of the corresponding peaks, in relation with a standard solution. The total lipids and 40 free FAs were analyzed for the 28 freshwater fish composite samples. For each sample, the free fat was extracted, purified, and esterified by reaction with boron trifluoride according to the NF EN ISO 5509 standards.46 The esters were then analyzed using a gas phase chromatograph equipped with a flame ionization detector according to the NF EN ISO 5508 standards47 and identified in the same way as for the marine fish samples. Data and Statistical Analysis. All statistical analyses were performed using the SAS software 9.3 (SAS Institute Inc., Cary, NC, USA). Normality of the distributions was tested with the Kolmorogov− Smirnov test and homoscedasticity with the Bartlett test. The Kruskal− Wallis test was used to compare fish PFAA contamination between the

MATERIALS AND METHODS

Collection of Fish Samples. Marine Fish Sampling. The list of marine fishes to be collected was based on the individual dietary consumption analysis of the 996 adult high-seafood consumers of the CALIPSO study,38 recruited in four coastal areas in metropolitan France between October and December 2004. The fish consumption habits of the population of these four areas and their surrounding 20−25 km, namely, La Rochelle in Gironde-Charente Maritime Sud, Le Havre in Normandy-Baie de Seine, Lorient in South Brittany, and Toulon in Mediterranean-Var, match the highest fish consumption habits in metropolitan France.38 Five primary samples were bought in each region for each species between January and April 2005 to take into account the purchase habits of the considered population (fresh or frozen products; purchase locations of fishmarket, supermarket, etc.). For example, if a fish species is purchased 80% of the time in a supermarket and 20% of the time in a fishmarket, then four of the five primary samples were purchased in a supermarket and one primary sample was purchased in a fishmarket. The sex and fish body weight and length were not available and consequently not taken into account in the sampling and analyses. However, it should be remembered that the purpose of the present study was to perform an exposure assessment study at populational scale and not to study the determinant factors associated with the observed levels of chemicals. The used sampling process respected the purchase habits of the study population, and it has been considered as fairly representative of what is bought and consumed by the CALIPSO population. Each sample finally considered for analysis was a composite one of 1000 g, made up of five 200 g portions of the primary sample raw edible parts (fillets without skin or entrails). Twenty-three composite samples were then constituted in La Rochelle, 22 in Le Havre, 26 in Lorient, and 23 in Toulon. The sampling period ranged between January and April 2005, and a total of 94 fish composite samples were prepared, corresponding to 29 species, which reflect the regional consumption and contamination differences. This sampling procedure allowed covering 89−96% of the total consumption of fish according to the four areas. Recruiting strategy, consumption survey development, and marine fish sampling methodology have already been described.38 Freshwater Fish Sampling. The list of freshwater fishes to be collected was based on the individual dietary consumption analysis of the 606 anglers or their family members of the ICAR-PCB study, initially designed to assess French freshwater fish contamination in PCBs along with angler’s PCB internal exposure (i.e., serum level). Seven hundred and forty-seven freshwater fishes were collected in 2008 and 2009 covering different seasons by ONEMA (French National Agency for Water and Aquatic Environments) in six major French rivers, each river being divided in three or four sections. The dietary consumption analysis and the freshwater fish sampling methodology have already been described elsewhere.39,40 Briefly, each sample had to contain at least 400 g of fish muscle for analysis purposes. If one fish was not enough to reach this amount, then several fishes from the same species and the same location were pooled to form a composite sample, as was the case for half of the samples. The consequence is the loss of some raw data such as sex, body weight, and body length, but as the fishes were sampled randomly, it was considered that they were fairly representative of what could be caught and consumed by the anglers. Although this study was designed for PCBs, we assessed that PCB and PFAA contamination levels in freshwater fishes were not statistically associated. Indeed, due to their physicochemical properties, PCBs are lipophilic and accumulate in fat, whereas PFAAs are amphiphibic and tend to accumulate in liver and blood.41,42 It is assumed that our samples offer a representative view of freshwater fish contamination in metropolitan France. The 16 most consumed fish species among the remaining samples after multiple analyses (for PCBs, dioxins, hexachlorobenzene, etc.) were selected, resulting in 387 samples being analyzed for PFAAs. The samples for lipid and FA analysis were extracted from the same ONEMA freshwater fish sample library constituted between 2008 and 2010. The number of fish included per composite sample of the same freshwater fish species was determined based on availability of material in the library and was also linked to the weight of individual fish, more fishes being needed per composite for small fish. Among the 7594

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Journal of Agricultural and Food Chemistry

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Table 1. PFAA Contamination of Fresh and Frozen Marine Fish LB-UB If LB Value Different from UB Value (Nanograms per Gram ww)a species anchovy, Engraulis encrasicolus monkfish, Lophius piscatorius, Lophius budegassa catshark Scyliorhinus canicula, Scyliorhinus stellaris cod, Gadus morhua common dab Limanda limanda, Microstomus kitt orange roughy, Hoplostethus atlanticus plaice/witch Pleuronectes platessa, Glyptocephalus cynoglossus goatfish, Mullus barbatus barbatus, Mullus surmelutus grenadier, Coryphaenoides rupestris gurnard Trigla lucerna haddock Melanogrammus aeglefinus hake Merluccius merluccius halibut Hippoglossus hippoglossus, Reinhardtius hippoglossoides John Dory, Zeus faber ling, Molva molva, Molva dypterygia mackerel, Scomber scombrus pollack, Pollachius pollachius pout, Trisopterus luscus ray ,Raja clavata, Raja naevus, Raja circularis saithe, Pollachius virens salmon, Salmo salar sardine, Sardina pilchardus scorpionfish, Scorpaena porcus, Scorpaena scrofa, Helicolenus dactylopterus seabass, Dicentrarchus labrax sea bream, Spondyliosoma cantharus, Sparus aurata, Pagellus bogaraveo sole, Solea solea swordfish, Xiphias gladius tuna, Thunnus thunnus whiting, Merlangius merlangus mean (std) min−max a

n

PFBA

PFPA

1 4

0−0.08 0.45−0.46

0−0.07 0.14−0.16

4

0.17

4 4

PFHpA

PFOA

PFNA

PFDA

PFUnA

0−0.05 0.4−0.43

0−0.06 0.36−0.39

0−0.07 0.98−0.99

0−0.06 0.09−0.11

0−0.09 0.06−0.08

0−0.09 0.16−0.16

0.14−0.16

0.25−0.27

0.26−0.28

0.41−0.43

0.06−0.08

0.04−0.07

0.14−0.15

0.34−0.38 0.17

0.11−0.13 0.12−0.13

0.43 0.33−0.34

0.36 0.28−0.29

1.05−1.06 0.8−0.81

0.15 0.09−0.1

0.13−0.13 0.09−0.09

0.19−0.19 0.03−0.06

3

0.44

0.32−0.33

1.2

0.73

1.69

0.19

0.35−0.35

0.13−0.15

2

0.48

0.46

0.87

0.6

1.21

0.16

0.17−0.17

0.01−0.04

3

0.74−0.77

0.48−0.5

0.65−0.66

0.43−0.45

0.87−0.89

0.15−0.19

0.1−0.1

0.02−0.07

4

0.26

0.07−0.08

0.19

0.22

0.3−0.31

0.05−0.07

0.03−0.05

0.07−0.07

1 2

0.27 0.6

0.03 0.23

0−0.05 1.01

0−0.06 0.4

0−0.01 1.3

0−0.04 0.17

0−0.05 0.3−0.3

0.07−0.07 0.07−0.07

4 4

0.41 0.08−0.12

0.14−0.15 0.05−0.09

0.33−0.34 0.13−0.15

0.26−0.28 0.1−0.13

0.74−0.76 0.29−0.33

0.05−0.06 0.03−0.06

0.07−0.07 0.03−0.06

0−0.05 0.14−0.14

2 4

0.37 0.21

0.25 0.04−0.05

0.53 0.13

0.51 0.17

0.48 0.29−0.3

0.08−0.1 0.03−0.07

0.08−0.08 0.03−0.05

0.24−0.24 0.09−0.09

4 3 1 4

0.12−0.17 0.19 0.51 0.14−0.16

0.32−0.38 0.21 0.32 0.11−0.12

0.34−0.38 0.67 1.06 0.36−0.37

0.22−0.26 0.55 0.88 0.28−0.3

0.31−0.34 1.58 1.44 0.88−0.91

0.03−0.06 0.19 0.12 0.13

0.02−0.05 0.15−0.15 0.04−0.04 0.22−0.22

0.21−0.23 0.13−0.13 0.04−0.04 0.74−0.74

4 4 4 1

0.11−0.14 0.06−0.13 0.67−0.71 0.29

0.07−0.09 0.09−0.12 0−0.09 0.23

0.16−0.17 0.26−0.27 0.11−0.14 1.6

0.15 0.18−0.19 0.1−0.13 0.16

0.47−0.48 0.66−0.68 0.26−0.29 0.92

0.04−0.06 0.05−0.07 0−0.07 0.12

0.05−0.05 0.05−0.06 0.02−0.09 0.55−0.55

0.15−0.15 0−0.14 0.42−0.54 0.18−0.18

4 4

0.44 0.23−0.25

0.25−0.28 0.25−0.29

0.42−0.44 0.35−0.37

0.35−0.38 0.3−0.35

0.76−0.79 0.41−0.44

0.21−0.24 0.07−0.09

0.13−0.14 0.05−0.08

1.56−1.56 0−0.07

4 4 4 4

0.8 0.06−0.14 0−0.09 0.34−0.35

0.12−0.13 0−0.07 0−0.04 0.13−0.14

0.5 0−0.08 0.08−0.1 0.41−0.42

0.39−0.4 0.04−0.08 0.06−0.07 0.32−0.33

0.92 0.18−0.22 0.21−0.24 0.92−0.93

0.27 0−0.06 0−0.04 0.07−0.1

0.11−0.11 0−0.08 0.01−0.03 0.07−0.09

0.04−0.06 0−0.08 0.35−0.4 0.05−0.05

0.31 (0.22) 0.33 (0.21) 0−0.8 0.08−0.8

0.16 (0.18) 0.18 (0.12) 0−0.48 0.03−0.5

0.44 (0.39) 0.46 (0.38) 0−1.6 0.05−1.6

0.3 (0.21) 0.32 (0.2) 0−0.88 0.06−0.88

0.7 (0.46) 0.72 (0.45) 0−1.69 0.01−1.69

0.09 (0.07) 0.11 (0.06) 0−0.27 0.04−0.27

0.1 (0.12) 0.12 (0.11) 0−0.55 0.03−0.55

0.18 (0.31) 0.21 (0.3) 0−1.56 0.04−1.56

LB UB LB UB

PFHxA

Std, standard deviation; min, minimum contamination value; max, maximum contamination value.

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coastal sites or major river sections. Ward’s method has been applied for the hierarchical cluster analysis to define three groups of freshwater fishes (low, medium, and high levels of PFAA contamination). A multiple regression analysis was conducted to assess the association between PFAA contamination and content of lipids, FAs, n-3 PUFAs, and n-6 PUFAs. Differences were considered as significant for an α risk below 5%. The percentage of left-censored data (i.e., data below the limit of detection, LOD) for the PFAA contamination was above 60%, and the LOD was low enough for the WHO GEMS/Food-EURO workshop recommendations to be applied.48 Two scenarios were defined. In the lower bound (LB) scenario, the values below the LOD were replaced by 0, and in the upper bound (UB) scenario, the values below the LOD were replaced by the value of the LOD.

RESULTS AND DISCUSSION

PFAA Contamination. The 15 PFAAs analyzed in both marine and freshwater fishes were considered to describe PFAA contamination. The results are expressed with the sum of the 15 following PFAAs: PFBA, PFPA, PFHxA, PFHpA, PFOA, PFNA, PFDA, PFUnA, PFTrDA, PFTeDA, PFBS, PFHxS, PFHpS, PFOS, and PFDS. The percentage of censored data ranged from 9.5% (for PFOS) to 100% (for PFBS and PFHpS) for marine fishes and from 0.26% (for PFOS) to 100% (for PFPA) for freshwater fishes with mean percentages of, respectively, 69.9 and 64.3%. Contamination under UB hypothesis is about 4% higher

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Journal of Agricultural and Food Chemistry

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Table 2. PFAA Contamination of Fresh and Frozen Marine Fish LB-UB If LB Value Different from UB Value (Nanograms per Gram wet weight)a species anchovy, Engraulis encrasicolus monkfish, Lophius piscatorius, Lophius budegassa catshark, Scyliorhinus canicula, Scyliorhinus stellaris cod, Gadus morhua common dab, Limanda limanda, Microstomus kitt orange roughy, Hoplostethus atlanticus plaice/witch Pleuronectes platessa, Glyptocephalus cynoglossus goatfish, Mullus barbatus barbatus, Mullus surmelutus grenadier, Coryphaenoides rupestris gurnard, Trigla lucerna haddock, Melanogrammus aeglefinus hake, Merluccius merluccius halibut, Hippoglossus hippoglossus, Reinhardtius hippoglossoides John dory, Zeus faber ling, Molva molva, Molva dypterygia mackerel, Scomber scombrus pollack, Pollachius pollachius pout, Trisopterus luscus ray, Raja clavata, Raja naevus, Raja circularis saithe, Pollachius virens salmon, Salmo salar sardine, Sardina pilchardus scorpionfish, Scorpaena porcus, Scorpaena scrofa, Helicolenus dactylopterus seabass, Dicentrarchus labrax sea bream, Spondyliosoma cantharus, Sparus aurata, Pagellus bogaraveo sole, Solea solea swordfish, Xiphias gladius tuna, Thunnus thunnus whiting, Merlangius merlangus mean (std) min−max

n

PFTrDA

PFTeDA

PFBSb

PFHxS

PFHpSb

PFOS

PFDS

ΣPFAAs

1 4

0−0.07 0.02−0.05

0−0.07 0−0.06

0.03 0.02

0−0.03 0−0.02

0.05 0.03

0.34 0.43

0−0.05 0−0.03

0.34−1.19 3.1−3.41

4

0.12−0.13

0−0.05

0.02

0.02−0.04

0.03

1.01

0−0.03

2.62−2.92

4 4

0−0.08 0−0.04

0−0.04 0−0.04

0.02 0.02

0−0.02 0−0.02

0.01 0.03

0.63 0.37

0−0.01 0−0.03

3.38−3.63 2.29−2.55

3

0−0.05

0−0.04

0.02

0−0.03

0.03

0.18

0−0.03

5.23−5.45

2

0−0.04

0−0.07

0.02

0−0.02

0.03

1.15

0−0.03

5.1−5.34

3

0−0.05

0−0.04

0.03

0−0.03

0.04

0.96

0−0.04

4.4−4.81

4

0.02−0.05

0−0.03

0.02

0−0.02

0.03

0.05

0−0.03

1.26−1.5

1 2

0−0.05 0−0.04

0−0.01 0−0.06

0.03 0.02

0.03 0−0.02

0.04 0.03

0.97 0.6

0−0.04 0−0.03

1.37−1.73 4.68−4.88

4 4

0−0.04 0−0.06

0−0.03 0−0.05

0.02 0.02

0−0.02 0−0.03

0.03 0.04

0.08 0.33

0−0.03 0−0.04

2.07−2.39 1.19−1.64

2 4

0−0.05 0.01−0.05

0−0.04 0−0.04

0.02 0.02

0.02−0.04 0−0.02

0.03 0.03

1.04 0.14

0−0.03 0−0.03

3.59−3.8 1.15−1.4

4 3 1 4

0−0.05 0−0.08 0−0.05 0.12−0.14

0−0.05 0−0.05 0−0.04 0−0.05

0.02 0.02 0.03 0.02

0−0.03 0−0.02 0−0.03 0−0.02

0.04 0.01 0.04 0.03

1.01 0.71 0.25 1.24

0−0.04 0−0.01 0−0.04 0.17−0.19

2.58−3.12 4.38−4.56 4.67−4.89 4.38−4.63

4 4 4 1

0−0.08 0−0.13 0−0.05 0−0.05

0.01−0.07 0−0.04 0.03−0.07 0−0.01

0.02 0.03 0.02 0.02

0−0.02 0−0.03 0−0.03 0−0.03

0.01 0.02 0.04 0.04

0.18 0.05−0.07 1.4 0.1

0−0.01 0−0.01 0−0.04 0−0.04

1.38−1.67 1.39−1.98 3.01−3.71 4.14−4.32

4 4

0.06−0.1 0−0.06

0−0.04 0−0.03

0.02 0.02

0−0.03 0−0.03

0.04 0.04

2.6 0.03

0−0.04 0−0.04

6.79−7.13 1.7−2.2

4 4 4 4

0−0.04 0−0.07 0−0.1 0−0.04

0−0.04 0−0.05 0−0.05 0−0.05

0.02 0.03 0.03 0.02

0−0.02 0−0.03 0−0.03 0−0.02

0.03 0.05 0.01 0.03

0.62 0−0.03 0.15−0.16 0.32

0−0.03 0−0.05 0−0.01 0−0.03

3.77−4.01 0.28−1.11 0.86−1.38 2.64−2.9

LB UB LB UB

0.01 (0.03) 0.06 (0.03) 0−0.12 0.04−0.14

0.00 (0.01) 0.04 (0.01) 0−0.03 0.01−0.07

0 (0) 0.02 (0.00) 0−0 0.02−0.03

0.00 (0.01) 0.03 (0.01) 0−0.03 0.02−0.04

0 (0) 0.03 (0.01) 0−0 0.01−0.05

0.58 (0.57) 0.59 (0.57) 0−2.6 0.03−2.6

0.01 (0.03) 0.04 (0.03) 0−0.17 0.01−0.19

2.89 (1.66) 3.25 (1.56) 0.28−6.79 1.11−7.13

a

Std, standard deviation; min, minimum contamination value; max, maximum contamination value bNull contamination level under LB hypothesis. Only UB contamination level is shown.

than in LB hypothesis for freshwater fishes and about 17% for marine fishes. This difference between freshwater and marine fishes is linked to the level of PFAA contamination: the lower the contamination is, the higher the censored-data part under UB is. Indeed, marine fishes are about 18 times less contaminated than freshwater fishes in mean (Tables 1−3). The LB contamination range is 0.3−6.8 ng g−1 for marine fishes and 9.4−168.4 ng g−1 for freshwater fishes (1.1−7.1 and 10.3−169.9 ng g−1 under the UB hypothesis). Swordfish Xiphias gladius and brown trout Salmo trutta fario appear to be the less contaminated species, both being marine fishes, whereas seabass Dicentrarchus labrax and

gudgeon Gobio gobio are the most contaminated ones, both being freshwater fishes. The differences between marine and freshwater species are based not only on contamination levels but also on their contamination profile. Whereas PFOS is clearly the prevailing PFAA for the freshwater fishes (LB, 74% of the total contamination), for marine fishes the latter is shared between PFOA (24%), PFOS (20%), PFHxA (15%), PFHpA (11%), and PFBA (11%). The percentages between the LB and UB hypotheses are similar. These differences are to date not well documented. Nevertheless, our results are consistent with the literature,49−51 7596

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PFBA

PFPAa PFHxA

PFHpA

PFOA

PFNA

PFDA

PFUnA

PFTrDA

PFTeDA

7597

a

Null contamination level under LB hypothesis. Only UB contamination level is shown.

barbel, Barbus barbus 5 0−0.57 0.25 0−0.38 0−0.18 0−0.1 1.19−1.21 5.26 43.64 36.3 0.8−0.91 bleak, Alburnus alburnus 9 0−0.14 0.24 0−0.32 0−0.08 0−0.04 0.01−0.03 1.24 0.85 0.16−0.19 0−0.08 brown trout, Salmo 31 0.02−0.12 0.17 0.07−0.21 0.01−0.07 0.04−0.11 0.08−0.13 1.94 0.95 0.29−0.32 0.03−0.05 trutta fario chub, Squalius cephalus 9 0−0.51 0.22 2.2−2.47 0.22−0.35 1.08−1.15 0.68−0.7 3.71 18.32 5.03 0.06−0.22 7 0−0.12 0.18 0−0.14 0−0.1 0.03−0.08 0.03−0.1 0.82 0.39 0−0.08 0−0.08 common carp, Cyprinus carpio common roach, 67 0.02−0.18 0.13 0.11−0.24 0.01−0.09 0.07−0.12 0.34−0.38 1.18 12.58 12.44−12.49 0.14−0.19 Rutilus rutilus minnow, Phoxinus phox1 0−0.56 0.24 0−0.37 0−0.17 0−0.1 0.19 7.07 6.17 0.42 0−0.23 inus European eel, Anguilla 137 0.07−0.36 0.27 0.05−0.34 0.01−0.18 0.09−0.2 0.12−0.26 2.12 1.84 0.22−0.33 0.09−0.18 anguilla European perch, Perca 9 0−0.12 0.09 0.03−0.13 0−0.05 0.02−0.07 0.12−0.13 2.79 2.51 0.42−0.43 0.1−0.16 f luviatilis freshwater bream, Abra34 0.03−0.18 0.12 0.08−0.21 0.01−0.08 0.05−0.09 0.11−0.13 1−1.02 0.46−0.47 0.11−0.17 0.07−0.12 mis brama gudgeon, Gobio gobio 5 0−0.58 0.25 3.88−4.1 0.33−0.43 1.75−1.81 0.21−0.24 8.27 23.19 1.18 0.06−0.25 northern pike, Esox lu8 0−0.13 0.14 0−0.13 0−0.08 0−0.04 0.04−0.07 1.46 2.87 0.46−0.48 0.02−0.04 cius white bream, Blicca 22 0.01−0.16 0.14 0−0.15 0−0.09 0.04−0.08 0.06−0.11 0.97−0.99 1.13−1.14 0.14−0.23 0.07−0.14 bjoerkna thicklip grey mullet, 6 0−0.14 0.13 0−0.21 0−0.11 0.01−0.05 0.15* 1.27 0.56 0−0.04 0−0.01 Chelon labrosus wels catfish, Silurus gla14 0−0.15 0.13 0−0.13 0−0.08 0.01−0.05 0.08−0.1 1.27 2.01 0.33−0.34 0.02−0.08 nis western vairone, Telestes 1 0−0.61 0.26 0−0.41 0−0.19 0−0.1 0−0.08 2.52 2.45 0.38 0−0.25 souf f ia pike-perch, Sander lucio22 0−0.17 0.11 0−0.14 0−0.08 0−0.05 0.03−0.08 1.34 0.82 0.02−0.11 0.01−0.08 perca fishes with low level of PFAA contamination and n > 5 (freshwater bream, northern pike, thicklip grey mullet, wels catfish, brown trout) mean LB 0.01 0.00 0.03 0.00 0.02 0.09 1.15 1.26 0.23 0.04 UB 0.14 0.14 0.18 0.08 0.07 0.12 1.17 1.27 0.26 0.07 std LB 0.01 0.00 0.04 0.00 0.02 0.04 0.28 0.94 0.19 0.04 UB 0.02 0.02 0.05 0.01 0.03 0.03 0.25 0.94 0.18 0.06 fishes with medium level of PFAA contamination and n > 5 (European perch, chub, common roach, European eel, silver bream, common carp, pike-perch, bleak) mean LB 0.01 0.00 0.30 0.03 0.16 0.17 1.65 4.76 2.30 0.06 UB 0.22 0.17 0.49 0.13 0.22 0.22 1.69 4.77 2.36 0.14 std LB 0.02 0.00 0.77 0.08 0.37 0.23 1.00 6.76 4.44 0.05 UB 0.14 0.07 0.80 0.10 0.38 0.22 1.00 6.76 4.43 0.06 fishes with high level of PFAA contamination and n > 5 (gudgeon, barbel) mean LB 0.00 0.00 1.94 0.16 0.88 0.70 6.76 33.42 18.74 0.43 UB 0.58 0.25 2.24 0.30 0.95 0.73 6.76 33.42 18.74 0.58 std LB 0.00 0.00 2.74 0.23 1.24 0.69 2.13 14.46 24.84 0.52 UB 0.01 0.00 2.63 0.18 1.21 0.68 2.13 14.46 24.84 0.46

n

Table 3. PFAA Contamination of Fresh Freshwater Fish LB-UB (Nanograms per Gram ww)

0.21−0.3

0.07

0.04 0.17 0.04 0.04 0.23 0.37 0.28 0.26 0.27 0.31 0.12 0.12

0.00 0.09 0.00 0.02 0.00 0.08 0.00 0.03 0.00 0.06 0.00 0.00

0.06−0.16

0.36−0.39 0.02−0.18

0.06 0.08

0.08

0.1−0.24

0.07

0−0.17

0.11−0.2

0.08

0.06

0.67−0.77

0.16

0.01−0.12

0.4

0.06

0.1

0.06−0.18

0.07

0.01−0.16

0.05−0.16 0.67−0.76

0.07 0.07

0.12

0.19−0.22 0.02−0.41 0.06−0.17

PFHxS

0.06 0.06 0.09

PFBSa

0.30 0.40 0.43 0.30

0.15 0.22 0.15 0.12

0.02 0.08 0.03 0.02

0.08−0.11

0−0.2

0−0.07

0−0.06

0.09−0.13

0.6 0−0.08

0.07−0.12

0.28−0.3

0.08−0.21

0.68

0.24−0.3

0−0.17 0.42−0.46

0−0.19 0.02−0.11 0−0.08

PFHpS

84.88 84.88 61.06 61.06

37.80 37.80 19.23 19.23

13.05 13.05 6.25 6.24

31.2

41.78

7.78

13.71

35.6

128.06 14.73

22.3

82.52

39.55

134.81

17.63

35.5 32.28

41.7 28.11 7.27

PFOS

0.36 0.42 0.19 0.12

0.16 0.23 0.07 0.06

0.05 0.09 0.04 0.04

0.26−0.27

0−0.25

0.04−0.08

0−0.04

0.13−0.16

0.5 0.06−0.12

0.11−0.14

0.27−0.28

0.1−0.25

0−0.23

0.1−0.17

0.14−0.28 0.1−0.14

0.23−0.34 0.16−0.25 0.04−0.08

PFDS

148.9 150.6 27.6 27.3

47.8 49.1 20.1 20.1

16 17 6.2 6.1

33.8−34.8

47.1−49.7

11−11.9

15.7−16.7

38.4−39.5

168.4−169.9 19.3−20.3

24.5−25.5

88.8−89.5

44.6−46.6

149.7−151.7

44.6−45.7

67−68.9 34.6−35.7

129.3−131.3 30.4−32 9.4−10.3

ΣPFAAs

Journal of Agricultural and Food Chemistry Article

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Figure 1. Mean marine fish PFAA contamination by coastal area (ng g−1 ww), LB hypothesis.

Figure 2. Mean European eel PFAA contamination by river section (ng g−1 ww), LB hypothesis.

which also finds a higher PFAA contamination in freshwater fish than in marine ones. One of the hypotheses about this observation is the water contamination level difference between rivers/lakes and seas/oceans, where the pollutants are diluted in a larger amount of water. Whereas the contamination levels in seawaters seem to range from a few picograms per liter to a few nanograms per liter,52 except in coastal waters and industrialized bays such as Tokyo Bay, where a maximum level of 192 ng L−1 was found for PFOA,53 the levels in rivers can reach from nanograms per liter to micrograms per liter,54−56 a thousand times higher than in seawaters. Another hypothesis is a difference in the bioaccumulation potency between marine and freshwater fishes. However, comprehensive studies are still lacking in this field. No association was found between feeding behavior, living depth (demersal, pelagic, etc.), or climate (temperate, subtropical environment, etc.) and PFAA contamination levels. Moreover, it seems that marine fish PFAA contamination levels are different among the four coastal areas. Indeed, the fresh and frozen fishes collected in Lorient and La Rochelle are twice more contaminated than those collected in Le Havre or Toulon as presented in Figure 1. Even if the Kruskal−Wallis test revealed a significant difference (p < 0.001), it remains difficult to draw a conclusion, as some species are specific from some sampling sites. For example, the gurnard Trigla lucerne was sampled only in Lorient, as it was the only site among the four where it was sufficiently consumed.

Spatial trend of freshwater fish PFAA contamination was also investigated. The European eel Anguilla anguilla was chosen to illustrate this part of the study because of the importance of the size of this group (n = 137). European eel contamination was mostly driven by PFOS as is the case for the freshwater fishes in general, except for one sample caught in the Garonne river, which shows a contamination split among PFOS (52%), PFDA (26%), and PFUnA (18%). With regard to the sum of the 15 PFAAs, the contamination was significantly different among the 18 river sections (Kruskal−Wallis test, p < 0.0001) (Figure 2). The results are deliberately presented by river section and not by major river because of the contamination heterogeneity within some rivers such as the Seine. This heterogeneity could be explained by the presence of a point source of PFAA discharge such as wastewater treatment plants and plants producing or processing PFASs, for instance. Our colleagues from the Nancy laboratory of hydrology are currently working on this subject.57,58 Table 4 presents a few PFOS contamination data from the literature to be compared to the results of the present study. It can be noted that the PFAA contamination data from this study are consistent with the existent data, with PFOS being the most frequently detected PFAA, often dominating the contamination pattern. In Babut and co-workers’ study, gudgeon, common roach, European eel, and barbel Barbus barbus were the species with the highest PFAA levels.59 Our fishes from the low contamination level group are 2.7 times more contaminated when 7598

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LC-PUFAs, n-6 LC-PUFAs, and PUFAs, common carp Cyprinus carpio, the second oiliest fish, is relatively poor in PUFAs. Our data are consistent for European eel, wels catfish, pike-perch, trout, common carp, and bream Abramis brama in recent German and Polish studies.62,63 The differences between the studies could be explained by the difference of sampling season, fish size, and intraspecies variability. PFAA Contamination and LC-PUFA Balance. According to the multiple regression analysis, no positive or negative relationship has been highlighted between PFAA contamination and muscle protein rate, lipid content, and n-3 and n-6 LC-PUFA composition of the studied fishes (R2 = 0.26, p = 0.86), which is not surprising considering the physicochemical properties of PFAAs. PFAAs are amphiphibic and are mostly found in the liver and blood,64,65 not in fat tissues, as are organic persistent compounds such as PCB or dioxins. As PFAAs seem to bind preferentially to serum proteins rather than to muscle proteins, the results would surely be interesting to measure these contaminants in the liver and blood. The most interesting fishes with high n-3 LC-PUFA content and low PFAA contamination are the swordfish, anchovy, halibut, salmon, and mackerel, all of them being marine fishes, whereas the potentially risky fishes to consume with high PFAA contamination and low n-3 LC-PUFA content are common carp, pike-perch Sander lucioperca, European perch Perca fluviatilis, thicklip grey mullet Chelon labrosus, and common roach Rutilus rutilus, all of them being freshwater fishes. If consumed regularly, these fishes could largely contribute to increase the PFAA level found in human serum. This hypothesis is consistent with Höltzer’s study, which observed that anglers who consume fish two to three times per month have a higher PFOS serum concentration. Those fishes, caught in Lake Möhne, present a PFAA contamination similar to of the fishes of the present study.61 Our data tried to reflect an extended view of PFAA contamination along with n-3 and n-6 LC-PUFA composition of marine and freshwater fishes in metropolitan France. However, for some species such as western vairone Telestes souffia (n = 1), minnow Phoxinus phoxinus (n = 1), or marine fishes (n = 1−4), the size of the group is a limit of this study, even if the samples are composite ones made up of a homogeneous mix of several individual fishes. This concern leads to the question of the spatial trend of PFAA contamination. In this study, we chose to work on mean PFAA contamination of fishes because our FA composition data were available only by species, but there are obviously some differences between the river sections or the coastal areas, probably due to the differences in environmental contamination. Further studies could include a comparison between river sediment/water contamination and the corresponding fish contamination. Another weakness of our study is the lack of information available concerning the age, weight, and seasonality effect during the freshwater fish sampling. The French agency for food, environmental, and occupational health and safety (Anses) recently published an overview of all their recommendations since 2002 concerning the risk−benefit balance of fishery and aquaculture products for the general population, elderly, immunodepressed people, children, and pregnant or breastfeeding and child-bearing age women.66 However, these recommendations take into account only dioxins, methylmercury, and PCBs. It could be suggested to add pike-perch, European perch, thicklip grey mullet, and common roach in the list of species that should be consumed with limitation, common carp already being in this list, as they do not present such a positive profile as far as the lowest n-3 LC-PUFA levels and the highest PFAA level were

Table 4. PFOS Contamination Level of Freshwater Fishes (Nanograms per Gram wet weight) species

n

median

mean

low level medium level high level Rhône−Mediterranean drainage basin

93 282 10 823

13.71 33.89 84.88 5

13.05 37.8 84.88

Germany

perch (Perca f luviatilis) pike (Esox lucius) eel (Anguilla anguilla) roach (Rutilus rutilus)

15 6 5 10

96 37 77 6.1

US

northern pike (Esox lucius)

21

9.6

China

grey mullet catfish catfish

5 4 21

country France

reference this study

Babut et al.59

Hölzer et al.61

9.9

Xiao et al.67

1.2 0.49 0.51

Zhao et al.60

the median value is considered and 17 times more contaminated for the high contamination level group. However, the authors mentioned that their fishes were globally less contaminated than anywhere in France. Our thicklip grey mullet samples (mean PFOS level = 13.7 ng g−1 ww) and our wels catfish Silurus glanis samples (mean PFOS level = 7.8 ng g−1 ww) were surprisingly 10 and 15 times more contaminated in mean by PFOS than those purchased in local markets in Xiamen, China,60 even if China is still known for their PFAS production. A German study collected and analyzed European perch (n = 15), northern pike Esox lucius (n = 6), European eel (n = 5), and roach (n = 10) fillets for PFOS contamination in Lake Möhne.61 The median contaminations were, respectively, 96, 37, 77, and 6.1 ng g−1 ww. Our mean PFOS contaminations were 83 ng g−1 ww for European perch (n = 9), 15 ng g−1 ww for northern pike (n = 8), 40 ng g−1 ww for European eel (n = 137), and 18 ng g−1 ww for roach (n = 67). Globally, our data were consistent with those published in the literature, even if some fishes were probably sampled in “hotspots” of environmental contamination. Lipid and Fatty Acid Composition. Table 5 shows the marine and freshwater fish composition for lipids, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), n-3 LC-PUFA, n-6 LC-PUFA, and total PUFA for the 29 marine fish species and for 11 of the 16 freshwater fish species. Results for marine fish were already published and compared with the existing literature data.44 Marine fish n-3 LC-PUFA level distribution is wider than the freshwater fish one, with ranges of, respectively, 46−4472 mg for 100 g ww (mean = 708 mg for 100 g ww) and 4−3895 mg for 100 g ww (mean = 368 mg for 100 g ww). As expected, for the marine fish, the oiliest species are also the richest in PUFA. The three oiliest marine fishes are salmon Salmo salar (13.5% of lipids ww in mean), swordfish (12.4% of lipids in ww in mean), and halibut Hippoglossus hippoglossus (11.7% of lipids in ww in mean), which are also the richest in PUFAs and n-3 LC-PUFAs, including EPA. The DHA-rich marine fishes are salmon (2164 mg for 100 g ww), swordfish (1750 mg for 100 g ww), and mackerel Scomber scombrus (1404 mg for 100 g ww), and the n-6 LC-PUFA-rich marine fishes are anchovy Engraulis encrasicolus (682 mg for 100 g ww), salmon (671 mg for 100 g ww), and swordfish (5410 mg for 100 g ww). For freshwater fishes, lipid and PUFA amounts are not so clearly related. Indeed, if European eel, the oiliest freshwater fish, is also the richest in n-3 7599

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Table 5. Mean Free Fatty Acid Composition of Fresh and Frozen Marine Fish and Fresh Freshwater Fish (Milligrams per 100 g ww)a species marine fishb anchovy monkfish catshark cod common dab European eel orange roughy plaice goatfish grenadier gurnard haddock hake halibut John Dory ling mackerel pollack pout ray saithe salmon sardine scorpionfish seabass sea bream sole swordfish tuna whiting

n 1 4 4 4 4 1 3 2 3 4 1 2 4 4 2 4 4 3 1 4 4 4 4 1 4 4 4 4 4 4

mean (std) min−max

freshwater fish bleak brown trout common carp common roach European eel European perch bream northern pike thicklip grey mullet wels catfish pike-perch mean (std) min−max a

lipids (g/100 g) 7.51 0.21 0.55 0.30 0.72 20.4 5.78 0.37 3.75 0.44 0.73 0.25 0.59 11.7 0.59 0.33 7.07 0.27 0.29 0.61 1.04 13.5 5.72 2.27 2.99 4.89 0.40 12.4 0.73 0.25 3.56 (5.05) 0.2 120.4

2 5 3 6 6 1 5 2 2 3 3

9.69 11.03 16.74 6.08 46.38 4.39 7.59 2.08 8.44 4.32 1.57 10.76(12.59) 1.57 46.38

C20:5 n-3 (EPA)

C22:6 n-3 (DHA)

LC-PUFA n-3

LC-PUFA n-6

PUFA

701 26 113 28 84 432 471 46 348 41 43 18 28 969 32 45 662 15 36 17 71 1112 638 121 357 497 14 1265 35 15

1365 37 66 75 131 716 742 41 669 78 3 60 123 1400 156 65 1404 76 30 156 173 2164 1269 507 617 773 72 1750 131 69

3241 66 195 112 250 1880 1940 97 1147 160 46 84 180 3960 203 112 2585 97 77 195 262 4472 2270 890 1090 1507 109 3764 179 93

682 7 23 8 30 1285 201 15 148 8 10 10 14 191 13 4 259 5 14 23 126 671 130 57 125 350 19 541 31 5

3923 71 219 121 281 3205 2141 111 1295 168 54 94 193 4186 217 117 2845 103 91 216 391 5146 2407 949 1221 1859 128 4331 211 98

276 (361) 14 1265

399 (535) 3 2164

708 (1195) 46 4472

167 (286) 4 1285

1,213 (1563) 54 5146

131 50 45 155 958 165 228 59 57 132 41

196 108 54 190 632 274 247 204 39 228 132

556 271 168 530 3895 563 740 332 131 542 209

868 296 300 322 2511 309 594 127 47 275 91

1464 583 516 885 6702 894 1376 467 208 850 305

184(264) 41 958

209(160) 39 632

368(231) 4 3895

294(252) 3 2511

429(352) 1 6702

Std, standard deviation; min, minimum contamination value; max, maximum contamination value. bFrom Sirot et al.44

■

found compared to other fish species analyzed in this study. As a further step, additional data on marine and fish species could be interpreted in terms of fish consumption recommendation by an expert committee and the food safety recommendations to French consumers updated, if needed, according to the last recommendations of the French agency for food, environmental, and occupational health and safety.

AUTHOR INFORMATION

Corresponding Author

*(A.Y.) Mail: LUNAM Université, Oniris, Laboratoire d’Etude des Residus et Contaminants dans les Aliments (LABERCA), USC INRA 1329, Nantes 44307, France. Phone: +33 (0) 611169833. E-mail: [email protected]. 7600

dx.doi.org/10.1021/jf501113j | J. Agric. Food Chem. 2014, 62, 7593−7603

Journal of Agricultural and Food Chemistry

Article

Funding

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This work was partially supported by the ANR (Agence Nationale de la Recherche/French National Research Agency) within the framework of the CONTREPERF program “Emerging perfluorinated contaminants: contribution to the human exposure assessment, to the study of their metabolism and to the characterization of their toxicological impact” (Grant ANR-10-CESA-008). For the ICAR-PCB study, samples provided from ONEMA national PCB action plan and freshwater fish analyses were funded by the French Ministry of Health. The CALIPSO study was funded by the French Ministry of Agriculture. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Florence Ramdin for her kind help. ABBREVIATIONS USED DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; FA, fatty acid; LB, lower bound; LOD, limit of detection; LC-PUFA, long-chain polyunsaturated fatty acid; ONEMA, French national agency for water and aquatic environments; PCB, polychlorobiphenyl; PFAA, perfluoroalkyl acid; PFBA, perfluorobutanoic acid; PFBS, perfluorobutanesulfonate; PFDA, perfluorodecanoic acid; PFDS, perfluorodecanesulfonate; PFHxA, perfluorohexanoic acid; PFHxS, perfluorohexanesulfonate; PFHpA, perfluoropentanoic acid; PFHpS, perfluoropentanesulfonate; PFNA, perfluorononanoic acid; PFOA, perfluorooctanoic acid; PFOS, perfluorooctanesulfonate; PFPA, perfluoropentanoic acid; PFTeDA, perfluorotetradecanoic acid; PFTrDA, perfluorotridecanoic acid; PFUnA, perfluoroundecanoic acid

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