Chemosphere 91 (2013) 802–808

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Chemosphere

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Occurrence of perfluorinated alkylated substances in breast milkof French women and relation with socio-demographical and clinicalparameters: Results of the ELFE pilot study

0045-6535/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.chemosphere.2013.01.088

⇑ Corresponding author. Address: LABERCA, Oniris, Site de la Chantrerie, Route deGachet, BP 50707, 44307 Nantes Cedex 3, France. Tel.: +33 2 40 68 78 80; fax: +33 240 68 78 78.

E-mail address: laberca@oniris-nantes.fr (J.-P. Antignac).

Jean-Philippe Antignac a,b,⇑, Bruno Veyrand a, Hanane Kadar a, Philippe Marchand a, Amivi Oleko c,Bruno Le Bizec a, Stéphanie Vandentorren c

a LUNAM Université, Oniris, USC 1329 Laboratoire d’Etude des Résidus et Contaminants dans les Aliments (LABERCA), Nantes, Franceb Institut National de la Recherche Agronomique, INRA, Centre de recherche Angers-Nantes, Site de la Géraudière, Nantes, Francec Institut de Veille Sanitaire (InVS), Saint-Maurice, France

h i g h l i g h t s

" We analyze 14 perfluorinated compounds in a set of 48 breast milk samples collected from French women." PFOS, PFOA, and PFHxS appeared as major contributors to the total PFAS exposure." No statistically significant relation was observed between these exposure levels and developmental outcomes.

a r t i c l e i n f o

Article history:Received 5 September 2012Received in revised form 8 January 2013Accepted 19 January 2013Available online 7 March 2013

Keywords:Perfluorinated alkylated substancesBreast milkPerinatal exposureEndocrine disruption

a b s t r a c t

A previously developed and validated methodology based on liquid chromatography coupled to high res-olution mass spectrometry was used for determine the concentration levels of 14 perfluoroalkylated sub-stances (PFASs) in a set of 48 breast milk samples collected from French women in the frame of the ELFEpilot study. In accordance with other similar studies conducted at european and international levels,PFOS, PFOA, and PFHxS were detected and quantified in most of the analyzed samples (90%, 98% and100%, respectively), and appeared as major contributors to the total PFAS exposure (38%, 37%, 25%,respectively), whereas the other targeted PFAS were very rarely, if not, found at the limits of detectionof the method. Also in agreement with other published data, the concentration levels measured for thedetected substances varied from <0.05 to 0.33 lg/L for PFOS (median = 0.079), from <0.05 to 0.22 lg/Lfor PFOA (median = 0.075), and from 0.04 to 0.07 lg/L for PFHxS (median = 0.050). On the basis of thisrelatively limited data set, no statistically significant relation was observed between these exposure lev-els and developmental outcomes, in particular the weight at birth. Similarly, no relation was observedbetween the measured PFAS levels and various socio-demographical parameters including the consump-tion of seafood, alcohol, smoking, or socio-economical level. These results suggest a need for furtherresearch and better knowledge regarding the sources, pharmacokinetics, and factors of exposure for othersubstances belonging to this class of emerging contaminants.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Perfluroalkylated substances encompass a large set of chemicalsfrom anthropic origin that are structurally characterized by a fullyfluorinated carbon chain attached to a polar moiety, the latterbeing constituted by various possible functional groups, e.g.

carboxylate, sulfonate, sulfonamide. (Lehmler, 2005). Due to theiramphiphilic physico-chemical properties (i.e. both hydrophobicand lipophobic), these compounds present the macroscopic char-acteristics to repel both water and oil (Rayne and Forest, 2009).They also present a very high chemical, thermal, and biological sta-bility due to the strength of the C–F bound (Fromel and Knepper,2010). These properties are exploited at industrial scale mainlyas anti-sticking material or surfactant related products. Thus, PFASare used in carpets, textiles, leather, paper, cardboard, some foodpacking materials, electronic and photographic devices, cleaningagents cosmetics, etc. (Lewandowski et al., 2006). They are also

J.-P. Antignac et al. / Chemosphere 91 (2013) 802–808 803

used as essential processing aid in the manufacture of certain fluo-ropolymers such as polytetrafluoroethylene (PTFE), and to a lesserextent in other industrial applications as antistatic additives orflame retardants in fire-fighting foams. Consequently, consumersfrom industrialized countries are today in contact with thesechemicals in their daily life, through wide range of manufacturedproducts (Fromme et al., 2009). In parallel, PFAS may be releasedinto the environment at each step of their living cycle. Finally thepresence of PFAS was reported in various environmental compart-ments (Ahrens, 2011), several components of the food chain(D’Hollander et al., 2010), as well as human biological fluids andtissues (Vestergren and Cousins, 2009).

PFAS are organic substances recognized to be, in some extent,persistent in the environment, susceptible to bio-accumulate, andresponsible of adverse effects on animal and human health. As aconsequence, perfluorooctane sulfonate (PFOS) and perfluorooc-tane sulfonyl fluoride (PFOSF) were listed in May 2009 as ‘‘underrestricted use’’ compounds in Annex B of the Stockholm Conven-tion on persistent organic pollutants (Wang et al., 2009). In addi-tion to an evidenced hepatotoxicity which was mainly observedfor high doses of PFOS and PFOA on laboratory animal models(Kennedy et al., 2004), PFAS are also considered as endocrine dis-ruptor chemicals (EDCs) at low doses (Jensen and Leffers, 2008;White et al., 2011), with a specific debate related to their impacton the development and reproductive functions (Lau et al., 2004;Olsen et al., 2009). A recent concern associated to PFAS is notablyfocused on the potential impact of early exposures, i.e. during pre-natal and neonatal periods (Das et al., 2008; Liu et al., 2010b). In-deed, in utero exposure to PFAS in rodents was found to reducetheir postnatal survival rate (Luebker et al., 2005), alter somegrowth and developmental outcomes (White et al., 2007; Yahiaet al., 2010), and impair neurodevelopmental parameters (Johans-son et al., 2009). Thus, the question of a possible deleterious impactof a PFAS exposure for human foetus and newborn is posed. Amongother elements, this kind of investigation imply the availability ofaccurate prenatal (e.g. from cord blood samples) and newborn

Table 1Overview of the PFOS, PFOA and PFHxS concentration levels (median values and range, in

Reference Population (Country) Number of samples

Fujii et al. (2012) Japan 28Korea 24China 19

Haug et al. (2011) Norway 19

Kadar et al. (2011) France 30

Kim et al. (2011)a,b Korea 35

Karrman et al. (2010) Spain 10

Liu et al. (2010a) China 24 (pools)

Llorca et al. (2010) Spain 20

Roosens et al. (2010) Belgium 22 (pools)Bernsmann and Fürst (2008) Germany 203

Tao et al. (2008) USA 45Cambodgia 24Vietnam 40Indonesia 20Philippines 24Malaysia 13India 39Japan 24

Volkel et al. (2008) Germany (Munich) 19Germany (Leipzig) 38Hungary 13

Karrman et al. (2007) Sweden 12

Nakata et al. (2007) Japan 51

So et al. (2006) China 19

(e.g. from breast milk samples) exposure data. Such data were al-ready provided from different countries (see Table 1), but werepractically inexistent for France.

A synthesis of these published data related to the occurrence ofPFAS in Human breast milk is given in Table 1. Globally, the con-centration levels observed for PFOS and PFOA are in the severaldozens to several hundreds of lg/L range. These two particularcompounds are the most commonly monitored and detected PFAS,with measured concentrations always higher for PFOS than forPFOA. At first sight, these PFAS concentrations in Human breastmilk can be considered as very low, and notably significantly lowerthan those reported in human serum. But because of both a re-duced body weight and an exclusive consumption with finally ahigh volume intake, the exposure of breastfed newborns to PFASstill represents a matter of concern (Liu et al., 2011). In addition,since a good correlation between the circulating levels in motherserum, cord blood, and breast milk has been demonstrated (Kimet al., 2011a,b), the PFAS concentration levels measured in breastmilk have to be also considered as an indicator of the foetal expo-sure. Other representatives of this class of substances are some-times reported with lower occurrence, including for instancePFHxS, PFNA, PFDA, or PFUnA. Because the pharmacokinetics andtoxicological characteristics of all these detected PFAS have notbeen evaluated yet, the determination of such extended contami-nation profiles appears of valuable interest, especially for thosecompounds presenting a more pronounced bioaccumulative char-acter (number of carbon atoms higher than seven) (Conder et al.,2008). The potential benefit of such profiles (including the differ-entiation of linear and branched forms), as signatures of a localsource of contamination with subsequent interpretation in termsof risk analysis, is a second element to encourage such extendedmonitoring (Benskin et al., 2010). However, some methodologicallimitations makes this approach uncommon in the existing studies.

In this context, the purpose of the present study was to deter-mine the concentration levels of 14 PFAS in a set of 48 breast milksamples collected in the frame of the ELFE pilot study. ELFE is a

lg/L) determined in Human breast milk and reported in the literature.

PFOS (lg/L) PFOA (lg/L) PFHxS (lg/L)

n.d. 0.089 (<0.040–0.194) n.d.n.d. 0.062 (<0.040–0.173) n.d.n.d. 0.051 (<0.040–0.122) n.d.

0.087 (0.040–0.250) 0.025 (<0.018–0.830) n.d.

0.074 (0.024–0.171) 0.057 (0.018–0.102) n.d.

0.06 (n.a.) 0.05 (n.a.) n.d.

0.11 (0.070–0.220) n.d. (0.070–0.220) n.d.

0.049 (0.006–0.137) 0.034 (<LOD-0.814) n.d.

0.084 (0.028–0.865) n.d. n.d.

2.9 (<0.400–28.2) 0.3 (<0.300–3.50) n.d.0.082 (0.050–0.284) 0.09 (0.025–0.610) n.d.

0.106 (<0.032–0.617) 0.036 (<0.030–0.161) 0.012 (<0.012–0.064)0.04 (0.017–0.327) n.d. n.d.0.058 (0.017–0.393) n.d. 0.004 (<0.002–0.027)0.067 (0.025–0.256) n.d. 0.013 (<0.002–0.059)0.104 (0.027–0.208) n.d. 0.007 (<0.002–0.013)0.111 (0.049–0.350) n.d. n.d.0.039 (<0.011–0.120) n.d. n.d.0.196 (0.140–0.523) 0.078 (<0.042–0.170) 0.0006 (<0.002–0.018)

0.113 (0.028–0.239) n.d. n.d.0.123 (0.033–0.309) n.d. n.d.0.33 (0.096–0.639) n.d. n.d.

0.166 (0.060–0.470) n.a. (<0.209–0.492) 0.07 (0.031–0.172)

n.d. (0.008–0.401) n.d. (<LOD-0.339) n.d. (<LOD-0.025)

0.100 (0.045–0.360) 0.11 (0.047–0.210) 0.011 (0.004–0.100)

804 J.-P. Antignac et al. / Chemosphere 91 (2013) 802–808

French national cohort aiming to follow-up a total of 20,000 chil-dren from their birth to 18 years old, in order to investigate a widerange of socio-demographical, morphological, nutritional, and bio-logical parameters. One aspect of this project is related to the influ-ence of environmental factors, in particular chemical exposure, onseveral developmental outcomes. A pilot study was then con-ducted in order (1) to evaluate the feasibility of the global projectat various levels (logistical, technical, financial, etc.) and (2) to gen-erate preliminary data in the scope of prioritizing some biomarkersof exposure with higher relevance compared to other ones. The re-sults of these PFAS measurements in breast milk are described, aswell as preliminary observations related to the possible link be-tween these exposure levels and some clinical end-points charac-terizing the mothers and/or their newborn.

2. Materials and methods

2.1. Reagents and chemicals

Mixtures of nine perfluoroalkyl carboxylic acids (PFBA, PFPeA,PFHxA, PFHpA, PFOA, PFNA, PFDA, PFUnA and PFDoA), five perfluo-roalkane sulfonates (PFBS, PFHxS, PFHpS, PFOS and PFDS) as well asseveral mass-labeled internal standards (13C4-PFBA, 13C4-PFOA,13C9-PFNA, 13C2-PFDA, 13C7-PFUnA, 13C2-PFDoA, 18O2-PFHxS and13C4-PFOS) were purchased from Wellington Laboratories (Guelph,Ontario, Canada). Mix working solutions containing the 14 targetnative PFAS at 0.1, 0.01, and 0.001 ng/lL, as well as a mix contain-ing eight mass-labeled (13C and/or 18O) internal standards at0.01 ng/lL were prepared by appropriate dilutions of commercialstandards in methanol. All these solutions were stored in the darkat �20 �C. The concentration levels covered by the prepared cali-bration solutions ranged from 25 to 2000 pg/mL. Methanol andacetone (picograde quality) were from UGC Promochem (Wesel,Germany). Ammonium acetate, glacial acetic acid and ammoniac(32%) were purchased from Merck (Darmstadt, Germany). Deion-ised water (>18 mO cm) was obtained from nanopure system(Barnstead, Germany). Formic acid and 500 mg single use envicarb(Envicarb�) cartridges were acquired from Supelco (Sigma–Aldrich, Saint-Quentin Fallavier, France). Oasis HLB 500 mgcartridges were from Waters (Guyancourt, France).

2.2. Sample collection

The recruitment took place at home using a specific bottle in glassgiven to participants at the maternity. All participants were joinedby phone one month after leaving the maternity to explain carefullyhow to properly collect breast milk and ship specimen for storage ina centralized biobank before analysis. The expected target volume ofmilk collected was 150 mL. The milk sample could be obtained dur-ing several lactation sessions either using manual pressing of thebreast and direct collection in collecting flasks, or using breastpumps and further decant of the milk in the collecting flasks. In aver-age, 15 aliquot samples of 10 mL were collected for each participat-ing mother. Aliquots were transferred and added up to the stockcontainer stored in the freezer (�18 �C). This pooled sample con-tainer was shipped by mail in a waterproof pre-labeled box to thebiobank where samples were stored at �20 �C until they wereshipped with dry ice to the laboratory for measurement. A total of48 samples with a volume greater than 100 mL were finaly collected.

2.3. Questionnaires

Pilot counties were chosen for regional diversities in terms ofpopulation and urbanization. There were five different counties:Seine-Saint Denis, Ardèche, Isère, Loire, and Savoie. A total of 38

public and private maternities were concerned. Criteria ofinclusion were (i) babies born on the 1st, 2nd, 3rd and 4th of Octo-ber 2007, (ii) at least 22 weeks of amenorrhea, and (iii) maximumof two living twins delivered (no multiple birth greater than 2). Thedata collection included a questionnaire consisted of a 1 h face-to-face interview with the midwife; a collection of medical data usingthe newborn’s and mother’s medical files in order to obtain infor-mation about pregnancy, the prenatal period and the mother’s andnewborn’s health at delivery, basic demographic, questions aboutheight, weight, smoking status and food habits (in particular fishconsumption) were also obtained by a self-administered question-naire for the mother.

2.4. Sample preparation

The analytical strategy used for isolating and measuring PFASfrom milk samples has been described elsewhere (Kadar et al.,2011). Briefly, a preliminary protein precipitation step with9 mL of acetone was applied to 3 mL initial breast milk samplealiquots, followed by a two stages purification by Solid PhaseExtraction (SPE), using Oasis� HLB and carbon graphitized(Envicarb�) cartridges, respectively. Final extracts were reconsti-tuted in 200 lL of a fluorometholone solution as external stan-dard in a methanol/water mixture (30/70v/v). mass-labeledPFAS surrogates (13C and/or 18O) were added prior to extractionas internal standards used for quantification according to the iso-tope dilution method.

2.5. LC-HRMS measurement

The system used included a 1200 series HPLC pump (AgilentPalo Alto, CA, USA) equipped with a binary low-pressure mixingLC-pump (G1312B), with a built-in vacuum degasser (G1379B), a50 lL loop injection, a temperature controlled autosampler(G1367D), and a column oven (G1316B). The system was fittedto a reverse phase column Gemini C18 (3 lm, 50 � 2.0 mm)equipped with a guard column (3 lm, 10 � 2.0 mm) (Phenomenex,Torrance CA, USA). The mobile phase consisted of methanol (Sol-vent A) and ammonium acetate 20 mM (Solvent B). The elutiongradient started with 30% A for 2 min, followed by a 7 min lineargradient to 100%, then 5 min hold at 100%, and returned back to30% in 3 min. The flow rate was 0.6 mL/min and the injection vol-ume was set at 20 lL. The column heater was used to ensure a sta-ble column temperature of 40 �C.

The HPLC system was interfaced with a linear ion trap coupledto an orbital trap (LTQ-Orbitrap™) instrument (Thermo Scientific,Germany) operating in negative electrospray ionization mode.Mass spectra were acquired in full scan mode from m/z 200 to900 using a mass resolution of 30,000 FWHM at 400 m/z, in cen-troid mode. Quantitative sample analysis was performed using ex-tracted mass chromatograms from full scan. The following massspectrometer parameters were applied: capillary voltage was setat �14 V, source voltage at 4 kV and capillary temperature at280 �C. Nitrogen was used as sheath and auxiliary gas at flow ratesof 40 and 10 (arbitrary unit) respectively.

3. Results and discussion

3.1. Occurrence of perfluorinated compounds in breast milk

A summary of the concentration levels determined for thetarget PFAS in the analyzed breast milk samples are reported inTable 2. PFOS, PFOA, and PFHxS have been detected and quantifiedin most of these samples (90%, 98% and 100%, respectively). PFBAwas also detected in 17% of the samples. Conversely, PFNA, PFHxA,

J.-P. Antignac et al. / Chemosphere 91 (2013) 802–808 805

and PFHpA were only detected in one out of the 48 analyzed sam-ples. All other targeted PFAS (PFPeA, PFDA, PFUnA, PFDoA, PFBS,PFDS, and PFHpS) were not found at the limits of detection (LOD)of the method (that were estimated at 0.1 lg/L for PFDS and PFU-nA, and from 0.05 to 0.07 lg/L for other substances). Consideringthese very low detection rates observed for several PFAS and sub-sequent inconsistency in terms of representativity, all further dataanalyses were restricted to PFOS, PFOA and PFHxS. The fact thatPFOS and PFOA appear as the two main detected compounds inbiological matrices in general, and in breast milk in particular, isa matter of consensus among the different available studies (Ta-ble 1). The detection of PFHxS is also reported, but only in abouthalf of the published studies. The other PFAS representatives areclearly more rarely detected. Among the different possible optionsfor managing the non-detected values, i.e. replacement by zero(lower bound approach), LOD (upper bound), ½ LOD (mediumbound), or suppression, etc. the last one was retained in the pres-ent case considering the only few non-detected samples for PFOS(n = 5) and PFOA (n = 1).

As illustrated in Fig. 1, PFOS was found as the most contributingPFAS to the global contamination profile (38%), with measuredconcentration varying from 0.050 (LOD) to 0.330 lg/L (mean andmedian value of 0.092 and 0.079 lg/L, respectively). PFOA ap-peared as the second most abundant compound (37%), with con-centrations levels ranging from 0.050 (LOD) to 0.224 lg/L (meanand median value of 0.082 and 0.075 lg/L, respectively). The med-ian contribution of PFHxS to the PFAS contamination profiles wasthen estimated to 25%. The concentration levels observed for this

Table 2Summary of the concentration levels determined for the target PFAS detected in the 48 a

PFOS PFOA PFHxS

Detected (n, (%)) 43 (90) 47 (98) 48 (100)Mean 0.092 0.082 0.049Median 0.079 0.075 0.050Min <LODa <LODa 0.040Max 0.330 0.224 0.066

a LOD = 0.05 lg/L.b LOD = 0.07 lg/L.

Fig. 1. Detail and distribution frequency of the concentration levels determined

last compound appeared in a narrow range compared to the twoprevious compounds for which a higher interindividual variabilitywas noticed (i.e. from 0.044 to 0.066 lg/L, with mean and medianvalues found at 0.05 lg/L). Globally, these concentration levels ap-pear in the same range as other reported values for PFOS and PFOA(Table 1). The values observed for PFHxS appeared however nota-bly higher compared to other available data, but very closed tothose reported by the study of Karrman et al. (2007) in Sweden.It can be then hypothesized that the observed occurrence of partic-ular PFAS in human biological fluids is depending on local sourcesof exposure (particular environmental and/or food factors).

On the basis of all measured data (Fig. 2A), a statistically signif-icant correlation was found between the occurrence values mea-sured for PFOS and PFOA (RPearson = 0.5214 p < 0.0001). However,the dispersion and relatively limited number of the present dataimpose a ponderation with regard to this observation. Indeed, thiscorrelation becomes non significant (RPearson = 0.2613, p = 0.1130)if the four most elevated values are discarded (Fig. 2B). In addition,a similar relation was noticed between the concentrations mea-sured for PFHxS and those determined for PFOS (RPearson = 0.3594,p = 0.018) but not for PFOA (RPearson = �0.0230, p = 0.878). Finally,the hypothesis of the existence of distinct sources of exposure forthe different PFAS can be committed, which implies some difficultyto extrapolate individual global PFAS body-burden from the onlyinternal doses measured for PFOS or PFOA. Besides this hypothesis,the notion of chemical signatures associated to various sources ofPFAS exposure could be further explored, for instance by consider-ing the proportion of isomeric forms, the PFOS/PFOA ratio, or more

nalyzed breast milk samples (lg/L).

PFBA PFHxA PFHpA PFNA

8 (17) 1 (2) 1 (2) 1 (2)0.081 – – –0.076 – – –<LODb <LODa <LODb <LODa

0.134 0.053 0.074 0.064

for PFOS, PFOA and PFHxS in the 48 analyzed breast milk samples (lg/L).

(A)

PFOA = .05196 + .35139 * PFOSCorrelation: r = .52144; p<0.0001

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35PFOS (µg/L)

0.00

0.05

0.10

0.15

0.20

0.25

0.30

PFO

A (µ

g/L)

PFOA = .04552 + .41093 * PFOSCorrelation: r = .026128; p=0.1130

0.04 0.05 0.06 0.07 0.08 0.09 0.10 0.11 0.12

PFOS (µg/L)

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

PFO

A (µ

g/L)

(B)

Fig. 2. Relation between the concentration levels in breast milk observed fordifferent PFAS compounds. [A]: correlation between the concentration levels ofPFOS and PFOA. [B]: loading plot of the principal component analysis realised on thebasis of the PFOS, PFOA and PFHxS measurements performed in all analyzedsamples (n = 48).

(A)

(B)

(C)

Sum PFOS+PFOA+PFHxS = .24818 - .0143 * Fat %Correlation: r = -.1601; p=0.2770

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0Fat %

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Sum

PFO

S+PF

OA

+PFH

xS (µ

g/L)

Sum PFOS+PFOA+PFHxS = .30250 - .0027 * Mother's AgeCorrelation: r = -.1078; p=0.4760

22 24 26 28 30 32 34 36 38 40 42Mother's Age

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Sum

PFO

S+PF

OA

+PFH

xS (µ

g/L)

Mean ± SEM

Sum PFOS+PFOA+PFHxS : F(3,42) = 1.6571, p = 0.1908

0.05

0.10

0.15

0.20

0.25

0.30

FOS+

PFO

A+P

FHxS

(µg/

L)

806 J.-P. Antignac et al. / Chemosphere 91 (2013) 802–808

globally the carboxylic acids/sulfonate compounds ratio or againthe long chain/short chain PFAS ratio, as additional informativeparameters to be monitored.

0 (n=15) 1 (n=16) 2 (n=7) 3 (n=8)Parity

0.00

Sum

P

Fig. 3. Relation between the observed PFAS concentration levels in breast milk andother chemical or socio-demographical data. [A]: correlation between the measuredPFAS levels in breast milk and the milk fat content. [B]: correlation between themeasured PFAS levels in breast milk and the age of the donors. [C]: mean ± std errorof the measured PFAS levels in breast milk depending on the parity of the donors.

3.2. Relation between PFAS concentration levels and other chemical,socio-demographical, and clinical parameters

A first set of statistical analyses was performed in order to as-sess the potential relation between the measured PFAS concentra-tion levels in breast milk and some factors known to have aninfluence on the levels of persistent organic pollutants in Human.However, the relatively limited present data set undoubtedly re-duces the statistical power of these analyses that have to be con-sidered accordingly. A significant correlation is usually observedbetween the levels of most of lipophilic substances (such as diox-ins, PCB or PBDE) and the milk fat content, due to the bioaccumu-lation of these POPs in this adipose compartment. In the presentcase, this relation was not found significant for PFAS (RPearson =�0.1601, p = 0.2770) as illustrated in Fig. 3A. A correlation is alsocommonly reported for the same previously mentioned classes ofPOPs between their concentration in Human and the age of theindividuals, also due to a lifelong accumulation in the body (againespecially in fat). Conversely, the correlation between the PFAS lev-els measured in the present samples and the age of the motherswas found not significant (RPearson = �0.1078, p = 0.4760) as illus-trated in Fig. 3B. Similarly, no statistically significant relation wasnoticed between the PFAS levels measured in breast milk and the

parity of the mothers (Fig. 3C, p = 0.1908), even if a decreasingtrend may be observed as sometimes reported for PFC but almostfor other classes of POPs. All these conclusions were not modifiedif non parametric tests are used (RSpearman). Finally, all these obser-vations would tend to modulate the persistent character of PFAS,and point out some discrepancy with the relatively long half life(several years) usually admitted for these substances in Human(Olsen et al., 2007). Besides this issue, further research regardingthe pharmacokinetics of PFAS appears to be necessary in order totake into account the different sub-classes of substances with dis-tinct physico-chemical properties, e.g. short chain versus longchain PFAS, since the later is believed to have a higher bioaccumu-lation potential.

A second set of analyses was performed in order to investigatethe possible relation between the measured PFAS exposure levels

2000

2200

2400

2600

2800

3000

3200

3400

3600

3800

4000

4200

4400

Weight at birth (g)

34

35

36

37

38

39

40

41

42

Ges

tatio

nal A

ge (w

eeks

)Sum PFOS+PFOA+PFHxS = .37748 - .5E-4 * Weight at birth

Correlation: r = -.2158; p=0.1500

2000 2400 2800 3200 3600 4000 4400

Weight at birth (g)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Sum

PFO

S+PF

OA

+PFH

xS (µ

g/L) (A)

(B)

Mean ± SEM

Q1 (n=11) Q2 (n=11) Q3 (n=12) Q4 (n=12)

Sum (PFOS+PFOA+PFHxS) level in breast milk (quartile)

2800

2900

3000

3100

3200

3300

3400

3500

3600

3700

3800

Wei

ght a

t birt

h (g

)

F(3,42) = 1.532, p = 0.2202

(C)

Mean ± SEM

Female (n=22) Male (n=22)

Gender

0.14

0.16

0.18

0.20

0.22

0.24

0.26

Sum

PFO

S+PF

OA

+PFH

xS (µ

g/L) (D)

Fig. 4. Relation between the observed PFAS concentration levels in breast milk and other clinical data. [A]: correlation between the measured PFAS levels in breast milk andthe weight at birth. [B]: correlation between the gestational age and the weight at birth. [C]: mean ± std error of the weight at birth depending on the PFAS exposure level. [D]:mean ± std error of the measured PFAS levels in breast milk depending on the new-born gender.

J.-P. Antignac et al. / Chemosphere 91 (2013) 802–808 807

and some developmental parameters. On the basis of the presentdata, no correlation (on the basis of Pearson or Sperman correlationcoefficients) was observed between the concentration levels ofPFAS in breast milk and the weight at birth (Fig. 4A) of the new-borns. According to the relation found between the weight at birthand the gestational age (Fig. 4B), this conclusion is not modifiedafter adjusting the data on the last parameter, or if two pre-termindividuals (gestational age below 37 weeks) are removed fromthe data set. Similarly, the consideration of individuals presentingthe lowest versus highest PFAS levels did not reveal any statisti-cally significant association with the birth weight, even if a slightdecreased birth weight was noticed for the last quartile of the PFASdistribution (Fig. 4C). In addition, no relation was observed be-tween the measured PFAS exposure levels and the gender of thenewborn (Fig. 4D). Finally, these results do not give more consis-tency to the hypothesized impact of PFAS exposure to the develop-mental functions in Human, which still remains a debated issue(Steenland et al., 2010).

A last set of analyses was performed in order to test the poten-tial relation between the measured PFAS exposure levels and sev-eral socio-demographical factors. Again, no statistically significantrelation was found between these concentrations of PFAS in breastmilk and the consumption of seafood products, alcohol, or smok-ing. Similarly, no influence of the socio-economical level of themother was observed on the PFAS exposure levels. Globally, theseresults indicate that the main factors determining the humaninternal exposure to PFAS largely remains unidentified and that

further research is needed regarding the sources and patterns ofcontamination to these substances.

4. Conclusion

A previously developed and validated methodology based on li-quid chromatography coupled to high resolution mass spectrome-try was used to determine the concentration levels of 14perfluoroalkylated substances in a set of 48 breast milk samplescollected from French women in the frame of the ELFE pilot study.The observed qualitative contamination profiles in these analyzedbreast milk samples are globally in accordance with similar studiesconducted at European and international level. PFOS, PFOA, andPFHxS were detected and quantified in practically all these sam-ples and appeared as the major contributors to the total PFAS expo-sure, while other targeted PFAS were very rarely, if not, found atthe limits of detection of the method. The global concentration lev-els measured for the detected substances were in the several doz-ens to several hundreds of lg/L range, also appears consistent withother published data. No statistically significant relation was ob-served between these exposure levels and developmental out-comes or various socio-demographical parameters. Several issuesmay be pointed out from these results as requiring more researcheffort. The absence of correlation between the concentration levelsmeasured for the different targeted PFAS representatives, theclearly lower concentrations reported for these substances in

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breast milk compared to those reported in serum, or the distinctbehavior of PFAS compared to other persistent organic pollutantsin terms of bioaccumulation, are example of issues meriting tobe further investigated from a pharmacokinetic point of view.The identification and monitoring of other PFAS compounds possi-bly contributing even higher to the global human exposure, in par-ticular precursor forms such as fluorotelomers or polyfluoroalkylsphosphates, would need to be initiated.

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