Trans-Placental Transfer of Thirteen Perfluorinated Compounds and

ARTICLE pubs.acs.org/est

Trans-Placental Transfer of Thirteen Perfluorinated Compounds and Relations with Fetal Thyroid Hormones Sunmi Kim,† Kyungho Choi,†,* Kyunghee Ji,† Jihyeon Seo,† Younglim Kho,‡ Jeongim Park,§ Sungkyoon Kim,† Seokhwan Park,|| Incheol Hwang,^ Jongkwan Jeon,# Hyeran Yang,3 and John P. GiesyO,[,z,2 †

School of Public Health, Seoul National University, Seoul, 151-742, Korea School of Human and Environmental Sciences, Eulji University, Seongnam, Gyeonggi, 461-713, Korea § College of Natural Sciences, Soonchunhyang University, Asan, Chungnam, 336-745, Korea School of Applied Sciences, Seowon University, Cheongju, Chungbuk, 361-742, Korea ^ Soonchunhyang University Gumi Hospital, Gumi, Gyeongbuk, 730-706, Korea # Seoul National University Hospital, Seoul, 110-744, Korea 3 Seoul Metropolitan Institute of Health and Environment, Seoul, 137-734, Korea O Toxicology Centre and Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, S7J 5B3, Canada [ Department of Zoology, Center for Integrative Toxicology, Michigan State University, East Lansing, Michigan 48824, United States z Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong SAR, PR China 2 Zoology Department, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia

)

‡

bS Supporting Information ABSTRACT: While the results of animal studies have shown that perfluorinated compounds (PFCs) can modulate concentrations of thyroid hormones in blood, limited information is available on relationships between concentrations of PFCs in human blood serum and fetal thyroid hormones. The relationship between concentrations of PFCs in blood and fetal thyroid hormone concentrations or birth weight, and ratios of major PFCs between maternal and fetal serum were determined. Concentrations of PFCs were measured in blood serum of pregnant women (n = 44), fetal cord blood serum (n = 43) and breast milk (n = 35). Total concentrations of thyroxin (T4), triiodothyronin (T3) and thyroid stimulating hormone (TSH) in blood serum were also quantified. The ratios of major PFCs in maternal versus fetal serum were 1:1.93, 1.02, 0.72, and 0.48 for perfluorotridecanoic acid (PFTrDA), perfluorooctanoic acid (PFOA), perfluorohexane sulfonate (PFHxS), and perfluorooctane sulfonate (PFOS), respectively. Fetal PFOS, PFOA, PFTrDA and maternal PFTrDA were correlated with fetal total T4 concentrations, but after adjusting for major covariates, most of the relationships were no longer statistically significant. However, the significant negative correlations between maternal PFOS and fetal T3, and maternal PFTrDA and fetal T4 and T3 remained. Since thyroid hormones are crucial in the early development of the fetus, its clinical implication should be evaluated. Given the observed trans-placental transfer of PFCs, efforts should be also made to elucidate the exposure sources among pregnant women.

’ INTRODUCTION Perfluorinated compounds (PFCs) have been extensively used for numerous applications, including surface coatings for fabrics, furniture and food packaging.1 Some PFCs are persistent and bioaccumulative, and some of the more volatile PFCs including perfluorosulfonic acid and perfluorinated carboxylic acids precursors, are capable of long-range transport to remote regions.2
4 PFCs are ubiquitous in the environment and have been detected in both wildlife and humans worldwide.5
7 Several studies have indicated widespread exposure to PFCs in humans, r 2011 American Chemical Society

not only due to occupational exposure, but also in the general population. There are also reports of detection of PFCs in biologically sensitive human populations, such as pregnant women and children. For instance, among pregnant Danish women (n = 1399, studied between 1996 and 2002), mean Received: April 20, 2010 Accepted: August 1, 2011 Revised: July 29, 2011 Published: August 01, 2011 7465

dx.doi.org/10.1021/es202408a | Environ. Sci. Technol. 2011, 45, 7465–7472

Environmental Science & Technology blood plasma concentrations of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) were 35.3 and 5.6 ng/mL, respectively.8 In Canada, mean concentrations of PFOS and PFOA were 36.9 and 2.2 ng/mL, respectively, in maternal plasma obtained between 1994 and 2001 (n = 10).9 More recently, mean blood serum concentrations of PFOS and PFOA in blood serum of pregnant Canadian women (n = 101, between 2004 and 2005) were 18.3 and 2.5 ng/mL, respectively.10 In fetal blood sera collected during a hospital-based cross-sectional study of singleton births (n = 293) between 2004 and 2005 in the U.S., mean concentrations of PFOS and PFOA were 4.9 and 1.6 ng/ mL, respectively.11 In a Danish cohort study, mean PFOS and PFOA concentrations in cord plasma were 11.0 and 3.7 ng/mL (n = 50), respectively.8 Based on the mother-infant pair studies, it appears that certain PFCs are transferred to a considerable extent through the placental barrier to the fetus. However, to date these studies have been limited almost exclusively to measurements of PFOS and PFOA.8,10 Human milk also contains PFCs and could serve as an important exposure pathway to infants. Nine PFCs were detected in breast milk (n = 184) collected from seven Asian countries during 1999 to 2005, with an estimated mean daily intake of PFOS through breastfeeding at 11.8 ( 10.6 ng/kg/d, which was 7- to 12-fold greater than the estimated adult dietary intakes reported from Germany, Canada and Spain.12 In Sweden, concentrations of PFCs in human milk collected in 2004 were approximately 1% of the concentrations in blood serum and the amount of PFCs transferred by lactation would be approximately 200 ng/d.13 After a phase-out of perfluorooctanesulfonyl fluoride-based materials by 3M Company (St. Paul, MN) in 2000, the concentrations of several PFCs such as PFOS, perfluorohexane sulfonate (PFHxS), or PFOA in the general U.S. population have been declining.14,15 Widespread exposure to PFCs among human populations raises concerns about its potential public health consequences. The association between PFC exposure and fetal growth is one such concern. Although the relationship was not strong, concentrations of both PFOS and PFOA were reported to be negatively correlated with a clinical observation like birth weight in several cross-sectional studies.8,11,16 However, in other studies no association was observed.10,17 Thus, the relationship and a potential mechanism that might explain such a relationship if indeed it does exist remains to be elucidated. PFCs cause an imbalance in thyroid hormone in vitro and in vivo.18
21 Total concentrations of thyroxine (T4) and triiodothyronine (T3) were less in blood serum of rats exposed to PFOS, but thyroid stimulating hormone (TSH) was not affected.19,22 Population-based studies, generally in occupational settings have generally not found associations between concentrations of thyroid hormones and PFCs in blood,23 but such associations were reported in a few studies. Concentrations of PFOA were negatively correlated with concentrations of free T4 and positively correlated with total concentrations of T3 in blood plasma of fluorochemical workers. However, the extent of hormonal changes was within the reference range.24 Only one study, to date, found thyroid disease to be associated with concentrations of PFOA and PFOS among members of the general population of the U.S.25 However, there have been no reports that suggested associations between concentrations of PFCs and concentrations of thyroid hormone in the general population.26 Because the thyroid hormone system is crucial for human neurodevelopment and cognitive functioning, the possibilities of

ARTICLE

Table 1. Characteristics of Mothers and Infants N

variable

range

mean

SD

median

32

4.9

32

57.2

9.6

57.5

Pregnant Women (n = 44) age (year)

44

22
44

prepregnancy

44

38.0
78.0

weight (kg) height (cm)

44

148
171

BMI (kg/m2)

44

15.2
31.0

161 22.0

4.5 3.8

161

parity

44

1
3

2

0.7

1

gestational age at delivery (weeks)

44

33
41

39

1.6

39

gestational age at blood

39

20
41

35

6.1

37

21.8

sampling (weeks) Infants (n = 43)a sex

43

birth weight (kg)

43 2.22
4.01

male: 19, female :24 3.20

0.45

3.21

a

A total of 43 infants including 5 pairs of twins were born to the participating pregnant women. Among them, the number of matching mother-fetus pairs was 35 including 4 pairs of twins.

even subtle effects due to fetal exposure to certain environmental contaminants are of potential public health concern.27 Since there have been no reports that showed significant correlations between prenatal or trans-placental exposure to PFC and fetal thyroid hormone concentrations, this study was designed to (1) determine trans-placental transfer of PFCs, and (2) investigate possible associations between concentrations of PFCs in maternal or cord blood serum and thyroid hormone concentrations and birth weights of infants. Concentrations of PFCs were measured in blood serum of women during pregnancy and breast milk of the same women during lactation, and cord blood serum of matching fetuses.

’ MATERIALS AND METHODS Study Population. Pregnant women were recruited at three hospitals located in Seoul, Cheongju and Gumi, South Korea between August, 2008 and March, 2009. Blood samples were drawn from 44 women, mostly during the third trimester of pregnancy, but several subjects were sampled earlier (n = 7, sampled during week 20
25 of pregnancy). In addition, cord blood (n = 43) was drawn at delivery from the umbilical cord vein, 35 of which were collected from matching mother-infant pairs (Table 1). Breast milk (n = 35) was collected during the mother’s checkup-visit to the hospital, approximately one month after the delivery. Matching maternal blood-cord blood-milk samples were obtained from 26 mother-infant pairs. Blood serum was separated on site and stored in polypropylene cryovials at
70 C until analysis. Milk was stored at
20 C after collection. Participating women completed a detailed questionnaire, which included queries regarding current or previous pregnancy history, medical history and demographic parameters. Characteristics of infants were also gathered. The Institutional Review Board of the School of Public Health, Seoul National University approved the study and informed consent was obtained from the participating women. In addition, all samples and data were processed blind. 7466

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Table 2. Serum PFC Concentrations (ng/mL) in Pregnant Women by the Characteristics of Study Populationa PFCs median (IQR) variable all

N 44

n > LOD age

44

20
29

14

PFHxS 0.55

40
49

28 2

Primiparous

44

yes

25 19

BMI(kg/m2)

44

underweight

7

normal

29 7 1

0.44

PFDA 0.31

PFUnDA 0.60

PFTrDA 0.24

(1.15
1.91)

(0.23
0.62)

(0.24
0.39)

(0.50
0.99)

(0.17
0.31)

44

29

44

44

37

41

27

43

0.61 0.54 (0.46
0.89) 0.82 0.54 0.57

0.09* (0.07
0.10) 0.09* (0.06
0.10) 0.18† 0.09 (0.06
0.11) 0.08

2.02*† (1.57
3.66) 2.91* (2.25
4.16) 7.85† 2.66 (2.16
4.05) 2.75

1.43*† (1.03
1.87) 1.45* (1.24
1.81) 3.41† 1.65 (1.12
1.95) 1.37

0.40

0.25*

(0.15
0.79) 0.44

(0.17
0.38) 0.33*

(0.23
0.58) 0.72 0.50

(0.24
0.38) 1.06† 0.33

(0.15
0.63) 0.39

(0.24
0.38) 0.29

0.58 (0.42
0.97) 0.57 (0.52
0.99) 1.52 0.92 (0.52
0.99) 0.56

0.19 (0.15
0.28) 0. 25 (0.18
0.38) 0.56 0.25 (0.17
0.31) 0.24

(0.41
0.84)

(0.06
0.10)

(2.06
4.48)

(1.16
1.75)

(0.25
0.58)

(0.24
0.43)

(0.46
0.85)

(0.17
0.31)

0.67 (0.46
0.85)

0.09 (0.07
0.09)

3.29 (1.88
4.05)

1.75 (1.26
1.87)

0.58 (0.39
1.00)

0.37 (0.24
0.38)

0.77 (0.50
1.07)

0.21 (0.16
0.28)

0.54 0.57 (0.31
0.84)

obese

1.46

PFNA

(2.08
4.36)

(0.46
0.89) overweight

2.93

PFOA

(0.06
0.10)

(0.48
0.90) no

0.09

PFOS

(0.46
0.85)

(0.41
0.76) 30
39

PFHpS

0.84

0.09 (0.06
0.11) 0.08 (0.06
0.10) 0.10

2.66 (2.09
4.13) 2.69 (1.52
4.85) 4.19

1.49 (1.12
1.91) 1.34 (1.16
1.95) 1.37

0.47

0.30

(0.21
0.62) 0.42

0.35

(0.30
0.57) 0.38

(0.23
0.40) (0.24
0.47) 0.25

0.73 (0.53
1.03) 0.51 (0.38
0.65) 0.46

0.26 (0.19
0.33) 0.20 (0.15
0.43) 0.18

a

Median values are presented. Values in parentheses are interquatile range (IQR) showing the 25th and 75th centile values. In case where 10) were shown here. Comparison among groups was conducted only when the frequency of detection greater than LOD was >80%. LOD: 0.03 ng/mL for PFOA; 0.04 ng/mL for PFHpS and PFOS; 0.05 ng/mL for PFTeDA; 0.06 ng/mL for PFHxS; 0.07 ng/mL for PFDS, PFTrDA, and MePFOSAA; 0.12 ng/mL for EtPFOSAA; 0.13 ng/mL for PFNA; 0.14 ng/mL for PFDA; 0.20 ng/mL for PFDoDA; 0.27 ng/mL for PFUnDA. Non-detects were included in the calculation as a proxy value of a LOD/sqrt(2) for PFHxS, PFOS, PFOA, PFNA, PFDA and PFTrDA. PFDS, PFTeDA, and EtPFOSAA were detected below LOD in all samples. PFDoDA and MePFOSAA were detected in only one sample (0.21 and 0.10 ng/mL). Different symbol (*, †, or #) means significant differences among groups (p < 0.05) based on Bonferroni’s ANOVA for logtransformed PFCs.

Chemicals and Sample Preparation. Thirteen PFCs, including four perfluoroalkyl sulfonates, seven carboxylates, and two sulfonamides were analyzed in blood serum. These compounds included PFHxS, perfluoroheptane sulfonic acid (PFHpS), PFOS, perfluorodecane sulfonic acid (PFDS), PFOA, perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoroundecanoic acid (PFUnDA), perfluorododecanoic acid (PFDoDA), perfluorotridecanoic acid (PFTrDA), perfluorotetradecanoic acid (PFTeDA), 2-(N-methyl-perfluorooctane sulfonamido) acetic acid (MePFOSAA) and 2-(N-ethyl-perfluorooctane sulfonamido) acetic acid (EtPFOSAA). In breast milk, PFHxS, PFOS, PFOA, PFNA, PFDA, PFUnDA, PFDoDA, MePFOSAA, and EtPFOSAA were measured. Sample preparation for PFCs was performed according to the methods outlined in Hansen et al.,28 Reagen et al.,29 and Naile et al.30 with minor modifications (For details, see Supporting Information). Liquid Chromatography and Mass Spectrometry Conditions. An HPLC system (Series 1100, Agilent Technologies, Palo Alto, CA) was used for analysis of PFCs. PFCs were separated on a 2.0  150 mm Luna C18 column (Phenomenex, Torrance, CA). Injection volume was 5 μL and the flow rate was 200 μL/min. Analytes were separated in isocratic mode with mobile phases 15%

of A (2 mM ammonium formate in water, pH 3) and 85% of B (acetonitrile) (v/v). Identification and quantification of analytes were accomplished by use of an API 4000 triple quadruple mass spectrometer (Applied Biosystems, Foster city, CA), operated in the electrospray ionization (ESI) negative mode with multiple reaction monitoring (MRM). The ESI conditions for analysis of the targeted PFCs were optimized to the following conditions: ion source voltage
4.5 kV, ESI temperature 400 C, curtain gas 15 psi, nebulizer gas (GS1 40 psi, and GS2 60 psi), entrance potential
10.0 V, dwell time 45 ms. The settings for declustering potentials and collision energies were optimized individually for each chemical. The mass analyzer was operated in the MRM mode: PFOA (m/z 413f369), PFNA (m/z 463f419), PFDA (m/z 513f469), PFUnDA (m/z 563f519), PFDoDA (m/z 613f569), PFHxS (m/z 399f80), PFOS (m/z 499f80 and 499f99), MePFOSAA (m/z 570f419), EtPFOSAA (m/z 584f419), 13C4
PFOA (m/z 417f372), 13C4
PFOS (m/z 503f80 and 503f99). Quantification and Method Validation. Quantification was based on peak areas relative to the corresponding isotopically labeled internal standards. 13C4
PFOS was used as a surrogate for the sulfonates and sulfonamides, while 13C4
PFOA was used 7467

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Table 3. Cord Serum PFC Concentrations (ng/mL) in Infants by the Characteristics of Study Populationa PFCs median (IQR) variable all

N 43

n > LOD maternal age

43

20
29

15

PFHxS 0.34

40
49

26 2

Primiparous

42

yes

26 16

infant sex

43

male

19

female

24

1.26

PFOA 1.15

PFNA 0.45

PFDA 0.19

PFTrDA 0.47

PFTeDA 0.07

(0.05
0.08)

(0.81
1.82)

(0.95
1.86)

(0.23
0.66)

(0.16
0.20)

(0.36
0.73)

(0.06
0.11)

43

14

43

43

27

17

43

14

0.30 0.37 (0.29
0.61) 0.61 0.35 (0.27
0.59)

no

0.06

PFOS

(0.27
0.51)

(0.22
0.35) 30
39

PFHpS

0.31

0.05 (0.05
0.05) 0.07 (0.05
0.09) 0.06 0.06 (0.05
0.09) 0.06

0.84*

1.10

(0.50
1.19) 1.52†

(0.83
1.27) 1.37

(1.08
2.01) 1.95† 1.26

(0.95
2.02) 1.67 1.33*

(0.73
1.91)

(1.08
2.01) 1.06†

1.27

0.26

0.15

(0.16
0.56) 0.46

(0.15
0.15) 0.18

(0.23
0.67) 0.44 0.38

(0.16
0.23) 0.19 0.19

(0.23
0.57) 0.55

(0.17
0.24) 0.17

0.37* (0.23
0.51) 0.52† (0.40
0.79) 1.38# 0.48 (0.37
0.68) 0.48

0.06 (0.06
0.06) 0.07 (0.06
0.10) 0.11 0.06 (0.06
0.09) 0.10

(0.27
0.45)

(0.04
0.08)

(0.92
1.55)

(0.88
1.42)

(0.44
0.66)

(0.15
0.19)

(0.32
0.80)

(0.06
0.11)

0.35 (0.27
0.59)

0.05 (0.05
0.07)

1.26 (0.73
1.86)

1.13 (0.95
1.89)

0.26 (0.21
0.55)

0.17 (0.16
0.19)

0.51 (0.42
0.73)

0.06 (0.05
0.09)

0.32 (0.27
0.48)

0.07 (0.05
0.12)

1.24

1.18

(0.83
1.64)

(0.90
1.67)

0.50

0.20

(0.37
0.73)

(0.18
0.27)

0.39 (0.26
0.72)

0.09 (0.06
0.11)

Maternal to Cord Ratio MS:US

1:0.72

1:0.90

1:0.48

1:1.02

1:1.16

1:0.48

1:1.92

-

a

Median values are presented. Values in parentheses are inter-quatile range (IQR) showing the 25th and 75th centile values. In case where 10) were shown here. Comparison among groups was conducted only when the frequency of detection greater than the LOD was >80%. LOD: 0.03 ng/mL for PFOA; 0.04 ng/mL for PFHpS and PFOS; 0.05 ng/mL for PFTeDA; 0.06 ng/mL for PFHxS; 0.07 ng/mL for PFDS, PFTrDA, and MePFOSAA; 0.12 ng/mL for EtPFOSAA; 0.13 ng/mL for PFNA; 0.14 ng/mL for PFDA; 0.20 ng/mL for PFDoDA; 0.27 ng/mL for PFUnDA. Non-detects were included in the calculation as a proxy value of a LOD/sqrt(2) for PFHxS, PFOS, PFOA, and PFTrDA. PFDS and EtPFOSAA were detected below LOD in all samples. PFDoDA, MePFOSAA and PFUnDA were detected in only 1, 3, and 5 samples with median of 0.21 ng/mL, 0.10 ng/mL and 0.38 ng/mL, respectively. Different symbol (*, †, or #) means significant differences among groups (p < 0.05) based on Student’s t-test or Bonferroni’s ANOVA for log-transformed PFCs. MS:US means mean ratio of concentration of each PFC between in maternal serum and umbilical cord serum.

for the carboxylates. For serum, calibration standards were prepared in commercially available bovine serum (Sigma-Aldrich, St. Louse, MO) by spiking with each PFC standard at 0.01, 0.05, 0.1, 0.5, 1, 5, or 10 ng/mL. For milk, since no PFC-free milk was available, standards were prepared in water by spiking 0.005, 0.01, 0.05, 0.1, 0.5, 1, or 5 ng/mL of each PFC. The linear regression equation of the relation between the analytes amount and the detector response, and the standard error (SE) of this regression equation was obtained, and then the limit of detection (LOD) was calculated following NIOSH methods.32 The accuracy was tested at a lesser (0.5 ng/mL) and a greater concentration (5.0 ng/mL) for serum, and at lesser (0.1 ng/mL) and greater concentration (1.0 ng/mL) for milk. Each batch, containing six spiked samples, was prepared at two concentrations. Percent recovery was then calculated. The overall precision of the analytical procedure was measured as the pooled coefficient of variation (CVs) determined from replicate analysis of standards within the range of the lesser and greater concentrations (eq 1). sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ð fi CV 2i Þ ð1Þ pooled CV ¼ fi

∑ ∑

Where: fi is number of degrees of freedom and CVi is each CV of two concentrations. Accuracy and precision of analysis for

each compound were generally acceptable, and are shown in the Supporting Information (See Tables S1
4). Details about thyroid hormone analysis and statistical analysis can be found in the Supporting Information. Due to potential interferences in quantification of PFOS by use of the more sensitive 499-80 transition by the presence of bile acids in human blood,33 the less sensitive transition of 499-99 was also used because it is not subject to such interferences. When the results obtained from the two sets of transition ions were compared, it was found that there were no interferences from bile acids and results based on either transition would be accurate. To be conservative, the results based on the 499-99 transition were reported.

’ RESULTS Concentrations of PFCs in Maternal and Cord Sera and Breast Milk. PFHxS, PFOS and PFOA were detected in 100%,

and PFDA and PFTrDA were detected in >90% of maternal serum samples (n = 44) (Table 2). The median concentration was the greatest for PFOS (2.72 ng/mL), followed by PFOA (1.46 ng/mL), PFUnDA (0.60 ng/mL), and PFHxS (0.55 ng/ mL). Statistically significant differences (p < 0.05) in maternal PFC concentrations were observed by age group for PFHpS, PFOS, PFDA and PFTrDA of which concentrations were 7468

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Table 4. Breast Milk PFC Concentrations (ng/mL) of Lactating Mothers Stratified by Characteristics of the Study Populationa PFCs median (IQR) variable all

n 35

n > LOD age

35

20
29

13

30
39

21

PFOS 0.06

40
49

1 35

yes

19

no

16

BMI (kg/m2)

35

underweight

6

(0.03
0.07)

11

29

0.05 (0.00
0.07)

0.05 (0.05
0.06)

0.06

25

0.09

0.06

0.05

(0.00
0.07)

(0.03
0.06)

0.07 (0.00
0.12)

0.05 (0.04
0.07)

0.06 0.080 (0.031
0.098)

overweight

4

0.05 (0.03
0.06)

0.17

(0.05
0.08) normal

0.05

(0.00
0.10)

(0.00
0.10) Primiparous

PFOA

0.072 (0.012
0.130)

0.05 (0.05
0.06) 0.057 (0.029
0.069) 0.067 (0.042
0.115)

Serum to Milk Ratio MS:BM

1:0.03

1:0.04

a

Median values are presented. Values in parentheses are inter-quatile range (IQR) showing the 25th and 75th centile values. Limit of detection (LOD) 0.02 ng/mL for for PFOA and PFUnDA; 0.03 ng/mL for PFDA; 0.05 ng/mL for PFHxS and PFOS; 0.06 ng/mL for PFNA and PFDoDA; 0.09 ng/mL for MePFOSAA; 0.16 ng/mL for EtPFOSAA. Non-detects were included in the calculation as a proxy value of an LOD/sqrt(2) for PFOA. Other PFCs excluded in this table were less than LOD in all milk samples. MS:BM means mean ratios between breast milk and serum concentration using for each PFC which was calculated for each mother.

generally greater among older women. Concentrations of PFOS that were detected among Korean pregnant women were up to 13.6 fold less than those of Canadian mothers (n = 10),9 while those for PFOA were 1.8 to 3.8 fold less than for Danish (n = 1399),8 or Canadian mothers,9 respectively. The gestational week when blood samples were drawn did not influence concentrations of PFCs in maternal blood serum except for PFHxS (See Table S5, Supporting Information). PFHxS, PFOS, PFOA, and PFTrDA were detected in 100% of cord blood serum samples (n = 43) with median concentrations of 0.34, 1.26, 1.15, and 0.47 ng/mL, respectively (Table 3). The concentrations of PFOS that were detected among Korean fetuses were 3.9
8.7 fold less than those reported for US (n = 293),11 and Danish fetuses (n = 50).8 The PFOA concentrations of Korean fetuses were similar to those reported for U.S.,11 but 3.2 fold less than those of Danish fetuses.9 Concentrations of PFCs in cord blood serum were generally less than those detected in maternal blood serum. PFOS and PFOA were the dominant PFCs in cord serum, followed by PFTrDA and PFNA.

Figure 1. Ratio of fetal cord to maternal blood serum for each PFC. The blue dotted line denotes mean ratio and blue circle denotes 95% confidence interval. The black line in box denotes median ratio, and black circle denotes 5th and 95th centiles of the ratio.

PFOS and PFTrDA in cord serum were detected with statistically significant greater concentrations among the fetuses of older women. In general, neither primiparous status nor infant sex was associated with PFC concentrations in cord serum, except for PFOA. PFOA concentrations were greater among primiparous women than nonprimipara (Table 3). PFOS and PFOA constituted the major proportion of the total amount of PFCs in maternal blood serum and cord blood serum, and the sum of PFOS and PFOA comprised 71% and 68% of ∑PFCs in maternal blood serum and cord blood serum, respectively. In breast milk, only three PFCs were detected at concentrations greater than the LOD. PFOA and PFOS were detected in 83% and 31% of samples, but PFHxS was detected in only two samples. Median concentrations of PFOS (0.06 ng/mL) and PFOA (0.05 ng/mL) were similar. No maternal characteristics that were studied were associated with milk PFCs concentrations. Concentrations ranged between 3% and 4% of those detected in maternal blood serum (Table 4). Concentrations of all PFCs detected at frequencies >80% in both maternal and cord blood serum were significantly, positively correlated (See Figure S4, Supporting Information). Ratios of concentrations of PFCs between maternal and cord blood serum were least for PFOS and PFDA (1:0.48), followed by PFHxS (1:0.72), PFHpS (1:0.92), PFOA (1:1.02), PFNA (1:1.18), and PFTrDA (1:1.93) (Figure 1). Concentrations of PFOA in breast milk were significantly but weakly correlated with concentrations of PFOA in maternal blood serum (See Table S6, Supporting Information). Associations between PFCs Concentrations and Fetal Thyroid Hormones or Birth Weight. In cord blood serum, concentrations of some PFCs were negatively correlated with concentrations of total T4 and T3. Total concentrations of T4 in cord blood serum were significantly and negatively correlated with concentrations of PFOS, PFTrDA, and ∑PFCs in cord blood serum (Table 5), whereas concentrations of T3 and TSH were not correlated with any PFCs that were measured. Concentrations of PFOS, PFTrDA, and ∑PFCs in maternal serum were negatively associated with concentrations of thyroid hormones in fetal blood serum. Concentrations of PFTrDA in maternal blood serum were negatively correlated with concentrations of T3 and T4 in cord blood serum. Concentrations of 7469

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Table 5. Correlations between PFC Concentrations (ng/mL) in Fetal Cord or Maternal Serum and Concentrations of Thyroid Hormones in Cord Blood Serum Samplesa thyroid hormones in fetal cord blood serum

PFCs in fetal cord blood serum

PFCs in maternal blood serum

N

PFHxS

PFOS

PFOA

PFTrDA

∑PFCs

N

PFHxS

PFOS

PFOA

PFTrDA

∑PFCs

34


0.228


0.212


0.276


0.223


0.285

32


0.270


0.422b


0.202


0.391b


0.416b

34


0.178


0.157


0.240


0.190


0.242

32


0.261


0.414


0.238


0.380


0.413b

35


0.280


0.344b


0.297


0.391b


0.350b

33


0.046


0.293

0.058


0.511c

35


0.111


0.048


0.157


0.254


0.180


0.128

33

0.030


0.181


0.071


0.441b


0.130

not-adjusted

31


0.185


0.090

0.030

adjustede

31


0.069


0.088

0.089

0.038


0.106

29


0.019

0.045

0.290

0.261

0.117

0.083


0.051

29

0.091

0.109

0.443b

0.288

0.218

T3 not-adjusted d

adjusted

b

b

T4 not-adjusted e

adjusted TSH

Units in ng/dL for T3, μg/dl for T4 and μIU/mL for TSH. b p < 0.05. c p < 0.01. Pearson correlation tests were performed among the logarithms of fetal thyroid hormones and PFCs with- and without adjustment for influential covariates upon fetal thyroid hormones, which were selected from our preliminary analyses (multivariate model), as follows. d Maternal age and gestational age for T3. e Maternal age, gestational age and maternal BMI for T4 and TSH. a

PFOS and ∑PFCs in maternal blood serum were also negatively correlated with fetal T3 concentration. After adjusting covariates that had asssociations with T4 and those that were determined to be strong independent predictors of thyroid hormone concentrations in the study population, statistical significance between the fetal PFCs and the hormones disappeared, but negative trend remained. In addition concentrations of PFOS and PFTrDA were significantly negatively associated with fetal T4 or T3 even after adjustment for covariates (Table 5). After adjusting for maternal age, gestational age and BMI (See Table S7, Supporting Information), there were no statistically significant correlations between birth weight and concentrations of PFCs in maternal (n = 31) or cord serum (n = 43).

’ DISCUSSION Serum PFCs and Thyroid Hormones. Although the range of concentrations of thyroid hormones was comparable to those reported previously,34 a potential association between concentrations of PFCs in maternal or fetal cord serum and concentrations of thyroid hormone in fetal blood serum was observed. PFOS has been reported to affect concentrations of T4 or T3 without influencing the regulatory functions of thyroid hormone homeostasis in animal studies.19,35 Recently it was also shown that both in utero exposure and postnatal exposure through lactation caused hypothyroxinemia in rat pups.36 This is consistent with the stronger negative relationships between concentrations of thyroid hormones T3 and T4, and major PFCs in cord blood of twins (See Figure S5, Supporting Information). However, due to the limited sample size (n = 6), the biological significance of this observation is not clear. Among the general U.S. adult population (n = 3974), people with greater concentrations of PFOA or PFOS in serum were more likely to report treated thyroid disease.25 While the association observed in this study does not confirm causality of thyroid hormone balance by exposure to PFCs, the negative trends between concentrations of thyroid hormone and PFCs of both mother and fetus, as well as

among twins could be explained by several mechanisms. Such a trend is consistent with competition between endogenous fatty acids and structurally similar PFCs which has been reported for binding sites on albumin and other serum proteins.37 Since circulating thyroid hormones are bound to serum proteins such as transthyretin and PFOS has been shown to compete with T4 for binding sites on transthyretin, PFCs in the blood could increase the concentrations of free thyroid hormones, which are then subject to clearance, and hence leads to a reduction in total thyroid hormone concentrations in the blood.21 This mechanism is consistent with the fact that a similar trend in association was observed between concentrations of PFOS and PFTrDA in maternal blood serum and concentrations of fetal thyroid hormones. Given the observed efficiencies of trans-placental transfer for some PFCs, this observation is somewhat predictable. Serum PFCs and Birth Weight. Even though concentrations of PFOS and PFOA have previously been shown to be negatively associated with birth weight and length (n = 293),11 in this study no significant association was observed between concentrations of PFC in blood of mothers or the fetus and birth weight (See Table S7, Supporting Information). The results of larger-scale cross-sectional studies vary and have not been conclusive.8,16 PFOA, but not PFOS, has been suggested to affect fetal growth, in a study of 1400 pregnant women and their newborns from the Danish National Birth Cohort.8 More recently it has been reported that in utero exposure to relatively small concentrations of PFOS negatively affected birth weight, whereas PFOA did not.16 Trans-Placental Transfer of PFCs to Fetal Blood Serum. The observation that PFCs in maternal blood can pass the placental barrier to fetal blood that is reported here is consistent with the results of previous studies that have reported transplacental transfer of PFOS and PFOA.8,38 Both of these previous studies found that concentrations of PFOA in cord blood were more similar to concentrations in maternal blood than PFOS. Recently, in a study of Canadian mother-infant pairs (n = 101 for pregnant women, 105 for matching infants) reported ratios of concentrations in maternal blood serum to that in cord blood 7470

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Environmental Science & Technology serum for PFOA, PFNA, PFHxS, and PFOS were 1:0.87, 1:1.18, 1:1.25, and 1:0.44, respectively,10 which are comparable to ratios observed in this study, except for PFHxS. This divergence in PFHxS could be due to differences in the number of nondetects. In the present study, rate of detection for PFHxS in cord blood sera was 100%, whereas that of Monroy et al. was 20%.10 Competing mechanisms appear to be involved in the transplacental transfer of different PFCs. The trans-placental transfer ratio of PFCs did not scale linearly with the chain length of the target compounds. While PFTrDA has the greatest ratio, PFDA has a ratio less than those for PFOA and PFNA. Similarly, among the sulfonates, PFOS has a transfer ratio less than PFHxS or PFHpS. The transfer ratio of different PFCs can also be partly explained by their binding affinity to blood proteins like fatty acid binding protein (FABP). It has been reported that PFOS exhibited greater affinity of binding with FABP, compared to PFOA.39 Since passive diffusion through placental membranes may not be generally favored when a compound is bound to macromolecules such as FABP, compounds with greater affinity to blood protein binding may less easily cross the barrier. The relatively great efficiency of the trans-placental movement of PFCs has potential clinical implications since the fetus might be more susceptible to alteration in thyroid hormone homeostasis during critical periods of development due to control of availability of cholesterol and triglycerides, which support cellular differentiation and fetal development.17,40 Infant Exposure Assessment. While breast milk is expected to be a predominant source of PFCs exposure in breastfed infants, there have been few reports of concentrations of PFCs in human milk. Concentrations of PFCs observed in breast milk during the present study were comparable to those observed in Sweden13 but less than those observed in China.41 Currently, reference doses (RfDs) have not been suggested for PFCs, except for PFOS and PFOA, which are 25 and 333 ng/kg/d, respectively.42 Using the infant’s exposure factors for milk consumption of 600 g/d and body weight of 6 kg,42 the daily intake based on median concentrations measured in the current study were estimated to be 6 for PFOS and 5 ng/kg/d for PFOA. The resulting hazard quotients for PFOS and PFOA were 0.24 and 0.015, respectively (For details, see Supporting Information). Only one milk sample exceeded the hazard quotient of unity for PFOS. However, there are several uncertainties in this estimation. The limited sample size and variation in the exposure factors, such as, rapid change in body weight and feeding rate during the first half year of life, are among potential sources of uncertainty.

’ ASSOCIATED CONTENT

bS

Supporting Information. Detailed information on materials and methods, results and discussions. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Phone: 82-2-880-2738; fax: 82-2-888-4779; e-mail: kyungho@ snu.ac.kr.

’ ACKNOWLEDGMENT This research was supported by Korea Food & Drug Administration (08182KFDA499). The research was also supported by

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a Discovery Grant from the National Science and Engineering Research Council of Canada (Project # 326415-07) and a grant from the Western Economic Diversification Canada (Project # 6578 and 6807). The authors wish to acknowledge the support of an instrumentation grant from the Canada Foundation for Infrastructure. Prof. Giesy was supported by the Canada Research Chair program, an at large Chair Professorship at the Department of Biology and Chemistry and State Key Laboratory in Marine Pollution, City University of Hong Kong, The Einstein Professor Program of the Chinese Academy of Sciences and the Visiting Professor Program of King Saud University.

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