Serum levels of perfluoroalkyl compounds in human maternal and umbilical cord blood samples

ARTICLE IN PRESS Environmental Research 108 (2008) 56– 62

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Environmental Research journal homepage: www.elsevier.com/locate/envres

Serum levels of perfluoroalkyl compounds in human maternal and umbilical cord blood samples Rocio Monroy a, Katherine Morrison b, Koon Teo c, Stephanie Atkinson b, Cariton Kubwabo d, Brian Stewart d, Warren G. Foster a, a

Department of Obstetrics and Gynecology, McMaster University, HSC-3N52D, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5 Department of Pediatrics, McMaster University, HSC-3N52D, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5 c Department of Medicine, McMaster University, HSC-3N52D, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5 d Chemistry Research Division, Safe Environments Programme, Health Canada, Ottawa, Ontario, Canada b

a r t i c l e in f o

a b s t r a c t

Article history: Received 26 November 2007 Received in revised form 29 May 2008 Accepted 12 June 2008 Available online 22 July 2008

Perfluoroalkyl compounds (PFCs) are end-stage metabolic products from industrial flourochemicals used in the manufacture of plastics, textiles, and electronics that are widely distributed in the environment. The objective of the present study was to quantify exposure to perfluorooctane sulfonate (PFOS), perfluorooctanoate (PFOA), perfluorodecanoic acid (PFDeA), perfluorohexane sulfonate (PFHxS), perfluoroheptanoic acid (PFHpA), and perfluorononanoic acid (PFNA) in serum samples collected from pregnant women and the umbilical cord at delivery. Pregnant women (n ¼ 101) presenting for second trimester ultrasound were recruited and PFC residue levels were quantified in maternal serum at 24–28 weeks of pregnancy, at delivery, and in umbilical cord blood (UCB; n ¼ 105) by liquid chromatography–mass spectrometry. Paired t-test and multiple regression analysis were performed to determine the relationship between the concentrations of each analyte at different sample collection time points. PFOA and PFOS were detectable in all serum samples analyzed including the UCB. PFOS serum levels (mean7S.D.) were significantly higher (po0.001) in second trimester maternal serum (18.1710.9 ng/mL) than maternal serum levels at delivery (16.2710.4 ng/mL), which were higher than the levels found in UCB (7.375.8 ng/mL; po0.001). PFHxS was quantifiable in 46/101 (45.5%) maternal and 21/105 (20%) UCB samples with a mean concentration of 4.05712.3 and 5.05712.9 ng/mL, respectively. There was no association between serum PFCs at any time point studied and birth weight. Taken together our data demonstrate that although there is widespread exposure to PFCs during development, these exposures do not affect birth weight. & 2008 Elsevier Inc. All rights reserved.

Keywords: Perfluoroalkyl compounds Pregnancy Fetus Exposure

1. Introduction Perfluoroalkyl compounds (PFCs) such as perfluorooctanoate (PFOA), perfluorooctane sulfonate (PFOS), perfluorodecanoic acid (PFDeA), perfluorohexane sulfonate (PFHxS), perfluoroheptanoic acid (PFHpA), and perfluorononanoic acid (PFNA) are residual metabolic products found in high molecular weight industrial flourochemicals. These compounds have a wide range of commercial applications such as components of refrigerants, paints, surfactants, polymers, paper coatings, fire retardants, adhesives, cosmetics, and insecticides (Key et al., 1997). PFCs are well absorbed but poorly excreted and are not known to be metabolized (Harada et al., 2005; Johnson et al., 1984), having arithmetic and geometric mean (95% confidence interval) half-

 Corresponding author. Fax: +1 905 524 2911.

E-mail address: [email protected] (W.G. Foster). 0013-9351/$ - see front matter & 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.envres.2008.06.001

lives in humans of 3.5 (3.0–4.1) and 3.8 (3.1–4.4), 5.4 (3.9–6.9) and 4.8 (4.0–5.8), and 8.5 (6.4–10.6) and 7.3 (5.8–9.2) years for PFOA, PFOS, and PFHxS, respectively (Olsen et al., 2007). In blood samples from a population without occupational exposure, serum levels of PFOS and PFOA were 34.7 and 5.6 ng/mL, respectively (Olsen et al., 2005). Serum PFOS levels in 645 adult blood donors in the United States were 34.9 ppb (Olsen et al., 2003). In an adult Canadian population (n ¼ 56) without occupational exposure, the average serum PFOS concentration was 28.8 ng/mL (Kubwabo et al., 2004) similar to the level (28.4 ng/mL) reported in 65 human blood samples obtained from biological supply companies in the United States (Hansen et al., 2001). The National Health and Nutrition Examination Survey (NHANES) 2003–2004 of serum from United States residents indicated slight differences in PFOS concentration levels by gender, and ethnicity (Calafat et al., 2006). In this study, men had higher PFOS levels compared with women. Furthermore, PFOS and PFOA levels were higher in non-Hispanic whites compared

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with non-Hispanic blacks and Mexican Americans (Calafat et al., 2006). Comparison of data from the NHANES 2003–2004 and 1999–2000 surveys reveals that the concentrations of PFOS, PFOA, and PFHxS are declining in United States residents (Calafat et al., 2007). While PFCs exposure in adults, children, and elderly has been described, there is limited data regarding PFC exposure in pregnant woman and neonatal outcomes. Circulating PFOS concentrations in 15 pregnant women were highly correlated with umbilical cord blood (UCB) levels at delivery (Inoue et al., 2004). Similarly, PFOS and PFOA were detected in 99% and 100% of umbilical cord serum samples, respectively, collected from 299 singleton newborns in Baltimore with median concentrations of 5.0 and 1.6 ng/mL, respectively (Apelberg et al., 2007a). In a national survey of 1400 women and their infants from the Danish National Birth Cohort, PFOS and PFOA concentrations measured in maternal plasma were 35.3 and 5.6 ng/mL, respectively (Fei et al., 2007). These data indicate that PFOS can cross the placenta and enter the fetal circulation and is congruent with PFOS placental transfer from the dam to fetus during gestation in Sprague-Dawley rats (Luebker et al., 2005b). However, exposure to other PFCs has not been documented previously. Moreover, the effects of developmental exposure to PFCs are equivocal with reports of an inverse association with exposure to PFOA (Apelberg et al., 2007a; Fei et al., 2007) and PFOS (Apelberg et al., 2007a), whereas another study found no association between exposure to PFCs and birth weight (Grice et al., 2007). Furthermore, despite a weak inverse relationship between exposure to PFOA and birth weight, there was no increased risk associated with exposure to PFOS (Fei et al., 2007). The potential for PFCs to cause developmental toxicity is suggested by several independent animal studies (Fuentes et al., 2006; Grasty et al., 2003, 2005; Lau et al., 2003, 2006; Luebker et al., 2005a, b). Perinatal fetal toxicity following daily PFCs exposure during pregnancy has been documented in rodent models (Fuentes et al., 2006; Grasty et al., 2003; Lau et al., 2003; Luebker et al., 2005a, b). PFOS treatment (10 mg/kg) decreased birth weight (BW) in mice and rats (Thibodeaux et al., 2003). Recently, neonatal mortality and decreased BW were also induced by PFOA exposure during pregnancy in the 20 mg/kg dose group (Lau et al., 2006). However, it must be noted that the adverse effects documented in animal studies occurred with PFOS serum levels higher than the levels found in humans. For instance, decreased birth weights were found in pups from dams whose mean (7S.D.) serum PFOS levels were 26.2716.1 mg/mL (Luebker et al., 2005b), levels that are markedly greater (approximately 1000 times greater) than the concentrations measured in contemporary exposure studies (Apelberg et al., 2007a; Fei et al., 2007; Grice et al., 2007). Although there are substantial differences in the pharmacokinetics between experimental animals and humans such as absorption, distribution, metabolism, and excretion that affect species sensitivity to the agent (Butenhoff et al., 2004, 2006), human vulnerability to different PFOS levels cannot be discounted. Therefore, the purpose of the present study was twofold: to measure selected PFCs in maternal serum at midpregnancy and delivery and in UCB in healthy pregnant women from Hamilton, Ont., Canada, and to examine the relationship between exposure and birth weight in this study population.

57

participate in the Family Study with a recruitment rate of approximately 10%. Of women consenting to participate in the Family Study and meeting the inclusion criteria, maternal and UCB samples were collected from the first 101 study subjects to provide complete sample sets and form the basis of the present study. Briefly, after consent was obtained, study subjects were seen at their second trimester visit (24–28 weeks) to complete demographic and obstetrical history questionnaires. Blood was collected from these 101 women at the time of the glucose tolerance test (second trimester of pregnancy) and then again at delivery. Blood was collected into two 10 mL red-topped vacutainers, allowed to clot at 4 1C, and then centrifuged at 1500 rpm for 20 min. The serum was decanted into pre-cleaned Supelco glass vials for chemical analysis (Sigma-Aldrich Canada, Oakville, Ont., Canada). At delivery, UCB was collected from an umbilical vein immediately following delivery of the placenta. In the case of twins, one cord was marked with a clamp for ‘‘twin A’’ and the cord for ‘‘twin B’’ was left unclamped. The twins were subsequently identified as twin A and twin B for all future analyses. Within 1 h of collection, UCB samples were transported to the laboratory where they were allowed to clot for a minimum of 4 h at 4 1C, and were then centrifuged at 1500 rpm for 20 min. Serum was decanted into pre-cleaned Supelco glass vials for chemical analysis (Sigma-Aldrich). Post delivery, a chart review was performed to obtain mother and newborn demographic information such as age, occupation, education, weight, and height before pregnancy, obstetrical history, birth weight and gender of the new born. The study protocol was approved by the McMaster University Research Ethics Board. In addition, the determination of PFCs in maternal serum and UCB collected in this study was also approved by Health Canada’s Research Ethics Board.

2.2. Analytical methods The analytes were extracted from serum using an ion-pairing extraction procedure and analyzed by liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS) as previously described (Kubwabo et al., 2004). Analytes measured included PFOS, PFOA, PFDeA, PFHxS, PFHpA, and PFNA. The analytical laboratory successfully participated in international perfluorinated compounds intercalibration study using human serum and using LLE demonstrating the suitability of this technique.

2.3. Standards and reagents PFHpA (97%), PFOA (95%), and PFOS (potassium salt, 98%) were purchased from Fluka (Oakville, Ont., Canada). PFNA (97%) and PFDeA (98%) were purchased from Aldrich (Sigma-Aldrich). PFHxS (potassium salt, purity not specified) was provided by Miteni (Trissino, Italy). The internal standards 13C-perfluorooctanoic acid (13C-PFOA, 499%) and 13C-perfluorooctane sulfonate (13C-PFOS, 499%) were purchased from Wellington Laboratories (Guelph, Ont., Canada). Ammonium acetate (ACS grade) and sodium carbonate (99.9%) were purchased from BDH (BDH Chemicals, Toronto, Ont., Canada). Omnisolv acetonitrile and methyl-tert-butyl ether (MTBE) were obtained from EMD Chemicals (Gibbstown, NJ, USA). Tetrabutylammonium bisulfate (99%) was purchased from Fluka (Oakville, Ont., Canada). Sodium bicarbonate (ACS grade) was supplied by Fisher Scientific Company (Fair Lawn, NJ, USA). Goat serum was purchased from Aldrich (SigmaAldrich).

2.4. Sample extraction To a 15-mL polypropylene tube containing 1 mL of serum, 100 mL of 50 ng/mL of 13C-PFOA and 150 ng/mL of 13C-PFOS were added and gently mixed. One milliliter of 0.5 M tetrabutylammonium bisulfate solution (adjusted to pH 10) was added with gentle mixing to the spiked serum, followed by 2 mL of carbonate/ bicarbonate buffer, and the solution was again gently mixed. Five milliliters of MTBE was added to the spiked serum and the mixture was shaken for 30 min, using an Eberbach Reciprocating Shaker (Eberbach Corporation, Ann Arbor, MI, USA). The organic and aqueous phases were separated by centrifugation at 3500 rpm for 5 min. Four milliliters of the organic phase was transferred to a clean 15-mL polypropylene tube and the MTBE solvent was evaporated to just dryness under nitrogen at 30 1C, using an analytical nitrogen evaporator. The residue was then dissolved in 1 mL of 20% acetonitrile and was centrifuged at 10,000 rpm for 10 min. Two hundred microliters of the supernatant was transferred to a glass autosampler vial for analysis.

2. Materials and methods

3. LC–MS/MS 2.1. Study subjects and sample collection This study is nested in the ongoing large cohort study, Family Study, which started in 2004. In the Family Study, subjects were first contacted when they presented for an obstetrical ultrasound for standard prenatal care. From January 2004 until June 2005, 1058 subjects agreed to be contacted and screened to

The chromatographic separation of extracts was performed using a Finnigan Surveyor Plus HPLC System (Thermo Electron Corporation, San Jose, USA). Separation was achieved using a Zorbax 300SB-C18 column (150 mm  2.1 mm; 3 mm). An

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Opti-guard C18 column (10 mm  2.1 mm, 3 mm) was installed between the pump and the injector, in order to trap and delay PFCs originating from the solvents and the pumping system. The mobile phase consisted of 20% acetonitrile in 2 mM ammonium acetate (A) and 90% acetonitrile in 2 mM ammonium acetate (B). Solvents were degassed online, and the column temperature was maintained at 25 1C. The gradient elution started with 100% A for 1 min, followed by a 6 min linear gradient to 100% B, then 2 min hold at 100% B, and returned back to 100% A in 3 min, at a flow rate of 200 mL/min. The system was equilibrated for 3 min at the initial conditions before the next injection. Sample injection volume was 10 mL. Mass spectrometric experiments were performed using a Thermo Finnigan TSQ Quantum Ultra EMR triple quadrupole mass spectrometer (Thermo Electron Corporation). The samples were analyzed in negative ion electrospray ionization, using selective reaction monitoring mode. Xcalibur version 2 was used for data acquisition and processing. The following SRM transitions (m/z) were monitored: PFHxS (399-80.40), PFOS (499-80.40), 13C-PFOS (503-80), PFHpA (363-318.80), PFOA (413-368.80), 13C-PFOA (417-372.00), PFNA (463-418.80), and PFDeA (513468.80). Isomers were not determined separately.

3.1. Quality control and method performance

standard deviation associated with seven replicate analyses of each individual compound. Table 1 summarizes MDL, LOQ, and recovery data for PFHxS, PFOS, PFHpA, PFOA, PFNA, and PFDeA. 3.2. Statistical analysis All data were tested for normality using the Anderson–Darling normality test and the Kolmogorov Smironov test. All nonnormally distributed data were expressed as the median together with the interquartile range. Paired t-test and linear regression analysis were performed to determine the relationship between the concentrations of each analyte at different sample collection time points. Log transformation was done in non-normally distributed data and Wilcoxon statistical test was performed in non-parametrical analysis. The maternal serum levels contributed more than once in the case of twins only for the maternal vs. UCB PFOS and PFOA serum levels linear regression analysis where paired data was needed. However, during the birth weight analysis, all twin and pre-term baby data were removed from the analyses because these two conditions can affect the birth weight. To determine the effect of PFC exposure on birth weight, multiple stepwise regression analysis was performed. Covariate data including parity, gestational length, birth weight, and gender for each neonate and maternal Body Mass Index data were extracted from the study questionnaires or patient charts. Gestational age was obtained from the clinical chart. Neonates were classified as small for gestational age (SGA) if their birth weight was below the 10th percentile and large for gestational age (LGA) if the birth weight was above the 90th percentile for gestational age. Birth weight for gestational age was determined using the percentile scale published by the Canadian perinatal surveillance system (www.phac-aspc.gc.ca/rhs-ssg/bwga-pnag/ index.html). A po0.05 was considered as significant for all analyses performed. Data analysis was performed using Minitab 14 (Minitab Inc., State College, PA, USA) or SigmaStat 3.5 (Systat Software Inc., Point Richmond, CA, USA).

Solvents, extraction equipment, vials, method, and matrix blanks were checked for the presence of PFCs. Goat serum containing PFCs at concentrations below the method detection limit (MDL) was used as the surrogate matrix blank. Quality control goat serum samples fortified with PFHxS, PFOS, PFHpA, PFOA, PFNA, and PFDeA, at concentrations of 25 ng/mL were analyzed with every 5–10 samples to ensure the validity of the data generated; results within 720% of the theoretical value were considered acceptable to proceed with sample analysis. Matrix spikes (MS) and matrix spike duplicates (MSD) were prepared using human serum samples, extracted and analyzed to evaluate the matrix effect on the recovery efficiency and to measure the precision for each analyte. The background concentrations of the analytes in the sample matrix were determined in separate aliquots and the measured values in the MS and MSD were corrected for background concentrations. The calibration curves were obtained using a series of standard solutions over a concentration range from 1 to 50 ng/mL. Calibration curves demonstrated good linearity with a coefficient of correlation greater than 0.99 for each analyte. The MDL was determined according to the EPA Regulation 40 CFR part 136 (Appendix B) method. Seven replicates of goat serum spiked with the mixture of target analytes at 1 ng/mL were processed through the entire extraction procedure and analyzed. Standard deviation associated with the analysis multiplied by the Student’s t-value of 3.143 appropriate for a 99% confidence level provided the MDL. The limit of quantitation (LOQ) was calculated as 10 times the

The study population was composed of women presenting for their first obstetrical ultrasound assessment at McMaster University Medical Centre who enrolled in the Family Study and provided complete maternal and UCB samples. The majority of the study participants reported working (67.3%) in an office environment, whereas 17.8% reported that at the time of the study they were stay-at-home mothers. The mean maternal age was 32.775.2 years and the pregnancy studied was the second for 43% of study subjects (Table 2). Maternal blood samples (n ¼ 101) collected at 24–28 weeks of gestation and at the delivery as well as 105 serum samples from UCB (four twin pregnancies) collected at delivery were analyzed for PFOS, PFOA, PFHxS, PFHpA, PFNA, and PFDeA residue levels.

Table 1 Method detection limit (MDL), limit of quantification (LOQ), and percentage recovery for the target analytes PFHxS, PFOS, PFHpA, PFOA, PFNA, and PFDeA

Table 2 Characteristics of study participants

Analyte

MDL (ng/mL)

LOQ (ng/mL)

% recovery (RSD)

Variable

n

Median

Interquartile range

PFHxS PFOS PFHpA PFOA PFNA PFDeA

0.35 0.47 0.17 0.07 0.16 0.15

1.12 1.50 0.55 0.22 0.51 0.48

107 (2.57%) 106 (4.93%) 101 (0.90%) 103 (1.34%) 107 (0.84%) 122 (1.67%)

Pre-pregnancy weight (kg) Height (cm) BMI (kg/m2) No. of pregnanciesa Gestational age at delivery (weeks)

99 100 99 101 105

65.3 65 24.2 2.0 39.0

56.9–74.7 63–67 21.4–29.7 2.0–3.0 38.0–40.2

4. Results

a

Including current pregnancy.

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Table 3 Mean (7S.D.), median, and range of PFC levels (ng/mL) in maternal serum at 24–28 weeks gestation, delivery, and UCB samples

PFOA PFOS PFNA PFHxS

Maternal serum at 24–28 weeks

Maternal serum at delivery

Umbilical cord blood

n (%)

Mean7S.D.; median (range)

n (%)

Mean7S.D.; median (range)

n (%)

Mean7S.D.; median (range)

101 101 101 47

2.5471.65; 2.13 (1.46–3.14) 18.31710.95; 16.6 (10.8–22.9) 0.8670.81; 0.73 (0.58–0.96) 4.128711.43; 1.82 (1.44–3.06)

101 101 101 46

2.2471.61; 1.81 (1.33–2.64) 16.19710.43; 14.54 (9.19–20.22) 0.8070.93; 0.69 (0.542–0.87) 4.053712.30; 1.62 (1.33–2.66)

105 105 28 21

1.9471.54; 1.58 (1.09–2.37) 7.1975.73; 6.08 (3.92–9.11) 0.9471.04; 0.72 (0.61–0.80) 5.05712.92; 2.07 (1.46–2.77)

(100) (100) (100) (46.5)

(100) (100) (100) (45.5)

(100) (100) (26) (20)

The concentration of PFCs in serum was quantified by liquid chromatography–mass spectrometry/mass spectrometry as described in Section 2. PFHxS, perfluorohexane sulfonate; PFOA, perfluorooctanoate; PFNA, perfluorononanoic acid; PFHpA, perfluoroheptanoic acid; PFDeA, perfluorodecanoic acid. N, frequency of detection.

p<0.001 p<0.001

Serum concentration (ng/mL)

20

15

10

5

0 Delivery

24-28 weeks

UCB

4

p<0.001 Serum concentration (ng/mL)

PFOS and PFOA were detected in all serum samples examined whereas PFNA and PFHxS were quantified in approximately 85% and 46% of the maternal serum samples at 24–28 weeks of gestation and delivery, respectively, and less than 30% of UCB samples (Table 3). PFNA values less than the LOQ were assigned the mid-point between zero and the LOQ for statistical analysis. PFHpA and PFDeA were quantifiable in less than 5% of maternal and UCB serum samples. As the number of samples with levels of detection above the LOQ for PFHpA and PFDeA were low and the recovery percentage was higher that the expected (Table 1), no further analyses were carried out for these compounds. The serum concentrations of different PFCs varied significantly across samples collected at mid-pregnancy, delivery, and in UCB (Table 3). In particular, the concentration of PFOS was significantly higher (po0.001) in maternal serum samples collected between 24 and 28 weeks of pregnancy compared to those collected at delivery which in turn were significantly greater (po0.001) than the levels measured in UCB (Fig. 1). Furthermore, the concentration of PFOS in maternal serum samples collected between 24 and 28 weeks gestation and at delivery ranged from 10.8 to 22.9 ng/mL and 9.2 to 20.3 ng/mL, respectively, whereas those in UCB samples ranged from 3.9 to 9.2 ng/mL. PFOS levels in the UCB samples were predominately below 10 ng/mL (82.8% of samples), whereas maternal blood levels predominantly clustered between 10 and 30 ng/mL (64.3% of samples for both at 28 weeks and at delivery). The concentration of PFOA was significantly greater (po0.001) in maternal serum samples collected between 24 and 28 weeks of pregnancy compared with those collected at delivery which in turn were significantly greater (po0.001) than the levels measured in UCB (Table 3). In contrast to the other PFCs studied, the concentration of PFHxS in maternal serum at the two time points studied was not significantly different whereas the levels were significantly higher (po0.001) in the UCB compared with maternal serum collected at delivery. The relationship between PFOS and PFOA concentrations in maternal serum at the time of delivery and UCB was investigated by linear regression. The concentration of PFOS and PFOA in UCB were highly correlated with the concentration of PFOS (po0.001, r2 ¼ 0.832) and PFOA (po0.001, r2 ¼ 0.888) in maternal serum at the time of delivery, respectively. The mean ratio of PFOS levels in UCB/maternal serum at delivery was 0.4570.15 with a range of 0.18–1.09. The mean birth weight in this study was 3432.67516.8 g. Ninety percent (95/105) of the newborns were delivered at term and 78% (82/105) of the newborns had a birth weight that was considered appropriate for gestational age whereas 6% (6/105) of the neonates were determined to be SGA and 16% (17/105) were LGA. Birth weight was not affected by maternal serum PFOS, PFOA, PFHxS, or PFNA levels. The correlations between birth weight and maternal PFOS serum levels as well as birth weight and maternal PFOA serum levels were not statistically significant (Fig. 2a and b, respectively). Similarly, no

p<0.001

3

2

1

0

24-28 weeks

Delivery

UCB

Fig. 1. (a) Mean (7S.E.) PFOS and (b) median (95% CI) PFOA concentration in maternal serum collected between 24 and 28 weeks of pregnancy (n ¼ 101), at delivery (n ¼ 101), and UCB (n ¼ 101). In the case of twins, the serum PFOS and PFOA concentration for one twin A was used. Differences in the mean PFOS levels were determined by paired t-test and differences in median PFOA concentrations were performed on ranks with a po0.05 considered significant.

relationship between BW and UCB PFOS serum levels as well as BW and UCB PFOA serum levels could be found (Fig. 3a and b, respectively). Controlling potential confounding factors such as parity, gestational length, BMI, gender, and smoking status failed to change the relationship between PFOS, PFOA, PFHxS, or PFNA

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60

80 60 40 20 0

2000

2500

16 Maternal PFOA serum levels at delivery (ng/mL)

PFOS Levels in Umbilical cord blood at delivery (ng/mL)

Slope=0.000853 Std Error=0.00244 p value=0.727 R sqr= 0.00141

3000 3500 4000 Birth Weight (g)

4500

5000

12 10 8 6 4 2 0 2000

2500

3000 3500 4000 Birth Weight (g)

4500

40 30 20 10 0 2500

3000 3500 4000 Birth Weight (g)

4500

5000

14

Slope=0.000171 Std Error=0.000379 p value=0.653 R sqr= 0.00233

14

Slope=0.00186 Std Error=0.00137 p value=0.180 R sqr=0.0206

50

2000

PFOA Levels in Umbilical cord blood at delivery (ng/mL)

Maternal PFOS serum levels at delivery (ng/mL)

100

5000

Slope=0.000110 Std Error=0.000363 p value=0.763 R sqr=0.00105

12 10 8 6 4 2 0 2000

2500

3000 3500 4000 Birth Weight (g)

4500

5000

Fig. 2. (a) Linear regression analysis comparing BW vs. maternal PFOS serum levels. (b) Linear regression analysis comparing BW vs. maternal PFOA serum levels. n ¼ 89, twins and pre-term newborns were removed from analysis.

Fig. 3. (a) Linear regression analysis comparing BW vs. UCB PFOS serum levels. (b) Linear regression analysis comparing BW vs. UCB PFOA serum levels. n ¼ 89, twins and pre-term newborns were removed from analysis.

exposure and birth weight. Furthermore, maternal PFOS levels were not associated with maternal BMI and gestational length.

and PFOA were measured in 100% of the UCB samples examined. Therefore, PFOA as well as PFOS can cross the fetal–placental barrier during pregnancy resulting in exposure of the developing fetus. PFOA was detected in 100% in all three-sample sets. These results differ from those reported previously for a population of pregnant Japanese women where PFOA was detected in only 10% of the samples collected from mothers at delivery and none of the UCB samples (Inoue et al., 2004). The mean concentration of PFOS measured in maternal serum in the present study is lower than the 35.3 ng/mL reported from the Danish National Birth Cohort (Fei et al., 2007). Given the temporal decline in serum PFC levels that have been reported previously owing in part to different environmental levels and decreasing levels in the environment following the phase out of production for these chemicals, it is not surprising that the levels measured in the current study are lower than levels reported previously for samples collected between 1996 and 2002 (Fei et al., 2007). However, the PFOS concentration in UCB was higher in our study compared with those detected in a population of pregnant women from Baltimore, MD, USA (Apelberg et al., 2007a). Interestingly, in our study, UCB PFOS and PFOA concentrations were higher than those reported for the Baltimore cohort where median concentration of PFOS and PFOA were 4.9 and 1.6 ng/mL, respectively, and sample collection in our study was comparable both in sample collection time frame and methods used (Apelberg et al., 2007a). Divergent results may be due to

5. Discussion PFCs are industrial chemicals widely distributed in the environment. Results of the present study demonstrate that pregnant women are exposed to PFOA, PFOS, and PFNA. Additionally, a high proportion of the study population was also exposed to PFHxS. The concentration of PFOS and PFOA were significantly higher (po0.001) in samples collected at midpregnancy compared with delivery and UCB samples which could be explained by the physiological increase in maternal blood volume or renal clearance during pregnancy. However, the blood volume reaches a maximum increase at approximately 32–34 weeks of gestation with little change thereafter. Indeed, our results demonstrate that the timing of maternal blood sampling affects both the frequency of detection and the concentration measured. Moreover, our results reveal that PFCs levels, in general, tend to be higher in second trimester maternal samples compared with those collected at delivery or in UCB. Finally, there was no association between PFC exposure and birth weight in the present study. In the present study, PFOS, PFOA, and PFNA were quantified in 100% of the maternal serum samples studied whereas only PFOS

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differences in environmental levels of PFCs at the different study locations, the gestational week at blood collection or use of different assay techniques (Fei et al., 2007). The concentrations reported in the present study are similar to the levels reported in the NHANES 2003–2004 survey (Calafat et al., 2007) with the exception of PFHxS, which were greater in the current study. Moreover, the concentration of PHHxS was greater in the UCB compared to maternal serum at delivery. Taken together, these data indicate that the placenta provides an incomplete barrier to this compound, an observation that is harmonious with our findings. In a Canadian population with nonoccupational exposure, the PFOS level was 27.4 ng/mL (Kubwabo et al., 2004) compared to a US population (Olsen et al., 2003) with non-occupational exposure the PFOS levels were 35 ng/mL. Thus, in our population of pregnant women residing in the Hamilton area, maternal serum levels of PFOS were lower compared with non-pregnant adults with non-occupational exposure. We speculate that the concentrations of PFOS quantified in maternal serum may be lower due to increased blood volume and renal clearance that occurs in pregnancy but are most likely due to the decline in environmental levels of PFCs and differences in sample collection time frame for these studies. It should be noted that, in the present study, PFOA was found at concentrations above the LOQ in all samples studied, whereas PFOA was detected with lower frequency in maternal serum samples of pregnant Japanese women and not detected in the UCB (Inoue et al., 2004). The median of PFOA in maternal serum was 1.81 ng/mL, consistent with serum levels reported for Canadian and US populations: 3.4 and 6 ng/mL, respectively (Kubwabo et al., 2004). While PFOS and PFOA have been quantified in the serum of pregnant women previously, the present study expands the literature and provides the first evidence of developmental exposure to PFHxS and PFNA. Of the pregnant mothers in our sample, 100% were exposed to PFNA, whereas 26% of our study sample had quantifiable levels of PFHxS. In the present study, PFHxS and PFNA were quantified in UCB where the frequency of detection was one-half and one-third that of maternal samples, respectively. These data suggest that PFHxS and PFNA do not easily cross the placenta or that the placenta may provide a barrier to their transport. However, these data must be interpreted with caution as the levels measured in the maternal serum were only marginally above the level of quantification and differences could be due to analytical error. The median PFHxS level (1.8 ng/mL) measured in second trimester maternal serum samples of the present study is consistent with the level (2.4 ng/mL) measured in a US population with non-occupational exposure (Olsen et al., 2005). Serum PFHxS and PFNA levels were statistically greater in the UCB compared with maternal serum at delivery, but not different from maternal serum collected during the second trimester, suggesting that second trimester samples are representative of developmental exposure. In contrast, circulating PFOS and PFOA levels were significantly higher in maternal serum collected during the second trimester compared with delivery samples, which in turn were greater than in the UCB. Taken together, our results suggest that, of the sampling time points used in this study, the second trimester may be the most appropriate sampling window to characterize developmental exposure to PFCs. In the present study, multiple regression analysis controlling for potential confounders failed to reveal evidence that exposure to individual PFCs affected gestation length, birth weight, or birth gender, findings that are harmonious with some (Fei et al., 2007; Grice et al., 2007) but conflict with others (Apelberg et al., 2007a). Despite relatively low cord serum concentrations, small negative associations between both PFOS and PFOA concentrations and birth weight have been detected previously (Apelberg et al.,

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2007a). Similarly, an inverse association between maternal plasma PFOA levels and birth weight has recently been described (Fei et al., 2007) although neither PFOS nor PFOA levels were associated with increased risk for low birth weight in this study. Interestingly, no association between working in a PFOS-exposed job and birth weight has also been described (Apelberg et al., 2007b; Grice et al., 2007). Divergence between the findings of the present study and that from the National Danish Birth Cohort may be related to the smaller sample size and differences in general health of the participants studied in the present cohort. Unlike the National Danish Birth Cohort, the present study was conducted with study participants presenting for their routine obstetrical ultrasound and were otherwise healthy and thus may not be representative of the general population. It has been suggested that factors that affect maternal physiology may also affect both exposure and birth weight (Savitz, 2007). Furthermore, it has been proposed that PFOS and PFOA induced changes in birth weight, if replicated, may represent a modest shift in birth weight without any effect on the risk of low birth weight and thus may not indicate pathological change (Savitz, 2007). Regardless, further studies will be needed to clarify the relationship between exposure to PFCs and birth weight. In particular, the interaction between PFCs has not been addressed and potential mechanisms of action remain unknown. Moreover, the relevance of small changes in birth weight associated with exposure to environmental contaminants remains to be determined. In summary, results of the present study demonstrate that PFCs can be quantified in both maternal and UCB samples. It should be noted, however, that the concentration of PFOS and PFOA in maternal serum was significantly higher than in UCB. Consequently, of the sampling time points used, the second trimester was found to be the optimal sampling window for evaluation of developmental exposure to PFCs in the present study. Acknowledgments We are grateful for the help of our research nurses Julie Gross, Liz Helden, Sue Steele, and Mary Louise Beecroft for managing patient recruitment and sample collection for this study. Funding for the project was provided by the Canadian Institutes of Health Research and the American Chemistry Council. The sponsors had no part in the study design, data collection, analysis, interpretation, or writing of the manuscript. Salary support from the Canadian Institutes of Health Research Strategic Initiative in Research in Reproductive Health Sciences (CIHR/STIRRHS) to R.M. is gratefully acknowledged. Salary support (W.G.F.) was provided by the CIHR/Ontario Women’s Health Council. References Apelberg, B.J., Goldman, L.R., Calafat, A.M., Herbstman, J.B., Kuklenyik, Z., Heidler, J., Needham, L.L., Halden, R.U., Witter, F.R., 2007a. Determinants of fetal exposure to polyfluoroalkyl compounds in Baltimore, Maryland. Environ. Sci. Technol. 41, 3891–3897. Apelberg, B.J., Witter, F.R., Herbstman, J.B., Calafat, A.M., Halden, R.U., Needham, L.L., Goldman, L.R., 2007b. Cord serum concentrations of perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) in relation to weight and size at birth. Environ. Health Perspect. 115, 1670–1676. Butenhoff, J.L., Gaylor, D.W., Moore, J.A., Olsen, G.W., Rodricks, J., Mandel, J.H., Zobel, L.R., 2004. Characterization of risk for general population exposure to perfluorooctanoate. Regul. Toxicol. Pharm. 39, 363–380. Butenhoff, J.L., Olsen, G.W., Pfahles-Hutchens, A., 2006. The applicability of biomonitoring data for perfluorooctanesulfonate to the environmental public health continuum. Environ. Health Perspect. 114, 1776–1782. Calafat, A.M., Kuklenyik, Z., Caudill, S.P., Reidy, J.A., Needham, L.L., 2006. Perfluorochemicals in pooled serum samples from United States residents in 2001 and 2002. Environ. Sci. Technol. 40, 2128–2134.

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