Science of the Total Environment 596–597 (2017) 97–105

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Science of the Total Environment

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Concentration of perfluorinated compounds and cotinine in human foetal

organs, placenta, and maternal plasma
Linn Salto Mamsen a,⁎, Bo A.G. Jönsson b,1, Christian H. Lindh b, Rasmus H. Olesen c, Agnete Larsen c, Erik Ernst d,
Thomas W. Kelsey e, Claus Yding Andersen a
a
Laboratory of Reproductive Biology, Section 5712, The Juliane Marie Centre for Women, Children and Reproduction, University Hospital of Copenhagen, University of Copenhagen, Rigshospitalet,
2100 Copenhagen, Denmark
b
Division of Occupational and Environmental Medicine, Department of Laboratory Medicine, Lund University, 223 61 Lund, Sweden
c
Department of Biomedicine - Pharmacology, Aarhus University, 8000 Aarhus C, Denmark
d
Department of Obstetrics and Gynaecology, University Hospital of Aarhus, Skejby Sygehus, 8000 Aarhus, Denmark
e
School of Computer Science, University of St. Andrews, KY16 9SX St. Andrews, United Kingdom

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• In human foetal organs PFOS, PFOA,

PFNA, and PFUnDA were detected.
• Foetal concentrations of PFASs were sig-
nificantly and positively correlated with
foetal age.
• PFAS concentrations in foetal organs
occur at 5% to 27% of maternal plasma
concentrations.

a r t i c l e i n f o a b s t r a c t

Article history: Background: Perfluoroalkyl substances (PFASs) are bio-accumulative pollutants, and prenatal exposure to PFASs
Received 11 January 2017 is believed to impact human foetal development and may have long-term adverse health effects later in life. Ad-
Received in revised form 6 April 2017 ditionally, maternal cigarette smoking may be associated with PFAS levels. Foetal exposure has previously been
Accepted 7 April 2017 estimated from umbilical cord plasma, but the actual concentration in foetal organs has never been measured.
Available online xxxx
Objectives: The concentrations of 5 PFASs and cotinine – the primary metabolite of nicotine – were measured in
human foetuses, placentas, and maternal plasma to evaluate to what extent these compounds were transferred
Editor: D. Barcelo
from mother to foetus and to determine if the PFAS concentrations were associated with maternal cigarette smoking.
Keywords: Methods: Thirty-nine Danish women who underwent legal termination of pregnancy before gestational week 12
Prenatal exposure were included; 24 maternal blood samples were obtained together with 34 placental samples and 108 foetal or-
Perfluorinated compounds gans. PFASs and cotinine were assayed by liquid chromatography/triple quadrupole mass spectrometry.
Cigarette smoke Results: In foetal organs, the average concentrations of perfluorooctanesulphonic acid (PFOS), perfluorooctanoic
Maternal plasma acid (PFOA), perfluorononanoic acid (PFNA), perfluoroundecanoic acid (PFUnDa), and perfluorodecanoic acid
Placenta

Abbreviations: BMI, body mass index; EDTA, ethylenediamine tetraacetic acid; IS, internal standard; LC/MS/MS, liquid chromatography/triple quadrupole mass spectrometry; LOD,
limit of detection; pc, post conception; PCR, polymerase chain reaction; PFAS, perfluoroalkyl substance; PFASs, perfluoroalkyl substances; PFDA, perfluorodecanoic acid; PFNA,
perfluorononanoic acid; PFOA, perfluorooctanoate; PFOS, perfluorooctanesulphonate; PFUnDA, perfluoroundecanoic acid; TDI, tolerable daily intake.
⁎ Corresponding author at: Blegdamsvej 9, 2100 Copenhagen, Denmark.
E-mail addresses: linn.salto.mamsen@regionh.dk (L.S. Mamsen), Christian.lindh@med.lu.se (C.H. Lindh), raho@biomed.au.dk (R.H. Olesen), Al@biomed.au.dk (A. Larsen),
Erik.ernst@skejby.rm.dk (E. Ernst), twk@st-andrews.ac.uk (T.W. Kelsey), Yding@rh.regionh.dk (C.Y. Andersen).
1
Deceased.

http://dx.doi.org/10.1016/j.scitotenv.2017.04.058
0048-9697/© 2017 Elsevier B.V. All rights reserved.
98 L.S. Mamsen et al. / Science of the Total Environment 596–597 (2017) 97–105

(PFDA) were 0.6 ng/g, 0.2 ng/g, 0.1 ng/g, 0.1 ng/g, and 0.1 ng/g, respectively. A significant positive correlation was
found between the exposure duration, defined as foetal age, and foetal to maternal ratio for all five PFASs and
cotinine. Smokers presented 99 ng/g cotinine in plasma, 108 ng/g in placenta, and 61 ng/g in foetal organs. No
correlation between the maternal cotinine concentrations and PFAS concentrations was found.
Conclusions: PFASs were transferred from mother to foetus, however, with different efficiencies. The concentra-
tions of PFOS, PFOA, PFNA, PFUnDA, and PFDA in foetal organs were much lower than the maternal concentra-
tions. Furthermore, a significant correlation between the exposure duration and all of the evaluated PFASs was
found. The health-compromising concentrations of these substances during foetal development are unknown.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction
Perfluoroalkyl substances (PFASs) are slowly degradable pollutants
and belong to the group of water- and grease resistant fluorosurfactants
used in many industrial and consumer products, including outdoor
clothes, non-stick cookware, food packaging, electronics, stain-
resistant carpets and fire-fighting foams (Key et al., 1997; Jensen and
Leffers, 2008; De Solla et al., 2012). The PFASs include thousands of com-
pounds and some of these are suspected to impact foetal growth and
development and may disturb the endocrine system (de Cock et al.,
2014; Johnson et al., 2014; Bach et al., 2015). This study evaluates five
PFASs: perfluorooctanesulphonic acid (PFOS), perfluorooctanoic acid
(PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid
(PFDA) and perfluoroundecanoic acid (PFUnDA). Prenatal exposure to
PFOS and PFOA has been associated with an increased risk of congenital
cerebral palsy in Danish boys (Liew et al., 2014) and maternal exposure
to PFNA and PFDA has been associated with increased risk of pregnancy
loss (Jensen et al., 2015). In rodents, exposure to high doses of PFOS and
PFOA during pregnancy reduced postnatal survival, birth weight,
growth of the pups, and disturbed lactation (Lau et al., 2004; Olsen
et al., 2009). Further, both pre- and postnatal PFAS exposure has been
associated with hypothyroidism and significantly decreased T4 levels
in pups (Yu et al., 2009). PFASs, particularly PFOA and PFNA, may induce
synthesis of the oestrogen-responsive biomarker protein vitellogenin,
leading to an oestrogen-like activity in rainbow trout (Benninghoff
et al., 2011).
Notably, the half-lives of PFASs in rats are as short as a few days
(Kudo et al., 2002), compared to 200 days in the cynomolgus monkey
(Seacat et al., 2002), and 2.5–4.5 years in humans (Olsen et al., 2007;
Zhang et al., 2013), suggesting a large difference in the elimination ki-
netics between species, and animal models may, therefore, only reflect
the human situation to a limited extent.
Human foetal PFAS exposure has been estimated from concentra-
tions measured in maternal circulation and in umbilical cord blood in
newborns. Prenatal exposure to PFOA has been associated with a
dose-dependent decrease in birth weight (Johnson et al., 2014;
Lauritzen et al., 2017). An association between PFOS exposure and
birth weight has also been suggested, but reported results are conflicted
(Bach et al., 2015). In contrast, prenatal PFOS and PFOA exposure is sug-
gested to negatively affect thyroid function. In new-born boys, T4 levels
decreased with increasing prenatal exposure to PFOS and PFOA. Surpris-
ingly, the same study found the opposite effect in girls where the T4
level increased with increasing prenatal exposure to PFOA (de Cock
et al., 2014). PFOA may be cytotoxic to human breast cancer cells and
exert an oestrogen-like effect, although an anti-oestrogen effect was
found when cells were co-exposed to oestradiol (Henry and Fair,
2013). Information on the actual concentration of PFASs in human tis-
sues is limited, but a few reports do exist (Olsen et al., 2003a; Maestri
et al., 2006; Kärrman et al., 2010; Pérez et al., 2013). In general, lung tis-
sue is thought to accumulate the highest concentration of PFASs,
although PFOS and PFOA tend to accumulate predominantly in the
liver and bone structures, respectively (Pérez et al., 2013). In liver, the
concentration of PFOS was higher than that of PFOA (Maestri et al.,
2006; Kärrman et al., 2010; Pérez et al., 2013), which in turn was higher
than the concentrations of PFNA and PFDA (Pérez et al., 2013). One re-
port found similar concentrations of PFOA, PFNA and PFDA (Kärrman
et al., 2010). The evaluated PFASs were found in all human tissues
(Pérez et al., 2013). The tissue concentrations in human foetal organs
have not yet been reported.
Maternal cigarette smoking, together with other lifestyle parame-
ters, may impact maternal PFAS concentrations (Lauritzen et al.,
2016). Consequently, the present study included maternal smoking
and other lifestyle factors to evaluate if maternal lifestyle affects mater-
nal and foetal PFAS concentrations. Cotinine is the primary metabolite of
nicotine and is a valid biomarker used to discriminate smokers from
non-smokers (Benowitz et al., 2003). The adverse effects of maternal
smoking on the unborn child are well-known and widely described
(Mund et al., 2013). Plasma cotinine concentrations in newborns have
been reported to be approximately 60 ng/mL in children of heavy
smokers, 30 ng/mL in children from moderate smokers, and 3 ng/mL
in children from non-smokers (Ivorra et al., 2014). The actual concen-
trations in human foetuses have not previously been measured.
The present study is, to our knowledge, the first to measure the actu-
al concentrations of PFASs and cotinine in human first trimester foe-
tuses. These findings provide (i) important new knowledge on the
perfusion of PFASs and cotinine from the mother through the early pla-
cental barrier into foetal circulation and organs, (ii) an evaluation of
whether exposure duration is associated with foetal PFAS concentra-
tions, indicating a foetal accumulation over time and (iii) an indication
of whether maternal cigarette smoking affects PFAS accumulation in
the mother and foetus.
2. Materials and methods
2.1. Participating women
The participants were healthy women aged 18–46 years (mean ±
SEM, 26.4 ± 1.1), who had decided to terminate pregnancy for reasons
other than foetal abnormality. The exclusion criteria were age under
18 years, chronic diseases, or dependency on an interpreter. The project
was approved by the Research Ethics Committees of the Regional Capi-
tal (H-KF 01 258206); all participants received oral and written infor-
mation and gave their informed consent. The participants answered a
detailed questionnaire concerning lifestyle habits during pregnancy, in-
cluding smoking and drinking habits.
2.2. Human foetal tissues and maternal blood samples
Legal abortions were performed at the Department of Obstetrics and
Gynaecology, University Hospital Skejby, Denmark and at the Depart-
ment of Obstetrics and Gynaecology, Regional Hospital Randers,
Denmark, in collaboration with the Laboratory of Reproductive Biology,
Rigshospitalet, Denmark. All foetuses were morphologically normal.
Within 1 h after the surgical procedure, the foetal organs and placental
tissue were isolated, washed in sterile saline, snap frozen on dry ice, and
stored at −80 °C until analysis. Fatal organs and placental tissue proc-
essed for freezing more than 1 h after collection were not included in
the present study. Exposure duration was defined as the foetal age,
 L.S. Mamsen et al. / Science of the Total Environment 596–597 (2017) 97–105
which was measured by crown-rump lengths via ultrasound in connec-
tion with the surgical procedure. Gestational age was converted to age
post conception by subtracting two weeks.
Maternal blood samples were obtained in connection with anaes-
thesia prior to surgery, collected in ethylenediamine tetraacetic acid
(EDTA) tubes, and kept on ice until centrifugation (4000 × g for
15 min) to isolate plasma. Plasma was aliquoted into 200 μL
microinserts in 1.5 mL vials (Skandinaviska GenTec, Västra Frölunda,
Sweden) and stored at −20 °C until analysis.
2.3. Tissue processing
Foetal and placental samples were homogenized at a ratio of
three parts solution to one part tissue in 70% acetonitrile solution
containing isotopically labelled cotinine and PFASs as internal stan-
dards (IS). Homogenization was performed using a TissueLyser
(Qiagen, Copenhagen, Denmark) with a 0.5 mm stainless steel bead
for 1 min at 15 Hz, then shaken at room temperature (RT) for 30 min
followed by centrifugation at 1600 ×g. The supernatant was transferred
to 100 μL inserts fitted for 1.5 mL vials. The samples were transported at
−20 °C to the Division of Occupational and Environmental Medicine for
further analysis.
2.4. Analysis of PFASs and cotinine
The analyses of PFOS, PFOA, PFNA, PFUnDa, PFDA and cotinine in
plasma and placental and foetal tissue samples were performed by tri-
ple quadrupole mass spectrometry (LC/MS/MS) (QTRAP 5500; AB
Sciex, Foster City, CA, USA) coupled to a liquid chromatography system
(UFLCXR, Shimadzu Corporation, Kyoto, Japan) according to procedures
set out by Lindh et al. (Lindh et al., 2012). Plasma samples for calibration
standards were obtained from healthy volunteers at the laboratory in
Lund. Plasma was also used as a proxy matrix for the tissue samples.
The concentrations were quantified and samples with low amounts of
all compounds were selected for the calibration standards. Calibration
standards were prepared by adding a standard solution containing all
analysed compounds. Concentrations were determined by peak
area ratios between the analytes and the IS. The concentrations of
all compounds in the pooled serum used for preparation of standards
were quantified in each batch, and the calibration standards were
corrected for the concentration found in this sample. Additionally,
all values were corrected for the chemical blank. The limit of detection
(LOD) was defined as the concentration corresponding to three times
the standard deviation of the ratio of the peak at the same retention
time as the analysed compounds and the corresponding IS determined
in the chemical blank samples. The LODs were: 0.2 ng/g for PFOS,
0.04 ng/g for PFOA, 0.03 ng/g for PFNA, 0.05 ng/g for PFUnDa,
0.04 ng/g for PFDA and 0.5 ng/g for cotinine. However, concentrations
with values below the LOD that were obtained in the evaluation of the
mass spectrometry data were used in the statistical analysis, thus not
replaced with LOD/2 (Gyllenhammar et al., 2017). The tissue extraction
time was tested for 5, 15, 30, and 45 min. It was found that after 30 min
no additional compounds were extracted from the tissue; thus a 30 min
extraction time was subsequently used for all samples included in the
present study. To ensure that the study set-up was not contaminated
with additional compounds, blank samples (IS solution) were included
for all batches of tissue and were prepared and treated exactly the same
way as the actual samples (see Section 2.3.). The laboratory is part of the
Round Robin inter-comparison program for analyses of PFOS and PFOA
(Professor Dr. med. Hans Drexler, Institute and Out-Patient Clinic for
Occupational, Social and Environmental Medicine, University of
Erlangen-Nuremberg, Germany) with results within the program's tol-
erance limits. Further quality-assurance analysis was not made due to
limited foetal material.
99
2.5. Statistical methods
All statistical analyses were performed using the GraphPad Prism
6.07 program (GraphPad Software, Inc., CA, USA) and RStudio program
(RStudio software, Boston, Massachusetts, USA). The significance level
was defined as a probability lower than 0.05 (p = 0.05). To compare
PFAS plasma concentrations from smoking versus non-smoking
women, an unpaired parametric t-test was used when the concentra-
tions were normally distributed; when the concentrations were not
normally distributed, an unpaired non-parametric Mann-Whitney test
was used. Spearman's rank test was performed to test for correlation be-
tween PFASs and cotinine, PFASs and foetal age, and PFASs and lifestyle
parameters. Additionally, a linear regression model was used to test the
potential correlations between the PFAS concentrations and lifestyle pa-
rameters and maternal and foetal ages. A pairwise correlation model
was used to test if the lifestyle parameters correlated.
3. Results
Thirty-nine women were included in this study and a total of 34 pla-
cental samples, 108 foetal organs and 24 blood samples were obtained
from the participants (for the detailed distribution, see Fig. 1). The 108
organs were obtained from a total of 36 foetuses aged 37–68 days pc
(mean ± SEM, 52 ± 1.3) (Fig. 1, Table 1). Characteristics of the partici-
pating women are presented in Table 1.
3.1. Cotinine concentrations in plasma, placenta, and foetal organs
A plasma cut-off value of 3 ng/mL was used to discriminate between
smokers and non-smokers (Benowitz et al., 2009). Three participants
reported themselves as non-smokers but presented with cotinine con-
centrations above the cut-off value. They were grouped as smokers
and their questionnaire excluded from analysis. Women who smoked
had significantly higher cotinine concentrations (p b 0.001), as present-
ed as the mean ± SEM and range of concentrations, in plasma
(99.3 ng/g ± 26.9, 6.2–326.1 ng/g), placenta (107.8 ng/g ± 22.3, 3.8–
374.7), and foetal organs (61.1 ng/g ± 9.2, 1.1–336) than women who
did not smoke (Table 2). Cotinine was detected in all foetal organs ex-
posed to cigarette smoke (Fig. 2). Cotinine was hardly detectable in
the plasma of non-smokers (Fig. 2). Maternal plasma, placenta and foe-
tal organs obtained from the same women were available in 21 cases.
From each woman, the weighted mean cotinine concentration (ng/g)
in foetal organs was calculated and the overall mean presented
(Table 3). The weighted mean accounts for the respective masses of
the organs. The percentage was calculated as the concentration in foetal
organs or placenta in relation to the corresponding maternal plasma
(100%) (Table 3). Among smokers, a significant positive correlation
was found between foetal exposure duration and the foetal to maternal
cotinine ratio (p = 0.0168) (Fig. 3), indicating that the foetal cotinine
concentration increases with age. No association was found between
the number of cigarettes smoked and the cotinine concentration in foe-
tal tissues (p N 0.1) (data not shown).
Fig. 1. Study population, plasma and placenta samples, and included foetuses.
100 L.S. Mamsen et al. / Science of the Total Environment 596–597 (2017) 97–105

Table 1 3.3. Correlation between lifestyle and PFAS concentrations

Characteristics of the participating women (n = 39) together with foetal age.

% or mean ± SEM (range)a Maternal characteristics and life-style habits are presented in
Maternal age (years) 26.4 ± 1.1 (18–46)
Table 1. The concentrations of PFNA and PFUnDA showed a significant
Smoking during pregnancy 51.3% negative association with BMI (p = 0.0391, 95% CI: − 0.7126 to
Number of cigarettes per day 10 ± 1.0 (3–18) − 0.0118; p = 0.0085, 95% CI: − 0.7712 to − 0.1415, respectively)
Second hand smoke (h/day) 1.8 ± 0.4 (0–4) (Fig. 4), while there was no significant association between maternal
Non-smoking during pregnancy 48.7%
BMI and foetal PFAS concentrations (p N 0.1) or between PFASs and
Number of cigarettes per day 0.0
Second hand smoke (h/day) 0.0 maternal cigarette smoking, maternal age, or rural/urban residence
BMI 22.7 ± 0.6 (17.6–32.4) (p N 0.1).
Exercise (h/week) 1.1 ± 0.3 (0–5) Further, a positive correlation (over 50%) was found between first-
Soft drinks (dl/day) 0.8 ± 0.3 (0–5) hand smoking and second-hand smoking (r = 0.65, p b 0.0001),
Diet soft drinks (dl/day) 0.5 ± 0.2 (0–5)
Coffee (cups/day) 1.0 ± 0.2 (0–4)
second-hand smoking was also positively correlated with the father's
Tea (cups/day) 0.7 ± 0.2 (0–3.5) smoking habits (r = 0.50, p = 0.004) (Supp. Table 1). Alcohol consump-
Foetal age (days post conception) 52 ± 1.3 (37–68) tion correlated positively with soft drink consumption (r = 0.57, p =
a
Numbers may not sum total number of participants due to missing data in a few cases. 0.0002) (Supp. Table 1). Use of over the counter medication correlated
Three participants have been drinking alcohol during the present pregnancy on average positively with use of prescription medicine (r = 0.56, p = 0.0003)
0.5–1.4 drinks per week. Alcohol consumption did not correlate with any of the measured (Supp. Table 1). There was no significant correlation between any
lifestyle parameters (Supp. Table 1). other pairs of measured parameters (Supp. Table 1).

3.2. PFAS concentrations in maternal plasma, placenta, and foetal organs 4. Discussion

In the 21 cases where maternal plasma, placenta and foetal organs The evaluated five PFASs together with cotinine were detected
were obtained from the same women, a significant positive correlation in first trimester foetal organs. Further, the concentrations of these
between the foetal exposure duration and foetal to maternal ratio of all compounds increased with exposure duration, suggesting that these
five PFASs was found: PFOS p = 0.0008, 95% CI [0.33–0.86]; PFOA p = compounds may accumulate in the foetus during gestation. The concen-
0.0246, 95% CI [0.06–0.77], PFNA p = 0.0106, 95% CI [0.13–0.80]; trations of the five evaluated PFASs were significantly higher in mater-
PFUnDA p = 0.0197, 95% CI [0.08–0.77]; PFDA p = 0.0390, 95% CI nal circulation than in foetal organs. Further, a negative correlation
[0.01–0.75] (Fig. 3). This indicates that the PFAS concentrations in foetal between maternal BMI and the plasma concentrations of PFNA and
organs increase with foetal age. Further, a significant linear correlation PFUnDA was found supporting previous reports (Lauritzen et al.,
between PFOS (p = 0.001, R2 = 0.438), PFNA (p = 0.024, R2 = 0.240), 2016). Collectively, these data provide information on PFAS concentra-
and PFUnDA (p = 0.047, R2 = 0.192) and foetal age was found (Fig. 3). tions in foetal organs, placenta, and maternal plasma in the first trimes-
In maternal plasma, PFOS was detected at the highest concentrations ter of pregnancy.
followed by PFOA, PFNA, PFUnDA, and PFDA (Table 2). In maternal plas-
ma, the respective mean concentrations were: 8.2, 2.1, 1.1, 0.5, and
4.1. Exposure duration correlated positively with foetal concentrations of all
0.3 ng/g. In placenta, the respective mean concentrations were reduced
five PFASs
to: 1.3, 0.2, 0.1, 0.1, and 0.1. In foetal organs the respective mean concen-
trations were: 0.6, 0.2, 0.1, 0.1, and 0.1 (Table 2). All five measured
Exposure duration correlated positively with the foetal to maternal
PFASs were present in the evaluated foetal organs. No association be-
concentration ratio of the five PFASs, indicating that the evaluated
tween maternal plasma cotinine concentrations and plasma PFASs
PFASs accumulate in the foetus with age and may continue to accumu-
was found (p N 0.1).
late during pregnancy. It has been shown that phthalates and PFOS ac-
The evaluated PFASs in placentas and foetal organs were greatly re-
cumulate in human amniotic fluid during the second trimester with
duced compared to maternal plasma. The relative concentrations of
an approximate 10% increase per gestational week (Jensen et al.,
PFOS, PFOA, PFNA, and PFUnDA in placentas were 11–15% of the con-
2012), supporting the present findings. Given that the development of
centration found in maternal plasma and were further reduced to 5–
keratinized epidermal skin is first seen in human foetuses at week 22
13% in foetal organs (Table 3). PFDA was detected at relatively higher
pc (Hardman et al., 1999), prior to this age the foetal skin is permeable
concentration in the placenta (43%) and foetal organs (27%) compared
and chemicals from the amniotic fluid may be absorbed by this route.
to the concentrations of other PFASs (Table 3). PFOS in maternal plasma
Hence, in early pregnancy the foetus may be exposed to PFASs from
was significantly higher compared to the other measured PFASs. Inter-
both placental blood and from the amniotic fluid.
estingly, PFOS had the lowest relative concentration in foetal organs
(5%) (Table 3).
4.2. PFASs in human foetal organs in relation to maternal concentrations

Table 2 The five PFASs were detected in all evaluated foetal organs indicat-
Mean PFAS and cotinine concentrations in maternal plasma, placenta, and foetal organs. ing that foetuses are systemically exposed to these compounds. Al-
Cotinine was evaluated in the cases where the mother smoked (n = 14). though the majority of organogenesis is completed at approximately
PFAS Mean PFAS concentrations (ng/g)
six to eight weeks (Zhou et al., 2012), the organs are still at an early im-
(range ng/g) mature stage, and organ-specific accumulation is not expected at these
early ages. In adults, PFOA and PFOS accumulate in liver and bone struc-
Maternal plasma Placenta Foetal organs
(n = 24) (n = 34) (n = 108) tures (Pérez et al., 2013), and all five PFASs evaluated in the present
study have been detected in adult liver tissue (Kärrman et al., 2010),
PFOS 8.2 (2.5–16.7) 1.3 (0.3–3.1) 0.6 (bLOD—2.6)
PFOA 2.1 (0.6–8.0) 0.23 (0.04–0.45) 0.17 (bLOD—1.71) suggesting that the tissue accumulation found in foetuses persists into
PFNA 1.1 (0.4–2.9) 0.14 (0.04–0.29) 0.09 (bLOD—0.33) adulthood.
PFUnDA 0.5 (0.2–1.7) 0.06 (bLOD—0.13) 0.06 (bLOD—0.29) The placental concentrations of PFOS, PFOA, PFNA, and PFUnDA
PFDA 0.3 (0.1–0.9) 0.10 (bLOD—0.25) 0.08 (bLOD—0.35) were 11–15% of the concentrations found in the maternal circulation.
Cotinine 99 (6–326) 108 (4–375) 61 (1–336)
These concentrations were even lower in the foetal organs, dropping
 L.S. Mamsen et al. / Science of the Total Environment 596–597 (2017) 97–105 101

Fig. 2. Concentrations of cotinine and PFASs in maternal plasma (A) and in placentas and different foetal organs (B). Error bars represent min. and max. values. Maternal plasma cotinine
(n = 14); cotinine NS: non-smokers; S: smokers; CT: connective tissue. *Cotinine concentrations in foetuses exposed to maternal cigarette smoke (n = 21).

to 5–13% of maternal concentrations, indicating that foetal exposure to
these compounds was 7–20 times lower than maternal exposure. Of the
PFASs examined, PFDA was found at the lowest concentrations in ma-
ternal plasma, but showed the relative highest concentrations in both
placenta (43%) and foetal tissues (27%) compared to maternal plasma.
However, the measurements are close to the LOD, and should be
interpreted with caution. These data suggest that these five PFASs accu-
mulate in foetal tissue with different efficiencies. The differences in foe-
tal uptake may be due to differences in placental clearance or exposure
duration.
102 L.S. Mamsen et al. / Science of the Total Environment 596–597 (2017) 97–105

Table 3
In 21 cases all three classes of samples (maternal plasma, placenta, and foetal organs) were obtained. In each case the mean concentration (ng/g) of PFASs in all foetal organs were calcu-
lated and the overall mean presented. The percentages were calculated as the concentrations in foetal organs and placentas respectively in relation to the corresponding maternal plasma
(100%). Cotinine was evaluated in the cases where the mother smoked (n = 13) and presented as for the PFASs.

PFAS Mean PFAS concentrations (ng/g)

(range ng/g; mean concentration in % in relation to maternal plasma)

Maternal plasma Placenta Foetal organs

(n = 21) (n = 21) (n = 68)

PFOS 8.2 (2.5–16.7; 100%) 1.0 (0.3–2.6; 14%) 0.3 (bLOD—0.7; 5%)
PFOA 1.89 (0.55–4.47; 100%) 0.18 (0.04–0.44; 11%) 0.10 (bLOD—0.28; 7%)
PFNA 0.98 (0.41–1.64; 100%) 0.12 (0.04–0.20; 14%) 0.07 (bLOD—0.15; 9%)
PFUnDA 0.41 (0.19–1.07; 100%) 0.06 (bLOD—0.10; 15%) 0.05 (bLOD—0.12; 13%)
PFDA 0.28 (0.06–0.94; 100%) 0.09 (bLOD—0.19; 43%) 0.05 (bLOD—0.14; 27%)
Cotinine 98 (6–326; 100%) 79 (4–207; 106%) 43 (4–150; 52%)

Previously, foetal concentrations of PFOS, PFOA, and PFNA have been
estimated from the concentrations in the umbilical cord blood (Monroy
et al., 2008; de Cock et al., 2014; Manzano-Salgado et al., 2015). Our
results help qualify estimations of foetal exposure by providing actual
measured concentrations. The present study measured actual foetal
concentrations of five PFASs that were 3–12 times lower than concen-
trations previously estimated from umbilical cord blood, which may
either reflect that the concentrations of these PFASs increase in the foetus
during gestation (the present study did find a significantly positive
correlation between PFASs and exposure duration), or indicate that
the actual foetal exposure may be less than previously anticipated.
4.3. PFAS concentrations in maternal plasma
In maternal plasma, PFOS was present at the highest concentrations
followed by PFOA, PFNA, PFUnDa, and PFDA. In foetal organs, PFOS was
also present at the highest concentrations followed by PFOA, PFNA,
PFUnDa, and PFDA, reflecting the maternal pattern. The maternal PFAS

Fig. 3. A significant positive correlation between exposure duration (foetal age) and the foetal to maternal ratio of all five PFASs and cotinine were found, indicating that the fraction of the
maternal PFAS level present in the foetus increases with age. Additionally, significant linear correlations were found for PFOS, PFNA, and PFUnDA.
 L.S. Mamsen et al. / Science of the Total Environment 596–597 (2017) 97–105
Fig. 4. Concentrations of PFNA and PFUnDA in maternal plasma in relation to maternal body mass index (BMI). Solid lines are unadjusted regression lines, dotted lines are error bars. A
significant positive correlation between exposure duration and PFOS and PFNA were found.
concentrations support previous measurements from pregnant women
(Monroy et al., 2008; Okada et al., 2013, 2014; Cho et al., 2015;
Manzano-Salgado et al., 2015; Papadopoulou et al., 2015; Callan et al.,
2016; Wang et al., 2016). PFUnDA has been previously detected both
at higher and lower concentrations (Okada et al., 2013; Callan et al.,
2016; Wang et al., 2016). The literature is not conclusive regarding the
concentrations of PFOS and PFOA in plasma. In a study of 1400
women, plasma concentrations of PFOS and PFOA were 4 and 8 times
higher than the present study (Fei et al., 2007); whereas Hanssen and
colleagues reported 5 and 1.5 times the concentrations of the present
study (Hanssen et al., 2010). These differences may be explained by var-
iations in local exposure or by the year in which the samples were taken.
During the last decade, changing PFAS levels have been observed
(Glynn et al., 2012; Olsen et al., 2012).
A negative correlation was found between maternal BMI and mater-
nal PFNA and PFUnDA concentrations, although maternal BMI did not
correlate with foetal PFNA and PFUnDA concentrations, suggesting
that maternal BMI does not affect the PFNA and PFUnDA concentrations
in the foetus.
Maternal plasma concentrations of all five PFASs were higher in
women who smoked than in women who did not smoke, but this differ-
ence was not significant. Maternal cigarette smoking has been associat-
ed with higher maternal PFAS concentrations, although results are
conflicting (Cho et al., 2015; Lauritzen et al., 2016, 2017) and the effect
of maternal cigarette smoke may be questionable. Nevertheless, this
tendency was not reflected in placenta and foetal tissues, suggesting
that maternal smoking does not affect the concentrations of the evaluat-
ed PFASs in the foetus. Participating women were recruited from both
rural and urban areas, and no association was found between the loca-
tion of residence and plasma concentrations of PFAS, suggesting that ex-
posure to pollutants from urban living does not impact the evaluated
PFAS concentrations in pregnant women in Denmark.
4.4. Health compromising PFAS concentrations
Dietary intake has been suggested as the primary source of PFAS ex-
posure and 50% of the total PFAS intake is estimated to comes from PFOS
and PFOA (Domingo, 2012; Lauritzen et al., 2016) with the largest con-
tribution coming from meat, animal fat, and snacks (Halldorsson et al.,
2008). Additionally, PFDA and PFNA have been found in commercial
baby food (Domingo, 2012). The EFSA CONTAM panel has established
a tolerable daily intake (TDI) for PFOS and PFOA of 150 ng/kg/day and
15 μg/kg/day, respectively, based on the lowest non-observed adverse
effect level identified in animal exposure studies. The estimated intakes
of PFOS and PFOA in the present study were 2.7 and 2.1, respectively,
which is well below advised TDI. In the Swedish adult population, the
mean dietary PFAS exposure has been estimated to be 0.6–8.5
ng/kg/day (Domingo, 2012), which is also well below the estimated
103
health compromising levels and suggests a minimal health risk for Scan-
dinavian adults. PFOS and PFOA plasma concentrations in Swedish
women were 10.2 ng/mL and 2.9 ng/mL, respectively (Axmon et al.,
2014), which was slightly higher than the maternal plasma concentra-
tions in the present study. This can be interpreted to mean that neither
Swedish nor Danish adult women are exposed to health compromising
levels of these substances, given that uptake from sources other than
diet is insignificant. However, the health compromising levels of PFASs
during foetal life are, to our knowledge, not defined. Maternal plasma
PFOA concentrations above 3.9 ng/mL have been significantly associat-
ed with reduced birth weight (Fei et al., 2007), suggesting that health
compromising concentrations may be lower in the foetus than in adults.
4.5. PFASs and lifestyle
Of the evaluated self-reported lifestyle parameters, only smoking,
second-hand smoke, and the father's smoking habits were correlated,
indicating that smokers are more likely to be surrounded by other
smokers, and may therefore expose their foetus to more cigarette
smoke than their personal cigarette consumption indicates. In all
other aspects, the lifestyles of the participants were similar. The varia-
tion in PFAS concentrations between foetuses could not be explained
by the monitored lifestyle parameters: coffee, tea, soft drink, or diet
soft drink consumption, smoking habits or exercise. Significant positive
correlations were found between the measured foetal PFASs and foetal
exposure duration, suggesting that exposure duration may be the most
likely explanation for the variation between foetuses. Differences in pla-
cental clearance may also impact foetal PFAS concentrations.
4.6. Cotinine concentration in maternal plasma, placenta, and foetal organs
Among smokers, cotinine concentrations were significantly higher
than PFAS concentrations in foetal organs. Cotinine levels in the placen-
ta were 106% of the levels found in maternal plasma. The concentrations
in smoke-exposed foetal tissues were reduced to 52% of the concentra-
tion found in maternal plasma, indicating that cotinine readily crosses
the placenta and reaches the foetal organs. The mean cotinine concen-
tration in smoke-exposed foetuses was 61.1 ng/g, which was very
close to the concentration previously measured in serum from new-
borns of heavy smokers (59.1 ng/mL) (Ivorra et al., 2014). This may in-
dicate that, even though high concentrations of cotinine reach the
foetus, its clearance is rapid (half-life: 16 h, total clearance: 48 h
(Benowitz and Jacob, 1994)) compared to PFASs (≈4 years). However,
the ratio of foetal to maternal cotinine did increase with exposure dura-
tion, indicating that the foetal cotinine concentrations increase with age.
The highest cotinine concentration was detected in the foetal liver,
which was close to the same concentration found in the placenta, indi-
cating that cotinine may accumulate in the liver. The lowest cotinine
104 L.S. Mamsen et al. / Science of the Total Environment 596–597 (2017) 97–105
concentration was found in the ribs, likely reflecting that cotinine
reaches more perfused organs more readily than bony structures.
4.7. Limitations
The optimal tissue extraction time was tested and no increases in
compound concentrations were found after a 30 min extraction time.
A direct measurement of PFAS concentrations in foetal blood cannot
be performed, as it is not possible to obtain blood samples from foetuses
at these early developmental stages. Foetal organs were used instead,
and their PFAS content compared to concentrations in the maternal cir-
culation. Previously, PFOS has been measured in serum and liver tissue,
and a mean liver to serum ratio was reported to be 1.3 to 1 in adults
(34–70 years) (Olsen et al., 2003b). However, PFOS has been found at
higher concentrations in the liver compared to other organs (Pérez
et al., 2013).
5. Conclusions
In conclusion, the present study provides actual measurements of
five PFASs and cotinine concentrations in human foetuses, together
with comparisons to placental and maternal plasma samples. Foetal
age correlated positively with all evaluated PFASs, suggesting foetal ac-
cumulation over time. In maternal plasma, PFOS concentrations were
N4 times higher than PFOA concentrations, which were twice as high
as PFDA, PFUnDa and PFDA concentrations. The evaluated PFAS concen-
trations were reduced in the placenta, and further reduced in the foetus.
Taken together, the present data suggest that PFAS concentrations in
first trimester foetuses occur at 5% to 27% of maternal plasma concentra-
tions, and foetal concentrations increase with foetal age, although the
health compromising levels of these substances during foetal life is
unknown.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.scitotenv.2017.04.058.
Ethical approval
‘The Scientific Ethical Committee for the Capital Region’ [KF (01)
258206] and [KF (01) 170/99] has given their approval for this study.
All participants gave informed consent before taking part and have
given written consent that their data can be included in publications.
Authors' roles
L.S.M. was responsible for writing the paper, collected the human
foetuses and placenta samples, prepared samples for chemical analysis,
did the statistics, and interpreted the data. B.A.G.J. and C.H.L. were re-
sponsible for the design of the chemical analysis and interpreted the
data. R.O. did the scanning during the evacuation procedure. A.L.
assisted with collecting foetal material and placenta samples. E.E. was
responsible for the surgical procedure for terminating pregnancies,
consulted the participating women prior to the operation for comple-
tion of questionnaires, and obtained the blood samples. T.W.K.
interpreted the data, assisted in the statistical correlation analysis, and
assisted in writing the paper. C.Y.A. interpreted the data, assisted in
writing the paper and was responsible for the study design. All authors
approved the last version of the manuscript.
Financial interest declaration
The financial supporters had no role in the study design, collection
and analysis of data, data interpretation or in writing the report. The
corresponding author had full access to all data presented in the study
and had the final responsibility for the decision to submit this paper
for publication.
Conflict of interest
None declared.
Acknowledgements/funding
We thank The Research Pools of Rigshospitalet and the EU Interre-
gional Project ReproUnion for funding this study.
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