Human biomonitoring to evaluate exposure to toxic and essential trace elements during pregnancy. Part A. concentrations in maternal blood, urine and cord blood.

Accepted Manuscript Human biomonitoring to evaluate exposure to toxic and essential trace elements during pregnancy. Part A. concentrations in maternal blood, urine and cord blood. Beatrice Bocca, Flavia Ruggieri, Anna Pino, Joaquim Rovira, Gemma Calamandrei, María Ángeles Martínez, José L. Domingo, Alessandro Alimonti, Marta Schuhmacher PII:

S0013-9351(19)30396-2

DOI:

https://doi.org/10.1016/j.envres.2019.108599

Article Number: 108599 Reference:

YENRS 108599

To appear in:

Environmental Research

Received Date: 9 May 2019 Revised Date:

19 July 2019

Accepted Date: 19 July 2019

Please cite this article as: Bocca, B., Ruggieri, F., Pino, A., Rovira, J., Calamandrei, G., Martínez, Marí.Á., Domingo, José.L., Alimonti, A., Schuhmacher, M., Human biomonitoring to evaluate exposure to toxic and essential trace elements during pregnancy. Part A. concentrations in maternal blood, urine and cord blood., Environmental Research (2019), doi: https://doi.org/10.1016/j.envres.2019.108599. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Human biomonitoring to evaluate exposure to toxic and essential trace elements during pregnancy. Part A. Concentrations in maternal blood, urine and cord blood. Beatrice Boccaa*, Flavia Ruggieria, Anna Pinoa, Joaquim Rovirab,c, Gemma Calamandreia, María Ángeles Martínezc, José L. Domingob, Alessandro Alimontia, Marta Schuhmacherb,c a

Istituto Superiore di Sanità, Rome, Italy Laboratory of Toxicology and Environmental Health, School of Medicine, IISPV, Universitat Rovira i Virgili, Sant Llorenç 21, 43201 Reus, Catalonia, Spain c Environmental Engineering Laboratory, Departament d'Enginyeria Quimica, Universitat Rovira i Virgili, Av. Països Catalans 26, 43007 Tarragona, Catalonia, Spain

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b

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*Corresponding author

Abstract

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Exposures to toxic elements or deficiencies of essential elements during pregnancy may be associated to various birth complications or even diseases in early life. The aim of this paper was to assess the concentrations of selected toxic (As, Cd, Cr, Hg, Ni, Pb) and essential trace elements (Co, Cu, Mn, Se and Zn) in blood and urine samples of delivering women at different periods of gestation and cord blood, as well as to evaluate the placental permeability for these elements. A total of 53 women participating in the HEALS-EXHES study were enrolled. In particular, 48 blood samples from 1st trimester of pregnancy, 40 blood samples at delivery, and 31 cord blood at delivery were collected. Moreover, mothers’ urine were sampled at the 1st (53 samples), 2nd

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(53 samples) and 3rd trimester (49 samples) of pregnancy. Results showed that Hg and Mn levels in cord blood were about 2.0 times higher than in maternal blood, suggesting that these elements may be transferred from mother to fetus. The cord blood levels of As and Pb were lower (ca. the 65%) than those in maternal blood, showing that the placenta modulates the rate of transfer for these elements. Essential elements as Cu

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and Zn showed significantly lower levels in cord than in maternal blood suggesting that the transplacental transfer of these nutrients was very limited. In addition, correlation between paired maternal and cord blood

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samples for As, Hg and Pb was statistically significant indicating that the fetal body burden may reflect the maternal exposure. Cadmium, Co, Cr, Ni and Se levels did not show significant correlations between maternal and cord blood. Maternal urinary concentrations of trace elements, including As, Cr, Cu, Hg, Se and Zn decreased along pregnancy, which may cause variations in fetal exposure. The levels of toxic and essential elements in maternal blood and urine, as well as in cord blood, were for most elements at the lower end of the ranges found in the scientific literature not being of special concern for pregnant women and the unborn.

Keywords: Biomonitoring, Elements, Exposure, Mother/child pairs

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1. Introduction Pregnant women and their fetuses are more vulnerable to adverse effects from exposure to environmental toxic substances (Zheng et al., 2014). Exposure to contaminants during pregnancy may extend negative impacts in early childhood and may affect the later risk of diseases (Gluckman et al., 2008). The placenta establishes an interface between maternal circulation and the fetus by regulating the transport of

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nutrients and acting as a filter reducing the passage of potentially toxic substances to the fetus.

Nowadays, it has been widely demonstrated that several toxicants can totally or partially cross the placental barrier (Needham et al., 2011). Particularly, arsenic (As), cadmium (Cd), mercury (Hg) and lead (Pb) could extend the health risk to the fetus even at low levels through transplacental circulation (Caserta et

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al., 2013; Chen et al., 2014). The associations between prenatal exposure to trace elements of toxicological interest and physiological changes or diseases during pregnancy have been evaluated in recent decades. Various studies have shown that prenatal As, Cd and Pb exposures were inversely associated with

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anthropometric parameters of newborns, such as birth weight, birth length, and head circumference (AlSaleh et al., 2014; Sun et al., 2014; Shirai et al., 2010). Also, trace elements which are basically essential nutrients – such as copper (Cu), manganese (Mn), selenium (Se) and zinc (Zn) - and cofactors of the major antioxidant enzymes (including Cu/Zn and Mn-SODs and selenoproteins) were involved, when their supply is limited, in adverse pregnancy outcomes (Mistry and Williams, 2011). Moreover, following environmental or occupational exposure, the interaction of toxic elements as Cd and Pb with essential elements such as Cu,

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Zn and Se plays an integral role in metal toxicity. The final effect of elements may be increased or lessened by the metabolic status of these essential elements, which can be perturbed either through inborn genetic defects, or upon habits as smoking (Zhang et al., 2004). Therefore, ensuring an adequate intake of elements is essential to prevent deficiencies and meeting in

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utero accretion rates, while avoiding toxicity from excess exposure to toxic elements is also an issue for reducing birth morbidity and consequences in later life. The main goals of this study were: 1) to assess the levels of toxic (As, Cd, Cr, Hg, Ni, Pb) and essential (Co, Cu, Mn, Se, Zn) trace elements during pregnancy

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by analyzing blood and urine samples of childbearing women at 1st, 2nd and 3rd trimesters of pregnancy, and 2) to assess placental permeability to toxic and essential trace elements by measuring levels in paired maternal-cord blood samples. Results were evaluated taking also into account current thresholds and reference values for elements available so far. The relationships between elements concentrations and lifestyle, diet and health factors will be also examined via exposure-response relationships and presented in a forthcoming paper.

2. Methods 2.1. Participants

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ACCEPTED MANUSCRIPT For this study, 53 pregnant women participating at the HEALS-EXHES study, conducted in Reus (Tarragona, Spain), were involved in the current investigation and samples were obtained at “Sant Joan” University Hospital during a year, from March 2016 to March 2017. Women with histories of medical complications, fetal abnormalities, chromosomal malformations in the current pregnancy, or those with a major chronic illness were excluded from the present study. Maternal blood in lithium-heparin tubes metal free containers (vacutainer BD, Becton Dickinson Labware, Franklin Lakes, NJ, USA) was collected at the

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1st trimester of pregnancy (48 samples) and at delivery (40 samples), while cord blood samples were obtained immediately after delivery (31 samples). Maternal spot urine samples (ca. 5 mL) were collected at the 1st (53 samples), 2nd (53 samples) and 3rd trimester (49 samples) of pregnancy. Blood and urine samples were firstly stored at -20 °C and then shipped frozen to the Department of Environment and Health (unit of Human

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Exposure to Environmental Contaminants) of the Istituto Superiore di Sanità (Rome) for elemental analysis. During all pregnancy, from first to third trimester, women had undergone a face-to-face questionnaire interviews. The questionnaire covered some background personal data, diet and life-style characteristics. The

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present study was carried out in accordance with the protocol approved by the institutional review board of the Ethical Committee of Clinical Research of the Sant Joan Hospital. All mothers were voluntary. A written informed consent from each participant was obtained. 2.2 Sample preparation

One mL of blood was added to 2 mL of ultrapure HNO3 (Normatom, Leuven, Belgium) in 15 mL

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Falcon polystyrene plastic tubes and digested at 80 °C on a hot plate (Mod Block CPI International, Santa Rosa, CA, USA) for 3 hours, being then filled up to a final volume of 15 mL with high-purity deionized water. Urine samples were diluted 1:5 (v/v) with high-purity deionized water (Barnstead EASY-Pure II, Dubuque, IA, USA) before metal quantification. Samples were mixed with internal standards (69Ga and 115In)

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and analyzed by sector field inductively coupled plasma‐mass spectrometry (SF-ICP-MS, ThermoFischer, 114

Cd,

Cr,

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Cu,

As and

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Bremen, Germany) working at low resolution mode (LR, m/∆m =300) for not-interfered elements as 55

Hg,

Mn,

208 60

Pb; at medium resolution mode (MR, m/∆m=4000) for interfered ones such as 64

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202

Ni, and

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Zn; and at high resolution mode (HR, m/∆m=10,000) for the interfered

Co, 75

52

Se,

following previously reported methods (Bocca et al., 2016; Ruggieri et al., 2016). The analytical methods were fully developed and validated following the international standard ISO/IEC 17025. The method detection limit (MDL) was calculated as 3 times the standard deviation (SD) from 10 consecutive measurements of a representative sample. The MDL values in blood were the following (in µg/L): 0.3 for As; 0.2 for Cd; 0.01 for Co; 0.04 for Cr; 0.8 for Cu; 0.5 for Hg, Mn, Pb and Se; 0.3 for Ni; and 1.3 for Zn. The MDL values in urine were as follows (in µg/L): 0.5 for As; 0.1 for Cd, Co and Pb; 0.05 for Cr and Mn; 0.3 for Cu and Hg; 0.2 for Ni; 2.0 for Se and Zn. Analytical quality was assured by the repeated analysis of Certified Reference Materials (CRMs) Seronorm Trace Elements Blood and Urine (SERO AS, Billingstad, Norway). The measured values of the CRMs for all elements were between 85% and 103% in blood and

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ACCEPTED MANUSCRIPT ranged from 90% to 120% in urine. Precision for spiked blood and urine samples with elements were better than 10% in both matrices. 2.3 Data treatment Due to the not-normal distribution of data, concentrations of elements in maternal blood at the 1st trimester and at delivery, as well as those in cord blood are shown as 50th percentile (P50) and 95th percentile

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(P95) in Table 2. Regarding urine, metal concentrations were adjusted for sample creatinine, measured by the Jaffé’s method using a Cobas autoanalyser. Sample replicates and standards were analyzed. Correction of urinary metal content by creatinine was generally considered a valid way to normalized urine for individual hydration in order to better compare metal concentrations between groups, or among subjects (Leese et al.,

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2017). Anyway, some studies recognized that urinary creatinine was affected by age, sex, diet - particularly protein intake - and specific conditions like pregnancy. Consequently, adjustment by creatinine may provide higher values for urinary element concentrations than did the corresponding data expressed as µg/L (Barr et

creatinine-adjusted levels (Table 3).

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al., 2005). For this reason, we reported both values of urinary elements, for non-creatinine adjusted and

Wilcoxon signed rank test for blood samples and Friedman's two-way analysis of variance by ranks for urine samples were used to assess significant differences between two or more measurements. Spearman correlations were used to examine the relationships between maternal blood at delivery and cord blood metal concentrations. All statistical analyses were carried out with the statistical package IBM SPSS Statistic 24.

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Differences were considered as significant at p<0.05.

3. Results

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Socio-demographic and lifestyle characteristics of the 53 pregnant women are reported in Table 1. The mean age of the mothers was 34 years, ranging from 26 to 45 years. Most women were at the first (33%) and

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second (48%) pregnancy, with only few at the third or more gestations (19%). The mean body mass index (BMI) was 25.4, 27.3 and 29.4 kg/m2 in the 1st, 2nd and 3rd trimester, respectively. Most women had university degree (47%), while a lower percentage had a high (32%) or primary (21%) school degree. Mothers working during pregnancy represented 57% of the sample. In the 12 months before pregnancy, 21% of mothers smoked and 24% consumed alcohol. During pregnancy, the majority of mothers quit smoking (87%) and drinking (85%). Table 2 summarizes the median (P50), 95th percentile (P95) and minimum and maximum concentrations for each element in maternal blood (1st trimester and at delivery) and in cord blood. In turn, Table 3 shows the element levels - non-creatinine and creatinine-adjusted - in maternal urine at the three stages of pregnancy. All elements were above their respective MDL in all samples in both matrices. Figures 1a and 1b depict longitudinal patterns of toxic and essential elements, respectively, in maternal blood at 1st trimester and delivery and in cord blood of paired samples. The longitudinal trends for toxic and 4

ACCEPTED MANUSCRIPT essential elements in maternal urine at the three trimesters of pregnancy in paired samples are reported in Figure 2a and 2b, respectively. Figure 3a and 3b show correlations for toxic and essential elements, respectively, in paired samples of maternal blood at delivery and cord blood. With respect to As, the median concentrations in cord blood (1.2 µg/L) resulted significantly lower (p=0.045) than those in blood of mothers at delivery (1.8 µg/L) (Figure 1a). In urine, lower median As

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concentration was found in mothers at the late gestation period than at 1st and 2nd trimesters (Figure 2a). A strong significant correlation between As in paired samples of maternal (at delivery) and cord blood was found (ρ=0.8; p< 0.001) (Figure 3a).

Cadmium levels in maternal and cord blood samples were not statistically different, even if a mild

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decrease was observed in cord blood than in mothers’ blood (Figure 1a); maternal urine Cd levels adjusted for creatinine resulted similar at 3rd trimester (0.7 µg/g) and at 1st or 2nd trimester (0.9 µg/g, in both periods) (Figure 2a).

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The median Hg concentration in cord blood of 2.8 µg/L, resulted higher (p<0.001) than that at maternal blood at delivery (1.8 µg/L) (Figure 1a). The correlation between paired cord and maternal blood was highly significant (ρ=0.9; p<0.001) as shown in Figure 3a. In urine of mothers, Hg levels (both non-creatinine adjusted and creatinine-adjusted) significantly decreased (p<0.001) from 1st and 2nd to 3rd trimester of pregnancy (Table 3 and Figure 2a).

The median Cr and Ni levels were not significantly different in cord and maternal blood, even if few

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mothers presented higher levels of both elements in cord blood (Figure 1a). There were observable decrements of Cr and Ni levels in urine during the three trimesters of pregnancy (Table 3 and Figure 2a). No significant correlations between paired cord and maternal blood samples for Cr and Ni concentrations were detected (Figure 3a).

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In relation to Pb levels in maternal blood, they were higher at delivery than at 1st trimester of pregnancy (12 vs. 10 µg/L; p=0.002); and significantly lower in cord blood (7.9 µg/L) (Table 2 and Figure 1a). Correlation cord/maternal blood for Pb was highly significant (ρ=0.9; p<0.001) as shown in Figure 3a.

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Urinary Pb levels were comparable among the 1st, 2nd and 3rd trimester of pregnancy (Figure 2a). Cobalt did not show any significant trend in maternal and cord blood samples (Figure 1b), while Cu and Zn levels were higher at delivery respect to the 1st trimester (Cu: 1664 µg/L vs. 1302 µg/L; p<0.001; Zn: 6708 µg/L vs. 6147 µg/L; p=0.001). In contrast, cord blood Cu (623 µg/L) and Zn (2311 µg/L) levels were lower (p<0.001) than those in paired maternal blood at delivery (Table 2 and Figure 1b). No correlation between paired maternal and cord blood was observed for Co, Cu and Zn levels (Figure 3b). In urine of mothers, creatinine-adjusted Co presented no longitudinal trend among the three trimesters while Cu (p<0.001) and Zn (p=0.004) levels were lower at the 3rd trimester than at 1st and 2nd trimesters (Table 3 and Figure 2b). Manganese in blood of mothers was found to be higher (p<0.001) at delivery in relation to that at 1st trimester of pregnancy (16 µg/L vs. 10 µg/L); in addition, the highest Mn level was found in cord blood (28 5

ACCEPTED MANUSCRIPT µg/L) (Figure 1b). However, there was no correlation between paired maternal and cord blood Mn levels (Figure 3b). Non-creatinine adjusted and creatinine-adjusted Mn concentrations were comparable among the three trimesters of pregnancy (Figure 2b). The levels of Se in maternal blood (Figure 1b) showed a decrease at delivery with respect to levels found at 1st trimester (107 vs. 114 µg/L; p=0.007). The same Se decreasing trend in blood was observed in urine of mothers sampled at different pregnancy weeks (p=0.04 for non-creatinine adjusted data; p=0.002 for

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creatinine adjusted data) (Figure 2b). No correlations between paired maternal and cord blood samples were found for this element (Figure 3b).

In Table 4, other mother/child cohort studies previously performed in various countries, reporting data

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on the levels of toxic and essential elements in blood, cord blood and urine, are summarized.

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4. Discussion 4.1 Concentration of toxic elements during pregnancy

Recent studies have shown increased rates of fetal loss, preterm births, and neonatal mortality, as well as decreased birth weight at high levels of As exposure during pregnancy (Rahman et al., 2011; Ahmed et al., 2011). In utero As exposure was also associated with a higher risk of diarrhea and respiratory symptoms during the first year of life (Farzan et al., 2016). Paired maternal/cord blood levels of As were used as

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parameters for fetal As exposure, as this metalloid has been shown to easily cross the placental barrier. Maternal urine levels of As likely reflect also the burden experienced by the fetus (Concha et al. 1998; Hall et al., 2007). In the present study, we found lower median levels of As in cord blood (1.2 µg/L) with respect to blood of delivering mothers (1st trimester, 1.7 µg/L; delivery, 1.8 µg/L) (Figure 1a). In maternal urine,

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comparable As content was found at the 1st and 2nd trimesters of pregnancy, being lower in the late gestation period (31 vs. 44 vs. 20 µg/g of creatinine, respectively). Therefore, our results revealed the presence of As

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in cord blood, but the placenta seemed to provide a partial barrier against this element. As shown in Table 4, other cohorts showed As median values lower than current data: in Canada, ca. 2000 delivering women of the MIREC study had median blood As of 0.69 µg/L at the 3rd trimester; in Flanders, 235 mothers showed geometric mean blood As of 0.64 µg/L; in China, 212 pregnant women presented a median blood As of 0.52 µg/L; in South Africa, blood As median level was of 0.57 µg/L (Baeyens et al., 2014; Jin et al., 2014; Ettinger et al., 2017). Higher As levels in pregnant women and in cord blood were generally observed in environmental hot-spots settings, with specific As sources, likely mining activities or contaminated drinking water, and/or high fish consumption habits. For instance, among As-exposed pregnant women served drinking contaminated public water in Bangladesh As levels reached values of 11.9 µg/L in maternal blood and 15.7 µg/L in cord blood (Hall et al., 2007). A study conducted in Argentina, where the drinking water contained about 200 µg/L, the As concentration in cord blood (median, 9 µg/L) was almost as high as that in maternal blood (median, 11 µg/L) (Concha et al., 1998). We also found a significant correlation between As 6

ACCEPTED MANUSCRIPT in maternal blood at delivery and in cord blood (Figure 3a). Similar to our findings, significant correlations were reported in blood of mother/newborn pairs of South African, Flanders and Argentina populations, indicating that the developing fetus may be at risk for As exposure via placental transfer (Concha et al., 1998; Rudge et al., 2009; Baeyens, et al., 2014). Anyhow, the As ratio of cord/maternal blood (0.7) in our population was comparable than those found in South African and Flemish mothers (ca. 0.8 for both campaigns) and lower than that in high As-exposure areas such as Bangladesh (1.3) (Rudge et al., 2009;

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Rahman et al., 2011; Baeyens, et al., 2014). The measured urinary total As concentrations reflected exposure to inorganic and organic As from all sources during pregnancy. Urine As concentrations were comparable to those reported in pregnant women from other Mediterranean countries, with no differences between the 1st and 3rd trimesters (34 and 37 µg/g creatinine, respectively) (Fort et al., 2014). Rhaman et al. (2011) reported

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that the estimated risk of lower respiratory tract infection symptoms increased by 69% in infants of mothers with urinary mean As concentrations in the range of 262–977 µg/L (measured in early and late gestation) (Rahman et al., 2011). Considering the low mean As values in maternal urine found in the present study, we

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assumed that the possibility of indirect effects on the fetus can be excluded.

Regarding Cd, prenatal exposure to this element was associated to pregnancy-induced birth defects during embryogenesis, premature delivery and low birth weight (García-Esquinas et al., 2013). We found slightly lower levels of Cd in cord blood (min-max: 0.2-0.9 µg/L) than in maternal blood (min-max: 0.3-4.1 at 1st trimester) (Figure 1a), but no correlations between paired maternal/cord blood samples (Figure 3a). Previous studies, reported in Table 4, observed Cd concentrations similar to our study both in maternal blood

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(range of medians: 0.15-0.98 µg/L) and cord blood (range of medians: 0.02-0.70 µg/L). Some of these studies reported no correlation between cord and maternal blood levels of Cd (Rudge et al., 2009; Baeyens et al., 2014; Arbuckle et al., 2016). Among maternal blood, few mothers (7.5%) had blood concentration of Cd >1.0 µg/L at delivery, which is the reference level for blood Cd in healthy populations (Schulz et al., 2011).

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Moreover, all cord blood samples were lower than 1.0 µg/L. A protective capacity of the placenta against exposure to Cd more marked during the last trimester of pregnancy has also been reported. These alterations are thought to be mediated by metallothioneins (MT) that bind Cd to decrease its toxicity, and the Cd-MT

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complexes will avoid the Cd transport from the placenta to the fetus (Espart et al., 2018). Indeed, placental levels of this metal are related with maternal concentrations but not with cord blood levels. In turn, median Cd levels in urine of mothers (Table 3) were comparable among the three trimesters of gestation (ca. 0.50 µg/L or 0.80 µg/g creatinine), and also lower than the human biomonitoring guidance values (HBM-II) for Cd in urine of adults (4.0 µg/L), which described the Cd concentration at which - and above which - there is an increased risk of adverse health effects (Schulz et al., 2011). Slightly lower levels (P25-P75, 0.29-0.69 µg/g creatinine and 0.21-0.83 µg/g creatinine in 6th and 30th gestational week, respectively) were found in a previous study conducted in the same geographical area (Hernandez et al., 1996). Maternal exposure to Hg during pregnancy can affect the neurodevelopment of the fetus, with remarkable effects on behaviour, cognition, motor skills and the immune and reproduction systems later in life (Rice and Barone, 2000). We found median cord blood Hg levels significantly higher (2.8 µg/L) than 7

ACCEPTED MANUSCRIPT maternal blood collected at delivery (1.8 µg/L) (Table 2), as well as a strong positive correlation between paired maternal and cord blood samples (Figure 3a), revealing that the placenta may act as a significant route of exposure of the fetus to Hg. Previous studies (Table 4) of mother-infant pairs, performed in South Africa, Germany, Saudi Arabia, Spain and Korea, reported median cord blood Hg about two-fold or even three-fold higher than maternal Hg (Rudge et al., 2009; Kopp et al., 2012; Al-Saleh et al., 2014; García-Esquinas et al., 2013; Kim et al., 2015). Anyway, because Hg - as methyl-Hg (MeHg) - has a high affinity for fetal

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hemoglobin (Hb), the higher Hg levels in cord blood can simply represent a passive equilibrium due to binding of MeHg to Hb, rather than an active transfer of the metal (Björnberg et al., 2003). A total Hg blood safe-concentration was set at 3.5 µg/L in maternal blood, and at 5.8 µg/L in cord blood, by using the US EPA reference dose (RfD) for chronic oral exposure to MeHg (Mortensen et al., 2014). In the scientific literature,

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levels of Hg above 5.8 µg/L in cord blood have been associated with IQ decrease (Trasande et al., 2006). In the current study, 13% and 25% of maternal blood samples at 1rst trimester and at delivery, respectively, exceeded the level of 3.5 µg/L. Among cord blood, only one sample had Hg levels above 5.8 µg/L,

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suggesting a non-appreciable risk of deleterious effects of Hg for the developing fetus. Regarding measurement of Hg in urine of pregnancy women, a mild reduction was observed (Table 3 and Figure 2a) in the 3rd trimester of pregnancy (0.7 µg/g creatinine). These mothers’ urinary Hg levels were very far from the reference HBM-I values for total Hg in urine (i.e., 25 µg/L or 20 µg/g creatinine), a threshold value below which no adverse health effects are expected (Schulz et al., 2011).

Some epidemiological studies showed increased risk of congenital malformations, DNA damage and

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low birth weight in infants born from women living near Cr contaminated areas (Eizaguirre-García et al., 2000; Li et al. 2008). A very large prospective birth cohort study in China provided evidence of a positive association between higher Cr levels during pregnancy and the risk of preterm birth (Pan et al., 2017). We noticed only few mothers with higher Cr levels in cord blood than in maternal blood (Figure 1a), and no

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correlation in paired cord/maternal samples (Figure 3a). In another study, only the 29% of Cr passed the placenta in pregnant women with metal-on-metal hip arthroplasty, suggesting that the placenta exerts a modulatory effect on the rate of Cr transfer (Ziaee et al., 2007). In urine, we found significantly lower Cr

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concentrations in the late gestation (0.2 µg/g) than at the 1st (0.6 µg/g) or 2nd trimester (0.4 µg/g) (Table 3 and Figure 2a). These values were much lower than those observed in pregnant women of China (1.86 µg/g creatinine) and comparable to those reported in Western Australia (0.44 µg/g creatinine) (Table 4) (Callan et al., 2013; Pan et al., 2017).

With respect to Ni, the European Commission suggested a link between exposure to Ni compounds and reproductive toxicity (Committee for Compounds Toxic to Reproduction, 2003). Such toxicological effect can be plausible since Ni is known to be readily transferred across the placenta (Odland et al., 1999). Anyway, the median values found in this study in maternal urine were various times lower than those found in Ni-exposed delivering women that presented weak or no association with the risk of spontaneous abortion and genital malformations (Vaktskjold et al., 2008). Moreover, the observed median Ni values in maternal blood (0.6 µg/L) and in cord blood (0.7 µg/L) were lower than the levels reported in a mothers-child study 8

ACCEPTED MANUSCRIPT conducted in China (maternal blood: 1.4 ng/g; cord blood: 0.9 ng/g) (Hu et al., 2015). These authors also found that increasing Ni exposure was not associated with a decrease in infant birth weight. In contrast to Cd, Pb has the ability to cross freely the placental barrier. Therefore, it can enter the blood of the fetus. Since Pb can also cross the blood brain barrier, neurological development is of great concern when prenatal exposure to Pb occurs (Agency for Toxic Substances and Disease Registry, 2007). In the present survey, we found median Pb concentrations significantly lower in cord blood samples than in

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maternal blood at delivery (7.9 vs. 12 µg/L) (Table 2 and Figure 1a). Furthermore, a strong correlation between paired mother blood and cord blood Pb concentrations was observed (Figure 3a). These findings suggested that Pb partially crossed the placental barrier at a low transfer rate (cord/maternal blood ratio of ca. 0.7). Higher blood levels were observed in other countries like Spain (Cañas et al., 2014), UK, China and

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Saudi Arabia (Table 4) than those found in the present study. Also higher cord blood levels were observed in Spain (Llop et al., 2011; García-Esquinas et al., 2013) and in other countries (Table 4). In addition to differences in exogenous exposure, blood Pb concentrations can vary because of changes in hematocrit and

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Ca levels, plasma volume, and mobilization of Pb from bones during pregnancy (Schuhmacher et al., 1996; Gulson et al., 2016). Also in the Flemish population, lower content in cord blood than that in maternal blood was found, being related to the placenta allowing transport of Pb from mother to fetus (Baeyens et al., 2014). Gilbert and Weiss (2006) reported that there was sufficient and compelling scientific evidence for the CDC to lower the blood Pb action level for children to 20 µg/L, in order to ensure that children are protected from the detrimental neurobehavioral effects of this metal (Gilbert and Weiss, 2006). Recently, the European Food

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Safety Agency (EFSA) established that an increase of 12 µg/L in blood could produce a decrease of the IQ score by one unit, without threshold below which neurodevelopmental toxicity could be defended (European Food Safety Authority, 2010). In addition, evidence of low cognitive development indices in Saudi infants at the age of six months has been notice at prenatal Pb exposure levels ≥ 25 µg/L (Al-Saleh et al., 2009). In

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addition, decrease of psychomotor scores has been recently reported in 2-year old children with cord blood lead levels much lower than 50 ug/L (Polanska K et al., 2018). If we adopt the 20 µg/L as considered as safe level, only two cord blood samples in the current study had Pb blood levels higher than the level of possible

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developmental toxicity. However, these results should be interpreted with caution because there is broad recognition that no level of Pb exposure is “safe” in fetus and during the development. Regarding Pb in urine of mothers, there were no differences in excretion rates during the trimesters of pregnancy as elsewhere reported (Gulson et al., 2000). We suggested that renal function of pregnant women was not affected by toxicity mechanism of Pb, and values were even lower than those in healthy pregnant women (1.8 µg/L) (Wibowo et al., 2014) and in childbearing women from China and Japan (Table 4). 4.2 Concentration of essential elements during pregnancy Cobalt is an essential trace metal used in the formation of vitamin B12 and 85% of the human body content of Co is in this form; consequently, very few studies enrolling women in pregnancy focused on this element. A prospective birth cohort study showed maternal serum Co concentrations during pregnancy 9

ACCEPTED MANUSCRIPT negatively associated with the risks of pregnancy-induced hypertension syndrome (Liang et al., 2018). A case study reported extremely high blood levels of Co (up to 138 µg/L) in pregnant women with implantation of metal-on-metal hip devices, but no signs of intoxication and developmental toxicity were reported (Fritzsche et al., 2012). In the present study, median concentrations of Co in blood of mothers and cord blood (ca. 0.3 µg/L) were lower respect to previously reported data (Table 4) in maternal and cord blood samples of South African pregnant women (Rudge et al., 2009), and slightly higher than those observed in

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blood of delivering women in Norway and Western Australia (Hansen et al., 2011; Callan et al., 2013). Median concentrations of urinary Co during the three trimesters of gestation (0.9-1.3 µg/g creatinine) (Table 3) overlapped those found during the 1st and 3rd trimester of pregnancy in Spanish women (0.7 and 1.6 µg/g creatinine respectively) (Fort et al., 2015).

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Copper and Zn levels significantly increased in maternal blood at delivery respect to the 1st trimester of pregnancy (Table 2 and Figure 1b), which was probably due to the increased mothers’ metabolic demand of these nutrients as already reported in Norwegian pregnant women (Hansen et al., 2011). In addition, the

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levels of both elements were significantly reduced in cord blood in comparison to maternal blood (Table 2 and Figure 1b), indicating a limited transplacental passage of these elements from mother to fetus. Regarding Cu, similar significant lower contents of this element in cord blood respect to maternal blood were reported in Germany, Flanders and South Africa (Table 4) (Rudge et al., 2009; Baeyens et al., 2014; Kopp et al., 2012). In urine, Cu declined from the 1st trimester of pregnancy to the 3rd one (26 vs. 16 µg/g creatinine) (Table 3 and Figure 2b). It was demonstrated that the newborn is dependent on stored Cu, which could not be

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adequate in premature infants (Perveen et al., 2002). Copper deficiency can lead to anemia, neutropenia, bone disease and growth retardation in pediatric patients. Anyhow, the current Cu urinary concentration in pregnant women was well within the Italian reference ranges for women (P50-P95 values of 9.3-27 µg/L) (Bocca et al., 2016).

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For Zn, the median concentration in cord blood (2311 µg/L) were more than 2.5 times lower than in maternal blood (Figure 1b) at 1st trimester and delivery (6145 µg/L and 6708 µg/L, respectively), as previously reported in other mother/child pairs (Table 4) (Rudge et al., 2009; Kopp et al., 2012). In urine, Zn

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(like Cu) declined significantly from 1st trimester to 3rd trimester of pregnancy (497 vs. 383 µg/g creatinine) (Table 3 and Figure 2b). It is known that circulating Zn levels decline during pregnancy due to hemodilution, decreased levels of Zn-binding protein, and hormonal changes (Shah and Sachdev, 2006). The main characteristics following a Zn deficiency included weight loss, failure to thrive, and enhanced susceptibility to infections; the Zn supplementation may have a positive effect on neonatal immune status and infant morbidity from infectious diseases. It is hard to define accurately the Zn status in the population of this study, also because the median urinary Zn levels overlapped reference values in healthy adults women (P50, 293 µg/L) (Bocca et al., 2016). Median blood Mn showed increased levels over the course of pregnancy (1st trimester, 10 µg/L; delivery, 16 µg/L), being the highest in cord blood (28 µg/L) as showed in Table 2 and Figure 1b. Maternal blood Mn levels could increase during pregnancy because of the increased intestinal absorption, and possibly 10

ACCEPTED MANUSCRIPT increased binding capacity towards Hb to ensure the metal sufficiency (Abbassi-Ghanavati et al., 2009). Other studies on Mn levels in childbearing women reported significant increases depending on the gestational week, with the highest Mn levels found at the 3rd trimester of pregnancy (Mora et al., 2014; Arbuckle et al., 2016). Regarding the significant higher Mn levels in cord blood compared to maternal blood, it could reflect the active transport of this element from mother to fetus. However, because we did not find a correlation between paired maternal/cord blood Mn concentrations (Figure 3b), other reasons might explain

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the higher Mn in cord blood. For example, the lower or restricted elimination of Mn by the fetus or the inability of the fetus to utilize this element (Nandakumaran et al., 2015). Another reason for the higher Mn concentration in cord blood can rely on the accumulation of this element in the red blood cells, which represent the main fraction in the umbilical cord blood. As reported in Table 4, Mn cord blood levels were

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ca. 2-3 times more than in maternal blood also in various other countries (Baeyens et al., 2014; Rudge et al., 2009; Kopp et al., 2012; Arbuckle et al., 2016; Takser et al., 2004). For Mn, both deficiency and excess can mean detrimental health effects, and like Hg and Pb, the brain is the critical target organ for Mn. Although

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the risk of Mn-induced neurotoxicity during pre- and postnatal brain development is not fully understood, an association between Mn uptake during pregnancy, as well as an early psychomotor development of children was reported (Takser et al., 2004). Normative concentrations for blood Mn during pregnancy should be established. Nowadays, a normal range of blood Mn concentrations at 4-15 µg/L has been established only for the general population (Agency for Toxic Substances and Disease Registry, 2012). The wide range of blood Mn concentrations observed in our mothers’ blood (max, 33 µg/L) and cord blood (max, 77 µg/L)

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might reflect different diets, use of supplements, metals (as Fe) deficiency status, etc. Moreover, it is widely believed that blood Mn levels in pregnant women and neonates are higher than the normally accepted levels for general populations, due to a compromised processing mechanism, which requires further investigation (Agency for Toxic Substances and Disease Registry, 2012).

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As observed for Mn, both Se excess and deficiency may be detrimental to health. Selenium is involved as a constituent part of selenoenzymes protecting the body against oxidative insult and it has a crucial involvement in the thyroid hormone metabolism. Deficiencies in Se are associated to pregnancy

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outcomes such as preterm delivery, miscarriage, and preeclampsia. In addition, Se may be able to reduce the toxic effects of Cd and Pb in pregnant women (Tomislav et al., 2008). In contrast, at very high concentrations, Se can act as a pro-oxidant, causing oxidative damage to cells and tissues. In this context, opinions considerably diverge about the upper and lower safe Se limits, as well as the specific outcomes linked to altered Se status, also because this element exists in various inorganic and organic forms, having considerable and even extreme variations in both toxicological and physiological properties (Vinceti et al., 2015). In the current study, median Se levels (Figure 1b) were slightly lower in cord blood (100 µg/L) than in maternal blood at delivery (107 µg/L) and at 1st trimester (114 µg/L). These values were in line to those found (Table 4) in Norway, Western Australia, South Africa, China and Japan (Sakamoto et al., 2012). Maternal Se blood values were also lower than reference levels reported for this element in Italian adults (P50, 153 µg/L) (Oggiano et al., 2018). In maternal urine, we found a significant decrease in the body burden 11

ACCEPTED MANUSCRIPT of mothers, especially in the late gestation (Table 3 and Figure 2b). These Se values during the entire pregnancy period were lower than those found in a control adult population in Italy (P50, 35 µg/L) (Oggiano, et al., 2018). In this setting, the dietary Se supplementation during pregnancy and lactation has been suggested to ensure adequate Se transfer via placenta and mammary gland, as well as during infancy (Dylewski et al., 2002).

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5. Conclusions

In this study, we estimated pregnancy exposure to toxic and essential elements by measuring their concentrations in maternal blood (from 1rst trimester and at delivery) and cord blood, and in mothers’ urine

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(at 1rst, 2nd and 3rd trimester of pregnancy). Maternal blood levels of Cu, Mn, Pb and Zn increased over the course of pregnancy. Cord blood, in comparison to maternal blood at delivery, presented significantly higher Hg and Mn levels, and lower As and Pb concentrations. In turn, essential elements such as Cu and Zn were

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ca. two times lower in cord blood than in maternal blood. A significant correlation for As, Hg and Pb was observed in paired maternal/cord blood samples. In maternal urine, decreased concentrations of many elements as As, Cr, Cu, Hg, Se and Zn with the increase of weeks of gestation were found. Altogether, the current results suggest that Hg and Mn accumulated in cord blood and can be transferred to fetus, while As and Pb were moderately transferred to fetus, and essential elements such as Cu

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and Zn were preferentially stored in mothers. Clearly, physiological and metabolic changes during gestation altered the concentration of toxic and essential elements in this study. In addition, the levels of toxic and essential elements were relatively low respect to reference values and without clinical significance for the mothers and the unborns. Further research is ongoing to elucidate sources of exposure and predictor factors that may influence

Acknowledgements

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levels of elements during pregnancy.

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This work was supported by the European Commission through the HEALS Project (FP7-ENV-2013603946). J. Rovira received funds from Health Department of Catalonia Government, trough "Pla Estratègic de Recerca i Innovació en Salut" (PERIS 2016-2020) fellowship.

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Ziaee, H., Daniel, J., Datta, A.K., Blunt, S., McMinn, D.J., 2007. Transplacental transfer of cobalt and chromium in patients with metal-on-metal hip arthroplasty: a controlled study. J. Bone. Joint. Surg. Br. 89(3): 301-305.

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Table 1. Socio-demographic and lifestyle characteristics of pregnant women Variables

Mean ± SD or %

Age (years)

34.1 ± 5.1

2

1st trimester 2nd trimester (kg/m2) 3th trimester (kg/m2)

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BMI (kg/m ) 25.4±5.5 27.3±5.3 29.4±4.9 33

2nd child

48

3rd child or more

19

Educational level (%) 21

secondary school

32

university

47

no Smoke 1-year before pregnancy (%) yes no Smoke during pregnancy (%) yes no

57 43

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yes

21

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Work during pregnancy (%)

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primary school

79

13 87

Alcohol 1-year before pregnancy (%) yes

24

no

75

Alcohol during pregnancy (%)

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1st child

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Parity (%)

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15

no

85

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SC

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yes

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Table 2. Concentration of trace elements in maternal blood (MB) at 1st trimester and at delivery and in cord blood (CB)

Elements

MDL

p-valuea

Toxic

Cd

0.2

Cr

0.04

Hg

0.5

Ni

0.3

Pb

0.5

1.7 (5.3) 0.3-8.5 0.5 (2.0) 0.3-4.1 0.4 (0.7) 0.1-0.8 2.0 (7.7) 0.5-15 0.6 (1.5) 0.4-1.9 10 (28) 3.8-52

Essential

1.2 (4.3) 0.3-5.9 0.5 (0.7) 0.2-0.9 0.6 (2.0) 0.1-2.5 2.8 (7.7) 0.7-8.7 0.7 (1.3) 0.5-1.7 7.9 (21) 2.8-32

ns ns ns ns ns 0.002

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0.01

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0.3 (0.7) 0.3 (0.5) ns 0.1-0.9 0.1-0.6 1664 1302 (1842) 623 (768) 0.8 (2312) 892<0.001 Cu 738-2189 386-813 2626 10 (16) 16 (27) 28 (71) 0.5 <0.001 Mn 3.8-26 5.6-33 7.7-77 114 (162) 107 (137) 100 (159) 0.5 0.007 Se 74-213 37-156 40-170 6708 6147 (7794) 2311 (2879) 1.3 (8480) 0.001 Zn 3963-9295 1489-3049 4071-9064 a Wilcoxon signed rank test: 1st trimester maternal blood vs. delivery maternal blood; b Wilcoxon signed rank test: delivery maternal blood vs. delivery cord blood. ns: not significant (p>0.05) MDL: Method Detection Limit Co

0.2 (0.6) 0.1-0.8

1.8 (5.6) 0.3-7.3 0.4 (1.4) 0.3-2.5 0.5 (0.9) 0.2-1.2 1.8 (5.9) 0.5-9.0 0.6 (1.2) 0.4-1.9 12 (35) 5.2-41

0.045 ns

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0.3

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As

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CB (µg/L) Delivery (n=31) P50 (P95) p-valueb Min-Max

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MB (µg/L) Delivery 1 trimester (n=48) (n=40) P50 (P95) P50 (P95) Min-Max Min-Max st

ns

<0.001 ns

<0.001

ns <0.001 <0.001 ns <0.001

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Table 3. Concentration of trace elements in maternal urine (MU) at three trimesters of pregnancy MU Non-creatinine adjusted (µg/L) rd

1 trimester (n=53) P50 (P95) Min-Max

2 trimester (n=539 P50 (P95) Min-Max

3 trimester (n=49) P50 (P95) Min-Max

24 (119) 1.3-425 0.5 (1.7) 0.1-1.9 0.3 (2.0) 0.07-2.8 0.6 (2.5) 0.3-4.4 1.4 (3.2) 0.2-3.9 0.7 (3.2) 0.1-3.5

20 (127) 1.4-285 0.5 (1.3) 0.1-2.3 0.2 (0. 8) 0.06-2.8 0.7 (2.3) 0.3-7.0 1.1 (4.6) 0.2-6.3 0.8 (2.9) 0.1-4.0

18 (192) 1.3-765 0.5 (1.5) 0.1-1.6 0.2 (0.6) 0.07-1.0 0.5 (2.2) 0.3-4.1 1.2 (4.4) 0.3-9.9 0.7 (2.4) 0.2-3.5

p-valuea

Toxic

Cd

0.1

Cr

0.05

Hg

0.3

Ni

0.2

Pb

0.1

Essential

ns ns

31 (179) 5.3-238 0.9 (3.2) 0.3-5.6 0.6 (4.8) 0.07-21 1.1 (4.9) 0.4-5.5 2.3 (8.9) 0.3-18 1.4 (5.3) 0.2-6.0

M AN U

0.5

0.001

<0.001 ns

ns

TE D

As

Creatinine adjusted (µg/g) 1 trimester 2nd trimester 3rd trimester (n=53) (n=53) (n=49) P50 (P95) P50 (P95) P50 (P95) Min-Max Min-Max Min-Max st

RI PT

Elements MDL

nd

SC

st

44 (360) 5.6-895 0.9 (3.0) 0.3-7.8 0.4 (3.4) 0.06-6.7 1.5 (7.1) 0.3-24 2.3 (8.0) 0.4-11 1.3 (5.6) 0.3-14

AC C

0.1

EP

0.4 (1.5) 0.5 (2.6) 0.7 (2.3) 0.9 (3.9) 1.3 (4.4) ns 0.1-2.3 0.1-3.2 0.1-3.4 0.1-6.5 0.1-6.2 15 (42) 13 (31) 14 (33) 26 (95) 27 (73) 0.3 ns Cu 2.0-65 2.7-52 1.7-52 12-469 8.6-148 0.2 (0.7) 0.2 (0.4) 0.2 (0.4) 0.4 (1.4) 0.4 (1.3) 0.05 ns Mn 0.06-0.9 0.06-0.5 0.07-1.4 0.07-7.3 0.04-2.1 19 (68) 16 (49) 15 (48) 32 (71) 26 (71) 2.0 0.036 Se 1.5-108 1.8-80 2.6-66 8.7-157 6.6-129 290 (826) 322 (850) 272 (1176) 497 (1610) 566 (2006) 2.0 ns Zn 11-1663 37-1644 24-1672 19-6842 146-2532 a Friedman's two-way analysis of variance by ranks: 1st trimester vs. 2nd trimester vs. 3rd trimester maternal urine in µg/L; b Friedman's two-way analysis of variance by ranks: 1st trimester vs. 2nd trimester vs. 3rd trimester maternal urine in µg/g creatinine. ns: not significant (p>0.05) MDL: Method Detection Limit Co

20 (157) 2.1-454 0.7 (1.6) 0.1-3.9 0.2 (0.8) 0.09-2.5 0.7 (3.8) 0.2-7.0 1.7 (4.6) 0.5-5.7 1.2 (2.9) 0.3-3.7 0.9 (3.3) 0.1-6.9 16 (32) 4.1-60 0.3 (0.9) 0.06-1.6 21 (37) 5.1-71 383 (992) 83-1412

p-valueb 0.011 ns <0.001 0.001 ns ns

ns <0.001 ns 0.002 0.004

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Table 4. Data on toxic and essential elements (as medians) in maternal blood (MB), cord blood (CB) and maternal urine (MU) of pregnant women reported in previous studies

Cd

Cr Hg

a

0.54

0.46

RI PT

2001 235 215 62 173 211 78 2001 235 215 209 62 50 79 163 1579 140 78 4286 173 7290 2001 215 62 50 79 211

MU µg/g creatinine

13.2

SC

Canada Flanders China South Africa Western Australia North Norway Japan Canada Flanders China China South Africa Germany South Korea North Norway Saudi Arabia Spain Japan UK Western Australia China Canada China South Africa Germany South Korea North Norway

CB µg/L

76.9a

0.20 0.31a 0.47 0.48 0.15 0.34 0.59 0.15 0.98 0.60

M AN U

As

MB µg/L 0.69 0.64a 0.52 0.57 1.26 1.40a

TE D

Women n.

0.07a 0.15 0.02

0.70 0.27 0.77a

0.29 <1.0 0.56 0.26 0.65 0.44 2.66 1.20

0.10

0.04

EP

Country

AC C

Toxic elements

0.44 1.86 0.80 1.2 1.48 5.58

References Ettinger et al., 2017 Baeyens et al., 2014 Jin et al., 2014 Rudge et al., 2009 Callan et al., 2013 Hansen et al., 2011 Shirai et al., 2010 Arbuckle et al., 2016 Baeyens et al., 2014 Jin et al., 2014 Sun et al., 2014 Rudge et al., 2009 Kopp et al., 2012 Kim et al., 2015 Hansen et al., 2011 Al-Saleh et al., 2014 García-Esquinas et al. 2013 Shirai et al., 2010 Taylor et al., 2014 Callan et al., 2013 Pan et al., 2017 Arbuckle et al., 2016 Jin et al., 2014 Rudge et al., 2009 Kopp et al., 2012 Kim et al., 2015 Hansen et al., 2011

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South Africa Western Australia North Norway Flanders South Africa Germany Western Australia North Norway Japan Canada Flanders South Africa Germany Western Australia North Norway

62 173 211 235 62 50 173 211 78 2001 235 62 50 173 211

Cu

Mn

32.3 10.3 8.8

RI PT

Co

7.67 8.6a

0.66

SC

Women n.

2.32

M AN U

Country

34.1 MB µg/L 0.60 0.18 0.10 1312a 1730 1120 1252 1650

2.87 7.66

TE D

Essential elements

Ni Pb

1.95 4.61 1.86 <2.0 5.59 11.1a 24.5 40.5 11.5 10.2 7.50 25.4 18.9

EP

1579 140 4134 173 2001 235 215 209 50 79 210 1579 140 78 4285

AC C

Saudi Arabia Spain UK Western Australia Canada Flanders China China Germany South Korea North Norway Saudi Arabia Spain Japan UK

20.5 13.8

CB µg/L 0.27

0.48a MU µg/g creatinine 1.17

600a 657 470 10.4 12.8a

12.6 12.1a 16.8 17.0 9.14 10.7

31.9 31.2a 34.9 28.8 1.22

Al-Saleh et al., 2014 García-Esquinas et al., 2013 Taylor et al., 2014 Callan et al., 2013 Arbuckle et al., 2016 Baeyens et al., 2014 Jin et al., 2014 Sun et al., 2014 Kopp et al., 2012 Kim et al., 2015 Hansen et al., 2011 Al-Saleh et al., 2014 García-Esquinas et al., 2013 Shirai et al., 2010 Taylor et al., 2014 References Rudge et al., 2009 Callan et al., 2013 Hansen et al., 2011 Baeyens et al., 2014 Rudge et al., 2009 Kopp et al., 2012 Callan et al., 2013 Hansen et al., 2011 Shirai et al., 2010 Arbuckle et al., 2016 Baeyens et al., 2014 Rudge et al., 2009 Kopp et al., 2012 Callan et al., 2013 Hansen et al., 2011

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104 88 85

111 25.6

131 6290 5120 2330 5110

126 2548 1340

37.6a 5.07

396

393a

EP

TE D

M AN U

geometric mean

AC C

a

62 173 211 78 209 62 50 173 211 78

RI PT

Zn

South Africa Western Australia Norway Japan China South Africa Germany Western Australia North Norway Japan

SC

Se

Rudge et al., 2009 Callan et al., 2013 Hansen et al., 2011 Shirai et al., 2010 Sun et al., 2014 Rudge et al., 2009 Kopp et al., 2012 Callan et al., 2013 Hansen et al., 2011 Shirai et al., 2010

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Figure 1a. Longitudinal pattern of toxic elements in maternal blood (MB 1st trimester, MB delivery) and cord blood (CB delivery) in paired samples As

10

2.0

µg/L

6 4

1.5 1.0 0.5

0

0.0

MB 1 trimester

MB delivery

SC

2

RI PT

2.5

8

MB 1 trimester

CB delivery

16

Cr

MB delivery

CB delivery

Hg

M AN U

3.0

14

2.5

12

1.5

10

µg/L

µg/L

2.0

8 6

1.0

4

TE D

0.5

2

0.0

0

MB 1 trimesterMB 1 trimester MB delivery

1.6

60

Ni

delivery MBCB delivery

CB delivery

Pb

50

1.2 40

0.8 0.6

µg/L

1.0

µg/L

EP

1.4

AC C

µg/L

Cd

3.0

30 20

0.4 0.2 0.0

10 0

trimester MB 1 trimesterMB 1 MB delivery

MBCB delivery delivery

CB delivery

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Figure 1b. Longitudinal pattern of essential elements in maternal blood (MB 1st trimester, MB delivery) and cord blood (CB delivery) in paired samples Co

1.0

Cu

0.6

1500

µg/L

0.4

1000

0.2

500

0.0

0

MB delivery

CB delivery

Mn

100

MB 1 trimester

M AN U

MB 1 trimester

SC

2000

µg/L

0.8

RI PT

2500

250

80

MB delivery

CB delivery

Se

200

60

µg/L

µg/L

150

40 20 0

Zn

10000

µg/L 4000 2000

CB delivery

AC C

8000 6000

MB delivery

EP

MB 1 trimester

TE D

100

0

MB 1 trimester

MB delivery

CB delivery

50 0

MB 1 trimester

MB delivery

CB delivery

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Figure 2a. Longitudinal pattern of toxic elements in maternal urine (MU) at 1st trimester, 2nd trimester and 3rd trimester in paired samples As

Cd

1000

9.0 8.0

400

5.0 4.0 3.0 2.0

200

1.0 0

0.0

1 trimester

2 trimester

3 trimester

1 trimester

Cr

30

2 trimester

3 trimester

Hg

M AN U

25

SC

6.0

600

RI PT

7.0

µg/g

µg/g

800

25

20

20

µg/g

µg/g

15 10

15 10

5

5

1 trimester

14

6 4 2 0

AC C

µg/g

12

1 trimester

2 trimester

1 trimester

2 trimester

3 trimester

Pb 16 14 12 10

µg/g

16

8

3 trimester

EP

18

10

2 trimester

Ni

20

0

TE D

0

8 6 4 2 0

3 trimester

1 trimester

2 trimester

3 trimester

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Figure 2b. Longitudinal pattern of essential elements in maternal urine (MU) at 1st trimester, 2nd trimester and 3rd trimester in paired samples Co

Cu

500

8.0 7.0

4.0

300 200

3.0 2.0

100

1.0 0.0

1 trimester

2 trimester

2 trimester

3 trimester

Se

M AN U

180

7.0

160

6.0

140 120

µg/g

5.0

µg/g

1 trimester

Mn

8.0

4.0 3.0

100 80 60

2.0

40

1.0

1 trimester

6000 5000

1000 0

AC C

4000

2000

3 trimester

EP

7000

3000

2 trimester Zn

8000

TE D

20

0.0

µg/g

0

3 trimester

SC

µg/g

µg/g

5.0

RI PT

400

6.0

1 trimester

2 trimester

3 trimester

0

1 trimester

2 trimester

3 trimester

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AC C

EP

TE D

M AN U

SC

RI PT

Figure 3a. Correlations between paired maternal blood (MB) and cord blood (CB) samples for toxic elements

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AC C

EP

TE D

M AN U

SC

RI PT

Figure 3b. Correlations between paired maternal blood (MB) and cord blood (CB) samples for essential elements

ACCEPTED MANUSCRIPT Human biomonitoring to evaluate exposure to toxic and essential trace elements during pregnancy. Part A. Concentrations in maternal blood, urine and in cord blood. By Beatrice Bocca et al.

Changes during gestation altered toxic and essential elements levels Hg and Mn accumulated in cord blood and may be transferred to fetus As and Pb were moderately transferred to fetus

AC C

EP

TE D

M AN U

SC

Essential elements such as Cu and Zn were preferentially stored in mothers

RI PT

Highlights