Assessment of Bisphenol A Levels in Preschool Children: Results of a Human Biomonitoring Study in Ankara, Turkey

Objective: There is general concern regarding environmental chemical exposure and the impact it may have on human health. This is particularly important for vulnerable populations such as infants and children during critical periods of development. Bisphenol A (BPA) is an endocrine disrupting chemical used worldwide over the last 30 years in many consumer products. Evidence points to widespread human exposure to BPA. The aim of this study was to evaluate the exposure of Turkish preschool children to BPA. Methods: This study was conducted as a preliminary investigation of BPA in urine, collected from 3-6 year old children living in Ankara. After spot urine samples were taken from preschool children, free BPA, β-D-glucuronide and total BPA were determined using high-performance liquid chromatography tandem mass spectrometry and adjusted by creatinine concentration. Results: Preschool children from Ankara (n=125; males n=70, females n=55; mean age: 4.50±1.26) were recruited. BPA was detected in 76.8% of children from Ankara city, with urinary concentrations ranging from < limit of quantification to 18.36 μg/g creatinine. Total BPA levels were not statistically different between boys (1.26 μg/g creatinine) and girls (2.24 μg/g creatinine) (p>0.05). Conclusion: This study is an important contribution to the limited information about childhood exposure to BPA. The estimated daily BPA intake in this study is substantially lower than the European Food Safety Authority derived tolerable daily intake of 4 μg/kg BW/day.


Introduction
There is general concern regarding environmental chemical exposure and its impact on human health, but this is particularly important for vulnerable populations, such as infants and children during sensitive periods of development. In 1997 the leaders of the G8 countries stated, "We acknowledge that, throughout the world, children face significant threats to health from an array of environmental hazards. The protection of human health remains a fundamental objective of environmental policies to achieve sustainable development. We increasingly understand that the health and well-being of our families depends upon a clean and healthy environment. Nowhere is this more true than in the case of children, who are particularly vulnerable to pollution" (1). In addition, one of the biggest concerns of the World Health Organization (WHO) for children is exposure to chemicals during the intrauterine and childhood periods and associated health problems that arise later in life (2). In recent years, exposure to environmental pollution with chemicals known to act as endocrine disruptors (EDs) has been implicated in the incidence of many diseases and disorders.
Bisphenol A (BPA), with approximately 3.6 million tons annual global production (3), is an ED. The United States Environmental Protection Agency (EPA) estimates that more than 400,000 kilograms of BPA are leached into the environment every year (4). Due to the widespread use of BPA, over 90% of tested humans have detectable BPA, with the highest levels found in infants and children (5).
Childhood exposure to BPA occurs through specific exposure routes including mouthing, food intake, the use of BPA-containing products, inhalation, dermal contact and ingestion. Children are more susceptible to chemicals such as BPA than the general population due to their rapid development and increased food intake per kg body weight (2). BPA exposure has been linked to a range of adverse human health outcomes including decreased fertility, behavioural effects, disruption of endocrine function, altered development and increased prevalence of metabolic diseases (4,5). For example, relationships between BPA exposure and altered neurobehavioral outcomes including hyperactivity, attention problems, anxiety, and depression, in children have been reported by several human studies (6,7,8,9,10,11). Following the demonstration of a wide variety of adverse effects associated with BPA exposure in humans and laboratory animals over the last two decades, the Canadian Ministry of Health banned the import and marketing of infant feeding bottles made of polycarbonate in 2008, as BPA is used in the production process. In 2011, the European Union banned BPA use in the production of polycarbonate baby bottles and prohibited the sale and import of BPAcontaining products that come in contact with food for children aged 0-3 (12). The same restrictions have been applied in Turkey since 2011.
Numerous studies estimate exposures to BPA using urinary biomonitoring. Most have focused on adults from different societies to quantify human exposure to BPA. These studies have shown large variations between participants and studies, but very limited data are available for young children (13,14). To our knowledge, data regarding human exposure to BPA in Turkey are scarce (15). The primary aim of this study was to quantify exposure of preschool children to BPA.

Study Population
Urine samples were collected from preschool children (3-6 years  Each child provided a single spot urine sample collected in a 125 mL glass screw cap culture tube lined with polytetrafluoroethylene that had been previously cleaned with hexane. The sample was divided into aliquots. Urine samples from healthy children were collected in the evening, before a meal, to determine the spot urine concentrations of both free BPA and BPA-GLU.
The samples were kept cool until transportation to the study center and were immediately frozen at -20 °C until analysis.

Sample Preparation
Sample preparation, chromatographic and mass spectrometric (MS) conditions were used as described previously (16). Briefly, 13 C 12 -BPA was used as a stable internal standard and added to the samples at the beginning of the extraction. The BPA and BPA-GLU in 500 μL urine samples were purified by protein precipitation/dilution with 500 μL of acetonitrile and 50 μL of 13 C 12 -BPA and 13 C 12 -BPA-GLU. After protein precipitation, samples were centrifuged at 2250 rpm at 25 °C for 10 minutes. Total BPA values were calculated as reported previously. All other analytical data such as QA/QC assurance, matrix effects and data repeatability have been reported previously (16).

Instrumental Analysis
Identification and quantification of free BPA and BPA-GLU were performed with an Agilent 1200 Series 6460 (Agilent Technologies, CA, USA) triple quadrupole MS with Jet-Stream atmospheric pressure electrospray ionization source and Mass Hunter data acquisition/Quantitation software. The HPLC system was equipped with a binary pump, vacuum degasser, low carryover autosampler and thermoregulated column compartment. Twenty microliters of the extract was injected onto an Agilent (Agilent Technologies, CA, USA) Pursuit 3 pentafluorophenyl propyl column (100×3.0 mm, 3 μm particle size). The mobile phases A and B consisted of 2 mM ammonium acetate in water and methanol respectively. The limits of detection for free BPA and BPA-GLU were 0.03 ng/mL and 0.10 ng/mL and the limits of quantification (LOQ) were 0.08 ng/mL and 0.33 ng/mL respectively. The tandem MS-MS was operated with negative electrospray ionization in the selected reaction monitoring (SRM) mode. Nitrogen was used as both curtain and collision gas. The monitored quantifier SRM transitions were 227.1>132.8 for free BPA, 403.1>113.1 for BPA-GLU, and 239.2>224.1 for 13 C 12 -BPA (internal standard).

Creatinine Analysis in Urine
Both the free BPA and BPA-GLU values obtained in this study were corrected for creatinine. To assess the impact of creatinine adjustment on the total variance of spot urine samples, urine creatinine levels were analyzed using a modified method developed and validated for creatinine analysis by Park et al (17). Briefly, a 10 μL aliquot of urine was diluted with milli-Q water (1000-fold) and 100 μL (5 mg/L) of creatinine-d3 (internal standard, 5 mg/mL) was added. Creatinine was analyzed with LC-MS/MS in electrospray positive ionization mode and the SRM transitions monitored were 114.1>86.1 for creatinine and 117.2>89.2 for creatinine-d3. One microliter of the extract was injected onto an Agilent (Agilent Technologies, CA, USA) Zorbax SB-C18 chromatographic column (3 x 50 mm, 3.5 μm particle sizes). The mobile phases A (water) and B (methanol) both contained 2 mM ammonium acetate. The analysis for creatinine was achieved using isocratic conditions (80%B).

Estimated Daily Intake (EDI) Calculation
To understand the magnitude of BPA exposure in the children, EDI was calculated based on the assumption of urine excretion volumes of 0.4 L (ages 3-4 years) and 0.5 L (ages 5-6 years) for 24 hours for children (18). The daily exposure doses of BPA were estimated using the following equation:

Statistical Analysis
The statistical evaluations of the data were performed with Statistical Package for the Social Sciences, version 11.5 for Windows (IBM Inc., Armonk, NY, USA). Data were summarized as minimum, maximum, median, mean, geometric mean (GM), and standard deviation for total and each group. The normality of the data distribution was assessed with the Shapiro-Wilk test. The Mann-Whitney U test was used for multiple comparisons between groups. A p value less than 0.05 were accepted as statistically significant.

Results
In this study, free BPA and glucuronide conjugate of BPA (BPA-GLU) were measured in 125 preschool children (55 females, mean age 4.42±1.09 years and 70 males, mean age 4.56±1.39 years) who lived in Ankara. Table 1 presents the distribution of the main characteristics of the study populations. Urinary total BPA concentrations (adjusted for creatinine) in females and males are presented in Table 2. Total BPA was determined in 76.8% of the analyzed urine samples and BPA concentrations were equal to or above the LOQ of 0.08 ng/mL. Total urinary concentrations of BPA in Turkish preschool children ranged from LOQ-18.36 µg/g creatinine, with a mean concentration of 1.79 µg/g creatinine. The mean concentrations of total BPA in female and male groups were 2.24 µg/g creatinine and 1.26 µg/g creatinine, respectively, and there was no statistically significant difference (p=0.202). However, when the children were divided by age into <4 years and >4 years the mean BPA values of the <4 years-old females were statistically higher than the males of the same age (p=0.005) (Figure 1,

Discussion
BPA is a high trade volume chemical because it is widely used in many consumer products and exposure is almost inevitable in daily life. In addition to being the first study to evaluate BPA exposure in preschool children in Turkey, the results of this study are important for providing basic data on BPA concentrations in the human population in Turkey. Studies assessing BPA exposure show that because of a dramatic increase in the use of BPA-containing products in daily life, BPA and its metabolites are present at detectable levels in nearly every person's blood, tissue and urine. In order to assess the exposure of humans to BPA, measurement of their urinary concentration of free species, in this case BPA, and target compound conjugates, in this case conjugated BPA, is essential (22,23). BPA in biological samples is found as both free and conjugated BPA. Among the conjugated BPAs, BPA-GLU is a sufficiently specific and stable compound that can be regarded as a biomarker to evaluate BPA exposure (16). Varying levels of BPA and BPA-GLU are detected in urine samples depending on nutrition and lifestyle.
Although there is a general concern about possible effects of exposure to environmental chemicals on human health, these concerns are especially important for susceptible groups such as babies and children, during critical stages of their development. One of the biggest concerns of the WHO regarding infants is health problems that will show    up later in life because of exposure to chemicals during the intrauterine and childhood periods. In particular, ED chemicals make important alterations in cellular pathways that provide a basis for these diseases (2). Hormones are the chemicals that regulate physiological homeostasis and functions of our body. These regimens are carried out in very small doses at the "picogram" level. Therefore, as a result of continuous exposure to EDs such as BPA, minor changes in hormone levels may cause major changes in biological function, particularly over the long term (2).
BPA is an ED (24) and is ubiquitous in the environment due to its widespread use in many consumer products globally over the past 30 years including in toys, baby bottles, plastic storage containers, heating containers for food and beverages, the lining of metal cans, medical equipment, consumer electronics and dental sealants, to give but a sample of the products containing BPA. A recent hypothesis states that BPA exposure may lead to many health risks (25), particularly obesity (26) and poor reproductive health (27). As exposure to this compound during a critical period, such as childhood, will provide a basis for exposure-related health problems, it is vital to determine the extent of BPA exposure in childhood both for the health of the individual and for future healthcare planning.  (7). Similarly, the 3-5 year age group (GM 3.55 μg/L) had a higher urinary BPA concentration than the 6-8 (GM 2.72 μg/L), 9-11 (GM 2.22 μg/L), and 12-14 (GM 2.42 μg/L) year age groups in the German Environmental Survey for Children (29). These results indicate that younger people, particularly infants and children below the age of six, are subjected to greater exposure risk. Similar results were obtained in our study.
In this study, no significant associations between the consumption of various canned foods and beverages and BPA levels were found (p>0.05) ( Table 6). Urinary BPA levels of children consuming their food from heated plastic containers tended to be higher, but it was not statistically significant (p>0.05). Dental materials made of BPA derivatives such as BPA-dimethacrylate and BPA-diglycidyldimethacrylate, have been used as an alternative to mercury amalgams in dentistry. Therefore, in this study, whether the children had white dental filling was also evaluated. Composite restorations were not associated with urinary BPA concentrations in our study (p>0.05). Further, there were no statistical associations between BPA levels and the use of plastic materials and toys (p>0.05).
A few studies have determined the BPA exposure level of individuals in Turkey. In 2014, mean urinary BPA values were 0.61 µg/g creatinine in 200 people from Mersin city (15). In a further study, BPA amounts were quantified for 26 female children aged 4-8 years having the endocrine condition Idiopathic Central Precocious Puberty (ICPP) and 21 healthy controls. The average BPA concentration was 1.62 µg/g creatinine in the healthy group, whereas this value was 8.34 µg/g for the ICPP group (30). As a result, the estrogenic effects of BPA may be an etiologic factor for ICPP. Similarly, a study was performed on newly diagnosed ICPP patients (n=42; mean age 7.4±0.68 years) and peripheral  In a very recently completed study, BPA was detected in 100% of 40 maternal urine samples (GM; 0.12 μg/L), their 1-2-month-old infant urine samples (GM; 0.13 μg/L) and breast milk (GM; 0.12 μg/L). However, these BPA concentrations were relatively low compared to previous studies (32). In another recent study, urinary BPA levels of 50 children with type 1 diabetes mellitus and of 50 healthy children, all aged between 5 and 18 years, were measured using HPLC (38). In this study, urinary BPA levels of children with type 1 diabetes mellitus and healthy children were found to be 27.71±17.53 μg/g creatinine and 25.37±17.89 μg/g creatinine respectively. These values are somewhat higher than the values found in our study and other previous studies. This may be due to the HPLC method used to determine urinary BPA levels. Since HPLC is not a low-precision chromatographic method for determining BPA levels, it is not currently preferred by researchers to determine low BPA levels in biological materials.

Study Limitations
We believe that the present study makes an important contribution to the limited information about exposure to BPA during childhood. Although 125 children from Ankara were included in this study, this number is not sufficient for this type of population biomonitoring study. However, our results might be evaluated as preliminary finding for Turkish children. In order to provide a better understanding of exposure to BPA, studies on a larger population are needed and daily exposure levels from different sources should be determined.   developmental periods should be undertaken to evaluate the likely long-term impact of these EDs. It is believed that the results of this study will enhance awareness not only for BPA, but also for other chemical compounds, amongst health care professionals with an empahsis on pre-school aged children.