Human body burden of bisphenol A: a case study of lactating mothers in Florianopolis, Brazil

Exclusive breast milk is the diet recommended by the World Health Organization (WHO) until 6 months of age. However, breastfeeding has the potential of transferring certain toxic chemicals from the mother to the infant. Bisphenol A (BPA) is a synthetic chemical used as a monomer in polycarbonate plastics and epoxy resins. Information on BPA concentration in the breast milk of lactating mothers is very limited; thus, this study aimed to determine the concentration of BPA in the colostrum of 64 post-partum women at a university-affiliated tertiary hospital in South Brazil. The results showed that all the breast milk samples contained a high concentration of BPA with a median value of 34.18 ng/mL. Furthermore, the concentration of BPA in mothers was influenced by the consumption of foods packaged in plastic packaging, especially when the plastic is heated (p = 0.0182). The total daily intake of BPA in breastfed infants was 19.5 µg/kg/day and 28.5 µg/kg/day was recorded at the 95th percentile of body weight per day, which is higher than the maximum daily intake estimated by the European Authority of Food Safety. These data showed a high concentration of BPA in the breastmilk of the lactating mothers which might be through the use of plastic containers as food/drink packages. This is of public health importance as the high concentration of BPA in their breast milk can be an indicator of potentially serious health problems in these mothers and much more in the babies breastfed with BPA-contaminated breast milk.


Introduction
Exclusive breast milk is the recommended nutrition by the World Health Organization (WHO) until 6 months of age, followed by introducing foods with continual breastfeeding. Lactogenesis, the process of milk production and secretion, can be divided into two stages: lactogenesis I and II. Lactogenesis I begins during gestation and is characterized by the development of the breast, while lactogenesis II starts after birth and signifies the start of milk secretion (Preusting et al. 2017). The process of breastfeeding is very important to both the breastfeeding mother and the child. During this process, chemicals, microorganisms, and immunity can be transferred from mother to child which makes breastfeeding an important route of infants' exposure to many chemical agents some of which can be of public health importance (Braun 2016). To help solve this problem, more research is needed to assess the child's exposure to these chemical agents, strengthen the measures put in place to reduce this exposure, and demonstrate the harmful effects of ingesting these products. In this article, we demonstrate a chemical (Bisphenol A) found in the breast milk of mothers from Florianopolis-Brazil, proving that the lack of public policies in the country regarding the consumption of products from this chemical by lactating mothers has consequences for their breastfeeding children. We hope that with the data of the present study, the current legislation in South Brazil will become more stringent.
Bisphenol A (BPA), also called 2,2-bis (4-hydroxyphenyl) propane, is a white crystalline compound with a molecular weight of 228.29 g/mol, a melting point of 156 °C, and has low solubility in water. BPA has been widely used as a monomer in plastic polycarbonate and epoxy resin in food packaging, dental sealants, and ducts (Huang et al. 2012). In addition, it is also used for coating cans, automotive equipment, electrical and electronic equipment, in construction, and as a coating for drinking water pipes, among many others. Its high industrial production (up to 2.7 billion kilograms per year) and also its widespread domestic use have made BPA one of the most ubiquitous environmental chemicals in the world, thus making it one of the Persistent Organic Pollutants (Haffner and Schecter 2014). The release of BPA into the environment can occur during the manufacture, transport, and processing of chemical products and after consumption as well, through the discharge of effluents from municipal water treatment plants, leaching from sanitary landfills, combustion of domestic waste and the natural decomposition of plastics in the environment (US EPA 2010).
Both freshwater and marine water can be contaminated with BPA, through the migration of BPA-based products, effluents from water treatment plants, and also through landfills. More and more studies have been carried out to collect data and analyze BPA in the aquatic environment (Huang et al. 2012). In soil, despite having a short halflife (between 1 and 10 days), the presence of BPA can present a significant concern due to its ubiquity. (Fox et al. 2007). Regarding atmospheric contamination, a positive correlation was found between BPA and 1,3,5-triphenylbenzene, a substance used as a tracer for plastic burning in urban regions, indicating that the burning of plastics in open-air domestic waste is a source significant amount of atmospheric BPA emission; therefore, this contamination can be caused by the combustion process (Huang et al. 2012). In addition, another study demonstrated that BPA concentrations in indoor air in homes were higher than concentrations in outdoor air, indicating that household items and furniture materials are also potential sources of atmospheric BPA emissions (Wilson et al. 2001).
BPA is a known endocrine disruptor, and its estrogenic and antiandrogenic activities have been described in glucocorticoids and thyroid hormones (Skledar and Mašič 2016). The primary source of human exposure to BPA is through the oral route, by eating contaminated food and drinks, although air, dust, and water are other possible sources of exposure. BPA can penetrate food from the internal epoxy resin linings of canned foods and consumer products, such as polycarbonate tableware, food storage containers, water bottles, and baby bottles. The degree to which BPA passes from polycarbonate vials to liquid or food depends on the product's temperature and the container. The greater the temperature change, the greater the release of BPA (Dualde et al. 2019). Human exposure to BPA also occurs through environmental means such as ambient air, indoor air, drinking water, soil, and dust, in addition to the use of consumer products. Even with various forms of exposure, food intake is the main source, hence the importance of monitoring the levels of BPA contamination in food (Huang et al. 2012), as sumarized in Fig. 1.
Reports have shown high human exposure to BPA with documentation of the high occurrence of the analyte in biological samples, especially urine samples (Azzouz et al. 2016). Exposure to BPA can cause numerous adverse health effects such as diabetes, obesity, cardiovascular diseases, reduce basal testosterone secretion, and decrease fertility in males (Rochester 2013;Eladak et al. 2015;Alabi et al. 2021).
It has been shown that a high concentration of urinary BPA during pregnancy increases the possibility of giving birth to children with lower birth weights (Huo et al. 2015). BPA has also been shown to cross the human placenta barrier and present a risk to fetal development (Corbel et al. 2014). Developmental periods, especially prenatal, birth, and childhood, are the most sensitive developmental stages to the toxic effect of BPA, and because it is an endocrine disruptor, it can cause endocrine changes and metabolic disorders (Braun 2016). Public health concern about infant exposure to BPA is on the increase. According to an expert report by the Food Agriculture Organization (FAO) and WHO, it is estimated that the average exposure of exclusively breastfed babies (0 to 6 months) to BPA is 0.3 µg/ kg body weight per day. Exposure estimates are generally higher for babies fed formula in bottles containing polycarbonate compared to bottles without polycarbonate. The most significant exposure occurs in children from 0 to 6 months of age who are formula-fed, suffering an intake of 2.4 µg/kg of body weight per day of BPA. Once solid foods are introduced (at 6-36 months), exposure to BPA decreases proportionally with increasing body weight (WHO 2010).
In Brazil, Resolution RDC N ° 41, 2011 was enacted to prohibit the use of plastics in infant bottles (Ministério Da Saúde 2011); however, lactating mothers may be exposed to BPA due to contact with plastic products and packaging intended for adults thereby exposing their infants to BPA or its metabolites through breastfeeding. Since breastfeeding is a potential source of an infant's exposure to BPA, there is a need to constantly monitor the exposure of lactating mothers to BPA which can invariably transfer to the lactating infants.
Despite the high rate of the use of plastic containers by women of reproductive age and lactating mothers in many countries, information about the body burden of BPA in lactating mothers is scarce in the literature and not available at all in Brazil, hence, no data on infant's exposure to BPA through breastfeeding. Therefore, this study aimed at determining the concentration of BPA in the colostrum of mothers hospitalized at University Hospital Professor Polydoro Ernani de São Thiago through the enzyme-linked immunosorbent assay and to establish a possible association(s) between the use of plastics by the mother and BPA-contaminated breast milk.

Participants
This study was approved by the University Hospital Professor Polydoro Ernani de São Thiago (HU-UFSC) and the Ethical Committee of The Federal University of Santa Catarina (CAAE 68,008,317.4.0000.0121). Participants from Florianopolis, Brazil, were recruited immediately after childbirth between January and April of 2019. To participate, patients accepted all study procedures and signed the written informed consent. Mothers infected with human immunodeficiency virus (HIV) 1-2, human t-lymphotropic virus (HTLV), or any other clinical impairment for breastfeeding were excluded. A total of 80 participants were included in the study (Fig. 2).

Information gathering
A well-designed questionnaire was used to collect the necessary information from the participants while their medical records were used to obtain medical information about them. The mothers were asked about pre-and post-gestational, sociocultural, and socio-economic information. Also, information about their usage of plastics and plastic materials for food and drink was collected. Clinical data such as Capurro (method for assessing the newborn's gestational age), type of delivery, newborn weight at birth, and duration of labor (the onset of labour was considered when contractions became frequent and regular) were obtained from the patients' medical records. After birth, the participants were followed up monthly by telephone for up to 6 months and were instructed to attend monthly visits to their pediatrician of choice (private healthcare network or Basic Health Unit) and receive information about breastfeeding importance. Information on their children's growth and/or possible complications was also collected. The breastfeeding status was defined as follows: -Exclusive breastfeeding; -Mixed breastfeeding (formula and breast milk); -Only formula.

Sample collection and storage
At least 2 mL of breast milk was collected between 24 and 48 h after birth by manual milking and transferred to clear BPA-free plastic tubes. The samples were kept at − 80 °C throughout the study.

Fat extraction and determination of bisphenol A in breast milk
Only 64 of the 80 breast milk samples collected were centrifuged at 11,200 g for 30 min at 4 °C, and the liquid intermediate layer was collected using a micropipette. The other 16 were used as a test to standardize the methodology. BPA concentrations were analyzed in the 64 fatless breast milk samples through a sandwich-type immunoenzymatic assay kit (ELISA) manufactured by MyBioSource (San Diego, California, USA). The analysis was performed according to the protocols provided by the manufacturer. In summary, the samples and the calibration curve were added to corresponding wells, incubated, and subsequently, the wells were washed. Thereafter, the liquid biotinylated human BPA antibody was added to each well and again incubated and washed. Afterwards, the enzymeconjugated liquid was added to all wells, except the blank, incubated, and washed. Finally, the color reagent was added to each well, incubated, the stop solution was added, and the absorbance was read in a spectrophotometer at 450 nm. Concentrations were determined by interpolation on a calibration curve, and the sensitivity of the assay was 0.23 ng/mL. A comparison was also made between the concentration of BPA in breast milk and the nutritional status of the patient according to the table developed by Atalah et al. (1997).

Evaluation of the newborn's exposure to BPA
To obtain an estimated exposure of breastfed children to BPA, information on milk consumption and the total BPA concentrations in the participant's breast milk were combined. Exposure to BPA in breastfed newborns was estimated using the deterministic approach. In this approach, the estimated daily consumption was calculated according to the following equation: where C is the breast milk's BPA concentration, and M is the average milk consumption rate (amount of human milk consumed per day) (Dualde et al. 2019), so the M value was 480 mL/kg/day for girls and 495 mL/kg/day for boys. Breast milk intake of 150 mL/kg of newborn body weight per day was used. Also, exposure to BPA was calculated using the upper percentile (the upper percentile is reported as the mean plus two standard deviations) of breast milk intake as recommended by the US Environmental Protection Agency (EPA) for children from birth to less than 1 month of age (220 mL/kg of body weight of the newborn per day), (US EPA 2011).

Determination of the maximum permissible BPA concentration in breast milk
The maximum allowable concentration of BPA in breast milk, so that the intake of the newborn does not exceed the tolerable daily intake (TDI) of 4 μg/kg/day established by the European Food Safety Authority (EFSA), (EFSA 2015), was calculated thus: where C is the maximum allowable BPA concentration in breast milk, TDI is equivalent to 4 μg/kg/day, and M is the average rate of milk consumption per day.
We used the average weight of children born at 40 weeks of gestation in Brazil (3.2 kg for girls and 3.3 kg for boys) (Pedreira et al. 2011) and 150 mL/kg/day as the average amount of milk ingested by children up to 1 month of age (US EPA 2011).

Statistical analysis
All analyses were performed using MedCalc v19.0.4 software (MedCalc, Ostend, Belgium). The results were expressed as medians and range or relative frequency. Differences between groups were assessed using the Mann-Whitney U tests for numerical variables, and Chi-square for analyses of the association of qualitative variables. Logistic regression was used to test the variables. For this purpose, BPA concentrations were divided into two groups: high and low. The high concentration group was those who presented values above the median and the low ones, values below the median. The odds ratios were calculated (OR), as well as their 95% confidence intervals (95% CI). The differences were considered statistically significant when P ≤ 0.05.
By determining BPA in human milk, a possible association(s) between the BPA exposure populations that are highly exposed to environmental chemicals can be established and identifying these populations is crucial for creating public policies in the country. Table 1 shows the characteristics of the women from HU-UFSC who participated in this study. All the 80 women were in their post-gestational period with a median age of 27 years old. Most of the women were white (65%), followed by 35% black and only 2% indigenous. Normal delivery (64%) is the main type of delivery with a median birth weight of 3.18 kg for newborns.

Clinical data
The response of the participants to the use of plastic materials for food and drink before and during pregnancy, extending to the lactation period, is shown in Fig. 3. The majority of the participants have the habit of packing hot food/liquid in plastic containers (51%) and heating food/liquid in plastic containers in the microwave (50%). A total of 14% of the participants get their hot liquids such as coffee and tea in plastic containers once a day while 21% eat canned food/ drink once a day.

Breastfeeding status
After 6 months of telephone follow-up with the mothers, the breastfeeding status was determined as 78% of the mothers practiced exclusive breastfeeding, 8% practiced mixed breastfeeding, and 14% used formula only.

Determination of bisphenol A
Enzyme-linked immunosorbent assay used for the detection of BPA in the breast milk of the nursing mothers was sensitive, being able to detect BPA in all the samples tested as shown in Fig. 3 and more detailed in Supplementary material 1. The concentration of BPA in breast milk was also associated with breastfeeding to assess it as a disruptor of lactogenesis II (Fig. 4). In this study, a high concentration of BPA in breast milk did not affect the outcome of breastfeeding. Furthermore, a comparison made between the concentration of BPA in breast milk and the nutritional status of the mothers (Fig. 5) showed no significant association ( Table 2).
The logistic regression of the following variables, gestational age, participants who ate canned food, heated food in a plastic container in the microwave, drank hot liquids in plastic packaged food before or after cooling in plastic containers, type of delivery, and birth weight (P = 0.005), showed that mothers who packed food in plastic containers and did not wait to cool down before packaging had 22.05 times (95% CI = 1.69 to 287.64; P = 0.0182) more chances of having high BPA concentrations compared to mothers who did not package food in plastic containers. In addition, women who prepared their food in a plastic container but allowed it to cool down before packaging were 9.81 times (95% CI = 1.05 to 91.06; P = 0.0445) more likely to have high BPA concentrations in breast milk compared to mothers who did not package food in plastic materials. The relationship between BPA concentration and skin color (white, black or indigenous) was also evaluated; however, there was no statistically significant difference between the groups. In addition, the Mann-Whitney U test was performed to assess the relationship between age and BPA concentration. For this, the median age (26 years) was used and the patients were separated into two groups (up to 26 years and above 26 years). In this case, patients aged up to 26 years had a statistically lower BPA concentration than patients older than 26 years (P = 0.0023).

Evaluation of newborn's exposure to BPA
In the deterministic approach, the average consumption of BPA by the newborn was calculated. Figure 6 shows the average daily intake of BPA by newborns and the upper percentile of milk consumption. The median concentration of BPA intake by the newborn up to the first month was 15.4 µg/kg/day and 22.5 µg/kg-day for the upper percentile of milk consumption which is above the maximum acceptable daily intake of 4 μg/kg/day established by European Food Safety Authority (EFSA 2015).

Evaluation of the maximum acceptable BPA concentration in breast milk
The maximum permissible BPA concentration in breast milk (C) in the present study was calculated to be 8 ng/mL (Fig. 7). The data showed that all the participants in this study had breast milk BPA concentrations above the calculated upper limit.

Discussion
BPA is an industrial chemical that is widely used for the production of polycarbonate plastic used to make materials that come into contact with food, such as baby bottles and food containers, and epoxy resins used as a protective coating for canned food and drinks and coating on metal lids for glass jars and bottles. These result in consumer exposure to BPA through diet (WHO 2010). Some studies have evaluated the amount of BPA that can be leached from packaging materials, demonstrating human exposure. Nerin and colleagues analyzed microwave-safe plastic food container packaging. BPA was found at a concentration of 30 μg/g of plastic and the level of migration to food was 6.5 μg/g of food. In addition, heating can further increase the level of BPA leaching (Nerín et al. 2003). Brotons and colleagues performed one of the first studies that analyzed the migration of BPA in the lining of cans, resulting in a BPA leaching range of 4 to 23 μg per can (Brotons et al. 1995). Takao and colleagues also analyzed the level of BPA after heating the cans, resulting in an average increase of 18.2 × in the concentration of BPA in the water contained within the container (Takao et al. 2002).  Breastfeeding is fast becoming an important route of infant exposure to BPA, hence the urgent need for biomonitoring of breastmilk BPA in lactating mothers.
This study reported the presence of a high concentration of BPA in the breast milk of lactating mothers in Florianopolis with a median of 34.18 ng/mL. This concentration is higher than what has been previously reported in other countries. In addition, all newborns were consuming BPA above the maximum permissible limit; thus, they were more exposed to BPA than the safe limit and consequently more susceptible to the consequences of exposure to BPA. In this first moment, the exposure of the newborn to BPA, due to the high rates of exclusive breastfeeding, occurs through breastfeeding. However, even formula-fed babies are ingesting BPA due to the container that stores the milk. The situation becomes even more worrying when we wonder how long these newborns will be exposed, as they will all continue to consume large concentrations of BPA until 6 months, either through breast milk or infant formula, at which time they are highly susceptible. After that, even with the ban on children's plastic packaging, they can continue to consume breast milk, artificial milk, water, and food stored in plastic, extending into adulthood.
To the best of our knowledge, this study is the first in Brazil to analyze the concentration of BPA in the breast milk of lactating mothers. Even though the law that prohibits the use of BPA in baby bottles has been enacted in Brazil, the result of the present study showed that this is not enough to prevent infants' exposure to this xenobiotic. The lack of public policies in Brazil which prevents exposure to BPA is worrisome since newborns are exposed to BPA through breast milk and they are the most sensitive risk group to the toxic effects of BPA exposure. For example, newborns, exposed both during prenatal care, due to the BPA's ability to cross the placental barrier, and after birth, through breast milk, can develop a series of cerebral consequences, such as disturbances in development and interruption of dopaminergic activity (Van Den Dries et al. 2020). Studies on prenatal and perinatal exposure in rodents that have been exposed to relevant human doses of BPA have reported increased growth of epithelial tissue, decreased apoptosis, decreased latency, and increased incidence of pre-neoplastic and neoplastic lesions in the mammary gland. Other studies also in rodents have linked exposure to BPA to neurobehavioral deficits, reproductive disorders, and liver tumours (Tucker et al. 2018).
The result of the logistic regression showed that women who packaged food in plastic material are more likely to have high concentrations of BPA, an indication of a positive association between the use of plastic materials and breast milk BPA concentration. There is, therefore, a need for the restriction of the use of plastic materials by women of reproductive age, pregnant, and lactating mothers. BPA has been associated with female infertility, as it has been detected more frequently in infertile women. Its deleterious effects are more critical during perinatal exposure; however, both prenatal, perinatal, and postnatal exposure can impair several stages of ovarian development. Animal studies have also seen that BPA, because it causes hormonal disruption, has been associated with an increased risk of endometriosis, polycystic ovary syndrome, and affecting ovarian function (Pivonello et al. 2020). Changes in eating habits, such as reduced consumption of canned foods and drinks or food stored in plastic packaging as well as the replacement of plastic bottles and jars with glass, should be encouraged. Although BPA is omnipresent in the environment, however, a decrease in consumption can mitigate the consequences of ingesting high concentrations of BPA.
Furthermore, no association was found between breast milk BPA concentration and lactogenesis II in the present study. Instead, the study showed a high percentage of mothers who continued to breastfeed within the first 6 months of birth. This observation might be due to a breastfeeding incentive program called Central do Incentivo ao Aleitamento Materno (CIAM) at HU-UFSC, where the participants were recruited. CIAM assists all mothers admitted to the HU-UFSC at the time of delivery so that they can breastfeed and continue exclusive breastfeeding after being discharged from the hospital for at least six months. During the study, to overcome issues related to the gathering of information on plastic daily use, a more detailed questionnaire was created to facilitate the measurement, which later corroborated the results of BPA levels in the mothers' breast milk. With these results, we suggest that the mothers and their newborns were exposed to high concentrations of BPA, through the ingestion of breast milk, which shows that the public policy adopted in the country is not enough to avoid exposure. More stringent legislation is needed that expands the bans on the use of BPA in the development of products intended for adults as well, not just children.

Conclusion
The present study found high concentrations of BPA in the breast milk of mothers in a hospital in Florianopolis, Brazil, compared to the reports in the literature from other countries, potentially translating to the high exposure of newborns to BPA. Furthermore, all the participants in this study had BPA concentrations in breast milk above the calculated maximum permissible BPA concentration in breast milk, and all the newborns were ingesting more BPA than the maximum acceptable daily intake. The results further showed that packaging food in plastics increases the chances of having a high concentration of BPA in breast milk. However, exposure to BPA did not alter the mother's nutritional status or lactogenesis II. These data are of public health importance, and hence, public policies that reduce both adult and children's exposure to BPA must be enacted in Brazil. Further study is needed to assess the concentration of BPA in lactating mothers in other hospitals in Brazil to gain holistic data on the body burden of BPA in Brazil.

Policy suggestions
This study was developed after a long review of the literature, which showed the difference between exposure to BPA among countries, according to the public policies adopted. In Brazil, the use of BPA is currently prohibited only for the production of children's packaging, which raised the question of whether this is enough. After analyzing the concentration in breast milk, we were able to answer this question and we saw that it is not, considering that we found high concentrations of BPA in mothers' breast milk in this study. The data of the present study, therefore, suggest that the present legislation needs revision to include a ban on any BPA-containing products used by pregnant or lactating mothers.
Author contribution All authors contributed to the study's conception and design. Material preparation, data collection, and analysis were performed by Carolina Dumke de Siqueira. The first draft of the manuscript was written by Carolina Dumke de Siqueira and all authors commented on previous versions of the manuscript. Alabi Okunola Adenrele, Ana Carolina Rabello de Moraes, and Fabíola Branco Filippin-Monteiro edited the manuscript. Fabíola Branco Filippin-Monteiro also supervised the manuscript. All authors read and approved the final manuscript.
Funding This study was supported by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) scholarship to Carolina Dumke de Siqueira, and financially supported by Fundação de Amparo à Pesquisa do Estado de Santa Catarina (FAPESC).

Data availability
Datasets generated during the current study are not publicly available to maintain patient anonymity but are available from the corresponding author upon reasonable request.

Declarations
Ethics approval This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of The Federal University of Santa Catarina (CAAE 68008317.4.0000.0121).

Consent to participate
All authors consent to participate in the works of the manuscript.

Competing interests
The authors declare no competing interests.