Associations of lifestyle factors with phthalate metabolites, bisphenol A, parabens, and triclosan concentrations in breast milk of Korean mothers

Associations of lifestyle factors with phthalate metabolites, bisphenol A, parabens, and triclosan concentrations in breast milk of Korean mothers

Journal Pre-proof Associations of lifestyle factors with phthalate metabolites, bisphenol A, parabens, and triclosan concentrations in breast milk of ...

2MB Sizes 0 Downloads 7 Views

Journal Pre-proof Associations of lifestyle factors with phthalate metabolites, bisphenol A, parabens, and triclosan concentrations in breast milk of Korean mothers Ju Hee Kim, Dohyeong Kim, Seung-Min Moon, Eun Jung Yang PII:

S0045-6535(20)30342-8

DOI:

https://doi.org/10.1016/j.chemosphere.2020.126149

Reference:

CHEM 126149

To appear in:

ECSN

Received Date: 9 November 2019 Revised Date:

5 February 2020

Accepted Date: 6 February 2020

Please cite this article as: Kim, J.H., Kim, D., Moon, S.-M., Yang, E.J., Associations of lifestyle factors with phthalate metabolites, bisphenol A, parabens, and triclosan concentrations in breast milk of Korean mothers, Chemosphere (2020), doi: https://doi.org/10.1016/j.chemosphere.2020.126149. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier Ltd.

Ju Hee Kim: Conceptualization, Methodology, Validation, Writing- Original draft preparation, Supervision, Project administration, Funding acquisition. Dohyeong Kim: Methodology, Investigation, Writing-Original Draft, Writing- Review & Editing. Seung-Min Moon: Software, Formal analysis, Visualization. Eun Jung Yang: Investigation, Resources, Data Curation, Project administration

Title Page

Associations of lifestyle factors with phthalate metabolites, bisphenol A, parabens, and triclosan concentrations in breast milk of Korean mothers Ju Hee Kima, Dohyeong Kimb*, Seung-Min Moonc, Eun Jung Yanga a

College of Nursing Science, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul, 02447, South Korea

b

School of Economic, Political and Policy Sciences, University of Texas at Dallas, 800 W Campbell Rd, Richardson, TX, 75080, United States

c

Department of Public Administration, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul, 03722, South Korea

*Corresponding author. School of Economic, Political and Policy Sciences, University of Texas at Dallas, 800 W Campbell Rd, Richardson, TX, 75080, United States. Email address: [email protected] (D. Kim), [email protected] (J. Kim), [email protected] (S. Moon), [email protected] (E. Yang)

1

Associations of lifestyle factors with phthalate metabolites, bisphenol A, parabens, and triclosan concentrations in breast milk of Korean mothers Abstract The toxicity of endocrine disruptors depends on the synergistic interactions of biological, environmental, and behavioral factors. The specific effects of diet, consumer product use, and behaviors, however, are debated in the literature, particularly with regard to endocrine disruptors found in breast milk. This study aimed to measure the levels of phthalate metabolites, bisphenol A, parabens, and triclosan in breast milk and to investigate their associations with various lifestyle factors. The breast milk samples as well as surveys were collected from 221 first-time mothers throughout South Korea and each sample was analyzed for the presence of 15 endocrine disruptors. Phthalate metabolites were detected in 5.4–83.3 % of the samples, with median concentrations of 0.08–1.72 µg/L, while bisphenol A, parabens, and triclosan were detected in 25.8–88.2 % of the samples, with median concentrations of 0.12–1.47 µg/L. High levels of endocrine disruptors were associated with frequent consumption of fish and cup noodles; the use of plastic and disposable food containers; the use of air fresheners, lotions and make-up; the purchase of new furniture; and socioeconomic status. We also observed the potential role of moderate walking activity on the reduction of these chemicals in breast milk. Our data provide evidence of the potential effects of diet, consumer products, and behavior on the presence of phthalate metabolites, bisphenol A, parabens, and triclosan in breast milk. Future studies should include community or regional impact on a mothers’ exposure to endocrine disruptors, to assess the joint contribution of both individual and neighborhood factors. Keywords: breast milk; endocrine disruptors; lifestyle; diet; consumer product; behavior 2

1. Introduction

Over the last fifty years, chemicals such as phthalates, bisphenol A (BPA), parabens, and triclosan (TCS) have been used in a variety of consumer products, personal care products, and medical items (Centers for Disease Control and Prevention, 2019). Recently however, they have been banned or limited in many products because of their suspected toxicity and roles as endocrine disruptors (National Research Council (US) Committee on the Health Risks of Phthalates, 2008; FDA, 2019), and there is evidence that they have a negative impact on human health. Phthalates are known to cause reproductive developmental damage, neurodevelopmental problems, growth retardation, asthma, and allergies (Cho, 2012; Jurewicz et al., 2013; Polanska et al., 2016; Wassenaar et al., 2017; Kim et al., 2018; Rowdhwal and Chen, 2018). BPA is a class of manufactured chemicals with structural similarities to the hormone 17ß- estradiol (Zimmers et al., 2014) which may cause damage to the pituitary-thyroid axis, immune system problems, adverse reproductive and developmental effects, changes in thyroid hormone levels, and increased risk of type 2 diabetes, infertility, impaired neuropsychological development, low birth weights, and adverse birth outcomes (Lee et al., 2014; Huo et al., 2015; Andrianou et al., 2016; Song et al., 2016; Ziv-Gal and Flaws, 2016). Moreover, strong endocrine activities of BPA substitutes, such as bisphenol S, bisphenol F, and bisphenol B, have been substantiated in the literature (Rochester and Bolden, 2015; Serra et al., 2019). Animal and human studies have reported that parabens and triclosan are associated with endocrine disruption, especially changes in thyroid hormones, and interference with ion channels (Boberg et al., 2010; Koeppe et al., 2013; Giulivo et al., 2016; Yueh and Tukey, 2016; Weatherly and Gosse, 2017). These chemicals enter the human body through the ingestion of foods, dermal absorption from the skin, and air inhalation (Center for the Evaluation of Risks to Human Reproduction, 2000). Most chemicals that enter the body are released to the urine within a few hours, but some accumulate in the body and are detected in serum, saliva, breast milk, and the placenta, where they 3

may affect the individual’s health directly or indirectly (Hines et al., 2009; Lin et al., 2011; Larsson et al., 2014; Lee et al., 2018). Pregnant women, lactating mothers, and infants are more vulnerable than others to these risks due to their physiological characteristics. Fetuses and infants in particular have rapid cellular differentiation and incomplete metabolism, which makes it difficult for them to eliminate harmful chemicals from their systems (Birnbaum, 1994; Kim et al., 2015). Many previous studies have reported that pregnant women may transmit toxic chemicals to the fetus through the placenta, and lactating mothers transmit them to their newborns through their milk (Balakrishnan et al., 2010; Morck et al., 2010; Lin et al., 2011). Furthermore, it has been described that mycoestrogen such as Zearalenone can cross the human placental barrier (Warth et al., 2019), and the combination of exposure to xenoestrogens, even at low dose, can be potentially related to synergistic adverse effects such as neurodevelopmental delay and breast cancer later in life (Fucic et al., 2012; Kim et al, 2018; Vejdovszky et al., 2017). Breast milk is particularly useful sample in the study of toxin exposure, because it can be noninvasively sampled and used to assess the environmental exposure of breastfed infants (Kim et al., 2015). The chemicals in breast milk have been analyzed in many different countries, using a variety of sample sizes and analytical methods. For example, since 2000, studies related to phthalate analysis have been conducted in Canada, Denmark, France, Germany, Italy, Korea, Sweden, Taiwan, and the USA, using anywhere from 3 to 130 samples (Fromme et al., 2011; Lin et al., 2011; Guerranti et al., 2013; Arbuckle et al., 2016; Bubba et al., 2018; Kim et al., 2018), while BPA analysis was performed in France, Japan, Korea, Spain, and the USA, using 3 to 127 samples (Yi et al., 2010; Migeot et al., 2013; Mendonca et al., 2014; Rodríguez-Gómez et al., 2014; Zimmers et al., 2014; Deceuninck et al., 2015; Nakao et al., 2015; Kim et al., 2018; Lee et al., 2018). However, these studies did not have adequate sample sizes to estimate the potential risk of the chemicals, and the analysis of the chemicals in the breast milk focused on either phthalates or bisphenol A. 4

Therefore, it is necessary to use larger sample sizes and examine a variety of chemicals in breast milk to assess the exposure of breastfed infants to chemicals. As breast milk may be the only source of nutrition for breastfed infants, it is important to accurately measure their daily exposure to chemicals to understand the potential risks. However, in most previous studies, the daily chemical intake was estimated using the reference values of each country, rather than actually measuring the amount of breastfeeding and the infants’ weight gain (Guerranti et al., 2013; Kim et al., 2015; Bubba et al., 2018). Furthermore, exposure to chemicals occurs mainly through diet, use of personal care products, and exposure to household goods, and the consequences are especially important for infants. However, studies that examined the association between chemicals and diet or lifestyle have been conducted primarily using urine samples, and few have used breast milk samples (Larsson et al., 2014). Only one recent study reported that the consumption of whipped cream and purified water was significantly associated with the presence of mono-isobutyl phthalate (MiBP) and mono-n-butyl phthalate (MnBP) in breast milk (Kim et al., 2015). It is therefore important for breastfeeding mothers to be informed about chemical exposures from their diet and lifestyle. Therefore, in this study, we used nationwide data to identify the concentrations of 15 chemicals in the breast milk of Korean mothers, including 10 main phthalate metabolites, BPA, three parabens, and TCS, using a larger sample size than in previous studies. We then examined the relationship between healthy behaviors such as exercise, hand washing, and ventilation, as well as diet and consumer products, and the concentrations of a diverse range of chemicals in the breast milk. In addition, our study estimated the daily intake of phthalate, BPA, parabens, and TCS of the breastfed infants by requesting a qualified professional to measure the infant’s weight and the amount of breast milk the infant received through nursing to increase the sensitivity and accuracy.

2. Materials and methods

5

2.1. Study population and sample collection

The study population was made up of mothers who were receiving lactation coaching at the nationwide breastfeeding clinics across Korea. For representative sampling, the country was divided into four regions (Seoul metropolitan, Chungcheong, Honam, and Yeongnam region), and the stratified samples taken for each region was estimated based on the birth rate of each region between 2015 and 2017 in Korea. The study population was limited to primipara mothers who birthed a single infant (Guerranti et al., 2013), who were currently breastfeeding, and who had lived in their current residence for more than one year. Mothers who had inflammation, such as mastitis, were excluded from the study. Breast milk sampling and individual interviews with subjects were conducted from July 2 to September 9, 2018. The qualified nurses collected 20 mL of breast hind milk using hand expression to avoid exposure to endocrine disruptors from plastic materials (Arbuckle et al., 2016), and the breast milk samples were stored in polypropylene tubes that did not contain endocrine disruptors. Samples were transported to the experimental laboratory and stored at -70 ℃ until they were analyzed. Initially, 223 breast milk samples were collected, but two samples were excluded due to disqualification. A total of 221 breast milk samples were analyzed. The questionnaire administered to each mother included 84 questions covering demographic information (area of residence, age, weight, height, education level, monthly income, occupation, residence type, residence period); obstetrical information (postpartum complications, dietary supplements taken during pregnancy, parity, type of delivery); dietary, lifestyle, and behavior information (food, drinking, smoking, exposure to secondhand smoke, level of physical activity, house ventilation, hand washing); and neonatal information (age, sex, birth weight, current weight, height). Moderate walking activity was defined as walking at moderate intensity during the past week, 50-70% of your maximum heart rate. In order to engage in an ethical research process and to protect the rights and safety of the 6

subjects, this study was subjected to review by the Institutional Review Board of Kyung Hee university (KHSIRB-18-029) and was approved.

2.2. Analysis of chemicals

BPA, methylparaben (MP), ethylparaben (EP), propylparaben (PP), TCS, and 10 phthalate metabolites [mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono-(2-ethyl-5-carboxypentyl) phthalate (MECPP), mono-n-butyl phthalate (MnBP), monobenzyl phthalate (MBzP), mono-(carboxyoctyl) phthalate (MCOP), mono-isobutyl phthalate (MiBP), mono-isononyl phthalate (MiNP), mono-2-ethylhexyl phthalate (MEHP), monoethyl phthalate (MEP)] in breast milk samples were investigated. The chemical analysis followed previous reports, with some modifications (Calafat et al., 2004; Ye et al., 2008). The chemical analysis was conducted in two

steps: five phenols first (BPA, TCS, and three parabens) and then 10 phthalate metabolites. A total of 15 analytes and isotope-labeled internal standards were purchased from Cambridge Isotope Laboratories (Andover, MA, US) (13C12-BPA, 13C12-triclosan, 13C4-MEHHP, 13C4-MEOHP, 13C4-MECPP, 13

C4-MnBP, 13C4-MBzP, 13C4-MCOP, 13C2-MiBP, 13C2-MiNP, 13C2-MEHP, and 13C2-MEP) and Toronto

Research Chemicals (North York, ON, Canada) (D4-methylparaben, D4-ethylparaben, and D4propylparaben). Reagents for chemical analysis were acetonitrile, water, n-hexane, acetic acid, phosphoric acid (HPLC grade, ≥ 98.0 %), β-glucuronidase/sulfatase from Helix pomatia (Sigma-Aldrich, St. Louis, MI, US) for BPA, TCS, parabens, and β-glucuronidase from E. coli K12 (Hoffmann-La Roche, Basel, Switzerland) for phthalate metabolites. These enzymes were applied to control hydrolysis in preprocessing, taking into account the different metabolic processes of phenols and phthalates (Silva et al., 2003).

7

The samples were prepared using the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) approach. First, 2.0 mL of each sample was placed in a test tube and kept at -20 ℃ until use. Next, 100 µL (200 µL for phthalate metabolites) of 1 M phosphoric acid buffer solution was added to the sample, which was thawed at room temperature and shaken. Then 2.0 mL of internal standards and enzyme buffer solutions were added and incubated at 37 ℃ for 90 minutes. Next, 10 mL of acetonitrile was added and the sample was shaken for 10 minutes. Subsequently, 3.0 g (1.0 g for phthalate metabolites) of magnesium sulfate, 1.0 g of sodium citrate tribasic dehydrate, and 1.0 g (0.5 g for phthalate metabolites) of sodium citrate dibasic sesquihydrate were added, after which the sample was shaken for 10 minutes and centrifuged at 2,800 rpm for 20 minutes. After n-hexane (C18 powder for phthalate metabolites) was added 4.0 mL to the separated 4mL upper layer solution, the sample was stirred and centrifuged for 10 minutes. Then 5.0 mL of the separated upper layer solution was concentrated with nitrogen, after which the water/acetonitrile (50/50, v/v) solution was reconstituted into 1.0 mL samples (water: acetonitrile=1:1 for volume). This solution was filtered with a 0.2-µm membrane filter (PTFE, polytetrafluoroethylene) and used as a sample for the analysis. The standard solution was added to the blanks at the same concentration and prepared under the same conditions as the samples. Both liquid chromatography (LC, Agilent 1290 Infinity system, Agilent Technologies, Santa Clara, CA, US) and tandem mass spectrometry (MS, Agilent 6490 Triple Quadrope, Agilent Technologies, Santa Clara, CA, US) were used simultaneously rather than the gas chromatography method, as it requires a relatively large sample volume (Ye et al., 2008). The LC column was an ACE 5 C18 (2.1 mm * 150 mm, 5.0 µm) HPLC-column (ACE® Advanced Chromatography Technologies, Scotland, UK). Solvent A was water and B was acetonitrile for the mobile phases. The LC conditions included a flow rate of 0.2 mL/min, column temperature of 30 ℃, and injection volume of 5 µL. In addition, electrospray ionization and the multiple reaction monitoring mode were applied in the MS condition (see Supplement 4 for details).

8

The analytical method followed a quality assurance protocol by measuring blank samples and performing internal quality controls for each batch of breast milk samples. The internal quality control included tests of linearity, accuracy, precision, and detection limits. For each analyte, the calibration curve consisted of five points that covered the concentration range in pooled breast milk and R2 was > 0.99 in the linearity tests of all analytes. The accuracy test was performed to evaluate the recovery rate at each of the three concentration levels (low, medium, and high) by spiking reference materials to the pooled breast milk sample. For all analytes, the recovery percentages from 93 to 115 %. The precision test included a comparison of inter- and intra-day samples. The maximum value of the relative standard deviation (RSD) was ≤ 10.0 % for both the intra- and inter-day tests of phenols and phthalate metabolites. The limit of detections (LODs) were calculated by multiplying the standard deviation of the results by 3.14 and adding a standard solution of 7 concentrations to the matrix (US EPA, 1984). The LOD for each analyte was estimated as follows: MEHP, 0.139 µg/L; MEP, 0.131 µg/L; MiBP, 0.188 µg/L; MiNP, 0.043 µg/L; MnBP, 0.282 µg/L; MBzP, 0.082 µg/L; MEHHP, 0.139 µg/L; MEOHP, 0.154 µg/L; MECPP, 0.113 µg/L; MCOP, 0.040 µg/L; BPA, 0.076 µg/L; TCS, 0.035 µg/L; EP, 0.035 µg/L; MP, 0.101 µg/L; and PP, 0.134 µg/L. For the external quality control, 30 samples were sent to different university laboratories and analyzed following the same standard operating procedure (LC-MS/MS), with all compounds showing ≥ 0.8 intra-class correlation values, except for one compound (MnBP, ICC value = 0.53; p-value = .054).

2.3. Estimation of daily chemical intake and risk assessment

The nurses measured each infant’s body weight and the amount of daily breastfeeding on the day the breast milk was sampled. The daily intake of phthalate metabolites, BPA, parabens, and TCS for the

9

breastfed infants was estimated using the following equation that was based on previous studies (Fromme et al., 2011; Kim et al., 2015).

Daily intake (µg/kg bw-day) = (BC (µg/L) * DBI (mL)/1000)/ body weight (kg)

where BC = chemical concentration in breast milk and DBI = daily breast milk intake amount for the infants. It is generally accepted that phthalates exist in both diester and monoester forms in breast milk, with a higher concentration of the diester than the monoester (Hines et al., 2009; Kim et al., 2015). Therefore, we estimated the concentration of diester by referring to the following ratios of parents / metabolites from previous studies: 0.10–3.73 for the DiBP/MiBP ratio, 0.39–2.78 for the DnBP/MnBP ratio, 1.70–18.4 for the DEHP/MEHP ratio, 0.98–2.13 for the BzBP/MBzP ratio, 2.63 for the diisononylphthalate (DiNP)/MiNP ratio, and 0.44 for the DEP/MEP ratio (Hogberg et al., 2008; Fromme et al., 2011; Arbuckle et al., 2016). To calculate the risk assessment, we compared our data for each of the chemicals with the tolerable daily intake (TDI) value recommended by the U.S. FDA, U.S. EPA, European Food Safety Authority (EFSA), and World Health Organization (WHO). For DEHP, DiNP, DnBP, and BBzB, TDI values of 50, 15, 10, and 500 µg/kg bw-day were established, respectively (European Food Safety Authority, 2005; Giovanoulis et al., 2018). The TDI value for DEP was chosen as 500 µg/kg bw-day (WHO, 2003), while that for BPA was 10 µg/kg bw-day. No TDI value of DiBP existed, but as the TDI value of DiBP is known to be similar to that of DnBP, the TDI value for DiBP was chosen as 10 µg/kg bw-day (Kortenkamp and Faust, 2010; Giovanoulis et al., 2018). The EC Scientific Committee for Food (SCF) evaluated parabens in 1994 and assigned a temporary acceptable daily intake value of 10 mg/kg bw for the sum of MP, EP, and propyl p-hydroxybenzoic acid esters, and their sodium salts. The estimated

10

worst-case aggregate internal exposure values for MP, EP, and PP were 3.1, 0.2, and 1.2 mg/kg bw-day (National Institute for Public Health and the Environment, 2018).

2.4. Statistical analysis

For undetected samples (the values below the LOD), we assigned a proxy value as LOD divided by the square root of 2 (Hornung and Reed, 1990). The normality of distribution was tested by the oneway Kolmogorov-Smirnov test. The Spearman correlation test was conducted to determine the correlations between the levels of chemicals in breast milk samples. A bivariate relationship among phthalate metabolites, BPA, parabens, and TCS exposure and food consumption, usage of consumer products, and health behaviors were tested using the Wilcoxon-Mann-Whitney test. All statistical analyses were conducted using Stata 14.0 (Stata Corp, College Station, TX, USA).

3. Results

3.1. Characteristics of the study population

Participants were between 19 and 42 years old (mean 31 years), with a maternal pre-pregnancy BMI ranging between 16.0 and 33.3 kg/m2 (mean 21.3 kg/m2). The mean maternal gestational weight gain was 5.7 kg (a range of -10.0–28.0 kg), and the mean weeks of gestation was 39.2 weeks (a range of 34.0– 41.6 weeks). Mean neonatal age, birth weight, and birth height values (and a range) were 34 days (3–108 days), 3.2 kg (2.2–4.3 kg), and 50.4 cm (40–57 cm), respectively. The amount of breast milk consumed per day ranged from 200 to 1260 mL (mean 723 mL). The details of the socioeconomic and maternal characteristics of the subjects are shown in Table 1. 11

3.2. Concentration of chemicals

Phthalate metabolites (MEP, MnBP, MiBP, MBzP, MEHP, and MiNP) were detected in 5.4–83.3 % of the samples, with median concentrations of 0.08–1.72 µg/L, and geometric means of 0.10–1.44 µg/L. MEHHP, MEOHP, MECPP, and MCOP were not detected. BPA, MP, EP, PP, and TCS were detected in 25.8–88.2 % of the samples, with median concentrations of 0.12–1.47 µg/L, and geometric means of 0.15–0.95 µg/L (see Table 2 for details). The chemicals we tested for in the breast milk samples were highly positively correlated with one another (see Supplement 2 for details).

3.3 Bivariate association between chemicals and risk factors

The results of the bivariate association analysis for the chemicals and maternal characteristics, food consumption, use of consumer products, and health behaviors are presented in Table 3. More importantly, the levels of MnBP were significantly positively associated with air freshener use (p = .045), while the levels of MiBP were significantly associated with air freshener use (p = .037) and the purchase of new furniture last 1 year (p = .010). The levels of MEHP were significantly positively associated with the purchase of new furniture within last 1 year (p = .025), while the levels of MiNP were marginally associated with consumption of ice cream (p= .058) and hand washing (p = .055). EP was significantly positively associated with frequent consumption of cup noodles (p = .006), and moderate walking activity last 1 week (p = .016). MEP was significantly associated with frequent consumption of fish (p = .049), air freshener use (p = .050), and the purchase of new furniture last 1 year (p < .001), while MBzP was significantly associated with use of plastic food containers (p =. 008). TCS was positively associated with use of disposable food containers (p = .034), use of lotion (p = .003), use of make-up (p = .035), and 12

moderate walking (p = .032), while MP was positively associated with use of plastic food containers (p =. 046), the use of air freshener (p = .034), and use of lotion (p = .050). PP was positively associated with the frequent consumption of fish (p = .034), and the use of disposable containers (p = .008). Figure 1 shows the box plots for selected risk factors that were statistically significant determinants for some chemicals, such as consumption of cup noodles, moderate walking activity (for EP), use of air fresheners (MiBP), and purchase of new furniture (MEHP).

13

Table 1. Socioeconomic and maternal characteristics of the subjects

Variables

Categories

N (%) / Mean (SD)

Median (Min-Max)

31 (3)

31 (19-42)

Maternal pre-pregnancy BMI (kg/m2)

21.3 (3.1)

20.6 (16.0-33.3)

Gestational weight gain (kg)

5.7 (5.4)

-10.0-28.0

Maternal age (years)

Maternal education

Household income ($/month)

Employment status

Residence area


24 (11%)

≥college

197 (89%)

≤ 5,000

124 (57%)

> 5,000

95 (43%)

No

53 (24%)

Yes

167 (76%)

Metropolitan

80 (36%)

Non-metropolitan

141(64%)

Neonatal age (day)

34 (25.9)

29 (3-108)

Birth weight (kg)

3.2 (0.4)

3.2 (2.2-4.3)

Birth height (cm)

50.4 (2.3)

50.0 (40.0-57.0)

14

Table 2. Phthalate metabolites, BPA, triclosan, and parabens found in breast milk Parent compound DEP DnBP DIBP BBzP DEHP DINP

Measured biomarker

MEP (µg/L) MnBP (µg/L) MiBP (µg/L) MBzP (µg/L) MEHP (µg/L) MiNP (µg/L) BPA (µg/L) Triclosan (µg/L) Methyl-paraben (µg/L) Ethyl-paraben (µg/L) Propyl-paraben (µg/L)

Percentile LODa

N (% > LODa)

GMb (SDC)

5th

25th

50th

75th

95th

0.13 0.28 0.19 0.08 0.14 0.04 0.08 0.04 0.10 0.04 0.13

102 (46) 161 (72) 153 (69) 12 (5) 184 (83) 158 (71) 0.076 0.035 0.101 0.035 0.134

0.17 (2.37) 0.83 (3.16) 0.47 (3.19) 0.06 (1.37) 1.44 (6.00) 0.10 (2.84) 0.12 (2.91) 0.04 (2.62) 0.33 (5.61) 0.46 (5.23) 0.21 (3.30)




0.27 1.86 0.93 -
1.96 8.46 7.44 0.10 24.38 0.61 0.88 0.29 10.34 4.60 2.17

a

Level of detection Geometric Mean c Standard deviation b

15

Table 3a. Bivariate associations for the chemicals and maternal characteristics, diet, use of consumer products, and behaviors (with proxy value) Variables Maternal age (years) Maternal education General characteristics

Maternal pre-pregnancy BMI Household income ($/month) Residence area

Obstetrical characteristics

Delivery weeks Delivery type Neonatal age (day)

Neonatal characteristics

Neonatal sex Birth weight (kg) Fish Fast food

Food consumption

Cup noodles Ice cream Canned food Plastic food containers Disposable food container Air Freshener

Consumer products Lotion Skin make-up New furniture last 1 year Moderate walking activity last 1 week Health behavior

Hand washing during the day Household ventilation

Categories

N

<35 ≥35
178 43 24 197 197 24 124 95 141 80 14 207 116 105 112 109 114 107 14 207 190 28 114 105 37 181 142 76 84 135 196 23 165 54 119 98 205 13 177 37 110 108 129 91 182 33 207 13

obesity(≥25) <5,000 ≥5,000 non-metropolitan metropolitan preterm normal term C-section vaginal delivery <30 ≥30 female male <2.5 kg ≥2.5 kg Eat frequently Do not eat often Eat frequently Do not eat often Eat frequently Do not eat often Eat frequently Do not eat often Eat frequently Do not eat often yes no yes no yes no yes no yes No yes No yes no several times a day never/seldom daily or sometimes never/ seldom

MnBP (µg/L) Median (IQR) 0.90(0.20-1.89) 0.88(0.20-1.40) 1.52(0.67-2.08)* 0.85(0.20-1.70) 0.89(0.20-1.86) 0.62(0.25-2.07) 0.80(0.20-1.43)* 0.98(0.39-2.20) 1.20(0.39-2.32)** 0.55(0.20-1.09) 0.62(0.20-1.70) 0.90(0.20-1.86) 0.90(0.39-1.87) 0.87(0.20-1.70) 0.89(0.20-1.73) 0.88(0.29-1.89) 0.96(0.20-1.76) 0.84(0.20-1.89) 1.15(0.29-2.53) 0.89(0.20-1.82) 0.90(0.20-1.91) 0.77(0.39-1.15) 0.87(0.20-1.70) 0.90(0.34-1.89) 1.12(0.20-1.56) 0.87(0.20-1.87) 0.98(0.20-1.89) 0.83(0.25-1.69) 1.08(0.30-1.76) 0.84(0.20-1.91) 0.88(0.20-1.73) 1.16(0.20-2.43) 0.89(0.29-1.87) 0.87(0.20-1.76) 1.08(0.39-1.97)* 0.78(0.20-1.68) 0.88(0.20-1.76) 1.44(0.64-1.87) 0.88(0.20-1.70) 0.90(0.34-1.87) 1.08(0.20-2.08) 0.84(0.20-1.50) 0.98(0.34-2.05) 0.78(0.20-1.65) 0.86(0.20-1.86) 1.29(0.39-1.70) 0.89(0.20-1.82) 1.16(0.33-1.97)

* Significant at the 0.05 level ** Significant at the 0.01 level

16

MiBP (µg/L) Median (IQR) 0.52(0.13-1.01) 0.35(0.13-0.70) 0.76(0.21-1.02) 0.46(0.13-0.88) 0.49(0.13-0.93) 0.52(0.18-0.94) 0.39(0.13-0.77)* 0.54(0.13-1.09) 0.60(0.20-1.09)** 0.29(0.13-0.54) 0.29(0.13-0.98) 0.50(0.13-0.93) 0.52(0.13-0.98) 0.47(0.13-0.86) 0.52(0.13-1.02) 0.46(0.13-0.81) 0.49(0.13-0.88) 0.43(0.13-1.05) 0.50(0.13-1.24) 0.49(0.13-0.92) 0.51(0.13-1.01) 0.31(0.13-0.58) 0.53(0.13-0.98) 0.42(0.13-0.92) 0.54(0.13-0.88) 0.47(0.13-0.95) 0.54(0.13-1.00) 0.37(0.13-0.73) 0.54(0.13-0.90) 0.41(0.13-0.98) 0.47(0.13-0.94) 0.56(0.13-1.12) 0.51(0.13-0.95) 0.39(0.13-0.74) 0.56(0.13-1.03)* 0.36(0.13-0.69) 0.47(0.13-0.92) 0.64(0.36-0.98) 0.49(0.13-0.95) 0.39(0.13-0.81) 0.57(0.21-1.07)* 0.36(0.13-0.73) 0.55(0.13-1.03) 0.39(0.13-0.74) 0.46(0.13-0.88) 0.58(0.20-1.01) 0.48(0.13-0.95) 0.59(0.24-0.84)

MEHP (µg/L) Median (IQR) 1.80(0.35-4.88) 0.90(0.28-5.67) 1.71(0.75-4.88) 1.72(0.34-4.89) 1.76(0.35-4.93) 0.95(0.39-2.94) 1.76(0.34-4.12) 1.67(0.33-6.03) 1.91(0.35-6.00) 1.32(0.34-3.78) 1.04(0.34-3.09) 1.76(0.35-5.11) 1.41(0.33-5.55) 2.06(0.42-4.34) 2.22(0.70-6.10)** 1.23(0.22-3.47) 1.86(0.69-5.81) 1.39(0.17-4.09) 1.45(0.23-4.88) 1.75(0.35-4.89) 1.71(0.33-4.88) 1.78(0.66-5.78) 1.75(0.37-3.90) 1.72(0.33-5.11) 2.18(0.71-5.10) 1.67(0.33-4.88) 1.80(0.33-5.68) 1.41(0.40-3.58) 1.74(0.39-5.55) 1.78(0.33-4.88) 1.69(0.35-4.47) 2.99(0.22-15.56) 1.67(0.35-4.51) 1.91(0.30-6.04) 1.90(0.35-4.89) 1.53(0.33-4.88) 1.72(0.34-4.88) 2.15(1.06-3.90) 1.71(0.33-4.42) 2.08(0.60-6.00) 1.86(0.76-5.68)* 1.38(0.19-4.04) 1.72(0.33-4.26) 1.76(0.51-5.11) 1.71(0.34-4.88) 1.90(0.54-4.89) 1.72(0.33-4.89) 1.59(0.76-4.88)

MiNP (µg/L) Median (IQR) 0.08(0.03-0.23) 0.08(0.03-0.21) 0.10(0.04-0.24) 0.08(0.03-0.22) 0.08(0.03-0.23) 0.09(0.03-0.18) 0.11(0.04-0.25) 0.08(0.03-0.15) 0.11(0.05-0.26)* 0.07(0.03-0.14) 0.08(0.03-0.12) 0.08(0.03-0.23) 0.10(0.05-0.24) 0.08(0.03-0.17) 0.11(0.05-0.24) 0.08(0.03-0.19) 0.08(0.03-0.19) 0.08(0.03-0.23) 0.11(0.03-0.22) 0.08(0.03-0.23) 0.08(0.03-0.22) 0.07(0.03-0.20) 0.08(0.03-0.23) 0.08(0.03-0.19) 0.07(0.04-0.24) 0.08(0.03-0.20) 0.10(0.03-0.24) 0.07(0.03-0.19) 0.09(0.03-0.23) 0.08(0.03-0.21) 0.08(0.03-0.20) 0.14(0.03-0.35) 0.08(0.03-0.21) 0.08(0.03-0.22) 0.08(0.03-0.21) 0.08(0.03-0.21) 0.08(0.03-0.21) 0.07(0.03-0.28) 0.08(0.03-0.22) 0.08(0.03-0.27) 0.10(0.03-0.24) 0.08(0.03-0.19) 0.09(0.04-0.23) 0.07(0.03-0.21) 0.08(0.03-0.18) 0.14(0.50-0.31) 0.08(0.03-0.22) 0.14(0.10-0.23)

Ethyl-Paraben (µg/L) Median (IQR) 0.61(0.11-1.54) 0.78(0.26-2.62) 1.01(0.38-2.40) 0.54(0.13-1.54) 0.53(0.11-1.54)** 1.17(0.56-2.57) 0.69(0.16-1.82) 0.57(0.12-1.38) 0.61(0.11-1.78) 0.62(0.22-1.39) 0.76(0.50-1.54) 0.59(0.14-1.62) 0.78(0.23-2.08)* 0.43(0.09-1.30) 1.27(0.57-2.62)** 0.23(0.06-0.62) 0.67(0.23-1.46) 0.57(0.09-1.65) 0.63(0.12-1.45) 0.62(0.14-1.64) 0.60(0.11-1.46) 0.64(0.29-2.12) 0.65(0.16-1.62) 0.53(0.10-1.30) 1.10(0.53-2.15)** 0.46(0.10-1.39) 0.70(0.19-1.78) 0.43(0.10-1.15) 0.67(0.13-2.05) 0.57(0.14-1.39) 0.63(0.15-1.55) 0.32(0.06-2.07) 0.63(0.11-1.48) 0.49(0.16-2.07) 0.65(0.16-1.65) 0.44(0.11-1.30) 0.61(0.15-1.62) 0.50(0.04-0.77) 0.59(0.14-1.50) 0.69(0.13-1.38) 0.65(0.09-1.78) 0.53(0.15-1.46) 0.44(0.09-1.27)* 0.78(0.24-1.78) 0.51(0.11-1.54) 0.91(0.50-1.91) 0.63(0.14-1.64) 0.54(0.09-0.91)

Table 3b. Bivariate associations for the chemicals and maternal characteristics, diet, use of consumer products, and behaviors (with original value) Categories

Variables

<35 ≥35
178 43 24 197 197 24 124 95 141 80 14 207 116 105 112 109 114 107 14 207 190 28 114 105 37 181 142 76 84 135 196 23 165 54 119 98 205 13 177 37

yes

110

64

58.2

Moderate walking activity last 1 week Hand washing during the day

no yes no several times a day never/seldom

108 129 91 182 33

37 58 44 80 17

34.3 48.4 45.0 44.0 51.5

Household ventilation

daily or sometimes never/ seldom

207 13

94 8

45.4 61.5

Maternal education Maternal prepregnancy BMI Household income ($/month) Residence area Obstetrical characteristics

Delivery weeks Delivery type Neonatal age (day)

Neonatal characteristics

Neonatal sex Birth weight (kg) Fish Fast food

Food consumption

Cup noodles Ice cream Canned food Plastic food containers Disposable food container Air freshener

Consumer products

Lotion Skin make-up New furniture last 1 year

Health behavior

MEP (%) 45.5 48.8 62.5 44.2 47.2 37.5 41.1 52.6 56.7 27.5 35.7 46.9 44.0 48.6 48.2 44.0 50.0 42.1 57.1 45.4 48.4 28.6 45.6 46.7 37.8 47.5 48.6 40.8 44.1 47.4 46.4 43.5 49.1 37.0 52.1 38.8 44.4 69.2 47.5 40.5

N>LOD 81 21 15 87 93 9 51 50 80 22 5 97 51 51 54 48 57 45 8 94 92 8 52 49 14 86 69 31 37 64 91 10 81 20 62 38 91 9 84 15

Maternal age (years)

General characteristics

N

p-value 0.694 0.089 0.368 0.091* <0.001** 0.418 0.493 0.533 0.236 0.394 0.049* 0.876 0.282 0.271 0.628 0.788 0.123 0.050* 0.081 0.443 <0.001** 0.619 0.422 0.258

N>LOD 10 2 0 12 10 2 4 8 10 2 1 11 10 2 8 4 7 5 0 12 10 2 6 6 2 10 7 5 6 6 8 4 9 3 6 5 11 0 7 4

MBzP (%) 5.6 4.7 0.0 6.1 5.1 8.3 3.2 8.4 7.1 2.5 7.1 5.3 8.6 1.9 7.1 3.7 6.1 4.7 0.0 5.8 5.3 7.1 5.3 5.7 5.4 5.5 4.9 6.6 7.1 4.4 4.1 17.4 5.5 5.6 5.0 5.1 5.4 0.0 4.0 10.8

8

7.3

4 8 4 10 1

3.7 6.2 4.4 5.5 3.0

12 0

5.8 0.0

* Significant at the 0.05 level ** Significant at the 0.01 level

17

p-value 0.802 0.214 0.506 0.094 0.148 0.770 0.028* 0.255 0.630 0.354 0.684 0.883 0.977 0.611 0.394 0.008** 0.977 0.984 0.391 0.086 0.248 0.561 0.554 0.372

N>LOD 90 17 12 95 94 13 59 46 68 39 8 99 55 52 48 59 49 58 9 98 91 15 53 54 17 90 70 37 43 64 93 14 78 29 56 50 98 8 86 19

BPA (%) 50.6 39.5 50.0 48.2 47.7 54.2 47.6 48.4 48.2 48.8 57.1 47.8 47.4 49.5 42.9 54.1 43.0 54.0 64.3 47.3 47.9 53.6 46.5 51.4 46.0 49.7 49.3 48.7 51.2 47.4 47.5 60.9 47.3 53.7 47.1 51.0 47.8 61.5 48.6 51.4

56

50.9

51 61 46 92 13

47.2 47.3 50.6 50.6 39.4

102 4

49.3 30.8

p-value 0.194 0.869 0.550 0.902 0.940 0.500 0.754 0.094 0.095 0.220 0.575 0.465 0.675 0.931 0.586 0.223 0.412 0.561 0.337 0.760 0.586 0.633 0.238 0.195

N>LOD 48 9 5 52 50 7 33 24 39 18 5 52 27 30 22 35 25 32 5 52 48 9 32 25 12 45 36 21 18 39 49 8 37 20 27 30 49 8 42 15

Triclosan (%) 27.0 20.9 20.8 26.4 25.4 29.2 26.6 25.3 27.7 22.5 35.7 25.1 23.3 28.6 19.6 32.1 21.9 29.9 35.7 25.1 25.3 32.1 28.1 23.8 32.4 24.9 25.4 27.6 21.4 28.9 25.0 34.8 22.4 37.0 22.7 30.6 23.9 61.5 23.7 40.5

28

25.5

29 26 30 50 6

26.9 20.2 33.0 27.5 18.2

54 3

26.1 23.1

p-value 0.417 0.556 0.689 0.822 0.399 0.381 0.369 0.034* 0.176 0.381 0.439 0.473 0.340 0.715 0.221 0.312 0.034* 0.187 0.003** 0.035* 0.814 0.032* 0.263 0.810

Methyl-Paraben N>LOD (%) p-value 104 58.4 0.807 26 60.5 14 58.3 0.959 116 58.9 117 59.4 0.623 13 54.2 76 61.3 0.412 53 55.8 86 61.0 0.384 44 55.0 8 122 72 58 78 52 57 73 9 121 107 21 70 59 25 103 87 41 47 82 111 18 92 37 62 65 117 11 102 25

57.1 58.9 62.1 55.2 69.6 47.7 50.0 68.2 64.3 58.5 56.3 75.0 61.4 56.2 67.6 56.9 61.3 54.0 56.0 60.7 56.6 78.3 55.8 68.5 52.1 66.3 57.1 84.6 57.6 67.6

59

53.6

69 72 57 106 22

63.9 55.8 62.6 58.2 66.7

125 5

60.4 38.5

0.895 0.303 0.001** 0.006** 0.668 0.061 0.433 0.230 0.296 0.484 0.046* 0.098 0.034* 0.050* 0.263 0.124 0.312 0.364 0.119

Propyl-Paraben N>LOD (%) p-value 74 41.6 0.755 19 44.2 11 45.8 0.693 82 41.6 82 41.6 0.693 11 45.8 57 46.2 0.130 34 35.8 63 44.7 0.299 30 37.5 6 87 53 40 54 39 40 53 8 85 75 17 49 43 15 76 61 30 35 57 78 14 61 31 43 48 85 6 71 20

42.9 42.0 45.7 38.1 48.2 35.8 35.1 49.5 57.1 41.1 39.5 60.7 43.0 41.0 40.5 42.0 43.0 37.5 41.7 42.2 39.8 60.9 37.0 57.4 36.1 49.0 41.5 46.2 40.1 54.1

44

40.0

47 51 41 76 16

43.5 39.5 45.1 41.8 48.5

88 5

38.5 42.5

0.952 0.253 0.061 0.030* 0.238 0.034* 0.761 0.871 0.619 0.935 0.053 0.008** 0.056 0.739 0.119 0.598 0.414 0.472 0.774

Figure 1. Boxplots for the concentrations of ethylparaben (EP), mono-isobutyl phthalate (MiBP), and mono-2-ethylhexyl phthalate (MEHP) (µg/L) by consumption of food (cup noodles), behavior (moderate walking activity), and use of household products (air freshener and new furniture). The outliers are excluded from each boxplot. All differences are significant at the 0.05 level. All units are in µg/L. 18

3.4 Estimated daily intake of chemicals and risk assessment

The median (min-max) daily intake for MEP, MnBP, MiBP, MBzP, MEHP, and MiNP were calculated as 3.48 (0.10–9.07), 0.21 (0.02–2.88), 1.32 (0.10–9.84), 0.04 (0.01–0.13), 0.62 (0.10– 102.87), and 0.02 (0.00–0.56) µg/kg bw/day, respectively. The estimated daily values for DEP, DnBP, DiBP, BBzP, DEHP, and DiNP were 1.53, 0.08–0.58, 0.13–4.92, 0.03–0.09, 0.61–6.72, and 0.05 µg/kg bw/day, respectively. The median (min-max) daily intake for BPA, MP, EP, PP, and TCS were 0.04 (0.01–1.53), 0.14 (0.01–47.91), 0.13 (0.00–2.52), 0.08 (0.02–8.24), and 0.02 (0.01–0.98) µg/kg bw/day, respectively. To perform a risk assessment of chemical exposures from breast milk, we compared the daily chemical intake to the TDI value. Using the diester intake to assess the risk of phthalate monoesters is a conservative approach, given that monoesters are generally more toxic than diesters (Kim et al., 2015). Among the chemicals, the DEHP concentrations for 8 breast milk samples, along with two samples for MEHP and one sample for DiBP, exceeded the TDI values, as shown in Figure 2.

19

Figure 2. Distributions of the estimated daily intake rates for chemical concentrations in breast milk samples, along with their tolerable daily intake (TDI). The box indicates the interquartile range (IQR) between Q1 (first quartile) and Q3 (third quartile) and the whiskers show the range of non-outliers with dots indicating outliers. Outliners are determined to be any values smaller than Q1-1.5×IQR or larger than Q3+1.5×IQR. DEHP was calculated using published literature ratios. All units are in µg/kg bw/day.

20

4. Discussion

This study attempted to measure the concentrations of different chemicals in breast milk and to determine the association between these chemicals and the breastfeeding mothers’ diet and environment. The detection rates and concentrations of phthalate metabolites, BPA, parabens, and TCS in the breast milk of the Korean mothers tested was lower than in previous reports. The median concentrations of MEHP and MEP were 1.72 and 0.28 µg/L, which are lower than the values reported from most of the previous studies, although those of MEHP were higher than those of a Swedish study (Hogberg et al., 2008) and a Canadian study (Arbuckle et al., 2016). The median levels of MiBP, MBzP, and MiNP were 0.49, 0.19, and 0.08, which are lower than those of previous studies. The median level of BPA was 0.25 µg/L, lower than in previous studies conducted in the USA, Spain, Korea, and Japan. The median level of TCS was 0.12 µg/L, lower than in previous studies conducted in Australia and Sweden (Allmyr et al., 2006; Toms et al., 2011) (see Supplement 3 for details). The reason for a lower detection rates in this study is presumably due to a difference in LOD. For instance, the LOD for BPA in this study was 0.08 µg/L, while the LODs in the previous studies varied from 0.003 to 0.3 µg/L (Deceuninck et al.,2015; Lee et al., 2018; Ye et al., 2008). In addition, the samples in this study were collected in 2018, after global policies limiting the use of endocrine-disrupting chemicals went into effect (Bever et al., 2018). The U.S. FDA banned TCS in washing products in 2016 and in medical products in 2017 (FDA, 2016, 2019). Few studies have measured the amounts of parabens in breast milk, and the median levels of MP, EP, and PP were not consistently comparable with the previous studies. Phthalate secondary metabolites such as MEHHP, MEOHP, and MECPP were not observed in the present study except in one sample. These data are in close agreement with previous studies, in which MEHHP and MEOHP were not detected above the LOD in the breast milk samples (Fromme et al., 2011; Lin et al., 2011; Kim et al., 2018). The secondary metabolites were not detected or were only detected at low concentrations in our study because they are too hydrophilic to be found in the breast milk and easily 21

excreted in the urine (Latini et al., 2009; Kim et al., 2015). The concentrations of chemicals in the breast milk in the present study were lower than those found in previous studies that analyzed the urine of pregnant and postpartum women, mostly because phthalate metabolites, BPA, parabens, and TCS are easily hydrolyzed, oxidized, glucuronidated, or sulfonated, and are excreted mainly via the urine. The present study confirmed the association of 11 endocrine disrupting chemicals with socioeconomic characteristics, as well as with diet, use of consumer products, and behavior. Few previous studies have investigated the relationship among these chemicals in breast milk and diet, use of consumer products, and behaviors. The results related to socioeconomic characteristics support the results of earlier studies reporting that these chemicals are associated with maternal education, maternal pre-pregnancy BMI, household income, residence area, delivery type, neonatal age, and neonatal sex (Mendonca et al., 2014; Cullen et al., 2017; Lee et al., 2017; Liao et al., 2018; Polinski et al., 2018). Regarding diet, the consumption of fish was associated with the presence of phthalate metabolites and parabens in the present study, which is consistent with previous results (Serrano et al., 2014; Bai et al., 2015), except for Dong et al. (2017) reporting fish is negatively correlated with phthalate exposure. This can be explained by the differences in dietary culture, such as a preference for boiling food and the storage of unpackaged fish in ceramic rather than plastic containers (Dong et al., 2017). Our study is the first to reveal that the consumption of cup noodles was strongly associated with EP as it is used as an antifungal preservative in instant foods, including cup noodles. Regarding consumer products, the use of plastic or disposable food containers, air fresheners, lotions, make-up, and new furniture was associated with the presence of phthalate metabolites, parabens, and TCS in this study, in accordance with previous studies (Larsson et al., 2014; Hines et al., 2015; Fisher et al., 2017; Tratnik et al., 2019). In particular, Larsson et al. (2014), who investigated potential predictors of chemical exposures in Swedish mothers and their children, confirmed that the presence of high molecular weight phthalate was associated with the consumption of certain foods, whereas the presence of parabens was associated with use of cosmetics and personal care products; these results 22

are in close agreement with those of the present study. Most previous studies reported that physical activity was negatively associated with concentrations of phthalate metabolites in urine (Lee et al., 2017; Wenzel et al., 2018), which was consistent with our finding that the concentrations of EP and triclosan were lower in the participants who engaged in moderate walking than in those who did not. As phthalate is volatile, higher levels of physical activity presumably make it more likely for phthalates to be discharged in urine or breast milk. According to our study, moderate walking could be recommended to reduce the concentrations of chemicals in breast milk. As breast milk or infant formula is the only source of food for neonate and infants, the first months of life requires higher energy intake per kilogram body (EFSA SC, 2017). The concentration of endocrine disruptors in breast milk, measured in this study, provides information on the degree to which newborns are exposed to these xenoestrogens. In the case of phthalates, we calculated the daily phthalate diester intake, on the basis of the measured phthalate metabolite concentrations in breast milk and considering the diester-metabolite ratio reported in previous studies (Fromme et al., 2011; Arbuckle et al., 2016). The daily phthalate intake results of the present study differed from the results of previous studies. For example, the daily MEHP and DEHP intake found in the present study were higher than those in Kim et al. (2015) and Bubba et al. (2018), and similar to the result of Fromme et al. (2011). For MnBP, the daily intake in the present study was lower than that reported by Kim et al. (2015). The median daily TCS intake recorded in the present study was substantially lower than that reported by Tom et al. (2011) or (Dayan, 2007). The discrepancy with the previous study may reflect country-specific or regional factors that cannot be directly measured. In this study, the DEHP concentrations for 8 breast milk samples, along with two samples for MEHP and one sample for DiBP, exceeded the TDI values. However, considering that these TDI values are the basis obtained from urine samples in adults, the real risk is thought to be much lower (European Food Safety Authority, 2005). Recently, the EFSA Scientific Committee proposed to use a higher consumption value of 260mL/kg bw/day when performing an exposure assessment for infants below 16 weeks of age (EFSA SC, 2017). When applying this criterion, the estimated daily chemical intake rates would be 23

considered to be minimal. The present study, however, has some limitations. As it was a cross-sectional study that analyzed chemicals in breast milk, it was difficult to clarify a causal relationship between a chemical’s presence and the factors that caused it to be present. Therefore, epidemiological studies are needed to determine the pathway of exposure through a long-term cohort study that tracks pregnant women and continues tracking their infants as they grow. Also, the present study used a one-to-one statistical analysis, including analysis of one chemical, one diet, and the use of one daily product. However, health impacts from exposure to chemicals may not be the result of a single chemical but may be the synergistic consequences of several chemicals and multiple behavioral or environmental factors. Therefore, future studies will require analysis methods and research designs that simultaneously measure the effects of various chemicals exposed through various routes, such as ingestion, inhalation, and skin absorption (Preindl et al., 2019). Furthermore, no TDI standard for phthalate metabolites exists, so the risk was evaluated by referring to the TDI standard of the phthalate diester, and the results may be over or underestimated. In addition, the TDI values for the phthalates, BPA, parabens, and TCS are based on adult standards, and are not applicable to breastfeeding infants. Therefore, it is necessary to determine the TDI in the breast milk from the accumulated evidence of multiple followup studies.

5. Conclusion

In this study we have analyzed the concentrations of 15 analytes (including phthalate metabolites, BPA, parabens, and TCS) in Korean breast milk samples and proved that these concentrations were associated with diet, use of consumer products, and behavior. In particular, the consumption of fish and cup noodles, and the use of plastic and disposable food containers, air fresheners, make-up, and new furniture were associated with the presence of these chemicals in breast milk. Moderate walking activity was found a significant factor that could reduce the levels of these 24

chemicals. These findings demonstrate the importance of lifestyle factors that can reduce the level of exposure to xenoestrogens and their adverse health effects. Pregnant women and breastfeeding mothers would benefit by being informed of the sources of exposure to these substances and changing their lifestyle accordingly during their everyday lives.

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (Ministry of Science, ICT) (NRF-2018R1C1B6004256). The authors thank the subjects who provided the breast milk and the nurses who participated in collecting the breast milk samples.

Conflict of interest statement

The authors declare that they have no conflict of interest.

References

Allmyr, M., McLachlan, M.S., Sandborgh-Englund, G., Adolfsson-Erici, M., 2006. Determination of triclosan as its pentafluorobenzoyl ester in human plasma and milk using electron capture negative ionization mass spectrometry. Anal Chem 78(18), 6542-6546. Andrianou, X., Gängler, S., Piciu, A., Charisiadis, P., Zira, C., Aristidou, K., Piciu, D., Hauser, R., Makris, K., 2016. Human exposures to bisphenol A, bisphenol F and chlorinated bisphenol A derivatives and thyroid function. PLoS One 11(10), e0155237. Arbuckle, T., Fisher, M., MacPherson, S., Lang, C., Provencher, G., LeBlanc, A., Hauser, R., Feeley, M., Ayotte, P., Neisa, A., Ramsay, T., Tawagi, G., 2016. Maternal and early life exposure to 25

phthalates: the plastics and personal-care products use in pregnancy (P4) study. Sci Tot Environ 551-552(1), 344-356. Bai, P.Y., Wittert, G.A., Taylor, A.W., Martin, S.A., Milne, R.W., Shi, Z., 2015. The association of socio-demographic status, lifestyle factors and dietary patterns with total urinary phthalates in Australian men. PLoS One 10(4), e0122140. Balakrishnan, B., Henare, K., Thorstensen, E., Ponnampalam, A., Mitchell, M., 2010. Transfer of bisphenol A across the human placenta. Am J Obstet Gynecol 202(393), e391-397. Bever, C.S., Rand, A.A., Nording, M., Taft, D., Kalanetra, K.M., Mills, D.A., Breck, M.A., Smilowitz, J.T., German, J.B., Hammock, B.D., 2018. Effects of triclosan in breast milk on the infant fecal microbiome. Chemosphere 203, 467-473. Birnbaum, L.S., 1994. Endocrine effects of prenatal exposure to PCBs, dioxins, and other xenobiotics: implications for policy and future research. Environ Health Perspect 102(8), 676-679. Boberg, J., Taxvig, C., Christiansen, S., Hass, U., 2010. Possible endocrine disrupting effects of parabens and their metabolites. Reprod Toxicol 30(2), 301-312. Bubba, M., Ancillotti, C., Checchini, L., Fibbi, D., Rossini, D., Ciofi, L., Rivoira, L., Profeti, C., Orlandini, S., Furlanetto, S., 2018. Determination of phthalate diesters and monoesters in human milk and infant formula by fat extraction, size-exclusion chromatography clean-up and gas chromatography-mass spectrometry detection. J Pharm Biomed Anal 148(30), 6-16. Calafat, A., Slakman, A., Silva, M., Herbert, A., Needham, L., 2004. Automated solid phase extraction and quantitative analysis of human milk for 13 phthalate metabolites. J Chromatogr B 805(1), 49-56. Center for the Evaluation of Risks to Human Reproduction, 2000. NTP-CERHR expert panel report on di(2-ethylhexyl)phthalate. . NTP-CERHR-DEHP-00. U.S. Department of Health and Human Services, National Toxicoloty Program, Alexandria, VA. Centers for Disease Control and Prevention, 2019. National report on human exposure to environmental chemicals: updated tables. Atlanta, GA. 26

Cho, H., 2012. Epigenetic control of endocrine disrupting chemicals on gynecological disease: focused on phthalates. Korean J Obstet Gynecol 55(9), 619-628. Cullen, E., Evans, D., Griffin, C., Burke, P., Mannion, R., Burns, D., Flanagan, A., Kellegher, A., Schoeters, G., Govarts, E., Biot, P., Casteleyn, L., Castano, A., Kolossa-Gehring, M., Esteban, M., Schwedler, G., Koch, H., Angerer, J., Knudsen, L., Joas, R., Joas, A., Dumez, B., Sepai, O., Exley, K., Aerts, D., 2017. Urinary phthalate concentrations in mothers and their children in Ireland: results of the DEMOCOPHES human biomonitoring study. Int J Environ Res Public Health 14(12), 1456. Dayan, A.D., 2007. Risk assessment of triclosan [Irgasan®] in human breast milk. Food Chem Toxicol 45(1), 125-129. Deceuninck, Y., Bichon, E., Marchand, P., Boquien, C., Legrand, A., Boscher, C., Antignac, J., Bizec, B., 2015. Determination of bisphenol A and related substitutes/analogues in human breast milk using gas chromatography-tandem mass spectrometry. Anal Bioanal Chem 407(9), 24852497. Dong, R., Zhou, T., Zhao, S., Zhang, H., Zhang, M., Chen, J., Wang, M., Wu, M., Li, S., Chen, B., 2017. Food consumption survey of Shanghai adults in 2012 and its associations with phthalate metabolites in urine. Environ int 101, 80-88. European Food Safety Authority, 2005. Opinion of the scientific panel on food additives, flavourings, processing aids and materials in contact with food (AFC). EFSA Journal 3(9), 1-20. FDA, 2016. FDA issues final rule on safety and effectiveness of antibacterial soaps. FDA News Release. FDA, 2019. FDA issues final rule on safety and effectiveness of consumer hand sanitizers. FDA News Release. Fisher, M., MacPherson, S., Braun, J., Hauser, R., Walker, M., Feeley, M., Mallick, R., Bérubé, R., Arbuckle, T., 2017. Paraben concentrations in maternal urine and breast milk and its association with personal care product use. Environ Sci Technol 51(7), 4009-4017. 27

Fromme, H., Gruber, L., Seckin, E., Raab, U., Zimmermann, S., Kiranoglu, M., Schlummer, M., Schwegler, U., Smolic, S., Volkel, W., 2011. Phthalates and their metabolites in breast milk — results from the Bavarian Monitoring of Breast Milk (BAMBI). Environ Int 37(4), 715-722. Giovanoulis, G., Alves, A., Papadopoulou, E., Cousins, A.P., Schutze, A., Koch, H.M., Haug, L.S., Covaci, A., Magner, J., Voorspoels, S., 2016. Evaluation of exposure to phthalate esters and DINCH in urine and nails from a Norwegian study population. Environ Res 151, 80-90. Giovanoulis, G., Bui, T., Xu, F., Papadopoulou, E., Padilla-Sanchez, J.A., Covaci, A., Haug, L.S., Cousins, A.P., Magner, J., Cousins, I.T., de Wit, C.A., 2018. Multi-pathway human exposure assessment of phthalate esters and DINCH. Environ Int 112, 115-126. Giulivo, M., Lopez de Alda, M., Capri, E., Barcelo, D., 2016. Human exposure to endocrine disrupting compounds: their role in reproductive systems, metabolic syndrome and breast cancer. A review. Environ Res 151, 251-264. Guerranti, C., Sbordoni, I., Fanello, E.L., Borghini, F., Corsi, I., Focardi, S.E., 2013. Levels of phthalates in human milk samples from central Italy. Microchem J 107, 178-181. Hines, E., Calafat, A., Silva, M., Mendola, P., Fenton, S., 2009. Concentrations of phthalate metabolites in milk, urine, saliva, and serum of lactating North Carolina women. Environ Health Perspect 117(1), 86-92. Hines, E.P., Mendola, P., von Ehrenstein, O.S., Ye, X., Calafat, A.M., Fenton, S.E., 2015. Concentrations of environmental phenols and parabens in milk, urine and serum of lactating North Carolina women. Reprod Toxicol 54, 120-128. Hogberg, J., Hanberg, A., Berglund, M., Skerfving, S., Remberger, M., Calafat, A., Filipsson, A., Jansson, B., Johansson, N., Appelgren, M., Hakansson, H., 2008. Phthalate diesters and their metabolites in human breast milk, blood or serum, and urine as biomarkers of exposure in vulnerable populations. Environ Health Perspect 116(3), 334-339. Hornung, R., Reed, L., 1990. Estimation of Average Concentration in the Presence of Nondetectable Values. Appl Occup Environ Hyg 5(1), 46-51. 28

Huo, W., Xia, W., Wan, Y., Zhang, B., Zhou, A., Zhang, Y., Huang, K., Zhu, Y., Wu, C., Peng, Y., Jiang, M., Hu, J., Chang, H., Xu, B., Li, Y., Xu, S., 2015. Maternal urinary bisphenol A levels and infant low birth weight: a nested case–control study of the Health Baby Cohort in China. Environ Int 85, 96-103. Jurewicz, J., Polańska, K., Hanke, W., 2013. Exposure to widespread environmental toxicants and children’s cognitive development and behavioral problems. Int J Occup Med Environ Health 26(2), 185-204. Kim, S., Eom, S., Kim, H., Lee, J., Choi, G., Choi, S., Kim, S., Kim, S., Cho, G., Kim, Y., Suh, E., Kim, S., Kim, S., Kim, G., Moon, H., Park, J., Kim, S., Choi, K., Eun, S., 2018. Association between maternal exposure to major phthalates, heavy metals, and persistent organic pollutants, and the neurodevelopmental performances of their children at 1 to 2 years of ageCHECK cohort study. Sci Total Environ 624(15), 377-384. Kim, S., Lee, J., Park, J., Kim, H., Cho, G., Kim, G., Eun, S., Lee, J., Choi, G., Suh, E., Choi, S., Kim, S., Kim, Y., Kim, S., Kim, S., Kim, S., Eom, S., Moon, H., Choi, K., 2015. Concentrations of phthalate metabolites in breast milk in Korea: estimating exposure to phthalates and potential risks among breast-fed infants. Sci Total Environ 508(1), 13-19. Koeppe, E., Ferguson, K., Colacino, J., Meeker, J., 2013. Relationship between urinary triclosan and paraben concentrations and serum thyroid measures in NHANES 2007-2008. Sci Total Environ 445-446, 299-305. Kortenkamp, A., Faust, M., 2010. Combined exposures to anti‐androgenic chemicals: steps towards cumulative risk assessment. Int J Androl 33(2), 463-474. Larsson, K., Björklund, K., Palm, B., Wennberg, M., Kaj, L., Lindh, C., Jönsson, B., Berglund, M., 2014. Exposure determinats of phthalates, parabens, bisphenol A and triclosan in Swedish mothers and their children. Environ Int 73, 323-333. Latini, G., Wittassek, M., Del Vecchio, A., Presta, G., De Felice, C., Angerer, J., 2009. Lactational exposure to phthalates in Southern Italy. Environ Int 35(2), 236-239. 29

Lee, B., Park, H., Hong, Y., Ha, M., Kim, Y., Chang, N., Kim, B., Kim, Y., Yu, S., Ha, E., 2014. Prenatal bisphenol A and birth outcomes: MOCEH (Mothers and Children's Environmental Health) study. Int J Hyg Environ Health 217(2-3), 328-334. Lee, J., Choi, G., Park, J., Moon, H., Choi, G., Lee, J., Suh, E., Kim, H., Eun, S., Kim, G., Cho, G., Kim, S., Kim, S., Kim, S., Kim, S., Eom, S., Choi, S., Kim, Y., Kim, S., 2018. Bisphenol A distribution in serum, urine, placenta, breast milk, and umbilical cord serum in a birth panel of mother–neonate pairs. Sci Total Environ 626(1), 1494-1501. Lee, K., Kho, Y., Kim, P., Park, S., Lee, J., 2017. Urinary levels of phthalate metabolites and associations with demographic characteristics in Korean adults. Environ Sci Pollut Res 24(17), 14669-14681. Liao, C., Liu, W., Zhang, J., Shi, W., Wang, X., Cai, J., Zou, Z., Lu, R., Sun, C., Wang, H., Huang, C., Zhao, Z., 2018. Associations of urinary phthalate metabolites with residential characteristics, lifestyles, and dietary habits among young children in Shanghai, China. Sci Total Environ 616-617, 1288-1297. Lin, S., Ku, H., Su, P., Chen, J., Huang, P., Angerer, J., Wang, S.L., 2011. Phthalate exposure in pregnant women and their children in central Taiwan. Chemosphere 82(7), 947-955. Liu, C., Zhang, Y., Benning, J.L., Little, J.C., 2015. The effect of ventilation on indoor exposure to semivolatile organic compounds. Indoor Air 25(3), 285-296. Mendonca, K., Hauser, R., Calafat, A., Arbuckle, T., Duty, S., 2014. Bisphenol A concentrations in maternal breast milk and infant urine. Int Arch Occup Environ Health 87(1), 13-20. Migeot, V., Dupuis, A., Cariot, A., Albouy-Llaty, M., Pierre, F., Rabouan, S., 2013. Bisphenol A and its chlorinated derivatives in human colostrum. Environ Sci Technol 47(23), 13791-13797. Morck, T., Sorda, G., Bechi, N., Rasmussen, B., Nielsen, J., Ietta, F., Rytting, E., Mathiesen, L., Paulesu, L., Knudsen, L., 2010. Placental transport and in vitro effects of bisphenol A. Reprod Toxicol 30(1), 131-137. Nakao, T., Akiyama, E., Kakutani, H., Mizuno, A., Aozasa, O., Akai, Y., Ohta, S., 2015. Levels of 30

tetrabromobisphenol A, tribromobisphenol A, dibromobisphenol A, monobromobisphenol A, and bisphenol a in Japanese breast milk. Chem Res Toxicol 28(4), 722-728. National Institute for Public Health and the Environment, 2018. Exposure to and toxicity of methyl-, ethyl- and propylparaben: a literature review with a focus on endocrine-disrupting properties. RIVM Report 2017-0028, The Netherlans. National Research Council (US) Committee on the Health Risks of Phthalates, 2008. Phthalates and Cumulative Risk Assessment: The Tasks Ahead. National Academies Press (US), Washington, DC. Pei, J., Sun, Y., Yin, Y., 2018. The effect of air change rate and temperature on phthalate concentration in house dust. Sci Total Environ 639, 760-768. Polanska, K., Ligocka, D., Sobala, W., Hanke, W., 2016. Effect of environmental phthalate exposure on pregnancy duration and birth outcomes. Int J Occup Med Environ Health 29(4), 683-697. Polinski, K., Dabelea, D., Hamman, R., Adgate, J., Calafat, A., Ye, X., Starling, A., 2018. Distribution and predictors of urinary concentrations of phthalate metabolites and phenols among pregnant women in the Healthy Start Study. Environ Res 162, 308-317. Rodríguez-Gómez, R., Zafra-Gómez, A., Camino-Sánchez, F., Ballesteros, O., Navalón, A., 2014. Gas chromatography and ultra high performance liquid chromatography tandem mass spectrometry methods for the determination of selected endocrine disrupting chemicals in human breast milk after stir-bar sorptive extraction. J Chromatog A 1349, 69-79. Rowdhwal, S., Chen, J., 2018. Toxic effects of di-2-ethylhexyl phthalate: an overview. BioMed Res Int 2018. Serrano, S., Braun, J., Trasande, L., Dills, R., Sathyanarayana, S., 2014. Phthalates and diet: a review of the food monitoring and epidemiology data. Environ Health 13(1), 43. Song, Y., Chou, E., Baecker, A., You, N., Song, Y., Sun, Q., Liu, S., 2016. Endocrine‐disrupting chemicals, risk of type 2 diabetes, and diabetes‐related metabolic traits: a systematic review and meta‐analysis. J Diabetes 8(4), 516-532. 31

Toms, L.M., Allmyr, M., Mueller, J.F., Adolfsson-Erici, M., McLachlan, M., Murby, J., Harden, F.A., 2011. Triclosan in individual human milk samples from Australia. Chemosphere 85(11), 1682-1686. Tratnik, J., Kosjek, T., Heath, E., Mazej, D., Ćehić, S., Karakitsios, S., Sarigiannis, D., Horvat, M., 2019. Urinary bisphenol A in children, mothers and fathers from Slovenia: overall results and determinants of exposure. Environ Res 168, 32-40. Wassenaar, P., Trasande, L., Legler, J., 2017. Systematic review and meta-analysis of early life exposure to di(2-ethylhexyl) phthalate and obesity related outcomes in rodents. Chemosphere 188, 174-181. Weatherly, L., Gosse, J., 2017. Triclosan exposure, transformation, and human health effects. J Toxicol Environ Health B Crit Rev 20(8), 447-469. Wenzel, A.G., Brock, J.W., Cruze, L., Newman, R.B., Unal, E.R., Wolf, B.J., Somerville, S.E., Kucklick, J.R., 2018. Prevalence and predictors of phthalate exposure in pregnant women in Charleston, SC. Chemosphere 193, 394-402. WHO, 2003. Concise international chemical assessment document 52: diethyl phythalate. Geneva, Switzerland. WHO, 2007. Biomonitoring of human milk for POPs. Technical note. World Health Organization, Geneva, Switzerland. Wormuth, M., Scheringer, M., Vollenweider, M., Hungerbuhler, K., 2006. What are the sources of exposure to eight frequently used phthalic acid esters in Europeans? Risk Anal 26(3), 803-824. Ye, X., Bishop, A., Needham, L., Calafat, A., 2008. Automated on-line column-switching HPLCMS/MS method with peak focusing for measuring parabens, triclosan, and other environmental phenols in human milk. Anal Chim Acta 622(1-2), 150-156. Yi, B., Kim, C., Yang, M., 2010. Biological monitoring of bisphenol A with HLPC/FLD and LC/MS/MS assays. J Chromatogr B 878(27), 2606-2610. Yueh, M., Tukey, R., 2016. Triclosan: a widespread environmental toxicant with many biological 32

effects. Annu Rev Pharmacol Toxicol 56, 251-272. Zimmers, S., Browne, E., O'keefe, P., Anderton, D., Kramer, L., Reckhow, D., Arcaro, K., 2014. Determination of free bisphenol A (BPA) concentrations in breast milk of U.S. women using a sensitive LC/MS/MS method. Chemosphere 104, 237-243. Ziv-Gal, A., Flaws, J., 2016. Evidence for bisphenol A-induced female infertility: a review (2007– 2016). Fertil Steril 106(4), 827-856.

33

Highlights •

Phthalate metabolites, bisphenol A, parabens, and triclosan detected in breast milk



High levels of endocrine disruptors in mothers associated with lifestyle factors



Diet, consumer products, and behavior affect maternal toxic exposure in Korea

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: