Nov 12, 2012 - urine samples collected from the U.S. and China were analyzed for free and .... MA). d3-Creatinine (99%) was purchased from CDN Isotopes.
Article pubs.acs.org/est
Widespread Occurrence and Distribution of Bisphenol A Diglycidyl Ether (BADGE) and its Derivatives in Human Urine from the United States and China Lei Wang,†,‡ Yinghong Wu,§ Wei Zhang,§ and Kurunthachalam Kannan*,†,¶ †
Wadsworth Center, New York State Department of Health, and Department of Environmental Health Sciences, School of Public Health, State University of New York at Albany, Empire State Plaza, P.O. Box 509, Albany, New York 12210-0509, United States ‡ Ministry of Education Key Laboratory of Pollution Processes and Environmental Criteria/Tianjin Key Laboratory of Environmental Remediation and Pollution Control, Nankai University, Tianjin 300071, China § Tianjin Centers for Disease Control and Prevention, Tianjin 300171, China ¶ International Joint Research Center for Persistent Toxic Substances, State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China S Supporting Information *
ABSTRACT: Despite reports of the occurrence of bisphenol A diglycidyl ether (BADGE) and its derivatives in canned foods and consumer products, biomonitoring studies of human exposure to these compounds are lacking. In this study, 127 urine samples collected from the U.S. and China were analyzed for free and total (free plus conjugated) concentrations of BADGE and its three derivatives, bisphenol A (2,3dihydroxypropyl) glycidyl ether [BADGE·H2O], bisphenol A (3-chloro-2-hydroxypropyl) (2,3-dihydroxypropyl) ether [BADGE·HCl·H2O], and bisphenol A bis (2,3-dihydroxypropyl) ether [BADGE·2H2O], using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/ MS). BADGE and its three derivatives (collectively referred to as BADGEs) were found in 100% of the urine samples analyzed. Total urinary concentrations of BADGEs in the U.S. ranged from 1.24 to 9.03 ng/mL, with a GM concentration of 3 ng/mL. Concentrations of BADGEs in urine from adults (GM: 1.36 ng/mL) and children (1.02 ng/mL) in China were 3-fold lower than the concentrations found in the U.S. Both free and conjugated forms of BADGEs were present in urine, and the proportion of free form was inversely related to the total concentration of BADGEs. Among the four BADGEs measured in urine, BADGE·2H2O was the predominant compound, accounting for 45−60% of the total BADGEs concentration, followed by BADGE (17−24%). The distribution of the four BADGEs varied, depending on age, gender, and ethnicity of the adults and children. Daily intake (DI) and effective daily intake (DIE) of BADGEs were estimated based on urinary concentrations, and their respective values were 69.4 and 9.16 ng/kg-bw/day for the U.S. population and 28.4 and 5.69 ng/kg-bw/day for the Chinese population. The concentrations of BADGEs in U.S. urine were 3- to 4-fold higher than the corresponding concentrations of bisphenol A.
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INTRODUCTION Bisphenol A diglycidyl ether (BADGE) is a building block of epoxy resin, which is widely used in the coatings of food and beverage cans and in adhesives.1,2 BADGE also is used as a monomer in the production of epoxy-based polymers and as an additive for the elimination of surplus hydrochloric acid in polyvinyl chloride (PVC) production.1 The annual global production of BADGE was 957 000 t in 2003.2 BADGE and its hydrolytic and chlorinated derivatives, such as bisphenol A (2,3dihydroxypropyl) glycidyl ether [BADGE·H2O], bisphenol A (3-chloro-2-hydroxypropyl) (2,3-dihydroxypropyl) ether [BADGE·HCl·H2O], and bisphenol A bis(2,3-dihydroxypropyl) ether [BADGE·2H2O] (collectively referred to as BADGEs in this study), are present in many canned foods at © 2012 American Chemical Society
concentrations on the order of several milligrams per kilogram.3−6 BADGE and its derivatives can react with food matrix, especially proteins,3−5 which might lead to an underestimated dietary intake of these compounds. A migration limit of 9 mg/kg for BADGE and its hydrolytic derivatives and 1 mg/kg for the chlorinated derivatives has been proposed by the European Commission for food contact applications.7 BADGE has been reported to form DNA adducts,8 and to elicit teratogenic and mutagenic effects in vitro.9,10 In addition Received: Revised: Accepted: Published: 12968
October 5, 2012 November 9, 2012 November 12, 2012 November 12, 2012 dx.doi.org/10.1021/es304050f | Environ. Sci. Technol. 2012, 46, 12968−12976
Environmental Science & Technology
Article
Figure 1. Chemical structures of bisphenol A diglycidyl ether (BADGE) and its three derivatives, i.e., bisphenol A (2,3-dihydroxypropyl) glycidyl ether [BADGE·H2O], bisphenol A (3-chloro-2-hydroxypropyl) (2,3-dihydroxypropyl) ether [BADGE·HCl·H2O], and bisphenol A bis (2,3dihydroxypropyl) ether [BADGE·2H2O], as well as bisphenol A (BPA) and 13C-isotopically labeled 2-hydroxy-4-methoxybenzophenone (13C12−BP3). Potential formation route of BADGE derivatives in biological systems is shown.
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MATERIALS AND METHODS Chemicals. Analytical standards of BADGE (≥95%) and its derivatives, BADGE·H2O (≥95%), BADGE·HCl·H2O (≥95%), and BADGE·2H2O (≥97%), as well as creatinine (99%) and βglucuronidase from Helix pomatia (145 700 units/mL βglucuronidase and 887 units/mL sulfatase), were purchased from Sigma-Aldrich (St. Louis, MO). 13C-isotopically labeled 2hydroxy-4-methoxybenzophenone (13C12−BP-3) (99%) was purchased from Cambridge Isotope Laboratories (Andover, MA). d3-Creatinine (99%) was purchased from CDN Isotopes (Pointe-Claire, Quebec, Canada). The molecular structures and selected physicochemical properties of the target compounds are shown in Figure 1 and Supporting Information Table S1, respectively. Milli-Q water was prepared using an ultrapure water system (Barnstead International, Dubuque, IA). The stock solutions of target analytes and internal standards were prepared at 1 mg/mL in methanol and stored at −20 °C. Sample Collection. Urine samples from the U.S. were collected from healthy volunteers in Albany, New York, from May to July 2011 (n = 31), and those from Chinese adults were collected from Shanghai during August and September 2010 (n = 26). Information on age and gender is provided in Table S2. In addition, 70 urine samples were collected from children aged 9−10 yrs (38 male and 32 female) during March and April 2012 in Tianjin, China. All urine samples were collected in polypropylene (PP) tubes and stored at −80 °C before analysis. Institutional Review Board approvals were obtained from the New York State Department of Health (NYSDOH) for the analysis of human specimens. Sample Preparation. Urine samples were extracted using a liquid−liquid extraction (LLE) method. Five hundred microliters of urine samples were transferred into a 15-mL PP tube, and 50 μL of 13C12−BP-3 (100 ng/mL; i.e., 5 ng) were spiked. 13 C12−BP-3 was used as an internal standard, owing to its structural similarity to and chromatographic elution with
to genotoxic effects of BADGEs, cytotoxicity, developmental, and reproductive toxicity have been reported in laboratory animals.9,11−13 Male workers exposed to BADGE had lower levels of follicle stimulating hormone than the reference population.14 Following oral and/or dermal exposure, BADGE is metabolized to bis-diols by epoxide hydrolase.15,16 As a stable metabolite of BADGE, BADGE·2H2O was also reported as an endocrine disruptor, with potentials even greater than those of bisphenol A (BPA), a known endocrinedisrupting chemical.17 Further, antagonistic activity of the chlorohydroxy derivative of BADGE toward androgen has been reported.18 Studies also have shown that BADGE was not transformed into BPA by the mammalian metabolic system.15,16,19,20 However, it is believed that BPA in canned food is released primarily from BADGE-based epoxy resin coating.21 Although the occurrence of BADGEs in foodstuffs has been reported,3−6,22 human biomonitoring studies, which report concentrations in body tissues or fluids, are still lacking. Moreover, the forms and compositions of BADGE can change in human bodies through the formation of conjugated metabolites (e.g., glucuronidation and/or sulfation) or as hydrolytic and hydrochloro derivatives by the action of epoxide hydrolase. No earlier studies have reported the occurrence of BADGE and its derivatives in human tissues/fluids. In this study, concentrations of free and total forms (i.e., free plus conjugated) of BADGE and its three derivatives, BADGE·H2O, BADGE·HCl·H2O, and BADGE·2H2O, were determined in 127 urine samples collected from the U.S. and China. The objectives of this study were to (i) determine the occurrence and distribution of BADGEs in urine from the general populations in the U.S. and China; (ii) elucidate differences in the forms and profiles of BADGEs; and (iii) develop an index, based on the form/composition of BADGEs in urine, for the assessment of effective daily intake. 12969
dx.doi.org/10.1021/es304050f | Environ. Sci. Technol. 2012, 46, 12968−12976
Environmental Science & Technology
Article
concentrations of BADGE, BADGE·H2O, and BADGE·2H2O, respectively, in urine samples. Six urine samples were selected randomly, and four BADGEs as well as 13C12−BP-3 (5 ng each) were spiked and passed through the entire analytical procedure. Recoveries of all target compounds in spiked matrices ranged from 72% to 107% (corrected by 13C12−BP-3 recoveries). The results are presented in detail in Table S4. Recoveries of target compounds spiked into procedural blanks ranged from 82% to 102% (Table S4). Duplicate analysis of selected samples showed a coefficient of variation of 44 for creatinine and 117 > 47 for d3creatinine. The MS/MS collision energy was 25 eV, ion source temperature was 400 °C, and cone voltage was 4500 V. Quality Assurance and Quality Control (QA/QC). Contamination arising from laboratory materials and solvents was evaluated by the analysis of procedural blanks with every batch of samples. BADGE and BADGE·2H2O were detected in procedural blanks at concentrations of 0.02 and 0.03 ng/mL, respectively, which were well below the concentrations found in most urine samples. Values of 0.03, 0.01, and 0.03 were subtracted, as background values, from the measured
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RESULTS AND DISCUSSION Concentrations of Free and Total BADGEs in Urine. Of the 127 urine specimens analyzed, BADGEs were found in all (100%) urine samples (Table 1, Figure S2), indicating a widespread human exposure to these chemicals. The sum of concentrations of the four BADGEs in “free” form in urine from the U.S. ranged from 0.308 to 2.85 ng/mL, with a GM concentration of 0.913 ng/mL. For adults and children from China, the sum concentration of the four BADGEs in “free” form was slightly lower than the concentration measured in the U.S. samples. Nevertheless, total BADGEs (free plus conjugated forms) concentrations were significantly different between the U.S. and China. The GM concentration of the sum of the four BADGEs was 3.00 ng/mL for urine from the U.S., which was 2−3 times higher than the GM concentration found for adults (1.33 ng/mL) and children (1.07 ng/mL) from China. The distribution of free and conjugated forms varied among the four target analytes, which is discussed in detail below. Among the four BADGEs determined, BADGE·2H2O was the predominant compound found in urine samples, irrespective of “free” or “total” concentrations measured, with a detection rate of 100% (Table 1). Based on the total GM concentrations of the four BADGEs determined (Table 1), the distribution of BADGEs was calculated (Figure S3). In urine samples from the U.S., BADGE·2H2O accounted for ∼45% of the total BADGEs followed by BADGE (~24%) and BADGE·H2O (~17%). For adults and children from China, the proportion of BADGE·2H2O in total BADGEs concentration was ∼57% and ∼60%, respectively. In addition to the direct intake of BADGE·2H2O from foods, in vivo metabolism of BADGE and other precursors can contribute to the formation of BADGE·2H2O in human bodies.15,16,19,20 The proportion of BADGE·H2O·HCl is relatively low, especially in urine from China, indicating a lower level of chloride derivatives of BADGEs in human urine. 12970
dx.doi.org/10.1021/es304050f | Environ. Sci. Technol. 2012, 46, 12968−12976
0.594 0.552 0.333−5.816 100
1.073 1.018 0.585−6.778 100
Creatinine also was determined in urine samples from the U.S. and China. The GM concentrations of creatinine in urine from the volunteers in the U.S. and adults in China were 1.02 and 1.09 g/L, respectively, whereas the GM creatinine concentration in children from China was 0.45 g/L. The levels of creatinine in urine were in the normal range for the corresponding population groups, to some extent indicating the representativeness of the samples. Forms (Free versus Conjugated) of Urinary BADGEs. Distribution of BADGEs in urine, including forms (free or conjugated) and composition (precursor or derivatives) varied widely. The form distribution coefficient ( f i) was calculated as the percentage of free species in total concentration, as shown in eq 1 fi =
GM = geometric mean; to obtain GM, ((LOQ)/(√2)) was assigned to samples with values
