Migration of Parabens, Bisphenols, Benzophenone-Type UV Filters

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Migration of Parabens, Bisphenols, Benzophenone-Type UV Filters, Triclosan, and Triclocarban from Teethers and Its Implications for Infant Exposure Alexandros G. Asimakopoulos,†,§ Madhavan Elangovan,†,§ and Kurunthachalam Kannan*,†,‡ †

Wadsworth Center, New York State Department of Health, and Department of Environmental Health Sciences, School of Public Health, Empire State Plaza, Albany, New York 12201-0509, United States ‡ Biochemistry Department, Faculty of Science and Bioactive Natural Products Research Group, King Abdulaziz University, Jeddah, Saudi Arabia S Supporting Information *

ABSTRACT: Parabens (p-hydroxybenzoic acid esters), bisphenols, benzophenonetype UV filters, triclosan, and triclocarban are used in a variety of consumer products, including baby teethers. Nevertheless, the exposure of infants to these chemicals through the use of teethers is still unknown. In this study, 59 teethers, encompassing three types, namely solid plastic, gel-filled, and water-filled (most labeled “bisphenol A-free”), were collected from the U.S. market and analyzed for 26 potential endocrine-disrupting chemicals (EDCs) from intact surfaces through migration/leaching tests performed with Milli-Q water and methanol. The total amount of the sum of six parent parabens (Σ6 Parabens) leached from teethers ranged from 2.0 to 1990 ng, whereas that of their four transformation products (Σ4 Parabens) ranged from 0.47 to 839 ng. The total amount of the sum of nine bisphenols (Σ9 bisphenols) and 5 benzophenones (Σ5 benzophenones) leached from teethers ranged from 1.93 to 213 ng and 0.59 to 297 ng, respectively. Triclosan and triclocarban were found in the extracts of teethers at approximately 10-fold less amounts than were bisphenols and benzophenones. Based on the amount leached into Milli-Q water, daily intake of these chemicals was estimated from the use of teethers by infants at 12 months of age. This is the first study to document the occurrence and migration of a wide range EDCs from intact surfaces of baby teethers.

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INTRODUCTION Parabens (p-hydroxybenzoic acid esters; preservatives), bisphenols (BPs; intermediates in the production of epoxy resins and polycarbonate plastics), benzophenone-type UV filters (BzPs; sunscreen agents), and triclosan (TCS) and triclocarban (TCC) (antimicrobials) are used in a variety of consumer products, including cosmetics.1−10 Owing to the potential endocrine-disrupting properties of these chemicals, exposure of humans, especially infants, is a concern. Baby teethers are one source of exposure to several endocrine-disrupting chemicals (EDCs) of infants. Nevertheless, very little is known about the occurrence and migration of various EDCs from teethers.11−13 By chewing and sucking teethers, infants soothe the pain associated with the growth of their first set of teeth (which normally sprout during 3−7 months of age). Further, mouthing is believed to be important for an infant’s psychological development in exploring his or her surroundings through tasting objects.14 In this study, we determined parabens, BPs, BzPs, TCS, and TCC in teethers. Parabens are widely used as preservatives in cosmetics, pharmaceuticals, and foodstuffs and are found in numerous baby products, including skin-care lotions and wipes. The occurrence of methyl- (MeP), ethyl- (EtP), and propyl© XXXX American Chemical Society

(PrP) parabens in 10 samples of baby teethers has been shown.11 BPs are used in polycarbonate plastics and resins, and the most popular derivative is 2,2-bis(4-hydroxyphenyl)propane (bisphenol A; BPA). In 2012, the U.S. Food and Drug Administration (FDA) banned the use of BPA in baby bottles and children’s drinking cups.15 In 2011, the European Commission restricted the use of BPA in plastic infant feeding bottles.16,17 According to Toy Safety Directive 2009/48/EC, the specific migration limit for BPA used in toys intended for use by children under 3 years of age, or in other toys intended to be placed in the mouth, was 0.1 mg/L.18 BzPs are used in cosmetics and as additives in plastics to prevent degradation by UV light.19 The most popular derivative of this class is oxybenzone, or 2-hydroxy-4-methoxybenzophenone (benzophenone-3; BP-3). Antimicrobial agents TCS and TCC are added to many consumer products, such as toothpaste, to reduce or prevent microbial growth.20 To our knowledge, Received: August 15, 2016 Revised: November 11, 2016 Accepted: November 15, 2016

A

DOI: 10.1021/acs.est.6b04128 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Chemicals and Materials. Baby teethers were analyzed for 10 parabens (MeP, EtP, PrP, butyl- [BuP], benzyl- [BezP] heptyl- [HeP] parabens, 4-hydroxy benzoic acid [4-HB], 3,4dihydroxy benzoic acid [3,4-DHB], methyl-protocatechuic acid [OH−MeP], and ethyl-protocatechuic acid [OH-EtP]); nine BPs (BPA, 4,4′-(hexafluoroisopropylidene)-diphenol [BPAF], 4,4′-(1-phenylethylidene) bisphenol [BPAP], 4,4′-sulfonyldiphenol [BPS], 4,4′-dihydroxydiphenylmethane [BPF], 4,4′(1,4-phenylenediisopropylidene) bisphenol [BPP], 4,4′-cyclohexylidenebisphenol [BPZ], 2,2-bis(4-hydroxyphenyl)butane [BPB], and 1,3-bis(2-(4-hydroxyphenyl)-2-propyl)benzene [BPM]); five BzPs (BP-3, 2,4-dihydroxybenzophenone [BP1], 2,2-dihydroxy-4-methoxybenzophenone [BP-8], 2,2,4,4tetrahydroxybenzophenone [BP-2], and 4-hydroxybenzophenone [4-OH-BP]); and the antimicrobial agents TCS and TCC. Parabens were further grouped as parent parabens (MeP, EtP, PrP, BuP, BezP, and HeP; collectively, Σ6Parabens) and transformation products (4-HB, 3,4-DHB, OH−MeP, OH-EtP; collectively, Σ4Parabens). Analytical standards of BPA (≥97%), BPAF (∼97%), BPAP (∼99%), BPS (∼98%), BPF (∼98%), BPP (∼99%), BPZ (∼98%), and BPM (≥99.0%) were purchased from SigmaAldrich (St. Louis, MO). An analytical standard of BPB (∼98%) was obtained from TCI America (Portland, OR). 13Cisotopically labeled BPA (13C12−BPA ≥ 99%), BPS (13C12− BPS ≥ 99%), and BPAF (13C12−BPAF ≥ 99%) were purchased from Cambridge Isotope Laboratories (Andover, MA). Analytical standards of MeP (∼98%), BuP (∼98%), and 4HB (∼98%) were purchased from Cambridge Isotope Laboratories (Andover, MA). 3,4-DHB (≥97%), OH−MeP (∼97%), and OH-EtP (∼97%) were purchased from SigmaAldrich (St. Louis, MO). 13C6-4-HB was purchased from Cambridge Isotope Laboratories (Andover, MA). Analytical standards of EtP, PrP, BezP, and HeP were purchased from AccuStandard, Inc. (New Haven, CT). 2D4-HeP and 2D4-BezP were obtained from C/D/N Isotopes, Inc. (Pointe-Claire, Quebec, Canada), 13C6-MeP, 13C6-EtP, 13C6−PrP, and 13C6− BuP were purchased from Sigma-Aldrich (St. Louis, MO). Analytical standards of BP-3 (∼98%), BP-1 (∼99%), BP-8 (∼98%), BP-2 (∼97%), and 4-OH-BP (∼98%) were purchased from Sigma-Aldrich (St. Louis, MO). 13C-isotopically labeled BP-3 (13C6−BP-3) was purchased from Cambridge Isotope Laboratories (Andover, MA). Analytical standards of TCS (≥97%) and TCC (99%) were purchased from Sigma-Aldrich (St. Louis, MO). 13C6-TCC was obtained from Wellington Laboratories, Inc. (Guelph, Ontario, Canada). Formic acid (98.2%) was obtained from Sigma-Aldrich (St. Louis, MO). Ammonium acetate (98%), hydrochloric acid (HCl, 37%), ammonium hydroxide (NH4OH, 29.5% assayed as NH3), and all organic solvents (analytical grade) used in the experiments were purchased from Mallinckrodt Baker (Phillipsburg, NJ). Milli-Q water was purified by an ultrapure water system (Barnstead International, Dubuque, IA). An oscillator shaker (Eberbach Corp., Ann Arbor, MI) was used for shaking purposes. A rotary evaporator (Büchi Rotavapor Model R-200, Flawil, Switzerland), mounted on a heating bath (Büchi Heating Bath B-490, Flawil, Switzerland), and a MultiVap 118 nitrogen evaporator (Organomation Associates Inc., West Berlin, MA) were used for evaporation and condensation purposes. Wide-mouth amber glass jars (500 mL) with Teflonlined screw caps were obtained from I-CHEM (Rockwood, TN). Conical autosampler glass vials (1.5 mL) with assembled screw caps (with PTFE/silicone septum) were obtained from

studies that describe the occurrence and leaching of these chemicals in teethers have not been conducted. Plastic teethers are marketed in a wide variety of colors, shapes, materials, and textures. On the basis of their design, we grouped teethers as solid plastic, gel-filled, and water-filled. Teethers, when used refrigerated or frozen, are considered to provide extra relief to an infant’s sore gums. If the teether is water- or gel-filled, microbial growth can occur, and, to prevent this, preservatives, such as parabens, are added.11,12 To date, little information is available on the exposure of infants to the aforementioned chemical classes through the use of teethers. The only study that measured plastic pieces and gel-filled material from five teethers showed the presence of MeP, EtP, and PrP, at several hundred μg g−1 concentrations.12 Leaching of these chemicals into an aqueous solution, however, has not been studied thus far. In our study, 59 teethers were collected from the U.S. market, and the majority of these products were sold in the United States; products that originated from Thailand, Hong Kong, and the United Kingdom also were collected. Leaching and migration of parabens, BPs, BzPs, TCS, and TCC from the surfaces of teethers were examined through extraction with Milli-Q water and methanol. The objectives of this study were to (a) determine concentrations and profiles of migration of 26 EDCs from teethers; (b) establish correlations among the target chemicals in teethers; (c) develop a “fingerprint” for the identification of three different categories of teethers, using principal component analysis (PCA); and (d) estimate daily intake of the target EDCs by infants through the use of teethers. This is the first study to assess the migration of four classes of EDCs from intact surfaces of baby teethers.

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MATERIALS AND METHODS Sample Collection and Preparation. Baby teethers were collected through online ordering between January and February 2016. All teethers were made of plastic and were grouped into three categories: solid plastic, gel-filled, and waterfilled. Overall, 59 teethers (7 solid plastic, 21 gel-filled, and 31 water-filled) from 23 different brand names were purchased. The majority of the teethers were sold in the United States (n = 53), and the remaining were from Thailand (n = 3), Hong Kong (n = 1), and the United Kingdom (n = 2). Of the seven solid plastic teethers, six were labeled “BPA-free” (one teether had a silicone piece, and another teether was labeled “designed for teeth cleaning”). Of the 21 gel-filled teethers, 18 were labeled “BPA-free,” one teether was labeled “non-toxic,” one teether was labeled “non-toxic gel,” while another teether had the label “complies with phthalates regulations.” Of the 31 water-filled teethers, 24 were labeled “BPA-free.” The samples were removed from their packaging and placed in clear polypropylene bags. The samples were then stored at room temperature in the dark until two consecutive migration trials were performed with Milli-Q water and methanol. The migration in water is of importance, as the salivary fluid consists of approximately 99% water,21 and during the mouthing of teethers, saliva is exposed to chemicals that are leached from products. For numerous chemicals, water is a good stimulant for leaching studies. In fact, leaching of chemicals in water has been shown to represent all contact routes of exposure (except for inhalation).22 We also applied methanol extraction, following leaching tests with water, to determine occurrence and the worst-case scenario for the leaching of chemicals from teethers. B

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described earlier.10 Briefly, the analysis of BPs and BzPs was achieved using a Shimadzu Prominence Modular HPLC system (Shimadzu Corporation, Kyoto, Japan), interfaced with an API 3200 electrospray triple quadrupole mass spectrometer (ESIMS/MS; Applied Biosystems) that was operated in the negative ionization mode. A Betasil C18 column (2.1 mm × 100 mm, 5 μm; Thermo Electron Corp., Waltham, MA), serially connected to a Javelin guard column (Betasil C18, 2.1 mm × 20 mm, 5 μm; Thermo Electron Corp.), was used for separation. The mobile phase comprised methanol (A) and Milli-Q water that contained 0.1% (v/v) ammonium hydroxide (B). The analyses of parabens, TCS, and TCC were carried out using a Waters Acquity ultraperformance liquid chromatography (UPLC) system (Waters, Milford, MA) connected to an Applied Biosystems API 5500 electrospray triple quadrupole mass spectrometer (ESI−MS/MS; Applied Biosystems, Foster City, CA) that was operated in the negative ionization mode. A Kinetex C18 column (2.1 mm × 50 mm, 1.3 μm; Phenomenex Inc., Torrance, CA), serially connected to a SecurityGuard ULTRA C18 guard column (2.1 mm, sub-2 μm core−shell column; Phenomenex Inc.), was used for separation. The mobile phase comprised methanol (A) and Milli-Q water that contained 0.1% (v/v) formic acid (B). Tandem MS transitions for the target chemicals are provided in SI Table S1. Further details on HPLC mobile phase gradients, MS ion source parameters, and injection volumes are provided elsewhere.10 The limits of detection (LODs) and quantitation (LOQs) were calculated for each target analyte as 3 and 10 times the signal from the baseline noise (S/N ratio), respectively (SI Table S2). Quality Assurance/Quality Control (QA/QC). Contamination that arose from laboratory materials and solvents was evaluated and controlled by the analysis of procedural blanks. Nine (9) reagent blanks were prepared for each of the two leaching trials, one (1) for every batch of 5 to 7 actual samples. The average amounts found in reagent blanks ranged up to 3.20 ng for 4-HB in the water trial (SI Table S3). The amounts found in each of the reagent blanks were subtracted from the samples of the respective batch. All reagent blanks and extraction solvents were fortified with a known concentration of isotope-labeled internal standards (66.7 ng/mL in-vial) prior to extraction. Quantitation of target analytes was performed by the internal standard method. When using the internal standard method, we constructed a calibration curve for every target analyte from the ratio of the analyte response to the corresponding internal standard response in every measured standard solvent solution (methanol), plotted against the concentration (amount) of the spiked analyte. The isotopedilution standard was selected for every target analyte based on the chemical similarity (e.g., 13C or 2D-analog) and proximity to the chromatographic elution of the target analyte. Instrumental calibration from the LOQ of every target analyte to 100 ng/mL showed excellent correlation coefficients (r > 0.99). Throughout the analysis, seven pre-extraction matrix spikes were prepared by spiking known concentrations of target analytes (50 ng/mL in-vial) and passing them through the entire analytical procedure (without a teether sample). The precision of the method was demonstrated (relative to the corresponding isotope-labeled standard) at the same concentration (50 ng/mL in-vial) as relative standard deviation % (RSD %; n = 7) and was found for all target analytes to be MeP (55/59) ∼ BuP (54/59) ∼ 3,4-DHB (53/59) > OH−MeP (50/59) > HeP (40/59)> BezP (34/59) ∼ OH-EtP (32/59) (SI Table S5). Median total amounts for the sum of parent parabens (Σ6 parabens) and their transformation products (Σ4 parabens) that leached into water and methanol, collectively, were 27.4 and 11.7 ng, respectively (Table 1). The median amounts for MeP, EtP, PrP, and BuP leached into water were 7.07, 0.82, 0.88, and 0.07 ng, respectively, whereas those into methanol were 13.4, 1.38, 2.85, and 0.18 ng, respectively. Among paraben transformation products, the median amounts for 4-HB, 3,4-DHB, and OH−MeP leached into water were 9.74, 1.98, and 0.22 ng, respectively, and those into methanol were 5.18, 0.82, and 0.47 ng, respectively. The total amount of parent parabens that leached from gelfilled teethers (median: 138 ng) was approximately an order of magnitude higher than that from water-filled (median: 17.8 ng) and solid plastic teethers (median: 9.8 ng) (Figure 1). The median amount of MeP varied considerably in gel-filled teethers (SI Figure 3S). The transformation products of parabens leached into water and methanol did not exhibit differences among the three types of teethers; the rank order of median amount of Σ4 parabens leached was: gel-filled (14.6 ng) > water-filled (11.7 ng) ∼ solid plastic (11 ng) (Figure 1).

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RESULTS AND DISCUSSION Migration/Leaching of Target Chemicals. The amounts of 26 target chemicals leached into water and methanol from 59 baby teethers are shown in SI Tables S5−S7 and Table 1. Parabens were the major class of chemicals found in the water D

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Figure 2. Distribution profiles of the amounts of parabens, bisphenols, benzophenones, and antimicrobials leached from baby teethers following water and methanol extractions (based on median concentrations).

water-filled teethers (7.68 ng). BPA, BPF, and BPS have a log Kow value of 3.32, 2.91 and 1.70, respectively.32 The greater log Kow value of BPA explains the higher amounts of this compound in gel-filled teethers, which are filled with an organic gel. The rank order of detection rate of BzPs leached from teethers was: BP-3 (58/59) > BP-2 (44/59) ∼ 4-OH-BP (42/ 59) > BP-1 (29/59). BP-8 was not detected in any of the samples (SI Table S7). The median amount of BzPs (Σ5 ΒzPs) leached into water and methanol was 5.39 ng (Table 1), which was approximately 4- to 5-fold less than that found for parabens and BPs. The median amounts of BP-3, BP-2, 4-OH-BP, and BP-1 leached into water were 0.28, 0.11, 0.37, and 0.23 ng, respectively, and those into methanol were 2.69, 0.59, 1.83, and 2.92 (only 1 sample) ng, respectively. The total amounts of BzPs that migrated/leached from gelfilled teethers (median: 6.85 ng) were higher than those from solid plastic (median: 4.51 ng) and water-filled teethers (median: 3.59 ng) (Figure 1). BzPs absorb the UV light and protect plastic materials from photodegradation.33 Therefore, the total amounts of BzPs were equivalent to plastic content of the teethers. Gel-filled teethers maintained a higher content of plastic than did solid plastic teethers. The median weight of gelfilled teethers was 47.8 g, which was followed by water-filled (41.4 g) and solid plastic teethers (23.2 g). 4-OH-BP, in particular, was found at higher amounts in gel-filled teethers (3.22 ng), followed by solid plastic (0.79 ng) and water-filled teethers (0.42 ng). Higher amounts of 4-OH-BP were found in gel-filled teethers, which can be explained by this chemical’s use in translucent applications.33 BP-3 was found at higher amounts in solid plastic teethers (4.33 ng), which are usually opaque, followed by gel-filled (2.69 ng) and water-filled teethers (2.28 ng). The pattern of occurrence of BP-1 in leachates from teethers was similar to that found for BP-3; BP-1 was found at higher amounts in solid plastic teethers (0.35 ng), followed by gel-filled (0.26 ng) and water-filled teethers (0.14 ng). BP-2 was found at higher amounts in gel-filled teethers (0.78 ng), followed by water-filled (0.57 ng) and solid plastic (0.55 ng) teethers. BP-1 and BP-2 found in teether extracts were probably impurities of BP-3 and 4-OH-BP. BP-3 has been shown to be metabolized to BP-1, BP-2, and 4-OH-BP in biological systems.34 TCC (55/59) and TCS (54/59) were found in teethers at concentrations two to 3 orders of magnitude lower than those of parabens and BPs (SI Table S7). Nevertheless, TCS and TCC have demonstrated toxicity at trace amounts (pg).35,36 The median amount of TCS and TCC leached into water and methanol was 0.49 ng (Table 1). The median amounts of TCS

These results suggest that the transformation products are formed from parent parabens during the manufacture or storage of teethers. Alkyl protocatechuates that were found in teethers were potentially formed from photo-oxidation of parent parabens. Light-induced hydroxylation of MeP to OH− MeP has been shown.23 The mechanism of formation of 4-HB and 3,4-DHB from parabens in teethers, however, is still unclear, although biotransformation of parabens to 4-HB and 3,4-DHB has been documented in animal and human studies.24−27 The highest amount of parabens leached from teethers was 2068 ng, which was found in a gel-filled teether that was labeled “non-toxic.” All BPs were found in teethers, and the rank order of detection rate was: BPA (59/59) = BPS (59/59) > BPF (44/ 59) > BPAF (18/59) > BPB (15/59) ∼ BPZ (14/59) > BPAP (9/59) > BPM (5/59) > BPP (1/59) (SI Table S6). BPF, BPS, and BPA were the major BPs that were found in leachates from the teethers (SI Table S6). The median amount of total bisphenols (Σ9 BPs) leached into water and methanol from the teethers was 20.6 ng (Table 1), which was slightly less than those found for parabens (27.4 ng). Even though 48 of the 59 teethers were labeled “BPA-free,” our results suggest that the labels were misleading; BPA was found to migrate from all teethers analyzed in this study. The median amounts of BPA, BPS, and BPF leached into water were 2.28, 2.32, and 4.66 ng, respectively, and those into methanol were 8.41, 3.10, and 1.17 ng, respectively. In contrast to that found for parent parabens, the total amounts of BPs leached from all three types of teethers were similar; the rank order of the median amounts for Σ9 BPs was: gel-filled (21.5 ng) > solid plastic (19.8 ng) ∼ water-filled (19.5 ng) (Figure 1). BPS was found at higher amounts in solid plastic teethers (7.34 ng), followed by gel-filled (5.03 ng) and water-filled teethers (4.99 ng). BPS is relatively more heat and light stable than are the other bisphenols (such as BPA),28 and, therefore, its application is more appropriate on solid plastic teethers, which are designed for use at room temperature (rather than cold conditions). BPF was found at higher amounts in water-filled teethers (5.27 ng), followed by gel-filled (1.24 ng) and solid plastic teethers (1.17 ng). BPF-based resins have lower viscosity than do BPA resins29 and are more suitable for use in liquids with a fairly low viscosity such as aqueous solutions. BPF epoxy resins are used in water pipes, among the other applications.30 Materials with low viscosity, when in contact with water, reduce the formation of air-bubbles that can affect emulsion stability, promote oxidation, and encourage microbial growth.31 BPA was found at higher amounts in gelfilled teethers (13.8 ng), followed by solid plastic (10.5 ng) and E

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Figure 3. Principal component analysis of classification of teethers into three categories of solid, water-, and gel-filled based on the log transformed amount of parent parabens and transformation products, bisphenols, benzophenones, and antibacterial-antifungal agents (antimicrobials) leached from baby teethers into water and methanol extracts. The score plot demonstrates the classification, while the loadings plot demonstrates how the variables contribute to each of the principal components (PC1 and PC2).

between parent parabens and their transformation products. A significant correlation was found between the log-transformed amounts of TCS (Log TCS) and TCC (Log TCC) (r = 0.52, p = 0.0001), which indicated a common usage in teethers. A significant correlation also was found between the amounts of TCS and 4-HB leached from teethers (r = 0.38, p < 0.05), between amounts of BPS and BPA leached into methanol extracts (r = 0.49; p < 0.0001), between the amount of parabens (Log Σ10 Parabens) extracted into water and methanol (r = 0.65; p < 0.0001), and between the amount of BzPs (Log Σ5 BzPs) extracted into water and methanol (r = 0.39, p < 0.010). The correlations of parabens and BzPs suggest similarities in migration pattern, regardless of aqueous or organic nature of the solvent. Principal Component Analysis of Migration Patterns of Chemicals from Teethers. PCA is used to identify the patterns within a specific data set, with the aim of clustering similar observations. The desired outcome of the PCA is to project and visualize the data set on a two-dimensional space with minimal loss of information. For this purpose, it is necessary to decrease the number of variables to a few linear combinations of the data set. Each linear combination corresponds to a specific principal component. We used logtransformed values for total amounts of parent parabens, parabens’ transformation products, BPs, BzPs, and antimicrobials for PCA. The loading plot (Figure 3) shows which variables were influential for the PCA model and how these variables are correlated to each other. The points that are proximal in the loading plot (Figure 3) denote similar leaching profiles; when the value of one increases or decreases, then the value of the other will change in a similar manner. Thus, according to the loading plot, only antimicrobials are away from the other chemical classes studied. It is interesting to note that the generated score plot (Figure 3) is clustered based on the three categories of the teethers (solid plastic, gel-filled, and water-filled) analyzed. The score plot that was generated from PC1 and PC2 accounted for 89.8% of the variabilities. The eigenvalues for PC1 and PC2 were 41.1 and 11.9, respectively (both >1), which denoted a reliable model that can serve as a visualized “fingerprint” for the three different categories of teethers. The results of the PCA suggest that the profiles of chemicals migrated from the teethers differ, depending on the three categories analyzed. Assessment of Exposure to EDCs from Teethers. The estimated daily intake (EDI; ng/kg-bw per day) of parabens,

and TCC leached into water were 0.18 and 0.028 ng, respectively, and those into methanol were 0.32 and 0.092 ng, respectively. Solid plastic teethers leached higher amounts of TCS and TCC than did gel- and water-filled teethers (Figure 1). Solid plastic teethers are used at ambient temperatures, and there is the potential for increased microbial growth that is not seen in water- and gel-filled teethers, which are expected to be stored under cold conditions prior to use.37 For some water- and gelfilled teethers, manufacturers have instructed the consumers to keep them “in the refrigerator or freezer, when not in use.” Distribution Profiles of Migration and Correlations among Chemicals Leached from Teethers. The migration profile of the target chemicals analyzed in water and methanol extracts of the teethers is presented in SI Figure 4S. By taking into consideration the median values (SI Figure 4S), the amounts of most target compounds, except for 4-HB, 3,4-DHB, OH-EtP, BPF, BPP, and BPZ, leached into water were lower than those leached into methanol. Among several parabens, BPs, BzPs analyzed, MeP, BPA, and BP-3 were the most abundant compounds found in teether extracts (Figure 2). Our findings are in accordance with the previous findings from human biomonitoring studies,9,10 which suggested that these derivatives are widely used in many consumer products. The distribution of the amounts of target chemicals leached from each teether was right-skewed, and, therefore, the data were logarithm (log) transformed for the evaluation of intercorrelations. Significant intercorrelations between chemicals suggest combined usage and similar migration pattern. A significant correlation that was previously reported in human urine specimens9,10 also was demonstrated in the total migration amounts between MeP (Log MeP) and PrP (Log PrP) (r = 0.60, p < 0.0001). Other statistically significant correlations were found between MeP and EtP (r = 0.70, p < 0.0001), MeP and BuP (r = 0.46, p < 0.001), EtP and BuP (r = 0.53, p < 0.0001), PrP and BuP (r = 0.66, p < 0.0001), 4-HB and 3,4-DHB (r = 0.36, p < 0.01), 4-HB and OH−MeP (r = 0.50, p < 0.0001), PrP and OH−MeP (r = 0.56, p < 0.0001), MeP and 4-HB (r = 0.32, p = 0.010), PrP and 4-HB (r = 0.46, p < 0.001), BuP and 4-HB (r = 0.38, p < 0.010), EtP and OH− MeP (r = 0.47, p < 0.05), EtP and OH-EtP (r = 0.48, p < 0.05), and MeP and OH−MeP (r = 0.59, p < 0.0001). These correlations between parent parabens denote common applications in teethers, as most parabens are used in combination. The remaining correlations denote relationships F

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Table 2. Estimated Daily Intake (ng/kg-bw/day) of Parabens, Bisphenols, Benzophenones and Antimicrobial Agents from Mouthing of Baby Teethers for 1 h by 12 Month Old Infants Σ6 parabens(1)a average median maximum

Σ4 parabens(2)b

Σ9 BPs

Σ5 BzPs

Σ2 antimicrobial agentsc

Total (Σ26)

male

female

male

female

male

female

male

female

male

female

male

female

3.12 0.59 22.0

3.36 0.63 23.7

2.61 0.43 39.8

2.81 0.46 42.9

1.40 0.67 11.7

1.51 0.73 12.6

0.35 0.055 3.56

0.38 0.059 3.84

0.032 0.004 0.88

0.035 0.004 0.94

7.50 3.30 53.5

8.09 3.56 57.7

Σ6 Parabens(1): MeP+EtP+PrP+BuP+BzP+HeP. bΣ4 Parabens(2): 4-HB+3,4-DHB+OH−MeP+OH-EtP. cAntimicrobial agents represent TCS and TCC.

a

This is the first study to document the occurrence and migration profile of parabens, BPs, BzPs, and antimicrobial agents from the intact surfaces of baby teethers. The majority of the chemicals that were assessed were found with high detection frequencies. The finding of BPA in teethers labeled “BPA-free” was intriguing. Our findings can be used to develop appropriate policies to protect infants from exposure to potentially toxic chemicals found in baby teethers.

BPs, BzPs, and antimicrobials from the use of teethers directly from their package, without any prewashing step, was estimated for a 12-month-old infant. The maximum estimated mouthing time per day was reported to be generally between 60 and 90 min.14 The maximum daily mouthing on toys increases at 6−9 months of age and then decreases as children reach 5 years of age.14 At 12 months of age, the daily mouthing time on toys can reach up to 60 min.14 Thus, an estimate of intake can be obtained based on the results of a water migration test; our test involved the contact of teethers with Milli-Q water for 60 min. The exposure estimation for target EDCs was performed on the basis of eq 1:38

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.6b04128. Tables showing the tandem MS parameters for the analysis, the analytical method performance characteristics, and the concentrations of target chemicals found in baby teethers. Figures showing the migration trial protocols, differences between the median of the gelfilled teethers with the water-filled and solid teethers for selected parabens, and migration profiles of parabens, bisphenols, benzophenones and antimicrobial agents from baby teethers into water and methanol extracts (PDF)

⎛ ⎞ migration amount (ng) per day ng EDI⎜ ⎟= body weight (kg) ⎝ kg − bw/day ⎠ (1)

Males weigh 9.7 kg, and females weigh 9.0 kg at the 52.1% percentile (in the United States) at the age of 12 months.39 The body weight-normalized exposure doses of EDCs from the use of teethers for 60 min (based on median values) by an infant were 3.30−3.56 ng/kg-bw/day (Table 2). For parabens, an acceptable daily intake (ADI) of