ASSESSMENT OF PBDE EXPOSURE THROUGH MOUTHING ON TOYS - IN ... (2009)3. However, similar studies on PEs exist in the literature, such as the one of Niino et al. (2001 .... Greenpeace Feature âA toxic Toy Storyâ, July 5, 2005. 2.
ASSESSMENT OF PBDE EXPOSURE THROUGH MOUTHING ON TOYS - IN VITRO MIGRATION INTO SALIVA SIMULANT Ionas AC1*, Ballesteros Gómez AM2, Brandsma SH2, Leonards PEG2 and Covaci A1 1
2
Toxicological Centre, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium Institute for Environmental Studies, VU University, de Boelelaan 1085, 1081 HV Amsterdam, The Netherlands
Introduction Children usually spend a high amount of time indoors playing with toys. However, as our chemical industry has progressed, more and more toys contain a vast number of additives, some of which being toxic1. It is thus safe to assume that children are possibly exposed to these toxicants, if they leach out of the toys. In a recent study, we have analysed a wide array of toys for additives such as polybrominated diphenyl ethers (PBDEs), organophosphate esters (OPEs) and phthalate esters (PEs)2. Using models from the literature, we have tentatively assessed the exposure to these chemicals and found out that the most significant pathway of exposure was through mouthing (Fig. 1). Another worrisome fact was that the age group most at risk were the infants, thus at an age when they are most vulnerable and which is most critical in their development.
Figure 1: The estimated PBDE exposure according to the pathways considered, for all age groups2. The aim of this study was to more accurately assess the exposure of infants to PBDEs through mouthing activities, by determining the amount of these chemicals that can leach out of toys in simulated conditions. To our knowledge, only a brief, tentative study was published on this topic, in the supplementary information section of the study by Chen et al. (2009) 3. However, similar studies on PEs exist in the literature, such as the one of Niino et al. (2001, 2002)4,5 and Könemann et al. (1998)6. Materials and methods Materials. n-Hexane, acetone, toluene, tetrahydrofuran and iso-octane were purchased from J.T. Baker (Deventer, the Netherlands) and dichloromethane (DCM) for residue analysis from Promochem (Wesel, Germany). Anhydrous sodium sulphate was obtained from Merck (Darmstadt, Germany). All solvents and reagents were at least of analytical grade. Isotopically labelled (13C) BDE209, (99%, in toluene at 25 μg/mL) was supplied by Wellington Laboratories. Fluorinated PBDE congeners (F-BDE47 and F-BDE183) were bought from Chiron (Trondheim, Norway). PDBE congeners were also purchased as a mixture (BDE-MXE) in a nonane/toluene solution (48.6:51.4, v/v) from Wellington Laboratories, at concentrations ranging from 1 to 5 μg/mL. A certified reference material (CRM) of BFRs (PBDEs: 17, 28, 47, 49, 66, 74, 75, 85, 97, 99, 100, 101, 118, 119, 138, 139, 153, 154, 155, 173, 180, 181, 182, 183, 190, 197, 204, 207, 208, 209 and BB-209) in a polymeric matrix ( polypropylene: ERM-EC591) was purchased from IRMM (Geel, Belgium). Glass beads (G9268-100G, 425-600 µm) were acquired from Sigma Aldrich (St. Louis, MO, USA). Falcon™ conical centrifuge tubes (50 mL) were bought from Fisher Scientific (Waltham, MA, USA).
Simulation of mouthing/leaching into saliva methodology. The procedure used was adapted from similar studies on PEs4,5,6, which also included a comparison between the in vitro methodology and an in vivo assay, to serve as control. An Incubating Orbital shaker from VWR (Radnor, Pennsylvania, USA) was employed at a rotation speed of 250 rpm, at a temperature of 37 °C, as to mimic in vivo conditions as much as possible. The saliva simulant solution used was the one described in the British Standard BS 6684 (1987), with the following composition: 4.5 g NaCl, 0.3 g KCl, 0.3 g Na2SO4, 0.4 g NH4Cl, 0.2 g urea, 3.0 g lactic acid, dissolved in 1000 mL MiliQ water (resistivity 18.2 MΩ·cm) and adjusted to pH 6.8. The chosen pH is derived from literature data6, at a lower value because the pH of baby saliva is typically a bit lower than in adults. A volume of 30 mL of saliva simulant was used per assay, and was added to a 50 mL Falcon™ conical centrifuge tube, along with the sample. As the migration of chemicals in a solution occurs from the surface, the size of the sample was calculated in surface area rather than weight. A 10 cm2 total surface area was chosen as to correspond to the surface area of a child’s open mouth, as this is surface area typically available for mouthing at any one time7. Ideally, samples should be run in triplicate, for two exposure times (15 and 60 min, low and high exposure scenario, respectively). QA/QC. Controls were run for every step of the process to assess possible contamination or loss of analytes. A number of two procedural blanks were run with every batch of samples analysed. Samples. Two toy samples, with known PBDE concentrations in the order of tens of µg/g were tested as a proof of concept. Extraction. The method of extraction employed was liquid-liquid extraction, using 2 × 5 mL of a 1:1 v/v mixture of n-hexane and DCM. As soon as the organic phase was separated, it was spiked with internal standards and added over a solid-phase extraction cartridge with approximately 1 g of anhydrous sodium sulphate, to retain all traces of water. The extract was then evaporated, re-constituted in a 1:1 v/v mixture of iso-octane and toluene (200 µL) and transferred to injection vials. Instrumental analysis. The identification and quantification of the PBDEs was done on an Agilent GC 6890N (Agilent Technologies Netherlands BV, Amstelveen, the Netherlands) coupled to a 5975XL MS with a chemical ionisation source and equipped with a pulsed splitless inlet and an Agilent 7683 auto-sampler. F-BDE47, FBDE183 and 13C-BDE209 were used as internal standards. The separation of the analytes was carried out on an Agilent J&W DB-5HT (15 m × 0.25 mm × 0.1 μm film thickness). One microliter was injected at 275 °C in the pulsed splitless mode (pulse pressure 15 psi kept for 1.5 min). The oven temperature was programmed from 90 °C, for 1.5 min, then raised with 25 °C/min to 190 °C, then raised with 6.75 °C/min to 310 °C which was kept for 4 min. Methane was used as moderating gas (purity 4.5). Helium (purity 5.9) was used as a carrier gas with a ramped flow. The initial flow was 1 mL/min (for 20 min), then ramp 20 mL/min to 2 mL/min. The following mass fragments were monitored: m/z 79/81 (bromine trace), m/z 484.4/486.4 and m/z 494.4/496.4, corresponding to the ion [C6Br5O]− obtained by fragmentation of BDE209 and of 13C-BDE209, respectively. The temperatures of the interface, quadrupole and ion source were 300, 150 and 250 °C, respectively and the electron multiplier voltage was set at 1812 V. Results and discussion Method development. First, a 24 h preliminary test was conducted using the ERM-EC591 CRM, from which it was determined that the PBDE levels were close to the limit of solubility in aqueous media. Three methods of incubation/shaking were then tested: uninterrupted shaking (1 h)4, replenishing the artificial saliva solution after 30 min7 (1 h total shaking time) and uninterrupted shaking with glass beads added 6. The control against which these experimental conditions were tested was an incubation experiment using the same set-up and real human saliva, collected from nine human volunteers of different age and gender and pooled together. Adding glass beads or replenishing the artificial saliva did not significantly increase the observed migration rates, as described in the literature 8 (Fig. 1). The real saliva control did however display a slightly higher migration rate as compared to the other conditions, especially for the heavier PBDE congeners, while adding the glass beads seemed to marginally favour the release of the lighter PBDE congeners from the matrix (Fig. 2).
Amounts (ng)
Figure 2: One hour incubations with CRM and different experimental conditions Similar experiments were then run at different migration times: 16 h, 8 h, 4 h, 2 h, 1 h, 30 min, 15 min (all in triplicate), in order to observe the migration rate and behaviour of the analytes from the plastic matrix. The ERM-EC591 CRM contains PBDE congeners in amounts in the same order of magnitude as the amounts required to impart flame retardancy in a plastic item. The two toy samples analysed contain levels one order of magnitude lower and are representative for the scenario in which PBDEs are only present as a result of contamination during the manufacturing or recycling process. Five parents of infants and toddlers were interviewed about the mouthing behaviour of their children and, on the basis of the information they provided, two exposure scenarios were considered: a low exposure scenario (15 min mouthing time/day) and a high exposure scenario (or “favourite toy” scenario, 1 h mouthing time/day). PBDEs were detected in the low nanogram range even in the incubations for the low exposure scenario (15 min) with the CRM and in the plastic toy sample with the higher concentration (19 µg/g). In order to put the obtained values into perspective, a theoretical level of exposure was calculated using models from the literature3 and was compared with experimental values (Table 1). Theoretical Values Low exposure
High exposure
Experimental data Low exposure
High exposure
71 283 24 67 Table 1: Exposure levels (ng/day) both theoretically calculated and experimentally-derived The theoretical values match the experimental data reasonably well for both the high and the low exposure scenarios, although the experimental values tend to be 3-4 times lower than the theoretical ones. These differences show that more harmonisation between in vivo experiments and the theoretical assessment/modelling of PBDE migration in saliva is needed to provide more realistic exposure estimations. How this translates to exposure to certain age groups is yet to be determined, as in-depth data interpretation is still ongoing.
Acknowledgements ACI acknowledges the funding of his PhD scholarship from the European Union Seventh Framework Program (FP7/2007-2013) under grant agreement no 264600 (INFLAME project) and the volunteers who donated saliva required during the development of the migration method. References: 1. Greenpeace Feature “A toxic Toy Story”, July 5, 2005. 2. Ionas AC, Dirtu AC, Anthonissen T, Neels H, Covaci A. (2014); Downsides of the recycling process: Harmful organic chemicals in children’s toys. Environ Int. 65C:54–62 3. Chen S-J, Ma Y-J, Wang J, Chen D, Luo X-J, Mai B-X. (2009) Brominated Flame Retardants in Children’s Toys: Concentration, Composition, and Children’s Exposure and Risk Assessment. Environ Sci Technol. 43(11):4200–6. 4. Niino T, Ishibashi T, Itoh T, Sakai S, Ishiwata H, Yamada T, Onodera S. (2002) Comparison of Diisononyl Phthalate Migration from Polyvinyl Chloride Products into Human Saliva in Vivo and into Saliva Simulant in Vitro. J Heal Sci. 48(3):277–81 5. Niino T, Ishibashi T, Itho T, Sakai S, Ishiwata H, Yamada T, Onodera S. (2001) Monoester Formation by Hydrolysis of Dialkyl Phthalate Migrating from Polyvinyl Chloride Products in Human Saliva. J Health Sci. 47(3):318–22 6. Könemann WH. (1998) Phthalate release from soft PVC baby toys. RIVM Report 613320 002 7. Earls AO, Axford IP, Braybrook JH. (2003) Gas chromatography-mass spectrometry determination of the migration of phthalate plasticisers from polyvinyl chloride toys and childcare articles. J Chromatogr A. 983(1-2):237–46 8. US Consumer Product Safety Commission. (2001) Chronic Hazard Advisory Panel on Diisononyl Phthalate (DINP).
