Exposure assessment to parabens, bisphenol A and perfluoroalkyl compounds in children, women and men by hair analysis

1 Exposure assessment to parabens, bisphenol A and perfluoroalkyl 1 compounds in children, women and men by hair analysis 2 3 Julia Martín*, Juan Luis Santos, Irene Aparicio, Esteban Alonso 4 5 6 7 Corresponding author: Julia Martín 8 Address: Departmento de Química Analítica, Escuela Politécnica Superior, Universidad 9 de Sevilla, C/ Virgen de África, 7, E–41011 Sevilla, Spain 10 11 E-mail: [email protected] 12 Phone-number: +34-9-5455-6250 13 14 15 16 17 18 19 20 21 22 23 24 *Manuscript (double-spaced and continuously LINE and PAGE numbered)-for final publication Click here to view linked References 2 ABSTRACT: Population is continuously exposed to endocrine disrupting compounds 25 present in everyday products such as parabens, bisphenol A (BPA), and perfluoroalkyl 26 compounds (PFCs). The aims of this study were, first, to evaluate human exposure to 27 three parabens (methylparaben (MeP), ethylparaben (EtP) and propylparaben (PrP)), 28 BPA and six PFCs (perfluorobutanoic acid, perfluoropentanoic acid, perfluorohexanoic 29 acid, perfluoroheptanoic acid (PFHpA), perfluoroctanoic acid (PFOA) and 30 perfluorooctanesulfonic acid (PFOS) through the analysis of hair samples from children, 31 women and men and, then, to evaluate possible relationships between pollutant 32 concentration in hair and age, gender, smoking and dyeing habits or hair colour. Hair 33 samples were collected from 42 volunteers from Seville (Spain) (10 children, 16 women 34 and 16 men). Six of the monitored pollutants (MeP, EtP, PrP, BPA, PFHpA and PFOS) 35 were detected in at least 76% of the samples analysed. The highest concentrations and 36 frequency of detection (100% of the samples) corresponded to MeP and PrP (up to 37 14187 and 9009 ng/g, respectively). BPA was found in 83% of the samples at 38 concentrations in the range from 24 to 1427 ng/g whereas PFCs were detected at 39 concentrations in the range from 0.6 to 15.5 ng/g, being PFHpA and PFOS the ones 40 most frequently detected (86% and 76%, respectively). Concentrations of BPA and 41 parabens in adults were statistically higher than those in children. The results of this 42 study reveal the suitability of hair for biomonitoring endocrine disrupting compounds of 43 high concern (PFCs, parabens and BPA) to which population is internally or/and 44 externally but continuously exposed. 45 46 Keywords: Endocrine disrupting compounds; Parabens; Bisphenol A; Perfluoroalkyl 47 compounds; Hair analysis; Human biomonitoring 48 49 3 1. Introduction 50 In the last years, there has been an increasing concern about certain chemicals present in 51 everyday products that are suspected to involve human health risks (Alves et al., 2014; 52 Calafat et al., 2015). Endocrine disrupting chemicals (EDCs) such as bisphenol A 53 (BPA), parabens, perfluoroalkyl compounds (PFCs), phthalates and triclosan (Gore et 54 al., 2019; Katsikantami et al., 2016; Álvarez-Muñoz et al., 2018; Karzi et al., 55 2018a;2018b) are widely used in a variety of products including food packaging and 56 processing materials, consumer goods, and personal care products such as cosmetics, 57 soaps and fragrances. The main exposure routes to these pollutants are ingestion, 58 inhalation and dermal contact but also perinatal transmission through placenta and 59 breast milk, in the case of foetuses, newborns and babies. Nevertheless, exposition 60 routes are conditioned not only by the commercial applications of the contaminant but 61 also by its physical-chemical properties (Alves et al., 2014, Heffernan et al., 2015; 62 EFSA 2015; Pahigian et al., 2018). 63 BPA is extensively used in the production of epoxy resins and polycarbonate plastics 64 used in digital media, electronic equipment, medical devices, dental fillings, thermal 65 receipts, water pipes and toys (Dekant and Völkel, 2008; Halden, 2010; Tzatzarakis et 66 al., 2015). Humans can be exposed to BPA not only through the use of materials 67 containing BPA but also via food contaminated with BPA and/or derivatives through 68 plastic packaging materials (Tzatzarakis et al., 2017). In this regard, in 2011, the 69 Commission Regulation (EU) No 10/2011 (EU, 2011a), on plastic materials and articles 70 intended to come into contact with food, fixed a migration limit of 0.6 mg kg-1 for BPA 71 in food contact materials whereas the Commission Directive 2011/8/EU banned its use 72 in plastic infant feeding bottles (EU, 2011b). Epidemiological studies have associated 73 high urinary concentrations of BPA in adults and children with obesity and larger waist 74 4 circumference (Nicolucci et al., 2013; Wong and Durrani, 2017). Parabens are added as 75 antimicrobial preservatives to a wide variety of products especially personal care 76 products such as shampoo, creams, deodorants, hairspray and other cosmetics. They can 77 also be present in machine wash liquids/detergents, automotive care products, paints 78 and coating or adhesives, fragrances and air fresheners (ECHA, 2019). Therefore, the 79 main exposure route to parabens is dermal contact but also ingestion and inhalation 80 (ECHA, 2019). In the European Union, paraben concentration in cosmetics has been 81 limited to 0.8 % (w/w) for paraben mixtures and 0.4 % (w/w) for individual compounds 82 (European Union Regulation No. 1223/2009). 83 Methylparaben (MeP), ethylparaben (EtP) and propylparaben (PrP) are the parabens 84 most frequently used in cosmetics and food processing (Jiménes-Díaz et al., 2011; 85 Jackson, 1996) and the ones reported at the highest concentration levels in other 86 biological matrices (Raza et al., 2018; Jiménez-Díaz et al., 2014). PFCs are water, oil 87 and dirt repellents used in stain- and water-resistant coatings (carpets, clothing and 88 frying pan), in firefighting foams, in lubricants and paints, in food packaging and in 89 waxes and cleaners (Fromme et al., 2009; EPA, 2019). They are persistent and highly 90 bioaccumulative. PFCs have even been detected in drinking water to the point that in 91 February 2019, the United States Environmental Protection Agency has announced a 92 per- and polyfluoroalkyl substances action plan to protect public health from the 93 presence of these pollutants in drinking water (EPA, 2019). Exposure routes to PFCs 94 are inhalation (indoor air and dust) and ingestion of food, especially fish, eggs and meat 95 products, and drinking water, especially well water and tap water (Jian et al., 2017). 96 Biomonitoring in humans is commonly carried out through blood analysis (Angerer et 97 al., 2006, 2007) but, in the last years, special attention has been focused on non-invasive 98 matrices such as saliva, hair and nails, as alternatives to blood, since they offer 99 5 advantages with respect to sampling, handling, and ethical aspects, while ensuring 100 similar reliability and sensitivity (Schramm, 2008; Appenzeller et al., 2012; Król et al., 101 2013; Alves et al., 2014). Among them, hair is considered a promising biomarker for 102 biomonitoring of chemicals (Appenzeller et al., 2012) that present advantages such as 103 easy sampling, transport and storage and can provide information on short to long-term 104 exposure (from weeks to months or even years, depending on hair length) Moreover, 105 hair biomonitoring is suitable for adults but also for children, babies, elderly and/or sick 106 people. Hair analysis has allowed to assess past acute exposure to organophosphate 107 pesticides by hair segmental analysis (Tsatsakis et al., 2012) and to provide information 108 about “endogenous exposition” (sorption of the pollutants from blood) and “exogenous 109 exposition” (sorption or deposition of the pollutants from the atmosphere) (Zhang et al., 110 2007). Therefore, biomonitoring in hair samples provides information about integral 111 exposition to pollutants including inhalation from atmospheric pollution. 112 Nowadays, hair analysis is used in forensic and clinical analysis for the detection of 113 drugs of abuse, pharmaceuticals and inorganic compounds but its application to other 114 organic pollutants has been scarcely evaluated and limited to a few groups of pollutants 115 such as pesticides, polybrominated diphenyl ethers, polycyclic aromatic hydrocarbons, 116 dioxins and polychlorinated biphenyls (Appenzeller et al., 2012). 117 Therefore, the aim of this work was to evaluate the suitability of estimating the burden 118 to parabens, BPA and PFCs through the analysis of hair samples from children and 119 adults and to evaluate the existence of relationships between biomonitoring data and 120 age, gender, smoking habits and hair colour or dyeing habits. Target pollutants 121 monitored were three parabens (MeP, EtP and PrP), BPA and six PFCs 122 (perfluorobutanoic acid, perfluoropentanoic acid, perfluorohexanoic acid, 123 perfluoroheptanoic acid (PFHpA), perfluoroctanoic acid (PFOA) and 124 6 perfluorooctanesulfonic acid (PFOS). Analytical determination was carried out by a 125 previous reported method (Martín et al., 2016). Concentrations found in hair samples 126 have been compared to those reported in other biological matrices. To our knowledge, 127 this is the first simultaneous biomonitoring study of parabens, BPA and PFCs in human 128 hair. 129 130 2. Experimental 131 2.1. Chemicals and reagents 132 HPLC-grade acetone, methanol and water were supplied by Romil (Barcelona, Spain). 133 Analytical-grade acetic acid (HAc) (>99%), ammonium acetate and sodium 134 dodecylsulfate (SDS) were obtained from Panreac (Barcelona, Spain). High purity 135 standards of MeP, EtP, PrP, BPA, perfluorobutanoic acid (PFBuA), perfluoropentanoic 136 acid (PFPeA), perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), 137 PFOA, PFOS and propyl 4-hydroxybenzoate-13C6 (PrP-13C6) (t99%) were supplied 138 from Sigma-Aldrich (Steinheim, Germany). Bisphenol A-d14 (BPA-d14) (99.5%) was 139 obtained from Dr. Ehrenstorfer (Augsburg, Germany). Perfluorooctanoic acid-13C8 140 (PFOA-13C8) (99%) was supplied by Cambridge Isotope Laboratories (MA, USA). PrP-141 13C6, BPA-d14 and PFOA-13C8 were used as internal standards (I.S.). Individual stock 142 standard solutions were prepared at 1000 mg/L in MeOH and stored at -18 ºC. Working 143 solutions were prepared by dilution of the stock standard solutions in methanol. 144 145 2.2. Hair collection and pretreatment 146 Hair samples were collected from 42 volunteers (10 children, 16 women and 16 men) of 147 general population from Seville, Spain. Detailed information of the volunteers (age, 148 gender, hair colour and colouring, smoking habit and profession) is given in 149 7 Supplementary data (Table S1). Hair was cut on the posterior vertex as close to the 150 scalp as possible and wrapped in aluminium foil. Samples were stored in the dark at 151 room temperature until analysis. Paper and plastic containers were avoided to prevent 152 sample pollution. Hair samples were washed first with ultrapure water, then with SDS 153 (0.1%, w/v), and finally twice again with ultrapure water to remove endogenous 154 substances and chemicals adsorbed on hair surface. In each washing step, sonication for 155 5 min was applied. After that, hair samples were cut in small pieces (2-3 mm), dried at 156 room temperature and, when necessary, wrapped in aluminium foil and stored in the 157 dark at room temperature until analysis. 158 159 2.3. Hair treatment and LC-MS/MS analysis 160 Target compounds were extracted and analysed by a previously reported method 161 (Martín et al., 2016). Washed hair samples (100 mg) were transferred into 10 mL screw-162 cap glass centrifuge tubes and spiked with 50 ng of each I.S. Then, samples were 163 incubated with 2 mL of MeOH/HAc (85:15, v/v), at 38 °C overnight, to improve the 164 release of the target compounds from hair matrix. After incubation, samples were 165 cooled to room temperature and sonicated for 15 min after addition of acetone (3 mL). 166 The tubes were centrifuged at 2900 g for 10 min and the supernatants were transferred 167 to clean tubes and evaporated to dryness under a nitrogen stream. Extracts were 168 reconstituted in 500 μL of methanol and transferred to automatic LC injector vials. 169 Aliquots of 10 μL were injected into the LC-MS/MS system. 170 Analytical determination was performed on a 1200 Series LC system (Agilent, USA) 171 coupled to a 6410 Agilent triple quadrupole (QqQ) mass spectrometer (MS). 172 Chromatographic separation was carried out on an Agilent Zorbax Eclipse XDB–C18 173 Rapid Resolution HT (50 mm × 4.6 mm i.d.; 1.8 μm particle size) column. An 174 8 electrospray ionization source operating in negative-ion mode was used. MS parameters 175 were as follows: capillary voltage, 3,000 V; drying gas flow rate, 9 L min−1; drying gas 176 temperature, 350 °C; and nebulizer pressure, 40 psi. Instrument control and data 177 acquisition were carried out with MassHunter software (Agilent, USA). Separation was 178 performed by gradient elution with methanol (solvent A) and 5 mM ammonium acetate 179 aqueous solution (solvent B) at a flow rate of 0.6 mL min−1 with the column 180 thermostated at 25 °C. The elution program was as follows: 0–20 min, linear gradient 181 from 28 to 95% of solvent A, held for 2 min. In Tables S2 and S3 in Supplementary 182 material, MS/MS settings and validation parameters, respectively, are summarized. 183 More information can be found in a previous reported method (Martín et al., 2016) 184 185 2.4. Quality assurance and quality control 186 For each batch of 20 samples analysed (within one day), procedural blank, blank 187 samples spiked at 50, 200 and 500 ng/g for parabens and BPA and at 5, 20 and 50 ng/g 188 for PFCs and a standard solution containing a mixture of the target compounds at 100 189 ng mL-1 were processed and injected. The concentrations of all target chemicals in 190 procedural blanks were below the limits of quantitation (LOQs). 191 192 3. Results and discussion 193 3.1. Parabens, BPA and PFCs distribution in hair samples 194 In Table 1 can be seen the range of concentrations of each target compound and their 195 arithmetic and geometric means and median values. Individual concentrations are 196 shown in Table S1. 197 MeP, PrP and BPA were the compounds at the highest concentration levels (median 198 concentrations: 822, 256 and 195 ng/g, respectively) and the ones, in addition to PFHpA 199 9 and PFOS, most frequently detected (frequency of detection from 76 to 100% of the 200 analysed samples) (Table 1). The distribution pattern of parabens in the analysed hair 201 samples was as follow: MeP (mean 2821 ng/g, median 822 ng/g) > PrP (mean 1006 202 ng/g, median 256 ng/g) > EtP (mean 635 ng/g, median 47.2 ng/g), which is consistent 203 with the wide usage and long-term accumulation of these compounds and with the fact 204 that MeP and PrP are the most commonly used parabens in cosmetics and food 205 processing (Jimenez Díaz et al., 2011; Elder, 1984)). There was also observed 206 correlation between MeP (neperian logarithm (Ln) MeP) and PrP (Ln PrP) 207 concentrations (r2 = 0.54; p < 0.05) (Figure 1). This correlation can be explained by 208 their combined use in commercial formulations due to their synergistic effect. Their 209 concentrations have also been reported to be correlated in other samples such as 210 foodstuffs (Liao et al., 2013), dust (Wang et al., 2012) and in urine samples 211 (Asimakopolus et al., 2014). BPA was detected in 83 % of the analysed samples at 212 concentrations in the range from 24 to 1427 ng/g (mean value: 334 ng/g). Although 213 concentration levels of PFCs were lower (mean value: 2.0-8.9 ng/g) than those of 214 parabens (mean: 635-2821 ng/g) and BPA (mean: 334 ng/g), hair biomonitoring 215 revealed human exposure to these EDCs. At least one PFC was detected in each of the 216 42 hair samples analysed. The most frequently detected PFCs were those with the 217 longest alkyl chain: PFHpA, PFOA and PFOS (frequencies of detection: 86%, 57% and 218 76%, respectively). Correlation between the concentrations of PFOA and PFOS was 219 observed (r2 = 0.50; p < 0.05) as can be seen in Figure 1. Perfluoroalkyl acids of short 220 chain (PFBuA, PFPeA and PFHxA) were less frequently detected than those of longer 221 chain but, when detected, they were present at higher concentrations than those of 222 longer chain. A similar behaviour was reported for by Pérez et al. 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Total Environ. 636, 383–391. 585 Wong, K.H., Durrani, T.S., 2017. Exposures to Endocrine Disrupting Chemicals in 586 Consumer Products—A Guide for Pediatricians. Curr. Probl. Pediatr. Adolesc. 587 Health Care 47, 107–118. 588 Zhang, H., Chai, Z., Sun, H., 2007. Human hair as a potential biomonitor for assessing 589 persistent organic pollutants. Environ. Int. 33, 685–693. 590 591 592 25 FIGURE CAPTIONS 593 Figure 1. Correlations between MeP (Ln MeP) and PrP (Ln PrP) concentrations and 594 PFOA (Ln PFOA) and PFOS (Ln PFOS) concentrations in hair samples (n=42). 595 Figure 2. Distribution of EDCs in children (left), women (middle) and men (right). 596 Figure 3. Distribution of EDCs in smokers and non-smokers. 597 Figure 4. Distribution of EDCs in dyed and non-dyed hair. 598 0 5000 10000 15000 Children Women Men Concentration (ng/g) MeP 0 5000 10000 15000 Children Women Men Concentration (ng/g) EtP 0 5000 10000 15000 Children Women Men Concentration (ng/g) PrP 0 500 1000 1500 Children Women Men Concentration (ng/g) BPA 0 6 12 18 Children Women Men Concentration (ng/g) PFBuA 0 6 12 18 Children Women Men Concentration (ng/g) PFPeA 0 6 12 18 Children Women Men Concentration (ng/g) PFHxA 0 6 12 18 Children Women Men Concentration (ng/g) PFHpA 0 6 12 18 Children Women Men Concentration (ng/g) PFOA 0 6 12 18 Children Women Men Concentration (ng/g) PFOS Figure Click here to download Figure: Revised Fig. 2.pdf 0 5000 10000 15000 Non-smokers Smokers Concentration (ng/g) MeP 0 5000 10000 15000 Non-smokers Smokers Concentration (ng/g) EtP 0 5000 10000 15000 Non-smokers Smokers Concentration (ng/g) PrP 0 500 1000 1500 Non-smokers Smokers Concentration (ng/g) BPA 0 6 12 18 Non-smokers Smokers Concentration (ng/g) PFBuA 0 6 12 18 Non-smokers Smokers Concentration (ng/g) PFPeA 0 6 12 18 Non-smokers Smokers Concentration (ng/g) PFHxA 0 6 12 18 Non-smokers Smokers Concentration (ng/g) PFHpA 0 6 12 18 Non-smokers Smokers Concentration (ng/g) PFOA 0 6 12 18 Non-smokers Smokers Concentration (ng/g) PFOS Figure Click here to download Figure: Revised Fig. 3.pdf 0 5000 10000 15000 Non-Dyed Dyed Concentration (ng/g) MeP 0 5000 10000 15000 Non-Dyed Dyed Concentration (ng/g) EtP 0 5000 10000 15000 Non-Dyed Dyed Concentration (ng/g) PrP 0 500 1000 1500 Non-Dyed Dyed Concentration (ng/g) BPA 0 6 12 18 Non-Dyed Dyed Concentration (ng/g) PFBuA 0 6 12 18 Non-Dyed Dyed Concentration (ng/g) PFPeA 0 6 12 18 Non-Dyed Dyed Concentration (ng/g) PFHxA 0 6 12 18 Non-Dyed Dyed Concentration (ng/g) PFHpA 0 6 12 18 Non-Dyed Dyed Concentration (ng/g) PFOA 0 6 12 18 Non-Dyed Dyed Concentration (ng/g) PFOS Figure Click here to download Figure: Fig. 4.pdf Supplementary material for on-line publication only Click here to download Supplementary material for on-line publication only: Revised Supplementary material.doc