Article pubs.acs.org/est
Hair and Nails as Noninvasive Biomarkers of Human Exposure to Brominated and Organophosphate Flame Retardants Liang-Ying Liu,† Ka He,‡ Ronald A. Hites,† and Amina Salamova*,† †
School of Public and Environmental Affairs and ‡School of Public Health, Indiana University Bloomington, Bloomington, Indiana 47405, United States S Supporting Information *
ABSTRACT: After the phase-out of polybrominated diphenyl ethers (PBDEs), the use of alternative flame retardants (AFRs), such as FireMaster 550, and of organophosphate esters (OPEs) has increased. However, little is known about human exposure to these chemicals. This lack of biomonitoring studies is partially due to the absence of reliable noninvasive biomarkers of exposure. Human hair and nails can provide integrated exposure measurements, and as such, these matrices can potentially be used as noninvasive biomarkers of exposure to these flame retardants. Paired human hair, fingernail, toenail, and serum samples obtained from 50 adult participants recruited at Indiana University Bloomington campus were analyzed by gas chromatographic mass spectrometry for 36 PBDEs, 9 AFRs, and 12 OPEs. BDE-47, BDE-99, 2-ethylhexyl2,3,4,5-tetrabromobenzoate (TBB), di(2-ethylhexyl) tetrabromophthalate (TBPH), tris(1,3-dichloro-2-propyl)phosphate (TDCIPP), and triphenyl phosphate (TPHP) were the most abundant compounds detected in almost all hair, fingernail, and toenail samples. The concentrations followed the order OPEs > TBB+TBPH > Σpenta-BDE. PBDE levels in the hair and nail samples were significantly correlated with their levels in serum (P < 0.05), suggesting that human hair and nails can be used as biomarkers to assess human exposure to PBDEs.
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Considering the ubiquity of brominated flame retardants and OPEs in the environment, human exposure to these chemicals is inevitable, and there is an increasing number of studies reporting the levels of brominated flame retardants in humans from around the world.11−13 Furthermore, there have been a number of toxicological reports suggesting that endocrine disruption, reproductive toxicity, neurotoxicity, learning disabilities, and obesity are among the adverse effects of brominated flame retardant exposure.14−17 Data on the presence of OPEs or their metabolites in humans is limited to only a few hair18,19 and urine analyses.18,20,21 OPE toxicity data is also limited, but existing studies suggest that some of these chemicals may be potential thyroid disruptors, endocrine disruptors, as well as carcinogens.22−24 Despite these findings, epidemiological evidence of flame retardants’ adverse effects remains limited due to the difficulties of long-term human biomonitoring studies, including a lack of reliable, readily available, and minimally invasive biomarkers of exposure. Most of the studies on flame retardant concentrations in human tissues have focused on milk, blood, and urine.13,25 Milk can only be collected from lactating women, and its collection is somewhat invasive. Blood is a suitable matrix for most chemicals because blood plasma is in contact with all
INTRODUCTION Flame retardants are added to a variety of commercial and consumer products worldwide to delay ignition and to slow the spread of fire. Polybrominated diphenyl ethers (PBDEs) have been the most widely used class of brominated flame retardants up until recently, but the use of these compounds was discontinued because of their environmental ubiquity, persistence, and bioaccumulation.1 The major PBDE products, the so-called penta-, octa-, and deca-BDE mixtures, have been completely withdrawn from the market in the United States and Canada. However, due to continuing flammability standards, alternative (mostly brominated) flame retardants (AFRs), such as FireMaster 550, Firemaster BZ-54, and DP-45, have entered the market.1,2 The main components of FireMaster 550 are 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (TBB), di(2-ethylhexyl) tetrabromophthalate (TBPH), and triaryl phosphates, such as triphenyl phosphate (TPHP). Firemaster BZ-54 consists of ∼70% of TBB and ∼30% of TBPH. DP-45 contains only TBPH. Other replacements include hexabromobenzene (HBB), pentabromoethylbenzene (PBEB), 1,2-bis(2,4,6-tribromophenoxy)ethane (TBE), and decabromodiphenylethane (DBDPE). Like PBDEs, some of these replacement flame retardants have also become environmentally ubiquitous.3−6 In addition to AFRs, some organophosphate esters (OPEs) have gained an increased share of the flame retardant market.7 Like the brominated flame retardants, OPEs have now been widely found in indoor dust and air,8,9 water,4 outdoor air,3 sediment,6 and biota.10 © 2016 American Chemical Society
Received: Revised: Accepted: Published: 3065
October 15, 2015 January 25, 2016 February 13, 2016 March 1, 2016 DOI: 10.1021/acs.est.5b05073 Environ. Sci. Technol. 2016, 50, 3065−3073
Article
Environmental Science & Technology
determine if hair and nail samples can be used as biomarkers of human exposure to organic flame retardants. This is the first study to report the levels of organic flame retardants in the fingernails, toenails, hair, and the serum of individuals, all sampled simultaneously. This is also the first report on the levels of TBB and TBPH in hair samples and on the levels of organic flame retardants in nail samples.
body organs and tissues, and the concentrations of these chemicals in blood are in equilibrium with those in the organs. 26 However, for chemicals that undergo rapid metabolism in human tissues (like OPEs), blood and urine reflect only recent exposure. In addition, collecting blood samples is invasive, and collecting urine samples is somewhat complicated (especially if there is a need to collect 24-h urine). In fact, these problems are often cited as common reasons for participants’ refusal to provide biological specimens for research purposes.27 These problems are especially significant for the most vulnerable populations, such as children, pregnant women, and the elderly. Indeed, for infants and children, few data are available on their body burdens of flame retardants, with the exception of some studies on dry blood spots and umbilical cord blood.28−30 Clearly, the development of more convenient, noninvasive biomarkers of human exposure to flame retardants will provide essential benefits for the development of long-term biomonitoring studies. Human hair and nails are good examples of noninvasive matrices. Compared to other biological samples, these matrices are easily and inexpensively accessible and collectable, and they are simple to transport and store. Moreover, these matrices are stable, with low or no active metabolic activities, so that once the chemical is incorporated into the keratinous tissue of the hair or nail, the levels remain relatively unchanged. Because both of these matrices are slowly growing tissues, they provide an opportunity to measure exposure over a week to month time-window, and as such, they reflect both past and present exposures. Hair and nail samples have been used as indicators of human exposure to heavy metals and drugs for years.31 In addition, a few studies have explored the use of hair for assessing human exposure to persistent organic pollutants, specifically to flame retardants.32−35 Zheng et al.32 reported higher levels of PBDEs in the hair of e-waste recycling workers compared to nonoccupationally exposed residents in urban and rural China. Aleksa et al.33 detected PBDEs in newborn hair and suggested this approach could be used as a tool for determining in utero exposure. Two recent studies have started to investigate the potential of human hair as a biomarker of human exposure to OPEs.18,19 To establish the utility of hair or nail as a biomarker of exposure to organic flame retardants, it is essential to investigate the relationship between the levels of flame retardants in hair and nails, on the one hand, and in blood on the other. In this way, one can determine if these matrices are reliable biomarkers and reflect the body’s burden of these chemicals. Significant positive correlations have been observed for PBDE concentrations between hair and internal human tissues34,35 and between hair and internal tissues of hedgehogs and rats.35,36 However, there are no studies reporting TBB and TBPH concentrations in hair and no studies reporting any organic flame retardant concentrations in nail samples. In fact, only one study has assessed the possibility of using nails as a biomarker for organic compounds; in this case, the analytes of interest were perfluorooctane sulfate and perfluorooctanoic acid.37 The concentrations of these compounds in fingernails were significantly correlated with those in serum.37 The objectives of our study are to determine the concentrations of PBDEs, AFRs, and OPEs in paired human hair, fingernails, and toenails and to compare these levels to those measured in serum samples, all collected from a group of healthy adults living in the United States. The goal is to
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EXPERIMENT SECTION Sampling. Fifty apparently healthy participants (25 men and 25 women), aged 19−38, were recruited in July−August 2014 on Indiana University’s Bloomington campus for hair, fingernail, toenail, and blood collection. The study protocol was approved by the Indiana University Institutional Review Board, and all of participants signed an informed consent form. Blood samples were drawn by a trained professional into 10 mL BD Vacutainer serum tubes and centrifuged at 3000 rpm for 10 min immediately after collection to generate serum, which was transferred to an 8 mL amber glass vial and stored on ice before being transported to the laboratory, where the samples were stored at −20 °C until analysis. Hair samples were collected right after the blood draw. Hair (∼1−2 g) was cut at the back of the neck close to the scalp with stainless steel scissors that had been precleaned with ethyl alcohol. Hair strands were wrapped in aluminum foil, sealed in a Ziploc bag, and stored at −20 °C until analysis. Fingernail and toenail samples were collected by participants at home within 2 weeks after the blood draw. Nails were clipped from all fingers and toes with a stainless steel nail clipper that had been precleaned with ethyl alcohol; the samples were sealed in a foil-lined paper envelope and sent to the laboratory via regular mail. A questionnaire was completed before sample collection by each participant. The questions asked for information about age, gender, weight, height, dietary habits, indoor environment, and living habits (sedentary or not). Sample Treatment. Neither the hair nor nail samples were washed prior to extraction because a recent study suggested that there was no available medium that could exclusively remove external contaminants from hair.19 Thus, the hair and nail concentrations in this study represent both external and internal exposures. For analysis, we used a gas chromatographic mass spectrometry (GC−MS) method that was developed recently in our laboratory to simultaneously determine PBDEs, AFRs, and OPEs in hair and nail samples.38 Briefly, approximately 100 mg of hair or nails (from all the 10 fingers or toes) was weighed into a 50 mL screw-top tube. After addition of known amounts of surrogate recovery standards (BDE-77, BDE-166, and 13C12-BDE-209, tris(2-chloroethyl) phosphate-d12, and 13C18-triphenyl phosphate), the hair or nail samples were digested with HNO3/H2O2 (1:1 v/v) in a 60 °C water bath for 2 h. After the addition of 10 mL of HPLC-grade water, the digested mixture was extracted with 10 mL of a hexane: dichloromethane solution (4:1 v/v) three times. Serum samples (approximately 4−6 mL) were thawed overnight, transferred to a 50 mL screw-top tube, spiked with known amounts of the surrogate recovery standards, denaturized with HCl and 2-propanol, and extracted with 10 mL of a hexane:methyl t-butyl ether solution (1:1 v/v) three times. An aliquot of the serum extract was taken for gravimetric lipid measurement. Hair, nail, and serum extracts were subjected to the same cleanup method: The extract was rotary evaporated to approximately 1 mL with one solvent exchange with 25 mL 3066
DOI: 10.1021/acs.est.5b05073 Environ. Sci. Technol. 2016, 50, 3065−3073
Article
Environmental Science & Technology
Table 1. Summary of PBDE, AFR, and OPE Concentrations in Hair, Fingernail, Toenail, and Serum Samples (ng/g for hair, fingernails, and toenails and ng/g lipid for serum)a fingernail samples (n = 50)
hair samples (n = 50) % det BDE-28 BDE-47 BDE-99 BDE-100 BDE-153 BDE-154 BDE-209 ΣPBDE TBB TBPH PBBZ PBEB HBB TBE syn-DP anti-DP ΣAFR TCEP TCIPP TDCIPP TPHP ΣOPE
BDE-28 BDE-47 BDE-99 BDE-100 BDE-153 BDE-154 BDE-209 ΣPBDE TBB TBPH PBBZ PBEB HBB TBE syn-DP anti-DP ΣAFR TCEP TCIPP TDCIPP TPHP ΣOPE
98 100 100 100 66 98 100 100 98 94 54 32 62 6 38 14 100 68 88 90 98 98
range
geomean
median
0.23−8.6 1.2 5.2−880 43 2.2−1020 26 0.48−176 5.7 1.4−78 3.5 0.16−54 1.3 1.2−950 9.0 13−2220 107 7.6−4540 111 13−2600 109 0.33−4.9 0.59 0.10−2.6 0.29 0.11−9.0 0.53 2.5−3.8 3.2 0.10−4.0 0.22 0.36−3.7 0.83 0.23−5820 210 60−2740 250 100−9840 530 70−10490 390 70−4710 280 210−10800 1600 toenail samples (n = 50)
1.0 33 21 4.4 2.4 1.2 6.9 80 85 78 0.47 0.22 0.52 3.5 0.17 0.82 180 240 450 360 220 1530
% det BDE-28 BDE-47 BDE-99 BDE-100 BDE-153 BDE-154 BDE-209 ΣPBDE TBB TBPH PBBZ PBEB HBB TBE syn-DP anti-DP ΣAFR TCEP TCIPP TDCIPP TPHP ΣOPE
% det
range
geomean
median
86 100 100 98 46 92 100 100 94 80 8 24 66 12 26 24 98 8 32 50 74 84
0.21−8.5 3.9−910 2.1−1600 0.99−304 3.3−180 0.25−126 1.9−840 14−3160 13−2310 18−1990 0.72−2.9 0.20−1.1 0.24−5.3 1.5−7.2 0.24−2.3 0.59−5.4 0.39−4300 100−150 90−5150 75−2300 54−232900 120−233700
0.92 38 30 6.6 7.9 2.2 12 110 102 124 1.2 0.40 0.75 4.0 0.44 1.1 180 140 300 270 1980 2230
0.71 36 26 5.3 6.2 2.1 8.7 90 81 116 1.0 0.32 0.48 4.9 0.32 0.78 190 150 230 230 1080 1170
BDE-28 BDE-47 BDE-99 BDE-100 BDE-153 BDE-154 BDE-209 ΣPBDE TBB TBPH PBBZ PBEB HBB TBE syn-DP anti-DP ΣAFR TCEP TCIPP TDCIPP TPHP ΣOPE
94 100 100 98 50 94 100 100 96 86 4 12 46 14 26 12 98 20 36 66 74 90
geomean
median
0.22−8.5 0.60 4.5−890 26 2.1−1460 16 0.74−274 4.1 2.2−135 5.1 0.25−108 1.3 1.8−706 9.8 13−2900 75 11−1210 54 18−1120 77 0.90−1.3 1.1 0.25−1.1 0.53 0.20−3.0 0.48 0.75−8.7 2.6 0.22−1.3 0.51 0.70−3.4 1.7 5.4−2340 120 93−1860 230 74−2410 260 90−1410 320 110−59800 700 90−59800 1140 serum samples (n = 50)
range
0.54 23 14 3.8 4.2 1.2 7.7 67 40 74 1.1 0.42 0.35 3.1 0.61 2.0 110 190 220 300 370 770
% det
range
geomean
median
82 94 90 94 96 60 24 100 92 16 14 0 2 0 12 32 94 0 0 0 0 0
0.36−6.6 4.3−240 1.1−108 0.49−32 2.7−55 0.59−8.3 3.1−44 8.4−420 1.3−54 19−69 0.99−3.7
1.2 17 5.9 2.8 8.5 1.2 10 36 7.0 36 1.7
1.1 18 5.3 2.5 9.3 1.0 10 40 7.3 40 1.4
2.1
2.1
2.1
0.69−1.3 0.98−10 1.4−100
1.0 2.1 9.9
1.0 1.8 8.4
a ΣPBDE is the sum of BDE-28, -47, -99, -100, -153, -154, and -209 concentrations. ΣOPE is the sum of TCEP, TCIPP, TDCIPP, and TPHP concentrations. ΣAFR is the sum of PBBZ, PBEB, HBB, TBB, TBPH, TBE, syn-DP, and anti-DP concentrations.
of hexane and then fractionated on a column packed with 6 g of 2.5% (by weight) water-deactivated Florisil (Sigma-Aldrich, St. Louis, MO). The column was eluted consecutively with 35 mL of hexane (fraction 1), 35 mL of a hexane:dichloromethane solution (1:1 v/v, fraction 2), and 40 mL of a dichloromethane:acetone solution (1:1 v/v, fraction 3). All fractions were rotary evaporated to 1 mL. Fractions 2 and 3 had one and two solvent exchanges with 25 mL of hexane each. Each fraction
was transferred to a 4 mL amber glass vial and blown down to 1 mL with nitrogen. The first and second fractions were spiked with BDE-118, BDE-181, and BB-209 as internal standards and further blown down to 0.1 mL for the analysis of PBDEs and AFRs. The third fraction was spiked with anthracene-d10, dibenz[a]anthracene-d12, and perylene-d12 for the analysis of OPEs. 3067
DOI: 10.1021/acs.est.5b05073 Environ. Sci. Technol. 2016, 50, 3065−3073
Article
Environmental Science & Technology
Figure 1. Concentrations of selected PBDEs, AFRs, and OPEs in human hair, fingernails, toenails (in ng/g dry weight), and serum (ng/g lipid). No data is plotted for TDCIPP, TPHP, and ΣOPE in serum because they were not detected. TBB is 2-ethylhexyl-2,3,4,5-tetrabromobenzoate; TBPH is di(2-ethylhexyl) tetrabromophthalate; TDCIPP is tris(1,3-dichloro-2-propyl) phosphate; TPHP is triphenyl phosphate; ΣPBDE is the sum of BDE28, -47, -99, -100, -153, -154, and -209 concentrations; and ΣOPE is the sum of TCEP, TCIPP, TDCIPP, and TPHP. The boxes indicate the 25th and 75th percentiles, the whiskers indicate the 10th and 90th percentiles, the dots indicate the 5th and 95th percentiles, and the line in the box indicates the median.
Instrumental Analysis. Detailed information on the instrumental analysis is given elsewhere.38,39 The analysis of PBDEs and AFRs [see Table S1, Supporting Information (SI)] used the electron capture negative ionization mode with an Agilent 7890 series gas chromatograph (GC) coupled to an Agilent 5975C mass spectrometer (MS). An Agilent 6890 series GC coupled to an Agilent 5973 MS was used to analyze OPEs (Table S1, SI) in the electron impact mode. Quality Assurance and Quality Control. The accuracy and precision of the method were reported in our recent method development and validation paper.38 One procedural blank and one matrix spike recovery sample were included in each batch of 10 samples. The recoveries (average ± standard error) for the surrogate recovery standards for all hair and nail samples were 100 ± 7.4% for BDE-77, 86 ± 11% for BDE-166, 63 ± 14% for 13C12-BDE-209, 95 ± 19% for tris(2-chloroethyl) phosphate-d12, and 84 ± 18% for 13C18-triphenyl phosphate. The recoveries in the serum samples were 86 ± 12% for BDE77, 68 ± 10% for BDE-166, and 63 ± 8.6% for 13C12-BDE-209. Concentrations were neither recovery-corrected nor blankcorrected. The limits of quantitation were determined as the average blank levels plus two standard deviations, and measurements below these limits of quantitation were considered as nondetects.
study demonstrating the presence of TBB and TBPH in human hair, and our findings clearly suggest widespread human exposure to these chemicals. The rest of the AFRs were rarely detected, and their levels (
