Blood plasma concentrations of endocrine disrupting chemicals in Hong Kong populations

G Model HAZMAT-14857; No. of Pages 7 ARTICLE IN PRESS Journal of Hazardous Materials xxx (2013) xxx–xxx Contents lists available at SciVerse ScienceDirect Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat Blood plasma concentrations of endocrine disrupting chemicals in Hong Kong populations H.T. Wan, P.Y. Leung, Y.G. Zhao, X. Wei, M.H. Wong ∗ , Chris K.C. Wong ∗ Croucher Institute for Environmental Sciences, and Department of Biology, Hong Kong Baptist University, Hong Kong, PR China h i g h l i g h t s I I I I Over 150 human blood samples were analyzed for the presence of chemical pollutants. PFCs, BPA and phthalates were detectable over 90% of the blood samples. The mean plasma DEHP level was significantly higher in the young age group. PFCs were significantly higher in male than in female. a r t i c l e i n f o Article history: Received 27 September 2012 Received in revised form 29 November 2012 Accepted 16 January 2013 Available online xxx Keywords: Human plasma Perfluorinated compounds Plasticizers Bisphenol A Phthalates Internal exposure a b s t r a c t In this study we report the human plasma concentrations of some common endocrine disrupting chemicals (EDCs) in the Hong Kong population. We have analyzed 153 plasma samples for the contaminants by methods involving labeled standards spiked into the samples. Quantification was performed using high performance liquid chromatography tandem mass spectrometry for bisphenol-A (BPA) and perfluorinated compounds (PFCs), and gas chromatography mass spectrometry methods for phthalates. We found BPA, several types of PFCs and phthalates in over 90% of the plasma samples. Perfluorooctane sulfonate (PFOS) was the dominant PFC, followed by perfluroroctanoic acid (PFOA) and perfluorohexane sulfonate (PFHxS). Eight out of ten phthalates were detected, with bis(2-ethylhexyl) phthalate (DEHP) as the most abundant, followed by bis(2-methoxyethyl) phthalate (DMEP) and dioctyl phthalate (DnOP). The levels of PFOS, PFOA, PFHxS and perfluorohexanoic acid (PFHxA) were significantly higher in the male plasma samples (p < 0.05), while the mean plasma levels of DEHP and n-butyl benzyl phthalate (BBP) were significantly higher in the young age group (p < 0.02). The presence of the selected EDCs in human blood plasma indicates common exposure routes among different population cohorts. Although the plasma levels of the EDCs were comparable to other countries, regular monitoring of human blood EDC contamination levels is necessary to provide a time-trend database for the estimation of exposure risk and to formulate appropriate public health policy. © 2013 Published by Elsevier B.V. 1. Introduction In the past century the relentless advance of industrialization and technology and the consequent rapid growth of human populations have impacted on the environment in a way that is unprecedented in human history. The production of large amounts of synthetic industrial and biomedical chemicals in ∗ Co-corresponding authors at: Croucher Institute for Environmental Sciences, Department of Biology, 200 Waterloo Road, Kowloon Tong, Hong Kong. Tel.: +852 3411 7053; fax: +853 3411 5995. E-mail addresses: [email protected] (H.T. Wan), [email protected] (P.Y. Leung), [email protected] (Y.G. Zhao), sissywei [email protected] (X. Wei), [email protected] (M.H. Wong), [email protected] (C.K.C. Wong). addition to unwanted pollutants, has given rise to destructive consequences for our ecosystem and negative health effects on both wildlife and humans. A recent review has highlighted that about 40% of human deaths (62 million per year) is attributed to exposure to chemical pollutants. Some of the more worrying chemical contaminants are classified as endocrine disrupting chemicals (EDCs) because they are able to interfere with the synthesis, metabolism and action of endogenous hormones. They are known to exert different biological effects by means of diverse mechanisms. The adverse effects of EDCs have raised public concern due to epidemiological studies that correlate EDC exposure to many negative health outcomes in typical human populations, such as obesity, decreased fertility and immune dysfunction. A considerable number of EDCs are produced and used widely in our daily lives. Bisphenol A (BPA) is present in plastic water 0304-3894/$ – see front matter © 2013 Published by Elsevier B.V. https://dx.doi.org/10.1016/j.jhazmat.2013.01.034 Please cite this article in press as: H.T. Wan, et al., Blood plasma concentrations of endocrine disrupting chemicals in Hong Kong populations, J. Hazard. Mater. (2013), https://dx.doi.org/10.1016/j.jhazmat.2013.01.034 G Model HAZMAT-14857; No. of Pages 7 ARTICLE IN PRESS H.T. Wan et al. / Journal of Hazardous Materials xxx (2013) xxx–xxx 2 Table 1 Summary of plasma PFC concentrations. Mean SD Minimum Maximum Median % of samples > LOD PFOS PFOA PFHxS PFNA PFDA PFUdA PFHxA PFDoA PFBA 8.68 5.91 0.24 44.77 7.65 100.00 4.02 2.70 1.12 18.92 3.24 96.08 1.34 1.14 0.25 7.43 1.08 83.66 1.06 0.42 0.51 3.05 0.95 87.58 0.77 0.31 0.50 2.44 0.65 58.82 0.64 0.37 0.20 2.89 0.57 96.08 0.60 0.48 0.20 4.23 0.47 96.73 0.47 0.24 0.20 1.71 0.43 64.05 0.27 0.06 0.16 0.49 0.26 47.71 Nine species of PFCs family were found in plasma of Hong Kong donors’ blood samples and listed, according to the relative concentrations. bottles, food packaging, thermal printer paper and market fish [1–3]; phthalates are used in the production of cosmetics and plastic containers [4,5]; and perfluorinated compounds (PFCs) are found in indoor/outdoor dust and market fish [6–8], thereby indicating that contamination is ubiquitous. In the past 30 years numerous studies have measured levels of EDCs in many different environmental samples. Although pathways of exposure to EDCs can be diverse, the identification of all possible exogenous exposure is not yet a feasible task; and it is this factor which highlights a major limitation of the measurement of external exposure to EDCs. We maintain that there is a need for a transformational change in the approach to contaminants in which more emphasis be placed on correlating population-based data to reveal human-environment interactions. By doing so, researchers will be able to develop better predictive models of human response to toxicants. In this study we analyze a dataset of human blood samples in order to provide a framework of accumulated concentrations of EDCs. 2. Materials and methods 2.1. Sample collection A total of 153 whole blood samples (EDTA-treated) were collected between 2010 and 2011 from the Hong Kong Red Cross. Samples were separated equally into different groups by age (16–39 years and 40–63 years) and sex (male and female). The study was approved by the Hong Kong Red Cross and Hong Kong Baptist University and no personal information was disclosed. All whole blood samples were centrifuged at 3000 × g for 15 min to obtain the upper plasma layer. To minimize background contamination, all glassware and polypropylene tube were rinsed with methanol, n-hexane and ethyl acetate prior to use. All plasma samples collected were stored in sanitized polypropylene containers at −20 C until analysis. Aliquots of 500 ␮l plasma were thawed and transferred into a clean 15 ml polypropylene tube; thereafter the respective internal recovery standard was added (1 ng PFCs internal standard in methanol, 5 ng of deuterated-bisphenol A in methanol (d16 -BPA) or 5 ng of surrogate standards (diisooctyl phthalate in acetonitrile: THF (2:1)). Procedure blanks were tested every 10–15 samples to check for possible laboratory contaminations and interference. 2.2. Chemical materials for instrumental analysis A mixture of standard solution of perfluoroalkylcarboxylic acids (PFCAs) and perfluoroalkylsulfonates (PFASs) in addition to a masslabeled standard solution (used as the internal standard) were purchased from Wellington Laboratories (Ontario, Canada). Purities of the analytical standards were greater than 98%. Authentic standards of bisphenol A and bisphenol A-d16 (99% purities) were obtained from AccuStandard, CT, USA and Chiron, Trondheim, Norway. Methyl tert-butyl ether (MTBE), methanol, acetonitrile, and n-hexane were purchased from Tedia Company Inc. Pesticide grade ethyl acetate was purchased from LAB SCAN, UK. Tetrabutylammoniun hydroxide solution (TBA), sodium carbonate and sodium hydrogen carbonate were obtained from Sigma–Aldrich (MO, USA). An Agilent 1200 liquid chromatography (Waldbronn, Germany) equipped with a quaternary high-pressure gradient pump and an automatic sample injector was used for LC–MS/MS analysis for PFC and BPA. Chromatographic separation was performed using an Agilent C8 (2.1 mm × 12.5 mm, 5-␮m) guard column (ZORBAX Eclipse XDB-C8, Narrow-Bone) and a C18 ODS column (Agilent ZORBAX XDB-C18, 3.5-␮m × 2.1 mm × 50 mm, 3.5-␮m) for BPA and Zorbax Eclipse Plus C8 column (2.1 mm i.d. × 100 mm length, 3.5-␮m; Agilent Technologies) for PFCs. Tandem mass detection was conducted by an Agilent 6410B Triple Quadrupole mass spectrometer system equipped with an Agilent Mass-hunter Workstation and an electrospray ionization source. Table 2 Gender difference in plasma PFC concentrations (ng/ml). PFOS PFOA PFHxS PFNA PFDA PFUdA PFHxA PFDoA PFBA Male Mean SD Minimum Maximum % of samples > LOD 9.59* 7.07 0.33 44.77 100 4.50* 3.17 1.27 18.92 94 1.65* 1.38 0.25 7.43 90 1.07 0.45 0.51 3.05 83 0.80 0.36 0.50 2.44 51 0.57 0.38 0.30 2.89 94 0.70* 0.59 0.20 4.22 96 0.46 0.24 0.20 1.61 61 0.27 0.05 0.20 0.40 57 Female Mean SD Minimum Maximum % of samples > LOD 7.63 3.99 0.24 19.97 100 3.50 1.96 1.12 13.40 99 0.92 0.44 0.31 2.41 76 1.07 0.39 0.57 2.61 90 0.75 0.25 0.50 1.50 68 0.71 0.35 0.24 2.24 99 0.48 0.24 0.20 1.22 97 0.48 0.24 0.20 1.71 68 0.27 0.08 0.20 0.49 38 The plasma samples were analyzed separately according to gender. Male plasma PFOS, PFOA and PFHxS were found to have significantly higher levels (p < 0.05, <0.03 and <0.001) while plasma PFHxA levels were higher in the female group (p < 0.005). * Indicates significant differences among groups by analysis of variance (ANOVA) followed by Duncan’s multiple range test (significance at p < 0.05) SPSS16. Please cite this article in press as: H.T. Wan, et al., Blood plasma concentrations of endocrine disrupting chemicals in Hong Kong populations, J. Hazard. Mater. (2013), https://dx.doi.org/10.1016/j.jhazmat.2013.01.034 G Model HAZMAT-14857; No. of Pages 7 ARTICLE IN PRESS H.T. Wan et al. / Journal of Hazardous Materials xxx (2013) xxx–xxx 0.47 ± 0.23 66.07 0.47 ± 0.26 58.54 Plasma samples were separated into two groups according to age (between 16 and 39; 40–63). No significant differences were noted. PFBA PFDoA PFHxA 0.61 ± 0.54 95.54 0.57 ± 0.25 100 0.62 ± 0.33 99.11 0.67 ± 0.48 87.80 PFUdA PFDA 0.75 ± 0.30 62.50 0.86 ± 0.32 48.78 1.05 ± 0.41 90.18 1.10 ± 0.47 80.49 PFNA 8.85 ± 5.90 100 8.21 ± 5.99 100 16–39 (n = 112) % of samples > LOD 40–63 (n = 43) % of samples > LOD PFOA PFOS Age Table 3 Age difference in the mean of plasma PFC concentrations. We performed sample extraction and HPLC–MS/MS analysis as previously described but with a slight modification [1]. To state succinctly, 5 ml of acetonitrile was added to the samples, which were next shaken in an orbital shaker (HS501, IKA, Germany) for 30 min at 300 mot/min at room temperature, centrifuged at 4000 rpm for 5 min, and the supernatant transferred to a clean 50 ml polypropylene tube. The extraction was repeated twice and all the extracts pooled. The total extract (about 15 ml) was mixed with 7.5 ml n-hexane and shaken vigorously for 30 min to remove lipids [9] and were then concentrated to dryness under a gentle stream of nitrogen and reconstituted in 0.2 ml of methanol: H2 O (1:1) prior to LC/MS/MS analysis. Mobile phases were Milli-Q water and methanol. The MS/MS was operated in the negative ESI mode and MRM detection mode. The injection volume was 20 ␮l and the column temperature was maintained at 25 ◦ C throughout the chromatography. LOD was defined as 3-fold higher than the signal-to-noise ratio and was 0.15 ng/ml for BPA. LOQ was defined as 10-fold higher than the signal-to-noise ratio. Recoveries of BPA standard with 2.5 ng/ml, 5 ng/ml and 10 ng/ml spiking levels were 75–80%, 74–79% and 78–81%, respectively. The percentage of recovery of BPA-d16 standard with 5 ng/ml and 10 ng/ml spiking levels ranged between 76–78% and 78–79%, respectively. PFHxS 2.4. Determination of BPA 1.24 ± 0.99 85.71 1.65 ± 1.48 78.05 The method for the extraction and analysis of PFCs was performed as previously described [7], but to restate briefly, a plasma sample was mixed with 1 ml TBA, 2 ml TBA buffer and 5 ml MTBE, followed by shaking for 30 min at 300 mot/min at room temperature. After centrifugation at 3500 rpm for 15 min, the supernatant was transferred to a clean 50 ml polypropylene tube. The remaining aqueous phase was twice subjected to extraction. All three organic phases were pooled and were concentrated to dryness under a gentle stream of nitrogen and reconstituted with 1 ml of 10 mM Ammonium/Acetate: acetonitrile (6:4) prior to LC/MS/MS analysis. Standards of PFCs and labeled-PFCs used for calibration were both prepared in 10 mM ammonium acetate: acetonitrile (6:4) solution. The concentrations of spiked standard were 1 and 5 ng/ml. The detection of PFCs was performed using an Agilent 1200 highperformance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS, Aglient 1200 series, Aglient Technologies, CA, USA). A 30 ␮l aliquot of the extract was injected into a guard column (Zorbax Eclipse Plus-C8, 2.1 mm i.d. × 12.5 mm length, 5-␮m; Agilent Technologies), which was connected to a Zorbax Eclipse Plus C8 column (2.1 mm i.d. × 100 mm length, 3.5-␮m; Agilent Technologies). Instrumental parameters for PFC analysis referred to (2170 5041). LOD was defined as 3-fold higher than the signal-to-noise ratio and ranged from 0.2 to 0.4 ng/ml for nine PFCs. LOQ was defined as 10-fold higher than the signal-tonoise ratio. The values of matrix recoveries were 101.6%, 98.72%, 83.82%, 95.57%, 86.86%, 95.43%, 94.08%, 98.83% and 99.95% for perfluorobutanoic acid (PFBA), perfluorohexanoic acid (PFHxA), perfluroroctanoic acid (PFOA), perflurononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoroundecanoic acid (PFUdA), perfluorododecanoic acid (PFDoA), perfluorohexane sulfonate (PFHxS) and perfluorooctane sulfonate (PFOS), respectively. The matrix effects for the measurement of some PFCs were minimized by applying the “internal standard method” for the quantitative measurement. According to our test, the ratios between the native and internal standards were similar in the 10 mM ammonium acetate: acetonitrile (6:4) solution and the blood samples. No contamination of PFCs was found above the detection limit. 3.74 ± 2.33 96.43 4.79 ± 3.46 95.12 2.3. Determination of PFCs 0.26 ± 0.07 43.75 0.27 ± 0.06 58.54 3 Please cite this article in press as: H.T. Wan, et al., Blood plasma concentrations of endocrine disrupting chemicals in Hong Kong populations, J. Hazard. Mater. (2013), https://dx.doi.org/10.1016/j.jhazmat.2013.01.034 G Model HAZMAT-14857; No. of Pages 7 4 ARTICLE IN PRESS H.T. Wan et al. / Journal of Hazardous Materials xxx (2013) xxx–xxx No contamination of BPA was found above the detection limit. 2.5. Determination of phthalates All glassware and polypropylene tubes were thoroughly rinsed with pesticide-grade solvent prior to use. The plasma samples (0.5 ml) were thawed and diluted with 1 ml ultrapure water. Next, 5 ng of surrogate standards (diisooctyl phthalate in n-hexane) was added and the sample was extracted twice with 5 ml 1:1 MTBE: hexane and the extract was pooled together and concentrated to dryness by a stream of nitrogen gas. The resulting residue was reconstituted in 200 ␮l of n-hexane with 3 min of mixing vortex for analysis (Sample is transferred into eppendorf.). Afterwards, 5 ng of Anthracene-d10 (in n-hexane) was added as internal standard. 1 ␮l of the final solution was injected into the GC–MS system. The phthalates were determined by GC–MS analysis performed on an Agilent 6890 N GC/5975 N MSD instrument (Agilent Technologies, CA, USA). The separation was carried out in a fused silica capillary column (DB-17MS, 30 m × 0.25 mm, 0.25 micron, J&W Scientific, Folsom, CA, USA) with a carrier gas of ultrapure helium at a constant flow rate of 1.5 ml/min. The injection temperature was maintained at 270 ◦ C in splitless mode. A solvent delay of 4.5 min was selected. The GC oven temperature was programmed as follows: 110 ◦ C for 0.5 min then 20 ◦ C/min to 280 ◦ C for 1 min, and then 20 ◦ C/min to 320 ◦ C for 5 min. The mass spectrometer was operated in electron impact (EI) mode with electron energy of 70 eV. The target compounds were determined in both full scan and selected ion monitoring (SIM) modes. Thereafter, the presence of the compounds of interest was confirmed by the mass spectra obtained from the full scan acquisition mode with a scan range from m/z 50 to 500. The SIM mode was also used for quantitative determination. Three fragment ions were monitored for each compound and the most characteristic ion in the spectrum was selected for quantification; the other two ions were used for the purpose of confirmation. Phthalates were classified according to the retention times and mass spectra of the pure reference standards under the same analytical conditions. LOD was defined as 3-fold higher than the signal-to-noise ratio and ranged from 0.2 to 0.4 ng/ml for 8 phthalates. LOQ was defined as 10fold higher than the signal-to-noise ratio. Recoveries of phthalates standard with 62.5 ng/ml, 125 ng/ml, 250 ng/ml, and 500 ng/ml spiking levels were 71–72%, 73–75%, 81–84% and 72–85%, respectively. No contamination of BPA was found above the detection limit. samples, followed by PFHxA (96.73%) and PFOA (96.08%). The mean PFOS concentration was the highest (8.68 ng/ml ±5.91), followed by PFOA (4.02 ng/ml ±2.70) and PFHxS (1.34 ng/ml ±1.14). The data were compared with the studies reported in other countries such as Sweden (PFOS: 16 ng/ml, PFOA: 2.4 ng/ml, PFHxS: 1.5 ng/ml), the United States (PFOS: 13.2 ng/ml, PFOA: 4.13 g/ml, PFHxS: 1.96 ng/ml) and the 11 coastal cities (Dalian, Yingkou, Huludao, Qinhuangdao, Tsangshan, Weihai, Qingdao, Zhoushan, Ningbo, Fuzhou, Xiamen) in China (PFOS: 10.17 ng/ml, PFOA: 1.46 ng/ml, PFHxS: 0.92 ng/ml) [10–14]. The mean plasma PFOS concentration (8.68 ng/ml) detected in the present study, was around 1.5 times lower than that of the reports of the USA and Sweden, which suggests a lower exposure risk to the Hong Kong population. However, plasma PFOA levels in our study were two times higher than levels in China and Sweden, but were comparable to the USA. The levels of other types of PFCs were similar to other countries, resulting in levels less than 2 ng/ml. Previous reports have showed the association of gender with PFC concentrations in blood, in which males had higher plasma levels of PFOS and PFOA than females [10,14–16]. Therefore, in this study gender difference in the plasma concentrations of the nine PFCs was analyzed (Table 2). Plasma PFCs such as PFOS, PFOA, PFHxS, and PFHxA concentrations were significantly higher in males (p < 0.05), with the exception of PFUdA, which was significantly higher in the female plasma samples (p < 0.03). This observation is in general accord with some of the data of past studies thus indicating higher PFC levels detected in males [10,17]. A study from Karrman et al. suggested that a higher fish consumption rate may lead to a greater risk of PFOS exposure [10]. The general higher food consumption rate and the higher occupational exposure risk in males are the potential risk factors which result in higher plasma PFC levels. In addition to the analysis of gender difference in PFC accumulation, we have divided the samples into two age groups (aged 16–39; 40–63) to study the age effect on bioaccumulation (Table 3). No statistical difference between the two age groups was observed. Since the human elimination half-life of PFOS and PFOA is 3–5 years, respectively [18], the difference in the bio-accumulation of PFCs may not be evidence in these two age groups. Therefore, the ubiquitous occurrence of PFCs may be a major causal factor in the general accumulation profiles of PFCs in the Hong Kong population. Even in the aquatic food source, our previous study showed that PFCs were detected in over 70% of freshwater and marine fish species collected from Hong Kong markets [7]. 3.2. Plasticizer contaminants were ubiquitous 2.6. Statistical analysis Statistical evaluations were conducted by SPSS16. All data were tested to be normally distributed and independent through the use of the Normal Plots in SPSS at 5% significance. Differences among groups were tested for statistical significance by analyzing the variance (ANOVA), followed by Duncan’s multiple range test (significance at p < 0.05) SPSS16. Results are presented as the mean ± SEM. Groups were considered significantly different if p < 0.05. The mean plasma concentrations of BPA and phthalates are summarized in Tables 4 and 5. The MRM transitions in LC–MS/MS for BPA and the fragment ions in GC–MS for phthalates are tabulated in Supplementary Table 2. Among 153 human plasma samples, 94.44% of them contained BPA. The mean plasma BPA concentrations were 0.95 ± 0.56 ng/ml, and ranged from 0.16 to 3.38 ng/ml. Table 4 Summary of plasma BPA concentration. 3. Results and discussion 3.1. Trend of PFC exposure profile in relation to gender The concentrations of different PFCs in the plasma samples are summarized in Table 1. All 153 samples were scanned for the nine targeted PFCs (Supplementary Table 1). The multiple reaction monitoring (MRM) transitions in LC–MS/MS for PFCs are tabulated in Supplementary Table 2. PFOS were detectable in all All Samples (ng/ml) Mean SD Minimum Maximum Median % of samples > LOD 0.95 0.56 0.16 3.38 0.85 94.44 BPA Concentrations (ng/ml) Female Male 0.90 0.57 0.16 3.38 0.75 92.68 0.99 0.57 0.16 3.04 0.87 93.878 No statistical differences were noted based on gender. Please cite this article in press as: H.T. Wan, et al., Blood plasma concentrations of endocrine disrupting chemicals in Hong Kong populations, J. Hazard. Mater. (2013), https://dx.doi.org/10.1016/j.jhazmat.2013.01.034 ARTICLE IN PRESS G Model HAZMAT-14857; No. of Pages 7 H.T. Wan et al. / Journal of Hazardous Materials xxx (2013) xxx–xxx 5 Table 5 Summary of plasma phthalate concentrations (ng/ml). Mean SD Minimum Maximum Median % of samples > LOD DEHP DMEP DnOP DiBP DBP DMP DEP BBP Total phthalates 11.13 4.41 3.51 28.45 10.39 95.56 11.01 6.57 2.08 39.58 9.55 100.00 6.53 3.95 1.00 21.89 5.73 99.44 5.84 2.64 1.09 15.98 5.82 96.67 4.19 2.39 0.77 12.50 3.89 98.89 2.45 1.17 1.00 9.16 2.29 70.56 2.23 0.92 1.02 5.91 2.12 55.00 1.30 0.37 0.82 1.97 1.24 53.33 41.63 10.62 20.19 84.98 40.82 The plasma concentrations of eight phthalates and total phthalates are shown. Total phthalates is represented by the summation of all measured phthalates in the human plasma. The data were comparable to most studies on plasma or serum BPA levels in human blood samples, with mean BPA concentrations lower than 1 ng/ml [19–23]. However, a study in Korea reported a higher maternal blood BPA level (66.48 ng/ml) [24] which is 20 times higher than the present (and other) studies. In addition to BPA, all samples were analyzed for plasma phthalate concentrations (Supplementary Table 3). Our data showed that bis(2-methoxyethyl) phthalate (DMEP) was detected in all of the samples and DEHP, DnOP, diisobutyl phthalate (DiBP) and dibutyl phthalate (DBP) were detected in over 95% of the samples, whereas the concentrations of diisodecyl phthalate (DiDP) and dihexyl phthalate (DHP) were below the limit of detection (LOD). The mean concentration of DEHP was the highest among the eight phthalates (11.13 ng/ml ±3.95), followed by DMEP (11.01 ng/ml ±6.57), DnOP (6.53 ng/ml ±3.95) and DiBP (5.84 ng/ml ±2.64). The mean concentration of total phthalates in all of the human samples was 41.63 ng/ml ±10.62, ranging from 20.19 ng/ml to 84.98 ng/ml. Compared to a study in Sweden [25], the plasma DEHP, DnOP, DBP and DEP levels of our samples were 1.9–7.2 times higher. The relatively high levels of phthalates detected in the plasma samples might be attributable to food contamination in which phthalates were used illegally in beverage and different food items. This illegal use of phthalates in beverages as food additives was reported in Taiwan in May 2011. DEHP and DiNP were purposely added as clouding agents to substitute palm oil, reduce production costs, and increase the shelf-life of the products [26]. Over 900 different food Table 6 Age difference in plasma BPA concentrations (ng/ml). Age 16–39 (n = 112) 40–63 (n = 43) Mean SD Median % of samples > LOD 1.04 0.58 0.95 95.50 0.89 0.57 0.75 85.37 Samples were analyzed separately according to the two age groups. No significant difference was found between the groups. products (i.e. beverages, fruit jam and health supplements) were reported to be contaminated and were found to be sold in Hong Kong. This scandal raised worldwide attention to food safety and concern about the unexpected phthalate exposure. Nevertheless, the ongoing wide application of phthalates in cosmetics, paints, cutlery and dishes, toys and clothing could also be a contributing factor in the increase in phthalate exposure levels [17,26,27]. As with the PFCs, we analyzed gender and age difference in the levels of plasticizers in our samples (Tables 6–8). The plasma levels of BPA did not show significant differences between gender and the two age groups. However, the plasma concentrations of DEHP, DMP and BBP in relation to age differences were observed. Plasma DEHP and BBP were found at significantly higher concentrations in the young age group (p < 0.001 and <0.005, respectively). Both types of phthalates are commonly used in PVC and BBP is also applied in the production of artificial leather. The higher detected levels of Table 7 Age difference in the mean of plasma phthalate concentrations (ng/ml). DEHP 16–39 (n = 112) 40–63 (n = 41) DMEP * 11.06 ± 4.28 10.71 ± 4.39 DnOP 10.59 ± 5.48 11.89 ± 8.05 DiBP 7.00 ± 3.42 7.34 ± 5.20 5.28 ± 2.55 5.81 ± 2.61 DBP DMP 4.12 ± 2.39 4.54 ± 2.49 2.33 ± 0.79 2.99 ± 1.91* DEP 2.23 ± 0.76 2.06 ± 0.94 BBP Total phthalates * 1.40 ± 0.36 1.13 ± 0.35 41.10 ± 9.70 43.32 ± 12.14 Plasma DEHP and BBP levels were significantly higher in the young age group (p < 0.001 and <0.005, respectively). Meanwhile, the plasma DMP level was higher in the older age group (p < 0.02). * Indicates significant differences among groups by analysis of variance (ANOVA) followed by Duncan’s Multiple Range test (significance at p < 0.05) SPSS16. Table 8 Gender difference in plasma phthalates concentrations (ng/ml). DEHP DMEP Male Mean SD Minimum Maximum Median % of Samples > LOD DnOP DiBP 11.51 4.36 3.51 28.45 10.50 96.94 11.08 5.66 2.71 28.05 10.56 100.00 6.14 3.65 1.00 21.66 6.22 98.98 5.91 2.68 1.33 15.98 5.20 97.96 Female Mean SD Minimum Maximum Median % of samples > LOD 10.67 4.45 3.67 27.43 9.56 93.90 10.93 7.56 2.08 39.58 9.23 100.00 7.00 4.26 1.03 21.89 6.88 100.00 5.75 2.61 1.09 10.55 5.37 95.12 DBP DMP DEP BBP Total phthalates 4.41 2.64 0.90 12.50 4.12 97.96 2.30 0.84 1.03 5.73 2.29 74.49 2.27 0.96 1.02 5.91 2.19 62.25 1.28 0.38 0.82 1.97 1.26 51.14 42.32 10.64 20.19 68.15 42.43 – 3.94 2.06 0.77 9.84 3.54 100.00 2.65 1.50 1.00 9.16 2.47 65.85 2.16 0.87 1.09 3.93 2.08 46.34 1.34 0.35 0.84 1.96 1.26 48.78 40.76 10.66 20.53 84.98 39.58 – Samples were analyzed according to the two age groups. No significant difference among the groups was noted. Please cite this article in press as: H.T. Wan, et al., Blood plasma concentrations of endocrine disrupting chemicals in Hong Kong populations, J. Hazard. Mater. (2013), https://dx.doi.org/10.1016/j.jhazmat.2013.01.034 G Model HAZMAT-14857; No. of Pages 7 6 ARTICLE IN PRESS H.T. Wan et al. / Journal of Hazardous Materials xxx (2013) xxx–xxx DEHP and BBP in the young age group may be due to the more frequent use of the related consumer products, such as bags, shoes and coats. On the other hand, plasma DMP was significantly higher in the older age group (p < 0.02). This low molecular weight phthalate is often used as a solvent in fragranced household cleaning products and coatings for enteric-coated tablets, which are more likely to be used by the elderly. Other than consumer products, phthalates can also be found in various types of food, including seafood, poultry, vegetables, and fruits [28]. In this study no statistical differences in plasma phthalate levels were identified between males and females, thus indicating the ubiquitous occurrence of, and exposure to, phthalates. We suggest that the approach of present study can provide the most relevant information to highlight the immediate determinants of actual risk posed by EDC in humans. One must exercise caution with regard to the relevance of plasma data in predicting a linear relationship between environmental toxicants and health consequences in human populations. The general pathological manifestations of PFC intoxication (i.e. PFOS and PFOA) include hepatomegaly and reduced male fertility [29,30]. While BPA and phthalates are known to impede reproductive and developmental toxicity, especially in infants and children [31], at present there is no study that correlates levels of plasma pollutants to immediate health consequences. A pathological manifestation of EDC exposure may not be overtly apparent but what has clearly been proved is that the occurrence and the accumulation of these contaminants can certainly interfere with normal body function. 4. Conclusion Our study has identified the strong presence of PFCs, BPA and phthalates in the blood samples of most Hong Kong citizens. The observed characterization of the contamination profile using human blood may indicate a general exposure route to these contaminants. The blood plasma concentrations of BPA and PFCs were lower or comparable with data from other countries; however, plasma phthalate concentrations were higher than that of Sweden. The illegal and unexpected use of phthalates as food additives in greater China and the wide applications of phthalates in daily commodities may attribute to the relatively high levels of plasma phthalates. Nevertheless the identification of the sources of EDC exposure and their avoidance may therefore be an effective way to reduce the body loadings of plasma phthalates. 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Please cite this article in press as: H.T. Wan, et al., Blood plasma concentrations of endocrine disrupting chemicals in Hong Kong populations, J. Hazard. Mater. (2013), https://dx.doi.org/10.1016/j.jhazmat.2013.01.034