DSpace at VNU: Occurrence of Perchlorate and Thiocyanate in Human Serum From E-Waste Recycling and Reference Sites in Vietnam: Association With Thyroid Hormone and Iodide Levels

DSpace at VNU: Occurrence of Perchlorate and Thiocyanate in Human Serum From E-Waste Recycling and Reference Sites in Vi...

Arch Environ Contam Toxicol (2014) 67:29–41 DOI 10.1007/s00244-014-0021-y Occurrence of Perchlorate and Thiocyanate in Human Serum From E-Waste Recycling and Reference Sites in Vietnam: Association With Thyroid Hormone and Iodide Levels Akifumi Eguchi • Tatsuya Kunisue • Qian Wu • Pham Thi Kim Trang • Pham Hung Viet Kurunthachalam Kannan • Shinsuke Tanabe • Received: 23 October 2013 / Accepted: 18 March 2014 / Published online: 10 April 2014 Ó Springer Science+Business Media New York 2014 Abstract Perchlorate (ClO4-) and thiocyanate (SCN-) interfere with iodide (I-) uptake by the sodium/iodide symporter, and thereby these anions may affect the production of thyroid hormones (THs) in the thyroid gland Although human exposure to perchlorate and thiocyanate has been studied in the United States and Europe, few investigations have been performed in Asian countries In this study, we determined concentrations of perchlorate, thiocyanate, and iodide in 131 serum samples collected from locations in Northern Vietnam, Bui Dau (BD; electrical and electronic waste [e-waste] recycling site) and Doung Quang (DQ; rural site) and examined the association between serum levels of these anions with levels of THs The median concentrations of perchlorate, thiocyanate, and iodide detected in the serum of Vietnamese subjects were 0.104, 2020, and 3.11 ng mL-1, respectively Perchlorate levels were significantly greater in serum of the BD population (median 0.116 ng mL-1) than those in the DQ population (median 0.086 ng mL-1), which A Eguchi Á T Kunisue Á S Tanabe Center for Marine Environmental Studies, Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan T Kunisue (&) Faculty of Agriculture, Tottori University, 4-101 Koyamaminami, Tottori 680-8553, Japan e-mail: kunisue@muses.tottori-u.ac.jp Q Wu Á K Kannan New York State Department of Health and Department of Environmental Health Sciences, School of Public Health, Wadsworth Center, State University of New York at Albany, P.O Box 509, Albany, NY 12201-0509, USA P T K Trang Á P H Viet Center for Environmental Technology and Sustainable Development, Hanoi University of Science, 334 Nguyen Trai, Hanoi, Vietnam indicated greater exposure from e-waste recycling operations by the former Serum concentrations of thiocyanate were not significantly different between the BD and DQ populations, but increased levels of this anion were observed among smokers Iodide was a significant positive predictor of serum levels of FT3 and TT3 and a significant negative predictor of thyroid-stimulating hormone in males When the association between serum levels of perchlorate or thiocyanate and THs was assessed using a stepwise multiple linear regression model, no significant correlations were found In addition to greater concentrations of perchlorate detected in the e-waste recycling population, however, given that lower concentrations of iodide were observed in the serum of Vietnamese females, detailed risk assessments on TH homeostasis for females inhabiting e-waste recycling sites, especially for pregnant women and their neonates, are required Perchlorate (ClO4-) is an anionic compound, and its salts are used as oxidizing agents in rocket propellants, explosives, and fireworks and as dopant materials in the production of polyvinyl chloride (PVC) (Interstate Technology and Regulatory Council 2005) Perchlorate also occurs naturally in some fertilizers (Urbansky et al 2001), and is presumably generated in the atmosphere (Rao et al 2010) It has been reported in the United States that such sources and high-hydrophilic property of perchlorate led to the widespread presence of this anion in the aquatic environment and in drinking water (Blount et al 2010) In addition, perchlorate has been detected in some food items and vegetables (Sanchez et al 2005) Therefore, studies performed in the United States have shown that perchlorate is found in various human bodily fluids such as urine (Blount et al 2006), breast milk (Kirk et al 2007), saliva (Oldi and Kannan 2009), and blood (Oldi and Kannan 2009; Blount 123 30 et al 2009) Nevertheless, few studies on human exposure to perchlorate are available in Asian countries where perchlorate salts are produced and used It is known that thiocyanate (SCN-) is formed by way of the detoxification process of hydrogen cyanide contained in cigarette smoke (Tuncel et al 1994) This anionic compound is also a metabolite of cyanogenic glucosides present in plant foods such as cabbage, broccoli, and mustard (VanEtten et al 1969; Foss and Lund-Larsen 1986; Bertelsen and Hegedus 1994) Thus, it is thought that intake of cigarette smoke and plant foods is a main exposure source of thiocyanate for humans In Europe and the United States, the detection of thiocyanate in human urine and serum has been reported (Foss and Lund-Larsen 1986; Blount et al 2006) Iodide (I-) is essential for the production of thyroid hormones (THs) (Bianco et al 2002) Both perchlorate and thiocyanate can competitively inhibit iodide uptake by the thyroid gland (TG) by way of the sodium/iodide (Na?/ I-) symporter (NIS) consequently decreasing the synthesis of tri-iodothyronine (T3) and thyroxine (T4) (Tonacchera et al 2004; Dohan et al 2007) The United States National Academy of Sciences and the United States Environmental Protection Agency (USEPA) have adopted an oral reference dose (RfD) of 0.7 mg perchlorate/kg body weight (bw)/d based on a study of inhibition of iodide uptake by perchlorate into the TG (Greer et al 2002; Zewdie et al 2010) Exposure to perchlorate was associated with increased serum TSH levels in adult women with urinary iodine levels \100 lg L-1 (Blount et al 2006) It was also shown that concentrations of T3 and T4 decreased significantly in TG and serum of rats coadministered with perchlorate and an iodide-deficient diet (Kunisue et al 2010, 2011a) Although no RfD for thiocyanate is available, an in vivo study has shown that a single dose of thiocyanate to pregnant mice led to decreased concentrations of free T3 and T4 and to increased concentrations of TSH in plasma of their pups, which was associated with decreased iodine levels in the pup TG (Ghorbel et al 2008) Thus, the adverse effects of these two anions on TH homeostasis can be exacerbated under iodide-deficient conditions In Vietnam, it was reported that urinary concentrations of iodine in school-aged children were in the range of 20–49 lg L-1, \100 lg L-1, indicating insufficient iodide intake by this population (de Benoist et al 2008; Zimmermann et al 2008) Considering the above-mentioned information, it is likely that Vietnamese people may be at high risk of TH disruption by perchlorate and thiocyanate; however, human exposure to these anions has been never investigated in this country In addition, primitive methods of recycling of electrical and electronic wastes (e-wastes) in Vietnam have raised considerable concern because 123 Arch Environ Contam Toxicol (2014) 67:29–41 hazardous substances, such as heavy metals and persistent organohalogen compounds (Silicon Valley Toxics Coalition and Basel Action Network 2002), can be released into the environment Our research group recently suggested that Vietnamese workers in an e-waste site are occupationally exposed to polychlorinated biphenyls and polybrominated diphenyl ethers during recycling activities (Tue et al 2010) Given that perchlorate is contained in PVC and lithium-ion batteries as a dopant material, it is probable that residents and workers in such e-waste recycling sites are exposed to this anion The present study was aimed at determining the serum concentrations of perchlorate and thiocyanate in residents at an e-waste recycling site and a rural reference site in Northern Vietnam We examined the relationship between serum concentrations of THs and perchlorate/thiocyanate, to assess the effects of these anions on TH homeostasis in the Vietnamese population Materials and Methods Chemicals and Devices Ammonium perchlorate ([99.9 %) and methylamine (40 weight % solution in water) were purchased from SigmaAldrich (St Louis, Missouri, USA) Potassium iodide and thiocyanate solutions ([99.5 %) were from AccuStandard Inc (New Haven, Connecticut, USA) Isotopically labelled sodium perchlorate (Cl18O4-, [90 %) and potassium thiocyanate (S13CN-, [95 %) were purchased from Cambridge Isotope Laboratories (Andover, Massachusetts, USA) Vivaspin centrifugal-filtration devices (CFDs) were obtained from Sartorius Stedim Biotech (Goettingen, Germany) Samples Human serum samples (n = 131) were collected from 50 males and 81 females age 10–64 years in Bui Dau (BD, e-waste recycling site, n = 83) and Duong Quang (DQ, rural site, n = 48), Vietnam, during January 2010 to January 2011 (Fig 1) These 131 donors were informed beforehand about the purpose of the study at local government health stations where volunteers registered their consent to participate, and they consented to participation in our study All of the participants were randomly selected without arbitrary criteria Informed consent was obtained from all 131 donors, and this study was approved by the Ethical Committee of Ehime University, Japan Age, body mass index (BMI), living site, and health conditions (smoking habits, dietary habits, and Arch Environ Contam Toxicol (2014) 67:29–41 31 Fig Map of Vietnam showing serum sampling locations Table Cohort characteristics and demographics of the Vietnamese populations from e-waste recycling and rural sites Characteristics and demographics All participants (n = 131) E-waste recycling workers (n = 83) Residents in reference site (n = 48) Age (year) 36.2 ± 12.2 35.0 ± 13.4 37.0 ± 11.5 BMI (kg/m ) 20.4 ± 2.47 20.3 ± 2.50 20.4 ± 2.47 Male Female 50 (38 %) 81 (62 %) 35 (42 %) 48 (58 %) 15 (30 %) 33 (70 %) Pregnant (4.9 %) (4.2 %) (6.1 %) Smokera 30 (23 %) 23 (28 %) (15 %) 1–3 times/week 58 (44 %) 37 (45 %) 21 (44 %) 4–6 times/week 73 (56 %) 46 (55 %) 27 (56 %) times/week 119 (91 %) 79 (95 %) 40 (83 %) 1–3 times/week 12 (9 %) (5 %) (17 %) 1–3 times/week 24 (18 %) 19 (23 %) (10 %) 4–6 times/week 107 (82 %) 64 (77 %) 43 (90 %) 131 (100 %) 83 (100 %) 48 (100 %) Fish consumption Freshwater fish Marine fish Meat consumption Vegetable consumption a No females were smokers [6 times/week pregnancy status) were recorded by interview with the participants, and these data are listed in Table The interview was performed by volunteers from the Center for Environmental Technology and Sustainable Development, Hanoi University of Science, according to a standardized questionnaire Whole blood samples were collected by a certified physician, and serum samples were obtained by way of centrifugation after heparin treatment, frozen in liquid nitrogen, transported to Japan, and stored at -25 °C or -80 °C in the Environmental Specimen Bank (es-Bank) of Ehime University (Tanabe 2006) until analysis Analysis of Perchlorate, Thiocyanate, and Iodide Perchlorate, thiocyanate, and iodide were analyzed according to the procedures reported previously (Oldi and Kannan 2009; Zhang et al 2010) Each serum sample was thawed at room temperature, and an aliquot of 0.5 mL was transferred to a Vivaspin CFD Two hundred microliters of an internal standard mixture containing 0.1 ng (100 lL [1 ng mL-1]) of Cl18O4- and 10 ng (100 lL [100 ng mL-1]) of S13CN- and 300 lL of Milli-Q water were added The diluted sample was vortexed to 123 32 incorporate the internal standard into the sample matrix The Vivaspin CFD was then centrifuged for 30 at 4,0009g The filtrate was transferred to a sample vial for the following instrumental analysis Instrumental Analysis The instrumental analysis was performed using an Agilent 1100 Series high-performance liquid chromatograph (Agilent Technologies, Santa Clara, California, USA) coupled with a Micromass Quattro LC tandem mass spectrometer (Waters, Milford, Massachusetts, USA) One hundred microliters of the filtrate were injected using a Gilson 215 liquid handler (Gilson, Middleton, Wisconsin, USA) and a Gilson 819 injection module equipped with a 100-ll injection loop Separation of perchlorate, iodide, and thiocyanate in the sample was performed using an IonPac AS-21 column (guard column 50 mm mm, regular column 250 mm mm; Dionex, Sunnyvale, California, USA) An isocratic mobile phase of 200 mM aqueous methylamine was used at a flow rate of 0.3 mL/min Electrospray negative ionization was employed in the multiple reaction monitoring mode with the following mass transitions to identify and quantify perchlorate, Cl18O4-, thiocyanate, S13CN-, and iodide: 99 (35ClO4-) [ 83 (35ClO3-), 101 (37ClO4-) [ 85 (37ClO3-), 107 (35Cl18O4-) [ 89 (35Cl18O3-), 58 (SCN-) [ 58 (SCN-), 59 (S13CN-) [ 59 (S13CN-), and 127 (127I-) [ 127 (127I-) Relative responses of the native standard to the isotope-labeled internal standard and the ratios of 35Cl to 37 Cl (for perchlorate) were used for the confirmation of target analytes The ratios (35Cl:37Cl) were considered acceptable at 3.12 (± 25 %) Data acquisition and calculation were accomplished using Micromass MassLynx 3.5 (Waters) Quality Assurance and Quality Control Recoveries of internal standards Cl18O4-and S13CN- spiked into serum samples (n = 131) were in the range of 60–110 % and 88–135 %, respectively A 10-point calibration standard (in Milli-Q water) comprising concentrations ranging from 0.01 to 50 ng mL-1 for perchlorate, 2–2,000 ng mL-1 for thiocyanate, and 0.02–100 ng mL-1 for iodide were injected with each batch of 30 samples The regression coefficients for calibration curves were[0.999 for all target analytes A laboratory reagent blank and an instrument blank were run with each batch of samples Blanks contained trace levels of iodide, which were subtracted from concentrations of iodide detected in samples The limits of quantitation (LOQs) for perchlorate, thiocyanate, and iodide in serum samples were 0.05, 0.5, and 0.5 ng mL-1, respectively 123 Arch Environ Contam Toxicol (2014) 67:29–41 Perchlorate-Equivalent Concentration An in vitro study using Chinese hamster ovary cells expressing human NIS reported that the relative potency of ClO4- to inhibit 125I- uptake by the NIS was 15 times greater than that of SCN- on a molar basis (Tonacchera et al 2004) Based on the potency factor, we calculated perchlorate-equivalent concentrations (PECs) in serum sample, using the following formula: PEC (lmol L-1) = [ClO4-] (lmol L-1) ? [SCN-] (lmol L-1)/15 TH Analysis Concentrations of THs in serum were analyzed by electrochemiluminescence immunoassay method as described in our previous study (Kunisue et al 2011b) TSH, total T3 (TT3), total T4 (TT4), free T3 (FT3), and free T4 (FT4) were measured using Elecsys kits (Roche Diagnostics, Mannheim, New York, USA) and Modular Analytics E170 systems (Hitachi, Tokyo, Japan) The expected reference intervals in euthyroid humans are within 0.270–4.2 lIU mL-1 for TSH, 0.80 –2.0 ng mL-1 for TT3, 45–117 ng mL-1 for TT4, 2.0 –4.4 pg mL-1 for FT3, and 9.7–17 pg mL-1 for FT4 (Roche Diagnostics GmbH 2008) Statistical Analysis Statistical analyses were performed using R program Version 2.15.1 with its graphical user interface EZR (Saitama Medical Center, Jichi Medical University, The R Foundation for Statistical Computing, version 2.13.0) (Kanda 2013) This interface is a modified version of the R commander (version 1.6-3) and is designed to add frequently used biostatistical functions Steel–Dwass test (Kruskal–Wallis post hoc test) was used to assess differences in serum concentrations of anions and THs between the residents of e-waste recycling and reference sites or among females, nonsmoking males, and smoking males Spearman’s rank correlation coefficients were calculated to assess the strength of relationships between serum concentrations of each anion and age or BMI of the donors Associations between serum levels of THs and perchlorate, thiocyanate, or iodide were assessed using a stepwise multiple linear regression model The normality of distribution for each parameter was assessed by Shapiro–Wilk test Although the data for TT4 and FT4 were normally distributed in serum, the concentrations of TSH, TT3, FT3, perchlorate, thiocyanate and iodide were not normally distributed; hence, these data were log-transformed for the regression analyses Characteristics, such as age, residential area, BMI, pregnancy status, and dietary habits (meat, freshwater, and marine fish consumption), which can Arch Environ Contam Toxicol (2014) 67:29–41 influence TH levels, were used as explanatory variables A p-value of \0.05 denoted significance For any model, the parameters that optimize the approximation of the likelihood can be found numerically Following this, the optimized likelihoods from different models can be compared through Akaike’s information criterion (AIC) as follows: AIC = 2k-2ln L In this model, k is the number of parameters, and L is the maximized likelihood The model with the smallest AIC was selected, thus providing a tradeoff between model complexity (preferring models with fewer parameters) and the maximized likelihood of the model Results Perchlorate Perchlorate was detected in 129 of 131 serum samples with the concentrations ranging from 0.050 to 1.25 ng mL-1 (median 0.104 ng mL-1), whereas the levels in two donors were lower than the LOQ (Table 2) Serum concentrations of perchlorate in BD (e-waste recycling site) residents (median 0.116 ng mL-1) were significantly greater (p \ 0.05) than those in DQ (rural site) residents (median 0.086 ng mL-1) No significant relationships were found between serum concentrations of perchlorate and smoking, dietary habits, sex, age, or BMI (Table 2, Fig 2) Thiocyanate Thiocyanate was found in all 131 serum samples analyzed (median 2,020 ng mL-1) (Table 2) Significantly greater concentrations of thiocyanate were found in males (median 2,850 ng mL-1) than in females (median 1,760 ng mL-1) It is of note among both BD and DQ residents, male smokers had significantly greater thiocyanate levels than did male nonsmokers (all females were nonsmokers) (Fig 3) No significant associations were found between thiocyanate levels and location of sampling, dietary habits, or BMI, but an age-dependent increase in serum concentrations of thiocyanate was observed (Fig 2) Iodide Iodide was also found in all 131 serum samples analyzed in this study (median 3.11 ng mL-1) (Table 2) Significantly greater (p \ 0.05) concentrations of iodide were found in the serum of males (median 3.38 ng mL-1) and those who consumed less fish (median 3.27 ng mL-1) compared with those of females (median: 2.91 ng mL-1) and frequent fish eaters (median 2.49 ng mL-1) Other demographic characteristics, such as smoking, meat consumption, age, and 33 BMI, were not significantly associated with serum iodide levels PEC The PECs calculated from serum concentrations of perchlorate and thiocyanate in the Vietnamese populations were in the range of 0.365–23.1 lmol L-1 (median 2.28 lmol L-1) (Table 2), and thiocyanate levels accounted for [99 % of the PEC values Therefore, the associations between serum PECs and demographic characteristics of donors were identical with the trends observed for thiocyanate levels described previously, i.e., greater PECs were found in males and smokers than in females and nonsmokers Association of Serum TH Levels With Perchlorate, Thiocyanate, and Iodide Concentrations Concentrations of THs measured in Vietnamese sera are listed in Table Serum concentrations of FT3 and TT3 in BD residents (e-waste site) were significantly lower (p \ 0.05) than those in DQ residents (Table 3) To examine the relationship between TH levels and concentrations of perchlorate, thiocyanate, and iodide in sera, we used a stepwise multiple linear regression model Because serum concentrations of thiocyanate and iodide differed significantly between males and females, as described previously, subsequent analyses were separated by sex The multilinear regression analyses showed that iodide was a significant positive predictor of FT3 (p \ 0.01) and TT3 (p \ 0.01) and a significant negative predictor of TSH (p \ 0.01) in males (Table 4) In contrast, no significant associations between serum concentrations of THs and perchlorate, thiocyanate, or PEC were found for either males or females Discussion Perchlorate To our knowledge, this is the first study to determine serum levels of perchlorate in Vietnamese populations Interestingly, serum concentrations of perchlorate in BD residents were significantly greater than those in DQ residents (Table 2), and the measured concentrations were comparable with those reported for Albany and New York City, United States (Oldi and Kannan 2009); however, the levels in BD residents were relatively lower than those reported for New Jersey and Lansing, United States (Oldi and Kannan 2009; Blount et al 2009) (Fig 4) These results indicate the presence of perchlorate sources in the e-waste 123 34 Table Serum concentrations of perchlorate, thiocyanate, and iodide and PEC and selected demographic characteristics Arch Environ Contam Toxicol (2014) 67:29–41 Concentrations and characteristics Perchlorate (ng mL-1) Thiocyanate (ng mL-1) Iodide (ng mL-1) PEC (lmol L-1) Total (n = 131) Min \0.050a 317 1.26 0.365 1st Qu 0.076 1,320 2.40 1.49 Median 0.104 2,020 3.11 2.28 3rd Qu 0.153 3,200 4.15 3.62 Max 1.25 20,100 13.1 23.1 0.365 Location DQ (rural) (n = 48) Min \0.050 317 1.32 1st Qu 0.070 1,230 2.16 1.37 Median 3rd Qu 0.086 0.120 1,750 2,890 2.89 4.69 2.03 3.27 Max 0.660 20,100 13.1 23.1 0.54 BD (e-waste (n = 83) Min \0.050 466 1.26 1st Qu 0.085 1,410 2.61 1.58 Median 0.120 2,140 3.25 2.52 3rd Qu 0.160 3,700 3.91 4.32 1.25* 12,600 12.2 14.9 0.365 b Max Sex Female (n = 81) Min \0.050 317 1.26 1st Qu 0.076 1,190 2.23 1.37 Median 0.106 1,760 2.91 2.03 3rd Qu 0.156 2,610 3.84 3.00 Max 1.25 8,850 7.95 10.2 Male (n = 50) Min 0.050 772 1.46 0.889 1st Qu 0.078 1,710 2.82 1.97 Median 0.101 2,850 3.38 3.27 3rd Qu 0.139 5,790 4.32 6.66 Max 0.464 20,100**c 13.1*c 23.1**c 0.365 Habit of eating marine fish times/week (n = 119) Min \0.050 317 1.26 1st Qu 0.077 1,330 2.44 2.41 Median 0.104 2,060 3.27 2.51 3rd Qu 0.151 3,200 4.23 3.51 Max 1.25 20,100 13.1*d 23.1 1–3 times/week (n = 12) Min \0.050 1,030 1.44 1.21 1st Qu Median 0.070 0.120 1,290 1,640 2.06 2.49 1.34 1.77 3rd Qu 0.170 3,280 3.13 2.53 Max 0.278 10,800 3.52 11.3 Min \0.050 317 1.26 0.365 1st Qu 0.077 1,330 2.44 2.41 Habit of eating freshwater fish 1–3 times/week (n = 58) 123 Arch Environ Contam Toxicol (2014) 67:29–41 Table continued 35 Perchlorate (ng mL-1) Thiocyanate (ng mL-1) Iodide (ng mL-1) PEC (lmol L-1) Median 0.104 2,060 3.27 2.51 3rd Qu 0.151 3,200 4.23 3.51 Max 1.25 20,100 13.1*e 23.1 1.21 Concentrations and characteristics [4 times/week (n = 73) Min \0.050 1,030 1.44 1st Qu 0.070 1,290 2.06 1.34 Median 0.120 1,640 2.49 1.77 3rd Qu 0.170 3,280 3.13 2.53 Max 0.278 10,800 3.52 11.3 Habit of eating meat Qu Quartile, Max Maximum, Min Minimum a Lower than detection limit b Significantly greater than rural site c Significantly greater than females d Significantly greater than marine fish eater e Significantly greater than freshwater fish eater *p \ 0.05 **p \ 0.01 1–3 times/week (n = 24) Min 0.062 670 1.40 0.750 1st Qu 0.092 1,300 2.14 1.47 Median 0.126 1,990 3.22 2.25 3rd Qu 0.199 4,680 4.03 5.29 Max 0.464 8,930 10.1 10.1 \0.050 317 1.26 0.365 [6 times/week (n = 107) Min 1st Qu 0.075 1,330 2.44 1.51 Median 0.100 2,060 3.10 2.33 3rd Qu 0.147 3,050 4.15 3.45 Max 1.25 20,100 13.1 23.1 recycling site and the specific exposure of BD residents to perchlorate Given that perchlorate is used in PVC and lithium-ion batteries as a dopant material (Interstate Technology and Regulatory Council 2005), BD residents might be exposed to relatively high levels of this anion during e-waste recycling operations Based on the serum concentrations of perchlorate detected in this study, we estimated the perchlorate exposure dose for the Vietnamese populations, using the following equation, developed by Gibbs (2006), as follows: Log E = [log(C/99.5) -1.052]/0.9193, where E is the estimated exposure dose (mg/kg bw/d), C is the serum perchlorate level (lg L-1), and 99.5 is the molar mass of perchlorate The median exposure doses of perchlorate estimated for DQ and BD residents were 0.033 lg/kg/d and 0.048 lg/kg/d, respectively, and the values were order of magnitude lower than the USEPA RfD of 0.7 lg/kg/d (Zewdie et al 2010) However, the estimated exposure dose (0.81 lg/kg/d) for an e-waste recycling worker, who had the highest serum concentration of perchlorate, was comparable with the USEPA RfD, indicating that some individuals might be at risk from perchlorate exposure Further studies are needed to identify hot spots in the e-waste recycling site and exposure sources of perchlorate for BD residents in Vietnam Thiocyanate Greater serum concentrations of thiocyanate in males than in females were found (p \ 0.05) Especially at both the BD and DQ sites, male smokers had greater levels of this anion than did male nonsmokers and females (all females were nonsmokers) (Fig 3), suggesting that smoking is a major exposure source of thiocyanate In previous Norwegian (Foss and Lund-Larsen 1986) and Danish (Laurberg et al 2004) studies, greater serum concentrations of thiocyanate in smokers than in nonsmokers have been reported (Fig 5) In fact, it is known that thiocyanate is formed by way of the detoxification process of the hydrogen cyanide contained in cigarette smoke (Tuncel et al 1994) In this study, a significant positive correlation between serum thiocyanate levels and donor age was observed (Fig 2) However, this correlation no longer became significant after exclusion of data for smokers (p = 0.24), whereas serum thiocyanate levels in smokers were positively correlated with age (p \ 0.05) (data not shown) Thus, it is probable that the age-dependent increase in serum concentrations of thiocyanate observed in this study (Fig 2) is attributed to the smoking habit As for nonsmokers, serum concentrations of thiocyanate were comparable with the data reported from Norway (Foss 123 36 1.2 Perchlorate (ngmL -1) Fig Relationships between serum anion levels and age or BMI of Vietnamese subjects Arch Environ Contam Toxicol (2014) 67:29–41 1.0 p = 0.055 1.2 p = 0.24 r = 0.17 1.0 r = 0.08 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 0.0 0.0 10 Iodide (ng mL-1) 12 10 30 40 50 60 12 r = 0.09 10 8 6 4 2 20000 15000 20 30 40 50 60 20000 r = 0.23 15000 18 20 22 24 26 20 22 24 26 20 22 24 26 p = 0.99 r = 0.01 16 p = 0.008 18 p = 0.13 r = 0.13 10000 10000 5000 5000 0 10 20 30 40 Age (yrs) and Lund-Larsen 1986) and Denmark (Laurberg et al 2004) and were greater than those reported from the United States (Blount et al 2009) (Fig 5) Given that thiocyanate is also a metabolite of cyanogenic glucosides present in plant foods such as cabbage, broccoli, and mustard (VanEtten et al 1969, 1976; Foss and Lund-Larsen 1986; Bertelsen and Hegedus 1994), the differences in serum concentrations of this anion observed between Vietnamese and American nonsmokers may be related to the frequency of vegetable ingestion Iodide Iodide concentrations found in Vietnamese serum samples ranged from 1.26 to 13.1 ng mL-1 (median 3.11 ng mL-1) 123 16 p = 0.32 10 Thiocyanate (ng mL-1) 20 50 60 16 18 BMI (Table 2) Normal range of serum concentrations of iodine in healthy subjects were defined to range from 50 to 100 ng mL-1, and approximately % (2.5–5 ng mL-1) of this form in the serum are thought to be inorganic iodine (i.e., 95 % as organic iodine derivatives) (Wagner et al 1961; Fisher et al 1965; Nagataki et al 1967; Sternthal et al 1980) Iodide concentrations in 71.7 % of serum samples (n = 94) analyzed in this study were[2.5 ng mL-1, but the levels in 28.2 % samples (n = 37) were less than the reference value The World Health Organization (World Health Organization, United Nations International Children’s Emergency Fund, International Council for Control of Iodine Deficiency Disorders 2001) has reported that the optimal values for a population’s urinary iodine levels is in the range of 100 and 200 ng mL-1 In Vietnam, however, it was recently reported Arch Environ Contam Toxicol (2014) 67:29–41 Female Male Nonsmoker b 10000 a 5000 consumption of iodized salt, although no such information on salt intake is available for Vietnam Considering that serum iodide levels in the Vietnamese population were not associated with meat consumption and were greater in those who ate less fish (Table 2), the intake of iodideenriched food items, e.g., seaweed (Tokudome et al 2004) other than meat and fish may be less among Vietnamese females Male smoker b 15000 Concentrations (ng mL-1) 37 a a a PEC PECs calculated from serum concentrations of perchlorate and thiocyanate among Vietnamese subjects ranged from 0.365 to 23.1 lmol L-1, and thiocyanate accounted for [99 % of the PEC values PECs observed in 84 % of the subjects (n = 110) exceeded the IC50 (1.22 lmol L-1) values reported for inhibition of iodide uptake in an in vitro study using Chinese hamster ovary cells expressing human NIS (Tonacchera et al 2004) Recently, in the calculation of PECs, Bruce et al (2013) adopted a relative potency of 17.6 for thiocyanate When we recalculated the PECs using this value (17.6), those in 79 % of Vietnamese subjects (n = 103) were greater than the IC50 (1.22 lmol L-1) These results imply that the PEC in Vietnamese populations can inhibit iodide uptake into the TG An epidemiological survey suggested that serum PEC values 6.7 lmol L-1 were associated with adverse effects on TH homeostasis in humans under iodine-deficient conditions (Gibbs 2006) Such differences in the threshold values estimated between in vitro and in vivo studies are mainly Rural area E-waste site Fig Concentrations of thiocyanate in the serum from Vietnamese females (nonsmokers), male nonsmokers, and male smokers Different letters (a and b) on the top of each column represent the statistical differences by Steel–Dwass test (p \ 0.05) that urinary concentrations of iodine in school-age children were in the range of 20–49 ng mL-1 (de Benoist et al 2008; Zimmermann et al 2008) These observations indicate that Vietnamese people consume a relatively low amount of iodide In the present study, serum concentrations of iodide in females were significantly lower than those in males (Table 2) In fact, serum iodide levels in 35.8 % of females (n = 28) were \2.5 ng mL-1 It has been reported that females generally consume less (iodized) salt than males (Brown et al 2009), and hence the sex difference in iodide levels observed in this study may be attributed to the Table Concentrations of thyroid hormones in Vietnamese serum analyzed in this study Cohort TT3 (ng mL-1) TT4 (ng mL-1) FT3 (pg mL-1) FT4 (ng mL-1) TSH (lIU mL-1) Total (n = 131) Min 0.50 43 2.3 0.85 0.022 1st Qu 1.1 63 3.1 1.2 1.0 Median 1.3 74 3.3 1.3 1.5 3rd Qu 1.6 83 3.5 1.4 2.2 Max 4.8 130 11 1.9 8.2 46 2.7 0.93 0.040 Location Rural (n = 48) Min 0.93 1st Qu 1.2 69 3.2 1.2 0.99 Median 1.3 79 3.4 1.3 1.5 3rd Qu 1.4 87 3.5 1.4 2.2 Max 4.8 130 11 1.7 8.2 43 2.3 0.85 0.022 E-waste (n = 83) Qu Quartile, Max Maximum, Min Minimum a Significantly lower than rural site *p \ 0.05 **p \ 0.01 Min 0.50 1st Qu 1.1 61 3.0 1.2 1.0 Median 1.2 71 3.3 1.3 1.4 3rd Qu 1.3 81 3.5 1.4 2.1 Max 2.7**a 120 8.2*a 1.9 4.6 123 38 Arch Environ Contam Toxicol (2014) 67:29–41 Table Association coefficients (b) between serum concentrations of anions and THs in the Vietnamese populations by single and multiple linear regression models Perchloratec Unadjusted ba Thiocyanetec Adjusted bb Unadjusted b Iodidec Adjusted b Unadjusted b PECc Adjusted b Unadjusted b Adjusted b Female (n = 81) TT4 –4.9 TTc3 FT4 FTc3 c TSH 2.7 2.9 0.22 0.067 –0.02 0.087 0.036 0.062 0.047 –0.02 0.078 –0.18 0.042 –0.13 0.69 –0.0011 0.012 0.072 –0.0039 –0.011 Male (n = 50) TT4 –1.8 5.1 –4.6 TTc3 0.035 0.041 0.16* FT4 0.074 0.028 0.08 FTc3 0.011 c TSH –0.03 0.29 0.19** 0.0032 –0.0029 –0.01 0.14 0.16** –0.00086 –0.04 –0.51** –0.45** –0.0039 Blank cell Data omitted by stepwise procedure a Unadjusted coefficients were calculated by single regression analysis Coefficients were adjusted by age, BMI, pregnancy status (yes/no), living site (e-waste/reference), and habit of eating freshwater fish (1–3 times/week vs times/week), marine fish (0 times/week vs 1–3 times/week), and meat (1–3 times/week vs [4 times/week) b c Values of TT3, FT3, TSH, perchlorate, thiocyanate, iodide, and PEC were log-transformed * p \0.05 ** p \0.01 reported that nitrate accounted for [75 % of PEC values calculated from urinary perchlorate, thiocyanate, and nitrate levels Thus, the effect of exposure of Vietnamese populations to perchlorate, thiocyanate, and nitrate on TH homeostasis is a concern and should be the subject of further investigation Associations of Serum TH Levels With Perchlorate, Thiocyanate, and Iodide Concentrations Fig Comparison of serum concentrations of perchlorate in Vietnamese subjects analyzed in this study with previous United States data = Oldi and Kannan (2009); = Blount et al (2009) due to the differences in kinetics of perchlorate and thiocyanate Approximately 12 % of the Vietnamese subjects (n = 16) analyzed in this study had the PECs greater than the threshold value of 6.7 lmol L-1 reported by Gibbs (2006) Although nitrate was not analyzed in this study, it was reported in the National Health and Nutrition Examination Survey subjects (Suh et al 2013; Bruce et al 2013) that this anion might have affected thyroid NIS status Suh et al (2013) showed that urinary nitrate was a significantly negative predictor of serum free T4, and Bruce et al (2013) 123 Associations between serum levels of THs and perchlorate and related anions were assessed using a stepwise multiple linear regression model No significant associations between serum concentrations of THs and PECs were found for either males or females (Table 4) The multiple regression analyses for the concentrations of perchlorate and thiocyanate individually yielded similar results (p [ 0.05) These observations indicated that serum concentrations of perchlorate and thiocyanate in most of the Vietnamese donors are lower than the levels that would affect TH homeostasis As described earlier, PECs in 87.8 % of the Vietnamese serum samples (n = 115) were lower than the threshold value of 6.7 lmol L-1 (Gibbs 2006) In addition, an epidemiological study reported significant upregulation of TSH and downregulation of TT4 in the serum of a population exposed to thiocyanate (mean serum concentration of thiocyanate 13,000 ng mL-1) Arch Environ Contam Toxicol (2014) 67:29–41 39 Fig Comparison of serum concentrations of thiocyanate in Vietnamese subjects analyzed in this study with previous United States and European data Blount et al (2009); Laurberg et al (2004); and Foss and Lund-Larsen (1986) compared with a reference population (5,300 ng mL-1) (Banerjee et al 1997) Thiocyanate levels in 84.7 % (n = 111) of the Vietnamese serum samples analyzed in this study were \5300 ng mL-1, the mean value reported in the reference population by Banerjee et al (1997) However, in light of greater concentrations of perchlorate detected in serum from the population involved in e-waste recycling activities and the insufficient iodide consumption by Vietnamese females, detailed risk assessments for females inhabiting e-waste recycling sites, especially pregnant women and their neonates, are required In this study, significant positive correlations of FT3 (p \ 0.01) and TT3 (p \ 0.01), as well as a significantly negative correlation of TSH (p \ 0.01) with serum iodide levels, were found for males but not females with low iodide levels, indicating that the amount of iodide uptake was related to the synthesis of T3 However, no significant correlations were found between serum concentrations of iodide and T4 or FT4 levels (Table 4) Although the reason is unclear, it is possible that the above-mentioned phenomenon is attributed to the deiodination of T4 to T3 by deiodinases in tissues and subsequent secretion of iodide into the bloodstream (Greenspan & Forsham 1983) The above-mentioned analyses had limitations, such as sample sizes and potential covariates, i.e., donor characteristics, investigated, and hence more comprehensive surveys using larger sample sets and potential covariates are required to assess in detail the health effects of these anions In summary, we determined serum concentrations of perchlorate and thiocyanate and examined their associations with TH homeostasis in Vietnamese populations for the first time Interestingly, greater concentrations of perchlorate were found in serum from a population residing in an e-waste recycling site than in a reference rural sit, located in Northern Vietnam Although the serum concentrations of thiocyanate were not significantly different between the two populations, increased levels of this anion were observed for smokers Perchlorate and thiocyanate levels detected in the Vietnamese populations were not correlated with serum concentrations of THs, and most of the subjects had anion levels lower than the threshold values for toxic effects as reported previously in epidemiological studies However, given that perchlorate exposure doses estimated for e-waste recycling workers 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Sci Technol 44:6947–6953 Zimmermann MB, Jooste PL, Pandav CS (2008) Iodine-deficiency disorders Lancet 372:1251–1262 123 ... concentrations of perchlorate and thiocyanate individually yielded similar results (p [ 0.05) These observations indicated that serum concentrations of perchlorate and thiocyanate in most of the... Associations of Serum TH Levels With Perchlorate, Thiocyanate, and Iodide Concentrations Fig Comparison of serum concentrations of perchlorate in Vietnamese subjects analyzed in this study with. .. determining the serum concentrations of perchlorate and thiocyanate in residents at an e-waste recycling site and a rural reference site in Northern Vietnam We examined the relationship between serum