Plasma mercury levels in Hong Kong residents: In relation to fish consumption

Science of the Total Environment 463–464 (2013) 1225–1229

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Plasma mercury levels in Hong Kong residents: In relation to fish consumption Peng Liang a, Yan-Yan Qin b, Chan Zhang c, Jin Zhang a, Yucheng Cao a, Sheng-Chun Wu b, Chris K.C. Wong b, Ming H. Wong a, b,⁎ a b c

School of Environment and Resources Science, Zhejiang Agriculture and Forest University, Lin'an, Zhejiang Province, PR China Croucher Institute for Environmental Sciences and Department of Biology, Hong Kong Baptist University, Hong Kong, China College of Law and Political Science, Zhejiang Agriculture and Forestry University, Lin'an, Zhejiang Province, PR China

H I G H L I G H T S • • • •

Fish consumption rate was positively correlated with MeHg concentration in plasma. Gender and age were not major determining factors on Hg distribution in plasma. EDIFish and EDIBlood estimated similar results on MeHg exposure for Hong Kong residents. Hong Kong residents had lower EDI values than the standard of JECFA and USEPA.

a r t i c l e

i n f o

Article history: Received 11 May 2012 Received in revised form 12 March 2013 Accepted 14 April 2013 Available online 13 May 2013 Guest Editors: Ravi naidu, Ming Wong Keywords: Methylmercury Blood plasma Human exposure

a b s t r a c t Mercury exposure is of particular concern since mercury is a neurotoxin and the developing fetus is most sensitive to its adverse effect. Human blood is routinely used as an indicator for the evaluation of human exposure to Hg. To investigate Hg species in human plasma for Hong Kong residents and the relationship between fish consumption and Hg species in plasma, 151 plasma samples were analyzed for Hg species. The mean values of total Hg (THg) and methyl-mercury (MeHg) concentration in plasma were 0.62 and 0.28 μg/L, respectively. No significant differences were observed between females and males as well as among age groups. Fish consumption rate was significantly positively correlated with MeHg concentrations in plasma, which demonstrated that plasma could be a biomarker for human MeHg exposure. Two methods were used to estimate human MeHg exposure. One was based on fish MeHg content and fish consumption rate (EDIFish), another was employed by converting MeHg concentration in blood to MeHg exposure amount (EDIBlood). A significant positive correlation was observed between EDIBlood and EDIFish, and no significant difference was found between EDIBlood and EDIFish. These results demonstrated that fish consumption was the major source of MeHg for humans. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Human exposure to Hg compounds has been of great public concern around the world due to mercury's high toxicity. Human blood is routinely used as an indicator for the evaluation of human exposure to many pollutants, including Hg (Fukata et al., 2005; Mahaffey et al., 2009). Human exposure to Hg has been linked to several human health diseases, e.g., neurological problems, and myocardial infarction (Li et al., 2008). Methylmercury (MeHg), divalent mercury (Hg 2+) and elementary mercury (Hg0), the major Hg species in human blood, are known to have different exposure pathways and metabolism processes in the human body. IHg (Hg2+ and Hg0) in human blood is mainly derived from dental amalgams, skin creams, medicinal intakes and ⁎ Corresponding author at: Croucher Institute for Environmental Sciences and Department of Biology, Hong Kong Baptist University, Hong Kong, China. Tel.: +852 34117746; fax: +852 3411 7743. E-mail address: [email protected] (M.H. Wong). 0048-9697/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scitotenv.2013.04.049

occupational exposures (Mahaffey et al., 2004). Seafood consumption is the predominant pathway for MeHg exposure in human blood, which is estimated to constitute up to 95% of the total dietary Hg intake for most of the population (USEPA, 2001). When IHg enters the blood, the plasma is considered as the target fluid for its accumulation (WHO/IPCS, 1991), however, information is lacking on MeHg in plasma and its correlation with the major MeHg exposure pathways, e.g., fish consumption. It is known that Hong Kong residents consume a large amount of fish. Dickman and Leung (1998) revealed that an average person in Hong Kong consumed about 175 g of fish per week. A previous study illustrated that in Hong Kong, the THg concentrations in 78.4% of cord blood samples exceeded the reference dose of 5.8 μg/L, which was attributable to the increase of fish consumption in pregnant mothers (Fok et al., 2007). So far, all previous studies on Hg exposure to Hong Kong residents were only based on the determination of THg concentrations in whole blood (Fok et al., 2007; Ip et al., 2004), in which fish consumption was

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the major source of Hg exposure (Dickman and Leung, 1998; FEHD, 2002). USEPA adopted the one-compartment MeHg biokinetic model to set the reference dose (RfD) value from the perspective of MeHg metabolism (USEPA, 2001). However, previous studies assumed that all of the Hg in fish would be absorbed by humans when estimating human MeHg exposure (Dickman and Leung, 1998; FEHD, 2002). This model was challenged for the overestimation of Hg exposures (Sirot et al., 2008), in other words, the reliability of this commonly used method was questionable. To address this, the objectives of this study were to find out: (1) the distribution of Hg species in human blood of Hong Kong residents; (2) the influence of fish consumption on Hg accumulation in human blood plasma; and (3) if the dietary exposure assessment method overestimates human MeHg exposure compared to MeHg biokinetic models. 2. Materials and methods 2.1. Human plasma samples All participants were eligible for blood donation upon health history inquiry and screening before recruitment in this study. All blood samples were collected during blood donation by the nurses at the Hong Kong Red Cross Blood Transfusion Service. Whole blood (450 mL) was collected from each participant and plasma was separated out for measurement of Hg. The plasma samples were put in plasma bags (JMS, Singapore PTE LTD) and transported to the laboratory and stored at −20 °C until further analysis. Two batches of human plasma samples were collected in this study. The first batch contained 60 samples with only the information of the donors' gender and age, therefore, these samples were analyzed as a preliminary dataset. On the other hand, there were 91 samples in the second batch with information on the donors' age, gender, weight, height and the amount of seafood intake per week. This information was obtained by short questionnaire. The second batch of human samples was adopted to examine the correlation between fish consumption and Hg accumulation in human plasma.

(GC–CVAFS detection) (Books Rand MERX), following USEPA method 1630 (USEPA, 2001). The IHg concentration in plasma was calculated by subtracting the MeHg level from the THg level. 2.3. QA/QC Two method blanks, four certified reference materials (CRMs) and 10% replicate samples accompanied each sample batch (up to 40 samples). The relative percentage of sample duplicate for THg and MeHg ranged from 0.99% to 13.6% and from 0.12% to 16.3%, respectively. TORT-2 (lobster hepatopancreas references materials) was used as CRMs as it was a biological reference material. The mean recovery of CRMs for THg and MeHg is 104% and 105%, respectively. 2.4. Estimation of Hg species in whole blood and erythrocytes It is known that Hg exists in both plasma and erythrocytes and the distributions of MeHg and IHg differ in both of these. The WHO (1990) stated that the erythrocyte/plasma ratio for MeHg (e/pMeHg) was about 20, but on the contrary the concentration of IHg in erythrocytes was almost equal to the IHg concentration in plasma (USEPA, 1997). In this study, the MeHg concentration in erythrocytes was calculated as the MeHg concentration in plasma multiplied by the e/pMeHg, while, the IHg concentration in erythrocytes involved the IHg in plasma being multiplied by e/pIHg. Furthermore, the erythrocyte THg concentration was established as the sum of MeHg and IHg concentrations. Moreover, the Hg species in whole blood was calculated with the following equation (Sirot et al., 2008): MeHgðIHgÞwhole blood ¼ MeHgðIHgÞerythrocytes  R þ MeHgðIHgÞplasma  ð1−RÞ ð1Þ where R is cells per 1 mL of whole blood. THg concentration in whole blood was also calculated as the sum of MeHg and IHg concentration in whole blood. 2.5. Human MeHg exposure estimate

2.2. Analysis of Hg THg concentration in human plasma was detected by a direct Hg analyzer (Milestone, DMA-80) based on thermal decomposition, amalgamation, and atomic absorption spectrophotometry, following the USEPA method 7473 (USEPA, 2007). MeHg concentration in human plasma was determined according to Liang et al. (2000), where a total of 0.3 mL plasma was added into a 50 mL glass centrifuge tube, placed in an oven and covered with a piece of paper. The samples were heated at 70 °C for about 5 h and allowed to cool to room temperature (25 ± 2 °C), followed by the addition of potassium hydroxide (KOH)/methanol solution (25%, 2 mL). The samples were heated for a further 3 h in an oven at 75 °C and left to cool to room temperature (25 ± 2 °C), and then 8 mL methylene chloride (CH2Cl2) was added and mixed thoroughly by swirling the tube. Two milliliters of hydrochloric acid (HCl) (12 N) was added slowly and the tube was swirled again and the sample was passed through the phase separating filter paper (Whatman 2200-090), where only the organic layer was directly collected into a pre-weighed centrifuge tube. Approximately 35 mL of Milli-Q water was added to the tube along with a bamboo skewer to prevent the CH2Cl2 from “bumping” during the back extraction. The tube was heated in a water bath at 45 °C until no visible solvent was left in order for solvent evaporation to occur. The temperature of the water bath was increased to 75 °C, and the sample was purged with nitrogen for 8 min to remove solvent residue and its volume was made up to 40 mL with double distilled water. MeHg was determined using aqueous ethylation, purge, trap, and gas chromatography–cold vapor-atomic fluorescence spectrometry

Two methods were used to estimate human MeHg exposure. The first was a direct estimation method based on fish MeHg content and fish consumption rate, where the estimated daily intake of MeHg through fish consumption (EDIFish) was calculated according to the following equation (Sirot et al., 2008): EDIFish ¼ C  M=BW

ð2Þ

where C M BW

is the MeHg concentration in fish sample (μg/kg); is the amount of daily fish intake (kg); and is the human body weight (kg).

Another method developed by USEPA (2001) was employed for estimating MeHg intake by converting MeHg concentration in human blood to the amount of MeHg exposure. This method used a onecompartment MeHg biokinetic model, based on the characteristics of MeHg metabolism: EDIBlood ¼ c  v  b=A  f  bw where c b v A

is the MeHg concentration in whole blood (μg/kg); is the elimination constant as 0.014 days −1; is the volume of blood in the body (mL); is the absorption factor as 0.95;

ð3Þ

P. Liang et al. / Science of the Total Environment 463–464 (2013) 1225–1229

f bw

is the fraction of absorbed dose taken up by blood as 0.059; and is the body weight (kg).

Among all the parameters, c was the whole blood MeHg concentration that was used for the calculations in this study; v and bw were obtained from questionnaire surveys, while the other parameters used in this equation were cited from USEPA (2001). 2.6. Data analyses Difference of Hg concentrations in human plasma was performed by one-way ANOVA (LSD test) using SPSS 16.0 for Windows. 3. Results and discussions 3.1. Human plasma Hg level in Hong Kong residents THg concentrations in human plasma ranged from 0.13 to 2.08 μg/L, with a mean value of 0.63 μg/L. As far as Hg species were concerned, MeHg concentrations in human plasma ranged from 0.05 to 1.56 μg/L with a mean value of 0.28 μg/L, contributing to 45.3 ± 15.0% of THg. In comparison, the concentrations of IHg in human plasma ranged from 0.04 to 1.40 μg/L with a mean value of 0.34 μg/L, contributing to 54.7 ± 15.0% of THg. 3.2. Difference in gender and age 3.2.1. Gender difference in human plasma Hg level Fig. 1 shows the THg, MeHg and IHg concentrations in human plasma for male and female groups. The concentrations of THg, MeHg and IHg in the plasma of females were higher than that of the males, but the differences were not significant (p > 0.05). These results indicate that gender may not be a major determining factor on Hg distribution in human plasma.

on Hg in whole blood (Kosatsky et al., 2000; Mahaffey and Mergler, 1998), which might be due to the influence of fish consumption rate. Our questionnaire survey showed a lower frequency of seafood intake in younger donors (10–19 years old, 3.67 times per week) than in the older donors (40–49 years old, 4.76 times per week), and thus higher Hg concentrations in plasma were found in the older cohort. 3.3. Fish consumption on Hg species in plasma Significant positive correlations (p b 0.01) were observed between the fish consumption rate and the THg (r = 0.29, p b 0.01) and MeHg concentrations (r = 0.305, p b 0.01) in human plasma, but not with IHg concentration (r = 0.13, p > 0.05). These results indicate that the higher the rate of fish consumption, the larger is the amount of MeHg accumulation in human plasma. The significant correlations between fish consumption rate and MeHg or THg concentrations in whole blood and erythrocytes have been found in previous studies (Cole et al., 2004; Mahaffey et al., 2009). However, it has been reported that MeHg intake via fish consumption is mainly distributed in erythrocytes, but not in plasma (WHO/IPCS, 1990). Hence, erythrocytes are recognized as a biomarker of human MeHg exposure via fish consumption (Sakamoto et al., 2010), while plasma is recognized as a biomarker of human IHg exposure via inhalation (WHO/IPCS, 1991). However, our results indicate that the MeHg concentrations in plasma also positively correlated with the fish consumption rate. The highest plasma MeHg concentration (1.55 μg/L) was found in a 45 year old female with a fish consumption rate of 18 times per week. On the other hand, two individuals reported that they did not eat any fish or fish products, but MeHg was still found in their plasma (0.21 and 0.25 μg/L). As a result, it is suspected that there may be other sources contributing to human MeHg exposure. 3.4. Estimation of Hg species in erythrocytes and whole blood The estimated values for THg, MeHg and IHg in erythrocytes and whole blood are shown in Table 1. THg concentrations in erythrocytes ranged from 1.06 to 31.3 μg/L, while MeHg concentrations in human erythrocytes ranged from 0.94 to 31.1 μg/L. Moreover, THg concentrations in whole blood ranged from 0.56 to 15.0 μg/L. The lowest MeHg concentration in whole blood was 0.45 μg/L, and the highest was 14.9 μg/L. The German Commission on Human Biological Monitoring (HBM) of the German Federal Environment Agency established the reference value for Hg in human blood, which was derived from human toxicology and epidemiology studies (Ewers et al., 1999). Two reference

2.5

2.5

2.0

2.0

Hg con centration (ug/L)

Hg con centration (ug/L)

3.2.2. Human plasma Hg level in different age groups Five and four age groups were adopted for the first and second batches respectively as follows: 10–19 (G1); 20–29 (G2); 30–39 (G3); 40–49 (G4); and 50–59 (G5) years old. Fig. 2 shows the differences of THg, MeHg and IHg concentrations among different age groups. No significant difference was observed among all groups for Hg species. However, in the second batch, THg and MeHg were generally lower in G1 than the other four groups, especially G4, although no significant differences were observed. These results were similar to the previous studies

1.5 1.0 .5

1227

1.5 1.0 .5 0.0

0.0 F-THg M-THg F-MeHg M-MeHg F-IHg M-IHg

F-THg M-THg F-MeHg M-MeHg F-IHg M-IHg

The first batch (n = 60)

The second batch (n = 91)

Fig. 1. THg, MeHg and IHg concentrations in human plasma of the male and female groups. The upper and lower boards of the box represent the 75th and 25th value, respectively, the upper and lower whiskers represent the 90th and 10th values, respectively, and the line in the box represents the median value. The data for the outliers are the plots outside of the boxes; F indicates female and M male.

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1.2 THg MeHg IHg

1.0 .8 .6 .4 .2

Hg concentrations (ug/L)

Hg concentrations (ug/L)

1.2

0.0

THg MeHg IHg

1.0 .8 .6 .4 .2 0.0

10-20

20-30

30-40

40-50

10-20

The first batch (n = 60)

20-30

30-40

40-50

50-60

The second batch (n = 91)

Fig. 2. THg, MeHg and IHg concentrations in the plasma samples of different age groups.

values were established as HBM I and HBM II: where the former had the Hg concentration in human blood set at 5 μg/L, which gave an implication of the likelihood of no adverse health effects being presented when below this value; whereas the Hg concentration for the latter was set at 15 μg/L, which indicated an increased risk of adverse health effects when above this value. Another reference dose for fetal blood Hg concentration was set at 5.8 μg/L, which was established by the USEPA based on Hg concentration in cord blood (USEPA, 2001). However, a higher Hg concentration in cord blood than in maternal blood was found due to the bioconcentration of Hg across the placenta with a geometric mean value of the ratio of 1.79 (n = 1057) (Fok et al., 2007). As a result, a lower reference dose of 3.24 (5.8/1.79) μg/L should be considered for the maternal blood Hg concentration. In the cohort of this study, 20 sample donors (13.2% of the surveyed population) had THg concentration in whole blood higher than the HBM I reference value. THg concentrations in the whole blood of 12 female donors exceeded 3.24 μg/L, and 7 of them were younger than 40 years of age. Nevertheless, the human blood Hg concentration for Hong Kong residents was believed to lie in a relatively safe range. It should be noted that THg concentration in whole blood was estimated in this study and that errors may exist within these estimations. 3.5. Comparison of Hg species in different studies Table 2 compares Hg concentrations in human blood obtained in the present study with those of other studies. The plasma Hg concentrations of Hong Kong residents were comparable to those of Germany's population (Halbach et al., 1998), but lower than those of Spain (Mendiola et al., 2011) and Turkey (Tezel et al., 2001). Tezel et al. (2001) showed that the mean THg concentration reached 50 μg/L in the plasma of dental students who worked with amalgam in Turkey. In addition, THg concentration in the plasma of Hg mine workers in China reached 39.8 μg/L (Li et al., 2008), therefore, it can be deduced that occupational exposure, such as dentists using amalgam, may increase THg concentration in plasma substantially. The estimated Hg concentrations in whole blood for Hong Kong residents were comparable with those of the residents in Greece (Vardavas et Table 1 The estimated value for THg, MeHg and IHg concentrations as well as %MeHg and %IHg in whole blood and erythrocytes.

Erythrocyte

Whole blood

THg MeHg IHg THg MeHg IHg

Min

5th

50th

95th

Max

SD

1.06 0.94 0.04 0.56 0.45 0.04

1.73 1.45 0.11 0.93 0.69 0.11

5.05 4.64 0.29 2.60 2.22 0.29

13.4 13.1 0.72 6.57 6.27 0.72

31.3 31.1 1.40 15.0 14.86 1.40

4.05 4.00 0.20 1.97 1.91 0.20

al., 2009), Canada (Kosatsky et al., 2000) and Korea (Kim and Lee, 2011), but lower than those in Iran (Farzin et al., 2008), Cambodia (Agusa et al., 2007) and Brazil (Dolbec et al., 2000). The MeHg concentration in the whole blood of Hong Kong residents was higher than that of American (Mahaffey et al., 2004) and Swedish women (Ask et al., 2002). Mahaffey et al. (2004) showed that the mean fish consumption rate in pregnant women in the USA was only 56 g per month compared with 2012 g per month for pregnant women in Hong Kong (Fok et al., 2007). It seems evident that the higher fish consumption rate may be a major reason as to why pregnant women in Hong Kong displayed higher MeHg concentration in their blood when compared with their US counterparts. 3.6. Human MeHg exposure According to the mariculture fish Hg concentration (Liang et al., 2011), the fish consumption rate and body weight information from the questionnaire survey, the estimated EDIFish for the cohort of the present study ranged from 0.07 to 2.16 μg/kg/week, with a mean value of 0.47 μg/kg/week (n = 89). This value amounted to 31.3 and 15.2% of the PTWI for women of childbearing age (1.6 μg/kg/week) and the general population (3.3 μg/kg/week), respectively (JECFA, 2003). The correlation between the fish consumption rate and the EDIFish was established based on our data: y ¼ 0:1015x þ 0:206

where y x

is EDIFish; and is the fish consumption rate.

Following this equation, the dietary exposure of people who consume seafood 5 times or more per week will most likely exceed the RfD of 0.7 μg/kg/week (USEPA, 2001). However, according to the PTWI of 1.6 μg/kg/week (JECFA, 2003), individuals who have fish 14 times or more per week will be at greater risk. It was estimated that a mean of 0.39 ± 0.30 μg/kg of MeHg was ingested per week by the cohort according to the MeHg metabolism in human blood. A significant positive correlation was observed between EDIBlood and EDIFish (r = 0.355, p b 0.001), but no significant difference was found between EDIBlood and EDIFish. These results demonstrate that fish consumption may be the major source of MeHg in humans, and the two estimation methods (dietary consumption and MeHg biokinetic models) produced similar results in estimating the MeHg exposure of Hong Kong residents.

P. Liang et al. / Science of the Total Environment 463–464 (2013) 1225–1229

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Table 2 Comparison with other studies for Hg concentration in human blood. Country

Population

Blood type

Hg species

50th

5th

95th

Sample size

Reference

Hong Kong Germany Spain Turkey China

General population General population General population Dental students Occupational workers General population General population Pregnant women Sport-fish eaters (≥1 meal/week) Sport-fish eaters (b1 meal/week) General population General population Pregnant women General population Riverine population General population Women Pregnant women (gestational week 36)

Plasma Plasma Plasma Plasma Serum

THg THg THg THg THg

0.21 0.12

1.23 0.32 10.6

THg THg THg

0.93

6.57

Whole blood Whole blood Whole blood Whole blood Whole blood Whole blood Whole blood Whole blood

THg THg THg THg THg MeHg MeHg MeHg

3.94

4.36

0.69 None 0.19

6.27 6.7 2.1

151 29 61 17 37 35 151 1057 60 72 101 1997 50 20 67 151 1709 148

This study Halbach et al. (1998) Mendiola et al. (2011) Tezel et al. (2001) Chen et al. (2006)

Whole blood Whole blood Whole blood

0.63 0.23 5.8 46.5 38.5 0.91 2.60 4.92 3.00 1.40 8.48 4.15 1.5 9.3 27.0 2.21 0.6 0.73

Hong Kong Hong Kong Canada Iran Korea Greece Cambodia Brazil Hong Kong USA Sweden

4. Conclusions This study investigated the distribution of Hg species in human plasma, where no significant differences were observed between the male and female groups as well as among all age groups. The fish consumption rate significantly and positively correlated with THg and MeHg concentrations in human plasma, which demonstrated that human plasma can serve as a biomarker for MeHg intake via fish consumption. The mean values of EDIFish and EDIBlood were 0.47 and 0.39 μg/kg/week, respectively. Both of them were lower than the PTWI of 1.6 μg/kg/week established by JECFA and the RfD of 0.7 μg/kg/week established by USEPA.

Acknowledgments The authors thank Ms. Sue Fung for improving the manuscript. Financial support from the Natural Science Foundation of Zhejiang Province (No. LQ12D03001), the Natural Science Foundation of Zhejiang A&F University (2011FK003), the Collaborative Research Fund (CRF) (HKBU 1/07C) of the Hong Kong Research Grants Council is gratefully acknowledged.

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