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Environmental Pollution 157 (2009) 396–403

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Environmental Pollution

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Relationship of urinary arsenic metabolites to intake estimates in residentsof the Red River Delta, Vietnam

Tetsuro Agusa a,b, Takashi Kunito c, Tu Binh Minh a,d, Pham Thi Kim Trang e, Hisato Iwata a, PhamHung Viet e, Shinsuke Tanabe a,*

a Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japanb Department of Legal Medicine, Shimane University Faculty of Medicine, Enya 89-1, Izumo 693-8501, Japanc Department of Environmental Sciences, Faculty of Science, Shinshu University, 3-1-1 Asahi, Matsumoto 390-8621, Japand Department of Biology and Chemistry (BCH), City University of Hong Kong, 83 Tat Chee Avenue, Kowloon Tong, Hong Kong, Chinae Center for Environmental Technology and Sustainable Development (CETASD), Hanoi National University, 334 Nguyen Trai Street, Thanh Xuan, Hanoi, Viet Nam

Positive correlations between estimated arsenic intake and urinary ino

rganic arsenic and its metabolites were observed in human from the RedRiver Delta, Vietnam.

a r t i c l e i n f o

Article history:Received 15 May 2008Received in revised form24 September 2008Accepted 25 September 2008

Keywords:ArsenicGroundwaterRiceHuman urineMethylation capacityRed River DeltaVietnam

* Corresponding author. Tel./fax: þ81 89 927 8171.E-mail address: shinsuke@agr.ehime-u.ac.jp (S. Ta

0269-7491/$ – see front matter � 2008 Elsevier Ltd.doi:10.1016/j.envpol.2008.09.043

a b s t r a c t

This study investigated the status of arsenic (As) exposure from groundwater and rice, and its methyl-ation capacity in residents from the Red River Delta, Vietnam. Arsenic levels in groundwater ranged from<1.8 to 486 mg/L. Remarkably, 86% of groundwater samples exceeded WHO drinking water guideline of10 mg/L. Also, estimated inorganic As intake from groundwater and rice were over Provisional TolerableWeekly Intake (15 mg/week/kg body wt.) by FAO/WHO for 92% of the residents examined. Inorganic Asand its metabolite (monomethylarsonic acid and dimethylarsinic acid) concentrations in human urinewere positively correlated with estimated inorganic As intake. These results suggest that residents inthese areas are exposed to As through consumption of groundwater and rice, and potential health risk ofAs is of great concern for these people. Urinary concentration ratios of dimethylarsinic acid to mono-methylarsonic acid in children were higher than those in adults, especially among men, indicatinggreater As methylation capacity in children.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Inorganic arsenic (As) is known to be a carcinogenic element(Goering et al., 1999), and also one of the hazardous watercontaminants (Nordstrom, 2002; Smedley and Kinniburgh, 2002).Especially, contamination in Bangladesh and West Bengal, India ismost serious in the world because about 36 million people havesignificant health risks by As exposure through consumption ofgroundwater in these areas (Nordstrom, 2002; Smedley and Kin-niburgh, 2002). Previous epidemiological studies conducted in As-contaminated areas such as Bangladesh, West Bengal in India, andTaiwan revealed a dose–response relationship between Asconcentrations in groundwater and occurence of cancers ofbladder, kidney, skin, liver, and lung and their related mortality(Tondel et al., 1999; Wu et al., 1989). Also, other As-related healtheffects such as melanosis, depigmentation, keratosis, and

nabe).

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hyperkeratosis were commonly observed in As-affected people(Das et al., 1995).

In contrast to these areas, little information is available on Aspollution in groundwater, human exposure and its epidemiology inSoutheast Asia. Berg et al. (2001) reported As pollution ingroundwater from Hanoi for the first time, and the level was up to3050 mg/L. Their findings suggest a possibility that human healtheffects by As exposure are of great concern in the Red River Deltabecause more than 11 million people in this region are usinggroundwater (Berg et al., 2001). Furthermore, As contamination ingroundwater from the Mekong River Delta, South Vietnam wasreported recently (Agusa et al., 2004, 2005, 2006, 2007; Iwata et al.,2007; Minh et al., 2005; Shinkai et al., 2007; Trang et al., 2005).However, there is little information on human exposure to As inthese areas. We have conducted monitoring of As contamination inwater and residents in Vietnam since 2001 (Agusa et al., 2004,2005, 2006, 2007; Iwata et al., 2007), but the influence of age andsex on the exposure status and methylation capacity of As are stillunclear in these residents. Thus, in the present study, our previous

T. Agusa et al. / Environmental Pollution 157 (2009) 396–403 397

data on groundwater and urine (Agusa et al., 2005) are reanalyzedto obtain detailed information on the influence of age and sex.Furthermore, As concentration in rice, the staple food of Viet-namese, was measured, and As intake from water and rice wereestimated to assess the human health risk by oral As exposure.

2. Materials and methods

2.1. Samples

Sampling of groundwater, rice, and human urine was conducted in Vinh Tru (VT)Commune, Ha Nam Province and in Cat Que (CQ) Commune, Ha Tay Province in theRed River Delta in December 2004. The data of groundwater and human urine werealready published in Agusa et al. (2005), and As in the rice was analyzed in thepresent study.

Groundwater samples were collected from 15 tube wells in VT and from 13 tubewells in CQ. Both locations were about 60 and 30 km far from Hanoi, respectively,and there is no other significant As pollution source such as industrial and miningactivities in these areas. In the five houses from VT, we collected groundwater fromtwo wells in each house; one was currently used and the other was not used at thattime. Well depth and period after well establishment were 38–50 m and 2–11.5years, respectively in CQ and 6.5–70 m and 1–20 years, respectively in VT. Asreference sample, pond water was also collected in CQ (n¼ 2) and VT (n¼ 1). Whiterice was collected from each home in VT (n¼ 10) to estimate As intake from theconsumption.

After informed consent was obtained from all the donors, human urine samples(n¼ 97) were correspondingly collected from the residents of each house equippedwith the tube well in an ethical manner (Table 1). Detailed information (age, bodyheight and weight, body mass index (BMI), occupation, residential years, anddrinking and smoking habits) about donors was obtained using a questionnaire. Inthis study, people aged >15 years and �15 years are considered as ‘‘adults’’ and‘‘children’’, respectively. Mean age, body height and weight, BMI, and residentialyears are shown in Table 1. About 41% of all subjects were local farmers (n¼ 40), and32% were students (n¼ 31). Other occupations included medical doctor (n¼ 4),nurse (n¼ 2), teacher (n¼ 3), manual worker (n¼ 5), retired worker (n¼ 5), baby-sitter (n¼ 1), public office worker (n¼ 1), and salesclerk (n¼ 1). All children (n¼ 21)were students. 16% and 30% of the donors have smoking and alcohol habits,respectively.

After collection, all samples were kept at �15 �C in the freezer at the Center forEnvironmental Technology and Sustainable Development (CETASD), Hanoi NationalUniversity. After freezing at the CETASD for 2–5 days, they were transported to Japanwithin 24 h and were kept at�25 �C in the Environmental Specimen Bank for globalmonitoring (es-BANK) at the Center for Marine Environmental Studies (CMES),Ehime University (Tanabe, 2006). We conducted chemical analyses within threemonths after the sample collection.

2.2. Analytical method

The groundwater sample acidified with nitric acid was used for total As analysis.The measurement of total As was performed with an inductively coupled plasmamass spectrometer (ICP-MS) (HP-4500, Hewlett–Packard, Avondale, PA, USA). Theurine sample from subject was filtered (DISMIC-25AS-045AN, 0.45 mm, ADVANTEC,Tokyo, Japan) and diluted five times by Milli-Q water with no chemical treatment.The rice was freeze-dried and then homogenized. As followed by Pizarro et al.

Table 1Details of donors from Cat Que Commune, Ha Tay Province and Vinh Tru Commune,Ha Nam Province in Vietnam.

Location n Ageb

(y)Residentialperiodb (y)

Heightb

(cm)Weightb

(kg)BMIb, c

Cat QueAdult

Female 22 35 (16–58) 34 (3–58) 154 (143–166) 46 (37–65) 19 (16–24)Male 24 31 (16–50) 30 (2–50) 163 (153–173) 54 (44–66) 20 (17–24)

Childa

Female 10 13 (10–15) 13 (10–15) 144 (130–160) 34 (23–44) 16 (13–17)

Vinh TruAdult

Female 18 35 (16–63) 33 (16–63) 153 (141–163) 43 (35–50) 18 (16–22)Male 12 39 (18–60) 31 (10–58) 166 (148–179) 54 (45–62) 20 (17–23)

Childa

Female 4 15 (14–15) 15 (14–15) 152 (142–168) 38 (31–48) 16 (14–18)Male 7 11 (10–13) 11 (10–13) 132 (126–142) 26 (20–34) 15 (13–20)

a Resident aged <15 years old was considered as child.b Mean and range.c Body mass index (BMI). BMI is calculated as body weight (kg)/body height (m)2.

(2003), arsenicals in powdered rice were extracted by Milli-Q water and methanol(1:1 v/v). Concentrations of arsenobetaine (AB), dimethylarsinic acid (DMA), mon-omethylarsonic acid (MMA), arsenite (AsIII), and arsenate (AsV) in urine and ricewere quantified by a high performance liquid chromatograph (LC10A Series, Shi-madzu, Kyoto, Japan) coupled with ICP-MS using an anion exchange column (ShodexAsahipak ES-502N 7C, 100� 7.6 mm i.d., Showa Denko, Tokyo, Japan) (Mandal et al.,2001). The column was equilibrated with the mobile phase (15 mM citric acid, pH2.0 with nitric acid) at a flow rate of 1.0 mL/min at 30 �C before analysis. Rubidiumwas added to the mobile phase as an internal standard to monitor interferenceduring analysis. The injection volume was 20 mL. Total As concentrations in urineand rice were represented as the sum of As compounds detected in these proce-dures. Concentrations of total As in groundwater and As compounds in human urinehave been reported in our previous studies (Agusa et al., 2005). Iron concentration ingroundwater was determined by atomic absorption spectrometry (AAS) (ShimadzuAA680, Shimadzu, Kyoto, Japan).

A certified reference sample NIES No. 18 human urine provided by the NationalInstitute for Environmental Studies (NIES), Japan was analyzed for confirmation ofthe methodological accuracy. Analyzed concentrations of AB and DMA were in goodagreement with the certified values with the recoveries being 90–106% (n¼ 12).Coefficient of variance in concentrations for each arsenical was less than 2% (n¼ 12).For quality assurance and control, we have also participated in an intercalibrationexercise organized by the Swiss Federal Institute of Aquatic Science and Technology(Eawag) in the frame of the ongoing cooperation of Vietnam and Switzerland in As-related surveys and research.

Urinary creatinine level was measured by enzymatic assay consisting ofconsecutive enzymatic steps via creatine amidinohydrolase, sarcosine oxidase andperoxidase at SRL Inc. (Tokyo, Japan). Concentrations of As compounds in urine areexpressed on a creatinine basis.

2.3. Statistical analyses

All statistical analyses were performed with Stat View (version 5.0, SAS� Insti-tute, Cary, NC, USA) and SPSS (version 12.0 for Windows, SPSS Japan, Tokyo, Japan).One-half of concentration of the respective limit of detection was substituted forthose values below the limit of detection and used in statistical analysis. All datawere tested for goodness of fit to a normal distribution with Kolmogorov–Smirnov’sone sample test. Concentrations of urinary As compounds were not normallydistributed, so data were log-transformed. Multivariate analysis of variance (MAN-OVA) was used to test the effect of age, sex, and estimated As intake on the levels andcompositions of urinary As species. Result of MANOVA was evaluated by Wilks’Lambda. Effect of sex, age, residential year, occupation, and alcohol and smokinghabits on As methylation were examined using multi-way analysis of variance(multi-way ANOVA). Tukey–Kramer post hoc test was conducted when results ofmulti-way ANOVA were significant. A linear regression analysis was used to measurethe strength of the association between estimated As intake and concentrations ofurinary As compounds in human. A p value of less than 0.05 was considered toindicate statistical significance.

3. Results and discussion

3.1. Arsenic concentrations in groundwater

Arsenic was detected in all the groundwater samples and theconcentration ranged from 1.8 to 486 mg/L (Table 2). Concentrationsof As in groundwater from CQ (mean, 209 mg/L) were higher thanthose from VT (mean, 163 mg/L), but the regional difference was notsignificant (p> 0.05). About 86% of the analyzed groundwatersamples had over the guideline value (10 mg/L) established by WHO(WHO, 2004) for drinking, suggesting the possible risk of As toxicityfor residents in these regions. On the contrary, concentrations of Asin pond water (1.5–7.2 mg/L) were quite low compared with those ingroundwater.

Among the studies on As in groundwater from north and southVietnam (Table 3), maximum concentration was 3050 mg/L fromGia Lam in the Red River Delta (Berg et al., 2001). Concentrations ofAs in groundwater from CQ and VT were much lower than thosefound by Berg et al. (2001), but were relatively high compared withthose in other studies in Vietnam (Table 3).

Concentrations of As in groundwater from wells for which thedepth was >15 m (mean, 240 mg/L) in VT were higher than thosefrom shallow wells (�15 m well depth; mean, 10.7 mg/L). A weakbut significant positive correlation between As concentrations ingroundwater and well depth was observed in VT (r¼ 0.579,p< 0.05 by Spearman’s rank correlation test), implying that As

Table 2Concentrations of total As in groundwater (mg/L) and As compounds in human urine (mg/g creatinine) from Cat Que Commune, Ha Tay Province and Vinh Tru Commune, HaNam Province in Vietnam.

Location Groundwater Human urine

Total As AB DMA MMA AsIII AsV SAs

Cat Quena 13/13 55/56 56/56 56/56 52/56 27/56 56/56Mean 209 10.1 (14.8) 38.7 (63.4) 5.8 (9.5) 5.5 (8.6) 2.8 (3.7) 62.5S.D. 65.5 12.8 (13.5) 11.3 (11.7) 3.0 (4.1) 3.3 (4.6) 3.4 (5.2) 19.6Min 132 <1.0 (0) 19.5 (27.0) 1.5 (3.1) <1.0 (0) <1.0 (0) 31.4Max 344 70.9 (61.1) 64.0 (89.2) 18.5 (22.2) 15.7 (19.0) 11.8 (20.7) 124Median 194 5.6 (11.2) 38.6 (63.3) 4.8 (8.8) 5.3 (8.4) <1.0 (0) 63.9GM 200 6.2 37.1 5.1 4.3 1.4 59.6

Vinh Truna 15/15 39/41 41/41 40/41 37/41 26/41 41/41Mean 163 9.0 (14.1) 38.2 (61.7) 5.3 (8.4) 6.3 (9.6) 5.1 (6.2) 63.7S.D. 163 8.0 (10.6) 15.5 (11.0) 3.4 (3.5) 4.3 (4.8) 7.3 (7.6) 29.0Min 1.8 <1.0 (0) 17.7 (24.5) <1.0 (0) <1.0 (0) <1.0 (0) 36.4Max 486 33.2 (45.8) 86.0 (77.5) 20.3 (16.0) 22.6 (25.7) 35.1 (34.0) 179Median 153 7.8 (11.7) 34.7 (63.2) 4.4 (8.0) 5.5 (10.3) 2.8 (4.9) 54.5GM 48.0 6.0 35.7 4.5 4.8 2.2 59.1

GM; geometric mean. Percentage of As compounds to SAs is given in parenthesis.a Number of samples with detectable concentration.

T. Agusa et al. / Environmental Pollution 157 (2009) 396–403398

concentrations may be high in deeper layer of the aquifer in VT.Depth-dependent variation in groundwater As concentrations wasalso reported in other As-contaminated regions (e.g., Harvey et al.,2002; Polya et al., 2005). This is probably due to the depth-dependent differences in geological characteristics and redoxconditions. However, such a trend was not observed in CQ, whichmight be due to the small range of well depth (38–50 m). Period ofwell usage was not correlated to As concentrations in the ground-water in this study (p> 0.05).

Concentrations of Fe in groundwater from VT and CQ were inthe range of <50–63,000 mg/L. In contrast to previous reports

Table 3Concentrations (mg/L) of total As in groundwater from different locations in Vietnam.

Location n Mean Min Max Median References

Red River DeltaCat Que Commune,

Ha Tay Province13 209 132 344 194 This study

Vinh Tru Commune,Ha Nam Province

15 163 1.8 486 153 This study

Van Duc Commune,Gia Lam Province

11 10.8 <0.1 38.2 5.0 Agusa et al. (2006)

Van Phuc Commune,Thanh Tri Province

14 44.0 <0.1 330 1.5 Agusa et al. (2006)

Dong Anh Province 48 31 <1 220 Berg et al. (2001)Gia Lam Province 55 127 2 3050 Berg et al. (2001)Thanh Tri Province 45 432 9 3010 Berg et al. (2001)Tu Liem Province 48 67 1 230 Berg et al. (2001)Luong Yen, Hanoi 6 22.8 Nga et al. (2003)Yen Phu, Hanoi 7 40.5 Nga et al. (2003)Mai Dich, Hanoi 3 1.1 Nga et al. (2003)Ngoc Ha, Hanoi 3 1.6 Nga et al. (2003)Ngoc Si Lien, Hanoi 3 1.4 Nga et al. (2003)Phap Van, Hanoi 5 67.3 Nga et al. (2003)Tuong Mai, Hanoi 4 44.5 Nga et al. (2003)Ha Dinh, Hanoi 5 92.6 Nga et al. (2003)Red River Delta 83 140 1.3 460 Trang et al. (2005)

Mekong River DeltaAn Giang Province 24 5.4 <0.1 71.2 Minh et al. (2005)Can Tho Province 42 3.7 <0.1 23 Minh et al. (2005)Don Thap Province 12 96.5 <0.1 411 Minh et al. (2005)Soc Trang Province 2 6.0 <0.1 12 Minh et al. (2005)Ben Tre Province 2 66.6 47.1 86.1 Minh et al. (2005)Ho Chi Minh 10 5.5 <0.1 32.7 Minh et al. (2005)Long An Province 6 13.5 <0.1 30.4 Minh et al. (2005)Tien Giang Province 10 7.7 <0.1 29.5 Minh et al. (2005)Vinh Long Province 10 1.5 <0.1 4.54 Minh et al. (2005)Mekong River Delta 111 39 <1 850 Trang et al. (2005)

(Agusa et al., 2006; Berg et al., 2001), no clear relationship betweenconcentrations of Fe and As in groundwater was observed in thisstudy (p> 0.05). Therefore, even though the investigated areas inthe present study and previous studies are located within the RedRiver Delta, association of As with Fe might be different among thesampling points.

3.2. Concentration and composition of As species in rice

Rice is the staple food in Asian countries, and therefore, if rice iscontaminated with As, the rice will be a significant source for Asintake in residents. Indeed, it was reported that irrigated waterfrom aquifer in the Bengal Delta has resulted in increased As levelsin paddy soil and rice grain (Meharg and Rahman, 2003). Hence, weanalyzed white rice samples (n¼ 10) to evaluate As intake via riceconsumption. The sum of the concentrations of all arsenicals (SAs)in the rice ranged from 180 to 252 ng/g dry wt. (mean, 225 ng/g drywt.) with the dominant As species being AsIII (mean, 63.0%), fol-lowed by residual As (mean, 32.9%) that was remaining arsenical(s)in rice after the extraction by mixture of Milli-Q water and meth-anol (Pizarro et al., 2003), and DMA (mean, 4.1%). AsV was not foundin the white rice in the present study, which may be explained bythe suggestion of Abedin et al. (2002); the rice plants are effectivelyexposed to AsIII but not to AsV because of the reducing condition inpaddy fields. Other arsenicals were below detection limits (<1 ng/gdry wt.). This result would indicate that consumption of the ricecauses human health risk for toxicity of inorganic As (AsIII) as wellas drinking of groundwater.

Total As concentrations in the rice from this study were gener-ally in the same range of those from other studies in variouscountries including As-affected areas (Das et al., 2004; Heikens,2006; Roychowdhury et al., 2003; Schoof et al., 1999; Williamset al., 2005). Phuong et al. (1999) also reported that concentrationof total As in rice collected from the Red River Delta in Vietnam was231 ng/g dry wt. (208 ng/g wet wt.), the levels being very close toour results.

For inorganic As, the concentrations (111–175 ng/g dry wt.) inour investigation were similar to or slightly higher than otherstudies (Heikens, 2006; Schoof et al., 1999; Williams et al., 2005).Zhu et al. (2008) described that inorganic As concentrations in ricefrom As-contaminated areas were up to 444 ng/g dry wt. (400 ng/gwet wt.), which was higher than those in the present study.According to Williams et al. (2005) and Schoof et al. (1999),

T. Agusa et al. / Environmental Pollution 157 (2009) 396–403 399

inorganic As (AsIII and AsV) was the major arsenicals in rice. Inaddition, Meharg et al. (2008) indicate a value of inorganic Asaround 70–80% for brown rice and around 50% for white rice. Ourresults of inorganic As (mean, 63.0%) were similar to these studies.

3.3. Dietary intake of As and risk assessment

Although we did not analyze As species in groundwater, mostpredominant arsenical would be inorganic (AsIII and AsV). For riskassessment of inorganic As exposure through the consumption ofgroundwater and rice by local residents, we estimated intake ofinorganic As by the following equation:

DI ¼ ACG� IGþ ACR � IR

where DI, dietary intake of inorganic As (mg/day); ACG, inorganicAs concentrations in groundwater (mg/L); IG, ingestion rate ofgroundwater (L); ACR, inorganic As concentration in rice (ng/gwet wt.); and IR, ingestion rate of rice (g/day). World Health Orga-nization estimates that water consumption is 2 L/day (WHO, 2004).However, this value could be higher in developing countries in thetropical region. Since Watanabe et al. (2004) showed that about 3 Lof groundwater was daily consumed by local people from Bangla-desh, this consumption rate was also used in the present study.Ingestion rate of rice (463 g/day) was cited from the statisticaldatabase in Food and Agriculture Organization (FAO) (FAO, 2005).

The inorganic As intake was estimated to be 682 mg/day (624and 58 mg/day from groundwater and rice, respectively) (Table 4).In this estimation, about 91% of total As intake was derived fromgroundwater drinking.

Although estimation methodology of the As intake wasdifferent among studies, intake of inorganic As in residents fromthe Red River Delta, Vietnam were comparable to those from As-contaminated regions in which skin lesions were observed inlocal people, but were much higher than those from non-contaminated regions (Table 4). Provisional Tolerable WeeklyIntake (PTWI; 15 mg/week/kg body wt.) for inorganic As estab-lished by FAO/WHO (WHO, 1989) corresponds to about 2.14 mg/day/kg body wt. In the present study, daily intake of As wereestimated to be over the guideline value for about 92% of subjects,suggesting that the health risk of As is of great concern in localpeople from the Red River Delta, Vietnam.

3.4. Concentration and composition of As species in human urine

Sum of the concentrations of As species in human urine was31.4–179 mg/g creatinine (Table 2), and there was no regionaldifference between CQ and VT. Dimethylarsinic acid was thepredominant compound (mean, 62.7%) in the urine of local people

Table 4Daily intake (mg/day) of inorganic As in different populations.

Country Mean Min Max Remarks References

Arsenic contaminated siteVietnama 682 67 1520 This studyChile 1389 475 1647 Adult males Diaz et al. (2004)Mexicob 1220 320 3100 Summer Del Razo et al. (2002)Mexicob 899 198 1713 Winter Del Razo et al. (2002)India 1c 708 Adult males Roychowdhury et al. (2003)Bangladeshb 674 Adult males Watanabe et al. (2004)India 2c 564 Adult males Roychowdhury et al. (2003)

Control site for the above siteChile 125 57 200 Adult males Diaz et al. (2004)Mexicob 69 18 147 Summer Del Razo et al. (2002)Mexicob 56 15 106 Winter Del Razo et al. (2002)

a As in water assumed as inorganic As.b Total As assumed as inorganic As.c 50% of total As assumed as inorganic As.

from VT and CQ. Also, inorganic arsenic such as AsIII (mean, 9.1%)and AsV (mean, 4.7%) was detected although the levels were low,indicating that residents are exposed to inorganic arsenicals. On theother hand, AB (mean, 14.5%), which may be derived fromconsumption of fish and shellfish, was also detected in the urine ofresidents. For all arsenic compounds, the differences in concen-trations in human urine between CQ and VT were not significant(p> 0.05).

Significant positive correlations between concentrations of totalAs in groundwater, and DMA, AsIII, AsV, and SAs in human urine wereobserved, but total groundwater As was not related with urinary AB(Agusa et al., 2005). By using data of daily intake of inorganic As (DI),we examined the relationships between DI and As compoundlevels in human urine. A significant positive correlation betweenDI and concentrations of SAs in human urine was observed(log SAs¼ 1.72�10�4�DIþ 1.66, R2¼ 0.155, p< 0.001). More-over, concentrations of DMA (log DMA¼ 1.75�10�4�DIþ 1.44,R2¼ 0.167, p< 0.001), AsV (log AsV¼ 6.59�10�4�DI� 0.224,R2¼ 0.164, p< 0.001), AsIII (log AsIII¼ 2.87�10�4�DIþ 0.455,R2¼ 0.068, p< 0.01), and inorganic As (log inorganicAs¼ 3.55�10�4�DIþ 0.651, R2¼ 0.166, p< 0.001) (Fig. 1) andpercentage of (%) inorganic As (% inorganic As¼ 0.007�DIþ 8.93,R2¼ 0.095, p< 0.01) in urine also showed positive correlations withDI. On the contrary, there were no significant correlations between DIand urinary AB concentration (p> 0.05, Fig.1) and composition. Theseresults suggest that this population is mainly exposed to inorganic As,and DMA is derived from methylation of inorganic As, while AB comesfrom consumption of fish and/or shellfish.

There is no available data on urinary As compounds in SoutheastAsia except a previous study from As-contaminated areas (KratieProvince) in the Mekong River Basin in Cambodia (Kubota et al.,2006). In their study, concentrations of As in groundwater were inthe range of 43–217 mg/L (mean, 116 mg/L and median, 104 mg/L) inSambok and <1–886 mg/L (mean, 114 mg/L and median, 8.1 mg/L) inKompong Kor, and the levels were lower than those in the presentstudy (mean, 184 mg/L and median, 193 mg/L). However, urinary Asconcentrations (mean, 201 mg/g creatinine) in residents from theseareas in Cambodia were much higher than those (GM, 59.4 mg/gcreatinine) in Vietnam. This implies that the frequency of usage ofgroundwater may be high for the Cambodian compared to theVietnamese. Results of chemical speciation of As in human urinewere also different between Cambodia and Vietnam. In bothstudies, DMA was the major As compound but AB was not detectedin all urine samples collected in Cambodia (Kubota et al., 2006),suggesting that As intake from fish and shellfish consumptionmight be negligible for the Cambodian people investigated byKubota et al. (2006).

3.5. Factors influencing concentration of As species in human urine

Influence of sex, age, and estimated intake of inorganic As onconcentrations of arsenicals in human urine was examined usingMANOVA. Estimated intake of inorganic As were categorized intotwo groups, ‘‘High’’ and ‘‘Low’’, based on the mean value (682 mg/day). Because occupation, and alcohol and smoking habits do notshow clear influence on urinary As concentrations and also resi-dential years were closely related to age (r¼ 0.884, p< 0.001),these variables were not included in MANOVA. Result of MANOVAfor As concentration is shown in Table 5. Urinary DMA levels weresignificantly influenced by age (p< 0.05) and age� sex (p< 0.001).Concentrations of DMA in urine of children were significantlyhigher than those of adults (p< 0.05). In males, children showedhigher concentrations of DMA in urine compared to adults, but thisdifference was not found in females (Fig. 2). Similar results werealso obtained for urinary SAs (Fig. 2); the SAs concentration wasalso influenced by the age� sex interaction (p< 0.05). Some

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tratio

n o

f A

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ou

nd

s

in

h

um

an

u

rin

e (u

g/g

creatin

in

e)

Estimated daily intake of inorganic As (ug/day)

Inorganic As

y = 1.75x10-4x + 1.44

R2

= 0.167p < 0.001

ABSAs

y = 1.72x10-4x + 1.66

R2

= 0.155p < 0.001

DMA

1.2

1.4

1.6

1.8

2.0

1.4

1.6

1.8

2.0

2.2

2.4

-0.5

0

0.5

1.0

1.5

2.0

0

0.3

0.6

0.9

1.2

1.5

1.8y = 3.55x10-4

x + 0.651R2

= 0.166p < 0.001

0 400 800 1200 16000 400 800 1200 1600

0 400 800 1200 16000 400 800 1200 1600

Fig. 1. Relationships between estimated daily intake of inorganic As and concentrations of As compounds in human urine from Vinh Tru (VT) Commune, Ha Nam Province and CatQue (CQ) Commune, Ha Tay Province in Vietnam.

T. Agusa et al. / Environmental Pollution 157 (2009) 396–403400

authors reported that concentrations of As in urine of children werehigher than those of adults in As-contaminated areas (Del Razoet al., 1997; Chowdhury et al., 2003; Meza et al., 2005). Also in thestudy from Cambodia, relatively high As concentrations wereobserved in children although authors did not mention it (Kubotaet al., 2006). Del Razo et al. (1997) have explained that the resultmay be due to the high consumption of water in children comparedto adults. Chowdhury et al. (2003) calculated ratios of As excretionrate to As ingestion rate for adults and children, and found that thevalue for children (mean, 0.69) was higher than adults (mean, 0.53).From these results, children may retain less As in their body thanadults (Chowdhury et al., 2003), but additional studies are neededto clarify the mechanisms.

For AsV (p< 0.05) and inorganic As (p< 0.05), higher concen-trations were observed in urine of donors with high intake ofinorganic As (Table 5). These results were consistent with rela-tionships between inorganic As intake and urinary concentrationsof AsV and inorganic As as mentioned above. Therefore, it can bestated that AsV and inorganic As concentrations in human urine arestrongly dependent on intake of inorganic As.

3.6. Evaluation of As metabolism

Inorganic As could be metabolized to monomethyl (1st step ofmethylation) and dimethyl As compounds (2nd step of methyla-tion) in the human body and thus the composition of urinary As

Table 5Results of multivariate analysis of variance (MANOVA) for influence of age, sex, and esti

Independent variable AB DMA MMA

F p F p F p

Age (adult/child) 1.528 0.220 6.057 0.016 0.385 0.537Sex (female/male) 2.402 0.125 0.374 0.543 0.796 0.375Intake (high/low) 0.024 0.878 2.803 0.098 1.637 0.204Age� sex 0.920 0.340 11.940 0.001 0.506 0.479Age� intake 2.015 0.160 0.189 0.665 1.578 0.213Sex� intake 0.002 0.968 0.080 0.778 0.871 0.353Age� sex� intake 0.084 0.773 0.119 0.732 0.723 0.398

may be a useful indicator of As methylation ability (Hopenhayn-Rich et al., 1996). Concentration ratios of MMA/inorganic As (M/I)and DMA/MMA (D/M) may indicate the methylation capacities at1st and 2nd steps, respectively. In the present study, GM of M/I andD/M was 0.64 and 7.35, respectively. Results of multi-way ANOVAon the impact of sex, age, residential year, occupation, and alcoholand smoking habits on As methylation showed that concentrationratio of D/M was significantly affected by age (p< 0.05); significanthigh D/M values were observed in children compared to adults(p< 0.05), especially in males (Fig. 3). Similarly, previous studies inMexico (Meza et al., 2005) and Bangladesh (Chowdhury et al.,2003) showed that concentration ratios of D/M for adults werelower than those for children (Fig. 3). Chung et al. (2002) investi-gated urinary As compound profiles among family members(father, mother, son, and daughter) in Chile, and found that fathershowed the lowest D/M value.

Influence of sex, age, and intake of inorganic As on urinary Ascompositions was examined using MANOVA. Only % inorganic Aswas associated with inorganic As intake (p< 0.05) possibly due tothe significant relation between concentration of inorganic As inurine and intake of inorganic As (p< 0.05) as mentioned above. Onthe other hand, % of other arsenicals were not influenced by sex,age, and intake of inorganic As.

Although our sample size is limited and influence of intake ofalready-methylated arsenicals (MMA and DMA) from food cannotbe eliminated, methylation capacity from MMA to DMA in adults

mated intake of inorganic arsenic on concentrations of urinary arsenicals.

AsIII AsV Sum As Inorganic As

F p F p F p F p

0.020 0.888 0.378 0.540 3.313 0.072 0.007 0.9350.161 0.690 2.371 0.127 0.186 0.667 0.158 0.6921.612 0.208 6.310 0.014 1.445 0.233 6.972 0.0100.725 0.397 0.746 0.390 6.833 0.011 1.539 0.2180.052 0.820 0.176 0.676 0.008 0.927 0.048 0.8280.824 0.367 0.042 0.839 0.039 0.844 0.068 0.7950.040 0.842 0.008 0.930 0.027 0.870 0.092 0.763

25 50 75 20 40

SAs DMA

0 100 0 60

Adult

Female-Adult

Child

Male-ChildMale-Adult

Female-Child

FemaleMale

Fig. 2. Concentrations of sum of As compounds (SAs) and dimethylarsinic acid (DMA)in human urine from Vinh Tru (VT) Commune, Ha Nam Province and Cat Que (CQ)Commune, Ha Tay Province in Vietnam. Bar represents geometric mean.

T. Agusa et al. / Environmental Pollution 157 (2009) 396–403 401

(particularly in males) might be lower than that in children. Moresamples and expression level of arsenic methyltransferase (AS3MT),which catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to trivalent arsenicals and may play a role inarsenic metabolism (Lin et al., 2002; Wood et al., 2006), should bestudied to evaluate the difference in methylation ability betweenadults and children.

3.7. Enigma of absence of As-related disease

Skin disorder is known to be caused by chronic As exposure(Abernathy et al., 1999; Tsunetoshi, 2000). However, such symptomwas not observed in the residents, although it was estimated thatintake of inorganic As are much high in the population of this study(Table 4). Previous studies have also reported that there was nosymptom of As poisoning in residents from the suburb of Hanoi,Vietnam, in spite of the high concentrations of As in groundwater(Agusa et al., 2006; Berg et al., 2001). In this regard, Berg et al.(2001) suggested that it might be related to short span of usage ofgroundwater. The tube wells in the contaminated areas wereinstalled only 1–10 years ago when they studied and chronic Aspoisoning usually occurs after 5–10 years of exposure throughconsumption of As-contaminated water. However, some wellsinvestigated in the present study were developed more than 10

Female-AdultFemale-Child

Male-AdultMale-Child

AdultChild

AdultChild

MotherDaughter

FatherSon

Vietnam1

Mexico2

Bangladesh3

Chile4

AdultChild

FemaleMale

0 2 4 6 8 10 12Concentration ratio of DMA/MMA

in human urine

Fig. 3. Comparison of concentration ratios of dimethylarsinic acid (DMA)/mono-methylarsonic acid (MMA) in human urine from As-contaminated areas.1, This study; 2,Meza et al. (2005); 3, Chowdhury et al. (2003); 4, Chung et al. (2002). Value of DMA/MMA corresponds to the 2nd methylation capacity of inorganic As (from MMA to DMA).Bar represents geometric mean for reference 1 and 2 and mean for reference 3 and 4.

years ago. Vietnam has rainy and dry seasons, and some localpeople use rainwater in rainy season. Therefore, we assume thatexposure of As in the residents may not be chronic, leading to thelower As exposure than the expectation based on the As levels ingroundwater. Since there are no available data on difference in Asexposure status in residents between rainy and dry seasons, furtherinvestigation should be conducted to test the hypothesis.

As other factors, nutritional status and genetic polymorphism mayalso influence the expression of As toxicity. In West Bengal, India,people consuming low nutritional foods exhibit high frequency of Aspoisoning compared to those with high nutritional foods despite of thesimilar As concentration in groundwater (Das et al., 1995). Low intakeof calcium, animal protein, folate, and fiber may increase susceptibilityto As-caused skin lesions (Mitra et al., 2004).

Furthermore, a large variation in the susceptibility to As toxicitybetween individuals and ethnic groups may be associated withgenetic factors in the As metabolism (Vahter, 2002). DNA repairgenes, xeroderma pigmentosum groups A (XPA) and D (XPD) maybe related to sensitivities on skin cancer and its precursor diseaseinduced by inorganic As exposure (Ahsan et al., 2003; Applebaumet al., 2007; Thirumaran et al., 2006). Single nucleotide poly-morphisms (SNPs) in AS3MT can be related to As methylationcapacity by in vitro (Wood et al., 2006) and population studies(Agusa et al., 2008; Hernandez et al., 2008; Lindberg et al., 2007;Meza et al., 2005; Schlawicke Engstrom et al., 2007). Arsenicreductase, glutathione-S-transferase omega (GSTO) is able toreduce pentavalent arsenicals (Zakharyan et al., 2001), and theeffects of its polymorphism on As metabolism have been reportedin previous studies (Marnell et al., 2003; Schmuck et al., 2005;Tanaka-Kagawa et al., 2003).

Although information on nutritional status and SNPs in thesegenes and other As metabolism-related genes is still lacking for theresidents in Vietnam, it can be stated that these factors might berelated to the apparent absence of As-related toxic effects in theresidents in CQ and VT. Epidemiological approach using quantita-tive data on As exposure from water and food and nutrition intake,diagnosis in As poisoning, and identification of genetic poly-morphisms is required in future.

Acknowledgements

We are grateful to Dr. A. Subramanian in the CMES, EhimeUniversity, Japan for critical reading of the manuscript. The authorswish to thank the staff of the Center for Environmental Technologyand Sustainable Development (CETASD), Hanoi National University,Vietnam and Mr. M. Muraoka in the CMES, Ehime University, Japanfor sample collection. We also acknowledge Ms. N. Tsunehiro andMr. M. Kunimoto, staff in the es-BANK at CMES, Ehime University,Japan for their support in sample management. This study wassupported by Japan Society for the Promotion of Science (JSPS) forthe cooperative research program under the Core UniversityProgram between JSPS and Vietnamese Academy of Science andTechnology, and grants from Research Revolution 2002 (RR2002)Project for Sustainable Coexistence of Human, Nature and the Earth(FY2002), Grants-in-Aid for Scientific Research (S) (No. 20221003)from JSPS, and 21st Century and Global COE Programs from theMinistry of Education, Culture, Sports, Science and Technology(MEXT), Japan and JSPS. The award of the JSPS Post DoctoralFellowship for Researchers in Japan to T. Agusa (No. 207871) is alsoacknowledged.

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