Article pubs.acs.org/est
Serum Dioxin Levels in Vietnamese Men more than 40 Years after Herbicide Spraying Ho Dung Manh,†,‡ Teruhiko Kido,§,* Rie Okamoto,§ Sun XianLiang,† Le Thai Anh,† Supratman Supratman,† Shoko Maruzeni,∥ Muneko Nishijo,∥ Hideaki Nakagawa,∥ Seijiro Honma,⊥ Takeshi Nakano,# Takumi Takasuga,▽ Dang Duc Nhu, ▲,○ Nguyen Ngoc Hung, ▲ and Le Ke Son■ †
Division of Health Sciences, Graduate School of Medical Science, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-8640, Japan ‡ Faculty of Environmental Engineering and Biotechnology, Lac Hong University, No.10 Huynh Van Nghe, Buu Long, Bien Hoa, Dong Nai, Vietnam § Faculty of Health Sciences, Institute of Medical Pharmaceutical and Health Sciences, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-8640, Japan ∥ Department of Public Health, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 920-8641, Japan ⊥ ASKA Pharma Medical Co. Ltd., 5-36-1 Shimosakunobe, Kawasaki 213-8522, Japan # Center for Advanced Science and Innovation, Osaka University, Osaka 560-0043, Japan ▽ Shimadzu Techno-Research Inc., Kyoto 604-8435, Japan ▲ 10-80 Division, Hanoi Medical University, No.1 Ton That Tung, Dong Da, Hanoi, Vietnam ■ Environment Administration, Ministry of Natural Resources and Environment, 67 Nguyen Du Street, Hanoi, Vietnam ABSTRACT: Recent studies have found elevated dioxin levels inside some U.S. military former air bases in Vietnam, known as hotspots. Many studies of Agent Orange have been done in U.S. veterans; however, there is little known about Vietnamese men. In 2010, we collected blood samples from 97 men in a hotspot and 85 men in an unsprayed area in Northern Vietnam. Serum concentrations of not only TCDD but also other dioxins (PCDDs), furans (PCDFs), and nonortho polychlorinated biphenyls (PCBs) were significantly higher in the hotspot than in the unsprayed area. In the hotspot, three subareas were demarcated, based on their proximity to the air base. The total toxic equivalents (TEQ) of PCDDs/PCDFs+PCBs was 41.7 pg/g lipid in the area closest to the air base, while it was around 29 pg/g lipid in the other two subareas. In the unsprayed area, the dioxin levels were no different between men who went to the South during the Vietnam War and those who remained in the North, with TEQs PCDDs/PCDFs+PCBs of around 13.6 pg/g lipid. Our findings suggested that people living close to the former U.S. air bases might have been exposed to both Agent Orange and other sources of dioxin-like compounds.
■
INTRODUCTION Polychlorinated dibenzodioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs), all known as dioxins or dioxin-like compounds, are widespread, persistent toxic chemicals in the environment. Dioxins are lipophilic compounds and accumulate in the human body through the food chain. Once in the body, they are stored in adipose tissue and take many years to be eliminated.1 Although the human body burden has been decreasing in recent years,2 dioxin levels remain elevated in some specific areas where historical incidents occurred. During Operation Ranch Hand (1961−1971), the U.S. military sprayed millions of liters of various herbicides south of the former Demilitarized Zone at the 17th parallel, which was established as a dividing line between North and South Vietnam as a result of the First Indochina War. A little over © 2014 American Chemical Society
10% of South Vietnam was sprayed for purposes of defoliation and crop destruction. The best-known herbicide was Agent Orange, which is a 1:1 mixture of n-Butyl esters of 2,4dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Other herbicides were used including Agent White (2,4-D; picloram), Agent Blue (cacodylic acid), Agent Purple (2,4-D; 2,4,5-T), Agent Green (2,4,5-T), and Agent Pink (2,4,5-T). Unfortunately, 2,4,5-T was contaminated with varying levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most toxic chemical in the dioxin group.3 As a result, TCDD in breast milk samples collected in 1970 in the Received: Revised: Accepted: Published: 3496
October 30, 2013 February 15, 2014 February 19, 2014 February 19, 2014 dx.doi.org/10.1021/es404853h | Environ. Sci. Technol. 2014, 48, 3496−3503
Environmental Science & Technology
Article
Figure 1. Map of Vietnam and Study Areas.
versus an unsprayed area. The purpose of this study was to determine serum dioxin concentrations in these elderly men and compare dioxin levels in the two areas.
South showed impressive levels as high as 1832 ppt (parts per trillion)the highest level in human milk observed to date.4 Two decades later, TCDD levels in pooled blood samples still proved to be much higher in the South than in the North.5,6 Also, TCDD levels in pooled adipose tissue from Hanoi of former North Vietnamese soldiers reached as high as 8.1 ppt, compared with 1.4 ppt in the general population pool from Hanoi in North Vietnam.6 Moreover, other PCDD/F congeners in adipose tissues7 and sediment samples8 were also found to be higher in the South than in the North, which suggested that part of this difference may be due to atmospheric deposition generated by combustion sources. The Hatfield Consultancy recently theorized that dioxin levels in former U.S. air bases, where the herbicide was spilled when applied by truck-mounted sprayers and also sprayed intensively around the perimeter, remain extremely elevated.9,10 The major air bases at Bien Hoa, Da Nang, and Phu Cat are known as hotspots.11,12 Tai et al.13 collected breast milk from young mothers who were born after the war and found that their dioxin levels were 4-fold higher in the hotspots and 3-fold higher in sprayed areas compared with unsprayed areas. Other studies have found an association between dioxin levels in breast milk and steroid hormone levels in Vietnamese primiparae,14−16 as well as an association with the neurodevelopment17 and body development18 of infants in the hotspot areas. Many studies of Agent Orange have been done in the U.S. veterans who were exposed for a short time while serving in Vietnam.19 Few studies have been conducted on Vietnamese men who were directly sprayed or lived in herbicide-sprayed areas during the war and may continue to be exposed up to now. Previous studies quantifying dioxin in the blood of Vietnamese people often used pooled samples;4,6,10 only a few studies used individual samples,20 because the analysis technique required volumes of blood as large as 100 mL, which are difficult to collect in Vietnam. Recently, with advancements in analytical techniques, we can now analyze dioxins in amounts of blood as small as 10 mL. In 2010, we conducted a study on Vietnamese men located in a hotspot area
■
METHODS Study Areas. The study areas are shown in Figure 1. Phu Cat air base, a dioxin hotspot located in Binh Dinh province in South Vietnam, was a former U.S. air base during Operation Ranch Hand. Records indicate that 17 000 drums of Agent Orange, 9,000 drums of Agent White, and 2900 drums of Agent Blue were stored at this air base.21 The Hatfield Consultancy9 has reported TCDD levels as high as 236 000 pg/g in soil taken at the herbicide storage area inside the air basefar higher than the standard in some countries of less than 1.000 pg-TEQ/g in soil. In the Phu Cat district, we selected three subareas (PC1, PC2, PC3) based on their proximity to the air base. The air base is inside PC1 (the Cat Tan and Ngo May communes, total area 40 km2), near PC2 (Cat Tuong and Cat Trinh communes, total area 80 km2), and a little farther from PC3 (Cat Hanh and Cat Lam communes, total area about 110 km2). The comparison site is an unsprayed area in the Kim Bang district, located in Ha Nam province in the northern part of the country. This site was not sprayed with herbicide during the Vietnam War. Both the hotspot and the unsprayed area are rural and there is no industrial zone nearby. Population. Between 2010 and 2011, 97 men in the hotspot area and 85 men in the unsprayed area were recruited for this study. All of the subjects were between 55 and 80 years of age and agreed to provide 10 mL of blood for testing. They were interviewed to obtain their demographic data, including age, residency, smoking habit, jobs, family income, and education. In the unsprayed area, we also asked if they had any history of exposure to herbicides, whether they had been directly sprayed by herbicide or had lived in herbicide-sprayed areas where subsequent defoliation could be recognized, and where they had been during the herbicide spraying period (1961−1971), and thereafter. In addition, the height and the 3497
dx.doi.org/10.1021/es404853h | Environ. Sci. Technol. 2014, 48, 3496−3503
Environmental Science & Technology
Article
All statistical analyses were performed using the JMP@9 software package (SAS Institute, Japan) and the R Statistical Environment (R Development Core Team, 2013).
weight of each participant were measured to calculate their body mass index (BMI, kg/m2). The purpose of the present study was thoroughly explained to them and written informed consent was obtained from each participant through their local people’s committee. This study was approved by the Medical Ethics Committee of Kanazawa University (Health Permission No. 89). Analysis of Blood Samples. Blood samples (10 mL) were collected from each participant by medical staff at the community health center. After centrifugation of the samples, the serum was separated into chemically cleaned containers, frozen in dry ice for several days, transported by air to Shimadzu Techno-Research Inc., in Kyoto, Japan, and stored at −30 °C until analysis. The analytical technique and quality control and assurance procedures were implemented following the guidelines contained in the publication “Provisional Manual on the Analysis of Dioxin in Human Blood’’ (Japan, Ministry of Health and Welfare, 2000).22 The serum samples were spiked with a mixture of 13C12-label PCDDs/PCDFs and PCBs as an internal standard. The lipid content was extracted with n-hexane by liquid extraction and then determined using the gravimetric method. It was then subjected to a series of purification operations consisting of alkaline digestion and multilayer silica gel column chromatography. Finally, an active carbon-dispersed silica gel column was used to separate and collect the PCDDs/PCDFs and nonortho PCBs. The dioxin/furan/nonortho PCB fraction was quantified using HRGC-HRMS (Hewlett-Packard 6890 Series and Micromass Autospec, Ultima). The selected ion monitoring (SIM) mode was used, and the resolution was maintained above 10 000. The spike recovery (calculated using a syringe spike) ranged between 95% and 104% for PCBs, 78% and 89% for PCDD/Fs.23 All dioxin, furan, and nonortho PCB congeners were calculated on a lipid basis, then converted to toxic equivalents (TEQs) using the international toxicity equivalency factors (TEFs) 2005 recommended by the World Health Organization (WHO).24 The detection limits of congeners varied depending on the samples. Generally, a serum sample of 5 g wet weight with 0.5% fat content (w/w) has detection limits, as follows: Te-PeCDD/Fs: 0.01 pg/g wet, 2 pg/g fat; Hx-HpCDD/Fs: 0.02 pg/g wet, 4 pg/g fat; OCDD/Fs: 0.05 pg/g wet, 10 pg/g fat; and PCB #81, #77, #126, #169: 0.1 pg/g wet, 20 pg/g fat. Serum concentrations of dioxin, furan, and nonortho PCB congeners below the limit of detection (LOD) were assigned a value equal to half the LOD. Statistical Analysis. We transformed the dioxin concentrations into log10 to improve normality. Student’s t test and the chi-square test were used to compare the two groups according to continuous or categorical variables. For more than two groups, ANOVA was used first and then Tukey’s posthoc test to find which pair was significantly different. We used multiple linear regressions to compare dioxin concentration levels between the areas after adjusting for age, BMI, smoking, and residency. Since the serum concentration used in the regression model was transformed into log10, the regression coefficient was presented as 10β, where β is the regression coefficient. For the categorical variables, this number describes the fold change in the dioxin concentration (an increase if 10β > 1, or a decrease if 10β < 1) for each category compared with the reference category.
■
RESULTS Characteristics of the Participants. Table 1 shows the demographic characteristics of the participants in the two areas.
Table 1. Demographic Characteristics of the Study Participantsa hotspot (n = 97)
unsprayed (n = 85)
continuous variables
mean
SD
mean
SD
p value
age (years) height (cm) weight (kg) BMI (kg/m2) residency (years) income (*1000 VND)a categorical variables former soldier smoking alcohol education
