participated. Altogether, 100 pooled breast milk samples were collected and analyzed (Malisch and van Leeuwen 2003). This is the ﬁrst time the Hong Kong SAR has participated in the WHO-coordinated dioxin exposure study, and it provides the ﬁrst representative study of dioxins in human breast milk in South China. Materials and Methods Study population. Milk samples were collected from primiparous women who gave birth to a singleton from December 2001 through September 2002 in Hong Kong. A total of 316 eligible mothers were recruited by nurses dur- ing their postnatal visits at one of 16 Maternal and Child Health Centers distributed through- out Hong Kong. All participants (17–42 years of age) were Chinese, except for seven (one Indonesian, two Thais, and four Vietnamese). About half (n = 165) of the subjects were born in Hong Kong, 42% (n = 134) were born in mainland China, and 5.4% (n = 17) were born in other overseas countries (Indonesia, Singapore, Taiwan, Thailand, and Vietnam). Among the mothers born in Hong Kong, 87% (n = 144) had lived in Hong Kong since birth, and 13% (n = 21) had lived abroad for a period. For those who were born in mainland China, about half (n = 66) were born in cities in Guangdong province (adjacent to Hong Kong), and the rest (n = 68) in a wide range of provinces throughout China. Sixty-nine came to Hong Kong before delivery, and their mean duration of stay in Hong Kong was < 1 year. They were more likely than the other women in the study to have previous pregnancy expe- rience (30%), practice exclusive breast-feeding at the time of sampling (73%), have a male baby (61%), have a monthly household income of < US$1,280 (57%), and have edu- cational attainment of junior high school or lower (52%). Mothers who had mainly lived in Hong Kong regardless of their place of birth (n = 176; age, 31.1 ± 0.35 years, mean ± SE) were signiﬁcantly older than immigrants from main- land China (n = 55; age, 28.8 ± 0.50 years; t = 3.379, p < 0.001), mothers who only came to Hong Kong from China for delivery (n = 68; age, 27.3 ± 0.63 years; t = 5.473, p < 0.0005), and overseas-born mothers (n = 17; age, 27.2 ± 1.33 years; t = 3.260, p < 0.001). Most moth- ers (87%, n = 275) were never-smokers; 12% (n = 37) and 1.3% (n = 4) were ex-smokers or current smokers, respectively. The mothers who were ever-smokers (age, 26.9 ± 0.77 years) were significantly younger (t = 3.962, p < 0.0005) than were nonsmokers (age = 30.1 ± 0.29 years). All participants gave written consent before taking part in the study, which was approved by the ethics committees of the University of Hong Kong, the Chinese University of Hong Kong, and the Department of Health, Hong Kong SAR government. Data collection. In face-to-face interviews, participants provided information on area of residence, occupation, obstetric information, smoking history, and demographic character- istics, in response to a questionnaire translated from the third round of WHO PCDDs, PCDF, and PCB exposure studies. A semi- quantitative food frequency questionnaire was developed to assess the subjects’ habitual intake on potentially dioxin-related food groups, including a wide range of freshwater ﬁsh (n = 11), saltwater ﬁsh (n = 23), seafood and other ﬁsh products (n = 21), dairy prod- ucts (n = 21), eggs (n = 4), meat (n = 14), and poultry (n = 8). Usual frequency of consump- tion and amount per intake over the previous 5 years for each food listed on the question- naire were recorded. Dietary change over the course of pregnancy was ignored. Dietary intakes of each food group were estimated as grams per month. Sample collection. During the period between February and October 2002, a 30-mL sample of breast milk from each par- ticipant was collected using an electric breast pump (Medela, Baar, Switzerland) or hand expression when the baby was 2–6 weeks old. Because the prevalence of exclusive breast- feeding in Hong Kong at 4 weeks postpartum is only about 22% (Khin et al. 2002), we included mothers who partially breast-fed. Mothers’ degree of breast-feeding exclusivity was reported as 100% (exclusively), > 80% (high partial), 20–80% (medium partial), and < 20% (low partial). All breast-pump attachments that had contact with the milk samples during collec- tion were cleaned with tap water without detergent, rinsed with acetone, and autoclaved before use. No plastic milk bottles were used in sample collection to avoid the transfer of dioxins to the milk. Milk samples were trans- ported in a cool bag with ice packs from the site of collection to the laboratory. A 20-mL portion of each milk sample was stored with one tablet of potassium dichromate (no. 104858; Merck, Darmstadt, Germany) at –20°C for the gas chromatography/mass spec- trometry (GC/MS) analysis of dioxins and related compounds. Sample vials were capped with Teflon-lined caps. All sample vials and caps were cleaned with tap water, rinsed with acetone, and oven dried before use. Pooling. The WHO exposure study proto- col required milk samples to be grouped in pools representing expected low- or high-expo- sure groups in the region (WHO/EURO 2000). The advantages of using pooled samples include lower costs and that pool levels can be used to derive data on averages for population subgroups. Previous studies have suggested that Environmental Health Perspectives • VOLUME 114 | NUMBER 2 | February 2006 Human dioxin levels in Hong Kong human dioxin levels were strongly affected by the geographic distribution of the individuals, so mothers were ﬁrst classiﬁed into ﬁve groups according to the duration and period they had lived in Hong Kong, mainland China, and overseas countries. The ﬁve geographic classiﬁ- cations were as follows: a) pure Hong Kong: mothers who lived in Hong Kong since birth and never lived in mainland China or overseas (n = 120); b) mainland China: mothers living in mainland China since birth and who moved to Hong Kong within the last 2 years (n = 55); c) China immigrants: mothers born in main- land China but migrated to Hong Kong more than 2 years ago (n = 55); d) overseas: mothers who were either born overseas or born in Hong Kong but had lived overseas for a period (n = 30); and e) mixed residency: a group of mothers with any one of the above four resi- dential characteristics (n = 66). In each geo- graphic group, we further separated the mothers into two to ﬁve smaller pools by more detailed geographic classiﬁcation, smoking his- tory, or dietary preferences (Table 1). As a result, 13 pools of breast milk, each comprising samples from 12–42 mothers, were created to represent relatively homogeneous characteris- tics in terms of the mothers’ residential back- ground (Hong Kong, mainland China, China immigrant, overseas, and mixed), dietary habits (consumption of dairy products and seafood in pools 2–7, 12–13), or smoking (pool 1 only). However, other characteristics caused unavoid- able heterogeneity among the 13 pools, includ- ing mean age of mothers, gravidity, household income, and educational attainment (Table 2). Mothers’ body mass index before pregnancy and baby’s age at sampling did not vary signiﬁ- cantly among the 13 pools. Laboratory procedures. The individual samples were shipped with dry ice to the State Institute for Chemical and Veterinary Analysis of Food, Freiburg, Germany, for analyses of PCDD, PCDF, and related PCB content by GC/MS. This laboratory met all criteria for analyses of PCDDs, PCDFs, and dioxin-like PCBs in human milk during the fourth round of WHO/European Center for Environment and Health interlaboratory quality assessment studies and was therefore selected as the WHO reference laboratory. The GC/MS methods of analysis have been described in detail elsewhere (Malisch and van Leeuwen 2002). The limit of detection (i.e., the lowest limit for quantitative identiﬁcation, denoted by “<” in Table 3) was recorded for a sample if the mass concentration of a congener was undetected. Mass concentra- tion of the congener is reported as picogram per gram of milk fat, except for mono-ortho PCBs, for which nanogram per gram of milk fat is used. Calculation of TEQs. Concentrations of PCDDs, PCDFs, and PCBs were also expressed as TEQs, the sum of the mass 203