Environ. Sci. Technol. 2009, 43, 4830–4835
Dietary Intake and Human Milk Residues of Hexachlorocyclohexane Isomers in Two Chinese Cities Y A N X I N Y U , † S H U T A O , * ,† W E N X I N L I U , † XIAOXIA LU,† XUEJUN WANG,† AND MINGHUNG WONG‡ Laboratory for Earth Surface Processes, College of Urban and Environmental Sciences, Peking University, Beijing 100871, China, Croucher Institute for Environmental Sciences, and Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong, PR China
Received January 11, 2009. Revised manuscript received April 30, 2009. Accepted May 1, 2009.
Residues of hexachlorocyclohexane isomers (HCHs, including R-HCH, β-HCH, γ-HCH, and δ-HCH) in human milk of two populations from Beijing and Shenyang, China were studied. In addition to human milk samples from 76 women, 271 composite food samples covering major food categories were also collected for HCH analysis. The food consumption and socialdemographic characteristics of the studied populations were investigated and dietary intakes of HCHs of the milk donors on an individual basis were calculated. The dependences of HCH concentration in the human milk on food consumption, dietary intake of HCHs, and demographic characteristics were studied. It was found that β-HCH dominated the HCHs detected in the human milk. Although there were dramatic declines in HCHs in the human milk compared to historical data, the current levels (312 ( 377 ng/g fat and 360 ( 235 ng/g fat as the means and standard deviations for Beijing and Shenyang, respectively) were still much higher than those reported in other cities within China and around the world. It was revealed that the residual level of HCHs in the human milk was positively correlated (p < 0.001) to the quantities of food consumption. Milk, oil, vegetables, and fruits contributed a large portion of HCHs intake in Beijing, while cereals, milk, vegetables, oil, and meat were the most important dietary intake sources of HCHs in Shenyang. Both daily dietary intake of HCHs (p < 0.001) and body mass index (BMI, body weight divided by the squared height) (p < 0.01) were significantly correlated with human milk HCHs. A nonlinear model was developed to predict the residues of HCHs in human milk using both dietary intake and BMI as independent variables. Potential risk of the HCH exposure of breastfed infants is discussed.
Introduction Hexachlorocyclohexane isomers (HCHs, including R-HCH, β-HCH, γ-HCH, and δ-HCH) were extensively produced and applied in China in history (1, 2). High levels of HCHs have been found in various environmental media and human tissues (3, 4). Despite considerable decline in levels of HCHs * Corresponding author phone and fax: 0086-10-62751938; e-mail:
[email protected]. † Peking University. ‡ Hong Kong Baptist University. 4830
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since they were banned, it is expected that the enduring presence of HCHs in the environment will remain as a public concern for decades due to their high persistency. Humans are primarily exposed to HCHs through ingestion. It was estimated that dietary intake was responsible for 86.5% of the total intake of HCHs by the population in Tianjin, China (5). Therefore, contamination of food commodities is critical to public health. Human milk has been widely used as a good marker for assessing body burden of HCHs (4, 6-8). In addition, the presence of HCHs in human milk would impose a potential threat to infant’s health (9). Generally, the dietary intake depends on both dietary composition and contamination of various food items. Therefore, although association between frequency of dietary intake of certain food categories and organochlorine pesticides (OCPs) in human body were often reported, the dependence of intake on food habit varies from one population to another. For example, consumption of fish was often found to be correlated with concentrations of OCPs in human milk (4, 10). Consumption of animal meat or dietary products was also found to be a major reason causing accumulation of OCPs in many places (11, 12). Occasionally, a significant correlation between human milk residues of OCPs and consumption of vegetables or cereals was identified (13). Still, quantitative evidence on direct correlation between OCP uptake and residues in human body is desirable. In a previous paper, causal relationship between dietary intake and human milk residues of dichlorodiphenyltrichloroethane and metabolites were revealed (14). The objective of this paper was to address the association between dietary intake and human milk residues of HCHs. The target population was two subsets of mothers from Beijing and Shenyang, China. The hypothesis tested was that there is a direct correlation between dietary intake of HCHs and HCH residues in human milk.
Materials and Methods Sample Collection. All samples were collected from June 2005 to July 2007. Approximate 50 mL of human milk was donated by each participant and the samples were stored in chemical-free glass-bottles with potassium dichromate (1.4 mg/mL) spiked and stored at -18 °C. In total, 136 and 135 composite food samples (a mixture of at least four subsamples) were collected from randomly selected markets and supermarkets in Beijing and Shenyang, respectively. The samples were selected based on dietary composition of local population (15) and included fruits (apple, banana, pear, grape, and orange), vegetables (Chinese cabbage, cabbage, spinach, cucumber, carrot, green pepper, eggplant, lettuce, potato, and bean), cereals (rice and flour), fish (carp, grass carp, crucian, and bighead), meat (pork, chicken, beef, and mutton), eggs, milk (five brands), and oil (one brand). All nonliquid samples were freeze-dried (EYELA-FDU-830, Tokyo Rikakikai, Japan). Target Population. The studied populations were 40 women recruited randomly from Tiantan Hospital in Beijing and 36 women from Liaoning Maternity and Children Hospital and Shenyang Maternity and Children Hospital in Shenyang. A written informed consent was completed by each participant and the project protocol was approved by the Committee on the Use of Human and Animal Subjects in Teaching and Research (HASC) of Hong Kong. A nondietary determinant questionnaire and a dietary questionnaire were completed by each participant; food consumption frequencies during a one-year period prior to childbirth and the average amount of food consumed on each occasion were 10.1021/es900082v CCC: $40.75
2009 American Chemical Society
Published on Web 05/20/2009
TABLE 1. Levels of HCHs in the Human Milk from Beijing and Shenyang (ng/g Fat)a city Beijing (n ) 40)
Shenyang (n ) 36)
detection limit a
arithmetic mean ( SD geometric mean range detectable rate arithmetic mean ( SD geometric mean range detectable rate ng/g (f.w.)
r-HCH
β-HCH
γ-HCH
δ-HCH
ΣHCH
0.80 ( 0.56 0.67 0.26-3.0 100% 1.4 ( 1.7 1.0 0.21-11 100% 0.013
310 ( 380 210 17-2000 100% 360 ( 230 280 37-1100 100% 0.027
N.C. N.C. N.D.-9.3 12.5% 2.3 ( 2.1 2.4 0.77-8.9 80.6% 0.046
0.91 ( 0.92 0.72 0.18-3.5 90% N.D. N.D. N.D. 0% 0.015
310 ( 380 210 28-2000 360 ( 240 290 38-1100
N.D.: not detectable; N.C.: not calculated because of the low detectable rate.
included in the latter. Information on social-demographic characteristics of the target populations and food consumption is listed in the Supporting Information (Tables S1, S2, and S3). Reagents. Acetonitrile, n-hexane, and dichloromethane of analytical grade (Beijing Reagent, China) were distilled before use. 2,4,5,6-Tetrachloro-m-xylene (internal standard), 4,4′-dichlorobiphenyl (surrogate), and mixed standard of OCPs were purchased from J&K Chemical, USA. Granular anhydrous sodium sulfate (Beijing Reagent, China) was heated at 600 °C for 6 h. Florisil (60-80 mesh, Dikma Technologies, USA) was precleaned for 6 h at 650 °C and dried in an oven at 130 °C for at least 16 h before use. Silica gel (100-200 mesh, Beijing Reagent, China) was heated at 450 °C for 4 h and reactivated at 130 °C for 16 h immediately prior to use. Sample Extraction. U.S.EPA Method 3630 was used for extraction of animal-origin samples except eggs (16). Three grams of samples were homogenized with 5 g of anhydrous sodium sulfate and Soxhlet extracted in a mixture of n-hexane (20 mL) and dichloromethane (80 mL) at 55 °C for 24 h. The samples were extracted twice with n-hexane saturated acetonitrile (30 mL) for 1 min each time, 300 mL of 5% sodium sulfate and 30 mL of n-hexane for 10 min, and finally extracted with 30 mL of n-hexane for 10 min. Milk, eggs, and oil samples were extracted using the U.S. EPA-600/8-80-038 method (17). Five milliliter samples were extracted three times with 12 mL acetonitrile for 5 min each time, and shaken with 120 mL of 12% sodium sulfate solution for 5 min, and finally extracted with 30 mL of n-hexane for 15 min each time. The FDA 2905a (6/92) method was adopted for determinations of vegetables (40 g), fruits (40 g), and cereals (10 g) (18). The pulverized samples were extracted with acetonitrile (80 mL for vegetables or fruits, 25 mL for cereals) for 30 min, shaken for 10 min with 300 mL (vegetables) or 100 mL (fruits and cereals) of 12% sodium sulfate solution, filtered and extracted twice with 40 mL of n-hexane for 15 min each time. Sample Cleanup and Analysis. All extracts were concentrated to 1 mL first. The extract of all the samples except vegetables, fruits, and cereals was eluted with 20 mL of n-hexane (discarded) and 35 mL of dichloromethane in sequence at a rate of 2 mL/min on a chromatography column (30 cm × 10 mm i.d., silica gel). The extract of vegetables, fruits, and cereals was eluted with n-hexane (50 mL) and a mixture of n-hexane and dichloromethane (50 mL, v/v ) 3:7) in sequence on a chromatography column (30 cm × 10 mm i.d., 4 g of florisil and 6 g of silica gel). HCH isomers were measured using a gas chromatograph (GC, Agilent GC 6890) with a 30 m HP-5 column and a 63Ni electron capture detector. The oven temperature was maintained at 50 °C, raised to 150 °C at a rate of 10 °C/min, to 240 at 3 °C/min, and held for 5 min. The injector and the detector temperatures were 220 and 280 °C, respectively. HCH isomers were identified based on retention time and quantified by the internal standard (125 µL, 125 ppb, added
to 0.8 mL sample). The lipid contents were measured by gravimetric method (19). Quality Control. At least two procedural blanks and a standard solution were run simultaneously with every set of the sample analysis. Two or three replicates were measured for the majority of samples. Detection limits for four categories of the samples (fruits and vegetables, cereals, meat and fish, and milk and eggs) were 0.002-0.020, 0.004-0.040, 0.007-0.070, and 0.002-0.030 ng/g on the basis of fresh weight for R-HCH, β-HCH, γ-HCH, and δ-HCH, respectively (Supporting Information). The recoveries of the surrogate were 94, 94, 93, and 71% for the four sample categories, respectively. The average recoveries of the individual species in the standard solution spiked samples varied from 81.3 to 106%. Detailed information on the detection limits and recoveries for various categories samples is presented in the Supporting Information (Tables S4 and S5). Data Analysis. Statistical analysis was conducted using Statistica (v5.5, StatSoft) and a significant value of 0.05 was always applied. Monte Carlo simulation was performed using Matlab (v.6.0, The MathWorks).
Results and Discussion Residues of HCHs in Human Milk. The observed levels of HCHs in the human milk from Beijing and Shenyang are summarized in Table 1. The detailed data are provided in Table S6. Among the four isomers, R-HCH and β-HCH were detected in all the samples, while γ-HCH and δ-HCH were detected in 34 and 36 samples out of the 76 in total. Although there was no significant difference in ΣHCH between Beijing and Shenyang (p > 0.05), it is interesting to see that the detectable rate and mean concentration of γ-HCH in Beijing were significantly lower than those in Shenyang (p < 0.001), likely suggesting that more lindane was applied in Shenyang in the 1990s. δ-HCH was found in 90% of the samples in Beijing but not in a single case in Shenyang. Although the major compound in the technical HCHs was R-HCH (ca. 71%) and lindane was almost pure γ-HCH (ca. 99.9%) (20), the dominant compound observed in the human milk was β-HCH, which was the most persistent isomer among the four (21). It appears that the majority of the HCHs detected were those left from the historic application. This is similar to what was found for DDT and metabolites (14). The high variation in the measured HCHs was likely due to the difference in food consumption among the individuals which is to be addressed in the next section. Table 2 lists the concentrations of HCHs in the human milk reported in the literature for Beijing (1982), Shenyang (2002), and several other cities around the world (from 1991 to 2004). The concentrations of HCHs in various environmental media including human milk were very high in history (3, 4). Because of the ban, the measured concentrations of HCHs in the environment decreased significantly during the last two decades. Consequently, there were significant declines in HCHs in human milk. In Beijing, ΣHCH in the VOL. 43, NO. 13, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 2. Arithmetic Means of HCHs in Human Milk from the Literature for Comparison (ng/g fat)a city, country
sampling year
r-HCH
β-HCH
γ-HCH
δ-HCH
ΣHCH
reference
Beijing, China Shenyang, China Shijiazhuang, China Tangshan, China Canadab Turku, Finland Copenhagen, Danmark Wielkopolska, Poland Chennai, India New Delh, India Mumbai, India Kolkata, India
1982 2002 2002-2003 2002-2003 1991 1997-2001 1997-2001 2004 2002-2003 2004-2006 2004-2006 2004-2006
250 5.0 N.A. N.A. 0.31 0.19 0.51 N.A. 8.4 4.6 4.7 9.1
6590 550 109 21.2 22.6 11.6 18.4 13.3 5000 240 210 680
