Exposure assessment of breast-fed infants in the Czech Republic to indicator PCBs and selected chlorinated pesticides: Area-related differences

Chemosphere 78 (2010) 160–168

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Exposure assessment of breast-fed infants in the Czech Republic to indicator PCBs and selected chlorinated pesticides: Area-related differences Milena Cˇerná a,d,*, Vladimír Bencko b, Marek Brabec c,d, Jirˇí Šmíd d, Andrea Krsková d, Libor Jech e a

Charles University in Prague, Third Faculty of Medicine in Prague, Czech Republic Charles University in Prague, First Faculty of Medicine in Prague, Czech Republic c Institute of Computer Science, Academy of Sciences of the Czech Republic, Czech Republic d National Institute of Public Health, Prague, Czech Republic e Axys Varilab Ltd., Czech Republic b

a r t i c l e

i n f o

Article history: Received 18 May 2009 Received in revised form 24 September 2009 Accepted 27 September 2009 Available online 7 November 2009 Keywords: Human milk Czech Republic PCBs and OCPs Breast-fed infant exposure Statistical modeling of exposure Bayesian modeling

a b s t r a c t The aim of our study was to obtain data on the exposure of breast-fed infants to polychlorinated biphenyls (PCBs) and selected organochlorine pesticides (OCPs) in different urban areas of the Czech Republic. The PCB and OCP levels were determined in 90 human milk samples collected in seven urban areas in 1999–2000 according to the WHO protocol. The estimated daily intake (EDI) was calculated for each of the analytes and compared with the respective tolerable daily intake (TDI). Significant local differences in the sum of 35 PCB congeners analyzed (total PCBs) as well as in the most prominent indicator congeners 138, 153, and 180 values were observed, with the highest levels being found in breast milk samples from mothers living in the vicinity of a former plant using PCBs in Uherské Hradišteˇ (median and ranges 3410 and 1448–13 754 ng g1 lipid weight (lw)). Non-exposed mothers from the same area had about threefold lower levels (median and ranges 1073 and 757–2139 ng/g lw, respectively). The lowest levels of total PCBs were found in Telcˇ (median and ranges 480 and 293–731 ng g1 lw, respectively). In all study areas, EDIs for PCBs in breast-fed infants exceeded the TDI of 0.4 lg kg1 bw d1 recommended in the Czech Republic. EDI for HCB exceeded the recommended TDI of 0.16 lg kg1, EDI for DDT was slightly below TDI of 10 lg kg1 bw d1 and HCHs EDI was negligible. The database of analytical results from this study was used for Bayesian modeling of breast-fed infant exposure to PCBs and OCPs during the recommended 6 months of exclusive breastfeeding. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) are persistent organic pollutants that have worldwide distribution. As a result of their lipophilicity and long biological half-lives, they bioaccumulate and biomagnify in the food chain and enter the human body mainly through the intake of food rich in animal fat and marine fat (Grandjean et al., 1995) to accumulate in the human body fat. Human biomonitoring is a suitable tool to assess the human burden of these substances. Human milk, with a relatively high fat content is considered to be one of the preferable matrices for the monitoring of exposure and body burden. Concern for possible adverse health effects in breast-fed infants resulted in global breast milk monitoring studies. WHO coordinated altogether four international studies of persistent organic

* Corresponding author. Address: Charles University in Prague, Third Faculty of Medicine, Ruská 87, CZ 100 00 Prague 10, Czech Republic. Tel./fax: +420 267082378. ˇ erná). E-mail address: [email protected] (M. C 0045-6535/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2009.09.062

pollutants in breast milk based on pooled sample analysis (WHO/ ECEH, 1996). Human milk monitoring programs have also been implemented in many countries including the Czech Republic to assess the current exposure levels and body burden and to follow temporal trends (Fürst et al., 1994; Bencko et al., 1998; Schade ˇ erná et al., 2007; Lignell et al., 2009). and Heinzow, 1998; C In former Czechoslovakia, PCBs as technical mixtures marketed under the trade name Delor had been produced in an amount of about 21 500 tonnes in Eastern Slovakia from 1959 to 1984 when the production was banned (Taniyasu et al., 2003). Delor 106 high in hexa- and hepta-PCB congeners was produced for using in a paint factory in Uherské Hradišteˇ (Taniyasu et al., 2003; Pavúk et al., 2004). The first data on sum PCBs in Czech human milk samples were published in local journals since 1985 and reviewed in ˇ erná and Bencko (1999). Since 1994, the levels of indicator congC eners of PCBs and selected OCPs in human milk have been monitored continuously until now within the Czech Environmental ˇ erná et al., Health Monitoring System (Kliment et al., 1997; C 1999). Although the Czech Human Biomonitoring Project (CZˇ erná et al., 2003) HBM) showed a significantly decreasing trend (C

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in the levels of indicator PCB congeners in the Czech human milk samples, the PCB levels were still higher than those in most other European countries for the same time period (Malisch and van Leeuwen, 2003). Therefore, some other studies concerning PCBs were performed in the Czech Republic in the last decade to know more about the local differences in the exposure and body burden. One of them was a cross-sectional study monitoring polychlorinated dibenzodioxines (PCDDs), polychlorinated dibenzofuranes (PCDFs), non-ortho and mono-ortho dioxin-like PCBs, indicator PCBs IUPAC Nos. 28, 52, 101, 118, 138, 153 and 180, and selected OCPs in individual samples from seven regions. Whereas the results concerning PCDDs, PCDFs, and dioxin-like PCBs were published earlier (Bencko et al., 2004), the levels of individual indicator PCB congeners and OCPs are presented here for the first time. The main objectives of this part of the study were to map geographic differences in the breast milk levels of indicator PCBs and OCPs and to compare the exposure levels in breast-fed infants by estimating the daily intake and 6 month body burden of the examined xenobiotics.

2. Materials and methods

161

collected from two groups of breastfeeding mothers. One group (UH I), sampled in October 1999, consisted of mothers living in the Old Town in the vicinity of a former industrial plant, which had produced PCB paints in the 1970s and 1980s. The second group (UH II) consisted of mothers living in new quarters of Uherské Hradišteˇ situated far from the plant on the left bank of the Morava river. The protocol of the study was approved by the Ethical Committee of the Charles University in Prague. All study subjects provided written informed consent. The mothers were enrolled by local pediatricians on voluntary basis. The refusal rate varied between 25% and 30% of the addressed mothers. The sampling and donor interviews were organized according to the WHO/ECEH protocol (1996). Six to fifteen breast milk samples of about 50 ml of volume each were obtained by area. Self-administrated questionnaire data on maternal age, height, weight before pregnancy and after delivery, sampling time, duration of residence in the locality, smoking status, dietary habits (weekly consumption of animal origin food such as milk and dairy products, meat, fish and other seafood), occupational history, prescription medication use and newborn’s sex and birth weight were collected from each donating mother. Basic maternal and infant characteristics are presented in Table 1.

2.1. Breast milk sampling and survey respondents In the period from October 1999 through February 2001 altogether 90 breast milk samples were collected from primiparae between the second week and 2 months after delivery. Six to fifteen breast milk samples of about 50 ml of volume each were obtained in seven urban areas of the Czech Republic: Prague as the capital of the Czech Republic, Ústí nad Labem (UL) and Kolín as industrial areas, Liberec and Kladno as reference urban areas, Telcˇ as an agricultural area, and Uherské Hradišteˇ as an industrial and agricultural area (Fig. 1). Since Uherské Hradišteˇ was recognized as a hot spot within the 2nd WHO-coordinated study (WHO/ECEH, 1996; Bencko et al., 1998), breast milk samples in this area were

2.2. Sample analysis Breast milk samples collected in glass vessels were stored frozen (18 °C) until they were analyzed. Fifty milliliter of breast milk were spiked with 13C labeled internal standards in acetone and extracted first with 100 ml hexane–acetone 1:1 and then twice with 30 ml of hexane. The extract was dried and evaporated to constant weight for gravimetric fat determination. The sample was then cleaned up by GPC on a Biobead SX-3 column, layer silica gel column and carbon column according to EPA 1613 method (EPA, 1994). Two fractions were collected from the carbon column. The

Fig. 1. Locations of studied populations in the Czech Republic.

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162 Table 1 Characteristics of the mothers and infants groups examined. UH I

UH II

Prague

UL

Kolín

Liberec

Kladno

Telcˇ

N mother and child pairs

14

10

15

11

11

11

10

8

Age (y): Mean Median Ranges

26 25 20–36

28 27 22–35

26 29 23–30

26 25 18–36

26 27 21–30

25 26 20–31

24 24 18–32

24 24 20–30

Height (cm): Mean Median Ranges

167 168 158–176

166 164 163–173

168 170 158–178

165 168 153–178

167 170 156–176

169 170 157–182

168 168 155–178

168 169 158–176

Weight before pregnancy (kg): Mean Median Ranges

60 55 45–90

61 61 52–73

60 60 49–72

70 70 54–92

61 64 51–76

60 55 48–75

62 56 49–82

63 62 55–74

BMI (kg/m2) before pregnancy Mean Median Ranges

21.2 20.9 16.9–31.9

21.8 22.0 19.4–25.3

21.1 20.3 18.2–26.2

25.8 24.2 19.1–36.7

21.9 21.6 17.6–26.3

20.9 20.2 17.2–24.4

21.8 20.5 17–28.3

22.7 22 19.7–25.8

Weight before delivery (kg): Mean Median Ranges

74 72 55–107

72 72 59–90

74 73 64–100

81 80 64–101

76 73 62–95

75 67 58–93

77 74 61–100

78 76 69–94

Weight of child at birth (kg): Mean Median Ranges Gender of child (male/female):

3.3 3.3 2.3–4.0 7/8

3.3 3.4 2.3–3.6 7/3

3.3 3.4 2.1–4.3 6/9

3.3 3.5 1.8–3.9 7/4

3.4 3.5 2.9–4.1 7/4

3.0 3.1 2.2–3.6 4/7

3.3 3.3 2.8–3.9 4/6

3.7 3.7 2.7–4.5 6/2

first fraction eluted with 10 ml of dichloromethane was concentrated to 20 ll and used for PCB and OCP determination. The second fraction was eluted with 50 ml of toluene and used for analysis of PCDDs, PCDFs and non-ortho PCBs. These data were presented elsewhere (Bencko et al., 2004). The PCB and OCP analyses were carried out by GC-MS using a DB-5 column (length 60 m, i.d. 0.25 mm, 0.20 lm film) mounted in a Varian 3400 GC oven coupled to a Varian Saturn ion trap mass spectrometer. Samples were analyzed in batches of 10 with a blank. The certified cod oil standards (BCR CRM 349 and BCR CRM 598) were used for method validation. The uncertainty of measurement was ±20% at 95% confidence level. The following PCB congeners (IUPAC Nos.) were measured and summed to obtain the sum of 35 PCB congeners: 28, 47 + 48, 52, 56 + 60, 66, 74, 77, 84, 95, 101, 105, 99 + 113, 118 + 123, 126, 135, 136 + 144, 146, 149, 151, 153, 156, 157, 167, 169, 170, 171, 172, 174, 177, 178, 182 + 187, 183, 180, 189 and 193. The limit of detection (LOD) for individual PCB congeners was 0.004 ng per ml of milk volume. Analysis of OCPs targeted p,p0 - and o,p0 -dichlorodiphenyltrichloroethane (DDT), p,p0 -dichlorodiphenyldichloroethylene (DDE), hexachlorobenzene (HCB), and a-, b-, and c-hexachlorocyclohexanes (HCHs).

Based on the model (2) adjustment for maternal age and body mass index (BMI) heterogeneity as well as for inequality of child age at the moment of milk sampling, we estimated the mean 6 month exposure and its variability (using the Bayesian approach facilitated by Markov Chain Monte Carlo calculations implemented in Winbugs (Spiegelhalter et al., 2007). The data needed for the exposure calculations were obtained as follows. Distributions of maternal age and BMI and infant’s birth weight were estimated from the data collected in this study. The milk fat content t = 41 g L1 was taken from Kent et al., (2006). The WHO daily milk consumption data reported by Schutz et al. (1998) was used, i.e. 120 ml kg1 of infant’s body weight. Detailed weight data (the weight-for-age growth curve portion for the relevant age range) was taken from the most recent Czech anthropological survey (Vignerová and Lhotská, 2006; Vignerová et al., 2006). According to this survey the median body weight of male and female infants aged 0– 6 months is 3.0–8.6 kg, and 3.0–7.9, respectively. We should point out that in the Bayesian calculation we neglected variation in milk consumption and milk fat content. We assumed independence between PCB concentration and fat concentration as well as constant PCB concentration in milk fat throughout the time of breastfeeding.

3. Results 2.3. Statistics 3.1. Demographics Throughout the paper, we regard a p-value less than 0.05 as significant. Correlations were assessed with a nonparametric Spearman rank correlation test. For numerical summaries, we used mainly medians (due to the skewed nature and lognormal-like distribution of the measured concentrations). Formal statistical analyses (done both for assessing the effect of potential confounding covariates and adjusting for the covariates) used the lognormal assumption (see the model (2) description below).

The maternal and infant characteristics are summarized in Table 1. The average primiparous age was 26 years, age range 18–36 years. The average birth weight was 3.3 kg (range 1.8– 4.5 kg). No significant demographic differences (maternal age or BMI, infant birth weight or sex ratio) were found between areas. The maternal dietary habits did not differ substantially as all mothers but two lacto-ovo-vegetarians reported eating a mixed diet.

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3.2. Levels of PCBs and congener pattern Indicator PCBs 28, 118, 138, 153 and 180 were detected in all breast milk samples analyzed. PCB congeners 52 and 101 were not detected in any sample from the areas Kladno and Telcˇ (N = 6 and 8, respectively). PCB 52 was below the LOD in 10%, 13%, 27% and 47% of samples from Liberec, Prague, Kolín and UH I, respectively. In the other samples, the levels of PCBs 52 and 101 were above the LOD. Concentrations of indicator PCB congeners and selected OCPs in 90 breast milk samples are listed in Table 2. as means ± SD, medians and ranges. The sum of PCBs is expressed: (1) as the sum of the concentrations of all the 35 PCB congeners analyzed, (2) as the sum of the seven PCB indicator congeners, and (3) as the sum of the six indicator congeners without PCB 118. The obtained data distribution was right skewed. Therefore, we analyzed the data as lognormal. The concentrations of the contaminants analyzed showed a wide inter-individual variation. The sum of 35 PCB congeners ranged from 293 ng g1 lw in Telcˇ to 13 754 ng g1 lw in Uherské Hradišteˇ (UH I) for the first sampling period. The highest PCB values were found in UH I, followed by Kolín, and UL. Area-related differences in the median levels of one of the most abundant PCB indicator congener 153 are illustrated in Fig. 2. Significant age-related positive correlation was observed for the sum of 35 PCBs and the most abundant indicator congeners 138, 153 and 180. The correlation between these three indicator congeners was higher than 90%. The sum of analyzed congeners 28, 74, 118, 153, 138, 153, 170, 182, 183, and 180 constituted from 87% to 94% of the total PCB value. The levels of other PCB congeners analyzed were negligible. The most abundant indicator congeners 138, 153 and 180 contributed from 62% to 73% (arithmetic mean 67%) to the total PCB value. The only exception was UH I where the sum of the above mentioned congeners only made up about 53% of the total PCB value and the sum of the congeners 138, 153 and 180 only accounted for about 42% of total PCB value. 3.3. OCP levels Table 2 shows the levels of p,p0 -DDT, o,p0 -DDT, p,p0 -DDE, o,p0 DDE, DDE/DDT ratio, HCB, and b-HCH. The p,p0 -DDD and o,p0 DDD concentrations were very low in general and below the LOD in most breast milk samples. The concentrations of a and c-HCHs were below the LOD in almost all samples analyzed. Similarly to the PCB congeners, OCP levels varied considerably among areas. The median HCB values ranged from 357 ng g1 lw in UL to 91.8 ng g1 lw in Kladno. The concentrations of p,p0 -DDT were the highest (median 90.1 ng g1 lw) in UL again and the lowest (median 13.8 ng g1 lw) in Liberec. The median levels of p,p0 DDE, the main metabolite of DDT, reached around 600 ng g1 lw to slightly above in four out of eight study areas, with the highest median level of 678 ng g1 lw in UH 1 and the lowest median level of 169 ng g1 lw in Liberec. The DDE/DDT ratio reflects the history of DDT usage. In this study, it was the lowest in UL (median 6.95). The highest DDE/ DDT ratios were obtained in UH I and Prague (24.2 and 24.1, respectively).

163

EDIs of the sum of PCBs and of the most abundant indicator congeners in breast-fed infants are presented in Table 3. In all groups, the infant exposure to the sum of PCBs significantly exceeded the TDI of 0.4 lg kg1 bw d1 accepted in the Czech Republic (Ruprich, 2006). The highest exposure level was, as expected, obtained in UH I followed by UL. In fact, the highest infant exposure in the most exposed area (UH I) in 2000, i.e. 15 years after the ban of PCB production in former Czechoslovakia, did not differ substantially from EDI in the general population of breast-fed children before 1985 (Cˇerná and Bencko, 1999). The EDIs of OCPs presented in Table 3 shows that sum DDT did not exceed TDI of 10 lg kg1 bw d1 (FAO/WHO, 2000). EDI for HCB has not yet been precisely established; however, 0.16 lg kg1 bw d1 has been recommended as TDI with no carcinogenic effects (ICPS/WHO, 1997). This TDI was exceeded in almost all breast milk samples analyzed. For HCHs, no TDI limits have been established except for c-HCH (lindane) where an acceptable daily intake (ADI) of 5 lg kg1 bw has been recommended (FAO/WHO, 2002). However, as only the b-HCH values were above the LOD, EDI for the sum of HCHs could not be calculated. (2) We estimated PCB exposure per 6 months of breastfeeding using a formalized statistical model (Brabec et al., 2007). Because we found important effects of several covariates influencing PCB concentrations, we used the following statistical model to adjust for heterogeneity of the population under study. Specifically, we adjusted for unequal maternal age and BMI and infant’s age at maternal sampling

Y i ¼ exp½b0 þ b1 ðMAi  MAÞ þ b2 ðCAi  CAÞ þ b3 ðBMIi  BMIÞgi

gi  LNð0; r2 Þ; independently across i0 s where Yi is the particular congener’s concentration (in pg g1 lw) for ith mother–child pair, MAi the age (in years) of ith mother. Its mean across mothers in this study is MA ¼ 25:30. CAi the age (in weeks) of ith mother’s child. Its mean across mothers in this study is CA ¼ 7:30. BMIi the BMI of ith mother (computed from weight before pregnancy). Its mean across mothers in this study is BMI ¼ 21:93. b0, b1, b2, r are the unknown parameters to be estimated from observed data (i.e. measured concentrations) (Table 4) and gi is the random error. According to this model it is possible to estimate the integral infant exposure during the first 6 months of breastfeeding taking into account the above mentioned parameters. The estimates for PCB congeners 153 and 118 for the total of breast-fed infant are presented here as examples for an average infant in the group under study using this model: PCB 153: 1.55 mg (median), 0.309–7.89 mg (2.5th–97.5th percentiles) PCB 118: 0.113 mg (median), 0.025–0.53 mg (2.5th–97.5th percentiles). 4. Discussion 4.1. POP concentrations in breast milk samples

3.4. Infant exposure to PCBs and OCPs In this study we calculated the breast-fed infant exposure using two procedures: (1) We calculated EDI assuming a consumption of 120 ml of breast milk kg1 bw d1 with a lipid content of 3.5% (Schutz et al., 1998). The median value and ranges of the respective contaminant were used for the calculation.

The hexa- and hepta-chlorinated PCB congeners (PCBs 138, 153, 170, and 180) were detected in all breast milk samples. These congeners were also dominant in technical mixtures Delor 105 and 106 produced in the former Czechoslovakia (Taniyasu et al., 2003). As expected, their concentrations showed a clear rise with increasing maternal age. Very good correlation was found between the sum of 35 PCBs and PCB 153. As the most predominant PCB indicator congener in human milk, PCB 153 was used in this study

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164

Table 2 Concentrations of indicator PCB congeners and selected chlorinated pesticides (in ng g1 lw) in 90 individual human milk samples by location including basic statistical data. UH I N = 14

UH II N = 10

Prague N = 15

UL N = 11

Kolín N = 11

Liberec N = 11

Kladno N=6

Telcˇ N=8

Mean ± SD Median Ranges Mean ± SD Median Ranges n < LOD Mean ± SD Median Ranges Mean ± SD Median Ranges Mean ± SD Median Ranges Mean ± SD Median Ranges Mean ± SD Median Ranges Mean ± SD Median Ranges Mean ± SD

7.06 ± 5.52 5.07 1.33–18.97 0.69 ± 0.48 0.53 0.31–1.73 7 1.86 ± 1.10 1.57 0.57–4.39 69.9 ± 64.5 41.6 22.5–232 597 ± 444 437 162–1782 903 ± ± 680 646 274–2655 539 ± 393 455 75.5–1360 2046 ± 1494 1457 611–5814 2118 ± 1545

4.86 ± 4.95 3.03 1.55–15.5 2.87 ± 6.44 0.84 0.33–21.2 0 2.80 ± 5.19 1.26 0.55–17.5 23.4 ± 12.0 21.9 7.08–48.3 259 ± 101 232 124–410 326 ± 101 302 190–503 267 ± 130 201 171–546 863 ± 841 748 510–1383 887 ± 863

19.2 ± 15.7 16.7 0.71–47.0 1.15 ± 0.86 0.97 0.09–3.22 2 2.33 ± 1.37 2.15 0.38–4.27 34.4 ± 19.2 28.4 13.2–89.2 245 ± 103 219 86.5–496 376 ± 167 343 135–735 170 ± 116 127 61.7–464 813 ± 376 693 319–1648 848 ± 391

2.78 ± 0.77 2.63 1.71–3.90 1.16 ± 2.92 0,30 0.13–9.96 0 1.29 ± 0.62 1.15 0.59–2.40 29.1 ± 16.1 27.7 11.9–57.1 407 ± 190 363 175–686 516 ± 235 476 207–915 379 ± 198 318 142–758 1306 ± 614 1099 531–2363 1335 ± 627

3.15 ± 2.45 2.56 0.66–10.1 0.32 ± 0.15 0.30 0.15–0.62 3 0.80 ± 0.57 0.57 0.37–2.36 22.6 ± 17.6 16.9 7.22–70.1 271 ± 253 219 60.6–1013 348 ± 319 288 78.2–1279 232 ± 225 172 49.5–882 855 ± 798 701 193–3178 877 ± 814

2.09 ± 0.94 1.97 096–3.63 0.36 ± 0.26 0.24 0.12–0.88 1 0.78 ± 0.27 0.69 0.45–1.38 14.9 ± 6.00 14.0 6.12–24.4 138 ± 42.7 120 86.2–206 191 ± 59.2 177 113–280 133 ± 39.6 126 73.0–206 465 ± 140 408 274–684 480 ± 145

2.76 ± 1.45 2.22 1.41–5.00 ND

2.78 ± 0.64 2.78 1.82–3.75 ND

6 ND

8 ND

6 17.1 ± 7.48 17.3 8.74–28.0 158 ± 43.8 164 97.0–207 128 ± 44.8 142 57.1–176 127 ± 48.4 128 52.8–196 415 ± 133 441 202–556 432 ± 136

8 18.7 ± 7.37 17.5 11.2–30.0 125 ± 29.7 127 80.2–167 92.8 ± 24.5 97.5 54.1–122 80.1 ± 26.6 78.6 45.8–122 301 ± 64.2 313 191–405 320 ± 68

Median Ranges Mean ± SD Median Ranges Mean ± SD Median Ranges n < LOD Mean ± SD Median Ranges Mean ± SD Median Ranges

1495 646–6010 4897 ± 3614 3410 1448–13 754 22.5±.13.5 20.7 9.46–62.9

767 517–1412 1279 ± 491 1073 757–2139 29.9 ± ± 28.3 20.4 13.2–108

1137 544–2401 1951 ± 951 1601 780–3607 Not calculated

715 202–3257 1258 ± 1215 1036 305–4824 25.4 ± 16.3 19.9 8.70–62.7

327 204–425 488 ± 143 480 293–731 NA

185 ± 142 166 10.9–442 48.7 ± 76.8 22.2 10.7–264

263 ± 198 188 31.0–739 41.3 ± 39.2 32.6 10.4–147

418 280–701 677 ± 194 609 401–987 26.9 ± 13.8 26.0 11.4–49.6 1 147 ± 122 109 42.2–443 23.8 ± 34.4 13.8 7.15–126

460 211–565 636 ± 202 660 316–866 NA

192 ± 104 187 68.0–467 36.8 ± 33.2 28.4 11.6–141

718 332–1737 1091 ± 531 921 455–2264 35.7 ± 18.7 27.4 17.3–80.0 3 256 ± 139 217 95.5–538 38.2 ± 18.8 33.4 14.0–74.8

119 ± 98.1 91.8 45.9–303 43.7 ± 41.8 26.0 11.0–122

159 ± 101 126 54.8–316 52.8 ± 30.0 43.3 20.3–112

p,p0 -DDE

Mean ± SD Median Ranges

839 ± 527 678 320–2016

342 ± 244 297 52.9–894

754 ± 307 660 3.37–1136

595 ± 399 618 94.2–1428

261 ± 160 169 118–600

342 ± 131 410 116–437

589 ± 426 514 239–1592

sum DDT

Mean ± SD Median Ranges Mean ± ± SD Median Ranges Mean ± SD Median Ranges

884 ± 563 657 334–2045 28.6 ± 17.3 24.2 9.15–69.8 2.8 ± 1.1 2.7 1.2–4.7

391 ± 313 316 72–1158 10.6 ± 4.89 11.2 2.70–17.8 4.2 ± 1.7 4.0 1.5–7.5

792 ± 313 718 351–1210 22.6 ± 9.31 24.1 6.40–36.1 3.1 ± 1.2 3.0 1.5–5.7

636 ± 423 678 105–1479 19.3 ± 16.8 14.0 7.07–66.8 3.9 ± 2.4 3.1 1.3–8.8

284 ± 160 240 127–628 18.9 ± 11.3 18.4 1.20–44.0 4.1 ± 1.8 4.1 1.8–7.2

386 ± 149 446 135–515 13.8 ± 13.4 8.24 3.21–39.2 4.8 ± 2.1 4.0 3.3–8.9

642 ± 449 545 45 266–1704 12.3 ± 6.76 11.4 3.93–27.0 5.4 ± 1.1 5.6 3.6–6.8

Compound PCB 28

PCB 52

PCB 101

PCB 118

PCB 138

PCB 153

PCB 180

Sum of six indicator PCBs (without 118) Sum of seven indicator PCBs

R35 PCBs analyzed

b-HCH

HCB

p,p0 -DDT

DDE/DDT ratio

Lipid conc. (%)

7 748 ± 729 357 184–1717 97.7 ± 23.3 90.1 76.4–134 NA = 5 821 ± 559 628 349–1911 NA = 5 918 ± 579 727 426–2046 7.94 ± 3.44 6.95 4.51–14.2 3.8 ± 1.0 3.7 2.5–6.0

ND – not detected (levels below the LOD). NA – not analyzed (because of insufficient volume of samples).

as the indicator of both the PCB concentrations in breast milk and infant exposure. In addition to high inter-individual variation, significant local differences were found in PCB congener levels. The highest PCB concentrations of the sum of 35 PCB congeners as well as indicator congeners 138, 153, and 180 in UH I, i.e. in the vicinity of a former PCB paint factory confirmed the existence of the hot spot first recognized in 1996 (WHO/ECEH, 1996, Bencko et al., 1998). As UH I differed from other study areas in the PCB congener pattern, we suspected further PCB congeners other than those screened for in this study of contributing to the exposure and body burden in

the Old Town of Uherské Hradišteˇ (UH I). The paint additive Delor 106 used in the former local factory consisted mostly of hexa- and hepta-PCB congeners (Taniyasu et al., 2003), some of which (e.g. PCB 128 to 134, 137, 141, 159, 171, 173, and 174) were not analyzed in this study. The PCB levels in breast milk from mothers living in the same city but far from the source of pollution (UH II) were comparable with those obtained in other study areas. These results suggest that local PCB pollution was spatially limited. The 3rd WHO-coordinated exposure study conducted in 2000 also documented that the PCB levels in the Czech breast milk pooled samples were close to the upper limit of the range reported

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165

700 646 600

476

500

400 343 302 300

288

202 177

200

168 142 97, 5

100

0 Uherské Hradišt I

Ústí n. L.

Praha

Uherské Hradišt II

Kolín

Liberec

Kladno

Te l

CZ-HBM 2000

3rd WHO study 2000

Fig. 2. Median levels of the PCB indicator congener 153 (ng g1 lw) in breast milk samples analyzed in cross-sectional study 1999–2000; comparison with the CZ-HBM and the 3rd WHO study results in 2000. CZ-HBM: data from the Czech Human Biomonitoring Project based on the analysis of 408 breast milk samples collected in 2000 in four urban areas of the Czech Republic (Annual Report 2000, Nat. Inst. Publ. Health, Prague, 2001, in Czech) www.szu.cz 3rd WHO study: median value is based on the analysis of three pooled breast milk samples collected in 1999 and 2000 in urban area Kladno, Liberec and Uherské Hradišteˇ.

Table 3 Estimated exposure of breast-fed infants to PCBs and OCPs expressed in lg kg1 of body weight (assumed lipid content 3.5% and a daily consumption of 120 ml of breast milk kg1 bw according to Schutz et al. (1998)). Compound PCB 118 PCB 138 PCB 153 PCB 180 Sum of six indicator PCBs (without 118) Sum of seven indicator PCBs

R35 PCBs analyzed b-HCH HCB p,p0 -DDT p,p0 -DDE sum DDT

Median Ranges Median Ranges Median Ranges Median Ranges Median Ranges Median Ranges Median Ranges Median Ranges Median Ranges Median Ranges Median Ranges Median Ranges

UH I N = 14

UH II N = 10

Prague N = 15

UL N = 11

Kolín N = 11

Liberec N = 11

Kladno N=6

Telcˇ N=8

0.17 0.09–0.97 1.84 0.68–7.49 2.71 1.15–11.2 1.91 0.32–5.71 6.12 2.57–24.4 6.28 2.71–25.2 14.3 6.08–57.8 0.09 0.04–0.24 0.79 0.29–1.96 0.12 0.05–0.59 2.85 1.35–8.47 2.76 1.40–8.59

0.09 0.03–0.20 0.97 0.52–1.72 1.27 0.80–2.11 0.84 0.72–2.29 3.14 2.14–5.81 3.22 2.17–5.93 4.51 3.18–8.98 0.09 00.6–0.45 0.70 0.05–1.86 0.09 0.04–1.11 1.25 0.22–3.75 1.34 0.30–4.86

0.12 0.06–0.37 0.92 0.36–2.08 1.44 0.57–3.08 0.53 0.26–1.95 2.91 1.34–6.92 3.02 1.40–7.30 3.87 1.91–9.51 0.11 0.07–0.34 0.91 0.40–2.26 0.14 0.06–0.31 2.77 1.42–4.77 3.01 1.48–5.08

0.12 0.05–0.24 1.52 0.73–2.88 2.00 0.87–3.84 1.34 0.60–3.19 4.62 2.23–9.92 4.77 2.29–10.1 6.73 3.28–15.1 Not calculated

0.07 0.03–0.29 0.92 0.25–4.26 1.21 0.33–5.37 0.72 0.21–3.71 2.94 0.81–13.4 3.00 0.85–13.7 4.35 1.28–20.3 0.08 0.04–0.26 0.79 0.13–3.10 0.14 0.04–0.62 2.60 0.40–6.00 2.85 0.44–6.21

0.06 0.03–0.10 0.50 0.36–0.86 0.74 0.47–1.18 0.53 0.31–0.87 1.71 1.15–2.87 1.75 1.18–2.95 2.56 1.69–4.15 0.11 0.05–0.21 0.46 0.18–1.86 0.06 0.03–0.53 0.71 0.5–2.52 1.01 0.53–2.64

0.07 0.04–0.12 0.69 0.38–0.87 0.60 0.24–0.74 0.54 0.22–0.82 1.85 0.85–2.34 1.93 0.88–2.37 2.77 1.33–3.64 NA

0.07 0.05-.013 0.53 0.34–0.70 0.41 0.23–0.51 0.33 0.19–0.51 1.32 0.80–1.70 1.38 0.86–1.78 2.01 1.23–3.07 NA

0.39 0.19–1.27 0.11 0.05–0.51 1.72 0.49–1.84 1.87 0.57–2.16

0.53 0.23–1.33 0.18 0.09–0.47 2.16 1.00–6.69 2.29 1.12–7.16

1.50 0.77–7.21 0.38 0.32–0.56 2.64 1.46–8.03 3.05 1.79–8.59

NA – not analyzed (because of insufficient volume of samples).

for the most industrialized European countries (Malisch and Van Leeuwen, 2003; Colles et al., 2008). The median value (502 ng g1 lw) and ranges (496–1009 ng g1 lw) of the sum of indicator PCBs from the three pooled samples (Malisch and Van Leeuwen, 2003) with the upper limit concentration of 1009 ng g1 lw being reported for Uherské Hradišteˇ, were compa-

rable with our data obtained for individual breast milk samples. The PCBs concentrations detected in our study, at least in several areas, are even higher than those reported for the exposed population in Slovakia (Petrík et al., 2001; Malisch and Van Leeuwen, 2003). The decline rate observed in the late 1990s for the PCB background concentrations in breast milk in urban areas of the Czech

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166 Table 4 Estimated parameters in MLE and Bayesian models. MLE

PCB 118 + 123 b0 b1 b2 b3

r PCB 153 b0 b1 b2 b3

r

Bayesian estimate

Estimate

Uncertainty (standard error)

Estimate (posterior mean)

Uncertainty (posterior standard deviation)

10.1221 0.0604 0.0294 0.0145 0.6917

0.0901 0.0242 0.0134 0.0235

10.1200 0.0601 0.0293 0.0151 0.7023

0.0926 0.0247 0.0135 0.0239 0.0675

12.7293 0.0355 0.0238 0.0161 0.7633

0.0994 0.0267 0.0148 0.0260

12.7200 0.0351 0.0237 0.0168 0.7751

0.1022 0.0272 0.0149 0.0745 0.0745

MLE = Maximum likelihood estimate.

Republic (Cˇerná et al., 2003) is slowing down or even has recently stopped declining (Cˇerná et al., 2007; Malisch, 2006). p,p0 -DDT, p,p0 -DDE, HCB, and b-HCH were clearly quantified win each breast milk sample. DDT was first detected in human milk in 1951 (Laug et al., 1951). The use of this pesticide has been banned in Europe including the Czech Republic since the early 1970s. Therefore, its concentration in the human body including breast ˇ erná, 1995). However, this fammilk has a long downward trend (C ily of xenobiotics and their metabolites still exist and circulate in the food chain due to soil persistence, inappropriate waste disposal etc. and thus are detectable even in recent breast milk samples. The median levels of p,p0 -DDT in our study ranged between 13.8 and 90.1 ng g1 lw. The value was twice as high in UL than in other areas and the DDE to DDT ratio was the lowest in UL, most probably due to old local chemical industry stockpiles. The results possibly reflect relatively recent exposure to DDT in this area, as the relative proportions of DDT and DDE can be indicative of the length of time since exposure. However, this assumption is based on a limited number of samples analyzed because 5 out of 11 milk samples in this area were too small to be screened for all analytes. Furthermore, no significant differences were observed in the DDT concentrations in breast milk from four urban areas including UL in the CZ-HBM Project (Kliment et al., 2000). The DDT levels in this study (except for UL) are lower than reported in Northern Russia or Poland (Szyrwin´ska and Lulek, 2007), but higher than those detected, for example, in Norway (Polder et al., 2008a,b), or Spain (Bordajandi et al., 2008). Germany has reported DDT levels comparable to those in our study (Zietz et al., 2008). The p,p0 -DDE was the dominant compound among the OCPs analyzed. The levels of p,p0 -DDE, the major metabolite of DDT, reflect the historical use of DDT and the following food chain contamination. The levels of sum DDTs in this study were slightly lower than those previously found in the Czech breast milk samples (Schoula et al., 1996). HCB is a persistent organochlorine chemical that is both a pesticide and an industrial by-product. The importance of industrial sources of this pollutant is documented by the highest HCB median level (357 ng g1 lw) in UL. The increased HCB exposure in UL is likely to be connected with both the historical and recent chemical industry production in this area. In the remaining study areas, the median HCB levels ranged between 91.8 ng g1 lw in Kladno and 217 ng g1 lw in Prague. Similar results (median 249 ng g1 lw) were reported for breast milk from 26 primiparas in Prague sampled in 2000 (Cˇajka and Hajšlová, 2003). However, even in other of our study areas, the HCB concentrations were about three times higher than those found in 2000–2002 in Northern Russia (Polder et al., 2008a) or Poland in 2003 (Szyrwin´ska and Lulek, 2007), and about one order of magnitude higher than reported in Norway

(Polder et al., 2008b) or in Northern Germany (Zietz et al., 2008). Since HCB is believed to have a dioxin-like effect with a TEF = 0.0001 (Van Birgelen, 1998), the incremental exposure of the Czech population to HCB might contribute to adverse endocrine-disrupting effects. Nevertheless, a substantial downward trend in HCB levels has been recently observed in the Czech breast milk within the CZ-HBM Project, with a median value of 66 ng g1 lw recorded in 2007 (Summary Annual Report, 2008). HCH, a mixture of eight isomers, has insecticidal activity. Lindane (c-HCH) was also widely used in the Czech Republic. In this study, no detectable concentrations were found in breast milk samples, as a- and c-HCH are converted by biotransformation into the b-isomer in the body (Solomon and Weiss, 2002). Therefore, only b-HCH, the predominant persistent isomer, was detected, with the levels ranging between 19.9 and 27.4 ng g1 lw. However, b-HCH was not analyzed in the samples from Kladno and Telcˇ because of their small volume and the descriptive statistics was not calculated in UL where the b-HCH levels were below the LOD in 7 out of 11 samples. The median b-HCH concentrations in this study were similar to those reported in Northern Germany (Zietz et al., 2008) or in Poland (Szyrwin´ska and Lulek, 2007), about one order of magnitude lower compared to the data from Northern Russia in 2000 (Polder et al., 2008a), and about twice as high as found in Norway (Polder et al., 2008b). 4.2. Infant exposure In this study we calculated the EDI for a breast-fed infant, assuming a consumption of 120 ml of breast milk kg1 bw d1 with a lipid content of 3.5% (Schutz et al., 1998). However, others used different entry data for the EDI calculation, e.g. Polder et al. (2008b) assumed a daily consumption of 700 ml of breast milk and a body weight of 5 kg while Focant et al. (2002) departed from the data of Paul et al. (1988), i.e. a 7 kg infant receiving 600 ml of 3% fat breast milk per day. The calculation of the cumulative exposure during the 6 months of breastfeeding according the model used in this study (Brabec et al., 2007) takes into account the concentrations of the chemicals obtained in this study, the amount that the children consume, the 6 months of exclusive breastfeeding recommended by WHO, and the infant growth rate based on the Czech data. We have calculated the median cumulative 6 month exposure to the most abundant indicator PCB congener 153 to be about 1550 lg. For comparison, Dekoning and Karmaus (2000) estimated a PCB intake of 1528 lg in 13 weeks of nursing based on the sum of PCB congeners 118, 138, 153 and 180 of 550 ng g1 lw in breast milk (Lanting et al., 1998).

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EDIs for PCBs in this study were about two orders of magnitudes higher than those in Poland for the same time period 2000–2001 and about one order of magnitude higher than reported in Lower Saxony, Germany (Zietz et al., 2008), in Oslo (Johansen et al., 1994) or in Canada (Hoover, 1999). For DDT, a TDI of 10 lg kg1 bw d1 (FAO/WHO, 2000) was used. This TDI was not exceeded by EDI for any of our breast milk samples. However, at least in one infant per area in UH I and UL, EDI reached about 8 lg kg1 bw d1. EDIs for DDT sum in this study were comparable with the Polish data (Szyrwin´ska and Lulek, 2007), but they were slightly higher than in Canada (Hoover, 1999).

167

5. Conclusions Generally, the PCB levels detected in the Czech human milk samples, at least in those from industrial areas, are still among the highest in Europe. We found exposure of breast-fed infants to PCBs in the Czech Republic to be close to the upper limit of the European range whereas exposure to OCPs is comparable with the data reported in other European countries. The proposed statistical model for estimating infant exposure may help in health risk assessment and management. The remedial strategies should be directed towards reducing the PCBs intake through the food chain, especially in the hot spot areas. Acknowledgments

4.3. Possible adverse health effects of breast-fed infant exposure The health consequences of the potential temporary elevation of infant exposure via breastfeeding are uncertain. No health based guidance value for humans have been established yet for non-dioxin-like PCBs in the body (EFSA, 2005). Environmental exposure to PCBs has been suggested to be associated with subtle developmental changes in human infants. However, the effect was only seen in the most highly exposed infants (Brouwer et al., 1998; Jorissen, 2007). The association between PCB breast milk level and behavioral changes was observed for concentration in excess of 1.25 mg kg1 lw (Newsome et al., 1995). Considering that the median PCB concentrations in this study were higher than 1 mg kg1 lw in four out of seven urban areas, possible behavioral adverse effects could not be excluded. However, TDIs are based on a lifelong intake and are not directly applicable to breast-fed infants because of the relatively short nursing period and the rapid increase in amount of the infant adipose tissue (Baars et al., 2004). The intake of PCBs during a 6 month nursing period was estimated to be less than 5% of the lifetime intake (WHO, 1988). Therefore, it is to believe that EDIs identified in the Czech breast milk, even those higher than TDIs, probably do not pose unacceptable risk to infants provided that the lifelong-average intake is lover than TDI. However, the varying developmental organ sensitivity windows have to be taken into account. Breastfeeding is widely recognized as the best way of infant nutrition (LaKind et al., 2004; Pohl et al., 2004) and it is generally accepted that breastfeeding should be encouraged.

4.4. Advantages and limitations of the study The advantage of this study is that individualized data from seven different urban areas are available. These results were obtained almost contemporaneously with the pooled sample data of the 3rd WHO-coordinated study. The limitation of this study are the relatively small numbers of samples per urban area which do not allow statistical analysis of differences between areas, inter-individual variability in maternal body burden and outliers and thus the conclusions cannot be generalized to the general population of the Czech Republic. As the individualized breast milk fat percentages are known, our study would make it possible to calculate exposure of each infant. However, intraindividual variation in fat content was observed during the breastfeeding period, depending on circadian rhythm, acute nutritional status, sampling time of (at the beginning or at the end of breastfeeding), etc. In breast-fed infants, the PCB body burden is expected to rise rapidly from birth to 3 months of age and to sharply decrease immediately following weaning (LaKind et al., 2004).

The authors acknowledge all mothers who participate for this study, as well as the fieldworkers and other personnel of participating health facilities. The study was supported by the Ministry of the Environment, Research Project VaV 520/699 and partly by the Institutional Research Plan AV0Z10300504 ‘‘Computer Science for the Information Society: Models, Algorithms”, CASCADE NoE, EU contract FOOD-CT-2004-506319 and INTARESE, EU contract GOCE-CT-2005-018385. References Baars, A.J., Bakker, M.I., Baumann, R.A., Boon, P.E., Freier, J.I., Hoogenboom, L.A.P., Hooger Brugge, R., van Klaveren, J.D., Liem, A.K.D., Traag, W.A., de Vries, J., 2004. Dioxins, dioxin-like PCBs and non-dioxin like PCBs in foodstuffs: occurrence and dietary intake in The Netherlands. Toxicol. Lett. 151, 51–61. Bencko, V., Skulová, Z., Krecˇmerová, M., Djien Liem, A.K., 1998. Selected polyhalogenated hydrocarbons in breast milk. Toxicol. Lett. 96 (97), 341–345. ˇ erná, M., Jech, L., Šmíd, J., 2004. Exposure of breast-fed children in the Bencko, V., C Czech Republic to PCDDs, PCDFs, and dioxin like PCBs. Environ. Toxicol. Pharmacol. 18, 83–90. Bordajandi, L.R., Abad, E., Gonzáles, M.J., 2008. Occurrence of PCBs, PCDD/Fs, PBDEs and DDTs in Spanish breast milk: enantiomeric fraction of chiral PCBs. Chemosphere 70, 567–575. Brabec, M., Bencko, V., Cˇerná, M., 2007. Statistical issues in modeling exposure of breast-fed children to PCDDs and PCDFs. In: Poster for: Cascade 3rd Annual Meeting, Helsinki, Finland, April 17–19, p 14 (Book of Abstracts). Brouwer, A., Ahlborg, U.G., van Leeuwen, F.X.R., Feeley, M.M., 1998. Report of the WHO working group on the assessment of health risks for human infants from exposure to PCDDs, PCDFs and PCBs. Chemosphere 37, 1637–1643. ˇ ajka, T., Hajšlová, J., 2003. Polychlorinated biphenyls and organochlorine pesticides C in human milk from the locality Prague, Czech Republic: a comparative study. Bull. Environ. Contam. Toxicol. 70, 913–919. ˇ erná, M., 1995. Exposure of our population to contaminating substances in the C food chain – results published in 1980–1992. Hygiena 40 (3), 180–185 (in Czech). ˇ erná, M., Bencko, V., 1999. Polyhalogenated hydrocarbons: body burden of the C Czech and Slovak populations. I. Polychlorinated biphenyls. Cent. Eur. J. Publ. Health 7, 67–71. ˇ erná, M., Svobodník, J., C ˇ ízˇková, M., Kry´sl, S., Šmíd, J., 1999. Levels of PCBs, PCDDs C and PCDFs in human milk of mothers living in four districts of the Czech Republic. Cent. Eur. J. Publ. Health 8, 24–25. ˇ erná, M., Šmíd, J., Svobodník, J., Grabic, R., Crhová, Š., Kubínová, R., 2003. C Monitoring of selected polyhalogenated hydrocarbons in breast milk: Czech Republic, 1994–2001. Fresenius Environ. Bull. 12, 203–207. ˇ erná, M., Speˇvácˇková, V., Batáriová, A., Šmíd, J., C ˇ ejchanová, M., Ocˇadlíková, D., C Bavorová, H., Beneš, B., Kubínová, R., 2007. Human biomonitoring in the Czech Republic. Int. J. Hyg. Environ. Health 210, 495–499. Colles, A., Koppen, G., Hanot, V., Nelen, V., Dewolf, M.-C., Noël, E., Malisch, R., Kotz, A., Kypke, K., Biot, P., Vinkx, Ch., Schoeters, G., 2008. Fourth WHO-coordinated survey of human milk for persistent organic pollutants (POPs): Belgian results. Chemosphere 74, 907–914. Dekoning, E.P., Karmaus, W., 2000. PCB exposure in utero and via breast milk. A review. J. Exp. Anal. Environ. Epidemiol. 10, 285–293. EFSA, 2005. Opinion of the Scientific Panel on Contaminants in the Food Chain Related to the Presence of Non Dioxin-like Polychlorinated Biphenyls (PCB) in Feed and Food. Question Number: EFSA-Q-2003-114. EPA, 1994. Method 1613 Tetra through Octa-Chlorinated Dioxins and Furans by Isotope Dilution HRGC/HRMS. FAO/WHO, 2000. Joint FAO/WHO Meeting on Pesticide Residue, 2000. FAO/WHO, 2002. Joint FAO/WHO meeting on pesticide residue, 2000. Focant, J.-F., Pirard, C., Thielen, C., De Pauw, E., 2002. Levels and profiles of PCDDs, PCDFs and cPCBs in Belgian breast milk estimation of infant intake. Chemosphere 45, 763–770.

168

M. Cˇerná et al. / Chemosphere 78 (2010) 160–168

Fürst, P., Fürst, C., Wilmers, K., 1994. Human milk as a bioindicator for body burden of PCDDs, PCDFs, organochlorine pesticides, and PCBs. Environ. Health Perspect. 102 (Suppl. 1), 187–193. Grandjean, P., Weihe, B., Needham, L.L., Burse, V.W., Patterson Jr., D.G., Sampson, E.J., Jørgensen, P.J., Vahter, M., 1995. Relation of a seafood diet to mercury, selenium, arsenic, and polychlorinated biphenyls and other organochlorine concentrations in human milk. Environ. Res. 71, 29–39. Hoover, S.M., 1999. Exposure to persistent organochlorines in Canadian breast milk: a probabilistic assessment. Risk Anal. 19 (4), 527–545. ICPS/WHO: Environmental Health Criteria 1997, 195, Geneva. Johansen, H.R., Becher, J., Polder, A., Skaare, J.U., 1994. Congener-specific determination of polychlorinated biphenyls and organochlorine pesticides in human milk from Norwegian mothers living in Oslo. J. Toxicol. Environ. Health 42, 157–171. Jorissen, J., 2007. Literature review. Outcomes associated with postnatal exposure to polychlorinated biphenyls (PCBs) via breast milk. Adv. Neonatal Care 7, 230– 237. Kent, J.C., Mitoulas, L.R., Cregan, M.D., Ramsay, D.T., Doherty, D.A., Hartmann, P.E., 2006. Volume and frequency of breastfeeding and fat content of breast milk throughout the day. Pediatrics 117, e387–e395. Kliment, V., Kubínová, R., Kazmarová, H., Havlík, B., Šišma, P., Ruprich, J., Cˇerná, M., Kodl, M., 1997. System of monitoring the environmental impact on population health of the Czech Republic. Cent. Eur. J. Publ. Health 5, 107–116. ˇ erná, M., Kliment, V., Kubínová, R., Kazmarová, Kratzer, K., Šišma, P., Ruprich, J., C Gregu˚rková, M., 2000. Five years of the system of monitoring the environmental impact on population health of the Czech Republic. Cent. Eur. J. Publ. Health 8 (4), 198–205. LaKind, J.S., Wilkins, A.A., Berlin Jr., C.M., 2004. Environmental chemicals in human milk: a review of levels, infant exposures and health, and guidance for future research. Toxicol. Appl. Pharmcol. 198, 184–208. Lanting, C.I., Fidler, V., Huisman, M., Boersma, E.R., 1998. Determinants of polychlorinated biphenyl levels in plasma from 42-month-old children. Arch. Environ. Contam. Toxicol. 35, 135–139. Laug, E.P., Kunze, F.M., Pricket, C.S., 1951. Occurrence of DDT in human fat and milk. Arch. Ind. Hyg. 3, 245–246. Lignell, S., Aune, M., Darnerud, P.O., Cnattingius, S., Glynn, A., 2009. Persistent organochlorine and organobromine compounds in mother’s milk from Sweden 1996–2006: compound-specific temporal trends. Environ. Health 109, 760–767. Malisch, R., 2006. Fourth WHO-Coordinated Survey of Human Milk for Persistent Organic Pollutants. Report of Status and Results as Available on 11.08.06. Malisch, R., Van Leeuwen, F.X.R., 2003. Results of the WHO-coordinated exposure study on the levels of PCBs, PCDDs, and PCDFs in human milk. Organohalogen Compd., 60–65. Dioxin 2003, Boston, MA. Newsome, W.H., Davies, D., Doucet, J., 1995. PCB and organochlorine pesticides in Canadian human milk – 1992. Chemosphere 30 (11), 2143–2153. Paul, A.A., Black, A.E., Evans, J., Cole, T.J., Whitehead, R.G., 1988. Breast milk intake and growth in infants from two to ten months. J. Hum. Nutr. Diet 1, 437–450. Pavúk, M., Cerman, J.R., Lunch, C.F., Schecter, A., Petrík, J., Chovancová, J., Kocˇan, A., 2004. Environmental exposure to PCBs and cancer incidence in eastern Slovakia. Chemosphere 54, 1509–1520. Petrík, J., Drobná, B., Kocˇan, A., Chovancová, J., Pavúk, M., 2001. Polychlorinated biphenyls in human milk from Slovak mothers. Fresenius Environ. Bull. 10 (4), 342–348. Pohl, H.R., McClure, P., Rosa, C.T.D., 2004. Persistent chemicals found in breast milk and their possible interactions. Environ. Toxicol. Pharmacol. 18, 259–266.

Polder, A., Gabrielsen, G.W., Odland, J.Ø., Savinova, T.N., Tkachev, A., Løken, K.B., Skaare, J.U., 2008a. Spatial and temporal changes of chlorinated pesticides, PCBs, Dioxins (PCDDs/PCDFs) and brominated flame retardants in human breast milk from Northern Russia. Sci. Total Environ. 391, 41–54. Polder, A., Thomsen, C., Lindström, G., Loken, K.B., Skaare, J.U., 2008b. Levels and temporal trends of chlorinated pesticides, polychlorinated biphenyls and brominated flame retardants in individual human breast milk samples from Northern and Southern Norway. Chemosphere 73, 14–23. Ruprich, J., 2006. Health Consequences of the Human Exposure to Foreign Substances from Food Chains in 2005. Environmental Health Monitoring System, Annual Report, Nat. Inst. Publ. Health, Prague (in Czech). Schade, G., Heinzow, H., 1998. Organochlorine pesticides and polychlorinated biphenyls in human milk of mothers living in northern Germany: current extent of contamination, time trend from 1986 to 1997 and factors that influence the level of contamination. Sci. Total Environ. 215, 31–39. Schoula, R., Hajšlová, J., Bencko, V., Poustka, J., Holadová, K., Vízek, V., 1996. Occurrence of persistent organochlorine contaminants in human milk collected in several regions of the Czech Republic. Chemosphere 33 (8), 1481–1494. Schutz, D., Moy, G.G., Kaferstein, F.K., 1998. GEMS/Food International Dietary Survey: Infant Exposure to Certain Organochlorine Contaminants from Breast Milk – a Risk Assessment. Food Safety Issues, WHO. Solomon, G.M., Weiss, P.M., 2002. Chemical contaminants in breast milk: time trends and regional variability. Environ. Health Perspect. 110, A339–A347. Spiegelhalter, D.,Thomas, A., Best, N., Lunn, D., 2007. WinBugs User Manual. Version 1.4.3. Summary Annual Report, 2008. Environmental Health Monitoring System in the Czech Republic, Nat. Inst. Publ. Health, Prague, 2009. Szyrwin´ska, K., Lulek, J., 2007. Exposure to specific polychlorinated biphenyls and some chlorinated pesticides via breast milk in Poland. Chemosphere 66, 1895– 1903. Taniyasu, S., Kannan, K., Holoubek, I., Ansorgova, A., Horri, Y., Hanari, N., Yamashita, N., Aldous, K.M., 2003. Isomer-specific analysis of chlorinated biphenyls, naphtalenes and dibenzofurans in Delor: polychlorinated biphenyl preparations from the former Czechoslovakia. Environ. Pollut. 126, 169– 178. Van Birgelen, A.P.J.M., 1998. Hexachlorobenzene as a possible contributor to the dioxin activity of human milk. Environ. Health Perspect. 106, 683–688. Vignerová, J., Lhotská, L., 2006. A fresh look at growth assessment of infants and young children in the Czech Republic in context of international developments. Cent. Eur. J. Publ. Health 14, 97–100. Vignerová, J, Riedlová, J., Bláha, P., Kebzová, J., Krejcˇovsky´, L., Brabec, M., Hrušková, M., 2006. 6th Nation-Wide Anthropological Survey of Children and Adolescents, 2001 Czech Republic. Summary Results. Charles Univ., Fac. of Nat. Sci and Nat. Inst. Publ. Health, Prague, 2006. ISBN 80-86561-30-5. WHO, 1988. Environmental Health 29. PCBs, PCDDs and PCDFs in Breast Milk. Assessment of Health Risks. WHO, Copenhagen. WHO/ECEH, 1996. Levels of PCBs, PCDDs and PCDFs in Human Milk. Second Round of WHO-Coordinated Exposure Study. Environmental Health in Europe Series 3, p. 121. Zietz, B.P., Hoopmann, M., Funcke, M., Huppmann, R., Xuchenwirth, R., Gierden, E., 2008. Long-term biomonitoring of polychlorinated biphenyls and organochlorine pesticides in human milk from mothers living in northern Germany. Int. J. Hyg. Environ. Health 211, 624–638.