PCBs and other organochlorines in human tissue samples from the Welsh population: II—milk

Environmental Pollution 84 (1994) 79 87

PCBs A N D O T H E R O R G A N O C H L O R I N E S IN H U M A N T I S S U E S A M P L E S F R O M T H E W E L S H P O P U L A T I O N : II M I L K Raquel Duarte-Davidson, Susan C. Wilson & Kevin C. Jones* Institute of Environmental and Biological Sciences, Lancaster University, Lancaster, UK, LA1 4 YQ

(Received 15 June 1992: accepted 17 November 1992)

portant part of this relatively large survey is that it included, where possible, the collection of additional data on the donors age, body weight, smoking and eating habits, and area of residence, as well as information on the number of children and total lactation period. This information was used to investigate correlations between the above variables and the YPCB and Z D D T concentrations found in the milk of the donors. Individual PCB congeners disperse, degrade, bioaccumulate and metabolise at different rates in the environment. PCB toxicity and persistence is also congener specific and dependent on the molecular configuration of each congener. The congener distribution pattern found in the samples was therefore also examined, and comparisons were made between the pattern distribution of human milk and adipose tissue from the Welsh population (Duarte-Davidson et al., 1994).

Abstract Polyehlorinated biphenyl ( P C B ) and Y D D T (i.e. p,p'D D T + p,p'-DDE + p,p'-DDD) concentrations were determined f r o m the analysis o f 115 Welsh breast milk samples collected in 1990 and 1991. Fifty P C B congeners were screened, o f which 24 were identified in most samph, s. The P C B congener pattern was consistent between individual milk samples, with IUPA C congeners 28, 138, 153 and 180 being the most abundant and accounting jor an average of 50% o f the Z P C B concentrations determined. P C B concentrations varied between 2 and 70 ng g i whole ntilk, were positive O, correlated with age, and negativeO' correlated with the total lactation period and with the percent lipid content o f the milk. PCB pattern distributions d(ff~,red between milk and adipose tissue samples. Human milk had a higher proportion o f tri- (18 and 28), tetra- (44, 52 and 66) and pentaehlorinated biphenyls (101) compared to human adipose tissue. Y~DDT concentrations ranged J?ont 0.3 to 71 ng g ~ o f whole ntilk, with p,p'-DDE eontributing towards an average o f 92% o f the Y D D T concentrations. Z D D T levels were also positiveO, correlated with age and negativeO, associated with the lactation period, though these correlations were rather weak. No sign~'eant d('fferenees in the Y P C B and Y~DDT concentrations were noted between milk samples .from donors living in rural and urban locations, or between the subjects' body weight, smoking habits or diet.

MATERIALS AND METHODS Sample collection and storage One hundred and fifteen human milk samples were collected by individual mothers through 1990 and early 1991. Contact was made with 10~12 gynaecologists/ sisters from different maternity units throughout the Principality, covering rural, semi-rural and urban locations. A map of the sampling areas is shown in Fig. 1. All mothers that donated the milk samples were chosen randomly. They were all provided with the necessary pre-cleaned glass vials and questionnaires, together with instructions on how to collect and store the samples. They were asked to collect approximately 50-100mls of human milk by expressing the milk manually into the clean glass container. The samples were then kept frozen at - 2 0 ° C until required for analysis. Questionnaires were given to each mother to obtain information which included their age and body weight, number of children, total period of breastfeeding, diet and permanent address/post-code.

INTRODUCTION The presence of significant levels of halogenated aromatic hydrocarbons in human milk has raised concerns over the potential exposure of breast-fed infants to these compounds (WHO, 1988). Due to their lipophilic nature, these compounds tend to accumulate in human fatty tissues, and may be excreted in breast milk during lactation. The aim of this survey was to determine the typical levels of polychlorinated biphenyls ( P C B s ) p , p ' - D D T and its metabolites, p,p'-DDD and p,p'-DDE, in breast milk from the contemporary Welsh population. An im-

Sample preparation and analysis All samples were homogenised by shaking the milk for at least 20 rain. The milk was then freeze-dried for a minimum period of 48 h prior to analysis. Approximately 2 g of freeze-dried milk was then mixed with anhydrous sodium sulphate to bind any water that

* Author to whom correspondence should be addressed. Environ. Pollut. 0269-7491/94/$07.00 © 1994 Elsevier Science Ltd, England. Printed in Great Britain

79

R. Duarte-David~on, S. C. Wilson, K. C. Jones

80

whole milk for p,p'-DDT and its metabolites (approximately equivalent to 0-1 ng g Llipid).

~ I[~'MXw

ATLANTIC

Statistical analysis All statistical analysis was run in SYSTAT on a Macintosh SE/30 and carried out on values expressed in ng g~ lipid. The statistical tests used have been described previously (Duarte-Davidson et el., 1994).

h

F~NCE

• P~zxs

VERPOOL / /

(~e) ~

Wrexham

k DolgeUao ~ " (MB)

-

IRISH SEA ....

J

/

~oAberystwyth (MA)

WALES Llandrindod • Wells (MC)

~

MILFORD i HAVEN ~a~ -~r~- ~ ~

Abergavenny (MoK) (MF) Pontypridd ( M G 2 ) ~

Swansea

Mu ..ewport//

BRISTOL

Fig. 1. Map of sampling sites. might remain in the sample. Samples were analysed for 50 individual PCB congeners and Z D D T following the method described by Duarte-Davidson et el. (1991). This involved Soxhlet extraction with 170 ml n-hexane for a minimum period of 8 h, lipid determination, sulphuric acid digestion and column chromatography with alumina used as an adsorbent. Residues were then quantified as described previously (Duarte-Davidson et at., 1994). Surrogate PCB standards (congener IUPAC nos 40, 155 and 185) (Ballschmiter & Zell, 1980) were added to each sample and blank prior to extraction to monitor analytical recoveries. Mean (+ standard deviation) recoveries for these congeners were 72 (+ 11), 75 (+ 12) and 85 (+ 17)°/7, respectively, recoveries increasing with increased level of chlorination. All samples were analysed in replicate, and all replicate congeners fell within the relative standard deviation precisions levels in the quality control protocol specified previously (see Duarte-Davidson et el., 1994). Detection limits varied for individual PCB congeners and p,p'-DDT, p,p'-DDE and p,p'-DDD, but were in the order of 5 pg g~ of whole milk (approximately equivalent to 40 pg g ~ lipid) for PCBs and 0.01 ng g l

General linear model ( G L M ) The purpose of the G L M was to determine whether factors such as age, body weight, total period of breastfeeding, number of children, address of residence, occupation of the mother, and percent lipid content in the mother's human milk are good predictors of the EPCB and Z D D T concentrations found in the milk samples analysed. A backward elimination technique was used to remove any variable that had no significant contribution towards predicting ZPCB and E D D T levels. Principal components analysis ( PCA ) PCA was used to assess the similarity of the individual PCB congener patterns between different mothers in the Welsh population, and to assess any differences that may exist between the congener patterns in milk and adipose tissue from different Welsh individuals. R E S U L T S AND DISCUSSION The mother's ages ranged between 18 and 41 years, with a mean of 28; their body weight varied from 41 to 111 kg with a mean of 65 kg; the maximum number of children was eight; breast-feeding varied from 0.43 up to 232 weeks, with an average of 28 weeks; milk lipid content was 2.6 "/,, ranging from 0.12 to 6.8%; samples were collected from 10 maternity hospitals throughout Wales to include rural (Aberystwyth (MA), Dolgellau (MB), Llandridrod Wells (MC), and Rhyl (MD)), semirural (Abergavenny (MK), and Wrexham (ME) and urban (Pontypridd (M J), Pontypool (MG2), Swansea (MF), and Newport (MGI)) districts (Fig. 1). Crude dietary information was divided into two groups: women consuming a normal diet versus women consuming a lot of fish. YPCB levels in milk samples Table 1 summarises the congener specific PCB data which were obtained from the analysis of 115 human milk samples. The EPCB levels (i.e. the sum of the individual congeners analysed) varied between 2 and 70 ng g ~ of whole milk (equivalent to 0-14-1.7 /xg g extractable lipid). Mean values were 12 ng g ' of whole milk (i.e. 0.5 p,g g~ of lipid). The median concentrations for YPCBs (and for individual congeners) were lower than the mean values (Table 1), the data being log normally distributed. Although not directly comparable--due to differences in sampling, analytical procedures and methods of quantification our data are similar to the congener specific data summarised by Jensen (1989), who con-

P C B s and other organochlorines in human tissue samples--milk

81

Table 1. Summary data for PCB, p,p'-DDT, p,p'-DDE and p,p'-DDD levels obtained from the analysis of 115 human milk samples Congener"

Mean value (ng g i lipid)

SD

18 28 52 44 61/74' 66 101 99 110 151 149 118 153 105 138 187 183 128 156/202'/ 180 170 201 194/205'

16.81 34.17 17.02 10.81 20.72 15.36 9.27 21.60 5.51 4.94 10.00 21.98 78.72 14.20 68.52 22.16 6.82 4.19 15-85 76.31 23-40 7.19 4.93

32.12 37-83 28.59 15.23 12.81 26.25 14.39 23.94 8.90 9.58 30.59 20.31 41.43 17.61 39-48 35.45 3.53 3.50 13-49 37.39 12.63 8.55 4.90

15.92 22.79 7.58 5.42 18.58 7.46 4-43 17-56 2.96 1.47 2.22 18.35 68-62 10.38 59.57 13.90 6-22 5.07 13.15 69.97 20.74 5.37 3.99

YPCB

521-86

303.13

441.59

p,p'-DDE p,p'-DDD p,p'-DDT

453.36 6-44 60.39

604.91 14.25 49.36

490.19

659.64

YDDT

Median values Range (ng g i lipid) (ng g 1 lipid) nd h 166 nd 214 nd 155 nd 89 I 68 nd 212 nd 82 1191 nd 60 nd 66 nd 283 nd 197 18 275 nd 148 19 183 nd-228 nd-22 nd-22 4-113 1 210 nd 82 1 63 nd 23

Mean values (ng g t milk)

SD

Median values Range % contribution (ng g I milk) (ng g 1 milk) to XPCB levels

0-44 0.59 (i.27 0.16 0.50 0-24 0-16 0-47 0-10 (I-(/8 0.13 (/.56 1.91 0.33 1-66 0-58 0.16 0.11 0.43 1-89 0.57 0.18 0.24

0.34 0.36 0.37 0-13 0-42 0-22 0.22 (I.48 0.09 0.12 0.19 0-57 1.65 0.39 1.65 1.23 0.12 0.11 0.64 1.43 0.43 0.27 1.33

0.36 0.50 0.17 0.13 0.40 0.18 0.10 0.38 0-08 0-03 0.06 0.46 1.60 0.24 1.34 0.32 0.12 0-08 0.30 1-57 0.47 0.12 0.09

nd-2.76 nd 2.02 nd 2.82 nd 0.68 0.01 3.02 nd-l-61 nd-l-55 0.04-0.47 nd 0.55 nd-0-68 nd 0.91 nd-3.90 0-15 15.32 nd 2.47 0.22-16.14 nd 9.20 0.02-0.75 nd 0.81 0.03 6.40 0.01 9.78 nd 3.24 0.01 2.54 nd 14.34

140 1 697

11.66

8.30

9.23

2.05 70.12

309.92 3.35 19.49

35 6 014 nd 131 nd 504

10.51 0.14 0.86

11.28 0.18 1.91

7.21 0.08 0.45

0.17 68.44 nd-l.32 nd 19.39

346.73

44-6 649

11.52

11.85

8-10

0.30 71.42

3.2 6.5 3.3 2.1 4.0 2.9 1.8 4-1 1.1 0-9 1.9 4.2 15.1 2.7 13.1 4.2 1.3 0-8 3.0 14.6 4.5 1.4 0.9 100 92.5 1.3 12.3 100

"Congener numbering according to Ballschmiter and Zell (1980) hnd equals <40 pg g / o f PCBs. and <0-1 ng g i forp,p'-DDT, p,p'-DDE and p,p'-DDD expressed on a lipid basis. ' Congeners 61/74 and 194/205 co-elute on the GC-ECD and could not be separated by GC-MSD since they have the same number of chlorine atoms (tetra- and octachlorinated, respectively). a Congeners 156/202 were separated by GC-MSD, and congener 156 was identified as accounting for an average of ~95% of the total measured concentration. cluded that, when more t h a n six PCB c o n g e n e r peaks are used for q u a n t i f i c a t i o n , the average b a c k g r o u n d levels in h u m a n milk fat are typically between 0.5 a n d 1.5 # g g ~ lipid c o n t e n t . Collins et al. (1982) reported a m e a n value of 0-5 /zg g ~, a n d a range o f <0-01-2-1 /,g g~ on a lipid basis for 102 h u m a n milk samples analysed in the U K between 1979 a n d 1980. Their YPCB c o n c e n t r a t i o n s were quantified based o n A r o c l o r 1260, whilst o u r data were based o n a c o n g e n e r specific m e t h o d o f q u a n t i f i c a t i o n . M e a s u r e m e n t of the percentage c o n t r i b u t i o n s o f i n d i v i d u a l congeners in A r o c l o r 1260 (Safe et al., 1985; Schulz et al., 1989) suggests that o u r m e t h o d a c c o u n t s for a p p r o x i m a t e l y 60 70°/,, w/w o f the YPCB c o n c e n t r a t i o n s in that Aroclor. Based o n this, data o b t a i n e d from the analysis o f the 115 Welsh h u m a n milk samples would have a m e a n c o n c e n t r a t i o n of 0.74~0.87 # g g ~ of lipid c o n t e n t , a n d a l t h o u g h still n o t directly c o m p a r a b l e with the d a t a reported by Collins et al. (1982), it seems unlikely that there has been a s u b s t a n t i a l decrease in the c o n c e n t r a t i o n s of PCBs in h u m a n milk over the last decade, despite widespread restrictions in the use o f PCBs in the U K since the mid/late 1970s. T h e m a j o r c o n s t i t u e n t s f o u n d in the milk samples are in good agreement with those reported by other workers.

The mixture of PCBs between different milk samples was quite consistent, with up to 24 congeners being measured for most samples. The ZPCB content was d o m i n a t e d by a few major congeners (Table 2), of which the 12 most a b u n d a n t (18, 28, 52, 61/74, 99, 118, 138, 153, 156/202, 170, 180 and 187) accounted for 80% of the ZPCB content (Table 1). In general, the major constituents seem to be in good agreement with those reported by other authors for h u m a n milk samples (Table 3). If we assume that, over a total period of 6 m o n t h s of breast-feeding, on average a b a b y c o n s u m e s 850 ml milk per day ( I C R P , 1975), which is a p p r o x i m a t e l y e q u i v a l e n t to 120 ml milk being c o n s u m e d per kg b o d y weight (bw) ( W H O , 1988), then n e w b o r n babies in the Welsh p o p u l a t i o n would on average ingest 9 . 9 / , g YPCBs per day, a n d a range of 1.7 59.6/xg day ~, which roughly c o r r e s p o n d s to a daily intake of Table 2. Contribution of individual congeners to the ~PCB content in milk Proportion >10% 3-10% <30/,,

Congener 138, 153, 180 18, 28, 52, 61/74, 99, 118, 187, 156/202, 170 44, 66, 101, 110, 151, 105, 128, 149, 183, 201, 194/205

82

R. Duarte-Davidson, S. C Wilson, K. C Jones

Table 3. Summary of data from other studies on human breast milka

% contribution to XPCB h

Congener

Major congeners (> 10%)

74, 99, 118, 138, 153, 180

3 10%

28,52,74,99, 101, 105, 118, 156, 170, 180, 183, 187

Minor congeners (<3%)

28, 33, 44, 49, 52, 66, 101, 105, 110, 119, 128, 151, 146, 156, 157, 172, 177, 183, 187, 189, 194, 195, 196, 201,203, 205, 206, 209 among others

" Data from Safe el al. (1985), Mes and Marchand (1987), Mes et al. (1987), Borlakoglu et al. (1990), Georgii and Brunn (1990) and Noren et al. (1990). Note: the percent contribution will obviously be influenced by the total number of congeners determined by each analyst. The papers cited measured between 7 and 88 congeners. The data summarised here should, therefore, only be considered as a guide to the relative abundance of individual PCBs in human tissues. 1.4/~g kg ~ bw, with a range 0-3-8.4/,g kg l bw. Based on experimental studies and/or human case investigations, the average intake of PCBs through breastfeeding are at least one to two orders of magnitude below the levels estimated by the W H O Working Party to cause any adverse health effects on infants (WHO, 1988). Furthermore, the intakes of PCBs during a nursing period were estimated to correspond to less than 5% of the individual's lifetime body burden (WHO, 1988). Y D D T levels in human breast milk samples

Z D D T levels in human milk samples varied between 0-3 and 71.4 ng g 1 of whole milk (equivalent to 0.04-6.7 p g g ~ of lipid), with a mean value of 11.5 ng g whole milk (or 0-49 /~g g ~ lipid) (Table 1). p , p ' - D D E concentrations on average contributed towards 92% of the Y D D T levels in the milk lipid. These results are very similar to those found for human adipose tissue in the Welsh population (Duarte-Davidson et al., 1994), and are indicative of exposure of D D T metabolites through food contamination, rather than to direct contact of this compound. The p , p ' - D D E levels ranged from 0.03 to 6.0 #g g lipid, the mean value being 0 . 4 5 / , g g L lipid, p , p ' - D D D mean concentrations were 0-006 /xg g l, varying from <0.01 ng g t lipid to 0.13 /xg g ~ lipid, and p , p ' - D D T levels had a mean value of 0-06/xg g ~ lipid (<0.01 ng g to 0.5/xg g ~ lipid). As for PCB concentrations, median values were lower than the mean, the data being log normally distributed. Collins et al. (1982) reported a mean value of 1.6/xg g ~ fat basis content, with a range of <0.01-7-3 /xg g t of lipid for p,p'-DDE, and a mean value of 0.11 /xg g of lipid, with a range of <0.01-1-2 /xg ~ for p , p ' - D D T for the analysis of 102 h u m a n milk samples collected in the U K between 1979 and 1980. These values, although not directly comparable with the range found for the contemporary Welsh population suggests a general de-

cline in the mean concentrations of both p , p ' - D D T and p , p ' - D D E in U K human breast milk in the last decade. Given the assumption made earlier, a newborn baby will consume, on average, 8-9/xg p , p ' - D D E per day and a range of 0.2 58/xg day ~, which is roughly equivalent to a daily intake of 1.3 /xg kg 1 bw and a range of 0.02-8-2 kg ~ bw. The W H O acceptable daily intake (ADI) for p,p'-DDE was estimated as being 5 /xg kg bw (data obtained from Bush et al., 1985). On this basis most contemporary Welsh infants are exposed to below the A D I concentrations, except for 3% of the children whose mothers have p , p ' - D D E residues in the top concentration range. However, as found for PCB intakes, the Y D D T intake through breast-feeding will account for a very small proportion of the total lifetime intake of such compounds. A G L M was developed to determine any correlations that may exist between p , p ' - D D T and its metabolites. The logarithmic values of p,p'-DDT, p , p ' - D D E and p , p ' - D D D were used to obtain a normal distribution of these variables. Both log p , p ' - D D D and log p,p'-DDE made a statistically significant ( P < 0 . 0 1 ) contribution towards determining the log p , p ' - D D T levels in the model (r = 0-75). However, as found for Welsh adipose tissue samples (Duarte-Davidson et al., 1994), a stronger correlation (P<0.01) was found between log p,p'-DDT, and log p , p ' - D D D (r--0.82) levels, thus confirming that this metabolite is a better predictor of the log p , p ' - D D T concentrations in human milk. Kutz et al. (1976) reported that p , p ' - D D T is biochemically dechlorinated in human tissues to p,p'-DDD, which is then either metabolised further or excreted directly from the body, whilst p,p'-DDE storage in the tissues is derived by direct ingestion o f p , p ' - D D E previously degraded in the environment, rather than by the direct ingestion and degradation of p,p'-DDT. The greater correlation between p , p ' - D D T and p , p ' - D D D was therefore expected. The ratio in the concentrations o f p , p ' - D D E and p,p'D D T increases in h u m a n tissues with time following direct exposure to p , p ' - D D T (Jensen, 1983), due to the greater persistence of p , p ' - D D E relative to p , p ' - D D T in the environment. On a whole milk basis, the mean p,p'D D E / p , p ' - D D T ratio increased from 1.6 in 1963-1964; (Egan et al., 1965) to 13.7 in 1979-1980 (Collins et al., 1982). However, the mean ratio for 1990-1991 was 12.2, and although these data are not directly comparable, it does not show any clear downward trends in the p , p ' - D D T concentrations relative to p , p ' - D D E for this population in recent years. YPCB and Y D D T concentration differences between mothers

G L M s were developed to explain any correlations that might exist between YPCB and Z D D T levels found in the milk samples of the individual mothers in relation to their age, body weight, parity and total period of breast-feeding, diet, smoking habits, and area of residence (i.e. rural, semi-rural and urban locations). Two G L M s were developed to explain any contributions that the above factors might have on both

P C B s and other organochlorines in h u m a n tissue samples

52PCB and Z D D T concentrations. The logarithmic values of the YPCB levels (log EPCB) and YDDT levels (log Z D D T ) were used in the G L M s to improve the normality of the distribution of the ZPCB and 52DDT concentrations in the sample population. No correlations were found between the residue concentrations in the Welsh milk samples and the body weight of the donor. These results are in good agreement with those obtained by Stacey and Thomas (1975) and Takahashi et al. (1981), who found no significant differences between PCB and DDT concentrations in human milk with body weight. Polishuk et al. (1977), however, reported that overweight women (>72 kg) excreted lower quantities of YDDT and PCBs in their milk than did females of normal weight (<63 kg), this probably being due to a dilution effect caused by the larger amounts of fat deposits in heavier women (Jensen, 1983). Cigarette smoking is a potential source of intake of pesticides, mainly DDT, due to the use of pesticides in tobacco fields. Some authors (e.g. Dillon et aL, 1981) have reported higher Z D D T levels in the milk of smokers when compared to non-smokers. However, no statistical correlations were observed between smoking habits and log 52DDT (or log ZPCB) concentrations. This is in agreement with other authors who have also found very little or no significant correlations between the subjects organochlorine concentrations and smoking as reported for Z D D T by Kreiss et al. (1981), Collins et al. (1982) and Mussalo-Rauhamaa et al. (1984) in human blood, milk and adipose tissue, respectively. Populations living in rural, semi-rural and urban areas will be equally exposed to ]~PCB and Z D D T levels. These results are in agreement with those reported by others (e.g. Collins et al., 1982; Bush et al., 1984, 1985), who found very small or no differences in residue concentrations between urban and rural dwellers. Similarly, dietary habits did not lead to elevated levels in human milk. The main exposure to PCBs in the industrialised world (WHO, 1988) and to DDT, in countries where direct exposure of this compound has ceased (Jensen, 1983), has been estimated as being mainly due to dietary intake. Thus, the main dietary intake of PCBs in the UK have been estimated to be through the consumption of fish (29% of total daily intake), milk and dairy products (24%) and meat (15%) (Duarte-Davidson, 1992). Therefore, dietary habits may lead to elevated levels in human tissues on an individual level. For example, Kreiss et al. (1981) and Cordle et al. (1982) reported a positive correlation of PCB concentrations in human blood relative to fish consumption whilst Takahashi et al. (1981) found no significant correlation between intake of meat and dairy products and residues in human milk. The similarity between urban and rural PCB and D D T concentrations in the Welsh milk probably reflects the pre-eminence of dietary intake as the major exposure pathway. Furthermore, of the communities analysed, none tend to consume a predominant amount of foodstuffs rich in PCBs/DDT (such as fish) in their diet. However, the present study

83

milk

was not thorough enough to estimate any real effects caused by diet, and therefore, it would be necessary to get more complete information on 'dietary composition' than that obtained by our survey in order to determine any real effects caused by diet. The final G L M was as follows: Y = 5.29 + 0.03 Xi - 0-001

X 2 -

0-08

X 3 q-- 0.02

X4

(1)

where Y is the log EPCBs; X~ is age (years); X2 is total period of breast-feeding (in weeks); X~ is the percent (%) lipid content; and )(4 is 1/lipid weight (g). The above equation shows a positive correlation of log ZPCBs with age, and a negative correlation with the % lipid content of the milk samples and period of breast-feeding (r--0.66), the correlations all being significant (P<0-02). Three cases (i.e. three of the human milk samples analysed) did not fit into the model (i.e. they were outliers). Removing these samples improved the explanatory power of the model ( P < 0-01; r-- 0-73). The term )(4 (1) was introduced to reduce the error caused by the weighing of the lipid content present in the milk samples. The lipid content of the milk samples analysed varied between 0.02 and 1.11 g, with a mean of 0.35 g. These values are small due to the low lipid content found in milk (between 0-12 and 6.8% by weight). Measuring such amounts might be expected to contribute towards an error that is equal to the inverse weight of the lipid (g). That is, smaller amounts of lipid (by weight) cause a greater error when measuring log ZPCBs in the milk samples (because the ZPCBs are expressed in ng g~ of lipid). This is shown by the negative correlation that exists between EPCB levels and lipid weight (g) in Fig. 2. This error term (i.e. lipid weight) compensates for an increase in the error value for ZPCBs with decreasing amounts of lipid. Ideally, it is possible to reduce the effect of this error term by analysing larger amounts of milk samples, but often this was not available. As for the PCB data, a significant positive correlation (P<0-01) existed between log Z D D T levels and age, and a significant negative correlation between the log Z D D T levels with period of breast-feeding (P < 0.01; r -- 0.48), the G L M equation being as follows: Y-- 4-13 + 0.01 X~ - 0.01 X2

(2)

where Y is the log ZDDT; X~ is age (years); and )(2 is total period of breast-feeding (in weeks). Thus, the general trend is that of an increase in the log 5~PCB and log Z D D T levels with age, although this relationship is not as clear as for the Welsh adipose tissue samples previously analysed (Duarte-Davidson et aL, 1994). This is due to the narrower age range of the mothers which has evidently led to a reduced correlation with age between the individual milk samples analysed relative to the adipose samples. Results obtained by other workers for human milk samples varies. Thus, for example, Dewailly et al. (1989) reported a positive correlation of PCB levels with age, whilst Takahashi et al. (1981) found no association for either

84

R. Duarte-Davidson, S. C. Wilson, K. C. Jones

"/De

:;".

I= U

PCB congener patten and PCA PCA was carried out to look for patterns in the congener mixture of the breast milk samples, and to determine PCB pattern distribution differences between Welsh adipose and milk tissues.

t.

-v":i.";-:.

-.

t~ o

5

.

I

I

0.5

1.0

l:Lp:Ld

we:Lgh'c

(g)

Fig. 2. Scatterplot of log XPCBs versus lipid weight. Z D D T and YPCBs with increasing age. Finally, Bush et al. (1984) found that p,p'-DDE showed a positive correlation with the age of the mother, whilst other compounds, including PCBs showed no such correlation. The log YPCBs and log Z D D T concentrations in the mothers' milk tissues were not significantly associated with the number of children. Residue levels in human milk have been found to decrease with increased number of children (Noren, 1983: Yakushiji, 1988). However, the residue levels will depend not only in the number of children, but also on the period elapsed between childbirths, as residue levels in the mothers' tissues will gradually increase as a function of dietary intake (Yakushiji, 1988), and on the total period of breast-feeding, as this is the main excretory route for these lipophilic compounds from the mothers' body (Wickizer & Brilliant, 1981). YPCB and YDDT levels in the Welsh milk samples were negatively correlated with the total period of breast-feeding. This effect was mainly caused by mothers that had breast-ted for a long period of time (>100 weeks). ZPCB and E D D T levels were more dispersed (mean YPCB levels being 0.54 + 0-31 /xg g ~ lipid: mean Z D D T = 0.48 + 0.63 /zg g l lipid) for mothers breast-feeding for less than 100 weeks. Mothers that had breast-fed for a cumulative total of over 100 weeks showed significantly lower levels in their milk (5"PCB -- 0.32 + 0.11 p,g g~ lipid: Y D D T = 0 - 2 1 + 0 . 1 8 p , g g t l i p i d ) . Only a small subsample of the mothers (8%) had breast-fed for this prolonged period of time. Results obtained by other workers seem to vary. Some have found no significant PCB level decline over different monitoring periods of time, which varied from 14 to 96 weeks of total lactation (e.g. Noren, 1983: Mes et al., 1984; Bush et al., 1985), whilst others have reported either slight or statistically significant decreases in the PCB (e.g. Fooken & Butte, 1987; Yakushiji, 1988; Dewailly et al., 1989) and p,p'-DDT and p,p'D D E (e.g. Mes et al., 1984) concentrations over periods of lactation which varied from 14 to over 119 weeks. In summary, these results seem to suggest that very prolonged periods of breast-feeding will generally result in cumulative elimination of PCBs from the mothers' body, as reported for the Welsh population.

P C B pattern distribution between individual breast milk samples PCA on the milk data showed no differences in congener pattern distribution between the milk samples for different age groups as reported previously for adipose tissue samples (Duarte-Davidson et al., 1994), this probably being due to the narrower age range of the mothers relatively to that of the adipose tissues. There were no defined differences in pattern between milk samples obtained from mothers that have breastfed for different periods of time, or mothers that live in different areas. Thus, the data were not sensitive enough to show any preferential removal of certain congeners through breast milk that would lead to a change in the congener pattern with increased lactation period. P C B pattern distribution between human adipose and milk samples The aim of this section is to compare the congener pattern differences between human milk and adipose tissue samples. Results obtained from the adipose tissue samples have been published in detail elsewhere (Duarte-Davidson et al., 1994). PCB molecules which have a 2,4,5 chlorine substitution pattern in one ring and at least one 4-position in the other ring have been reported as being the most stable and persistent in mammals (Yakushiji, 1988). Increasing the number of chlorine atoms in the PCB molecule also tends to increase the stability of the congeners which are less persistent (Yakushiji, 1988). Thus, the stability of the individual PCB congeners is dependent on both the structure and the level of chlorination of the PCB compounds. Analysis of 75 Welsh adipose tissue samples (Duarte-Davidson et al., 1994) showed that most of the congeners that accounted for >3% of the total PCB content generally had the structure described above, these compounds being 74, 99, 118, 138, 153, 156, 170, 180, 187 and 194/205. Comparing these results with those obtained for the Welsh breast milk samples (Table 1) shows that the main contributors to the ZPCB content in these samples was very similar to those obtained for the fat samples, with the exception of three lower chlorinated congeners, 18, 28 and 52. These do not have the structure which is characteristic of the more persistent congeners, and may therefore be eliminated from the body more readily. To determine congener pattern differences between the two matrices a PCA was carried out on both the milk and adipose tissues, the aim of the PCA being to simplify the data into factors formed by congeners that were correlated with each other to allow for easier interpretation of the data. A total of five factors or principle components (PCs) were included in the model, which

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Fig. 3. Plot of the factor loadings for factor 5 versus factor 1.

together explained 80% of the variance accounted for by the original data set. Figure 3 represents a plot of factor 5 versus 1, and shows that congeners 18, 28, 44, 52, 66 and 101 were highly negatively associated to factor I, whilst 128, 156/202, 170, 180, 183 and 201 were highly positively associated with that factor. Similarly, 99 and 105 show a strong positive correlation with factor 5. Figure 4 is a PCA plot obtained to determine the pattern distribution of the congeners in milk and adipose tissue. Factors 5 and 1 were plotted against each

18

I

other for individual milk and adipose tissue samples. This graph shows that adipose samples have been clustered towards the right-hand side of the graph, indicating a predominance of the higher chlorinated compounds (mainly 128, 156/202, 170, 180, 183 and 201) in human fat. This contrasts with the pattern found for the milk samples which have a larger proportion of the lower chlorinated compounds, the contribution of each of these congeners varying more widely between individuals, causing a wider spread of the distribution. Figure 5 is a histogram showing the percent contri-

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86

R. Duarte-Davidson, S. C. Wilson, K. C. Jones

bution of some selected congeners towards the YPCB concentrations. These values represent the mean percent contribution calculated from all the samples analysed for both milk and adipose tissues. Again, lower chlorinated congeners are more predominant in milk samples, whilst higher chlorinated compounds show a greater percent contribution in the adipose tissues. Therefore, although the congener pattern found in milk samples was broadly similar to that for human adipose tissues, lower chlorinated congeners were present at higher concentrations in milk relative to adipose tissue. These congeners are tri- (18 and 28), tetra- (44, 52 and 66) and pentachlorinated biphenyls (101) which are probably mobilised more readily l¥om the body. All these results indicate that less persistent, lower chlorinated congeners which have shorter half lives and are more readily mobilised from the body are present in larger proportions in milk, whilst more stable higher chlorinated compounds are more readily retained in the fatty tissues. Bush et al. (1985) have suggested that mothers may be exposed to a continuous source of low chlorine content PCBs, which tend to dominate the mixture of congeners in some compartments of the contemporary environment (Jones et al., 1992).

then they would, on average, ingest 1-4 p,g YPCB kg r bw (with a range of 0-3-8.4 /.tg kg ~ bw), these values being at least two orders of magnitude below the levels estimated by the W H O to cause any adverse effects on infants. The p,p'-DDE daily intake was estimated as being 1.3 /.tg kg ~ (with a range of 0.02-8.2 /zg kg i). About 3'Vo of Welsh babies may exceed the acceptable daily intake for p , p ' - D D E estimated by the W H O . However, the total PCB and D D T intakes through breast-feeding will contribute towards a small fraction (<5%) of the lifetime body burden to such compounds. ZPCB and Y D D T levels were positively correlated with age and negatively associated to the total period ol breast-feeding, both effects being more significant for ZPCB concentrations. The effects observed in relation to the total lactation period was mainly caused by mothers who had breast-fed for very long periods of time (>100 weeks), indicating a long-term cumulative elimination of PCBs from the mothers' body. A clear correlation was also found between ZPCB concentrations and percent lipid content in the milk, possibly suggesting a dilution effect of the PCBs in the breast milk with increasing amounts of lipid. There were no significant differences in either YPCB or E D D T levels in milk with the donors' body weight, smoking and dietary habits. Neither were there any apparent broad regional differences in the residue concentrations in the Welsh milk samples. This was attributed to the fact that dietary intake is the main source of exposure ['or both D D T and PCBs in the general populations, this source being similar for both rural and urban locations. These results are in agreement with those found for human Welsh adipose tissue. Four individual PCB congeners (28, 138, 153 and 180) accounted on average for nearly 50°/,, of the ZPCB congeners analysed. The PCB congener pattern distribution was broadly similar for Welsh milk and adipose tissue. However, PCA techniques highlighted the main differences between the two matrices as being the presence of higher concentrations of tri- and tetrachlorihated biphenyls in the milk samples relative to the adipose samples.

CONCLUSIONS

ACKNOWLEDGEMENTS

One hundred and fifteen Welsh human breast milk samples were analysed for ZPCB and Z D D T concentrations. Mean ZPCB levels were 12 ng g ~ of whole milk, with a range of 2 70 ng g] of whole milk, whilst Y D D T levels had a mean value of 11 ng g ~ whole milk and a range of 0.3 71 ng g ] whole milk, p,p'-DDE on average accounting for 92% of the Z D D T concentrations found in the milk samples. The values obtained show a general downward trend in the concentrations o f p , p ' - D D T and p,p'-DDE levels in the U K breast milk in the past decade. However, there is no clear evidence of any substantial decline in the YPCB concentrations in the same period of time. Assuming that over a total period of 6 months of breast-feeding babies consume 120 ml milk kg ] bw,

The authors are grateful to the Welsh Office for their financial support, to the Welsh gynaecologists/sisters and all mothers for organising and donating the milk samples, and to B. J. Francis, from the Centre of Applied Statistics at Lancaster University, who provided useful information on the statistical methods necessary for the analysis of the samples. The views expressed here are those of the authors and do not necessarily represent those of the Welsh Office.

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REFERENCES Ballschmiter, K. & Zell, M. (1980). Analysis of PCBs by glass capillary gas chromatography. Fresenius. Z. Anal. Chem., 302. 20 31. Borlakoglu, J. T., Borlak, N. & Dils, R. R. (1990). Human

P C B s and other organochlorines in human tissue s a m p l e s - - m i l k

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Johnston, A. E. (1992). Evidence for a decline of PCBs and PAHs in rural vegetation and air in the United Kingdom. Nature, 356, 137-40. Kreiss, K. J., Zack, M. M., Kimbrough, R. D., Needham, L. L., Smerek, A. L. & Jones, B. T. (1981). Association of blood pressure and polychlorinated biphenyls levels. J. Amer Med. Assoc., 245, 2505 9. Kutz, F. W., Yobs, A. R. & Strassman, S. C. (1976). Organochlorine pesticide residues in human adipose tissue. Bull. Soc. Pharmacol. Environ. Pathol., 4, 17 19. Mes, J. & Marchand, L. (1987). Comparison of some specific polychlorinated biphenyl isomers in human and monkey milk. Bull. Environ. Contain. Toxicol., 39, 734~2. Mes, J., Doyle, J. A., Adams, B. R., Davies, D. J. & Turton, D. (1984). Polychlorinated biphenyls and organochlorine pesticides in milk and blood of Canadian females during lactation. Arch. Environ. Contam. Toxieol., 13, 217 23. Mes, J., Turton, D., Davies, D., Sun, W. F., Lau, P. Y. & Weber, D. (1987). The routine analysis of some specific isomers of polychlorinated biphenyl congeners in human milk. Intern. J. Environ. Anal. Chem., 28, 197 205. Mussalo-Rauhamaa, H., Pyysalo, H. & Moilanen, R. (1984). Influence of diet and other factors on the levels of organochlorine compounds in human adipose tissue in Finland. J. ToxicoL Environ. Health, 13, 689 704. Noren, K. (1983). Some aspects of the determination of organochlorine contaminants in human milk. Arch. Environ. Contain. Toxicol., 12, 277-83. Noren, K., Lunden, A., Sjovall, J. & Bergman, A. (1990). Coplanar polychlorinated biphenyls in Swedish human milk. Chemo~phere, 20, 935-41. Polishuk, Z., Ron, M., Wasserman, M., Cucos, S., Wasserman, D. & Lemeshch, C. (1977). Organochlorine compounds in human blood plasma and milk. Pest. Monit. J., 10, 121--9. Safe, S., Safe, L. & Mullin, M. (1985). Polychlorinated biphenyls: congener-specific analysis of a commercial mixture and a human milk extract. J. Agrie. Food. Chem., 33, 24~9. Schulz, D. E., Petrick, G. & Duinker, J. C. (1989) Complete characterization of polychlorinated biphenyl congeners in commercial Aroclor and Clophen mixtures by multidimensional gas chromatography-electron capture detection. Environ. Sci. TechnoL, 23, 852 9. Stacey, C. I. & Thomas, B. W. (1975). Organochlorine pesticide residues in human milk, Western Australia 1970-71. Pest. Monit. J., 9, 64 6. Takahashi, W., Saidin, D., Takei, G. & Wong, L. (1981). Organochlorine pesticide residues in human milk in Hawaii 1979-1980. Bull. Environ. Contain. ToxicoL, 27, 506-11. WHO (1988). Assessment of health risks in infants associated with exposure to PCBs, PCDDs and PCDFs in breast milk. Environmental Health Series no. 29. World Health Organisation, Copenhagen, Denmark. Wickizer, T. M. & Brilliant, L. B. (1981). Testing for polychlorinated biphenyl in human milk. Pediatrics, 68, 411 5. Yakushiji, T. (1988). Contamination, clearance and transfer of PCB from human milk. Rev. Environ. Contain. Toxicol., 101, 139-64.