Polychlorinated dibenzodioxins and dibenzofurans in human milk samples from Western Germany

Chemosphere, Vol.16, Nos.8/9, pp 1983-1988, 1987 Printed in Great Britain

0045-6535/87 $3.00 + .OO Pergamon Journals Ltd.

POLYCHLORINATED DIBENZODIOXINS AND DIBENZOFURANS IN HUMAN MILK SAMPLES F R O M W E S T E R N GERMANY

P. FUrst*, H.-A. Meemken, Chr. KrUger and W. Groebel Chem. Landesuntersuchungsamt

NW, Sperlichstr.

19, 4400 Muenster

INTRODUCTION In 1984 RAPPE and coworkers were the first to report on the isomer-speciflc determination of polychlorinated dibenzodioxins (PCDDs) and -dibenzofurans (PCDFs) in human milk samples from countries like Sweden and the Federal Republic of Germany which are not considered as uncommonly dioxln-contaminated (1). Due to the great resonance in the public the ministry of agriculture and evironment of Northrhine-Westfalia instructed us to analyse human milk samples from women living in NorthrhineWestfalia for residues of PCDDs and PCDFs. Since the beginning of 1985 more than 90 milk samples have been analysed for PCDDs and PCDFs in our laboratory. All the samples were obtained on a voluntary basis from nursing mothers living in Northrhine-Westfalia. To get an idea by which factors the amounts of dioxins and furans are influenced each mother was asked to fill in a questionnaire in which details should be given about age, weight and height of mother and baby, place of living, period of lactation etc.

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This paper presents our improved analytical procedure and the results as well as a first statistical evaluation of the data. EXPERIMENTAL Concentrate

Using the experience we have gained in the past few years concerning the analysis of organochlorine pesticides and PCBs in human milk in our laboratory we adapted this procedure to the determination of PCDDs and PCDFs (2). Fig. 1 illustrates the flow diagram of the analytical procedure. This procedure which was already presented by our group on the last dioxin-symposium in Bayreuth has been improved in the meantime to give better massfragmentograms for the lower chlorinated dioxlns and furans. The PCDDs and PCDFs are extracted together with fat using potassium oxalate, ethanol, ethylether and pentane (3). After addition of 13-C labeled ootachlorodibenzodioxin fat is remove~ by gel chromatography on Bio-Beads S-X5 with cyclohexane/ethylacetate 1+1 (4). Adsorption column 1983

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1984

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chromatography on florisil (5) and acid aluminia (6) is used to separate PCDDs and PCDFs from most of the organochlorine pesticides, PCBs, polychlorinated naphthalenes and other contaminants. The PCDDs and PCDFs are determined by HRGC/LRMS using a 60 m DB-5 fused silica column (J & W Scientific) in a Hewlett Packard 5987A system equipped with a splitless injector. MS-analysis is performed in SIM,mode (selected ion monltoring). Due to their structure the PCDDs and PCDFs can be determined very selectively and sensitively by use of negative chemical ionization (NCI) employing methane as s reagent gas. With the exception of 2.3.7.8"TCDD which shows only a poor

1985

response in the NCl-mode the detection limit for all other isomers found in human mllk is below I picogram. For the determination of 2.5.7.8-TCDD the MS is operated in electron impact mode (EI). 0ctachloronaphthalene is used as internal standard for the quantification based on a calibration run of standards. The calibration mixture which contains octachloronaphthalene as well as all 2.3.7.8-substituted dioxlns and furans including the labeled 0CDD at a concentration level of 3-6 pg/ul is injected once every day. The recovery rates for the 13-C labeled octachlorodlbenzodioxin are about 70-80 %. Representative fragmentograms of breast mllk samples are shown in Fig. 2 RESULTS AND DISCUSSION The results of our investigations are given in Tab. I. Snmmarlzlng these data it can be stated that all samples contain a typical pattern of PCDDs and PCDFs. 2.3.7.8-TCDD could not be detected in an T of the samples. At present the detection limit for this isomer is about 5 ng/kg (ppt) calculated on fat basis. It Is noteworthy that all isomers identified are 2.3.7.8 chlorine substituted. Whereas OCDD normally represents more than 50 % of the total dioxin amount the levels of the other isomers decrease with decreasing grade of chlorination. In all samples the hexachlorodibenzodioxin (hexa-CDD) levels were nearly of the same relative proportion. 1.2.3.6.7.8-hexa-CDD amounted about to 70 % of the sum of the three identified hexa-CDD isomers. The furan levels are always considerably lower than those of the dloxlns. 0ctachlorodibenzofuran could be detected in 62 samples. In most cases the levels Amount ng/kg (ppt) calc. on fat bas~s mean value*) range

Congener

1.2.3.4,6.7.8.9.-octachlorodibenzodioxin 1.2.3.4,6.7.8.-heptach|orodibenzodioxin 1.2.3.4.7.8.-hexachlorodibenzodioxin 1.2.3.6.7.8.-hexachlorodibenzodioxin 1.2.3.7.8.9.-hexachIorodibenzodioxin 1.2.3.7.8.-pentachlorodibenzodioxin 2.3.7.8.-tetrachlorodibenzodioxin 1.2.3.4.6.7.8.9.-octachlorodibenzofuran 1.2.3.4.6.7.6.-heptachlorodibenzofuran 1.2.3.4.7.8.-hexachlorodibenzofuran 1.2.3.6.7.8.-hexach|orodibenzofuran 2.3.4.6.7.8.-hexachlorodibenzofuran 1.2.3.7.8.-pentachlorodibenzofuran 2.3.4.7.8.-pentachlorodibenzofuran 2.3.7.8.-tetrachlorodibenzofuran

181.2 49.9 8.1 32.7 6.4 10.7

(n=92) (n=92) (n=87) (n=92) (n=86) (n=72)

22.8 6.4 8.2 6.6 3.3 1.8 22.9 2.6

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Tab. I PCDDs and PCDF in human milk

~5 86 20 28 25 9 7 67 g

*)Samples with levels below the detection l i m i t don't contribute to the mean values.

1 2 3 4 5 6

1.Z.3.4.6.7.B.9-octachlorodibenzodioxin 1.2.3.4.6.7,8-heptachIorodlbenzodioxin 1.2.3.4.7.8-hexachlorodibenzodioxin 1.2.3.6.7.8-hexachlorodibenzodioxin 1.2.3.7.8.9-hexachlorodlbenzodioxin 1.2.3.7.8-pentachlorodibenzodioxin

1

I 2

1.0000 .8111

2

3

4

5

6

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.39B0

.5768

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1.0000

.6416

.7065

.6193

.6472

3

.3980

.6416

1.0000

.8130

.7710

.6497

4

.5768

.7065

.8130

1.0000

.8423

.7924

1.0000

5

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.6193

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6

.6457

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.6497

.7924

.7587

.7587

1.0000

Tab. 2 Cross correlation table for PCDDs in human mllk

1986

were in the range from 1-15 ng/kg (ppt) calculated on fat weight. Some values, however, ranged up to 86 ppt. Similar to the hexa-CDDs the isomer distribution of the three hexachlorodibenzofurans (hexa-CDF) was found to be quite constant in all samples. It should be recognized that 2.3.4.7.8-pentachlorodibenzofuran (penta-CDF) represents the main compound of all furans detected in most of the samples. 1.2.3.7.8-penta-CDF could be determined in some samples with levels near the detection limit. I n a l l cases 2.3.7.8-tetrachlorodibenzofuran was detected it was found to be a peak of minor size. STATISTICAL EVALUATION Statistical calculations were performed to examine any possible correlations between the FCDDs and FCDFs identified. The cross-correlatlon table for the PCDDs in breast milk is shown in Tab. 2. It must be considered that all samples were randomly accessed, that is, they were taken at different times during the period of lactation. Nevertheless the statistical evaluation partially indicates relatively high correlation coefficients for single isomers. It is striking that the correlation coefficients for isomers differing in one chlorine are higher than those for compounds which differ in more than one chlorine. Whereas the cross-correlation coefficient between octachlorodibenzodioxin and 1.2.3.4.6.7.8.-heptachlorodibenzodioxin is 0.8111, it is only 0.39 to 0.59 between OCDD and the three hexachlorodibe~zodioxin-congeners. The high correlation coefficients between the three hexachlorodibenzcdioxins identified are very interesting. Although the absolute levels show a wide variation the relative proportion of the three congeners was found to be quite constant. The statistical evaluation for the polychlorinated dibenzcfurans shows a result that is very similar to that of the dioxins. Regarding the three hexachlorodibenzofurans identified we also observe relatively high correlation factors. This will be illustrated by Fig. 3. It shows the computer output of the calculation of the linear regression between 1.2.3.4.7.8.- and 1.2.3.6.7.8.-hexaCDF. In this case the correlation coefficient for these two congeners is 0.96. This graph illustrates the close relation between the levels of these congeners found in breast milk. We also observe this phenomenon for other dioxins and furans. So one may generally state that all breast milk samples analysed so far contain a typical pattern of PCDDs and FCDFs. Moreover in all samples the levels of the congeners identified were nearly of the same relative proportion. This is valid for the d i o x i n s a s well as for the furans. On the other hand a high dioxin level is not synonymous with a high furan level as can be seen on Fig. 4. The calculation of the linear regression between the sum of dloxins and the sum of furans reveals a correlation coefficient of only 0.446. So one can see that there is only a partially interdependence between these two groups. All milk samples were as well analysed for residues of organochlorine pesticides and FCBs. For this reason it was obvious to check for any possible interdependences between those contaminants and PCDDs and FCDFs respectively. Fig. 5 shows the computer plotted graph of the furans against the corresponding PCB-level of each sample calculated as Clophen A60. In this case the correlation coefficient is only 0.356. As regards FCBs and dioxins the statistical evaluation resulted in a correlation coefficient of 0.44, which is only slightly higher than that from the furans. From what was said one might cautiously conclude %/:at the sources for high dioxin-, furan- and PCB-levels must not necessarily be the same. From former investigations of breast milk samples it is known that the levels of organochlorine pesticides decrease with increasing period of lactation. So breast feeding seems to be a kind of detoxlcation process for the mother. 76 of the 92 mothers who sent their milk for analysis filled in the questionnaire. So it was possible to calculate the interdependences between the dloxin- and

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furan levels on the one hand and the period of lactation on the other. All statistical evaluations revealed negative cross correlation coefficients, that is to say, similar to the organochlorine pesticides the amount of dioxlns and furans decrease with increasing period of lactation. This will be exemplarily illustrated by Fig. 6. It shows the relation between octachlorodibenzodloxln and the period of lactation. In this case 33 samples from mothers feeding their first child, 37 samples from mothers feeding their second and 6 samples from mothers feeding their third child were analysed. Unfortunately we had no samples from one mother but different periods of lactation. To illustrate the influence of the period of lactation we performed a new estimation of the mean values (Fig. 7). It can be recognized that milk samples from women nursing their second child contain considerably lower levels of dioxlns than those samples from mothers nursing their first child. The corresponding calculation for the furans shows a very similar result. It is to ask if there are other factors having an influence on the amounts of dioxins and furans in breast milk. Plotting a graph of the dioxin levels of all samples dated from the first period of lactation versus the age of the mother one cannot observe any interdependence between these parameters. Breast milk samples from younger and older mothers don't show a difference in the magnitude of the dioxin level (Fig. 8). It is interesting to look at the influence ot the difference from optimum or ideal weight. Fig. 9 shows a computer plotted graph of this parameter versus the sum of dioxlns. All samples were originated from women who were nursing their first child. Optimum or ideal weight of the mother is defined as (height - 100) - 10 %. As indicated the highest dloxin levels were found in milk samples from

1988

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mothers who are considerably underweight. On the other hand milk from mothers being strongly overweight contain lower dloxin levels. The statistical evaluation of the correlation coefficient revealed a negative value. Subsequently one might conclude that the dloxln levels found in breast milk decrease with increasing overweight of the mother. This can be explained by the fact that overweight mothers have more fat tissue which lead to a higher dilution of the fat soluble dioxins and furans. Mothers being underweight contain the lipophylic compounds in a more concentrated manner. This results in a stronger mobilization of the dioxins and furans during breast feeding. The aim of our future investigations will be to substantiate the statistical conclusions by analysing more milk samples. Moreover we have already started to analyse foodstuffs to get an overview which sources may lead to a possible health hazard for the population. REFERENCES (I) Rappe, C. WHO Consultation on 0rganohalogen Compounds in Human Milk and Related Hazards, Bilthoven, 1985 (2) Fuerst, P., H.-A. Meemken and W. Groebel, Determination of Polychlorlnated Dibenzodioxins and Dibenzofurans in H,~man Milk, Dioxln 85, Bayreuth I!} Acker, L. and E. Schulte, Deutsche Lebensmittelrundschau, 66, 385 (1970) Specht, W. and M. Tillkes, Fressenius Z. Anal. Chem., 301, 300 (1980) Ballschmiter, K., in "Sachstand Dioxln", published by Umweltbundesamt, Berlin, 1983 (6) Smith, L.M, D.L. Stalling and J.L. Johnson, Anal. Chem. 56, 1830 (1984)