Polybrominated diphenylethers (PBDE) and hexabromocyclododecane (HBCD) in paired samples of blood serum and breast milk -a correlation study Polybrominated diphenylethers (PBDE) and hexabromocyclododecane (HBCD) in paired samples of blood serum and breast

Abstract

Postadress Box 622, 751 26 Uppsala Telefon 018-175500 Rapporttitel Polybrominated diphenylethers (PBDE) and hexabromocyclododecane (HBCD) in paired samples of blood serum and breast milk -a correlation study Beställare Naturvårdsverket 106 48 Stockholm Finansiering Nationell hälsorelaterad miljöövervakning Nyckelord för plats Uppsala Nyckelord för ämne PBDE (polybromerade difenyletrar, HBCD (hexabromcyklododekan) Tidpunkt för insamling av underlagsdata 2010 Sammanfattning I denna studie analyserades de bromerade flamskyddsmedlen PBDE och HBCD i parade prover av blodserum och modersmjölk från 30 förstföderskor i Uppsala. Proverna samlades in ca 3 veckor efter förlossningen, och syftet var att undersöka korrelationer mellan blodserum-och modersmjölkshalter för att utvärdera om halter av PBDE/HBCD i modersmjölk är en bra indikator på mammans kroppsbelastning och möjligtvis också på barnets exponering under fosterperioden. PBDE och HBCD analyserades med en metod baserad på gaskromatografisk separation och lågupplösande masspektrometisk detektion (ECNI-SIM). Halter under metodens kvantifieringsgräns (LOQ) användes i de statistiska analyserna eftersom de, trots större osäkerhet, ger värdefull information om spridningen i halter. I blodserum var medianhalten av BDE-209 högst (0,90 ng/g fett), följt av BDE-153 (0,72 ng/g fett) och BDE-47 (0,36 ng/g fett). I modersmjölk uppmättes de högsta halterna för BDE-153 (0,45 ng/g fett), följt av BDE-47 (0,46 ng/g fett), HBCD (0,22 ng/g fett) och BDE-100 (0,13 ng/g fett). Summan av de tio analyserade PBDE-kongenerna var ungefär dubbelt så hög i blodserum (median 2,8 ng/g fett) som i modersmjölk (median 1,5 ng/g fett). För BDE-66, BDE-99, BDE-138 och HBCD kunde inga korrelationer mellan blod-och mjölkhalter utvärderas eftersom halterna låg under LOQ i de flesta proverna. För övriga kongener var samtliga korrelationer signifikanta (p<0,05). Korrelationskoefficienterna (Pearson) för de tri-till hexabromerade kongenerna varierade mellan 0,83 och 0,98. Korrelationerna för de hexa-, hepta-och deka-brominerade kongenerna 66). De svagare korrelationerna för vissa kongener beror åtminstone delvis på större osäkerhet i analysresultaten (låga halter och många prover med halter under LOQ i blodserum och/eller modermjölk). Generellt minskade fördelningen av PBDE till modersmjölk med ökande bromeringsgrad. Mediankvoten mellan serum och mjölk varierade från 0,83 (BDE-47) till 17 (BDE-209). BDE-209 överförs i mycket liten utsträckning till mjölk. Sammanfattningsvis var korrelationerna mellan PBDE-halter i serum och modersmjölk signifikanta för samtliga analyserade kongener och starka för de tri-till hexabromerade kongenerna. Modersmjölks-halter av PBDE kan alltså användas som mått på mammans kroppsbelastning och sannolikt också på exponeringen av barnet under fosterperioden. Skillnaderna i absoluta nivåer i serum och modersmjölk kan vara viktiga att ta hänsyn till vid exponeringsbedömningar. 3 INTRODUCTION With funding from the Swedish Environmental Protection Agency (EPA), the Swedish National Food Agency (NFA) has made recurrent measurements of persistent halogenated organic pollutants (POP) in mother's milk from primiparae women in Uppsala since 1996. The study is called POPUP (Persistent Organic Pollutants in Uppsala Primiparas), and the aim is to estimate the body burdens of POP among pregnant and nursing women and to estimate temporal trends of the exposure of fetuses and breast-fed infants. Temporal trends of polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), chlorinated pesticides (e.g. DDT-compounds) and brominated flame retardants (polybrominated diphenylethers (PBDE) and hexabromocyclododecane (HBCD)) in breast milk between 1996 and 2010 have been established Lipid-adjusted breast milk levels of chlorinated POPs like PCBs, PCDD/Fs and p,p´-DDE can be used to estimate prenatal exposure since there is a high correlation between levels in breast milk, maternal blood and cord blood However, there are few studies on correlations between levels of brominated compounds in different matrices. The aim of the present study was to investigate correlations between blood serum and breast milk levels of PBDEs and HBCD in paired samples from 30 mothers in the POPUP cohort and to evaluate if the concentration of PBDE/HBCD in breast milk is a good indicator of maternal body burden and possibly also of prenatal exposure of the infant. MATERIALS AND METHODS Recruitment and sampling Women were randomly recruited among first-time mothers who were Swedish by birth and delivered at Uppsala University Hospital from January to December 2010 (N=30). The participating rate was 42%. 4 The participating mothers sampled milk at home during the third week after delivery (day 14-21 post partum). Milk was sampled during nursing using a manual mother's milk pump and/or a passive mother's milk sampler. The women were instructed to sample milk both at the beginning and at the end of the breast-feeding sessions. The goal was to sample 500 mL from each mother during 7 days of sampling. During the sampling week, the milk was stored in the home freezer in acetone-washed bottles. Newly sampled milk was poured on top of the frozen milk. At the end of the sampling week, a midwife visited the mother to collect the bottles. On that occasion, the participants also donated a blood sample. Blood sampling was done using 9 ml Vacutainer® or Vacuette® serum tubes and serum was stored at -20°C. Chemical analysis Extraction and clean-up Serum: Thawed serum (4 g) was mixed with methanol (4 ml) by vortexing in a 16 ml test tube. A mixture of diethyl ether and n-hexane (5 ml, 1+1 V/V) and 100 µl of a solution containing the internal surrogate standards, BDE-85 (2 pg/µl) and 13 C 12 BDE-209 (1 pg/µl) was added. The sample was extracted on a rotary mixer for 15 min and then centrifuged at 2500 rpm for 10 min. After centrifugation, the top layer, organic phase, was transferred to a test tube containing aqueous potassium chloride (4 ml, 1 % w/w). The denaturated serum was re-extracted with diethyl ether and n-hexane (5 ml, 1+1 V/V) and the organic phase was combined with the first extract. The pooled extracts and the potassium chloride solution was mixed on a rotary mixer for 15 min and then centrifuged at 2500 rpm for 5 min. The organic phase was transferred to a pre-weighed test tube. The potassium chloride solution was reextracted and the extracts were combined. The solvent was evaporated using a gentle stream of nitrogen and the lipid weight was determined gravimetrically. In order to remove the lipids and other polar materials the lipid extract was re-dissolved in nhexane (2 ml) using ultra-sonication and then treated with concentrated sulfuric acid (2 ml) by inverting the test tube 15 times and then centrifuged at 2000 rpm for 15 min. The organic phase was transferred to a test tube (5 ml) and the volume was reduced to 0.5 ml by using a gentle stream of nitrogen. In order to remove any remaining lipids the sample was transferred to an sulfuric acid impregnated silica gel column (8 mm id, 1 g, 1+2 w/w) and eluted with a mixture of dichloromethane and n-hexane (12 ml, 1+1 V/V). The eluent was reduced to 0.5 ml. The lipid-free extract was transferred to a pre-washed silica gel column (8 mm id, 2 g of 3 % deactivated silica gel) and eluted with n-hexane (8 ml). A second fraction, containing the 5 brominated flame retardants, was eluted with a mixture of dichloromethane and n-hexane (12 ml, 1+1 V/V). The second fraction was reduced to 1 ml using a rotary evaporator and transferred to a test tube where the solvent was changed to n-hexane. The final volume of the sample was adjusted to 100 µl using a gentle stream of nitrogen and then kept in an amber GC vial until analysis. Milk: Human milk (4 g) was mixed with n-hexane and acetone (10 ml, 1+1, V/V) and 100 µl of a solution containing the internal surrogate standards, BDE-85 (2 pg/µl) and 13 C 12 BDE-209 (1 pg/µl) was added. The sample was extracted on a rotary mixer for 15 min and then centrifuged at 2500 rpm for 10 min. After centrifugation, the top layer, organic phase, was transferred to a pre-weighed test tube. The extraction was repeated once with n-hexane and acetone (4 ml, 1+1 V/V) and the organic layers were combined. Ethanol (1 ml, 99.5 %) was added to the combined extract before the solvent was evaporated using a gentle stream of nitrogen. The lipid weight was determined gravimetrically. In order to remove the lipids and other polar materials the lipid extract was re-dissolved in nhexane (2 ml) using ultra-sonication and then treated with concentrated sulfuric acid (8 ml) by inverting the test tube 25 times and then centrifuged at 2000 rpm for 15 min. The organic phase was transferred to a test tube (5 ml) and the volume was reduced to 0.5 ml by using a gentle stream of nitrogen. The lipid-free extract was transferred to a pre-washed silica gel column (8 mm id, 4,5 g of 3 % deactivated silica gel) and eluted with n-hexane (17 ml). A second fraction, containing the brominated flame retardants, was eluted with a mixture of dichloromethane and n-hexane (25 ml, 1+1 V/V). The second fraction was reduced to 1 ml using a rotary evaporator and transferred to a test tube where the solvent was changed to n-hexane. The final volume of the sample was adjusted to 100 µl using a gentle stream of nitrogen and then kept in an amber GC vial until analysis Analysis on GC-LRMS The quantification of the analytes was performed using capillary gas chromatography and mass selective detection in electron capture negative ionization and selected ion monitoring modes (GC/LRMS/ECNI-SIM). The system used for quantification consisted of an Agilent 6890N GC equipped with an Agilent 5973N MS. The sample, 6 µl (2 x3 µl), was injected in pulsed splitless mode using a programmable temperature vaporizing (PTV) injector with an initial temperature of 70°C followed by rapid 6 heating to 300°C after injection. The analytes were separated on a DB-5MS (15 m x 0.25 mm x 0.10 μm, J&W Scientific) capillary column using a ramped carrier gas (helium) flow. The oven temperature was programmed from 60°C to 325°C including several ramps. The mass fragments m/z 79 and 81 were monitored fo