<p>Brominated flame retardants (BFRs) has for many years been used in products to reduce their</p><p>flammability, mainly in electronic products, textiles and construction materials.</p><p>In 2003, Sweden imported 300 tons of brominated flame retardants.</p><p>Leakage of these compounds has polluted natural environments. Fishes has shown increased</p><p>contents of these substances, especially fat fish, since brominated flame retardants tends to</p><p>accumulate in fatty tissues.</p><p>They are also regarded as persistent and that gives them the ability to travel long distances.</p><p>What also is really scary is that increased levels of brominated flame retardants have been</p><p>detected in human breast milk.</p><p>The knowledge of the brominated flame retardants is limited and not so much research has been</p><p>done in this field. There are many reasons though, to keep the research going. Partly their</p><p>structural resemblance to well-known toxics as PCB, but also their ability to accumulate in</p><p>biological systems and enrich in food chains.</p><p>In which way they affect humans we really don’t know yet. Experiments on mice have been done</p><p>and behavioural disturbances were observed as well as a decrease in learning capacity.</p><p>The BRFs chosen for this study are hexabromocyclododecan (HBCD) and a polybrominated</p><p>diphenyl ether (PBDE) called pentabromodiphenylether (BDE 99).</p><p>About 25 % of the flame retardants produced in the world every year, consists of the brominated</p><p>ones. If we look at the PBDEs it’s mainly tetrabromodiphenylether (BDE 47),</p><p>pentabromodiphenylether (BDE 99), octabromodiphenylether (BDE 205) and</p><p>decabromodiphenylether (BDE 209) that are common flame retardants.</p><p>As from July 1st 2006, PBDE was forbidden in electric and electronic products (15).</p><p>January 1st 2007, a Swedish prohibition concerning the use of BDE-209 was established. BDE-</p><p>209 is not allowed on the Swedish market if the content of substance exceed 0,1 percent of weight</p><p>(23).</p><p>BDE-99 is classified as environmentally dangerous and a healthrisk. It´s not only toxic for</p><p>waterliving organisms but also for humans if exposed during a long time (19).</p><p>We shall develop a well functioning, reproducible and economic method to analyse HBCD and</p><p>PBDE in sediment. It includes extraction, cleanup and sample analyses with a ECD (electron</p><p>capture detector) equipped capillary gas chromatograph.</p><p>Several analyses of adequate diluted standards has been analysed in a GC-ECD to find suitable</p><p>temperature gradients. Then we produced spiked sediment samples with known amounts of</p><p>standards. To homogenize the spiked samples we used a mixer. As a first step in the extraction we</p><p>used acetone as a solvent. Then we used cyclohexane and NaCl (2%) to separate the nonpolar and</p><p>polar substances from each other. Cleanup with concentrated sulphuric acid was done and for</p><p>some samples we also used Florisil. To concentrate the extract it was evaporated with N2(g).The</p><p>extract was then analysed in a GC-ECD and then the results was compared with the standards.</p><p>The result of this project showed that our method of extraction is useful. We managed to extract</p><p>50 % HBCD, 30 % BDE-99 and 60 % BDE-99 (dried sediment).</p>

Brominated flame retardants (BFRs) has for many years been used in products to reduce their flammability, mainly in electronic products, textiles and construction materials. In 2003, Sweden imported 300 tons of brominated flame retardants. Leakage of these compounds has polluted natural environments. Fishes has shown increased contents of these substances, especially fat fish, since brominated flame retardants tends to accumulate in fatty tissues. They are also regarded as persistent and that gives them the ability to travel long distances. What also is really scary is that increased levels of brominated flame retardants have been detected in human breast milk. The knowledge of the brominated flame retardants is limited and not so much research has been done in this field. There are many reasons though, to keep the research going. Partly their structural resemblance to well-known toxics as PCB, but also their ability to accumulate in biological systems and enrich in food chains. In which way they affect humans we really don’t know yet. Experiments on mice have been done and behavioural disturbances were observed as well as a decrease in learning capacity. The BRFs chosen for this study are hexabromocyclododecan (HBCD) and a polybrominated diphenyl ether (PBDE) called pentabromodiphenylether (BDE 99). About 25 % of the flame retardants produced in the world every year, consists of the brominated ones. If we look at the PBDEs it’s mainly tetrabromodiphenylether (BDE 47), pentabromodiphenylether (BDE 99), octabromodiphenylether (BDE 205) and decabromodiphenylether (BDE 209) that are common flame retardants. As from July 1st 2006, PBDE was forbidden in electric and electronic products (15). January 1st 2007, a Swedish prohibition concerning the use of BDE-209 was established. BDE- 209 is not allowed on the Swedish market if the content of substance exceed 0,1 percent of weight (23). BDE-99 is classified as environmentally dangerous and a healthrisk. It´s not only toxic for waterliving organisms but also for humans if exposed during a long time (19). We shall develop a well functioning, reproducible and economic method to analyse HBCD and PBDE in sediment. It includes extraction, cleanup and sample analyses with a ECD (electron capture detector) equipped capillary gas chromatograph. Several analyses of adequate diluted standards has been analysed in a GC-ECD to find suitable temperature gradients. Then we produced spiked sediment samples with known amounts of standards. To homogenize the spiked samples we used a mixer. As a first step in the extraction we used acetone as a solvent. Then we used cyclohexane and NaCl (2%) to separate the nonpolar and polar substances from each other. Cleanup with concentrated sulphuric acid was done and for some samples we also used Florisil. To concentrate the extract it was evaporated with N2(g).The extract was then analysed in a GC-ECD and then the results was compared with the standards. The result of this project showed that our method of extraction is useful. We managed to extract 50 % HBCD, 30 % BDE-99 and 60 % BDE-99 (dried sediment).

Das Ziel dieser Arbeit war die Untersuchung des enantiomerenspezifischen Umweltverhaltens des Flammschutzmittels Hexabromcyclododecan (HBCD). Zu Beginn erfolgte daher die Optimierung und Validierung eines enantiomerenspezifischen Analysenverfahrens für die Bestimmung von HBCD in Biota. Die errechneten mittleren Wiederfindungen lagen im Bereich von 100-102 % und die Nachweisgrenzen zwischen 0,131 und 0,255 pg g-1. Untersuchungen zur ubiquitären Verteilung von HBCD erfolgten an Eiern der Silbermöwe deutscher Nord- und Ostseeinseln (Probenahme 1988-2008). In allen Fällen dominierte alpha-HBCD das Diastereomerenmuster, wobei eine bevorzugte Anreicherung von (-)-alpha-HBCD sowie ein zeitlicher Trend aller Enantiomeren-Gehalte festgestellt wurde. Zur Klärung der Frage einer Bioakkumulation sowie -isomerisierung der HBCD-Stereoisomere erfolgten Langzeit-Fütterungsversuche an Spiegelkarpfen. Die Untersuchungen ergaben eine signifikante Akkumulation des jeweils gefütterten HBCD-Enantiomers, jedoch konnte die Hypothese der Bioisomerisierung nicht bestätigt werden. Ein weiterer Schwerpunkt lag in Untersuchungen zur cytochromabhängigen enantiomerenspezifischen Biotransformation von HBCD im Rahmen des Metabolismus an Lebermikrosomen diverser Spezies. Hier konnte gezeigt werden, dass HBCD dem Phase I-Metabolismus unterliegt und hydroxyliert wird. Dabei weist jedes HBCD-Enantiomer ein spezifisches Metabolitenmuster auf, was eine Zuordnung der hydroxylierten Verbindungen zum entsprechenden HBCD-Enantiomer erlaubt. Anhand von Zeitreihen sowie der Berechnung von Halbwertszeiten konnte der Verdacht eines enantiomerenspezifischen Metabolismus in Richtung einer Anreicherung von (-)-alpha- und (+)-gamma-HBCD bestätigt werden. Inkubationsansätze mit reinen Cytochrom (CYP)-Isoformen sowie molekülmechanische Berechnungen legen die Vermutung nahe, dass dem CYP3A4 eine Schlüsselrolle bei der Metabolisierung von HBCD zukommt. / The main emphasis of this thesis was on the enantio-specific environmental behaviour of the polybrominated flame retardant hexabromocyclododecane (HBCD). Initially, an enantio-specific analytical method for the determination of HBCD in biota was optimised and validated. The calculated mean recoveries ranged from 100 to 102 % and the limits of detection are in the range of 0.131 to 0.255 pg g-1. First investigations of the ubiquitous environmental distribution of HBCD were performed using herring gull eggs from different islands in the North and Baltic Sea (sampling 1988 to 2008). In all cases alpha-HBCD was the predominant diastereomer. Significant deviations from the racemic mixture revealed a preferred enrichment of the first eluting (-)-alpha-HBCD. In addition, a temporal trend of HBCD levels was observed. To clarify the issue of accumulation as well as bioisomerisation of HBCD stereoisomers, a long-term feeding study with mirror carps was performed. The results showed an accumulation of each initially fed HBCD enantiomer, but hypothesis of a bioisomerisation could not be confirmed. Another important focus of this work was to study the cytochrome-dependent enantio-specific biotransformation of HBCD enantiomers in various species of liver microsomes. It was shown that HBCD is subject to phase I metabolism. In the course of this process, HBCD is metabolised to hydroxylated products, whereas each HBCD enantiomer results in a specific metabolite pattern allowing the allocation of the corresponding hydroxylated compounds. Investigation of time series as well as the calculation of half-lives, the hypothesis of an enantio-specific metabolism towards an enrichment of (-)-alpha- and (+)-gamma-HBCD could be confirmed. Incubation mixtures with pure cytochrome (CYP) isoforms, as well as molecular mechanic calculations suggest that CYP3A4 plays a key role in the biotransformation processes of HBCD.

<p>The two additive brominated flame retardants, tetrabromoethylcyclohexane (TBECH) and hexabromocyclododecane (HBCD) are used to prevent fire to start and spread. They are simply mixed with material and are most likely to leach out in the environment, because of non-covalently binding to the material. TBECH can exist as four pairs of enantiomers, α-, β-, γ- and δ-TBECH. The technical HBCD can exist as three pairs of enantiomers, α-, β- and γ-HBCD and two meso forms δ- and ε-HBCD. None of these compounds are produced in Sweden, but they are imported to industries. TBECH has been found in Beluga blubber and can accumulate in zebrafish. HBCD has been found in water environments and can be toxic to and bioaccumulate in water-living animals.</p><p>In this study, a method was developed for separation and detection of α-, β-, γ- and δ-TBECH on HRGC/HRMS. All TBECH-isomers could be separated with the developed method. How much of the TBECH isomers that were recovered after applying existing extraction and clean-up procedures, normally applied for clean-up and extraction of PCBs and PCDD/Fs, was evaluated. Low recovered amounts (6.8-35.5 %) of TBECH-isomers added in known amounts to three different whale samples indicate severe evaporation losses and possibly photolytic degradation. None of the four enantiomers were detected in the three whale samples. For HBCD analysis, both the chromatography and MS/MS parameters were optimised for δ- and ε- HBCD yielding good chromatography and sensitivity. However, due to technical difficulties during the time-period of this project, no whale samples could be analysed for HBCD on UPLC/MS/MS. </p>

The two additive brominated flame retardants, tetrabromoethylcyclohexane (TBECH) and hexabromocyclododecane (HBCD) are used to prevent fire to start and spread. They are simply mixed with material and are most likely to leach out in the environment, because of non-covalently binding to the material. TBECH can exist as four pairs of enantiomers, α-, β-, γ- and δ-TBECH. The technical HBCD can exist as three pairs of enantiomers, α-, β- and γ-HBCD and two meso forms δ- and ε-HBCD. None of these compounds are produced in Sweden, but they are imported to industries. TBECH has been found in Beluga blubber and can accumulate in zebrafish. HBCD has been found in water environments and can be toxic to and bioaccumulate in water-living animals. In this study, a method was developed for separation and detection of α-, β-, γ- and δ-TBECH on HRGC/HRMS. All TBECH-isomers could be separated with the developed method. How much of the TBECH isomers that were recovered after applying existing extraction and clean-up procedures, normally applied for clean-up and extraction of PCBs and PCDD/Fs, was evaluated. Low recovered amounts (6.8-35.5 %) of TBECH-isomers added in known amounts to three different whale samples indicate severe evaporation losses and possibly photolytic degradation. None of the four enantiomers were detected in the three whale samples. For HBCD analysis, both the chromatography and MS/MS parameters were optimised for δ- and ε- HBCD yielding good chromatography and sensitivity. However, due to technical difficulties during the time-period of this project, no whale samples could be analysed for HBCD on UPLC/MS/MS.

L’hexabromocyclododécane (HBCD) a été utilisé comme additif dans des polystyrènes (PS). En 2013, l’HBCD a été inclus à la Convention de Stockholm comme Polluant Organique Persistant. Les denrées alimentaires d’origine animale (DAOA) constituent la source principale d’exposition de la population générale à ce polluant. Le niveau moyen d’exposition du consommateur ne représente pas un risque majeur pour la sécurité sanitaire des aliments (EFSA, 2011). Malgré des concentrations moyennes généralement proches d’un ng g-1 lipides dans les DAOA, des concentrations maximales supérieures à 2000 ng g-1 lipides dans certains échantillons d’œuf ont été rapportées. Plusieurs sources potentielles d’exposition orale des volailles existent. Les poules pondeuses pourraient être exposées majoritairement à l’isomère α-HBCD. Au vu de l’absence d’informations dans la littérature sur le devenir de l’α-HBCD chez la poule pondeuse, une expérimentation in vivo a été réalisée et a permis d’estimer la plupart des paramètres cinétiques du devenir de l’α-HBCD ingéré chez la poule pondeuse. Un modèle pharmacocinétique basé sur la physiologie (PBPK) de transfert de l’α-HBCD chez la poule pondeuse a été développé, calibré par des données de la littérature, soumis à analyse de sensibilité et plusieurs paramètres ont été ajustés sur des résultats in vivo. Une validation du modèle a été testée. Ce modèle a permis de déterminer l’influence des caractéristiques physiologiques de la poule pondeuse sur le devenir de l’α-HBCD en cas d’exposition à la molécule. Parmi les caractéristiques d’intérêt, le poids vif (PV) est celle qui influence le plus la concentration d’α-HBCD dans les tissus et les œufs. Un PV bas et des performances de ponte réduites prédisposent à une concentration plus élevée dans ces matrices. La mue produit une augmentation de la charge corporelle de la molécule. Ce modèle peut être extrapolé à d’autres animaux et molécules lipophiles après modification de certains paramètres / Hexabromocyclododecane (HBCD) has been used as an additive in polystyrene (PS). In 2013, HBCD was included in the Stockholm Convention as a Persistent Organic Pollutant. The foodstuffs of animal origin are the main source of exposure of the general population to this pollutant. The mean exposure level of the consumers to HBCD doesn’t represent a major risk to the food safety (EFSA, 2011). In spite of mean concentrations generally close to 1 ng g-1 lipids in the foodstuffs of animal origin, maximal concentrations higher than 2000 ng g-1 lipids have been reported in some egg samples. There are several potential sources of oral exposure of the hens to this pollutant. The hens could be exposed mainly to the α-HBCD isomer. Since the bibliographic synthesis highlights the absence of information about the α-HBCD kinetics in the laying hen, an in vivo experience has been realized and has allowed the estimation of the majority of the α-HBCD kinetic parameters in the laying hen. A physiologically based pharmacokinetic (PBPK) model has been developed, calibrated by literature data, submit to a sensitivity analysis and some parameters have been adjusted on the basis of experimental data. A validation of the model has been attempted. This model has allowed to determine the influence of the main physiological characteristics of the laying hen exposed to the molecule on the α-HBCD accumulation in tissues and transfer to eggs. Among the physiological characteristics of concern, the body weight (BW) is the main feature influencing the α-HBCD kinetics. If the hen is exposed to the α-HBCD, a low BW and low laying performances predispose the hen to have higher concentrations of the molecule in all the matrices. The molting stage raises the body charge of the molecule. This model can be extrapolated to other animals and lipophilic molecules after modification of some parameters

<p>Many modern products, especially electronic goods, are protected by brominated flame retardants (BFR). Some of the most common flame retardants are polybrominated diphenylethers (PBDE), tetrabromobisphenol-A (TBBP-A) and hexabromocyclododecane (HBCD). These compounds have been found in environmental samples and shown to have physiological effects on experimental animals. This thesis considers end-of-life aspects of brominated flame retardants. When spread in the environment, these compounds may be degraded into other forms. For example, if sludge contaminated with PBDE is used as an agricultural fertilizer, the PBDE could be degraded by sunlight to species of PBDE with lower degree of bromination and, to some extent, also form polybrominated dibenzofurans (PBDF). In addition, PBDF and polybrominated dibenzo-p-dioxins (PBDD) are formed during combustion of brominated flame retardants. When waste products with brominated flame retardants are co-combusted with household waste or other chlorinated fuel, polybrominated- chlorinated dibenzo-p-dioxins (PBCDD) and polybrominated- chlorinated dibenzofurans (PBCDF)will be formed. The bromin/chlorine composition of dioxins and furans is dependent on the bromine/chlorine ratio in the fuel, but the types of brominated flame retardants that are being combusted is less important. In the studies reported here, bromine levels higher than "normal" for household waste has been used. The results show that there is a pronounced increase in total dioxin levels in fluegas when when bromine is present, implying that waste containing brominated flame retardants should only be incinerated at combustion plants with effecient air pollution control devices.</p>

Humans are exposed to persistent organic pollutants (POPs) such as brominated flame retardants (BFRs, specifically polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecane (HBCD)) and perfluoroalkyl acids (PFAAs, specifically perfluoroalkane sulfonate (PFOS) and perfluorooctanoic acid (PFOA)). They are used in consumer products found in cars, offices, homes and day care centers. Diet was earlier thought to be a major human exposure route for legacy POPs, but does not account for body burdens found for many new POPs and indoor exposure from air and dust has been hypothesized as also important. In this thesis, BFRs in air and dust, and PFAAs in dust from different indoor microenvironments in Sweden were analysed, and the results used to estimate human exposure. BFRs and PFAAs were detected in dust from all microenvironments and PBDEs in all air samples. BFR and PFAA exposure occurs mostly in peoples’ homes with toddlers having higher intakes from dust ingestion than adults. Inhalation and dust ingestion play minor roles compared to diet for humans with median exposures, but in worst case scenarios, dust ingestion may be significant for a small part of the Swedish population. Sampling using home vacuum cleaner bag dust and researcher-collected above floor dust was compared. Correlations were seen for ∑OctaBDE and ∑DecaBDE but not for ∑PentaBDE and HBCD. Higher PBDE concentrations were found in above floor dust but higher HBCD concentrations were found in vacuum cleaner bag dust. BDE-47 concentrations were correlated between vacuum cleaner bag dust and breast milk, indicating exposure through dust ingestion. Similar concentrations of PBDEs were measured in indoor and outgoing air from day care centers, apartment and office buildings. Indoor air explained 54-92% of ∑PentaBDE and 24-86% of BDE-209 total emissions to outdoor air in Sweden, supporting the hypothesis that the indoor environment is polluting ambient air via ventilation systems. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Accepted. Paper 3: Accepted. Paper 4: Manuscript.</p>

Brominated flame retardants (BFRs) have been used in a variety of consumer products to enhance their fire safety. This has resulted in exposure of the environment, wildlife and humans causing risks of endocrine disruption. The use restrictions of many BFRs have resulted in the introduction of other brominated chemicals (emerging BFRs) on the market. Humans are exposed to BFRs primarily via diet and dust ingestion, but the importance of the different exposure pathways has scarcely been studied. Children in particular have not been studied well due to lack of biomonitoring data. In this thesis, a mother-toddler cohort (n=24) from Uppsala was studied for their exposure to tri-decabrominated diphenyl ethers (tri-decaBDEs), isomer-specific hexabromocyclododecanes (HBCDs) and emerging BFRs (EBFRs). A clean-up and fractionation method was developed for determination of the above-mentioned BFRs in the same sample without dividing the sample. Measures of external exposure from dietary intake and dust ingestion were estimated. These were compared to internal concentrations (serum, feces) to determine which exposure pathways were most important for explaining the different BFR concentrations and patterns found in mothers and toddlers. Taking all the results into account, the toddlers’ higher serum levels of tetra-pentaBDEs seem to be the result of previous breastfeeding and those of octa-decaBDEs from exposure to house dust. For mothers, diet was estimated to be the main exposure route of tri-hexaBDEs and HBCDs. Dust ingestion was estimated to be the main route for BDE-209 exposure in mothers. Significant correlations were found between the tetra-decaBDE concentrations in matched serum and feces samples indicating that feces could be used as a non-invasive sample matrix for biomonitoring of PBDEs in toddlers. EBFRs were detected in the feces of toddlers and in a few serum samples from both mothers and toddlers indicating that exposure to these replacement chemicals occurs. / <p><strong> </strong>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Submitted. Paper 4: Manuscript.</p>

Many modern products, especially electronic goods, are protected by brominated flame retardants (BFR). Some of the most common flame retardants are polybrominated diphenylethers (PBDE), tetrabromobisphenol-A (TBBP-A) and hexabromocyclododecane (HBCD). These compounds have been found in environmental samples and shown to have physiological effects on experimental animals. This thesis considers end-of-life aspects of brominated flame retardants. When spread in the environment, these compounds may be degraded into other forms. For example, if sludge contaminated with PBDE is used as an agricultural fertilizer, the PBDE could be degraded by sunlight to species of PBDE with lower degree of bromination and, to some extent, also form polybrominated dibenzofurans (PBDF). In addition, PBDF and polybrominated dibenzo-p-dioxins (PBDD) are formed during combustion of brominated flame retardants. When waste products with brominated flame retardants are co-combusted with household waste or other chlorinated fuel, polybrominated- chlorinated dibenzo-p-dioxins (PBCDD) and polybrominated- chlorinated dibenzofurans (PBCDF)will be formed. The bromin/chlorine composition of dioxins and furans is dependent on the bromine/chlorine ratio in the fuel, but the types of brominated flame retardants that are being combusted is less important. In the studies reported here, bromine levels higher than "normal" for household waste has been used. The results show that there is a pronounced increase in total dioxin levels in fluegas when when bromine is present, implying that waste containing brominated flame retardants should only be incinerated at combustion plants with effecient air pollution control devices.