The major objectives of this research were to examine the bioaccumulation parameters [depuration rates (kd), half life (t1/2) and biomagnification factor (BMF)] of individual isomers of hexabromocyclododecane (HBCD, C12H18Br6) in fish and to test the hypothesis of in vivo bioisomerization. This was done by exposing three groups of juvenile rainbow trout (Oncorhynchus Mykiss) to food fortified with known concentrations of an individual diastereoisomer (α, β, γ) for 56 days (uptake phase) followed by 112 days (depuration phase) of unfortified food. A fourth group of fish were exposed to unfortified food for the duration of the experiment. Fish (n=4) from all four aquaria were sacrificed on days 0, 7, 14, 56, 63, 70, 112 and 168 and muscle tissue was extracted and analyzed for diastereoisomer concentrations by high performance liquid chromatography tandem mass spectrometry (LC/MS/MS). Bioaccumulation of the γ diastereoisomer was linear during the uptake phase while the α and β diastereoisomers were found to increase exponentially with respective doubling times of 14.1 and 20.5 days. Both the β and γ diastereoisomers followed first order depuration kinetics with calculated t1/2’s of 94 ± 25 and 84 ± 51 (± 1 × standard error) days, respectively. The BMF for the α diastereoisomer (BMF = 4.1) was one and a half times greater than the β-diastereoisomer (BMF = 2.6) and about one fifth larger than the γ-diastereoisomer (BMF = 3.6). The large BMF for the α diastereoisomer is consistent with this diastereoisomer dominating higher trophic level organisms in wildlife. Although the BMF of the β diastereoisomer suggests that it will biomagnify, because it is present in small quantities in commercial mixtures it is rarely detected in environmental samples. Results from these studies also provide evidence of bioisomerization of the β and γ diastereoisomers. Most importantly, the α diastereoisomer which was recalcitrant to bioisomerization by juvenile rainbow trout in this study and known to be the dominant diastereosiomer in fish, was bioformed from both the β and γ diastereoisomers. To our knowledge, this is the first report of bioisomerization of a halogenated organic pollutant in biota. / May 2006

The major objectives of this research were to examine the bioaccumulation parameters [depuration rates (kd), half life (t1/2) and biomagnification factor (BMF)] of individual isomers of hexabromocyclododecane (HBCD, C12H18Br6) in fish and to test the hypothesis of in vivo bioisomerization. This was done by exposing three groups of juvenile rainbow trout (Oncorhynchus Mykiss) to food fortified with known concentrations of an individual diastereoisomer (α, β, γ) for 56 days (uptake phase) followed by 112 days (depuration phase) of unfortified food. A fourth group of fish were exposed to unfortified food for the duration of the experiment. Fish (n=4) from all four aquaria were sacrificed on days 0, 7, 14, 56, 63, 70, 112 and 168 and muscle tissue was extracted and analyzed for diastereoisomer concentrations by high performance liquid chromatography tandem mass spectrometry (LC/MS/MS). Bioaccumulation of the γ diastereoisomer was linear during the uptake phase while the α and β diastereoisomers were found to increase exponentially with respective doubling times of 14.1 and 20.5 days. Both the β and γ diastereoisomers followed first order depuration kinetics with calculated t1/2’s of 94 ± 25 and 84 ± 51 (± 1 × standard error) days, respectively. The BMF for the α diastereoisomer (BMF = 4.1) was one and a half times greater than the β-diastereoisomer (BMF = 2.6) and about one fifth larger than the γ-diastereoisomer (BMF = 3.6). The large BMF for the α diastereoisomer is consistent with this diastereoisomer dominating higher trophic level organisms in wildlife. Although the BMF of the β diastereoisomer suggests that it will biomagnify, because it is present in small quantities in commercial mixtures it is rarely detected in environmental samples. Results from these studies also provide evidence of bioisomerization of the β and γ diastereoisomers. Most importantly, the α diastereoisomer which was recalcitrant to bioisomerization by juvenile rainbow trout in this study and known to be the dominant diastereosiomer in fish, was bioformed from both the β and γ diastereoisomers. To our knowledge, this is the first report of bioisomerization of a halogenated organic pollutant in biota.

The major objectives of this research were to examine the bioaccumulation parameters [depuration rates (kd), half life (t1/2) and biomagnification factor (BMF)] of individual isomers of hexabromocyclododecane (HBCD, C12H18Br6) in fish and to test the hypothesis of in vivo bioisomerization. This was done by exposing three groups of juvenile rainbow trout (Oncorhynchus Mykiss) to food fortified with known concentrations of an individual diastereoisomer (α, β, γ) for 56 days (uptake phase) followed by 112 days (depuration phase) of unfortified food. A fourth group of fish were exposed to unfortified food for the duration of the experiment. Fish (n=4) from all four aquaria were sacrificed on days 0, 7, 14, 56, 63, 70, 112 and 168 and muscle tissue was extracted and analyzed for diastereoisomer concentrations by high performance liquid chromatography tandem mass spectrometry (LC/MS/MS). Bioaccumulation of the γ diastereoisomer was linear during the uptake phase while the α and β diastereoisomers were found to increase exponentially with respective doubling times of 14.1 and 20.5 days. Both the β and γ diastereoisomers followed first order depuration kinetics with calculated t1/2’s of 94 ± 25 and 84 ± 51 (± 1 × standard error) days, respectively. The BMF for the α diastereoisomer (BMF = 4.1) was one and a half times greater than the β-diastereoisomer (BMF = 2.6) and about one fifth larger than the γ-diastereoisomer (BMF = 3.6). The large BMF for the α diastereoisomer is consistent with this diastereoisomer dominating higher trophic level organisms in wildlife. Although the BMF of the β diastereoisomer suggests that it will biomagnify, because it is present in small quantities in commercial mixtures it is rarely detected in environmental samples. Results from these studies also provide evidence of bioisomerization of the β and γ diastereoisomers. Most importantly, the α diastereoisomer which was recalcitrant to bioisomerization by juvenile rainbow trout in this study and known to be the dominant diastereosiomer in fish, was bioformed from both the β and γ diastereoisomers. To our knowledge, this is the first report of bioisomerization of a halogenated organic pollutant in biota.

In rivers, association of organic contaminants with dissolved organic carbon may limit freely dissolved or bioavailable fractions and toxicity of organic contaminants. Consequently, assessment of toxicity of organic contaminants on the basis of their total chemical concentrations may lead to overestimation of risks to organic contaminants. Therefore, to achieve reliable and accurate risks assessment for organic contaminants, determination of bioavailability is important. The influence of humic acid on the bioavailability of organic contaminants in rivers was studied, using three chemicals with different properties as model contaminants, which at the start of the study were detected in wastewater effluents. It was hypothesized that in the presence of dissolved organic carbon, a fraction of the total concentration of an organic contaminant would not be bioavailable in river water. Therefore, the aim of the study was to determine bioavailability and its impact on toxicity. Bioavailability in the presence of humic acid was determined chemically and using a yeast estrogen screen assay. The chemical method comprised solid-phase extraction and liquid chromatography-mass spectrometry to determine freely dissolved and the fraction of the chemicals associated with dissolved organic carbon. The results indicated increased binding to dissolved organic carbon with the hydrophobicity of the test compounds except for perfluorooctane sulfonate. The dissolved organic carbon-water partition coefficient for ethinylestradiol was determined to be Log KDOC 2.36. Log KDOC values of 4.15 and 4.41 at 10 and 100 mg/L humic acid, respectively, were derived for hexabromocyclododecane indicating greater binding than ethinylestradiol due to the more hydrophobic character. The yeast estrogen screen was used as a biological method to measure the effect of humic acid on the bioavailability of ethinylestradiol and a more hydrophobic compound, dichlorodiphenyltrichloroethane. Results of the yeast estrogen screen indicated that the presence of humic acid had no effect on bioavailability of either of the chemicals.

Perfluorooctane sulfonate (PFOS) is widely used in industrial products and is potentially dangerous to the aquatic environment due to not being broken down whether by chemical or biological means, having a half-life of more than 41 years and disrupting hormones. Hexabromocyclododecane (HBCD) is the third most used brominated flame retardant and is of environmental concern as it bioaccumulates and magnifies in the food chain and is highly toxic to aquatic organisms. The purpose of this study was to examine the effect of PFOS and HBCD on the embryos of rainbow trout (Onchorhynchus mykiss) by analyzing lipid profiles with UHPLC/Q-TOF-MS. The fish embryos were treated with various concentrations of PFOS and HBCD (0.058-58 μg/l and 0.014-14 μg/l respectively) with DMSO as carrier solvent and then extracted after homogenization with 0.9% NaCl-solution followed by addition of ISTD mixture, methanol, methyl tert-butyl ether (MTBE) and MQ-water. The raw data was processed with MZmine-2.32. 153 lipids were identified with the main lipids consisting of glycerophospholipids and triacylglycerols. A two-tailed t-test was used to study the impact of the chemical exposure on the embryos, where p-values below 0.05 were lipids considered as significant change. The HBCD exposure caused significant change in various triacylglycerols, whereas PFOS exposure caused significant change in triacylglycerols as well as in glycerophospholipids such as PC(O-38:5) and LPC(20:4). The results were in alignment with previous studies.

<p>The dietary intake of polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecane (HBCD) have been estimated for Swedish children. A dietary survey performed in 2003, including 4, 8-9 and 11-12 year olds, and concentrations in individual food items were combined. The food included in the study was mainly of animal origin, consisting of fish and shellfish, dairy products, meat products, eggs, animal and vegetable fats and fats from miscellaneous food products. The medium-bound intake of PBDEs (9 congeners) were estimated to 23.0 ng/day, 30.9 ng/day and 27.7 ng/day for 4, 8-9 and 11-12 years olds respectively. The corresponding estimations for HBCD were 7.94 ng/day 10.7 ng/day and 9.46 ng/day for 4, 8-9 and 11-12 years olds respectively. These results show a higher daily intake for 8-9 year olds compared with the other age groups. However, when estimating the daily intake per kg bw, the intake decreases with age. BDE-47 contributed the most to the total intake of PBDEs, with approximately 40%. The food group contributing the most to the intake of PBDEs and HBCD was fish and shellfish, of which non-Baltic fatty fish was the largest contributor. There were no considerable differences between boys and girls in any of the aspects examined. The result from this study show a lower intake of PBDEs and HBCD in Swedish children compared with children in other studies made in Europe and the United States.</p> / <p>Bromerade flamskyddsmedel används för att skydda brännbara material från att fatta eld, till exempel skyddas textilier och plaster i bland annat elektronik, fordon och möbler. Två typer av bromerade flamskyddsmedel är polybromerade difenyletrar (PBDE) och hexabromocyklododekan (HBCD). Dessa är additiva flamskyddsmedel och blandas i materialet som ska skyddas men binder inte in i produkten och kan därför lätt läcka ut i miljön, vilket också har skett. Halter har påträffats i miljön och i biota långt från plaster där ämnena produceras eller används.</p><p>PBDE och HBCD har visats ha hormonstörande och neurotoxiska effekter i studier på råtta och mus. Thyroxinnivåerna sjunker vid exponering av PBDE och HBCD, vilket skulle kunna leda till sköldkörtelproblem och störd utveckling av bland annat hjärnan om exponering sker perinatalt. De neurotoxiska effekterna inkluderar inlärnings- och minnessvårigheter och ett förändrat beteende med hyper- och hypoaktivitet som följd.</p><p>Human exponering för PBDE och HBCD sker främst via födan och speciellt via animaliska produkter då dessa ämnen är lipofila, bioackumulerande och ofta biomagnifierande vilket gör att de påträffas i högre koncentrationer högre upp i trofinivåerna. Studier från bland annat Sverige och Finland visar att fisk och skaldjur är den största källan till intag av PBDE.</p><p>De flesta intagsberäkningar av PBDE och HBCD baseras på livsmedelskonsumtionen hos vuxna och visar följaktligen endast hur intaget ser ut för den delen av populationen. För barn, som är en av de känsligaste grupperna i populationen, finns inte många studier att tillgå, varken från Sverige eller andra delar av världen. I den här studien har därför intaget av PBDE (summan av 9 kongener) och HBCD beräknats för barn i Sverige.</p><p>I en rikstäckande kostundersökning utförd 2003 deltog barn i åldrarna 4, 8-9 och 11-12 år. De fick i en matdagbok ange sin konsumtion under fyra på varandra följande dagar. Data från denna undersökning kombinerades sedan med haltdata från olika livsmedel för att räkna ut intaget av PBDE och HBCD på individbasis. Undersökningen innefattade främst animaliska livsmedel och innehöll därför fisk och skaldjur, mejeriprodukter, köttprodukter, ägg, animaliskt och vegetabiliskt fett och fett från övriga livsmedel.</p><p>Resultaten visar att födointaget av PBDE var 23,0 ng/dag, 30,9 ng/dag och 27,7 ng/dag för 4, 8-9 respektive 11-12 åringar. Intaget av HBCD beräknades till 7,94 ng/dag, 10,7 ng/dag och 9,46 ng/dag för 4, 8-9 respektive 11-12 åringar. Detta visar att 8-9 åringar har det högsta dagliga intaget av PBDE och HBCD. När intaget beräknas på kroppsvikt däremot, har de yngsta barnen det högsta intaget som sedan sjunker med åldern. Fisk och skaldjur var den största källan till intaget av PBDE och HBCD, trots att konsumtionen av dessa livsmedel var relativt lågt. Det fanns ingen större skillnad mellan pojkar och flickor, varken i intag av PBDE eller av HBCD. Jämfört med de få studier som gjorts i andra länder, är det tydligt att svenska barn har ett lägre intag av PBDE och HBCD.</p><p>Undersökningen tyder också på att intaget av PBDE och HBCD hos svenska barn, utifrån de kunskaper vi har idag, inte utgör någon risk med avseende på de effekter av PBDE och HBCD som påträffats i toxikologiska studier. Däremot är barn i ett känsligt skede i livet och upprepad exponering samt exponering för flera miljögifter samtidigt skulle kunna påverka deras utveckling negativt.</p>

The dietary intake of polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecane (HBCD) have been estimated for Swedish children. A dietary survey performed in 2003, including 4, 8-9 and 11-12 year olds, and concentrations in individual food items were combined. The food included in the study was mainly of animal origin, consisting of fish and shellfish, dairy products, meat products, eggs, animal and vegetable fats and fats from miscellaneous food products. The medium-bound intake of PBDEs (9 congeners) were estimated to 23.0 ng/day, 30.9 ng/day and 27.7 ng/day for 4, 8-9 and 11-12 years olds respectively. The corresponding estimations for HBCD were 7.94 ng/day 10.7 ng/day and 9.46 ng/day for 4, 8-9 and 11-12 years olds respectively. These results show a higher daily intake for 8-9 year olds compared with the other age groups. However, when estimating the daily intake per kg bw, the intake decreases with age. BDE-47 contributed the most to the total intake of PBDEs, with approximately 40%. The food group contributing the most to the intake of PBDEs and HBCD was fish and shellfish, of which non-Baltic fatty fish was the largest contributor. There were no considerable differences between boys and girls in any of the aspects examined. The result from this study show a lower intake of PBDEs and HBCD in Swedish children compared with children in other studies made in Europe and the United States. / Bromerade flamskyddsmedel används för att skydda brännbara material från att fatta eld, till exempel skyddas textilier och plaster i bland annat elektronik, fordon och möbler. Två typer av bromerade flamskyddsmedel är polybromerade difenyletrar (PBDE) och hexabromocyklododekan (HBCD). Dessa är additiva flamskyddsmedel och blandas i materialet som ska skyddas men binder inte in i produkten och kan därför lätt läcka ut i miljön, vilket också har skett. Halter har påträffats i miljön och i biota långt från plaster där ämnena produceras eller används. PBDE och HBCD har visats ha hormonstörande och neurotoxiska effekter i studier på råtta och mus. Thyroxinnivåerna sjunker vid exponering av PBDE och HBCD, vilket skulle kunna leda till sköldkörtelproblem och störd utveckling av bland annat hjärnan om exponering sker perinatalt. De neurotoxiska effekterna inkluderar inlärnings- och minnessvårigheter och ett förändrat beteende med hyper- och hypoaktivitet som följd. Human exponering för PBDE och HBCD sker främst via födan och speciellt via animaliska produkter då dessa ämnen är lipofila, bioackumulerande och ofta biomagnifierande vilket gör att de påträffas i högre koncentrationer högre upp i trofinivåerna. Studier från bland annat Sverige och Finland visar att fisk och skaldjur är den största källan till intag av PBDE. De flesta intagsberäkningar av PBDE och HBCD baseras på livsmedelskonsumtionen hos vuxna och visar följaktligen endast hur intaget ser ut för den delen av populationen. För barn, som är en av de känsligaste grupperna i populationen, finns inte många studier att tillgå, varken från Sverige eller andra delar av världen. I den här studien har därför intaget av PBDE (summan av 9 kongener) och HBCD beräknats för barn i Sverige. I en rikstäckande kostundersökning utförd 2003 deltog barn i åldrarna 4, 8-9 och 11-12 år. De fick i en matdagbok ange sin konsumtion under fyra på varandra följande dagar. Data från denna undersökning kombinerades sedan med haltdata från olika livsmedel för att räkna ut intaget av PBDE och HBCD på individbasis. Undersökningen innefattade främst animaliska livsmedel och innehöll därför fisk och skaldjur, mejeriprodukter, köttprodukter, ägg, animaliskt och vegetabiliskt fett och fett från övriga livsmedel. Resultaten visar att födointaget av PBDE var 23,0 ng/dag, 30,9 ng/dag och 27,7 ng/dag för 4, 8-9 respektive 11-12 åringar. Intaget av HBCD beräknades till 7,94 ng/dag, 10,7 ng/dag och 9,46 ng/dag för 4, 8-9 respektive 11-12 åringar. Detta visar att 8-9 åringar har det högsta dagliga intaget av PBDE och HBCD. När intaget beräknas på kroppsvikt däremot, har de yngsta barnen det högsta intaget som sedan sjunker med åldern. Fisk och skaldjur var den största källan till intaget av PBDE och HBCD, trots att konsumtionen av dessa livsmedel var relativt lågt. Det fanns ingen större skillnad mellan pojkar och flickor, varken i intag av PBDE eller av HBCD. Jämfört med de få studier som gjorts i andra länder, är det tydligt att svenska barn har ett lägre intag av PBDE och HBCD. Undersökningen tyder också på att intaget av PBDE och HBCD hos svenska barn, utifrån de kunskaper vi har idag, inte utgör någon risk med avseende på de effekter av PBDE och HBCD som påträffats i toxikologiska studier. Däremot är barn i ett känsligt skede i livet och upprepad exponering samt exponering för flera miljögifter samtidigt skulle kunna påverka deras utveckling negativt.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第23183号 / 工博第4827号 / 新制||工||1754(附属図書館) / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 酒井 伸一, 教授 田中 宏明, 准教授 平井 康宏 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Brominated flame retardants (BFRs) are chemicals used in e.g. electronic equipment, textiles and plastics for the prevention of fire. Over recent decades, concern has been raised regarding some heavily used BFRs, since the levels in the environment have been increasing. In the present thesis, persistent, bioaccumulative, and toxic (PBT) properties were studied for a structurally varied set of BFRs selected to represent more than 60 organic BFRs. The studied BFRs include: 2,4,4'-tribromodiphenyl ether (BDE 28), 2,2',3,4,4',5',6-heptabromodiphenyl ether (BDE  183), decabromodiphenyl ether (BDE 209), hexabromocyclododecane (HBCD), tetrabromobisphenol A (TBBPA), tetrabromobisphenol A 2-hydroxyethyl ether (TBBPA OHEE), 2,4,6-tribromophenol (246BrPh), 1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane (TBECH), and hexabromobenzene (HxBrBz). It is likely that soil will act as a sink for BFRs. Therefore, studies of BFRs were performed on persistence in soil, and on bioaccumulation from soil in the earthworm Eisenia fetida. Large variation in the biodegradability in soil among the tested BFRs was observed. The studied brominated diphenyl ethers (BDE 28 and BDE 209) were very persistent under both aerobic and anaerobic conditions, while 246BrPh and TBECH degraded quickly in both aerobic and anaerobic soil. The bioaccumulation in earthworm from soil was high for HxBrBz, TBECH and for tested brominated diphenyl ethers with 4-6 bromine atoms (BDE 47, BDE 99, and BDE 153). Bioaccumulation was also studied in zebrafish after dietary exposure to a mixture of BFRs. It was shown that several metabolites were formed and retained in zebrafish, which highlights the importance of also searching for and identifying persistent degradation products. Maternal transfer was shown for all BFRs present in the female zebrafish. This shows that zebrafish young (fry) are exposed to these BFRs at approximately the same concentrations as female zebrafish during the early-life stages, when fish are usually most sensitive to organic contaminants. Toxicity of individual BFRs and a BFR mixture was studied in Nitocra spinipes using a silica gel-based system. Highest toxicities were observed for BDE 28, TBBPA, and TBBPA OHEE. In the mixture toxicity study, simultaneous exposure to low concentrations (individually causing no significant effect) of six BFRs significantly affected the survival of Nitocra spinipes. The results from the PBT studies presented in the thesis and literature data were compared with the criteria for PBT classification, as set in the European REACH legislation. Further, some BFRs with physico-chemical properties similar to those of identified PBTs were suggested to be prioritized for future PBT testing.

This diploma thesis was focused on the analysis of four compounds from brominated flame retardants group, namely pentabromoethylbenzene, hexabromocyclododecane, hexabromobenzene and tetrabromobisphenol A. Matrices were represented by four soil samples and four seat of fire samples. In addition, bisphenol A was also determined. First of all, process of extraction of analytes from matrices was optimized. Three different extraction techniques were tested (pressurized solvent extraction, microwave-assisted extraction and ultrasound-assisted extraction). Next step was to verify stability of target compounds in strongly acidic conditions and to optimize clean-up of extract by column chromatography. Gas chromatography with electron capture detection (GC-ECD) was chosen as an appropriate analytical method for the determination of brominated flame retardants. Bisphenol A was determined by gas chromatography with mass spectrometric detection (GC-MS). The derivatization of phenolic analytes before their final analytical determination was also essential.