Thesis (MTech (Chemistry))--Cape Peninsula University of Technology, 2008. / Includes bibliographical references (leaves 77-79). Also available online.

Brominated flame retardants (BFR) such as hexachlorocyclopentadienyl-dibromocyclooctane (HCDBCO), bis(2-ethylhexyl)tetrabromophthalate (BEHTBP), 1,2-bis(2,4,6-tribromophenoxy)ethane (BTBPE) and decabromodiphenylethane (DBDPE) are contaminants of environmental concern. These BFRs are replacement alternatives for some of the major production BFRs, which have been restricted from the marketplace due to their adverse health effects. Their presence in environmental matrices, including wild birds, suggests they should be tested for possible toxic effects. BFR alternatives have been detected in the eggs of colonial fish-eating birds, suggesting maternal transfer during ovogenesis and the potential for these chemicals to bioaccumulate through the food chain. However, information regarding the toxicity of HCDBCO, BEHTBP, BTBPE and DBDPE exposure in birds is lacking. This thesis consisted of a combined in vitro/in ovo approach to determine: 1) the concentration-dependent effects of these four BFR alternatives in chicken embryonic hepatocytes (CEH), and 2) the dose-dependent effects of HCDBCO and BTBPE in chicken embryos following injection into the air cell of eggs prior to incubation. Changes in the mRNA expression levels of genes previously found to be responsive to other BFRs were assessed in CEH and liver tissue, in addition to examining overt toxicity (i.e. cytotoxicity, pipping success). None of the BFRs tested were cytotoxic up to 60 µM HCDBCO, 60 µM BEHTBP, 1.4 µM BTBPE or 0.2 µM DBDPE in CEH. Injection doses up to 50 µg/g egg HCDBCO and 10 µg/g egg BTBPE had no effect on embryonic pipping success. The accumulation of HCDBCO and BTBPE was variable in liver and did not follow a linear uptake pattern with respect to injection dose, due in part to difficulties with the solubility of these chemicals in the dimethyl sulfoxide (DMSO) vehicle. In, CEH, HCDBCO caused a decrease in CYP1A4/5 mRNA at all concentrations tested, while CYP2H1 and CYP3A37 were induced only at 10 µM. In contrast, only TTR mRNA was down-regulated in hepatic tissue at all injection concentrations of HCDBCO. The highest concentration of BTBPE induced CYP1A4/5 mRNA to 115- and 18-fold in CEH, and 6.5- and 1.8-fold in liver tissue. In vitro and in ovo exposure to BTBPE caused a concentration-dependent decrease in DIO3 mRNA, while CYP3A37 was down-regulated 2-fold at 10 µg/g in liver tissue. In CEH, DBDPE induced CYP1A4/5 mRNA to a maximum of 29- and 59-fold at 0.2 µM, and increases in DIO1 mRNA and decreases in CYP3A37 mRNA were also observed. None of the gene targets were responsive to BEHTBP exposure in CEH. This is the first study to report on the toxicological and molecular effects of HCDBCO, BEHTBP, BTBPE and DBDPE in an avian species. Using this combined in vitro/in ovo approach has permitted the characterization of these four BFR alternatives by defining possible mechanisms of biological action in a model avian species, the chicken.

Polybrominated diphenyl ethers (PBDEs) have been intensively used as flame retardants (FRs) and have become ubiquitous environmental pollutants. PBDEs form hydroxylated PBDEs (OH-PBDEs) as metabolites. Further, some OH-PBDEs and methoxy-PBDEs (MeO-PBDEs) are natural products. These are all compounds of environmental and health concern and it is therefore important to confirm their identity and to assess their environmental levels and toxicities. Hence, it is vital to obtain authentic reference standards of individual PBDEs and OH/MeO-PBDEs. The thesis main aim was to develop synthesis methods of congener specific PBDEs, OH- and MeO-PBDEs. The second aim was to identify and quantify PBDEs, OH- and MeO-PBDEs in environmental samples. The third was to propose an abbreviation system for FRs. O-Arylation of brominated phenols, using either symmetrical or unsymmetrical brominated diphenyliodonium salts, was selected for synthesis of PBDEs and OH-/MeO-PBDEs. A total of 16 MeO-PBDEs, 11 OH-PBDEs, 1 diMeO-PBDE and 1 EtO-MeO-PBDE were synthesised. Three novel unsymmetrical diaryliodonium triflates were synthesised and used in synthesis. Optimisations were made to construct a reliable general method for congener specific PBDE synthesis, which was used in the synthesis of 8 representative PBDE congeners. The products were generally characterised by electron ionisation mass spectrometry (EIMS) and nuclear magnetic resonance (NMR) spectroscopy. Identification of PBDEs and OH-PBDEs in various matrixes was based on gas chromatographic and mass spectrometric analyses. Fourteen OH-PBDE congeners were identified in a pooled human blood sample. One previously uncharacterised natural PBDE analogue was identified as 6-OH-6’-MeO-BDE-194, and quantified in Swedish blue mussels. PBDE congeners and other BFRs were identified and quantified in workers and dust from a smelter in Sweden. A structured and practical abbreviation system was developed for halogen- and phosphorus containing FRs. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Submitted. Paper 4: Manuscript.</p>

Poly Brominated Diphenyl Ethers (PBDEs) are known flame retardants with highly persistent and lipophilic in nature. The continued usage of PBDE in various products amplifies the human burden of PBDEs. It is therefore, important to study the potential toxicological and/or biological effects of PBDE exposure in human. In this study we investigated the mode of action of PBDE induced toxicity in human liver by exposing human hepatocarcinoma cells in a time (24-72h) and dose (0-100μM) dependent manner. The highest test dose caused an inhibition in cell viability up to 50% after 72h, whereas lower doses (<50μM) showed slight increase in cell viability. Likewise, higher doses caused significant accumulation of intracellular ROS over time. Further, increase in caspase-3 enzyme levels and DNA fragmentation showed that, lower brominated PBDEs induce liver toxicity through accumulation of toxic metabolites and reactive oxygen species over time leading to caspase-mediated apoptotic cell death.

A thesis submitted in fulfilment of the requirements for the degree Magister Technologiae (Chemistry) in the Faculty of Applied Sciences, Department of Chemistry, Cape Peninsula University of Technology, 2008 / Green et al. attempted to synthesize linear naphthopyranquinones from a naphthyl dioxolane using a TiCl4 as a catalyst. They managed to synthesise an angular naphthopyran as well as a linear naphthopyran in low yield. They showed that reducing the steric strain at position 1 of the naphthyl dioxolane afforded a low percentage yield of the linear naphthopyran plus an angular one. This thesis describes the synthesis of linear naphthopyrans with an improved percentage yield using TiCl4 as a catalyst. This was achieved by placing a OMe group of less steric hinderance at position 1 and a Br atom at position 4 of a naphthyl dioxolane. The OMe group at position 1 was to allow isomerisation to occur at position 2, and the Br atom was to inhibit isomerisation at position 4, thereby inhibiting the formation of the angular naphthopyran.

<p>Brominated flame retardants (BFRs) are a diverse group of chemicals, which are used to slow down or inhibit the development of fires. BFRs are incorporated into a wide range of consumer products that are considered as potential fire hazards, such as TV-sets, household appliances, computers, and textiles. The production and use of BFRs is extensive and consists of mainly tetrabromobisphenol A (TBBPA), polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecan (HBCD). BFRs in general, but in particular the PBDEs, have led to both scientific and public concern since they have been found to bioaccumulate in humans and wildlife. The general population is targeted by the PBDEs due to their applications and via the food web. Occupational exposure occurs not only during direct handling of BFRs, but also during handling, repair and dismantling of flame retarded goods.</p><p>This thesis is aimed to assess occupational exposure to BFRs. It is mainly focused PBDEs and especially the PBDEs with high bromine content, such as decabromodiphenyl ether (BDE-209). The work has been accomplished by analysis of BFRs in indoor air at industries handling BFRs or flame retarded goods, and by analysis of blood drawn from workers with potential exposure to BFRs. A referent group, abattoir workers with no occupational exposure to PBDEs, was also investigated. Data from these cross-sectional investigations and from serum sampling during vacation in PBDE-exposed workers have been used for calculation of apparent halflives of hepta- to decaBDE in serum.</p><p>The results clearly show that the workers were exposed to PBDEs when handling PBDE containing products or goods. The serum PBDE levels in computer technicians were found to correlate to the estimated cumulative work hours with computers. Exceptionally high concentrations of BDE-209, almost up to 300 pmol/g lipid weight (l.w.) were observed in serum from rubber workers manufacturing or handling rubber compound that was flame retarded with a technical mixture of decabromodiphenyl ether (DecaBDE). Elevated concentrations of PBDEs with eight or nine bromine substituents were also observed. In an electronics dismantling plant, where elevated levels of PBDEs previously had been observed, reduced serum levels of some, but not all PBDE congeners were observed after industrial hygiene improvements. Notably, it was observed that the BDE-209 concentrations in referents with no occupational exposure were similar to the concentrations of 2,2’,4,4’- tetrabromodiphenyl ether (BDE-47), often referred to as the most abundant PBDE congener in humans and wildlife. Additionally, PBDEs with high bromine content were found to have a fast rate of elimination or transformation in humans, based on serum analysis. BDE-209 had an apparent half-life in serum of only 15 days.</p><p>The possibility of quantifying BFRs, such as PBDEs, in human serum at low levels of detection has been achieved by reducing the contamination of the samples and procedural blanks. Major efforts have been done to develop routines and clean up methodology to enable an almost contamination-free environment at the laboratory. The use of a clean room has decreased PBDE levels in the blanks to acceptable limits. The modifications of the original analytical method have made it possible to quantify almost all PBDE congeners of interest in one GC/MS run.</p><p>Occupational and general background exposure of BFRs to humans will continue as long as these chemicals are a part of our daily life and present as environmental contaminants. The present scientific knowledge of the potential health risks of these BFRs still needs to be further developed. It should be stressed that health effects to PBDEs have not been assessed in this work. It is the author's wish that this thesis should add another piece of knowledge to the puzzle of BFRs and BFR exposure to humans and that these data will be used in future risk assessments of PBDEs in particular.</p>

Vocal communication allows animals to express distress, territoriality, and most important, to attract mates. In the African Clawed frog, Xenopus laevis, vocal communication is unique, because not only do males advertise for mates using elaborate click vocalizations, but also females are able to advertise their reproductive readiness by eliciting a "rapping" call. Sex differences in vocal repertoire match sex differences in vocal circuitry. During development, the vocal circuitry in the male grows increasingly sensitive to circulating androgens. Androgens induce tremendous growth in the cartilage and musculature of the peripheral vocal organ, the larynx. Net addition of synapses and motor fibers soon follow providing communication from the motor nucleus in the hindbrain to the vocal organ. The laryngeal motor nucleus, n. IX-X, accumulates androgens that serve to protect n. IX-X neurons from programmed apoptosis. Females, who have low levels of circulating androgens, experience a profound net loss on n. IX-X neurons during this developmental critical period. Once the frogs reach sexual maturity males possess larger and more numerous n. IX-X neurons than females, as well as sizable sex differences in laryngeal robustness and physiology. These measurable sex differences yield vastly different vocal programs. Androgens continue to maintain a critical role in governing breeding season trophic effects and mediating call production. Because male X. laevis are so susceptible to the effects of androgens, they may also be sensitive to the actions of endocrine disrupting chemical agents. The vocal system of X. laevis and its androgen sensitivity thus provide an ideal model for studying changes imposed to the anatomy and physiology of the system by the brominated flame retardant, PBDE-209, a putative anti-androgen and common pollutant. The present studies investigate how PBDE-209 affects the male vocal system when animals are exposed during the androgen-sensitive critical period of vocal system development and during adulthood when the tissues are utilizing androgens to vocalize. PBDE-209 effectively reduces male n. IX-X number and size at higher concentrations after exposure during the organizational critical period. Similar dose-dependent effects were observed in adult n. IX-X neurons. Moreover, PBDE-209 inhibited male-typical vocalization by reducing the number of calls elicited as well as the average call amplitude. These data strongly suggest that PBDE-209 has cytotoxic effects that alter n. IX-X anatomy and function, and may be mediated through pathways that include blocking the androgens necessary for proper vocal system development.

Brominated flame retardants (BFRs) are a diverse group of chemicals, which are used to slow down or inhibit the development of fires. BFRs are incorporated into a wide range of consumer products that are considered as potential fire hazards, such as TV-sets, household appliances, computers, and textiles. The production and use of BFRs is extensive and consists of mainly tetrabromobisphenol A (TBBPA), polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecan (HBCD). BFRs in general, but in particular the PBDEs, have led to both scientific and public concern since they have been found to bioaccumulate in humans and wildlife. The general population is targeted by the PBDEs due to their applications and via the food web. Occupational exposure occurs not only during direct handling of BFRs, but also during handling, repair and dismantling of flame retarded goods. This thesis is aimed to assess occupational exposure to BFRs. It is mainly focused PBDEs and especially the PBDEs with high bromine content, such as decabromodiphenyl ether (BDE-209). The work has been accomplished by analysis of BFRs in indoor air at industries handling BFRs or flame retarded goods, and by analysis of blood drawn from workers with potential exposure to BFRs. A referent group, abattoir workers with no occupational exposure to PBDEs, was also investigated. Data from these cross-sectional investigations and from serum sampling during vacation in PBDE-exposed workers have been used for calculation of apparent halflives of hepta- to decaBDE in serum. The results clearly show that the workers were exposed to PBDEs when handling PBDE containing products or goods. The serum PBDE levels in computer technicians were found to correlate to the estimated cumulative work hours with computers. Exceptionally high concentrations of BDE-209, almost up to 300 pmol/g lipid weight (l.w.) were observed in serum from rubber workers manufacturing or handling rubber compound that was flame retarded with a technical mixture of decabromodiphenyl ether (DecaBDE). Elevated concentrations of PBDEs with eight or nine bromine substituents were also observed. In an electronics dismantling plant, where elevated levels of PBDEs previously had been observed, reduced serum levels of some, but not all PBDE congeners were observed after industrial hygiene improvements. Notably, it was observed that the BDE-209 concentrations in referents with no occupational exposure were similar to the concentrations of 2,2’,4,4’- tetrabromodiphenyl ether (BDE-47), often referred to as the most abundant PBDE congener in humans and wildlife. Additionally, PBDEs with high bromine content were found to have a fast rate of elimination or transformation in humans, based on serum analysis. BDE-209 had an apparent half-life in serum of only 15 days. The possibility of quantifying BFRs, such as PBDEs, in human serum at low levels of detection has been achieved by reducing the contamination of the samples and procedural blanks. Major efforts have been done to develop routines and clean up methodology to enable an almost contamination-free environment at the laboratory. The use of a clean room has decreased PBDE levels in the blanks to acceptable limits. The modifications of the original analytical method have made it possible to quantify almost all PBDE congeners of interest in one GC/MS run. Occupational and general background exposure of BFRs to humans will continue as long as these chemicals are a part of our daily life and present as environmental contaminants. The present scientific knowledge of the potential health risks of these BFRs still needs to be further developed. It should be stressed that health effects to PBDEs have not been assessed in this work. It is the author's wish that this thesis should add another piece of knowledge to the puzzle of BFRs and BFR exposure to humans and that these data will be used in future risk assessments of PBDEs in particular.

Poly Brominated Diphenyl Ethers (PBDEs) are known flame retardants with highly persistent and lipophilic in nature. The continued usage of PBDE in various products amplifies the human burden of PBDEs. It is therefore, important to study the potential toxicological and/or biological effects of PBDE exposure in human. In this study we investigated the mode of action of PBDE induced toxicity in human liver by exposing human hepatocarcinoma cells in a time (24-72h) and dose (0-100μM) dependent manner. The highest test dose caused an inhibition in cell viability up to 50% after 72h, whereas lower doses (<50μM) showed slight increase in cell viability. Likewise, higher doses caused significant accumulation of intracellular ROS over time. Further, increase in caspase-3 enzyme levels and DNA fragmentation showed that, lower brominated PBDEs induce liver toxicity through accumulation of toxic metabolites and reactive oxygen species over time leading to caspase-mediated apoptotic cell death.

Brominated flame retardants is a group of compounds present in numerous types of materials in our surroundings. Although their purpose is to slow the progression of a fire, many has been shown to be toxic to the environment. Novel brominated flame retardants have been introduced to the market as old ones have been removed. Reliable methods are crucial to be able to monitor how the novel brominated flame retardant spread and accumulate in the environment. To achieve this, a method validation of a cleanup method using multilayer silica followed by analysis by atmospheric pressure gas chromatography coupled to tandem mass spectroscopy was performed. This method had been previously used for polybrominated diphenyl ethers and the aim was to see if it could be used for analysis of novel brominated flame retardants as well. Spiking experiments showed generally good results, with recoveries of the native compound ranging from 40% to 174%. To apply the method on real matrix samples, eight samples of osprey eggs and five samples of adipose tissue of ringed seal was analyzed. Several novel brominated flame retardants were found, most abundant being the methoxylated polybrominated diphenyl ethers. Dominant congener was 2'-Methoxy-2,3',4,5'-tetrabromodiphenyl ether (2PMBDE#68) followed by 6-Methoxy-2,2',4,4'-tetrabromodiphenyl ether (6PMBDE#47), 5-Methoxy-2,2',4,4'-tetrabromodiphenyl ether (5PMBDE#47) and 5-Methoxy-2,2',4,4',6-pentabromodiphenyl ether (5PMBDE#100) with concentrations ranging from &lt;0,13-13 ng/g lipid weight in osprey eggs and &lt;0,003-249 ng/g lipid weight in ringed seal blubber. Also 1,2-Bis(2,4,6-tribromophenoxy)ethane and pentabromobenzene were found in both osprey eggs and ringed seal blubber. Hexabromobenzene was found in ringed seal blubber and 2,3,5,6-tetrabromo-p-xylene was identified in osprey eggs. / Bromerade flamskyddsmedel är en grupp ämnen närvarande i många olika typer av material i vår omgivning. Även om deras syfte är att bromsa förloppet vid en eldsvåda har många visat sig vara toxiska för miljön. Nya bromerade flamskyddsmedel har kommit ut på marknaden i takt med att gamla tagits bort. Pålitliga metoder är nödvändiga för att övervaka hur nya bromerade flamskyddsmedel sprids och ackumulerar i miljön. För att uppnå detta validerades en metod bestående av flerskiktskiseldioxid och analys med atmosfärstryck-gaskromatografi kopplat till tandem-masspektroskopi. Denna metod hade tidigare använts för analys av polybromerade difenyletrar och målet var att undersöka om samma metod kunde användas för analys ad nya bromerade flamskyddsmedel. Spikning gav generellt goda resultat - utbytet för de nativa ämnena var mellan 40% och 174%. För att pröva metoden på prov med riktigt matris analyserades åtta prov med fiskgjuseägg och fem prov med späck från vikare. Flera nya bromerade flamskyddsmedel hittades, mest förekommande var de metoxylerade polybromerade difenyletrarna. Dominerande kongen var 2'-metoxy-2,3',4,5'-tetrabromodifenyleter, följt av 6-metoxy-2,2',4,4'- tetrabromodifenyleter, 5-metoxy-2,2',4,4'- tetrabromodifenyleter och 5-metoxy-2,2',4,4',6- pentabromodifenyleter i koncentrationer från &lt;0,13-13 ng/g lipidvikt (fiskgjuseägg) och &lt;0,003-249 ng/g lipidvikt (sälspäck). Även 1,2-bis(2,4,6-tribromofenoxy)etan (fiskgjuseägg och sälspäck), pentabromobensen (fiskgjuseägg och sälspäck), hexabromobensen (sälspäck) och 2,3,5,6-tetrabromo-p-xylene (fiskgjuseägg) identifierades.