Synthesis of Organobromines As a Tool for Their Characterisation and Environmental Occurrence Assessment

Synthesis of organobromines as a tool for their characterisation and environmental occurrence assessment

Andreas Rydén

Department of Materials and Environmental Chemistry Stockholm University

Stockholm 2013

i Doctoral Thesis 2013 Department of Materials and Environmental Chemistry Stockholm University SE-106 91 Stockholm Sweden Abstract 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.

ISBN 978-91-7447-800-6

Printed in Sweden by US-AB, Stockholm 2013-10-21 Distributor: Department of Materials and Environmental Chemistry, Stockholm University

ii Tillägnad Frans

iii Table of contents Abstract...... ii Table of contents ...... iv List of papers ...... vi Abbreviations...... vii 1 Introduction ...... 1 1.1 Flame retardants ...... 3 1.2 Brominated flame retardants and analogues...... 4 2 PBDEs, their metabolites and natural analogues...... 8 2.1 Polybrominated diphenyl ethers ...... 8 2.1.1 Chemical structures and classes of PBDEs ...... 8 2.1.2 Physicochemical characteristics and reactivity ...... 9 2.1.3 Environmental occurrence and sources of exposure ...... 10 2.1.4 Biological fate and effects ...... 12 2.2 Hydroxylated and methoxylated PBDEs...... 13 2.2.1 Chemical structure and properties ...... 14 2.2.2 Environmental occurrence and biological effects...... 15 2.3 Aim of thesis...... 16 3. Synthesis of selected organobromines...... 18 3.1 Synthetic routes to PBDEs ...... 18 3.1.1 Bromination of diphenyl ether...... 18 3.1.2 Bromination of prefabricated PBDEs...... 20 3.1.3 Nucleophilic aromatic substitution...... 20 3.1.4 Ullmann ether synthesis...... 21 3.1.5 PBDEs from arylboronic acids...... 22 3.1.6 Synthesis via aminodiphenyl ethers ...... 22 3.1.7 Synthesis via diaryliodonium salts ...... 24 3.2 Synthesis of mono and di-functionalised PBDEs...... 35 3.2.1 Synthesis of MeO-PBDEs via S NAr...... 35 3.2.2 Synthesis of MeO-PBDEs via brominated diphenyliodonium salts...... 39 3.2.3 Synthesis of di-functionalised PBDEs...... 46 4 Identification and quantification of selected organobromines...... 49 4.1 Identification of OH-PBDEs as metabolites in human blood samples ...... 49 4.2 Identification and quantification of a phenolic di-functionalised PBDE in blue mussels ...... 52 4.3 Identification of BFRs in E-waste dust...... 54 4.4 BFRs in blood from personnel at a Swedish smelter facility ...55

iv 5 Definitions and novel abbreviations of flame retardants...... 57 5.1 Overarching definitions for flame retardants...... 57 5.2 Overarching abbreviations for flame retardants ...... 58 6 Future perspectives...... 60 7 Svensk sammanfattning...... 62 8 Acknowledgements ...... 64 9 Appendix A...... 66 10 Reference list ...... 67

v List of papers This thesis is based around the following articles, referred to in the text by their roman numerals. Hitherto unpublished manuscripts are also included.

Paper I Rydén, A., Nestor, G., Jakobsson, K., and Marsh, G., (2012), Synthesis and tentative identification of novel polybrominated diphenyl ether metabolites in human blood, Chemosphere , 88 , 1227-1234.

Paper II Rydén, A., Marsh, G., (2013), Synthesis of polybrominated diphenyl ethers via unsymmetrical diaryliodonium triflates, (manuscript)

Paper III Winnberg, U., Rydén, A., Löfstrand, K., Asplund, L., Bignert, A., Marsh, G., (2013), Novel octabrominated phenolic diphenyl ether identified in blue mussels from the Swedish west coast, (submitted manuscript)

Paper IV Rydén, A., Strid, A., Athanassiadis, I., Bergman, Å., (2013), Occurrence and potential human exposure to brominated flame retardants at a Swedish smelter handling electronic scrap, (manuscript)

Paper V Bergman, Å., Rydén, A., Law, R.J., de Boer, J., Covaci, A., Alaee, M., Birnbaum, L., Petreas, M., Rose, M., Sakai, S., Van den Eede, N., and van der Veen, I., (2012), A novel abbreviation standard for organobromine, organochlorine and organophosphorus flame retardants and some characteristics of the chemicals, Environ. Int ., 49 , 57-82.

Papers and manuscripts not included in this thesis are listed below.

Aydin, J., Rydén, A., Szabo, K.J., 2008. Chiral palladium-pincer complex catalyzed asymmetric condensation of sulfonimines and isocyanoacetate. Tetrahedron: Asymmetry 19 , 1867-1870.

Von Stedingk, H., Xie, H., Hatschek, T., Foukakis, T., Rydén, A., Bergh, J., Rydberg, P., (2013), Validation of a novel analytical procedure for quantification of the level of phosphoramide mustard formed in individuals being treated with cyclophosphamide. (manuscript)

The published papers are printed with the permission from the copyright holders.

vi Abbreviations

BAF bioaccumulation factor BB-209 decabromobiphenyl BDE brominated diphenyl ether BDMS bromodimethylsulfonium bromide BFRs brominated flame retardants BTBPE 1,2-bis(2,4,6-tribromophenoxy)ethane BTMA benzyltrimethylammonium tribromide b.w. body weight CDE chlorinated diphenyl ether CFRs chlorinated flame retardants DBDPE decabromodiphenyl ethane 4,4’-DDE 1,1-bis-(4-chlorophenyl)-2,2-dichloroethene DDT 2,2-bis(4-chlorophenyl)-1,1,1-trichloroethane DIPEA N,N -diisopropylethylamine DMAC N,N-dimethylacetamide DOM directed ortho metalation EAS electrophilic aromatic substitution ECHA European Chemicals Agency ECNI electron capture negative ionisation EFSA European Food Safety Authority EI electron ionisation formyl-PBDEs polybrominated phenoxybenzaldehydes FR flame retardant GC-EIMS gas chromatography electron ionisation mass spectrometry GCMS gas chromatography mass spectrometry HBB hexabromobenzene HBCDDs hexabromocyclododecanes IUPAC International Union of Pure and Applied Chemistry Log K OW octanol-water partition coefficient m-CPBA meta -chloroperoxybenzoic acid MeO-PBDEs methoxylated polybrominated diphenyl ethers NH 2-PBDEs amine functionalised polybrominated diphenyl ethers NMR nuclear magnetic resonance OECD Organisation for Economic Co-operation and Development OH-PBDEs hydroxylated polybrominated diphenyl ethers PBBs polybrominated biphenyls PBDFs polybrominated dibenzofurans PBT persistent, bioaccumulating and toxic PCB polychlorinated biphenyls PCDDs polychlorinated dibenzo-p-dioxins PCDEs polychlorinated diphenyl ethers PCDFs polychlorinated dibenzofurans PFRs phosphorus flame retardants POPRC Persistent Organic Pollutants Review Committee PRABs practical abbreviations RoHS Restriction of Hazardous Substances Directive SNAr nucleophilic aromatic substitution STABs structured abbreviations T3 triiodothyronine T4 thyroxine

vii 2,3,4,6-TEBP 2,3,4,6-tetrabromophenol TBBPA tetrabromobisphenol-A 2,4,6-TBP 2,4,6-tribromophenol TDBPP tris–(2,3-dibromopropyl)phosphate TDCIPP tris–(1,3-dichloro-iso-propyl)phosphate TfOH trifluoromethanesulfonic acid TFPA 2,2,2-trifluoroperoxyacetic acid

viii 1 Introduction

Chemicals are an essential part of our daily, comfortable life in modern society. Take a look around where you are seated right now. Chances are good that you will find one or more electronic devices close to you. Many of these devices are made of and/or contain synthetic compounds such as polyvinyl chloride plastics, plasticisers and flame retardants. The chair you are sitting in and the clothes you wear may be manufactured from synthetic materials. Even the thesis you are holding in your hands right now is entirely based on chemicals. Although many of the chemicals we use daily are beneficial to us and possess no obvious threat, some have been found to be of higher concern regarding (eco-)toxicological effects and ever so often we find out about this when they have already become widely distributed in our environment.

Awareness of environmental pollution rose in Sweden during the end of the 1950’s and early 1960’s when dramatic and unexplained changes in population of birds of prey in the Baltic Sea area was observed. The explanation came in the form of 2,2-bis(4-chlorophenyl)-1,1,1- trichloroethane (DDT), deliberately released into the environment since its introduction as a commercial insecticide in the 1940’s. DDT was found to be retained in birds of prey and marine animals in the 1960’s,1 but it was indicted as an environmental threat already in 1946.2 DDT is a PBT chemical (i.e . persistent, bioaccumulative and toxic) which explains why high levels are found in species at the top of the food chain. An important factor in the decrease in bird population was eggshell thinning that was in fact caused by 1,1-bis-(4-chlorophenyl)-2,2-dichloroethene ( 4,4’ -DDE), the primary biotransformation product of 4,4’-DDT.3

Polychlorinated biphenyls (PCBs), used for their excellent electrical insulating properties since their introduction on the market in the early 1930’s, was for the first time identified in white tailed sea eagle eggs in the mid 1960’s.1,4 The populations of Baltic seal species decreased and so did the population of European otter during the 1970’s. These changes were to a large extent caused by high levels of PCB in their diet which led to reproductive insufficiency.5-7 In attempts to understand the effects of PCBs on wildlife, minks have been used as a model animal in PCB exposure studies.8-12 Effects such as disturbed reproduction have been reported even from low doses of technical PCB mixtures.9 The effects manifest themselves as e.g. kits born with abnormalities, decreased survival amongst kits as well as reduced birth weight and growth.

1 In contrast to DDT, PCBs were not intentionally released to the environment. Today we know that chemicals spread globally and enter the ecosystem and biological systems whether intended or not. Accidents or leakages from waste disposal sites are just two possible discharge routes for industrial chemicals. A notable case referred to as the Yusho incident occurred in 1968 when 1800 people were poisoned by PCB contaminated rice oil in the Japanese island of Kyushu. 13 Severe health effects were reported after the incident including symptoms like chloracne, loss of hair and irregular menstrual cycles.14 Another frightening incident occurred in 1973 when polybrominated biphenyls (PBBs), a bioaccumulating and highly toxic brominated flame retardant marketed as “FireMaster BP-6”, were unintentionally fed to cattle in the state of Michigan, USA.15 The white crystalline PBB powder had been confused for being magnesium oxide, at the time frequently used as a digestion aiding additive to normal cattle feed. The effects of this unfortunate mix-up were disastrous. Cows became sick, milk production decreased and calves died. Almost 30,000 cattle and many other farm animals had to be quarantined and put down. For nine months no one knew the reason to these adverse effects and by the time the problem was finally tracked down to PBB, as many as 9 million people may have ingested contaminated meat and dairy products.16

DDT, PCBs and PBBs are no longer in use in most parts of the world and only after banning DDT and PCB in the 1970’s, a slow recovery could be seen in the population of birds of prey in the Baltic Sea area. Today their reproduction success is similar to before these chemicals entered our environment.17 PCB levels in food stuff are also decreasing according to studies monitoring PCB indicators in e.g. dairy products, egg products and fish. 18

Other unintentionally produced chemicals such as polychlorinated dibenzo- p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs), or as they are often referred to, “dioxins”, are highly toxic organohalogen compounds formed in e.g. incomplete combustion of chlorine-based chemicals with hydrocarbon and as by-products in the manufacture of compounds such as PCB and chlorinated phenols.19,20 Temporal trend studies have shown that the levels of PCDDs and PCDFs are decreasing globally and locally in e.g. human mothers’ milk. 21,22 This can be explained by regulations and improved source handling, leading to less emissions and consequently less exposure. 18 Also, one may hypothesise that better advice given to women, regarding food recommendations during pregnancy and nursing, contributes to the lower concentrations found in mothers’ milk.

The production, handling and use of synthetic chemicals are steadily increasing. Until today, well over 60 million chemicals or chemical mixtures have been synthesised or described in scientific literature. About 143.000 of

2 these have been pre-registered by the European Chemicals Agency (ECHA) for commercial use.23 These pre-registered chemicals include e.g. many, but not all, pharmaceuticals, pesticides and cosmetic products. The Organisation for Economic Co-operation and Development (OECD) have listed 4,636 that are considered to be high production volume chemicals with annual quantities surpassing 1,000 tonnes in at least one country.24 Yet, we know almost nothing about the environmental fate, toxicity, health effects and other characteristics of most these compounds. Only a handful of all man- made chemicals have been fully risk assessed as of today. Also, to characterise and assess present and future chemical mixtures and their potential transformation products and metabolites, authentic synthetic reference standards are needed. Not only for identification purposes but also to be able to fully assess their environmental fate and biological impact.

1.1 Flame retardants Fire may, just like chemicals, act as either friend or foe to humanity. Due to the potential hazards of uncontrolled fire, flame retardants (FRs) have been used as far back as 450 BC when Egyptians used solutions of potassium aluminium sulfate (Alum) to soak wood to reduce the flammability. 25 It was not until World War II that the FRs that we are used to today started to emerge on the market. In the 1970’s, flammability standards led to a more intense use of FRs in household items such as clothes, sofas and electronics. Today we divide FRs into 4 major groups: Inorganic, organophosphorus, nitrogen based and organohalogen FRs. The different groups of FRs work in several different ways. Inorganic metal salts exhibit their effect through the release of incombustible gas together with thermal quenching since the decomposition of metal salts is endothermic. Other FRs like the nitrogen and phosphorous based ones act by creating a non-combustible coating, thus limiting the fuel supply. Organohalogens and some phosphorus FRs interact chemically by releasing radicals which interferes with the self-propagating combustion process. 25

PCBs were in fact, apart from their use as insulating material in electrical equipment, applied as FRs from the 1930’s to the mid 1980’s. Historically several other chlorinated compounds have been used as FRs, sometimes combined with a phosphate group, such as the chlorinated and phosphorous FR (CFR/PFR) tris–(1,3-dichloro-iso-propyl)phosphate (TDCIPP). 25

The large number of established, emerging, potential and novel FRs has led to an ever increasing number of publications dealing with environmental issues regarding these chemicals. Due to the vast number of chemicals and mixtures it is difficult to keep track of them. Questions are raised regarding the status and name assignment of FRs. How many of the registered

3 brominated flame retardants (BFRs) are considered to be established, emerging, novel and potential FRs as of today? How should we abbreviate their complicated chemical names in literature and communication? These issues are addressed in Paper V, Chapter 5 and Chapter 6 of this thesis.

1.2 Brominated flame retardants and analogues Among the organohalogen FRs, bromine containing compounds are the most commonly used. A brominated isomer of above mentioned TDCIPP, tris– (2,3-dibromopropyl)phosphate (TDBPP, Figure 1.1) was put under the spotlight in the late 1970’s when it was revealed that children’s sleepwear were treated with this particular FR in up to 5% of the total fabric weight. The carcinogenic and mutagenic metabolite of TDBPP, namely 2,3- dibromopropanol, could be identified in the urine of exposed children. 26

Figure 1.1 TDCIPP (left), and the isomeric brominated analogue TDBPP (right) are phosphorus and halogenated FRs. TDBPP has been used in children’s sleepwear.

The first major review on BFRs was published in the mid 1990’s, dealing with toxicity and environmental occurrence, summarising the knowledge on these compounds at the time. 27 For more recent publications concerning BFRs the author recommends documents released by the European Food Safety Authority (EFSA), and more specifically their opinions on PBBs 28 , PBDEs 29 , hexabromocyclododecanes (HBCDDs) 30 , TBBPA and its derivatives 31 , and an opinion on emerging and novel BFRs in food32 . The BFRs mainly used and found in consumer products today are tetrabromobisphenol A (TBBPA), hexabromocyclododecan (HBCDD) and decabromodiphenyl ether (DecaBDE), which are briefly discussed below.

TBBPA

TBBPA (Figure 1.2) is manufactured from bisphenol A through a simple ortho selective bromination process, producing relatively pure TBBPA with possible traces of tribrominated bisphenol A.33 The global demand for TBBPA increased rapidly from 50,000 tonnes to 145,000 tonnes during the 1990’s. Global production volumes of more than 120,000 tonnes annually have been reported,34,35 but the present annual global production is unknown.

4 Most of the produced TBBPA originates from USA, China and Japan. Even though it is not produced within the EU, an estimated 40,000 tonnes TBBPA was imported annually both as pure substance and integrated in consumer products in 2006.31

Figure 1.2 TBBPA

Since TBBPA is phenolic, it is primarily used as a reactive FR meaning it is chemically bound to the matrix or material it is used in. The reactive form is used in e.g. circuit boards but TBBPA has also been reported to be used as an additive FR in plastics. 31 Ether derivatives of TBBPA such as tetrabromobisphenol A bis(2-hydroxyethyl) ether (TBBPA-BHEE) and TBBPA bis(2,3-dibromopropyl) ether (TBBPA-BDBPE) has also been used as novel additive FRs in goods such as polymers, kitchen hoods and electronics.36

Toxicity studies on rodents indicate TBBPA as an endocrine disruptor since it affects the thyroid hormone system by decreasing circulating thyroid hormone, thyroxine (T4), and increasing the levels of triiodothyronine (T3) in male rats. 37 Potential sources for human exposure to TBBPA are e.g. dust in homes and cars.

HBCDDs

It should be stated that the abbreviation HBCDD is used in this thesis even though HBCD is frequently used in the scientific literature as well. The issues with using multiple abbreviation systems in literature and scientific communication are further addressed in Paper V and Chapter 5.

Technical HBCDD (Figure 1.3) has been on the market since the 1960’s and is primarily used as an additive FR in polystyrene materials used in insulation in construction, but it has also been used in e.g. textiles. 30

Br Br

Br Br Br Figure 1.3 HBCDD

5 HBCDDs are manufactured by bromination of cis-1, trans-5, trans-9- cyclododecatriene which yields a mixture of the three enantiomeric pairs i.e. the so called α-HBCDD, β-HBCDD and γ-HBCDD. 38 The estimated production in EU was in the order of 6,000 tonnes annually in 2005, while the estimated global production in 2008 amounted to 13,400 tonnes. 30 In rodents exposed to technical HBCDD a decrease in T4 and T3 hormone has been reported, indicating that HBCDDs possess endocrine disrupting properties. 30

As HBCDD is an additive in materials and not chemically bound to it, leaching from the material is considered to be a possible route for HBCDD discharge in the environment. 39 Little is known about the effects on human health. However, we are indeed exposed to HBCDD in our daily life largely through ingestion of house-hold dust, which has been reported to contribute with as much as 23.9 % of the total exposure to HBCDDs for adults and 62.6 % for children.40 In late 2012, the Persistent Organic Pollutants Review Committee (POPRC) agreed to a proposal to include HBCDD in the Stockholm Convention’s Annex A for elimination, with some exemptions to give countries time to adjust and switch to substitutes.41

DecaBDE

It must first be stated that technical DecaBDE should not be confused with the specific PBDE congener decabromodiphenyl ether (BDE-209, Figure 1.4), which is a part of, but not the only congener in technical DecaBDE. Chemical structure, characteristics, environmental occurrence and biological impact of PBDEs are further discussed in Chapter 2.

Figure 1.4 BDE-209

The technical mixture DecaBDE has been produced commercially since the early 1970’s. Technical DecaBDE is manufactured by Lewis acid catalysed perbromination of diphenyl ether but the final product also contain traces of octa- and nonaBDE congeners. 29 Global production volumes have been estimated to up to 30,000 tonnes annually during the 1990’s and the global annual demand was in 2001 estimated to over 56,000 tonnes. 29,42

Today we know that DecaBDE does undergo debromination by photolysis, reduction and microbial and metabolic transformations to form lower brominated BDEs.43-48 These findings undermine the idea of DecaBDE as a

6 safer alternative to Penta- or OctaBDE mixtures, which have also been in use as FRs and are known to be bioaccumulating and toxic 29 .

In Europe, the use of DecaBDE in electronics is restricted from 2008 under the RoHS directive.49 In the US there is an ongoing phase-out while it is still unregulated in Asia.

In EU, the use of some established and hazardous BFRs has been banned since 2006 by the EU Directive RoHS. TDBPP, PBB and the two lower brominated technical PBDE mixtures, PentaBDE and OctaBDE are no longer in use. 25,50 Nevertheless, all of these substances are still present in the environment since they are persistent and included in products that are still in use.

Apart from established BFRs, novel BFRs are continuously being introduced and there are difficulties keeping track of current production volumes, import, export and use. Nevertheless, it is estimated that the cumulative production of BFRs exceeds 200,000 tonnes each year. 51 In just five years, this would amount to over 1,000,000 tonnes. This is a huge amount compared to the estimated total production of 1,000,000 tonnes of PCB since its introduction in the 1920’s. There are few reports regarding toxicity and environmental occurrence of novel BFRs and new challenges lay ahead regarding assessment of the environmental and biological impact of these chemicals.

7 2 PBDEs, their metabolites and natural analogues.

This chapter will focus on PBDEs, the metabolites formed from PBDEs in biological systems and their naturally occurring analogues. It is aimed to provide a background to PBDEs and PBDE analogues.

2.1 Polybrominated diphenyl ethers The three technical mixtures PentaBDE, OctaBDE and DecaBDE have been used in large volumes as additive FRs, which means that they are not chemically bound to the polymers. This makes PBDEs prone to leach out of goods and materials. 52 The production has been as high as 80,000 tonnes annually, but since then, legislations and other actions against PBDEs have affected the production volumes. Unfortunately more up-to-date figures are not available. 29

2.1.1 Chemical structures and classes of PBDEs PBDEs belong to a class of organobromine compounds consisting of two bromine substituted phenyl rings connected with an ether linkage. The general structure is shown in Figure 2.1. As a result of the symmetrical diphenyl ether backbone, a total of 209 congeners can be formed by altering the degree and position of bromine substituents. Like PCBs and PBBs, there are 3, 12, 24, 42, 46, 42, 24, 12, 3 and 1 theoretical isomers for monoBDE, diBDE, triBDE, tetraBDE, pentaBDE, hexaBDE, heptaBDE, octaBDE, nonaBDE, and decaBDE, respectively.

Figure 2.1 General structure of PBDEs where Σ (x+y) = 1 to 10. The ortho positions are 2,2’,6 and 6’, the meta positions are 3,3’,5 and 5’ and the para positions are 4 and 4’, relative to the ether linkage.

8 Conformational studies show that PBDEs can take on either a twisted or skewed non-planar conformation depending on the degree of bulky ortho bromine substituents (Figure 2.2). 53 A higher degree of bromination ortho to the ether linkage leads to a more skewed conformation as is the case with e.g. decaBDE.53

Figure 2.2 Twist ( θ1 = θ2 = 45˚) and skew ( θ1 = 0˚, θ2 = 90˚) conformation of diphenyl ether. Ortho -bromo substituted PBDEs adopt a more skew-like conformation.

Just like PCBs and PBBs, the preferred way of abbreviating the different congeners of PBDE, particularly in environmental chemistry literature, is to use the numbering system proposed by Ballschmitter and Zell. 54,55 For example, diphenyl ether with just one bromine substituent in ortho position to the ether linkage would be a monoBDE. Since the substituent is attached to the first available aromatic carbon next to the ether oxygen, this BDE would be abbreviated BDE-1 whereas a diphenyl ether where all 10 available aromatic positions is substituted with bromine would be a decaBDE or, using Ballschmitter and Zells system, BDE-209.

2.1.2 Physicochemical characteristics and reactivity Since the degree of bromination differs amongst PBDE congeners they also differ in their characteristics. The molecular weight ranges from 249.1 g/mole for the monoBDEs to 959.2 g/mole for the decaBDE. Log K OW - values range from approximately 5.9 to 8.3 for the tri- to heptaBDEs, while 56,57 decaBDE has been estimated to have a log K OW as high as 12.1. The relatively high log K OW -values indicate that PBDEs are hydrophobic compounds. Their limited water solubilities range between 130µg/L for BDE-15 to 1.5µg/L for the octaBDE congener BDE-183.58 The technical DecaBDE is estimated to have a water solubility of less than 0.1µg/L.59 The vapour pressures of PBDEs are low and decreasing with an increased number of bromine substituents resulting in decreasing volatility. In abiotic media lower brominated PBDEs, such as mono to pentaBDE, tend to be present to a larger extent in the vapour phase in contrast to higher brominated PBDEs, e.g. BDE-209, which typically are more absorbed to particulates.60 Tetra- to octaBDEs are relatively stable compounds while PBDEs with a lower or, above all, higher number of bromine substituents are more susceptible to exogenous transformations such as photolysis, reductive debromination and radical reactions. Generally, PBDEs are less sensitive to 61,62 nucleophilic aromatic substitution (SNAr) and oxidations. BDE-209 has been reported to decompose forming tri- to nonaBDEs and mono- to hexabrominated dibenzofurans (PBDFs) in several matrices such as

9 methanol/water, silica gel, sand, soil and sediment under the influence of UV-light in laboratory settings. 63-66 Microbial debromination of nonaBDEs has also been reported by Gerecke et al. 67 in anaerobic sludge. In the same study, technical decaBDE mixture was left for 238 days in activated sludge and a decrease of 30 % in the concentration of BDE-209 could be detected in the end of the experiment. From these results it may be hypothesised that naturally occurring debromination of BDE-209 might well be an important source of lower brominated diphenyl ethers.

It should be mentioned here that PBDEs also undergo metabolic transformations to form hydroxylated PBDEs (OH-PBDEs). These metabolites are further discussed in Chapter 2.1.4, Chapter 2.2 and Paper I.

2.1.3 Environmental occurrence and sources of exposure Environmental occurrence, concentration and time trends of PBDEs have been widely reviewed elsewhere,29,68-72 but a brief introduction and a few examples of PBDE occurrence in the environment, wildlife and humans are highlighted in this chapter.

PBDEs have been used in commercial products since the early 1970’s and their presence in FRs was addressed already in 1976. 73 Initial concerns about PBDEs and their environmental impact as contaminants were pointed out in the late 1970’s when it was discovered that environmental samples obtained near bromine industry facilities in the cities El Dorado and Magnolia, Arkansas, contained a total of 26 brominated organic compounds, including BDE-209. 74 In the early 1980’s a study showed that pike, sampled from the river Viskan in Sweden nowhere near any industry producing PBDEs, contained tri- to hexaBDEs in levels up to 88 mg/kg liver fat. 75 This was with great certainty caused by leakage from a nearby textile industry located upstream of the sampling site. Nevertheless, it stood clear that PBDEs were distributed in the environment and accumulated in fish, enabling their identification and quantification. The reported use during this time was a moderate 250 kg annually in Sweden (1978). 75 In the mid 1980’s, PBDEs were found in e.g. guillemots in the Baltic Sea, the North Sea and the Arctic Ocean area. 76 Guillemot muscle tissue samples from the Baltic area contained 370 ng/g fat, 2-5 times higher than in remote areas. The high levels of PBDEs in the Baltic Sea area may be explained by local sources, while PBDEs found in the Arctic Ocean and North Sea strongly indicate that PBDEs migrate and are globally distributed. PBDEs are known to undergo long-range transport to remote regions via the atmosphere similar as for PCBs.77 Using weekly samples from 1994, PBDEs were shown to undergo long-range transport as they were found in Arctic air samples in levels ranging 10-700 pg/m 3 in Yukon, Canada. 78,79 More recent data reported

10 PBDEs in the Canadian Arctic where the total PBDE concentrations ranged from 0.3 to 68 pg/m 3.80

Over the last two decades, a large amount of data has been reported on the topic of PBDE concentrations in biota, i.e. wildlife (fish, birds and mammals). The author recommends the EFSA scientific opinion on PBDEs in food 29 and references therein for a detailed overview.

Recent studies seem to indicate that the environmental occurrence of PBDEs is now declining slightly in e.g. air samples and vegetation. A trend study by Schuster et al. 81 monitoring European background air sample levels between the years 2000 and 2008 shows an overall decrease of PBDEs with a consistent decrease calculated for BDE-47, -49, -99, -100, -153 and -154. Archived UK pasture samples from 1930 to 2004 reveals PBDE trend of increasing concentrations during the 1970’s and 1980’s, high but stable levels during the late 1980’s and 1990’s and a decline in the early 2000’s, which corresponds well with restrictions in the use of technical Penta- and OctaBDE mixtures. 82 In studies on human breast milk, where similar temporal concentration trends are seen, the levels of PBDEs increased between 1970 and 2000.83-85 In more recent studies, declining temporal trends or a peak followed by a plateau of little variation has been reported in human matrices such as breast milk 86,87 , and in blood samples 88 .

The two major sources for human exposure to PBDEs are considered to be associated with food intake and dust ingestion from indoor environments. Because PBDEs are persistent, lipophilic and highly bioaccumulative they are found in fatty tissues in wildlife and thereby also in fatty food, particularly fat fish from higher trophic levels, which is thought to be the major source for dietary exposure amongst Europeans.89-91

Schecter et al. 92 measured concentrations of 13 PBDE congeners in food samples and estimated the levels of PBDE intake from food for the U.S. population. A conclusion drawn was that the measured levels in the food samples alone could not account for the PBDE concentrations in the general population, indicating other important sources in the total exposure. For example, the extensive use of PBDEs in indoor applications combined with small spaces and low air circulation leads to high concentrations, which makes indoor exposure a contributing factor to the total exposure.93,94 Ordinary indoor house dust has in fact been estimated to contribute to as much as 82% and 77% of the total PBDE exposure in adults and small children, respectively, in the US.95,96

Human exposure may also occur under working conditions. High levels of PBDEs, compared to control subjects, have been reported in blood samples from e.g. Swedish electronics dismantlers 97 , Swedish computer

11 technicians 98 , Chinese employees at waste dismantling facilities99-101 , and children working as informal scrap scavengers at a municipal waste disposal site in Nicaragua 102 , In the above mentioned study on Swedish electronic dismantlers by Sjödin et al. 97 , the concentration of BDE-183, one of the most abundant congeners in technical OctaBDE, was nearly 70 times higher than in control groups. Although occupational exposure may be problematic, it has been shown that hygiene measures such as process planning and sufficient ventilation in combination with good praxis in cleaning procedures reduce the risk of occupational exposure as reported by e.g. Thuresson et al. 103 and in Paper IV.

2.1.4 Biological fate and effects PBDEs are bioaccumulative with log bioaccumulation factors (BAFs) in e.g. blue mussels as high as 6.1 for BDE-47 and BDE-99 and 5.3 for BDE- 153.104 Field studies revealed that for the same congeners even higher log BAFs were reported and were 1-1.5 orders of magnitude higher than for PCBs. 105 Biomagnification of PBDEs has been studied and confirmed by comparing levels at different trophic levels in marine ecosystems. 106-109

In vivo PBDE metabolism have been studied in e.g. rats where both mono- and dihydroxylated PBDEs (OH-PBDEs, diOH-PBDEs) have been detected after administration of either congener specific PBDEs or mixtures thereof.110-113 In one of the above mentioned studies of rats, Malmberg et al. 110 reported sixteen OH-PBDE congers and two di-OH-PBDE congeners after exposure to a single dose of a mixture of BDE-47, BDE-99, BDE-100, BDE-153, BDE-154, BDE-183, and BDE-209. Most OH-PBDEs had the hydroxy group in either a meta - or para-position to the diphenyl ether bond, while naturally occurring OH-PBDEs, almost exclusively, are ortho -hydroxy substituted.114,115 It should be stated that the ortho -substituted 2’-OH-BDE- 28, 6-OH-BDE-47 and 2’-OH-BDE-66 have been identified as minor metabolites. 115 In another exposure study on pike ( Esox lucius ) six OH- PBDEs were reported as metabolites to isotope labelled BDE-47 ( 14 C BDE- 47). 116 OH-PBDEs have also been found to be retained in human serum,102,117-120 which suggests that they form through metabolic transformations of PBDEs in humans as well. Less is known about whether metabolic transformation of PBDEs to MeO-PBDEs takes place, although, in a recent study by Feng et al. 121 , BDE-209 exposed rainbow trout (Oncorhynchus mykiss ), were found to contain both debrominated PBDEs and a total of 7 MeO-PBDEs. The subject of metabolism is also discussed in Paper I where OH-PBDEs were identified as metabolites present in human blood.

For detailed information on the toxicological effects of PBDEs, the author recommends more comprehensive reviews on the topic e.g. the recently

12 published EFSA scientific opinion on PBDEs 29 , but also Darnerud et al.122 , Darnerud 123 and McDonald 124 . A few examples on effects and toxicology are given below.

The most recent data indicate associations between PBDE congeners and cryptorchidism and thyroid hormone suppression.125 PBDEs and their hydroxylated analogues, OH-PBDEs, also shares structural similarities with the thyroid hormone T4 (Figure 2.3) and PBDEs are known to act as endocrine disruptors in humans. 122,126-128 Results from a study on rats have supported the hypothesis that PBDEs can act as endocrine disruptors and interfere with sexual development and sexually dimorphic behaviour. 129 A study by Eriksson et al. 130 has shown that neonatal exposure to BDE-99 and BDE-47, the major congeners of technical PentaBDE, can cause permanent alterations in spontaneous behaviour in mice. BDE-99 also had an effect on learning capabilities and memory functions in adult mice. 130 BDE-183, the major congener of technical OctaBDE, has been shown to decrease spontaneous and habituation behaviour in mice exposed to 15.2 mg/kg b.w. 131 The neurotoxic potential of the major decaBDE congener BDE-209 has also been studied in dosed mice. Changes in learning and visual discrimination could be observed after exposures of 6 mg/kg b.w. 132 Human toxicity studies are rare, although it has been suggested that pregnant women, foetuses and small children would be the most sensitive group.124

Biological effects of PBDEs are also linked to metabolically formed OH- PBDE metabolites, which is further discussed in Chapter 2.2.2.

Figure 2.3 Structure of the thyroid hormone, thyroxine (T4).

2.2 Hydroxylated and methoxylated PBDEs The two structural analogues of PBDEs, OH-PBDEs and MeO-PBDEs, have been raising increased scientific interest in the recent decades, although these are less well researched. In contrast to PBDEs, OH- and MeO-PBDEs are not used as industrial chemicals. As discussed in Chapter 2.1.4, OH-PBDEs are known to be formed as metabolites from anthropogenic PBDEs. 133 OH- PBDEs can also be demethylated metabolites of MeO-PBDEs. 134 Furthermore, functionalised PBDE analogues, such as OH-PBDEs and MeO-PBDEs, are naturally produced in the marine environment. 135-139

13 2.2.1 Chemical structure and properties Structurally, OH- and MeO-PBDEs differ from PBDEs by the substitution of one hydroxy or methoxy group on either of the aromatic rings. Example structures of a OH-PBDE and a MeO-PBDE are shown in Figure 2.4. There are a vast number of theoretical congeners of which only a few have been synthesised and characterised. As an example, counting all the theoretical OH-tetraBDEs and OH-pentaBDEs, together adds up to a total of 396 possible congeners. Synthesis of OH- and MeO-PBDEs is discussed in detail in Chapter 3 and syntheses of specific congeners are reported in Paper I and II.

Figure 2.4 3’-OH-BDE-99 (left) and 3’-MeO-BDE-99 (right).

Due to the introduction of a hydroxy or methoxy functional group, an extension of the Ballschmiter and Zell nomenclature is frequently used in the abbreviation of these substances in accordance with the system used for methyl sulfone substituted and hydroxylated PCBs (MeSO 2-PCBs and OH- PCBs). 140 The nomenclature is exemplified above in Figure 2.3 by 3’-OH- BDE-99 and 3’-MeO-BDE-99.

OH-PBDEs are phenolic and thus slightly more soluble in water compared to PBDEs, especially at higher pH, i.e . when in its deprotonated form. Data regarding these functionalised PBDE analogues are scarce but MeO-PBDEs should, just as PBDEs, have low solubility in water.

PBDEs substituted with two or more hydroxy or methoxy functional groups or, as in some cases, one of each as illustrated in Figure 2.5, are less studied than above mentioned OH- and MeO-PBDEs. DiOH-PBDEs may, just as OH-PBDEs, be formed either metabolically 110 from PBDEs or naturally 141 by marine organisms. Syntheses of di-functionalised PBDE analogues are discussed further in Chapter 3 and Paper III.

Figure 2.5 Structure of a di-functionalised PBDE.

14 2.2.2 Environmental occurrence and biological effects OH-PBDEs were first reported to be present in the marine sponge ( Dysidea herbacea ) by Sharma and Vig 142 in the late 1960’s. Since then, ortho - substituted OH-PBDEs (Figure 2.6) are know to be formed as natural products in e.g. marine sponges (Dysidea herbacea )143-145 and/or its symbiont cyanobacteria (Oscillatoria spongeliae )143-145 , red algae (Ceramium tenuicorne )135,146 and green algae (Cladophora fascicularis )147 .

It has been hypothesised that marine sponges may benefit from the antimicrobial properties of OH-PBDEs which may protect them from bacterial infections. 148-150 Since certain OH-PBDEs and MeO-PBDEs are naturally produced in the marine environment, it is not surprising that they are also frequently found in marine wildlife. By analysing the abundance of natural radiocarbon ( 14 C), Teuten et al. 151 could determine that the two MeO- PBDE congeners 6-MeO-BDE-47 and 2’-MeO-BDE-68 found in whale blubber were in fact of natural origin. Teuten et al. 139 later reported the presence of the same two MeO-PBDE congeners in whale oil samples, collected in 1921 during the last voyage of the whaling ship Charles W. Morgan , which predates the industrial production of PBDE like compounds and further strengthens the evidence that these compounds are of natural origin. Identification of a naturally occurring di-functionalised PBDE is discussed in Chapter 4 and Paper III.

Figure 2.6 Examples of the natural products 6-OH-BDE-47 (left) and 6- MeO-BDE-47 (right)

While halogenated phenolic compounds (HPCs), such as pentachlorophenol (PCP) and OH-PCBs, are well known toxicants acting as e.g. inhibitors of the oxidative phosphorylation and as endocrine disrupters,125,152,153 less is known about OH-PBDEs and MeO-PBDEs. However, an experimental study on 6-OH-BDE47 has revealed acute toxicity in zebrafish embryos and adults, which is attributed to its potent uncoupling of the mitochondrial oxidative phosphorylation.154 The disrupting effect of 6-OH-BDE 47 occurs at nanomolar concentrations, which is about 10 times lower than effect concentrations shown for “classical” uncouplers such as PCP. 152 Preliminary studies have also revealed other OH-PBDEs that disrupt the oxidative phosphorylation and induce embryo- and cytotoxicity. Recently, several OH- PBDEs were reported to bind to the estrogen receptor and act as estrogens.155 Further, OH-PBDEs are thyroidogenic compounds, and may act as agonists

15 or antagonists depending on their structures. 156 Also, OH-PBDEs have been indicated to increase risks for developmental neurotoxicity compared to PBDEs.157 The ongoing research on OH-PBDE toxicity is in an intense phase and is rapidly increasing, while far less is published on MeO-PBDEs. The latter compounds are hitherto primarily studied from an occurrence point of view. It is, however, evident that the compounds synthesised as presented in Paper I, II and III of this thesis are of interest for further toxicological oriented studies.

2.3 Aim of thesis The importance of authentic reference standards cannot be stressed enough. Analysis, including identification and quantification, of chemicals is completely dependent on the availability of reference material. They are also needed for toxicological studies, understanding of the metabolic pathways and for reactivity assessments. Furthermore, to be able to fully characterise these chemicals relatively large amounts of each substance is required. Evaluated, selective and high-yielding methods open up for cost effective syntheses, both when it comes to authentic reference standards as well as isotope labelled standards.

The main objective of this thesis was to develop efficient synthetic routes to obtain congener specific PBDEs and PBDE analogues such as hydroxylated and methoxylated PBDEs (OH-PBDEs and MeO-PBDEs) in high purity. The second objective was to identify and quantify PBDEs, OH- and MeO- PBDEs in environmentally relevant samples. A third objective was to put forward a structured abbreviation system for chemicals of environmental concern, first and foremost for FRs.

A short introduction to the papers included in the thesis is given below.

Paper I: The goal was to synthesise hypothesised PBDE metabolites and subsequently use these as reference standards in an analytical study regarding the occurrence of hydroxylated PBDEs (OH-PBDEs) in blood samples from children working at a waste disposal site in Nicaragua.

Paper II: The synthetic work in Paper I motivated further optimisations of a synthetic route to analytical non-functionalised PBDE standards and MeO- PBDEs, which is presented in this paper.

Paper III: In this paper, two di-functionalised PBDE analogues were synthesised using the same strategy as in Paper II. The aim was to synthesise and use these as reference standards in identification and characterisation of a naturally occurring phenolic PBDE analogue in marine samples.

Paper IV: The aim was to search for FRs, including PBDEs, in electronic waste dust from a Swedish waste management facility. A secondary aim was to investigate if the results from the dust analyses were reflected in blood samples from workers at the same waste management facility.

Paper V: This paper suggests a novel abbreviation standard for brominated-, chlorinated- and phosphorus flame retardants (BFRs, CFRs and PFRs), aimed to simplify and promote scientific communication.

17 3. Synthesis of selected organobromines Chapter 3 provides a detailed background on previously used synthetic approaches towards PBDEs and their hydroxy- and methoxy-substituted analogues, OH-PBDEs and MeO-PBDEs. Furthermore, results within the work of this thesis, regarding syntheses of PBDE and PBDE analogues, are presented within this chapter and in Paper I, II and III.

3.1 Synthetic routes to PBDEs Possibly the very first reported synthesis of a PBDE can be traced back to 1871.158 The synthesised congener was 4,4’-dibromodiphenyl ether (BDE- 15) and it was obtained by adding elemental bromine (Br 2) to diphenyl ether. Since then, other ways of achieving congener specific PBDEs have been reported. One of the most recent methods is reported in Paper II and in this thesis. The most common routes to PBDEs are presented below.

3.1.1 Bromination of diphenyl ether The aromatic rings of diphenyl ether may be brominated via Electrophilic Aromatic Substitution (EAS) using elemental bromine, often in combination with a Lewis acid catalyst such as e.g. aluminium bromide (AlBr 3) or ferric bromide (FeBr 3). The latter Lewis acid is unstable and is therefore often formed in situ by the reaction of elemental iron filings (Fe) which in contact with bromine forms the desired Lewis acid catalyst. Elemental bromine is the most common brominating agent used on diphenyl ethers in the scientific literature, although a few exceptions exist when it comes to mono bromination. Two examples on the synthesis of 3-bromodiphenyl ether (BDE-3) are illustrated in Scheme 3.1. The first being bromination using N- bromosuccinimide (NBS) employing ammonium nitrate (NH 4NO 3) as a catalyst and the second using bromodimethylsulfonium bromide (BDMS). 159,160 The latter yielding only mono brominated product despite using more than one equivalent of the brominating agent. The higher brominated commercial mixtures such as technical Penta-, Octa- and DecaBDE were thus synthesised using the more common elemental bromine and Lewis acid method. 161

Scheme 3.1

When it comes to more congener specific syntheses of PBDEs, direct bromination of diphenyl ether has been successfully employed not only for above mentioned BDE-15 and BDE-3 but also in the synthesis of e.g. 2,2’,3,3’,4,4’,5,5’,6,6’-decabromodiphenyl ether (BDE-209) 161 and 2,2’,4,4’- tetrabromdiphenylether (BDE-47) 73 with isolation of the desired product in high yields and purity (Scheme 3.2). There are limitations to what congeners can be achieved by direct bromination of diphenyl ether, which are direct consequences of the ortho/para directing properties of the ether linkage similar to that observed in halogenation of alkyl phenyl ethers.

Scheme 3.2

Directed ortho metalation (DOM) was discovered independently in the late 1930’s by Gillman and Wittig.162,163 The concept builds on the fact that metalation occurs ortho to certain functionalised aromatics including alkyl phenyl ethers and diphenyl ethers. The oxygen in the ether linkage chelates the alkyl metal and directs ortho relative itself, as exemplified in Scheme 3.3 with an aromatic ether and alkyl lithium. Inductive effects also play a role by lowering the pKa of the adjacent ortho proton. An electrophile (E +) may then be used to quench the reaction and substitute the metal.

Scheme 3.3

The above mentioned DOM strategy opened up for a workaround to achieve 2,2’-dibromodiphenyl ether (BDE-4), exemplified in Scheme 3.4. Success in using this strategy has been reported via the use of butyl lithium to achieve an o,o’-dilithiodiphenyl ether intermediate which was subsequently quenched with bromine.164 The butyl lithium and the formed intermediate are

19 air and moist sensitive and failure to keep the reaction conditions inert may result in poor yields and formation of undesirable by-products.

Scheme 3.4

3.1.2 Bromination of prefabricated PBDEs Bromination of existing PBDEs, prepared by other measures than direct bromination, has been employed to yield a number of PBDE congeners with 2-8 bromine substituents and has been reported elsewhere.164-167 However, a compilation of reaction results are given in Scheme 3.5.

Scheme 3.5

3.1.3 Nucleophilic aromatic substitution Four specific PBDE congeners, BDE-99, BDE-153, BDE-154 and BDE-183 168 have all been prepared by Chen et a.l via S NAr reactions employing 2,5- dibromo-4-fluoronitrobenzene and various bromophenols as summarised in Scheme 3.6. The formed nitro-PBDEs were reduced to their corresponding brominated aminodiphenyl ether (NH 2-PBDEs) using tin and hydrochloric acid. The amino groups were then substituted for bromine by diazotisation followed by a Sandmeyer type reaction with cupper bromide (CuBr).

20 OH F Br O Br K2CO3 Brx Brx acetone Br NO2 Br NO2

2 steps O Br Brx Br Br

Phenol Product Yield

2,4-dibromophenol 2,2',4,4',5-pentaBDE (BDE-99) 82% 2,4,5-tribromophenol 2,2',4,4',5,5'-hexaBDE (BDE-153) 81% 2,4,6-tribromophenol 2,2',4,4',5,6'-hexaBDE (BDE-154) 48% 2,3,4,6-tetrabromophenol 2,2',3,4,4',5',6-heptaBDE (BDE-183) 12%

Scheme 3.6

3.1.4 Ullmann ether synthesis The original cupper-catalysed Ullmann reaction is known to be useful in the synthesis of biaryls. 169 A modified version, also known as Ullmann ether synthesis, has been employed in the synthesis of e.g. the above mentioned PBDE congeners BDE-2 and BDE-11, which subsequently were further brominated according to Scheme 3.5 above. 166 A general reaction mechanism has been proposed by Sperotto et al. 170 illustrated in Scheme 3.7 using the synthesis of BDE-11 as an example.

HO Br O Br Br KOH CuBr BDE-11 32%

Red. KBr H2O Elimination

Br Br Cu O Cu O

Br Br

Ox. Addition

Br Br

Scheme 3.7

21 3.1.5 PBDEs from arylboronic acids Arylboronic acids and aryl iodides are widely used in palladium-catalysed Suzuki couplings to yield biphenyls. 171 A similar approach has been reported in the synthesis of diaryl ethers and PBDEs, using phenols instead of the aryl iodide and cupper acetate instead of expensive palladium. 168,172 The synthesis of 3,4,4’,5-tetrabromodiphenyl ether (BDE-81) has been reported employing this strategy (Scheme 3.8). 168

Scheme 3.8

A drawback with arylboronic acids is their sensitivity to moisture. Since water is formed in the reaction some of the brominated arylboronic acid will hydrolyse to the corresponding phenol, which in turn may react with unreacted brominated arylboronic acid. Thus, failure in removing the formed water results in symmetrical diaryl ethers as a by-product. In the above mentioned synthesis of BDE-81, 4,4’-dibromodiphenyl ether was formed in a 10 % yield. 168

3.1.6 Synthesis via aminodiphenyl ethers 167,173 Several PBDEs have been prepared via perbromination of NH 2-PBDEs. Christiansson et al. 173 reported the preparation of three nonaBDE congeners, 2,2’,3,3’,4,4’,5,5’,6-nonabromodiphenyl ether (BDE-206), 2,2’,3,3’,4,4’,5,6,6’-nonabromodiphenyl ether (BDE-207) and 2,2’,3,3’,4,5,5’,6,6’-nonabromodiphenyl ether (BDE-208), by perbromination of ortho-, meta- and para-aminodiphenyl ether, respectively, followed by diazotisation with 3-methylbutyl nitrite and boron trifluoride diethyl etherate in THF and subsequent reduction of the diazonium salt using iron sulfate (FeSO 4). The reaction pathway is exemplified with meta -aminodiphenyl ether in Scheme 3.9.

Scheme 3.9

Using a similar approach, Teclechiel et al. 167 reported syntheses of the three octaBDEs, BDE-201, BDE-202 and BDE-204, starting from diaminodiphenyl ethers (Scheme 3.10). In the same publication, one octaBDE, BDE-198, was prepared from monoaminodiphenyl ether. BDE- 204 could also be prepared in higher yield using the monoaminodiphenyl ether as precursor instead of diaminodiphenyl ether (Scheme 3.10). 167

Scheme 3.10

Ortho selective bromination of 4,4’-diaminodiphenyl ether and selective bromination of 3,4’-diaminodiphenyl ether, followed by diazotisation and subsequent Sandmeyer reaction by treating the diazonium salt with cupper(I) bromide (CuBr) and cupper(II) bromide (CuBr 2) has also been reported to yield BDE-169 and BDE-191, respectively (Scheme 3.11).167 These two BDEs have been further brominated, in accordance to earlier discussed bromination of prefabricated PBDEs (cf. Scheme 3.5 and 3.11) to give BDE- 194 and BDE-196, respectively.167

23 O Br O Br 1.) Br2,AcOH r.t. H2N NH2 H2N NH2 Br Br

1.) BF3-Et2O, CH3CN 2.) CuBr,CuBr ,H O,CH CN Br Br 2 2 3 Br O Br Br O Br Br2, Fe

Br Br Br Br Br Br Br Br BDE-194 BDE-169 Br H N O H N O Br 2 1.) Br2,AcOH 2 r.t. NH2 Br Br NH2 Br

1.) BF3-Et2O, CH3CN 2.) CuBr,CuBr ,H O,CH CN Br Br Br 2 2 3 Br O Br Br O Br Br2, Fe

Br Br Br Br Br Br Br Br BDE-196 BDE-191

Scheme 3.11

3.1.7 Synthesis via diaryliodonium salts Congener specific polychlorinated diphenylether (PCDE) synthesis by O- arylation of chlorophenolates using chlorinated diphenyliodonium salts has been reported in the scientific litterature.174-178 The same strategy has also been employed for congener specific PBDE syntheses. A background on brominated diphenyliodonium salt syntheses follows below, including novel salts synthesised within this thesis, before in detail describing PBDE syntheses using these salts.

Synthesis of symmetrical brominated diphenyliodonium salts

Synthesis of most halogenated diphenyliodonium salts have been carried out using a method originally developed by Beringer et al. 179 By oxidising elemental iodine in a mixture of fuming nitric acid and sulfuric acid, a suspension of iodyl sulfate formed to which bromobenzene was added to yield 4,4’-dibromodiphenyliodonium chloride as shown in Scheme 3.12.178,179

Scheme 3.12

Marsh et al.165 reported the syntheses of two additional symmetrical tetrabrominated diphenyliodonium salts using the above mentioned conditions (Scheme 3.13).

Scheme 3.13

Teclechiel et al.180 later developed a slightly modified method for the preparation of three symmetrical hexabrominated diphenyliodonium salts. The concept was the same as above mentioned preparation, only with addition of extra sulfuric acid and longer reaction times (Scheme 3.14).

Scheme 3.14

Synthesis of unsymmetrical brominated diphenyliodonium salts

Little has been reported regarding syntheses of unsymmetrical brominated diphenyliodonium salts. However, Chen et al. 168 synthesised 4- methoxyphenyl(2,4,5-tribromophenyl)iodonium bromide by first preparing a trifluoroperoxyacetic acid (TFPA) solution from hydrogen peroxide and trifluoroacetic anhydride to which 1,2,4-tribromobenzene in dichloromethane was added. Addition of anisole and subsequent work-up with sodium bromide yielded the desired unsymmetrical diaryliodonium bromide (Scheme 3.15). It should be stated that the procedure is a multi step,

25 time consuming synthesis and gives a relatively low yield of 20%. Liu et al. 181 later reported the synthesis of 4-methoxyphenyl(2,4,5- tribromophenyl)iodonium bromide, using chromium trioxide instead of TFPA as oxidation agent with an increase in yield to 34%.

Scheme 3.15

Bielawski et al. 182 reported the synthesis of 4-bromophenyl(phenyl)iodonium triflate, starting from 1-bromo-4-iodobenzene and benzene according to Scheme 3.16. In addition, 3-bromophenyl(phenyl)iodonium tetrafluoroborate and 4-bromophenyl(phenyl)iodonium tetrafluoroborate have been prepared by Bielawski et al. 183 starting from iodobenzene and 3-bromophenylboronic acid or 4-bromophenylboronic acid, respectively (Scheme 3.16).

TfO- I I+ m-CPBA TfOH,CH Cl Br 2 2 Br 78 % - BF4 m-CPBA + I (HO)2B Br I Br BF3-Et2O r.t. 45 min 75 % - BF4 m-CPBA + I (HO)2B I BF3-Et2O

Br r.t. 45 min Br 78 % Scheme 3.16

In Paper I of this thesis, the novel polybrominated diphenyliodonium salt, phenyl(2,4,6-tribromophenyl)iodonium triflate, was synthesised using a synthetic approach previously reported by Bielawski et al. and Bielawski and Olofsson.182,184 A solution of 1,2,4-tribromo-5-iodobenzene, meta - chloroperoxybenzoic acid ( m-CPBA) in dichloromethane was treated with benzene followed by trifluoromethanesulfonic acid (TfOH) to yield the desired diaryliodonium triflate in satisfying yield in an off-white to slightly brown colour (Scheme 3.17).

26 Br Br

NH2 I t-BuONO Br Br I2 Br Br ACN/CCl4 71 %

Br TfO- Br 1.) Benzene (2 equiv.) I+ I 2.) TfOH (3 equiv, 0°C) mCPBA CH Cl Br Br 2 2 Br Br 70°C, 20h 80 %

Scheme 3.17

One observation we made, in the preparatory work for Paper II, was that the reaction mixture containing bromoiodobenzene, mCPBA and benzene often turned black or dark brown when adding the TfOH, even at sub zero temperatures. The yields varied and were not reproducible and the isolated products had a similar brown colour. For this reason a test reaction was performed, omitting the bromoiodobenzene. The experiment resulted in the observation that the mixture, now containing only benzene and m-CPBA, instantly turned black when adding TfOH. It was hypothesised that the benzene was sensitive to the TfOH-activated m-CPBA which possibly would oxidise the benzene. By changing the addition order, first adding TfOH to the solution of the iodoarene and m-CPBA and subsequently adding 6 equivalents of benzene in dichloromethane as described in Scheme 3.18, the salt could be isolated in higher yields and as a white solid with good reproducibility. It was further hypothesised, that the procedure applied gave the iodoarene some time to pre-oxidize, leaving less TfOH activated m- CPBA to react with the benzene. Furthermore, decreasing the reaction time to 1 h did not affect the yield negatively.

Scheme 3.18

In Paper II, two additional unsymmetrical diaryliodonium salts, phenyl(2,4,5-tribromophenyl)iodonium triflate and phenyl(2,3,4,6- tetrabromophenyl)iodonium triflate, were successfully prepared using the same conditions as for phenyl(2,4,6-tribromophenyl)iodonium triflate (Scheme 3.19).

Scheme 3.19

The yields in the syntheses of phenyl(2,4,6-tribromophenyl)iodonium triflate and phenyl(2,4,5-tribromophenyl)iodonium triflate in Paper II were 94% and 85%, respectively, which is higher than the previously reported yields of the corresponding symmetrical hydrogen sulfate salts, 2,2’,4,4’,6,6’- hexabromodiphenyliodonium hydrogen sulfate and 2,2’,4,4’,5,5’- hexabromodiphenyliodonium hydrogen sulfate with yields of 57 % and 55 % respectively (cf. Scheme 3.14). 180 Also, phenyl(2,3,4,6- tetrabromophenyl)iodonium triflate, for which corresponding symmetrical salts have not been reported, was synthesised in an acceptable yield of 69 %. The reaction times in Paper II is also considerably shorter with 1 h instead of reaction times of up to 72 h as was the case of reported brominated symmetrical diphenyliodonium salts.180

Attempts were also made to synthesise phenyl(2,3,4,5,6- pentabromophenyl)iodonium triflate from 1,2,3,4,5-pentabromo-6-iodo benzene using the same methodology. This however resulted in a black tar- like solution instantly upon the addition of benzene. The product proved hard to purify and contained only traces of desired unsymmetrical phenyl(2,3,4,5,6-pentabromophenyl)iodonium triflate. Sterical hindrance could be considered as a possible reason for this although bromines in both ortho -positions to iodine in 1,3,5-tribromo-2-iodobenzene had already been proven to be well tolerated (cf. Scheme 3.18). It may also by hypothesised that poor solubility or electron withdrawing effects from an increased number of bromine substituents may inhibit the oxidation of 1,2,3,4,5- pentabromo-6-iodo benzene. The fact that yields are lower when starting from 1,2,3,5-tetrabromo-4-iodobenzene (Scheme 3.19, 69%) than when starting from tribromo-iodo-benzenes indicates that this might be the case.

PBDE synthesis using symmetrical diaryliodonium salts

O-arylations of bromophenolates with symmetrical brominated diphenyliodonium salts have generated a vast number of congener specific PBDEs. Typically, a procedure developed by Crower et al. 185 has been widely used in PBDE synthesis. In this procedure, bromophenols are refluxed together with symmetrical brominated diphenyliodonium halides in

28 an alkaline aqueous solution as exemplified in Scheme 3.20. Using this approach, Marsh et al. 165 reported the synthesis of three monoBDEs, six diBDEs, eight triBDEs, six tetraBDEs, three pentaBDEs, one hexaBDE and two heptaBDE congeners using symmetrical non-, di- and tetrabrominated diphenyliodonium salts, as specified in Scheme 3.20.

X- 2 2 2' OH I+ O 3 3 3' NaOH (aq) Brx Bry Bry Brx Bry 4 6 4 6 6' 4' 100 °C, 5 5 5' 0.5-2h

Phenol (Brx) Salt (Bry) Product Name Yield

2- diphenyliodonium salt 2- BDE-1 44 % 3- diphenyliodonium salt 3- BDE-2 35 % 4- diphenyliodonium salt 4- BDE-3 18 % 2,4- diphenyliodonium salt 2,4- BDE-7 35 % 2- 4,4'- 2,4'- BDE-8 17 % 2,6- diphenyliodonium salt 2,6- BDE-10 37 % phenol 3,3',4,4'- 3,4- BDE-12 23 % 3- 4,4'- 3,4'- BDE-13 17 % 4- 4,4'- 4,4'- BDE-15 18 % 2- 2,2',4,4'- 2,2',4- BDE-17 39 % 3- 2,2',4,4'- 2,3',4- BDE-25 58 % 2,4- 4,4'- 2,4,4'- BDE-28 16 % 2,4,6- diphenyliodonium salt 2,4,6- BDE-30 25 % 2,6- 4,4'- 2,4',6- BDE-32 23 % 2- 3,3',4,4'- 2',3,4- BDE-33 58 % 3- 3,3',4,4'- 3,3',4- BDE-35 45 % 4- 3,3',4,4'- 3,4,4'- BDE-37 35 % 2,4- 2,2',4,4'- 2,2',4,4'- BDE-47 43 % 2,5- 2,2',4,4'- 2,2',4,5'- BDE-49 87 % 2,6- 2,2',4,4'- 2,2',4,6'- BDE-51 52 % 2,4- 3,3',4,4'- 2,3',4,4'- BDE-66 55 % 2,6- 3,3',4,4'- 2,3',4',6- BDE-71 43 % 2,4,6 4,4'- 2,4,4',6- BDE-75 23 % 2,4,6 2,2',4,4'- 2,2',4,4',6- BDE-100 36 % penta- diphenyliodonium salt 2,3,4,5,6- BDE-116 17 % 2,4,6- 3,3',4,4'- 2,3',4,4',6- BDE-119 8 % penta- 4,4'- 2,3,4,4',5,6- BDE-166 10 % penta- 2,2',4,4'- 2,2',3,4,4',5,6- BDE-181 11 % penta- 3,3',4,4'- 2,3,3',4,4',5,6- BDE-190 5 %

Scheme 3.20

Liu et al. 181 also reported the synthesis of BDE-75 but with a slightly lower yield of 21 % using the same conditons as Marsh et al.165 2,2’,3,4,4’- pentabromodiphenyl ether (BDE-85), 2,2’,3,4,4’,5’-hexabromodiphenyl ether (BDE-138) and 2,2',3,4,4',5',6-heptabromodiphenyl ether (BDE-183) were also synthesised in the scope of the publication by Liu et al. 181 in yields of 49%, 54% and 54%, respectively.

29 Teclechiel et al. 180 reported the synthesis of two hexa-, four hepta- and three octaBDEs by using hexabromodiphenyliodonium hydrogen sulfates and slightly modified conditions in the O-arylation of the bromophenols as depicted in Scheme 3.21.

Scheme 3.21

PBDE synthesis using unsymmetrical diaryliodonium salts

To the best knowledge of the author of this thesis, only two reactions have been previously reported in the scientific literature, using an unsymmetrical brominated diaryliodonium salt for congener specific PBDE syntheses. The first one was reported by Chen et al.168 who used 4-methoxyphenyl(2,4,5- tribromophenyl)iodonium bromide (cf. Scheme 3.15) in the O-arylation of 2,3,4,6-tribromophenol (BDE-183, 45%, Scheme 3.22). The other was reported by Liu et al. 181 , employing the same unsymmetrical salt in the O- arylation of 2,3,4-tetrabromophenol(BDE-138, Scheme 3.22). Liu et al.181 also reproduced the synthesis reported by Chen et al. 168 in a slightly higher yield (BDE-183, 54%, Scheme 3.22)

Scheme 3.22

The objective with the work described in paper II, apart from synthesising novel unsymmetrical brominated diphenyliodonium triflates, was to employ these salts in O-arylations of bromophenols in an attempt to improve the yield in congener specific PBDE syntheses. Our optimisation was based on an already published method reported by Jalalian et al .186 , where several deprotonated phenols were successfully O-arylated by diphenyliodonium triflate in THF. One entry from Jalalian et al. 186 particularly caught our interest as the chlorinated analogue to 2,3,4,5,6-pentabromodiphenyl ether (BDE-116), namely 2,3,4,5,6-pentachlorodiphenyl ether (CDE-116), was reported to form in high yields from pentachlorophenol and diphenyliodonium triflate (Scheme 3.23).

Scheme 3.23

It was decided to use phenyl(2,4,6-tribromophenyl)iodonium triflate to O- arylate 4-bromophenol, 2,4,6-tribromophenol and pentabromophenol in our optimisation of the arylation reaction since it was hypothesised it would be representative for further attempts with salts and phenols of lower or higher degrees of bromination. The outcome of our optimisation is shown in Scheme 3.24 below.

31 Br 6 6 1 OH O 5 1.) t-BuOK, THF 5 1 Brx Brx 4 2 2.) TfO- Br 4 2 Br Br 3 I+ 3

Br Br

Entry Phenol (Brx) Temp. (°C) Time Yield (%)

1 4- 40 20 min 62 2 4- 40 40 min 72 3 4- 40 1 h 84 4 4- 40 1.5 h 84 5 4- 70 1 h 89 6 2,4,6- 40 1 h 63 7 2,4,6- 40 1.5 h 63 8a 2,4,6- 40 1 h 47 9 2,4,6- 70 1 h 80 10 2,4,6- 70 1.5 h 80 11 penta- 70 1 h 45 12 penta- 70 1.5 h 45

a) N,N-Diisopropylethylamine (DIPEA) was used instead of t-BuOK Scheme 3.24

The conclusions drawn from our optimisations were that the reaction conditions reported by Jalalian et al. 186 seemingly did fit our needs quite well although we found it necessary to increase the reaction temperatures to maximise the yields. Increasing the reaction time beyond 1 h had no further effect on the yields regardless of the temperature. We also tried the base N,N -diisopropylethylamine (DIPEA), which in a pilot study showed promising results and yielded 1,3,5-tribromo-2-(4-bromophenoxy) benzene (BDE-75) in over 70% yield when refluxing the reaction mixture in dichloromethane. However, as can be seen in Scheme 3.24, Entry 8, the yield decreased to 47% using DIPEA in THF. Furthermore, the 47% yield is also lower than the yield of 63 % from the corresponding reaction using THF and t-BuOK (Scheme 3.24, Entry 6). The optimisation part is further discussed in Paper II but to further test our optimal conditions we tried additional reactions with other salts and bromophenols (Entries 4-8, Scheme 3.25). Entries 1-3 in Scheme 3.25 are the most successful entries from Scheme 3.24.

32 2 2 6 1 OH O 3 1.) t-BuOK, THF 3 1 5 Br 1 Br x - Brx y 4 6 2.) TfO 4 6 2 4 6 5 + I 5 5 3 1 Bry 2 4 3

Entry Phenol (Brx)Salt (Bry) Product Name Yield

1 4- 2,4,6- Br BDE-75 89 % O

Br Br Br

2 2,4,6- 2,4,6-Br Br BDE-155 80 % O

Br Br Br Br

3 2,3,4,5,6- 2,4,6- Br Br BDE-204 45 % Br O

Br Br Br Br Br 4 2,4- 2,4,5-Br BDE-99 94 % O Br

Br Br Br

5 2,4,5- 2,4,5- Br BDE-153 82 % O Br

Br Br Br Br 6 2,4,5- 2,3,4,6- Br Br BDE-183 83 % O

Br Br Br Br Br 7 2,3,4,5- 2,3,4,6- Br Br BDE-196 98 % Br O

Br Br Br Br Br 8 2,3,4,6- 2,3,4,6-Br Br BDE-197 64 % Br O

Br Br Br Br Br

Scheme 3.25

The yields in the O-arylations to achieve BDE-75, BDE-99, BDE-155, BDE- 183 and BDE-204 reported in Paper II are significantly higher than previously reported yields, when symmetrical and unsymmetrical brominated diphenyliodonium salts have been applied. A comparison is shown in Table 3.1. Furthermore, for the first time, BDE-153, BDE-196 and

33 BDE-197 were successfully synthesised using the route via brominated diphenyliodonium salts. To our knowledge, no other published methods have been available for the synthesis of BDE-197, one of the major congeners in technical octaBDE 187 .

Table 3.1 Reported yields in selected PBDE congener syntheses. Product Type of salt Yield Reference

BDE-75 symmetrical 23 % Marsh et al.165 BDE-75 unsymmetrical 89 % Paper II BDE-99 symmetrical 69 % Chen et al.168 BDE-99 unsymmetrical 94 % Paper II BDE-155 symmetrical 21 % Techlechiel et al.180 BDE-155 unsymmetrical 80 % Paper II BDE-183 symmetrical 59 % Techlechiel et al.180 BDE-183 unsymmetrical 54 % Liu et al.181 BDE-183 unsymmetrical 83 % Paper II BDE-204 symmetrical 13 % Techlechiel et al.180 BDE-204 unsymmetrical 44 % Paper II

Fortunately, we did not observe any O-arylation products containing the non-brominated phenyl ligand from the three unsymmetrical brominated diphenyliodonium salts employed. This is believed to be a consequence of both electronic and steric effects. The brominated arene is more electron deficient and thus more likely to transfer to the phenol. Also, the ortho-effect has previously been reported to favour transfer of an ortho-substituted aryl ligand over non-ortho-substituted aryl ligands in reactions between nucleophiles and diaryliodonium salts. 188,189 An explanation for this is that the bulkier ortho-substituted aryl ligand will occupy the equatorial position rather than the more crowded axial position in the reaction intermediate as exemplified in Scheme 3.26. The equatorial ortho-substituted ligand is then favoured for the subsequent reductive elimination since the axial phenyl ligand is too distant.

Scheme 3.26

The selection of unsymmetrical brominated diphenyliodonium triflates synthesised and subsequently employed to successfully arylate various

34 bromophenols in Paper II indicates that our unsymmetrical salt approach could most likely be used as a general method to prepare the majority of the 209 possible PBDE congeners. Unsymmetrical brominated diphenyliodonium triflates are generally fast and easy to prepare, very stable when stored dry, more soluble than corresponding symmetrical diaryliodonium halides and readily arylates even sterically hindered ortho- substituted bromophenols. Steric hindrance in ortho-position is also well tolerated in the diaryliodonium salts themselves, thus making it possible to synthesise a variety of authentic PBDE standards in high yields and purity in a relatively short time span.

3.2 Synthesis of mono and di-functionalised PBDEs Synthesis of OH-PBDEs and MeO-PBDEs is discussed jointly in this chapter because the synthetic approaches for achieving one of them also includes the possibility to transform one into the other via either O- methylation of OH-PBDEs using methyl iodide (CH 3I) or demethylation of MeO-PBDEs using boron tribromide (BBr 3) as exemplified in Scheme 3.27. MeO-PBDEs are important substances in analytical identification studies, as they are used as authentic reference standards to identify and quantify OH- PBDEs in methylated phenolic fractions of e.g. biological sample extracts.

Scheme 3.27

3.2.1 Synthesis of MeO-PBDEs via S NAr Syntheses of three ortho-MeO-PBDEs and one para-MeO-PBDE, and their corresponding OH-PBDEs, have been reported by Nikiforov et al. 190 using the nucleophilic aromatic substitution approach. In the above study, MeO- PBDEs were synthesised from brominated fluorobenzenes and monobrominated methoxyphenols in dimethyl formamide, using potassium carbonate as base. The MeO-PBDE structures and their abbreviated names are shown in Figure 3.1 below.

Figure 3.1

35 SNAr of 1,2-dibromo-4-nitrobenzene and 4-bromo-nitrobenzene with brominated 2-methoxyphenols have been used in syntheses of nitro-MeO- PBDEs as reported by Francesconi et al .191 . These nitro-substituted MeO- PBDEs could then be reduced to the corresponding NH2-MeO-PBDEs 191 (Scheme 3.28). Analogous to PBDE synthesis from NH 2-PBDEs, a diazotisation with tert -butyl nitrite followed by a Sandmeyer reaction or using cupper(II) bromide was reported to yield the desired MeO-PBDEs as exemplified in Scheme 3.28. 191

Scheme 3.28

Sharma and Vig 138 reported the synthesis of 6-MeO-2,2’,3,4,4’-pentaBDE (6-MeO-BDE-85) employing a similar synthetic approach with the difference of using 2,3,4-tribromo-6-methoxyphenol as the nucleophile substituting the chlorine in 2,4-dinitrochlorobenzene in an alkaline ethanol solution (Scheme 3.29). Subsequent reduction with sodium hydrosulfite (Na 2S2O4), diazotisation with sodium nitrite followed by a Sandmeyer type insertion of bromine gave the desired 6-MeO-BDE-85. Yields were unfortunately only stated for the reduction step.

OMe NO2 OMe NO2 OH Cl O NaOH EtOH Br Br NO2 Br Br NO2 Br Br ?%

OMe NH2 OMe Br O O Na2S2O4 NaNO2

CuBr2 Br Br NH2 Br Br Br Br 50 % Br 6-MeO-BDE-85, ? % Scheme 3.29

Marsh et al. 192 used a similar approach when coupling 2,4-dibromophenol with brominated fluorobenzaldehydes to yield brominated phenoxybenzaldehydes (formyl-PBDEs), but with sodium carbonate as base

36 in N,N -dimethylacetamide (DMAC) as shown in Scheme 3.30. The formyl- PBDEs were oxidized to the corresponding ester via Baeyer-Villiger oxidation using TFPA and a subsequent hydrolysis step with hydrochloric acid gave the desired OH-PBDEs (Scheme 3.30). 192

Scheme 3.30

In Marsh et al. 192 , the two congeners 2’-OH-BDE-7 and 2’-OH-BDE-28, were subsequently selectively monobrominated ortho to the hydroxy group using a bromine/ tert-butylamine complex and/or dibrominated ortho and para to the hydroxy group using benzyltrimethylammonium tribromide (BTMA Br 3) to give the three additional OH-BDE congeners as depicted in Scheme 3.31.

Scheme 3.31

Using the same S NAr- Baeyer-Villiger-hydrolysis strategy as above, Marsh et al. 192 also synthesised the para -substituted 4'-OH-BDE 17 starting from 2,4-dibromophenol and 3-bromo-4-fluorobenzaldehyde. 4'-OH-BDE-17 was used as a precursor for a subsequent non-selective bromination, which gave a crude mixture of three para -OH-PBDE congeners, as depicted in Scheme 3.32. The three congeners were isolated separately.

1 2 Br Br Br Br R R Product O O R2 Br2, AcOH H Br 4-OH-BDE-42 Br H 4'-OH-BDE-49 Br OH Br OH 1 Br Br 4-OH-BDE-90 R Scheme 3.32

In a more recent publication, Yu et al. 119 successfully synthesised two octabrominated and one nonabrominated ortho substituted MeO-PBDEs using a SNAr approach where O-arylation of 5-amino-2-methoxyphenol with 5-fluoro-2-nitroaniline gave 5-(5-amino-2-methoxyphenoxy)-2-nitroaniline as shown in Scheme 3.33. This OH- and MeO-PBDE precursor could then be converted by selective brominations, reductions, diazotisations, Sandmeyer reactions, demethylations and methylations to give 3 different OH- and MeO-PBDE congeners, also shown in Scheme 3.33. 119

Scheme 3.33

Sun et al. 193 employed a similar multi step strategy in the syntheses of an impressive additional 22 different MeO-PBDEs (summarised in Scheme 3.34) and 17 OH-PBDEs. Similarly to Yu et al. 119 , Sun et al. 193 started from 5-fluoro-2-nitroaniline but used four different phenols, namely 3- methoxyphenol, 5-amino-2-methoxyphenol, 2-amino-4-methoxyphenol and 3-amino-methoxyphenol, in their syntheses.

Scheme 3.34

3.2.2 Synthesis of MeO-PBDEs via brominated diphenyliodonium salts Similarly to PBDEs, MeO-PBDEs have also been synthesised via O- arylation of brominated methoxyphenols with brominated diphenyliodonium salts, using slightly modified conditions to those reported by Nilsson et al. 178 in the syntheses of PCDEs and methoxy-PCDEs (MeO-PCDEs), as summarised in Scheme 3.35 194 .

Scheme 3.35

Nikiforov et al. 190 reported the synthesis of two ortho substituted MeO- PBDEs by O-arylation of 4,5-dibromo-2-methoxyphenol and 2,4-dibromo-6- methoxyphenol, respectively, using 2,2’,4,4’-tetrabromodiphenyliodonium chloride according to Scheme 3.36.

Scheme 3.36

In Marsh et al. 192 three ortho substituted MeO-PBDEs and their corresponding OH-PBDEs were synthesised by O-arylation of pre- brominated 2-methoxyphenols using 2,2’,4,4’-tetrabromodiphenyliodonium iodide, employing potassium carbonate as base in DMAC, as outlined in Scheme 3.37. In addition, Marsh et al. 192 also reported the synthesis of the meta substituted 5-MeO-2,2’,4,4’-tetrabromdiphenyl ether (5-MeO-BDE 47) in 91 % yield from 2,4-dibromo-5-methoxyphenol and 2,2’,4,4’- tetrabromodiphenyliodonium chloride.

Scheme 3.37

In the same study, Marsh et al. 192 also O-arylated brominated ortho- hydroxybenzaldehydes using 2,2’,4,4’-tetrabromodiphenyliodonium chloride, employing sodium hydroxide as base in water and dioxane as shown in Scheme 3.38. The formyl-PBDEs were oxidised to the corresponding ester via Baeyer-Villiger oxidation using TFPA and subsequent hydrolysis with hydrochloric acid gave OH-PBDEs (Scheme 3.38).

Scheme 3.38

The two congeners 6’-OH-BDE-17 and 6-OH-BDE-47 were further selectively monobrominated ortho to the hydroxy group using a bromine/ tert-butylamine complex and/or dibrominated ortho and para to the hydroxy group using BTMA Br 3 to give the three additional OH-PBDE congeners as presented in Scheme 3.39192 .

Scheme 3.39

Also, in the study by Marsh et al. 192 a mixture of two meta-OH-PBDE congeners were prepared by O-arylation of 3-hydroxybenzaldehyde using 2,2’,4,4’-tetrabromodiphenyliodonium chloride followed by Baeyer-Villiger oxidation, hydrolysis and subsequent bromination according to Scheme 3.40

41 Br Cl- Br Br OHC OH I+ OHC O K2CO3, DMAC 18-crown-6 Br Br Br

1.) CF3CO3H, CH2Cl2 KH2PO4 2.) MeOH/HCl

Br Br Br Br HO O HO O HO O 1.) tBuNH2, Br2 CH Cl Br Br Br Br 2 2 Br 3-OH-BDE-47 3'-OH-BDE-28 Scheme 3.40

In Paper I, a total of 14 OH-PBDEs and 11 novel MeO-PBDE reference standards were synthesised. Five of the OH-PBDEs and two of the MeO- PBDEs were intermediate products in the total synthesis of our desired nine OH-PBDEs and their corresponding methyl ethers. The target compounds were four OH-pentaBDEs, four OH-hexaBDEs and one OH-heptaBDE and their corresponding MeO-PBDEs, which were all characterised by NMR spectroscopy and mass spectrometry. Of the nine target MeO/OH-PBDEs, eight were meta -functionalised and one was functionalised in the para position.

The synthetic approach was primarily based on the use of above mentioned symmetrical brominated diphenyliodonium salts 2,2’,4,4’-tetrabromo-, 2,2’,4,4’,5,5’-hexabromo- and 2,2’,4,4’,6,6’-hexabromodiphenyliodonium chlorides and iodides.180,192 However, the latter iodonium salt, yielding a 2,4,6-tribromo substitution in one of the phenyl rings when used as O- arylating agent, gave diphenyl ethers in unsatisfying yields. Also, the crude products contained by-products resulting in complicated purification procedures. Instead we prepared and used the unsymmetrical brominated diaryliodonium salt 2,4,6-tribromodiphenyliodonium triflate, reported in Paper I and II, which gave better results in the O-arylations of methoxyphenols and hydroxybenzaldehydes. A summary of the O-arylations performed in Paper I is shown in Scheme 3.41 and 3.42 below.

42 X- MeO OH I+ MeO O NaOH Brx Bry Brz' Brx Bry Dioxane/H2O

HO O BBr3 Brx Bry CH2Cl2

Entry3-Methoxyphenol Salt (Bry, Brz') X Product after demethylation Abbreviation 1 MeO OH 2,2',4,4'- Cl- Br 5'-OH-BDE-25 HO O

Br Br Br 2Br 2,2',4,4'- Cl- Br Br 3-OH-BDE-100 MeO OH HO O

Br Br Br Br Br

3Br 2,2',4,4',5,5'- Cl- Br Br 3-OH-BDE-154 MeO OH HO O

Br Br Br Br Br Br 4MeO OH 2,4,6- TfO- Br 5'-OH-BDE-69 HO O

Br Br Br Br Br 5MeO OH 2,4,6- TfO- Br Br 3-OH-BDE-155 HO O Br Br Br Br Br Br

Scheme 3.41

Scheme 3.42

The brominated 3-methoxyphenols shown in Scheme 3.41 were O-arylated using the conditions previously described by Marsh et al .192 i.e . by adding the brominated diphenyliodonium salt to a solution of sodium hydroxide in water and dioxane and stirring for 1-1.5 hours at 80 ºC. Subsequent demethylation of the isolated MeO-PBDEs using borontribromide yielded the corresponding OH-PBDEs. In entries 6, 7, and 8 in Scheme 3.42 we chose to use hydroxybenzaldehydes instead of methoxyphenols since our attempts to arylate 3-methoxyphenol and 3-bromo-5-methoxyphenol with symmetrical brominated diphenyliodonium salts gave poor yields and formation of non-separable by-products, most likely from radical side reactions. 5’-OH-BDE-25, 5’-OH-BDE-69, 3’-OH-BDE-29, 3’-OH-BDE-30 and 5’-OH-BDE-67 (cf. Scheme 3.41 and 3.42, entries 1, 4, 6, 7 and 8) were further selectively di-ortho brominated relative to the hydroxy group according to Scheme 3.43, using tert.-butylamine ( t-BuNH 2) and bromine similarly to the mono-ortho -brominations for simple phenols reported by Pearson et al. 195

Scheme 3.43

In paper II one ortho - and one para-MeO-PBDE were successfully prepared using the optimised conditions reported for non-functionalised PBDE synthesis (cf. Scheme 3.24 and 3.25) to investigate if the same method could be used in syntheses of functionalised PBDEs as shown in Scheme 3.44.

45 6 OH O 1.) t-BuOK, THF 5 R R 1 Br - y 2.) TfO 2 4 + 6 I 5 3 1 Bry 2 4 3 1h, 70°C

entry phenolsalt (y) product name yield

1 OMe 2,4,5- OMe 2'-MeO-BDE-74 52 % OH O Br

Br Br Br Br

2 Br 2,4,6-Br Br 4'-MeO.BDE-103 33 % OH O

MeO MeO Br Br Br Br Scheme 3.44

3.2.3 Synthesis of di-functionalised PBDEs Little has been reported regarding synthesis of di-functionalised PBDEs except for a few dihydroxylated diBDEs.196,197

In Paper III a multi-step synthesis to achieve two specific di-functionalised octaBDEs is reported. Our approach was to first synthesise phenyl(2,3,4,5- tetrabromo-6-methoxyphenyl)iodonium triflate, an unsymmetrical methoxy- functionalised brominated diphenyliodonium salt. The synthesis is outlined in Scheme 3.45 and a synthetic description is given below.

The commercially available precursor 2-nitrophenol ( 1) was selectively ortho/para brominated using BTMA-Br 3 and calcium carbonate in dichloromethane as described by Kajigaeshi et al. 198 . This gave 2,4-dibromo- 6-nitrophenol (2) in an almost quantitative yield (Scheme 3.45, Step 1). Subsequent quantitative O-methylation of the phenol was performed using methyl iodide in a solution of tetrabutylammonium hydroxide ((Bu) 4NOH) and sodium hydroxide in a two-phase system of water and dichloromethane (Scheme 3.45, Step 2). The product, 1,5-dibromo-2-methoxy-3-nitrobenzene (3) was first synthesised using an alternative route, where the first step was bromination of 2-nitrophenol using calcium dibromide (CaBr 2) in acetonitrile according to Kumar et al. 199 This reaction was followed by methylation with iodomethane and potassium carbonate in acetone similarly to conditions reported by Endo et al.200 However, this approach gave poor yields and was discarded. Compound 3 was then reduced to the corresponding aniline, 3,5-dibromo-2-methoxyaniline ( 4) in an acceptable yield, using tin chloride dihydrate. Compound 4 was perbrominated using N- bromosuccinimide and trifluoromethanesulfonic acid in acetonitrile to give 2,3,4,5-tetrabromo-6-methoxyaniline ( 5) similar to previously reported

46 bromination of phenols and anisoles. 201 Diazotisation of 5 and a subsequent Sandmeyer type iodination similarly to Ek et al.202 afforded 1,2,3,4- tetrabromo-5-iodo-6-methoxybenzene ( 7), which could be used in the preparation of the novel phenyl(2,3,4,5-tetrabromo-6- methoxyphenyl)iodonium triflate using conditions similar to previously reported for unsymmetrical brominated diphenyliodoniumsalts (Paper II). The over-all yield in this first part of the total synthesis was calculated to 29,5 %.

Scheme 3.45

The second part of the total synthesis was to synthesise two specific brominated phenols to be O-arylated with the novel phenyl(2,3,4,5- tetrabromo-6-methoxyphenyl)iodonium triflate. 2,3,4,5-Tetrabromo-6- methoxyphenol was synthesised by perbromination of 2-methoxyphenol using bromine and calcium carbonate (Scheme 3.46). 192 2,3,4,5-Tetrabromo- 6-ethoxyphenol was prepared accordingly starting from 2-ethoxyphenol. These reactions was solvent free and mixing was achieved through submersion in an ultra-sonic bath. 192

Scheme 3.46

Finally, the two phenols were O-arylated with the novel diaryliodonium triflate using conditions similar to those reported by Jalalian et al. 186 and those reported for PBDE syntheses in Paper II, to give 6,6’-dimethoxy- 2,2’,3,3’,4,4’,5,5’-octabromodiphenyl ether (6,6’-diMeO-BDE-194) and 6- ethoxy-6’-methoxy-2,2’,3,3’,4,4’,5,5’-octabromodiphenyl ether (6-EtO-6’- MeO-BDE-194) as shown in Scheme 3.47.

OH OMe OMe Br OMe Br O Br 1.) t-BuOK, THF 2.) Diaryliodoniumtriflate (7) Br Br Br Br Br Br Br Br Br 9 6,6'-diMeO-BDE194, 34% step 8

OH OMe OEt Br OEt Br O Br 1.) t-BuOK, THF 2.) Diaryliodoniumtriflate (7) Br Br Br Br Br Br Br Br Br 11 6-EtO-6'-MeO-BDE194, 29% Scheme 3.47

48 4 Identification and quantification of selected organobromines

This chapter covers the identification of OH-PBDEs, likely to originate from PBDE metabolism, in children working at a waste disposal site in Managua, Nicaragua (Paper I) and quantification of the naturally formed di- functionalised PBDE analogue 6-OH-6’-MeO-BDE194 in blue mussels from the Swedish west coast (Paper III). Chapter 4 also covers identification and quantification of individual PBDE congeners and other BFRs in dust samples at a Swedish smelter facility for printed circuit boards originating from electronic devices (Paper IV). The facility, Rönnskärsverken, has the capacity to process up to 120 000 tonnes of material each year. Also blood samples from potentially exposed employees were analysed for BFRs (Paper IV).

4.1 Identification of OH-PBDEs as metabolites in human blood samples The intention with Paper I was to identify novel OH-PBDE congeners present in human blood plasma. The serum sample used was from children working at a waste disposal site near Lake Managua, Nicaragua, in 2002. Athanasiadou and co-workers analysed the sample for PBDEs and those few OH-PBDE for which standards were available at that time.102 These children were actively working at the dump site and living at it, or close by, in an environment highly contaminated by PBDEs. The reported levels of PBDEs were some of the highest ever reported in human blood with the sum of BDE-28, BDE-47, BDE-66, BDE-85, BDE-99, BDE-100, BDE-153, BDE- 154, BDE-183 and BDE-209 in concentrations of up to 1,250 pmol/g fat.102 Further, 4’-OH-BDE-17, 6-OH-BDE-47, 3-OH-BDE-47, 4’-OH-BDE-49, 4- OH-BDE-42 and 4-OH-BDE-90 were identified and also quantified in serum from the children.102

The selection of which OH-PBDEs to look for was based on a study by Bergman et al.203 who used and matched mass spectra and retention times of detected OH-PBDEs (as methyl derivatives) in human blood samples (the pooled sample later used in Paper I) with mass spectra and retention times of detected OH-PBDEs (as methyl derivatives) from a rat metabolite study of major environmental PBDE congeners (BDE-47, -99, -100, -153, -154, -183 and -209) 110 . The degree of bromination and the position of the hydroxy functionality on the metabolically formed OH-PBDE congeners had been determined by full scan GC–EIMS by Malmberg et al .110 . Using this information and assuming that the metabolism of PBDEs is comparable

49 between humans and rats, we decided to analyse meta -OH-pentaBDEs, meta -OH-hexaBDEs and one para -OH-heptaBDE.

Due to a lack of commercially available standards, nine congener specific OH- and MeO-PBDEs were synthesised as described in Chapter 3 and reported in Paper I. This opened up for an extended identification analysis for additional OH-PBDEs, as their methylated derivatives (MeO-PBDEs), in the serum samples from the Nicaraguan children. A secondary objective was to conﬁrm the identity of the six OH-PBDEs previously reported as by Athanasiadou et al. 102 .

As reported in Paper I, a total of 14 OH-PBDEs were found to be present in the blood samples. Seven out of nine selected, and in-house synthesised meta-OH-PBDEs, were identified for the first time in human blood. In addition one para -OH-BDE was identified in human blood serum for the first time, by comparison to a commercially available authentic reference standard. Furthermore, it was possible to confirm the presence of the six OH-PBDEs previously reported by Athanasiadou et al. 102 . The fraction of the sample containing HPCs was methylated and the MeO-PBDEs were analysed by gas chromatography mass spectrometry (GCMS)-electron capture negative ionisation (ECNI) running in selected ion monitoring mode (SIM), scanning for bromine ( m/z 79, 81). One of the chromatograms recorded is presented in Figure 4.1 and a complete list of all identified OH- PBDE congeners, presented in order of elution, is given in Table 4.1.

Abundance 20000 10000 2000 1000 1500 500 0 0 human blood sample and standard mix. mix. standard and sample blood human 4.1 Figure 05 10 15 20 25 30 35 40 45 50 1 15.00 14.50 14.00 13.50 13.00 12.50 12.00 11.50 11.00 10.50 vraig hoaorm o mtyae HC fractio HPC methylated of chromatograms Overlaying

4’-MeO-BDE-17

6-MeO-BDE-47

3-MeO-BDE-47 4’-MeO-BDE-49

4-MeO-BDE-42

3’-MeO-BDE-100 and 3-MeO-BDE-100 3’-MeO-BDE-99 3-MeO-BDE-99 4-MeO-BDE-90 4’-MeO-BDE-101

3-MeO-BDE-155

3’-MeO-BDE-154 3-MeO-BDE-154 n of of n 3-MeO-BDE-153 DB-5column Plasma sample DB-5column Standard mix

4-MeO-BDE-187 5.50

Time

Table 4.1 OH-PBDE congeners detected, as their methyl ether derivatives, in human blood serum.

Diphenyl ether substitution Abbreviation 4'-Methoxy-2,2',4-triBDE 4'-MeO-BDE-17 6-Methoxy-2,2',4,4'-tetraBDE 6-MeO-BDE-47 3-Methoxy-2,2',4,4'-tetraBDE 3-MeO-BDE-47 4'-Methoxy-2,2',4,5'-tetraBDE 4'-MeO-BDE-49 4-Methoxy-2,2',3,4'-tetraBDE 4-MeO-BDE-42 3-Methoxy-2,2',4,4',6-pentaBDE 3-MeO-BDE-100 3'-Methoxy-2,2',4,4',6-pentaBDE 3'-MeO-BDE-100 3-Methoxy-2,2',4,4',5-pentaBDE 3-MeO-BDE-99 4-Methoxy-2,2',3,4',5-pentaBDE 4-MeO-BDE-90 4'-Methoxy-2,2',4,5,5'-pentaBDE 4'-MeO-BDE-101 3-Methoxy-2,2',4,4',5,6'-hexaBDE 3-MeO-BDE-154 3'-Methoxy-2,2',4,4',5,6'-hexaBDE 3'-MeO-BDE-154 3-Methoxy-2,2',4,4',5,5'-hexaBDE 3-MeO-BDE-153 4-Methoxy-2,2',3,4',5,5',6-heptaBDE 4-MeO-BDE-187

4.2 Identification and quantification of a phenolic di-functionalised PBDE in blue mussels As discussed in Chapter 2, naturally occurring OH-PBDEs tend to carry the hydroxy group in an ortho position relative to the diphenyl ether linkage. The same rule of thumb seems to hold true regarding di-functionalised PBDE analogues. Natural products of the type o,o ’-diOH-PBDEs, have previously been reported in e.g. marine sponges.144,204,205 In a recent study by Löfstrand et al. 206 on brominated phenols, anisoles and dioxins in blue mussels ( Mytilus edulis ), from the Baltic Sea, several unknown compounds were tentatively assigned as either diOH-PBDEs or OH-MeO-PBDEs based on fragmentation analysis of electron capture negative ionisation mass spectroscopy (ECNI-MS) of the methylated phenolic constituents extracted from mussel samples. The most abundant of the unknown compounds was thus tentatively identified as a phenolic derivative of a diMeO-octaBDE. Due to the lack of standards, no further conclusions were drawn at the time. In the planning of Paper III it was hypothesised that this diMeO-octaBDE originated as either 6,6’-dihydroxy-2,2’,3,3’,4,4’,5,5’-octabromodiphenyl ether (6,6’-diOH-BDE-194) and/or 6-hydroxy-6’-methoxy- 2,2’,3,3’,4,4’,5,5’-octabromodiphenyl ether (6-OH-6’-MeO-BDE-194) in the non-derivatised mussel samples.

52 OH OH OH OMe Br O Br Br O Br and/or Br Br Br Br Br Br Br Br Br Br Br Br 6,6'-diOH-BDE194 ? 6-OH-6'-MeO-BDE194 Figure 4.2 Two hypothesised di-functionalised PBDEs in blue mussels.

As reported in Chapter 3 and Paper III, 6,6’-diMeO-BDE-194 and 6-EtO-6’- MeO-BDE-194, were successfully synthesised. Further, in Paper III these synthesised standards were used to reveal the true identity of the di- functionalised octaBDE not only in the original sample acquired from Löfstrand et al. 206 , but also in recent blue mussels, sampled 2012 from the locations Fladen and Väderöarna along the Swedish West coast.

For qualitative identification of the di-functionalised octaBDE, the samples were run on a GCMS operating in ECNI full scan mode, comparing both retention times as well as fragmentation patterns between the sample after methylation of the fraction containing HPCs and the synthesised 6,6’- diMeO-BDE-194 standard (Figure 4.3). The methylated analyte was also quantified versus a standard curve of 6,6’-diMeO-BDE-194, based on its 2,3,4,5-tetrabromo-6-methoxyphenolate fragment ions (SIM, m/z 436.6, 438.6). The mean concentrations differed significantly between the mussels from the two sampling locations. The Fladen mussels contained a mean concentration of 4200 ng/g fat (± 2300) while the Väderöarna mussels contained 430 ng/g fat (± 120).

The structural verification was performed by comparing the retention time and GCMS electron ionisation (EI) full scan spectra of derivatives after ethylation of HPCs present in the appropriate phenolic fraction of a pooled mussel sample from Fladen, with the synthesised 6-EtO-6’-MeO-BDE-194 authentic reference standard. The retention times and fragmentation pattern, in which the same molecular ion (M + = 868) could be observed, matched between the pooled sample and the standard. Based on these results, the conclusion was drawn that the identity of the assumed di-functionalised octaBDE is 6-OH-6’-MeO-BDE-194 (Figure 4.5).

Figure 4.3 Overlaying chromatograms of methylated HPC fraction and 6,6’- diMeO-BDE-194 standard.

Figure 4.5 Structure of the identified di-functionalised PBDE analogue.

4.3 Identification of BFRs in E-waste dust In Paper IV, dust collected at the Swedish smelter, Rönnskärsverken, or more specifically at the test facility, was analysed for PBDEs and other BFRs, among which hexabromobenzene (HBB), 1,2-bis(2,4,6- tribromophenoxy)ethane (BTBPE), decabromobiphenyl (BB-209), decabromodiphenyl ethane (DBDPE), 2,4,6-tribromophenol (2,4,6-TBP) and TBBPA were found present. The facility is designed for cupper and lead production through recycling of electronic waste, e.g. from printed circuit boards. Before the smelting process, scrap electronics are shredded, and the fragmented material is transported and handled with large loaders and on conveyors, activities that will lead to distribution of dust that spreads throughout the facility and surroundings. Settled dust from 3 different locations inside the facility was sampled and subsequently extracted and processed using methods similar to those previously reported by Dodson et al. 207 in a study on house hold dust. The PBDE congeners and BFRs were identified using GCMS operating in ECNI mode, comparing retention times with commercially available standards. The results of the identification and subsequent quantification are presented in Table 1 (Paper IV).

54 It was obvious that BDE-209 and TBBPA were the two major BFRs in the collected dust samples. As mentioned in Chapter 1, DecaBDE and TBBPA are two of the most extensively used BFRs today, which explains the high concentrations in the analysed dust. HBCDDs, also discussed in Chapter 1, were not observed. This is not surprising since HBCDDs are mainly used in insulation materials and textiles. 30

Comparing the mean concentrations of BDE-209 with other studies from e- waste recycling sites in e.g. Thailand 208 and China 209,210 reveals that the dust from our study contains approximately 6-100 times more BDE-209. This may be explained by the difference in the type of e-waste processed and the higher throughput capacity of Rönnskärsverken with of up to 120.000 tonnes per year. Nevertheless, it is evident that the dust is a source for potential human exposure to BFRs unless not properly protected (cf. 4.4 below). The dust containing BFRs is a source of environmental contamination in the area of the facility. Since the plant store and handle the fragmented electronic scrap mainly indoors the discharges are limited to some extent. This was not the case in the late 1990’s when all the scrap was stored outdoors. 211 Thuresson et al. 211 concluded that the outdoor storage was a risk for contamination of this part of the Gulf of Bothnia. Unfortunately, no screening of BFR levels in biota from waters close to the smelter facility were done at that time, as suggested by the authors. This would have been of value for assessing the present contamination situation and a chance to see changes in the BFR contamination situation in biota from the gulf, today.

4.4 BFRs in blood from personnel at a Swedish smelter facility The potential occupational exposure to BFRs for workers at the above discussed cupper smelter facility was investigated and the results are presented in Paper IV. The exposure of personnel at the smelter test facility, expected to be the most exposed individuals, was compared to exposure in a control group (Paper IV). Blood samples from workers were collected and analysed for BFRs to see if any elevated concentrations were to be found among the workers. This is a study well in line with a number of previous studies where workplace exposures to BFRs have been studied by Bergman and coworkers.97,98,103,211,212 Among these previous studies is also one in which the exposure situation at the Swedish smelter facility was assessed around the turn of the century. 211 This study (Paper IV) is valuable for allowing the comparison of the internal exposure in the workers at the plant.

Extraction and cleanup procedures applied in Paper IV were similar to those reported by Hovander et al. 213 and the neutral and phenolic fractions of the samples were analysed using GCMS operating in ECNI mode, comparing

55 retention times with commercially available standards. Only a few congeners were quantified since the concentrations in the blood samples were generally low and in many cases under the limit of quantification. The most abundant congeners in the neutral fraction were BDE-28, BDE-47 and BDE-153 (all in the low pmol g -1 fat range) while the BDE-209, shown to be abundant in the dust at the facility, was below LOD. None of the other neutral FRs present in the dust, i.e. HBB, BTBPE, BB-209, and DBDPE could be detected in the samples.

In the fraction in which halogenated phenols were isolated, 2,4,6-TBP was found in concentrations of 6.1 pmol g -1 fat, similar to 6-OH-BDE-47 (6.0 pmol g -1 fat). However, the most abundant polybrominated phenol was 2,3,4,6-tetrabromophenol (2,3,4,6-TEBP), with a mean serum concentration of 50 pmol g -1 fat. This is a very interesting and novel result, only once reported before but then in fact in subjects handling e-waste. 214 It is not clear from where 2,3,4,6-TEBP originates, but the serum concentrations are so high that further studies must be undertaken. The 2,3,4,6-TEBP occurrence is particularly interesting when compared to TBBPA, that was not found in the serum from the workers in this study, even though dust contained high concentrations of TBBPA (cf. Chapter 4.3 and Paper IV).

It was concluded that the studied workers at Rönnskärsverken must be well protected against occupational exposure since the concentration of BFRs in their blood serum were similar of those in the control group. These results points towards that the measures taken at this specific facility are sufficient for the workers handling the electronic waste, which is not the case at many other facilities in e.g. China 99-101 were it can be concluded that the workers are not protected from BFR exposures.

56 5 Definitions and novel abbreviations of flame retardants.

A common problem in literature from toxicology-related sciences (including environmental chemistry) is the lack of coordination of abbreviations of environmental contaminants, in general. Luckily there is much of agreement in abbreviations of established pollutants, such as DDT, PCB, PCDDs etc., known as persistent organic pollutants listed by the Stockholm convention. The problem becomes evident as we go into the groups of emerging, novel and potential pollutants (cf. below). In these latter cases, abbreviations are put forward as letter combinations without structured plans thereof. The abbreviations may be a mix of IUPAC defined nomenclature, common names, commercial trade names and even personal abbreviations, invented on the fly. During a conference on brominated flame retardants (BFR-2010) in Kyoto, Japan, this issue was raised and the symposium delegates supported a recommendation that the BFR symposium board should seriously look into the task of bringing order to the present “anarchy”. The author of this thesis, being an environmental chemist, was invited by the board to develop an abbreviation standard for first and foremost BFRs. The result of this work is included in this thesis as Paper V.

5.1 Overarching definitions for flame retardants In Paper V, the issue of definitions of “established”, “emerging”, “novel” and “potential” FRs is addressed. This is however an issue of any class of environmentally relevant chemical, being a FR or not. Since “emerging” is used frequently in connection to both environmental pollutants and issues, there is a need for clarifications. While the status of a chemical may be defined as “established, emerging, novel or potential”, an emerging issue may differ from an emerging chemical depending on where the issue is raised, e.g. if raised in certain region with heavy contamination of arsenic, or regions/countries with recent use of e.g. hexachlorobenzene or endosulfan. The proposed definitions of the “established, emerging, novel or potential” are given below.

Established FRs are defined as chemicals known for their use as flame retardants, known to occur in the environment, in wildlife and humans and for which their (eco-)toxicity are well known. Extensive research is undertaken on this group of BFRs, and a large scientific database is available.

57 Emerging FRs are defined as chemicals which are applied as flame retardants that have been identified as anthropogenic chemicals in any of the environmental compartments, in wildlife, food or in humans. The databases for emerging FRs are in general poor but may in some cases be somewhat more extensive but always have obvious data gaps.

Novel FRs are defined as chemicals applied as flame retardants which have been confirmed to be present in materials or goods in concentrations above 0.1 %.

Potential FRs are defined as chemicals being suggested for applications as flame retardant for which there is no evidence that they are used for this purpose yet, e.g. known from patents.

5.2 Overarching abbreviations for flame retardants In Paper V, the main issues are related to the development of a chemically based and structured abbreviation strategy of FRs. One common present issue is the lack of information of the actual structures given in abbreviations proposed today. As mentioned in Chapter 2 this is not a problem for e.g. PBDEs and similar substituted diphenyl ethers or biphenyls where the congener numbering system was suggested a long time ago, by Ballschmiter et al .54,55 , a system well adopted nowadays. A common problem today is that some abbreviations are used for multiple chemicals or that one chemical is abbreviated in multiple ways.

In Paper V, we suggest that brominated, chlorinated and phosphorus containing FRs are abbreviated as BFRs, CFRs and PFRs, respectively. Further, these chemicals are abbreviated by following a set of rules that gives information on chemical structure and functional group content. This level in the strategy for finding proper abbreviations is suggested as, structured abbreviations (STABs). For further simplification we suggest practical abbreviations (PRABs) that are derived from above mentioned STABs. These are exemplified in Figure 5.1 using HBCDD as an example. As stated in Chapter 1 hexabromocyclododecane is also commonly abbreviated “HBCD”. This might be perceived as confusing as one could possibly mean hexabromocyclodecane (Figure 5.1).

Br Means Br Hx Hexa B Bromo Br c cyclo D Decane Br6 Br Br DD Dodecane Br Hexabromocyclododecane Hexabromocyclodecane STAB: HxBcDD STAB: HxBcD PRAB: HBCDD PRAB: HBCD Figure 5.1 Example of STABs and PRABs for two different organobromine compounds.

For more detailed information about the abbreviation system the author refers to Paper V. The paper was presented at a follow up session at BFR2013 in San Francisco, USA and was well received by participants in the field of environmental chemistry, and all other toxicology-related sciences present at the meeting.

59 6 Future perspectives

PBDEs are still, as presented in this thesis and as shown in Paper IV, present in materials, dust and biological samples and will be so for many years to come. Methods which will allow synthesis of all 209 PBDE congeners are in theory available, although they are not all published, and all 209 congeners can be bought as authentic reference standards from commercial sources. Even so, there is a need for researchers to obtain larger quantities of specific congeners for purposes other than analytical identifications such as toxicological studies, reactivity assessments and metabolism studies. The same holds true for OH- and MeO-PBDEs, where only a fraction of all theoretical congeners have yet been synthesised and with only a few of these being commercially available. In this thesis and in Paper II, a quick, efficient and novel approach to achieve congener specific PBDEs in high yields has been presented which opens up for researchers to prepare their own in house synthesised standards. In Paper I we show that OH-PBDEs are formed as metabolites in PBDE exposed humans, and in Paper III we prove the existence of a previously unidentified natural di-functionalised PBDE. The knowledge on these functionalised PBDEs is limited and our applied methods open up for further exploration of the synthesis of possible PBDE metabolites. In Paper I, unidentified peaks were observed in the pooled human blood samples, or more specifically in the fraction containing all HPCs, which could correspond to OH-PBDEs. However, since no authentic reference standards are available this calls for further syntheses and compound characterisation.

The presence of naturally occurring mono and di-functionalised PBDEs in wildlife samples motivates continued efforts to synthesise natural product as reference compounds for use in subsequent analysis. In the study by Löfstrand et al. 206 , which motivated the work in Paper III, several unknown and yet unidentified phenolic organobromine compounds could be observed in blue mussels. This is just one study amongst many that points out the lack of authentic standards for natural products.

Paper IV points out an emerging contaminant in human blood, 2,3,4,6- TEBP. Very little is known about its origin and the toxicological implication of the compound is not known. Since there are reasons to believe that this compound is an endocrine disrupting chemical 125 , further toxicological/endocrinological studies are required.

Paper V provides, not only suggested definitions of FR statuses and abbreviations of FRs, but also an overview of established compounds that has been or still are in use together with novel and potential FRs. In the

60 preparatory research for Paper V we identified a total of 55 BFRs, 18 CFRs and 23 PFRs but there is certainly more to be added to this list. Our proposed system for abbreviations of FRs allows for future additions. Of the 55 BFRs listed in Paper V only 6 matches the criteria to be considered established FRs, which means, well studied in regard of e.g. occurrence and toxicity. Twenty of the BFRs would be defined as emerging and 16 as novel. We know very little about the emerging and novel FRs which, according to the author of this thesis, calls for continued research regarding synthesis of authentic reference standards, reactivity, environmental fate, identification in environmental and biological samples and toxicology. Until we know more about the chemical cocktail that we are exposed to in our daily lives, we should strive for a conservative mindset in the legislation of chemicals and keep reminding decision makers that the so called precautionary principle is always the safest way to go.

It cannot really be stressed enough how important chemical synthesis is, including method development and characterisation of the synthesised compounds. There are so many other classes of chemicals apart from the BFRs that require authentic reference standards. Not the least these compounds are necessary for hazard identification, assessment, improved understanding of exposure of anthropogenic chemicals and their toxicokinetics.

61 7 Svensk sammanfattning Människor i dagens moderna samhälle är omgivna av syntetiska ämnen som i mångt och mycket underlättar våra liv. Allt ifrån det som finns i kläderna vi har på kroppen och rengöringsmedlen vi använder till avhandlingen du håller i din hand just nu utgörs av kemiska ämnen. Trots att många av de kemikalier vi använder är harmlösa så händer det att man upptäcker hälso- och miljörisker av en del, och ofta inte förrän kemikalien är vida spridd i produkter, varor och material och dessutom i vår miljö. Sedan 1960-talet har vi varit väl medvetna om vilka konsekvenser detta kan få. Oavsiktliga utsläpp av ämnen som polyklorerade bifenyler (PCB) och avsiktliga utsläpp av insekticider och pesticider som DDT och Hormoslyr har alla satt sina spår i vår omgivning. Östersjön har varit hårt drabbat av PCB och DDT, föroreningar som redan under 1960-talet orsakade en kraftig minskning av sälar och rovfåglar.

Polybromerade difenyletrar (PBDE:er), i form av tekniska blandningar såsom OktaBDE, NonaBDE och DekaBDE, har sedan 1970-talet använts som flammskyddsmedel i allt från möbelstoppning, textilier till hemelektronik. Denna klass av kemikalier liknar på många sätt PCB, framförallt genom att båda är halogenerade och har liknande kemiska strukturer.

Det är idag välkänt att PBDE:er är vitt spridda i vår miljö och att de ansamlas i fettvävnader i både djur och människor. De kan, som sådana, eller genom omvandling till hydroxylerade metaboliter (OH-PBDE:er), ha en toxisk verkan i biologiska system och orsaka rubbningar i bland annat hormonella system. För att kunna identifiera kemiska substanser i vår miljö samt i biologiska matriser så krävs det syntetiska autentiska referensstandarder. Metoder för substansspecifik syntes av PBDE:er är relativt väl beskrivet i vetenskaplig litteratur medan stora luckor finns att fylla då det gäller syntes av OH- PBDE:er.

I den här avhandlingen presenteras en ny generell metod för PBDE-syntes som snabbt ger rena standarder i höga utbyten. Vidare så presenteras syntes av ej tidigare karaktäriserade OH-PBDE:er och deras metylerade analoger (MeO-PBDE:er) samt de två di-funktionaliserade PBDE-analogerna, di- MeO-BDE-194 och EtOH-MeO-BDE-194.

De funktionaliserade PBDE-anolgerna har sedan använts som referensstandarder för att kunna identifiera PBDE metaboliter i blod från högexponerade barn i Nicaragua samt i jakten på att kunna slå fast identiteten på en naturprodukt som förekommer i blåmusslor från den

62 svenska västkusten. Vidare så presenteras identifiering av bromerade flamskyddsmedel i damm från nedmalt elektronikskrot från ett svensk smältverk såväl som i blodprover från anställda vid smältverket.

Utöver syntes- och analysresultat så framläggs även förslag på ett system för att förkorta de ofta väldigt komplicerade kemiska namnen på etablerade, tilltagande, nya samt potentiella flammskyddsmedel, med syftet att underlätta miljökemisk och övrig (eko-)toxikologisk forskning och vetenskaplig kommunikation.

63 8 Acknowledgements Med risk för att bli långrandig och sentimental. Nu kör vi!

Jag vill börja med att tacka dig Åke för att du fick mig intresserad av miljökemi och fick mig att förstå att man med fördel kan syssla med spännande organisk syntes även här. Jag är väldigt tacksam för allt stöd och för all tid du har lagt ner som har möjliggjort den här avhandlingen.

Göran, min handledare och partner in crime på synteslabbet. Du är en skön snubbe med mycket humor, även om man inte alltid förstår sig på den. Tack för att du har haft förtroende för mig och låtit mig pröva mina vingar när jag har kommit med galna organkemiska idéer och för att du står ut med att det spelas punk på labbet.

Ni som hängt ute i syntes-hoodsen har verkligen förgyllt tillvaron där ute genom åren. Lisa , du har stöttat när det har varit tunga dagar och dig kan man ”vara sig själv” med. Alltid hjälpsam och förstår när det är läge att vara lagom bitter! Maria S , reskamrat i Kina och har fått stå ut med att jag försover mig när vi ska hålla labbkurs men är alltid glad och hjälpsam ändå. Doctor Geezer, thanks for the much needed pub sessions! Johan E , dina tips och råd vid HPLC och omkristallisationer har varit till ovärderlig hjälp. Per , alltid på ”G”, alltid en bra rövarhistoria med efterföljande gapskratt i bakfickan och alltid ett bra bollplank för idéer. Jag gör vågen för dig! Ulrika, dig kan jag inte tacka nog mycket. Tack för allt slit du lagt ner och all pepp du bidragit med, utan din hjälp hade det blivit en tunnare avhandling! Tack också till Freddan och övriga exjobbare på stället genom åren.

Ioannis , tack för allt du har lärt mig om GCMS. Det har varit ett nöje att vårda “Agge” ute på syntes och du har alltid kommit med kloka råd när den har trilskats. Är även tacksam för hjälp med analyser, övrigt nördigt prylsnack och träning vi ägnat oss åt genom åren.

Alla ni på ”andra sidan”, nya som gamla. Ett extra stort tack riktas till Anna S, snacka om att kliva in som en räddare i nöden. Evigt tacksam! Johan F, ställer upp i vått och torrt och hänger alltid med på kiosken för köp av fika. Tack för att du bidrog till en grym vistelse i SFO . Linda L , var på din vakt, den kinesiska grisbollen kan komma flygande när som helst. Dennis , diazometan-kumpan. Hitesh, Hans, AK, Jessica, Cissi, Emelie, mr G, Henrik, Lillemor, Maggan, Birgit och Karin som fortfarande tittar in ibland, ni bidrar alla till god stämning här på miljökemi. Anita, du hör fortfarande ihop med oss, tack för allt du gjort för oss genom åren!

64 Naz-Jay, bästa kurskamraten! Vilka fantasiska år vi har haft. Galna danser, vilda fester och många betapet-matcher. Utan dig hade jag inte fixat grundutbildningen!

Alla mina underbara vänner som finns spridda i landet och världen, det är ni som gör att man orkar!

Till alla i min helt fantastiska och stora familj vill jag rikta ett särskilt stort tack för att ni alltid stöttat mig! Mamma & Pappa , tack för att ni alltid har trott på mig och varit stolta över vad jag har tagit mig för.

Helena , min Bopi! Jag kan inte fatta att jag har haft en sådan tur att jag träffat någon som dig. Ditt stöd och tålamod har betytt allt den här sista intensiva tiden. Och Frans , du har lyckats motivera mig att klara det här trots att du inte ens är född ännu. Snyggt jobbat!

Jag vill även uttrycka min tacksamhet till Ångpanneföreningen och K. A. Wallenbergs Stiftelse för stipendier som möjliggjort spännande resor och konferensdeltagande.

An extra big thank you goes out to all my co-authors in Paper I-V of this thesis. You made this possible!

The Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning ( FORMAS ), is acknowledged for their financial support through the funding of the projects with the following contract numbers: 2009-1297, 2009-1309, 2009-1303 and 2012-2116.

65 9 Appendix A Contribution to Paper I-V

I. Performed the major part of the synthetic and analytical work. Wrote the paper.

II. Performed the major part of the synthetic work and participated in writing the manuscript together with my co-author, Göran Marsh.

III. Performed some of the synthetic work and supervised the synthetic work done by diploma worker Ulrika Winnberg. Participated in the process of proofreading the manuscript.

IV. Performed analytical pilot study. Performed the work up of dust samples. Participated in writing the paper.

V. Participated in the creative process of inventing the abbreviation system together with my co-author, Åke Bergman. Created the tables and took part in proofreading the paper.

66 10 Reference list

1 Jensen, S., Johnels, A.G., Olsson, M., and Otterlind, G., (1969), DDT and PCB in marine animals from Swedish waters, Nature , 224 , 247- 250 2 Cottam, C. and Higgins, E., (1946), DDT and its effect on fish and wildlife, J. Econ. Entomol. , 39 , 44-52 3 Lundholm, C.E., (1997), DDE-induced eggshell thinning in birds: Effects of p,p'-DDE on the calcium and prostaglandin metabolism of eggshell gland, Comp. Biochem. Physiol. Part C , 188 , 113-128 4 Anonymous, (1966), Report of a new chemical hazard, New Scientist , 32 , 612 5 Bergman, A. and Olsson, M., (1985), Pathology of Baltic grey seal and ringed seal females with special reference to adrenocortical hyperplasia: Is environmental pollution the cause of a widely distributed disease syndrome?, Finnish Game Research , 44 , 47-62 6 Helle, E., Olsson, M., and Jensen, S., (1976), PCB levels correlated with pathological changes in seal uteri, Ambio , 5, 261-263 7 Roos, A., Greyerz, E., Olsson, M., and Sandegren, F., (2001), The Otter ( Lutra lutra ) in Sweden – Population trends in relation to DDT and total PCB concentrations during 1968-1998, Environ. Pollut. , 111 , 457-469 8 Aulerich, R.J. and Ringer, R.K., (1977), Current status of PCB toxicity to mink, and effects on their reproduction, Arch. Environ. Contam. Toxicol. , 6, 279-292 9 Jensen, S., Kihlström, J.E., Olsson, M., Lundberg, C., and Örberg, J., (1977), Effects of PCB and DDT on mink ( Mustela vision ) during the reproductive season, Ambio , 6, 239 10 Ringer, R.K., Aulerich, R.J., and Zabik, M., (1972), Effect of dietary polychlorinated biphenyls on growth and reproduction of mink, Prepr. Pap. Nat. Meet. , Div. Water, Air Waste Chem. , Amer. Chem. Soc. , 12 , 149-154 11 Platonow, N.S. and Karstad, L.H., (1973), Dietary effects of polychlorinated biphenyls on mink, Can J Comp Med , 37 , 391-400 12 Kihlström, J.E., Olsson, M., Jensen, S., Johansson, Å., Ahlbom, J., and Bergman, Å., (1992), Effects of PCB and different fractions of PCB on the reproduction of the mink ( Mustela vison ), Ambio , 21 , 563-569 13 Kuratsune, M., Yoshimura, H., Hori, Y., Okumura, M., and Masuda, Y., (1996), Yusho: A human disaster caused by PCBs and related compounds, Eds.: Kuratsune, M. and others, Yyushu University press, Japan

67 14 Reggiani, G. and Bruppacher, R., (1985), Symptoms, signs and findings in humans exposed to PCBs and their derivatives, Environ. Health Perspect. , 60 , 225-232 15 Carter, L., (4-16-1976), Michigan's PBB Incident: Chemical Mix-Up Leads to Disaster, Science , 192 , 240-243 16 Egginton, J., (2009), The Poisoning of Michigan, MICHIGAN ST University Press 17 Helander, B., Bignert, A., and Asplund, L., (2008), Using raptors as environmental sentinels: Monitoring the white-tailed sea eagle Haliaeetus albicilla in Sweden, Ambio , 37 , 425-431 18 EFSA, (2012), Update of the monitoring of levels of dioxins and PCBs in food and feed., The EFSA Journal , 10 , 2832 19 de Wit, C.A. and Strandell, M., (2000), Levels, sources and trends of dioxins and dioxin-like substances in the Swedish environment - The Swedish dioxin survey, Swedish Environmental Protection Agency, Stockholm, Sweden 20 Jensen, S. and Renberg, L., (1972), Contaminants in pentachlorophenol: Chlorinated dioxins and predioxins (chlorinated hydroxy-diphenyl ethers), Ambio , 1, 62-65 21 LaKind, J.S., (1-24-2007), Recent global trends and physiologic origins of dioxins and furans in human milk, J Expos Sci Environ Epidemiol , 17 , 510-524 22 Fång, J., Nyberg, E., Bignert, A., and Bergman, Å., (2013), Temporal trends of polychlorinated dibenzo-p-dioxins and dibenzofurans and dioxin-like polychlorinated biphenyls in mothers' milk from Sweden, 1972-2011, Environ. Int. , 60 , 224-231 23 ECHA, (2011), List of pre-registered substances. Helsinki, Finland, European Chemicals Agency. Available at: http://apps.echa.europa.eu/preregistered/pre-registered-sub.aspx 24 OECD, (10-8-2013), Search in OECD existing chemicals database 2013-10-08, http://webnet.oecd.org/hpv/ui/Search.aspx , OECD 25 WHO, (1997), Environmental health criteria 192. Flame retardants: A general introduction, International program on chemical safety, World Health Organization, Geneva, Switzerland 26 Blum, A., Gold, M.D., Ames, B.N., Kenyon, C., Jones, F.R., Hett, E.A., Dougherty, R.C., Horning, E.C., Dzidic, I., Carroll, D.I., Stillwell, R.N., and Thenot, J.-P., (1978), Children adsorb tris-BP flame retardant from sleepwear: urine contains the mutagenic metabolite, 2,3-dibromopropanol, Science , 201 , 1020-1023

68 27 Pijnenburg, A.M.C.M., Everts, J.W., de Boer, J., and Boon, J.P., (1995), Polybrominated biphenyl and diphenylether flame retardants: analysis, toxicity, and environmental occurrence, Reviews of Environmental Contamination and Toxicology , Springer-Verlag 28 EFSA, (2010), Scientific opinion on polybrominated biphenyls (PBBs) in food, EFSA Journal , 8, 1789 29 EFSA, (2011), Scientific opinion on polybrominated diphenyl ethers (PBDEs) in Food, EFSA Journal , 8, 1829 30 EFSA, (2011), Scientific opinion on hexabromocyclododecanes (HBCDDs) in Food, EFSA Journal , 9, 2296 31 EFSA, (2011), Scientific opinion on tetrabromobisphenol A (TBBPA) and its derivatives in food., EFSA Journal , 9, 2477 32 EFSA, (2012), Scientific opinion on emerging and novel brominated flame retardants (BFRs) in food., The EFSA Journal , 10 , 2908 33 McKinnie, Bonnie G., (1987), Process for tetrabromobisphenol A manufacture, 86-880598 34 Hakk, H., (2001), A survey of tetrabromobisphenol A, The Second International Workshop on Brominated Flame Retardants, BFR 2001 , 5-14- 0010, Stockholm, Sweden, 27-30 35 Arias, P., (2001), Brominated flame retardants - An overview, The Second International Workshop on Brominated Flame Retardants, BFR 2001 , 5-14-0010, Stockholm, Sweden, 17-20 36 Covaci, A., Harrad, S., Abdallah, M.A.E., Ali, N., Law, R.J., Herzke, D., and de Wit, C.A., (2011), Novel brominated flame retardants: A review of their analysis, environmental fate and behaviour, Environ. Int. , 37 , 532-556 37 Van der Ven, L.T.M., van de Kuil, T., Verhoef, A., Verwer, C.M., Lilienthal, H., Leonards, P.E.G., Schauer, U.M.D., Canton, R.F., Litens, S., De Jong, F.H., Visser, T.J., Dekant, W., Stern, N., Håkansson, H., Slob, W., van den Berg, M., Vos, J.G., and Piersma, A.H., (2008), Endocrine effects of tetrabromobisphenol-A (TBBPA) in Wistar rats as tested in a one-generation reproduction study and a subacute toxicity study, Toxicol. , 245 , 76-89 38 Law, R.J., Kohler, M., Heeb, N.V., Gerecke, A.C., Schmid, P., Voorspoels, S., Covaci, A., Becher, G., Janak, K., and Thomsen, C., (2005), Hexabromocyclododecane challenges scientists and regulators, Environ. Sci. Technol. , 39 , 281A-287A 39 Morf, L.S., Buser, A.M., Taverna, R., Bader, H.P., and Scheidegger, R., (5-28-2008), Dynamic substance flow analysis as a valuable risk evaluation tool - A case study for brominated flame retardants as an example of potential endocrine disrupters, CHIMIA International Journal for Chemistry , 62 , 424-431

69 40 Abdallah, M.A.-E., Harrad, S., and Covaci, A., (8-7-2008), Hexabromocyclododecanes and tetrabromobisphenol-A in indoor air and dust in Birmingham, UK: Implications for human exposure, Environ. Sci. Technol , 42 , 6855-6861 41 POPRC, (2012), Recommendation of the POPRC on hexabromocyclododecane, http://chm.pops.int/ 42 Arias, P., (1992), Brominated diphenyloxides as flame retardants - Bromine based chemicals, consultant report to the OECD, Paris 43 He, J., Robrock, K.R., and Alvarez-Cohen, L., (2006), Microbial reductive debromination of polybrominated diphenyl ethers (PBDEs), Environ. Sci. Technol. , 40 , 4429-4434 44 Stapleton, H.M., Brazil, B., Holbrook, D.R., Mitchelmore, C.L., Benedict, R., Konstantinov, A., and Potter, D., (2006), In vivo and in vitro debromination of decabromodiphenyl ether (BDE 209) by juvenile rainbow trout and common carp, Environ. Sci. Technol. , 40 , 4653-4658 45 Kierkegaard, A., Asplund, L., de Wit, C.A., McLachlan, M.S., Thomas, G.O., Sweetman, A.J., and Jones, K.C., (2007), Fate of higher brominated PBDEs in lactating cows, Environ. Sci. Technol. , 41 , 417-423 46 La Guardia, M.J., Hale, R.C., and Harvey, E., (2007), Evidence of debromination of decabromodiphenyl ether (BDE-209) in biota from a wastewater receiving stream, Environ. Sci. Technol. , 41 , 6663-6670 47 Stapleton, H.M. and Dodder, N.G., (2008), Photodegradation of decabromodiphenyl ether in house dust by natural sunlight, Environ. Toxicol. Chem. , 27 , 306-312 48 Alcock, R.E. and Busby, J., (4-1-2006), Risk migration and Scientific advance: The case of flame-retardant compounds, Risk Analysis , 26 , 369-381 49 The Court of Justice of the European Communities, (4-1-2008), Directive 2002/95/EC– Electrical and electronic equipment – Restriction of use of certain hazardous substances – Decabromodiphenyl ether ('DecaBDE') – Commission Decision 2005/717/EC – Exemption of DecaBDE from the prohibition on use – Actions for annulment – Commission's implementing powers – Infringement of the enabling provision, http://curia.europa.eu/en/transitpage.htm 50 Cox, P. and Efthymiou, P., (2003), Directive 2003/11/EC of the European parliament and of the council of February 6 2003 amending for the 24th time Council Directive 76/669/EEC relating to restrictions on the marketing and use of certain dangerous substances and preparations (pentabromodiphenyl ether, octabromodiphenyl ether), Official Journal of the European Union , OJ L 42 , 45-46

70 51 Birnbaum, L.S. and Staskal, D.F., (2004), Brominated flame retardants: Cause for concern?, Environ. Health Perspect. , 112 , 9-17 52 Sjödin, A., Patterson, D.G.Jr., and Bergman, Å., (2003), A review on human exposure to brominated flame retardants (BFRs) - particularly polybrominated diphenyl ethers (PBDEs), Environ. Int. , 29 , 829-839 53 Eriksson, J., Eriksson, L., and Jakobsson, E., (1999), Decabromodiphenyl ether, Acta Cryst. , C55 , 2169-2171 54 Ballschmiter, K. and Zell, M., (1980), Analysis of polychlorinated biphenyls (PCB) by glass capillary gas chromatography. Composition of technical aroclor- and clophen-PCB mixtures, Fresenius Z. Anal. Chem. , 302 , 20-31 55 Ballschmiter, K., Bacher, R., Mennel, A., Fischer, R., Riehle, U., and Swerev, M., (4-1-1992), The determination of chlorinated biphenyls, chlorinated dibenzodioxins, and chlorinated dibenzofurans by GC-MS, J. High Resol. Chromatogr. , 15 , 260-270 56 Braekevelt, E., Tittlemier, S.A., and Tomy, G.T., (2003), Direct measurement of octanol-water partition coefficients of some environmentally relevant brominated diphenyl ether congeners, Chemosphere , 51 , 563-567 57 Nyholm, J.R., Norman, A., Norrgren, L., Haglund, P., and Andersson, P.L., (2009), Uptake and biotransformation of structurally diverse brominated flame retardants in zebrafish (Danio rerio) after dietary exposure, Environ. Toxicol. Chem. , 28 , 1035-1042 58 Tittlemier, S.A., Halldorson, T., Stern, G.A., and Tomy, G.T., (2002), Vapor pressures, aqueous solubilities, and Henry's law constants of some brominated flame retardants, Environ. Toxicol. Chem. , 21 , 1804-1810 59 Hardy, M.L., (2002), A comparison of the properties of the major commercial PBDPO/PBDE product to those of major PBB and PCB products, Chemosphere , 46 , 717-728 60 Hale, R.C., La Guardia, M.J., Harvey, E., Gaylor, M.O., and Mainor, T.M., (2006), Brominated flame retardant concentrations and trends in abiotic media, Chemosphere , 64 , 181-186 61 Rahm, S., Green, N., Norrgran, J., and Bergman, Å., (2005), Hydrolysis of environmental contaminants as an experimental tool for indication of their persistency, Environ. Sci. Technol. , 39 , 3128-3133 62 Bastos, P.M., Eriksson, J., Vidarson, J., and Bergman, A., (2008), Oxidative transformation of polybrominated diphenyl ether congeners (PBDEs) and of hydroxylated PBDEs (OH-PBDEs), Environ. Sci. Pollut. Res. , 15 , 606-613

71 63 Eriksson, J., Marsh, G., and Bergman, Å., (2001), Photo decomposition of brominated diphenyl ethers in methanol/water, The Second International Workshop on Brominated Flame Retardants, BFR 2001 , Stockholm, Sweden, 203-206 64 Sellström, U., Söderström, G., de Wit, C., and Tysklind, M., (1998), Photolytic debromination of decabromodiphenyl ether (DeBDE), Organohalogen Compounds , 35 , Stockholm, Sweden, 447-450

65 Watanabe, I. and Tatsukawa, R., (1987), Formation of brominated dibenzofurans from the photolysis of flame retardant decabromobiphenyl ether in hexane solution by UV and sun light, Bull. Environ. Contam. Toxicol. , 39 , 953-959 66 Ohta, S., Nishimura, H., Nakao, T., Aozasa, O., and Miyata, H., (2001), Characterization of the photolysis of decabromodiphenyl ether and the levels of PBDEs as its photoproducts in atmospheric air of Japan, Organohalogen Compounds , 52 , Gyeongju, Korea, 321-324 67 Gerecke, A.C., Hartmann, P.C., Heeb, N.V., Kohler, H.P., Giger, W., Schmid, P., Zennegg, M., and Kohler, M., (2005), Anaerobic Degradation of decabromodiphenyl ether, Environ. Sci. Technol. , 39 , 1078- 1083 68 de Wit, C., (2002), An overview of brominated flame retardants in the environment, Chemosphere , 46 , 583-624 69 de Boer, J., de Boer, K., and Boon, J.P., (2000), Polybrominated biphenyls and diphenylethers, The handbook of environmental chemistry Vol. 3 Part K New types of persistent halogenated compounds , Ed.: Paasivirta, J., Springer-Verlag, 61-95 70 de Wit, C.A., Alaee, M., and Muir, D.C.G., (2006), Levels and trends of brominated flame retardants in the Arctic, Chemosphere , 64 , 209- 233 71 Law, R.J., Allchin, C.R., de Boer, J., Covaci, A., Herzke, D., Lepom, P., Morris, S., Tronczynski, J., and de Wit, C.A., (2006), Levels and trends of brominated flame retardants in the European environment, Chemosphere , 64 , 187-208 72 Law, R.J., Herzke, D., Harrad, S., Morris, S., Bersuder, P., and Allchin, C.R., (2008), Levels and trends of HBCD and BDEs in the European and Asian environments, with some information for other BFRs, Chemosphere , 73 , 223-241 73 Norström, Å., Andersson, K., and Rappe, C., (1976), Major components of some brominated aromatics used as flame retardants, Chemosphere , 5, 255-261

72 74 DeCarlo, V.J., (1979), Studies on brominated chemicals in the environment, Annals of the New York Academy of Sciences , 320 , 678-681 75 Andersson, Ö. and Blomkvist, G., (1981), Polybrominated aromatic pollutants found in fish in Sweden, Chemosphere , 10 , 1051-1060 76 Jansson, B., Asplund, L., and Olsson, M., (1987), Brominated flame retardants - Ubiquitous environmental pollutants?, Chemosphere , 16 , 2343- 2349 77 Wania, F., (2003), Assessing the potential of persistent organic chemicals for long-range transport and accumulation in polar regions, Environ. Sci. Technol. , 37 , 1344-1351 78 Alaee, M., Cannon, C., Muir, D., Blanchard, P., Brice, K., and Fellin, P., (2001), Spatial distribution and seasonal variation of PBDEs in Arctic and Great Lakes air, Organohalogen Compounds , 52 , 26-29 79 Alaee, M., (2001), Levels and trends of PBDEs in North American environment, The Second International Workshop on Brominated Flame Retardants, BFR 2001 , 5-14-0010, Stockholm, Sweden, 131-134 80 Shen, L., Wania, F., Lei, Y.D., Teixeira, C., Muir, D.C.G., and Xiao, H., (2006), Polychlorinated biphenyls and polybrominated diphenyl ethers in the North American atmosphere, Environ. Pollut. , 144 , 434-444 81 Schuster, J.K., Gioia, R., Breivik, K., Steinnes, E., Scheringer, M., and Jones, K.C., (2010), Trends in European background air reflect reductions in primary emissions of PCBs and PBDEs, Environ. Sci. Technol , 44 , 6760-6766 82 Hassanin, A., Johnston, A.E., Thomas, G.O., and Jones, K.C., (2005), Time trends of atmospheric PBDEs inferred from archived U.K. herbage, Environ. Sci. Technol. , 39 , 2436-2441 83 Meironyté, D., Norén, K., and Bergman, Å., (1999), Analysis of polybrominated diphenyl ethers in Swedish human milk. A time-related trend study, 1972-1997, Journal of Toxicology and Environmental Health Part A , 58 , 329-341 84 Akutsu, K., Kitagawa, M., Nakazawa, H., Makino, T., Iwazaki, K., Oda, H., and Hori, S., (2003), Time-trend (1973-2000) of polybrominated diphenyl ethers in Japanese mother's milk, Chemosphere , 53 , 645-654 85 Fürst, P., (2006), Dioxins, polychlorinated biphenyls and other organohalogen compounds in human milk. Levels, correlations, trends and exposure through breastfeeding, Mol Nutr Food Res , 50 , 922-933 86 Fängström, B., Athanassiadis, I., Odsjö, T., Noren, K., and Bergman, Å., (2008), Temporal trends of polybrominated diphenyl ethers and hexabromocyclododecane in milk from Stockholm mothers, 1980-2004, Mol Nutr Food Res , 52 , 187-193

73 87 Alivernini, S., Battistelli, C.L., and Turrio-Baldassarri, L., (2011), Human milk as a vector and an indicator of exposure to PCBs and PBDEs: Temporal trend of samples collected in Rome, Bull Environ Contam Toxicol , 87 , 21-25 88 Link, B., Gabrio, T., Mann, V., Schilling, B., Maisner, V., König, M., Flicker-Klein, A., Zöllner, I., and Fischer, G., (2012), Polybrominated diphenyl ethers (PBDE) in blood of children in Baden-Wurttemberg between 2002/03 and 2008/09, International Journal of Hygiene and Environmental Health , 215 , 224-228 89 Darnerud, P.O., Atuma, S., Aune, M., Bjerselius, R., Glynn, A., Grawe, K.P., and Becker, W., (2006), Dietary intake estimations of organohalogen contaminants (dioxins, PCB, PBDE and chlorinated pesticides, e.g. DDT) based on Swedish market basket data, Food and Chemical Toxicology , 44 , 1597-1606 90 Kiviranta, H., Ovaskainen, M.L., and Vartiainen, T., (2004), Market basket study on dietary intake of PCDD/Fs, PCBs, and PBDEs in Finland, Environ. Int. , 30 , 923-932 91 Bocio, A., Llobet, J.M., Domingo, J.L., Corbella, J., Teixidó, A., and Casas, C., (2003), Polybrominated diphenyl ethers (PBDEs) in foodstuffs: Human exposure through the diet, J. Agric. Food Chem. , 51 , 3191-3195 92 Schecter, A., Papke, O., Harris, T.R., Tung, K.C., Musumba, A., Olson, J., and Birnbaum, L., (2006), Polybrominated diphenyl ether (PBDE) levels in an expanded market basket survey of U.S. food and estimated PBDE dietary intake by age and sex, Environ. Health Perspect. , 114 , 1515- 1520 93 Wilford, B.H., Harner, T., Zhu, J., Shoeib, M., and Jones, K.C., (2004), Passive sampling survey of polybrominated diphenyl ether flame retardants in indoor and outdoor air in Ottawa, Canada: Implications for sources and exposure, Environ. Sci. Technol. , 38 , 5312-5318 94 Athanasios, B. and Constantini, S., (2012), Polybrominated diphenyl ethers (PBDEs) in the indoor and outdoor environments - A review on occurrence and human exposure, Environ. Pollut. , 169 , 217-229 95 Lorber, M., (2008), Exposure of Americans to polybrominated diphenyl ethers, J Expos Sci Environ Epidemiol , 18 , 2-19 96 Johnson-Restrepo, B. and Kannan, K., (2009), An assessment of sources and pathways of human exposure to polybrominated diphenyl ethers in the United States, Chemosphere , 76 , 542-548 97 Sjödin, A., Hagmar, L., Klasson Wehler, E., Kronholm-Diab, K., Jakobsson, E., and Bergman, Å., (1999), Flame retardant exposure: Polybrominated diphenyl ethers in blood from Swedish workers, Environ. Health Perspect. , 107 , 643-648

74 98 Jakobsson, K., Thuresson, K., Rylander, L., Sjödin, A., Hagmar, L., and Bergman, Å., (2002), Exposure to polybrominated diphenyl ethers and tetrabromobisphenol A among computer technicians, Chemosphere , 46 , 709- 716 99 Wu, K., Xu, X., Liu, J., Guo, Y., Li, Y., and Huo, X., (12-15-2009), Polybrominated diphenyl ethers in umbilical cord blood and relevant factors in neonates from Guiyu, China, Environ. Sci. Technol , 44 , 813-819 100 Huo, X., Peng, L., Xu, X., Zheng, L., Qiu, B., Qi, Z., Zhang, B., Han, D., and Piao, Z., (2007), Elevated blood lead levels of children in Guiyu, an electronic waste recycling town in China, Environ. Health Perspect. , 115 , 1113-1117 101 Qu, W., Bi, X., Sheng, G., Lu, S., Fu, J., Yuan, J., and Li, L., (2007), Exposure to polybrominated diphenyl ethers among workers at an electronic waste dismantling region in Guandong, China, Environ. Int. , 33 , 1029-1034 102 Athanasiadou, M., Cuadra, S.N., Marsh, G., Bergman, Å., and Jakobsson, K., (2008), Polybrominated diphenyl ethers (PBDEs) and bioaccumulative hydroxylated PBDE metabolites in young humans from Managua, Environ Health Perspect , 116 , 400-408 103 Thuresson, K., Bergman, Å., Rothenbacher, K., Herrmann, T., Sjölin, S., Hagmar, L., Päpke, O., and Jakobsson, K., (2006), Polybrominated diphenyl ether exposure to electronics recycling workers - a follow up study, Chemosphere , 64 , 1855-1861 104 Gustafsson, K., Björk, M., Burreau, S., and Gilek, M., (1999), Bioaccumulation kinetics of brominated flame retardants (polybrominated diphenyl ethers) in blue mussels ( Mytilus Edulis ), Environ. Toxicol. Chem. , 18 , 1218-1224 105 Booij, K., Zegers, B.N., and Boon, J.P., (2002), Levels of some polybrominated diphenyl ether (PBDE) flame retardants along the Dutch coast as derived from their accumulation in SPMDs and blue mussels (Mytilus edulis ), Chemosphere , 46 , 683-688 106 Haglund, P.S., Zook, D.R., Buser, H.R., and Hu, J., (1997), Identification and quantification of polybrominated diphenyl ethers and methoxy-polybrominated diphenyl ethers in Baltic biota, Environ. Sci. Technol. , 31 , 3281-3287 107 Burreau, S., Broman, D., and Zebühr, Y., (1999), Biomagnification quantification of PBDEs in fish using stable nitrogen isotopes, Organohalogen Compound , 40 , Venice, Italy, 363-366 108 Burreau, S., Broman, D., and Örn, U., (2000), Tissue distribution of 2,2',4,4' -tetrabromo[ 14 C]diphenyl ether ([ 14 C]-PBDE 47) in pike ( Esox lucius ) after dietary exposure - a time series study using whole body autoradiography, Chemosphere , 40 , 977-985

75 109 Boon, J.P., Lewis, W.E., Tjoen-a-choy, M.R., Allchin, C.R., Law, R.J., de Boer, J., ten Hallers-Tjabbes, C.C., and Zegers, B.N., (2002), Levels of polybrominated diphenyl ether (PBDE) flame retardants in animals representing different trophic levels of the North sea food web, Environ. Sci. Technol. , 36 , 4025-4032 110 Malmberg, T., Athanasiadou, M., Marsh, G., Brandt, I., and Bergman, Å., (2005), Identification of hydroxylated polybrominated diphenyl ether metabolites in blood plasma from polybrominated diphenyl ether exposed rats, Environ. Sci. Technol. , 39 , 5342-5348 111 Hakk, H., Larsen, G., and Klasson Wehler, E., (2002), Tissue disposition, excretion and metabolism of 2,2', 4,4', 5-pentabromodiphenyl ether (BDE-99) in the male Sprague-Dawley rat, Xenobiotica , 32 , 369-382 112 Chen, L.J., Lebetkin, E.H., Sanders, J.M., and Burka, L.T., (2006), Metabolism and disposition of 2,2',4,4',5-pentabromodiphenyl ether (BDE99) following a single or repeated administration to rats or mice, Xenobiotica , 36 , 515-534 113 Staskal, D.F., Hakk, H., Bauer, D., Diliberto, J.J., and Birnbaum, L.S., (2006), Toxicokinetics of polybrominated diphenyl ether congeners 47, 99, 100, and 153 in Mice, Toxicol. Sci. , 94 , 28-37 114 Hakk, H. and Letcher, R.J., (2003), Metabolism in the toxicokinetics and fate of brominated flame retardants - a review, Environ. Int. , 29 , 801- 828 115 Marsh, G., Athanasiadou, M., Athanassiadis, I., and Sandholm, A., (2006), Identification of hydroxylated metabolites in 2,2',4,4'- tetrabromodiphenyl ether exposed rats, Chemosphere , 63 , 690-697 116 Kierkegaard, A., Burreau, S., Marsh, G., Klasson Wehler, E., de Wit, C., and Asplund, L., (2001), Metabolism and distribution of 2,2',4,4'- tetrabromo[ 14 C]diphenyl ether in pike ( Esox lucius ) after dietary exposure, Organohalogen Compounds , 52 , Gyeongju, Korea, 58-61 117 Lacorte, S. and Ikonomou, M.G., (2009), Occurrence and congener specific profiles of polybrominated diphenyl ethers and their hydroxylated and methoxylated derivatives in breast milk from Catalonia, Chemosphere , 74 , 412-420 118 Qiu, X., Bigsby, R.M., and Hites, R.A., (2009), Hydroxylated metabolites of polybrominated diphenyl ethers in human blood samples from the United States, Environ. Health Perspect. , 117 , 93-98 119 Yu, Z., Zheng, K., Ren, G., Zheng, Y., Ma, S., Peng, P., Wu, M., Sheng, G., and Fu, J., (2010), Identification of hydroxylated octa- and nonabromodiphenyl ethers in human serum from electronic waste dismantling workers, Environ. Sci. Technol. , 44 , 3979-3985

76 120 Wan, Y., Choi, K., Kim, S., Ji, K., Chang, H., Wiseman, S., Jones, P.D., Khim, J.S., Park, S., Park, J., Lam, M.H.W., and Giesy, J.P., (2010), Hydroxylated polybrominated diphenyl ethers and bisphenol A in pregnant women and their matching fetuses: Placental transfer and potential risks, Environ. Sci. Technol. , 44 , 5233-5239 121 Feng, C., Xu, Y., He, Y., Luo, Q., Zha, J., and Wang, Z., (2010), Debrominated and methoxylated polybrominated diphenyl ether metabolites in rainbow trout (Oncorhynchus mykiss) after exposure to decabromodiphenyl ether, J. Environ. Sci. , 22 , 1425-1434 122 Darnerud, P.O., Eriksen, G., Jóhannesson, T., Larsen, P., and Viluksela, M., (2001), Polybrominated diphenyl ethers: occurrence, dietary exposure, and toxicology, Environ. Health Perspect. , 109 , 49-68 123 Darnerud, P.O., (2003), Toxic effects of brominated flame retardants in man and in wildlife, Environ. Int. , 29 , 841-853 124 McDonald, T.A., (2002), A perspective on the potential health risks of PBDEs, Chemosphere , 46 , 745-755 125 WHO/UNEP, (2013), State of the science of endocrine disrupting chemicals 126 Hooper, K. and McDonald, T.A., (2000), The PBDEs: An emerging environmental challenge and another reason for breast-milk monitoring programs, Environ. Health Perspect. , 108 , 387-392 127 Kuriyama, S.N., Wanner, A., Fidalgo-Neto, A.A., Talsness, C.E., Koerner, W., and Chahoud, I., (2007), Developmental exposure to low-dose PBDE-99: Tissue distribution and thyroid hormone levels, Toxicol. , 242 , 80- 90 128 Meeker, J.D., Johnson, P.I., Camann, D., and Hauser, R., (2009), Polybrominated diphenyl ether (PBDE) concentrations in house dust are related to hormone levels in men, Sci. Total Environ. , 407 , 3425-3429 129 Lilienthal, H., Hack, A., Roth-Haerer, A., Grande, S.W., and Talsness, C.E., (2006), Effects of developmental exposure to 2,2',4,4',5- pentabromodiphenyl ether (PBDE-99) on sex steroids, sexual development, and sexually dimorphic behavior in rats, Environ. Health Perspect. , 114 , 194-201 130 Eriksson, P., Jakobsson, E., and Fredriksson, A., (2001), Brominated flame retardants: A novel class of developmental neurotoxicants in our environment, Environ. Health Perspect. , 109 , 903-908 131 Viberg, H., Johansson, N., Fredriksson, A., Eriksson, J., Marsh, G., and Eriksson, P., (2006), Neonatal exposure to higher brominated diphenyl ethers, hepta-, octa-, or nonabromodiphenyl ether, impairs spontaneous behavior and learning and memory functions of adult mice, Toxicol. Sci. , 92 , 211-218

77 132 Rice, D.C., Thompson, W.D., Reeve, E.A., Onos, K.D., Assadollahzadeh, M., and Markowski, V.P., (2009), Behavioral changes in aging but not young mice after neonatal exposure to the polybrominated flame retardant DecaBDE, Environ. Health Perspect. , 117 , 1903-1911 133 Stapleton, H.M., Kelly, S.M., Pei, R., Letcher, R.J., and Gunsch, C., (2009), Metabolism of polybrominated diphenyl ethers (PBDEs) by human hepatocytes in vitro, Environ. Health Perspect. , 117 , 197-202 134 Wan, Y., Wiseman, S., Chang, H., Zhang, X., Jones, P.D., Hecker, M., Kannan, K., Tanabe, S., Hu, J., Lam, M.H.W., and Giesy, J.P., (2009), Origin of hydroxylated brominated diphenyl ethers: Natural compunds or man-made flame retardants?, Environ. Sci. Technol. , 43 , 7536-7542 135 Malmvärn, A., Zebühr, Y., Kautsky, L., Bergman, A., and Asplund, L., (2008), Hydroxylated and methoxylated polybrominated diphenyl ethers and polybrominated dibenzo-p-dioxins in red alga and cyanobacteria living in the Baltic Sea, Chemosphere , 72 , 910-916 136 Malmvärn, A., Eriksson, U., Kautsky, L., and Asplund, L., (2005), Cyanobacterria from the Baltic Sea - A producer of hydroxylated polybrominated diphenyl ethers (HO-PBDEs), methoxylated polybrominated diphenyl ethers (MeO-PBDEs) and polybrominated dibenzo-p-dioxins (PBDD)?, Organohalogen Compounds , Toronto, Canada, 1380-1383 137 Gribble, G.W., (2000), The natural production of organobromine compounds, Environ. Sci. Pollut. Res. Int. , 7, 37-49 138 Sharma, G.M. and Vig, B., (1972), Antimicrobial substances of sponges. VI. Structures of two antibacterial substances isolated from the marine sponge Dysidea Herbacea , Tetrahedron Lett. , 17 , 1715-1718 139 Teuten, E.L. and Reddy, C.M., (2007), Halogenated organic compounds in archived whale oil: A pre-industrial record, Environ. Pollut., 145 , 668-671 140 Letcher, R.J., Klasson Wehler, E., and Bergman, Å., (2000), Methyl sulfone and hydroxylated metabolites of polychlorinated biphenyls, New types of persistent halogenated compounds , Ed.: Paasivirta, J., The Handbook of Environmental Chemistry, 3, Springer-Verlag, 315-359 141 Kato, Y., Okada, S., Atobe, K., Endo, T., and Haraguchi, K., (2012), Selective determination of mono- and dihydroxylated analogs of polybrominated diphenyl ethers in marine sponges by liquid- chromatography tandem mass spectrometry, Anal Bioanal Chem , 404 , 197- 206 142 Sharma, G.M., Vig, B., and Burkholder, P.R., (1969), Antimicrobial substances of marine sponges IV, Food drugs from the sea. Procedings 1969 , Ed.: Youngken, H.W.Jr., Marine Technology Society, 307-310

78 143 Bowden, B.F., Towerzey, L., and Junk, P.C., (2000), A new brominated diphenyl ether from the marine sponge Dysidea herbcea , Aust. J. Chem. , 53 , 299-301 144 Fu, X., Schmitz, F.J., Govindan, M., and Ackerman, R.A., (1995), Enzyme inhibitors: New and known polybrominated phenols and diphenyl ethers from four indo-pacific Dysidea sponges, J. Nat. Prod. , 58 , 1384-1391 145 Handayani, D., Edrada, R.A., Proksch, P., Wray, V., Witte, L., van Soest, R.W.M., Kunzmann, A., and Soedarson, O., (1997), Four new bioactive polybrominated diphenyl ethers of the sponge Dysidea herbacea from West Sumatra, Indonesia, J. Nat. Prod. , 60 , 1313-1316 146 Malmvärn, A., Marsh, G., Kautsky, L., Athanasiadou, M., Bergman, Å., and Asplund, L., (2005), Hydroxylated and methoxylated brominated diphenyl ethers in the red algae Ceramium tenuicorne and blue mussels from the Baltic Sea, Environ. Sci. Technol. , 39 , 2990-2997 147 Kuniyoshi, M., Yamada, K., and Higa, T., (1985), A biological active diphenyl ether from the green alga Cladophora fascicularis, Experientia , 41 , 523-524 148 Faulkner, D.J., Unson, M.D., and Bewley, C.A., (1994), The chemistry of some sponges and their symbionts, Pure Appl. Chem. , 66 , 1983-1990 149 Unson, M.D., Holland, N.D., and Faulkner, D.J., (1994), A brominated secondary metabolite synthesized by the cyanobacterial symbiont of a marine sponge and accumulation of the crystalline metabolite in the sponge tissue, Mar. Biol. , 119 , 1-11 150 Unson, M.D. and Faulkner, D.J., (1993), Cyanobacterial symbiont biosynthesis of chlorinated metabolites from Dysidea herbacea (Porifera), Experientia , 49 , 349-353 151 Teuten, E.L., Xu, L., and Reddy, C.M., (2005), Two abundant bioaccumulated halogenated compounds are natural products, Science , 307 , 917-920 152 McKim, J.M., Schmieder, P.K., Carlson, R.W., Hunt, E.P., and Niemi, G.J., (1987), Use of respiratory-cardiovascular responses of rainbow trout ( Salmo gairdneri ) in identifying acute toxicity syndromes in fish: Part 1. Pentachlorophenol, 2,4-dinitrophenol, tricaine methanesulfonate and 1- octanol, Environ. Toxicol. Chem. , 6, 295-312 153 WHO, (1987), Environmental health criteria 71. Pentachlorophenol, International program on chemical safety, World Health Organization, Geneva, Switzerland

79 154 van Boxtel, A.L., Kamstra, J.H., Cenijn, P.H., Pieterse, B., Wagner, M.J., Antink, M., Krab, K., Burg, B.v.d., Marsh, G., Brouwer, A., and Legler, J., (2008), Microarray analysis reveals a mechanism of phenolic polybrominated diphenylether toxicity in zebrafish, Environ. Sci. Technol. , 42 , 1773-1779 155 Li, X., Gao, Y., Guo, L.H., and Jiang, G., (2013), Structure- dependent activities of hydroxylated polybrominated diphenyl ethers on human estrogen receptor, Toxicol. , 309 , 15-22 156 Ren, X.M., Guo, L.H., Gao, Y., Zhang, B.T., and Wan, B., (2013), Hydroxylated polybrominated diphenyl ethers exhibit different activities on thyroid hormone receptors depending on their degree of bromination, Toxicol. Appl. Pharmacol. , 268 , 256-263 157 Dingemans, M.M., van den Berg, M., and Westerink, R.H., (2011), Neurotoxicity of brominated flame retardants: (in)direct effects of parent and hydroxylated polybrominated diphenyl ethers on the (developing) nervous system, Environ. Health Perspect. , 119 , 900-907 158 Hoffmeister, W., (1871), Über Phenyläther und Diphenylenoxyd, Annalen der Chemie und Pharmacie , 191-217 159 Olah, G.A., Ohannesian, L., and Arvanaghi, M., (1986), Synthetic methods and reactions;127. Regioselective para halogenation of phenols, phenol ethers and anailines with halodimethylsulfonium halides, Synthesis , 10 , 868-970 160 Tanemura, K., Suzuki, T., Nishida, Y., Satsumabayashi, K., and Horaguchi, T., (2003), Halogenation of aromatic compounds by N-chloro-, N-bromo-, and N-iodosuccinimide, Chem. Lett. , 32 , 932-933 161 WHO, (1994), Environmental health criteria 162. Brominated diphenyl ethers, International program on chemical safety, World Health Organization, Geneva, Switzerland 162 Gilman, H. and Bebb, R.L., (1938), Relative reactivities of organometallic compounds. XX. Metalation, J. Am. Chem. Soc. , 61 , 109-112 163 Wittig, G. and Fuhrmann, G., (1940), Exchange reactions with phenyllithium. V. Behavior of the halogenated anisoles toward phenyllithium, Ber. Dtsch. Chem. Ges. B , 73B , 1197-1218 164 Kreisel, G., Rudolph, G., Schulze, D.K.W., and Poppitz, W., (1992), Bromierte Diphenylether - eine entscheidende Zwischenstufe auf dem Weg zu synthetischen Schmierstoffen, Monatsh. Chem. , 123 , 1153-1161 165 Marsh, G., Hu, J., Jakobsson, E., Rahm, S., and Bergman, Å., (1999), Synthesis and characterization of 32 polybrominated diphenyl ethers, Environ. Sci. Technol. , 33 , 3033-3037

80 166 Örn, U., Eriksson, L., Jakobsson, E., and Bergman, Å., (1996), Synthesis and characterization of polybrominated diphenyl ethers - unlabelled and radiolabelled tetra-, penta- and hexa-bromodiphenyl ethers, Acta Chem. Scand. , 50 , 802-807 167 Teclechiel, D., Christiansson, A., Bergman, Å., and Marsh, G., (2007), Synthesis of octabrominated diphenyl ethers from aminodiphenyl ethers, Environ. Sci. Technol. , 41 , 7459-7463 168 Chen, G., Konstantinov, A.D., Chittim, B.G., Joyce, E.M., Bols, N.C., and Bunce, N.J., (2001), Synthesis of polybrominated diphenyl ethers and their capacity to induce CYP1A by the Ah receptor mediated pathway, Environ. Sci. Technol. , 35 , 3749-3756 169 Fanta, P.E., (2-1-1946), The Ullmann Synthesis of Biaryls, Chemical Reviews , 38 , 139-196 170 Sperotto, E., van Klink, G.P.M., van Koten, G., and de Vries, J.G., (2010), The mechanism of the modified Ullmann reaction, Dalton Trans. , 39 , 10338-10351 171 Miyaura, N., Yanagi, T., and Suzuki, A., (1981), The palladium- catalyzed cross-coupling reaction of phenylboronic acid with haloarenes in the presence of bases, Synthetic Communications , 11 , 513-519 172 Chan, D.M.T., Monaco, K.L., Wang, R.-P., and Winters, M.P., (1998), New N- and O-arylations with phenylboronic acids and cupric acetate, Tetrahedron Lett. , 39 , 2933-2936 173 Christiansson, A., Teclechiel, D., Eriksson, J., Bergman, Å., and Marsh, G., (2006), Methods for synthesis of nonabromodiphenyl ethers and a chloro-nonabromodiphenyl ether, Chemosphere , 63 , 562-569 174 Nevalainen, T. and Koistinen, J., (1994), Synthesis, structure verification, and chromatographic relative retention times for polychlorinated diphenyl ethers, Environ. Sci. Technol. , 28 , 1341-2347 175 Hu, J., Kolehmainen, E., Nevalainen, T., and Kauppinen, R., (1994), 1H, 13 C NMR spectroscopy and conformational properties of 18 halogenated diphenyl ethers, Chemosphere , 28 , 1069-1078 176 Humppi, T., (1985), Preparation of polychlorinated phenoxyphenols as model compounds of impurities in technical chlorophenol formulations, Synthesis , 919-924 177 Garå, A., Andersson, K., Nilsson, C.A., and Norström, Å., (1981), Synthesis of halogenated diphenyl ethers and dibenzofurans - a discussion of specific isomers available, Chemosphere , 10 , 365-390 178 Nilsson, C.A., Norström, Å., Hansson, M., and Andersson, K., (1977), The synthesis of halogenated diphenyliodonium salts and their coupling products with halogenated phenols, Chemosphere , 6, 599-607

81 179 Beringer, F.M., Falk, R.A., Karniol, M., Lillien, I., Masullo, G., Mausner, M., and Sommer, E., (1959), Diaryliodonium salts. IX. The synthesis of substituted diphenyliodonium salts, J. Am. Chem. Soc. , 81 , 342- 351 180 Teclechiel, D., Sundström, M., and Marsh, G., (2008), Synthesis of polybrominated diphenyl ethers via symmetrical tetra- and hexabrominated diphenyliodonium salts, Chemosphere , 74 , 421-427 181 Liu, H., Bernhardsen, M., and Fiksdahl, A., (2006), Polybrominated diphenyl ethers (BDEs); preparation of reference standards and fluorinated internal analytical standards, Tetrahedron , 62 , 3564-3572 182 Bielawski, M., Zhu, M., and Olofsson, B., (2007), Efficient and general one-pot synthesis of diaryliodonium triflates: Optimization, scope and limitations, Adv. Synth. Catal. , 343 , 2610-2618 183 Bielawski, M., Aili, D., and Olofsson, B., (5-28-2008), Regiospecific One-pot synthesis of diaryliodonium tetrafluoroborates from arylboronic acids and aryl iodides, J. Org. Chem. , 73 , 4602-4607 184 Bielawski, M. and Olofsson, B., (2007), High-yielding one-pot synthesis of diaryliodonium triflates from arenes and iodine or aryl iodides, Chemical Communications , 2521-2523 185 Crowder, J.R., Glover, E.E., Grundon, M.F., and Kaempfen, H.X., (1963), Bisbenzylisoquinolines. Part IV. The preparation of diaryl ethers from diaryliodonium salts, J. Chem. Soc. , 4578-4584 186 Jalalian, N., Ishikawa, E.E., Silva, L.F., and Olofsson, B., (2-14- 2011), Room temperature, metal-free synthesis of diaryl ethers with use of diaryliodonium salts, Org. Lett. , 13 , 1552-1555 187 La Guardia, M.J., Hale, R.C., and Harvey, E., (2006), Detailed polybrominated diphenyl ether (PBDE) congener composition of the widely used Penta-, Octa-, and Deca-PBDE technical flame-retardant Mixtures, Environ. Sci. Technol. , 40 , 6247-6254 188 Lancer, K.M. and Wiegand, G.H., (10-1-1976), The ortho effect in the pyrolysis of iodonium halides. A case for a sterically controlled nucleophilic aromatic (SN) substitution reaction, J. Org. Chem. , 41 , 3360- 3364 189 Yamada, Y. and Okawara, M., (1972), Steric effect in the nucleophilic attack of bromide anion on diaryl- and aryl-2-thienyliodonium ions, Bulletin of the Chemical Society of Japan , 45 , 1860-1863 190 Nikiforov, V.A., Karavan, V.S., and Miltsov, S.A., (2003), Synthesis and characterization of methoxy-and hydroxy-polybromodiphenyl ethers, Organohalogen compounds , 61 , Boston, USA, 115-118

82 191 Francesconi, K.A. and Ghisalberti, E.L., (1985), Synthesis of some polybrominated diphenyl ethers found in marine sponges, Aust. J. Chem. , 38 , 1271-1277 192 Marsh, G., Stenutz, R., and Bergman, Å., (2003), Synthesis of hydroxylated and methoxylated polybrominated diphenyl ethers - Natural products and potential polybrominated diphenyl ether metabolites, Eur. J. Org. Chem. , 14 , 2566-2576 193 Sun, Y., Ren, G., Ma, S., Zheng, K., Yu, Z., Wu, M., Sheng, G., and Fu, J., (2013), Synthesis of Br 7-Br 9 hydroxylated/methoxylated polybrominated diphenyl ethers (OH/MeO-PBDEs) and analyses on mass spectra and GC data of the MeO-PBDEs, Chemosphere , 92 , 286-292 194 Utkina, N.K., Veselova, M.V., and Radchenko, O.S., (1993), Synthesis of polybrominated diphenyl ethers of marine origin, Chem. Nat. Compd. (Engl. Transl. ) , 29 , 291-293 195 Pearson, D.E., Wysong, R.D., and Breder, C.V., (1967), The ortho bromination of phenols, J. Org. Chem. , 32 , 2358-2360 196 Jin, Hong, Wang, Yuliang, Yang, Zhirong, Wu, Kaiqun, and Chen, Shuhua, (2006), Preparation of brominated 2,4'-dihydroxydiphenyl ethers as antibacterial agents 197 Chang, J.H., Nam, D.H., and Shin, H., (2008), Efficient synthesis of cylindol A and the proposed structure of cylindol B: the structure of cylindol B still in question, Bull. Korean Chem. Soc. , 29 , 1003-1006 198 Kajigaeshi, S., Kakinami, T., Tokiyama, H., Hirakawa, T., and Okamoto, T., (1987), Bromination of phenols by use of bezyltrimethylammonium tribromide, Chem. Lett. , 4, 627-630 199 Kumar, L., Mahajan, T., Sharma, V., and Agarwal, D.D., (2010), Environmentally-benign and rapid bromination of industrially-important aromatics using an aqueous CaBr 2-Br 2 system as an instant and renewable brominating reagent, Industrial & Engineering Chemistry Research , 50 , 705- 712 200 Endo, A., Yanagisawa, A., Abe, M., Tohma, S., Kan, T., and Fukuyama, T., (2002), Total synthesis of ecteinascidin 743, J. Am. Chem. Soc. , 124 , 6552-6554 201 Oberhauser, T., (1997), A new bromination method for phenols and anisols: NBS/HBF 4. Et 2O in CH 3CN, J. Org. Chem. , 62 , 4504-4506 202 Ek, F., Wistrand, L.G., and Frejd, T., (2003), Aromatic allylation via diazotization: Variation of the allylic moiety and a short route to a benzazepine derivative, J. Org. Chem. , 68 , 1911-1918 203 Bergman, Å., Athanasiadou, M., Fäldt, E., and Jakobsson, K., (2006), Hydroxylated PBDE metabolites in human blood, Organohalogen compounds , 68 , Oslo, Norway, 635-638

83 204 Carté, B. and Faulkner, D.J., (1981), Polybrominated diphenyl ethers from Dysidea herbacea , Dysidea Chlorea and Phyllospongia foliascens , Tetrahedron , 37 , 2335-2339 205 Norton, R.S., Croft, K.D., and Wells, R.J., (1981), Polybrominated oxydiphenol derivatives from the sponge Dysidea herbacea - Structure determination by analysis of 13 C spin-lattice and 13 C-1H coupling constants, Tetrahedron , 37 , 2341-2349 206 Löfstrand, K., Malmvärn, A., Haglund, P., Bignert, A., Bergman, Å., and Asplund, L., (2010), Brominated phenols, anisoles, and dioxins present in blue mussels from the Swedish coastline, Environ. Sci. Pollut. Res. , 17 , 1460-1468 207 Dodson, R.E., Perovich, L.J., Covaci, A., Van den Eede, N., Ionas, A.C., Dirtu, A.C., Brody, J.G., and Rudel, R.A., (2012), After the PBDE phase-out: A broad suite of flame retardants in repeat house dust samples from California, Environ. Sci. Technol. , 46 , 13056-13066 208 Muenhor, D., Harrad, S., Ali, N., and Covaci, A., (2010), Brominated flame retardants (BFRs) in air and dust from electronic waste storage facilities in Thailand, Environ. Int. , 36 , 690-698 209 Ma, J., Addink, R., Yun, S., Cheng, J., Wang, W., and Kannan, K., (2009), Polybrominated dibenzo-p-dioxins/dibenzofurans and polybrominated dethers in soil, vegetation, workshop-floor dust, and electronic shredder residue from an electronic waste recycling facility and in soils from a chemical industrial complex in Eastern China, Environ. Sci. Technol. , 43 , 7350-7356 210 Labunska, I., Harrad, S., Santillo, D., Johnston, P., and Brigden, K., (2013), Levels and distribution of polybrominated diphenyl ethers in soil, sediment and dust samples collected from various electronic waste recycling sites within Guiyu town, southern China, Environ. Sci. : Processes Impacts , 15 , 503-511 211 Thuresson, K., Jakobsson, K., Hagmar, L., Englyst, V., and Bergman, Å., (2002), Work related exposure to brominated flame retardants when recycling metals from printed circuit boards, Organohalogen compounds , 58 , Barcelona, Spain, 249-252 212 Thuresson, K., Bergman, Å., and Jakobsson, K., (2005), Occupational exposure to commercial decabromodiphenyl ether in workers manufacturing or handling flame retarded rubber, Environ. Sci. Technol. , 39 , 1980-1986 213 Hovander, L., Athanasiadou, M., Asplund, L., Jensen, S., and Klasson Wehler, E., (2000), Extraction and cleanup methods for analysis of phenolic and neutral organohalogens in plasma, J. Anal. Toxicol. , 24 , 696- 703

84 214 Eguchi, A., Nomiyama, K., Devanathan, G., Subramanian, A., Bulbule, K.A., Parthasarathy, P., Takahashi, S., and Tanabe, S., (2012), Different profiles of anthropogenic and naturally produced organohalogen compounds in serum from residents living near a coastal area and e-waste recycling workers in India, Environ. Int. , 47 , 8-16