TREBALL FINAL DE MÀSTER

Transcription

1 Analysis of flame retardants in marine organisms of Antarctic: Evaluation of long-range transport of these emerging pollutants. Treball realitzat per: Xuefei Yang Dirigit per: Dr. Ethel Eljarrat Esebag Dr. Alejandro Josa Garcia-Tornel Màster en: Ingeniería Ambiental Barcelona, 23 de junio del 2016 Departament d. Ingeniería Civil i Ambiental. TREBALL FINAL DE MÀSTER I

2 DEPOSIT DATE: June of 2016 ABSTRACT Persistent organic pollutants (POPs) are toxic chemicals that adversely affect human health and the environment around the world. The Stockholm Convention defined four criteria of POPs for persistence, bioaccumulation, potential for long-range transport and toxicity. Polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecane (HBCD) are the most used flame retardants (FRs) that are POPs regulated under the Stockholm Convention. Their production has been banned in Europe and North America. Hence new brominated FRs (BFRs) are used as substitutes; as well as chlorinated FRs, such as dechloranes. Over the years, presence of POPs has been detected in remote areas like Arctic and Antarctic. Although there are very few human impacts because of the geographic isolation and extreme climate, researches have demonstrated that POPs have reached this isolated continent and ocean area unfortunately. The present study investigates the occurrence of PBDEs, emerging BFRs and dechloranes in a total of 31 samples of 2 species of seals (Mirounga leonina and Arctocephalus gazella) collected in Livingston Island and Penguin Island (Antarctica). The samples correspond to different tissues: muscle, nervous system, fat and fur. The main aims of this study are: 1) to detect the presence of emerging FRs in these samples which can prove the transport capacity of long range with risks to the environment and living beings; 2) to assess the degree of exposure of these seals in this area with the levels of FRs found and compare them with other studies published in areas of the Antarctic. PBDEs, dechloranes and HBCDs were detected in some samples with the same order of magnitude (low ng/g lipid weight (lw)). In terms of examined PBDEs, BDE-28 and BDE-47 were the predominant pollutants in fat and fins samples with lower concentrations comparing with previous studies. As for dechloranes, Dec 602 showed the presence in fins, fat and nervous system samples while anti-dp was only detected in fat samples. In the case of HBCD, only γ-hbcd was found in fat samples. These results showed that Dec 602 and anti-dp, as well as PBDEs and HBCD, have the ability to be transported to long distance. II

3 ACKNOWLEDGEMENTS First of all, I would like to acknowledge my tutor in IDAEA-CSIC, Dr. Ethel Eljarrat Esebag, for her professionalism and for giving me the opportunity to make this TFM in her consulting by giving me all the help and information that I needed, and for the time she dedicated. I also want to extend my gratitude to my tutor of UPC, Dr. Alejandro Josa Garcia- Tornel, for his support and guidance during the thesis. And I want to thank Institute of IDAEA-CSIC for giving me the chance to finish my master thesis there and providing all I have needed during the experimental process. I wish to thank IDAEA scientists and staff for their friendship and support, specially to Òscar Aznar-Alemany for his guidance and patience during the experiment. I wish to thank all my friends from the Master because without your kindness and support, these two years could not have passed so enjoyably. Finally, my deepest appreciation goes to my family in China and my love for their infinite and unconditional love, encouragement and support. THANK YOU ALL SO MUCH! III

4 NOMENCLATURE ASPA Antarctic Specially Protected Area ASE accelerated solvent extraction BBB blood brain barrier BFR brominated flame retardant BSEF Bromine Science Environmental Forum DBDPE decabromodiphenyl ethane Dec 602 dechlorane 602 Dec 603 dechlorane 603 Dec 604 dechlorane 604 deca-bde decabrominated diphenyl ether DP dechlorane plus ECHA European Chemicals Agency EFSA European Food Safety Authority EPA Environmental Protection Agency EU European Union FR flame retardant GC gas chromatography HBB hexabromobenzene HBCD hexabromocyclododecane hepta-bde heptabrominated diphenyl ether hexa-bde hexabrominated diphenyl ether HFR halogenated flame retardant HPLC high-performance liquid chromatography LC liquid chromatography LOD limit of detection LPV low production volume LOQ limit of quantification lw lipid weight MeO-PBDE methoxylated polybrominated diphenyl ether MS mass spectrometry IV

5 MS/MS nona-bde NS octa-bde PBDE PBEB PCB penta-bde PLE POPs REACH SPE SRM TBBPA tetra-bde VCCEP tandem mass spectrometry nonabrominated diphenyl ether nervous system octabrominated diphenyl ether polybrominated diphenyl ether pentabromoethylbenzene polychlorinated biphenyl pentabrominated diphenyl ether pressured liquid extraction Persistent Organic Pollutants Registration, Evaluation, Authorisation and Restriction of Chemicals solid phase extraction selected reaction monitoring tetrabromobisphenol A tetrabrominated diphenyl ether Voluntary Children's Chemical Evaluation Program V

6 TABLE OF CONTENTS ABSTRACT... II ACKNOWLEDGEMENTS... III NOMENCLATURE... IV TABLE OF CONTENTS... VI CHAPTER 1 INTRODUCTION Persistent organic pollutants General characteristics of POPs Stockholm Convention on POPs Flame retardants FRs legislated in Stockholm Convention Alternatives of restricted FRs Natural compounds (MeO-PBDEs) Presence of FRs in remote environment Presence of PBDEs in remote environment Presence of dechloranes in remote environment CHAPTER 2 OBJECTIVES AND METHODS Objectives Study Area* Livingston Island Penguin Island Sampling Materials and methods Solvents, patterns, material and equipment Sample preparation Instrumental analysis Analytical parameters CHAPTER 3 RESULTS AND DISCUSSION PBDEs, MeO-PBDEs and NFRs in samples Dechloranes in samples HBCD in samples CHAPTER 4 CONCLUSION VI

7 REFERENCES ANNEX VII

8 CHAPTER 1 INTRODUCTION 1. Persistent organic pollutants 1.1. General characteristics of POPs Persistent organic pollutants (POPs) are organic compounds that persist in the environment, bioaccumulate through the food web, and pose a risk of causing adverse effects to human health and the environment. This group of priority pollutants consists of pesticides (such as DDT), industrial chemicals (such as polychlorinated biphenyls (PCBs)) and unintentional by-products of industrial processes (such as dioxins and furans). POPs are often halogenated and characterized by low water solubility and high lipid solubility, leading to their bioaccumulation in fatty tissues. They are also semivolatile, enabling them to move long distances in the atmosphere before deposition occurs. POPs are transported across international boundaries far from their sources, even to regions where they have never been used or produced. The ecosystems and indigenous people of the Arctic are particularly at risk because of the long-range environmental transportation and bio-magnification of these substances. Consequently, POPs pose a threat to the environment and to human health all over the globe Stockholm Convention on POPs To address global concern of POPs, the United States joined forces with 90 other countries and the European Community to sign a groundbreaking United Nations treaty in Stockholm. The Stockholm Convention on Persistent Organic Pollutants, which was adopted in 2001 and entered into force in 2004, is a global treaty whose purpose is to safeguard human health and the environment from highly harmful chemicals that persist in the environment and affect the well-being of humans as well as wildlife. The Convention requires parties to eliminate and/or reduce POPs, which have a potential of causing devastating effects such as cancer and diminished intelligence and have the ability to travel over great distances. Within the Stockholm Convention, there are four criteria of POPs for persistence, bioaccumulation, potential for long-range transport and toxicity: PERSISTENCE: evidence of a half-life of the chemical in water greater than two months, or a half-life in soil greater than six months, or a half-life in sediment greater than six months. 1

9 BIOACCUMULATION: bioaccumulation factor (BCF) in aquatic> 5000, or in the absence of data, log octanol-water partition coefficient (Kow) > 5 ADVERSE EFFECTS: evidence of adverse effects on human health and the environment POTENTIAL OF LONG RANGE TRANSPORT in the environment: evidence of detected levels of POPs in zones away from the sources of pollution. Table 1 shows the regulated POPs in Stockholm Convention. Annex of Stockholm Convention is shown in Annex. Annex A Elimination of production and use Annex B Restricted in production and use Annex C Unintentionallyproduced POPs Chemicals currently under review Table 1 POPs regulated under the Stockholm Convention Aldrin, chlordane, dieldrin, heptachlor, hexachlorobenzene, mirex, endrin, chlordecone, toxaphene, lindane, hexa- and hepta-bromodiphenyl ethers (commercial octabromodiphenyl ether), tetra- and penta-bromodiphenyl ethers (commercial pentabromodiphenyl ether), polychlorinated biphenyls, α- and β- hexachlorocyclorocyclohexane, α- and β-endoosulfans (technical endosulfan and its isomers), pentachlorobenzene, hexabromobiphenyl DDT, perfluorooctane sulfonic acid and its salts and perfluorooctane sulfonylfluoride Polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans, polychlorinated biphenyls, pentachlorobenzene, hexachlorobenzene Hexabromocyclododecane, short-chained chlorinated paraffins, chlorinated naphthalenes, hexachlorobutandiene, pentachlorophenol There are other legislations for the restriction of POPs. According to the DIRECTIVE 2003/11/EC, in order to protect health and the environment the placing on the market and the use of Penta-BDE and Octa-BDE and the placing on the market of articles containing one or both of these substances should be prohibited [83]. As laid down in Directive 2002/95/EC on the restriction of the use of certain hazardous substances in electrical and electronic equipment, new electrical and electronic equipment put on the market should not contain PBBs or PBDEs since July 2006 [84]. As of July 2008, Deca-BDE is also banned in electronics and electrical applications as decided by the European Court of Justice. However, according to the EU directive it is still allowed to use Deca-BDE in plastic. On June 2nd 2009 the European Chemicals Agency (ECHA) within the REACH framework decided to restrict the use of HBCD within the EU such that it can only be used when authorized for specific purposes. In the USA, Penta-BDE, Octa-BDE and Deca-BDE are included in the US Voluntary Children's Chemical Evaluation Program (VCCEP) with the goal to give information on the effects of chemicals to enable 2

10 consumers to make wise choices. Great Lakes Chemical Corp. (now Chemtura), the only US manufacturer of Penta-BDE and Octa-BDE, voluntarily phased out the production of both mixtures at the end of 2004 [85]. According to the US Environmental Protection Agency (EPA), the production, importation and sale of Deca-BDE was eliminated by announcing the Deca-BDE Phase-Out Initiative in December The European Commission has amended its Regulation on POPs through the Regulation (EU) 2016/293 [87], to ban HBCD. This substance, also known as HBCD, will now be listed in Annex 1 of Regulation (EC) No 850/2004 [88]. HBCD is also regulated in the EU under REACH regulation ( Moreover, the DBDPE is registered with regulations, HBB and PBEB are preregistered, and all these three BFRs are included in monitoring programs [89]. 2. Flame retardants Flame retardants (FRs), also known as fire retardants or fire-proofing agents, are functional materials that give fire retardation to the flammable polymer. When flammable materials are attacked by external fire source, they can effectively prevent or terminate the spread of flame after the processing for fire-retardancy. FRs play a huge role on the safety of human life and property, in consequence, production and promotion of fire retardants has been developed significantly. The appearance of FRs can be traced back to 450 BC, when Egyptians took the advantage of alum to achieve the flammability reduction of woods [1]. Nowadays, there is a wide range of FRs and according to their composition, they can be divided into four classes: inorganic, halogenated organic, nitrogen-containing and phosphorus-containing compounds [4]. Because of the lower decomposing temperatures, higher performance efficiency and low cost, brominated compounds are the most representative among halogenated FRs [2, 4]. Among FRs, brominated FRs (BFRs) are one of the most used families in a wide variety of indoor and outdoor products, such as household appliances, office electronics, textiles and furniture, where they form 5-30% of the product [2-4]. BFRs are incorporated as monomers, reactive or additives. Brominated styrenes and butadienes are examples of monomers, which are incorporated into polymers. Reactive BFRs such as tetrabromobisphenol A (TBBPA) are covalently bonded to polymer, while additive BFRs, such as polybrominated diphenyl ethers (PBDEs), polybrominated biphenyls (PBBs) and hexabromocyclododecanes (HBCDs) are blended with polymer. Since the early 70s, evidence of environmental concerns caused by using BFRs 3

11 appeared. At that time a commercial product of BFRs named Firemaster was in high quantities of production. However, the production was stopped in 1974 in the United States after an incident in Michigan, when Firemaster was accidentally fed to animal. The mistake was discovered after severe effects were observed among the exposed animals [5]. The production of octa- and deca-brominated biphenyls was stopped in 1979 in the United States (WHO 1994). And also evidence of the presence of BFRs in the environment at various locations which are far from the locations of production or of usage has increased. BFRs are detected in various types of samples: sewage sludge [6, 7, 8, 9], water [10, 11], soil and sediment [6-8, 12-14], indoor and outdoor air and dust samples [7, 13-18]. They are also detected in plants and wildlife, in human tissues, blood serum and breast milk of exposed occupational populations (i.e. individuals working in the production of BFRs or production, recycling) and in the general populations [6-8, 11, 13, 14, 16, 17, 19-32]. Moreover, toxicity and ecotoxicity of some BFRs have also been observed over these years FRs legislated in Stockholm Convention PBDEs One of the main kinds of additive BFRs are PBDEs. PBDEs are structurally similar to polychlorinated biphenyls (PCBs) (figure 1). Fig. 1 Chemical structures comparison between PBDE and PCB There are over 209 different PBDE compounds and some of them are mostly used in industry: Penta-BDE, Octa-BDE and Deca-BDE mixtures. Penta-BDE is mostly used in polyurethane foams while octa-bde is mainly used in rigid plastics such as ABS and high-impact polystyrene (table 1.3.1). The main contributors to penta-bdes are 2,2',4,4'-Tetrabromodiphenyl ether (BDE-47) and 2,2',4,4',5-pentabromodiphenyl ether (BDE-99). They are mainly used for epoxy resins, 4

12 phenolic resins, polyesters, polyurethane foams and fibers. In order to increase the furniture fire safety, most of the polyurethane and polyurethane foam with Penta-BDEs added were used in upholstery and furniture making. Octa-BDE has the form of white powder. The term octa-bde alone refers to isomers of octa-pbde congener numbers The melting point is about 200ºC and can decompose when the temperature is higher than 330 ºC. Its solubility in water is low and can dissolve in acetone (20 g/l), benzene (200 g/l) and methanol (2 g/l). It is mainly used in acrylonitrile butadiene styrene resin, polycarbonate and thermosetting plastics as telephones, fax machines, lamps and other small household electrical appliance housings, electrical connectors, automobile parts and some office appliances, production of raw materials. The additive amount is ranging from 12% to 18%. Deca-BDE is white crystalline powder, with PBDE congener number 209 (decabromodiphenyl ether) and nonabromodiphenyl ether being the most common. It is mainly added to circuit board and various textiles polyester (TV, computers and other electronic products). Deca-BDE dominates 80 percent of the PBDEs currently produced and is composed of around 97% pure Deca-BDE-209. It is mainly used in electronics and textiles [4]. Table 2 shows the application and common usage of three PBDE compounds. Table 2 Application and common usage of the three commercially available PBDE mixtures in different types of polymeric materials. (Source: Formulation Polymer Type Common Usage Penta-BDE Octa-BDE Deca-BDE Phenolic resins Polyvinylchloride(PVC) Polyurethane(PUR) Unsaturated polyester(upe) Rubber Textiles, paints/lacquers Acrylonitrile-butadiene-styrene(ABS) Polyamide(PA) Polybutylene terephthalate(pbt) Polystyrene(PS), high-impact PS(HIPS) Epoxy-resins Phenolic resins Polyacrylonitrile(PAN) Polyamide(PA) Polybutylene terephthalate(pbt) Polyethylene(PE), cross-linked PE(XPE) Polyethylene terephthalate(pet) Polypropylene(PP) Polystyrene(PS), high-impact PS(HIPS) Printed circuit boards Cable sheets Cushioning materials, packaging, padding Circuit board, coatings Transportation coatings Moulded parts Electrical connectors, automotive interior parts Electrical connectors and components TV cabinets and back covers, electrical appliance housings Circuit boards, protective coatings Printed circuit boards Panels, electrical components Electrical connectors, automotive interior parts Electrical connectors and components Cross-linked wire and cable, foam tubing, weather protection and moisture barriers Electrical components Conduits, electronic devices TV cabinets and back covers, electrical 5

13 Polyvinylchloride(PVC) Unsaturated polyester(upe) Rubber Textiles, paints/lacquers appliance housings Cable sheets Circuit boards, coatings Transportation coatings The main physical properties of PBDEs (Table 3) are related to solubility, density, color, odor, volatility, melting point, boiling point, thermal conductivity and electrical conductivity, etc., wherein, solubility, molecular density, thermal conductivity and electrical conductivity are the main properties that affect the application of PBDEs in products, restricting their behaviors of transportation and transformation in the environment and affecting the content distribution in various environmental media. The boiling point of PBDEs is ºC. They are closely integrated with the plastic molecules in electronic plastic housing through curing agent and can easily be released into the atmosphere, water and soil environment in the garbage dismantling process. They have low vapor pressure at the room temperature and are volatile, thus being released into the air with the capacity of long distance migration. Table 3 physical and chemical properties of PBDE, HBCD, some emerging BFRs and dechloranes Compounds Molecular Formula log K ow Solubility in water (25 ºC, mg/l) PBDEs C 12H xobr y (x+y = 10) HBCD C 12H 18Br DBDPE C 14H 4Br HBB C 6Br PBEB C 8H 5Br Dec 602 C 14H 4Cl 18O Dec 603 C 17H 4Cl Dec 604 C 13H 4Br 4Cl DP C 18H 12Cl The individual structure influences the chemical stability of the PBDE congeners. In general,pbde congeners with up to three bromine substituents and those with nine or ten bromine substituents are more sensitive to abiotic transformations. The highest stability is showed in those PBDE congeners with four to eight bromine substituents. Generally, PBDE congeners are persistent and bioaccumulative. But bioaccumulative properties of BDE-209 seem to be species dependent. BDE-209 can go through debromination reactions both in the abiotic environment and in biota, leading to 6

14 formation of PBDE congeners comprising seven to nine bromine atoms [36] HBCD HBCD is an effective brominated flame retardant used in extruded (EPS) and expandes (XPS) polystyrene foam mostly. Another small volume of HBCD is used in textiles and high-impact polystyrene (HIPS). The molecular structure of HBCD is shown in figure 2, and their physic-chemical properties in Table 3. Br Br Br Br Br Br Fig. 2 Chemical structure of HBCD The HBCD commercial mixture is composed of three main diastereomers denoted as alpha (α-hbcd), beta (β-hbcd) and gamma (γ-hbcd) with traces of others. Szabo et al. (2010) demonstrated a study of the toxic kinetic profiles of individual HBCD stereoisomers in vivo mice. γ-hbcd, as the predominant diastereomer in the HBCD mixture, undergoes rapid hepatic metabolism, fecal and urinary elimination while α- HBCD is more biologically persistent, resistant to metabolism [71,72] Alternatives of restricted FRs Emerging BFRs Over the years, the cognizance of the presence of the toxicity that affected the environment and human health caused by using FR has increased significantly. There for, novel BFRs (NBFRs) as replacements for the banned formulations (PBDEs and HBCDs) appeared in the market [70]. Decabromodiphenyl ethane (DBDPE), hexabromobenzene (HBB) and pentabromoethylbenzene (PBEB) are examples of NBFRs (Figure 3, Table 3). DBDPE is an alternative for deca-bde. Hardy et al. (2002) presented that because of the similarity of its structure with BDE-209, the properties of DBDPE are presumably similar, i.e. low volatility, low water solubility and a high Kow [73]. In China, DBDPE is the second highest current use additive BFR ( Europe does not produce DBDPE, but imports in 2001 were estimated to be between 1000 and 5000 tons, primarily to Germany [75,76]. There was a clear shift in consumption away from Deca-BDE to DBDPE [77]. Hexabromobenzene (HBB) is an additive FR, widely used in Japan to plastics, textiles 7

15 and woods [77]. The production of HBB in Japan was 270 in 1983 and the consumption did not increase like other BFRs with 350 tons/year constantly [78,79]. In China, 600 tons/year of HBB are produced at Shou Guang Longfa Chemical Co. Ltd. (Qindao, Shandong Province, HBB is marketed by Dayang Chemicals in China and also by the Japanese Nippoh Chemicals Corp as FR-B. Pentabromoethylbenzene (PBEB) is another additive FR which was produced in the US until 1986 [80]. PBEB is classified as a low production volume (LPV) chemical in the EU [81] and is included in the OSPAR list of chemicals, being ranked as persistent, liable to bioaccumulate and toxic, but not currently produced [82] Dechloranes Fig. 3 Chemical structures of DBDPE, HBB and PBEB Dechlorane Plus (DP), dechlorane 602 (dec 602), dechlorane 603 (dec 603) and dechlorane 604 (dec 604) are FRs used as substitutes for mirex since it was banned in the USA because of its toxicity. Between them, DP has properties similar to those of mirex [90]. OxyChem (Niagara Falls, NY, USA) manufactured DP firstly and it was mainly used in electrical wire and cable coatings, plastic roofing materials, computer connectors and other polymeric systems since the 1960s. Jiangsu Anpon Electrochemical Company located in East China has also manufactured it since 2003 [91]. The commercial DP is produced by the Diels-Alder reaction of two equivalents of hexachlorocyclopentadiene with one equivalent of cyclooctadiene. The syn- and antiisomer are formed in the approximate ratio of 1:3 [92,93] (figure 4). As an effective FR, DP has the advantage because of its thermal and photochemical stabilities, lower density and lower cost [95]. Source: Fig. 4 Chemical structures of the two stereoisomers of DP 8

16 Dec 602 (C14H4Cl12O), dec 603 (C17H8Cl12) and dec 604 (C13H4Cl6Br4) also entered into the market as FRs (figure 5, table 3). Dec 604 is used in Molykote AS 810 silicone grease (10-30 %) produced by Dow Corning [96] and it is also mentioned to be an impurity (2 %) in a commercial product of mirex [97]. In the case of dec 603, only some references to its presence as an impurity in some organochlorine pesticides such as aldrin and dieldrin [98] were found. Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl O Cl Cl Cl Cl Cl Cl Cl Cl Br Br Cl Cl Cl Cl Cl Cl Cl Cl Cl Br Br Dechlorane 602 Dechlorane 603 Dechlorane 604 Fig. 5 Chemical structures of Dechlorane 602, 603 and 604 Hoh et al. (2006) reported the first environmental detection of DP in the Great Lakes Basin in North America [99]. Following this, levels of DP were identified in water, air, soil and sediments [10, 98, 101, 102]. Levels of DP were also detected in different aquatic and terrestrial biota indicating their bioaccumulation and biomagnification potential [ ]. The information of levels of DP related compounds (dec 602, 603 and 604) in different matrices and their behavior in the environment is limited. 3. Natural compounds (MeO-PBDEs) Methoxylated polybrominated diphenyl ethers (MeO-PBDEs) are structural analogs to PBDEs; their chemical structure is showed in figure 6 and physic-chemical properties in table 3. MeO-PBDEs have been detected in algae, mollusks, marine sponges, fish, birds and mammals [ ]. It is proposed that MeO-PBDEs are synthesized by some algae, such as red algae [122, ], green algae, brown algae [126], and also by marine sponges [ ] and their associated cyanobacteria [ ]. Another synthesis pathway of MeO-PBDEs can be via biotransformation of hydroxylated PBDEs [131]. The biotransformation pathways have been partly elucidated by recent studies [ ]. MeO-PBDEs may be generated by microbial O-methylation products of hydroxylated PBDEs, as observed for the related phenols [134, 135]. 9

17 O Br 1-5 Br 1-5 OCH 3 Fig. 6 Chemical structure of MeO-PBDE Haglund et al. (1997) reported the first time of MeO-PBDEs analyzed in marine wildlife, seal and fish from the Baltic Sea [60]. Vetter et al. (2002) and Vetter (2006) indicated the presence of MeO-PBDEs in marine mammals at concentrations comparable to halogenated organic compounds of anthropogenic origin [65,66]. Vetter et al. (2002) also reported the MeO-PBDEs have been evidenced in marine environments with the tetrabrominated 2-MeO-BDE 68 and 6-MeO-BDE-47 being the most abundant compounds [66]. Dorneles et al. (2010) presented that MeO-PBDE concentrations were detected in liver samples from 51 cetaceans from Southeast Brazil with the concentration up to 250 μg/g lw [67]. 4. Presence of FRs in remote environment 4.1. Presence of PBDEs in remote environment Presence of PBDEs in Arctic PBDEs containing 2 to 10 bromines are ubiquitous in the Arctic, in both abiotic and biotic samples. Most new data for brominated compounds in the Arctic have been generated for the PBDEs, particularly the components of the Penta-BDE technical product, although more data are now available also for the higher brominated BDEs including several octa- and nona-bdes and BDE-209. The di-hepta-bdes are found in numerous abiotic (air, soil, moss, freshwater and marine sediments) and biotic matrices (zooplankton, invertebrates, fish, terrestrial birds, seabirds, terrestrial and marine mammals) from Alaska to the Barents Sea and the number of species studied has increased. C.A. de Wit et al. (2010) reported that there are some indications of elevated concentrations near emission sources, such as much higher PBDE concentrations in seabirds from the coast of the Gulf of Alaska, which is more populated, compared to seabirds from the Bering Sea, as well as higher concentrations in soils from inside a landfill compared to outside the landfill. Latitudinal trends for lower brominated PBDEs are seen in moss with declining concentrations of ΣPBDEs with increasing latitude. Higher concentrations of PBDEs are seen in some freshwater lake sediments on Svalbard as well as in Lake Ellasjøen on Bjørnøya, which may be due to inputs of seabird guano. Spatial trends of ΣPBDEs in several species indicate that the European Arctic is more contaminated than the North American Arctic, implicating similar atmospheric transport pathways for PBDEs as for organochlorines, i.e. from highly 10

18 populated eastern North America and western and central Europe [33]. Möller et al. (2010) presented the occurrence and distribution PBDEs in air and surface water from the Eastern Greenland Sea in the high Arctic to the Antarctica. As for atmospheric concentrations, BDE-47 and BDE-99 were resulted the most common BDEs which were detected in all samples. The highest airborne concentrations in the Arctic were found in the most southern stations indicating possible transport from Western Europe. Along the Atlantic transect towards the Antarctica, the highest concentrations were observed in the English Channel. Again this indicates Western Europe as source for PBDEs in the marine environment. In the southern hemisphere towards the Antarctica, the concentrations were relatively constant. Volatile BFRs like BDE-28 and BDE-47 were found almost exclusively in the gaseous phase while higher brominated PBDEs such as BDE-153 were found in the particulate phase. Seawater concentrations were dominated by BDE-47 and BDE-99, too. Coastal samples from East Greenland Sea showed the highest concentrations in the Arctic which might be resulted from melting snow and ice from Greenland coast. The highest concentrations in the Atlantic and Southern Ocean were again observed in the English Channel and in the coastal area of South Africa. The distribution between the particulate and dissolved phase followed the trends observed for atmospheric distribution [45]. These results indicated that long-range atmospheric and seawater transport could be the main pathway of PBDEs to remote areas although anthropogenic influence (e.g., from research station, tourism and biotic activities) could also influence the spatial distribution of PBDEs. Due to a great deal of information about level of PBDEs in Arctic area, we will focus on the presence of PBDEs in marine mammals in Arctic to compare with those in Antarctic. Average concentration of total PBDEs in marine mammal samples from Arctic environment is shown in table 4. As can be seen, PBDEs were detected in all the studied samples, with ΣPBDE concentration levels ranging from 3.2 to 72 ng/g wet weight (ww) Presence of PBDEs in Antarctic Over the years, the presence of PBDEs have been detected in remote areas like Arctic and Antarctic. Although there are very few human impacts (only from scientific stations) because of the geographic isolation and extreme climate of Antarctic Circle, researches have demonstrated that POPs have reached this isolated continent and ocean area unfortunately. 11

19 Indoor Dust, waste water sludge and sediments Hale et al. (2007) reported that indoor dust obtained from living quarters in the U.S.- McMurdo and New Zealand-operated Scott Antarctic bases contained substantial total PBDE concentrations, 9560 and 2240 ng/g, respectively. Before year 2002/03, domestic wastewaters were simply discharged directly to McMurdo Sound. To gain insight into past PBDE released from the two bases prior to implementation of secondary treatment, Hale et al. (2007) examined contemporary wastewater sludges from the McMurdo and Scott treatment plants. These sludges contained total PBDEs of 4690 and 637 ng/g dry weight (dw), respectively [119]. Hale et al. (2008) investigated levels of PBDEs in the indoor dust and treated wastewater effluent in the Antarctic environment. Indoor dust from McMurdo Station (USA) and Scott Base (New Zealand) exhibited total PBDE concentrations of 9,560 ng/g dw and 2,240 ng/g dw, respectively. The dominant congener in dust from Mcmurdo and Scott bases was BDE-209. Wastewater sludge from treatment plants of both stations contained 4,690 ng/g dw at McMurdo and 637 ng/g dw at Scott. Hale et al. (2008) also detected PBDEs in sediments, bottom-dwelling organisms and fish collected at different distances from the McMurdo outfall. In the main, with the increasing distances from the outfall, levels of PBDEs in these samples decreased [51]. Wang et al. (2012) detected the levels and distribution of PBDEs in soil and sediment from King George Island and Ardley Island with the average concentration 24.0 pg/g dw (range: pg/g dw) in soil and sediment [120]. Biota Various research teams have investigated the distribution of PBDEs in the Antarctic environment. Corsolini et al. (2004) reported PBDEs in blood samples of the Adélie penguin (Pygoscelis adeliae), the Emperor penguin (Aptenodytes forsteri) and the South Polar skua (Cataracta maccormicki) from three sites in the Ross Sea with the value: 664±1585 pg/g ww in Adélie penguins, 96.74±87.29 pg/g ww and 36.36±16.88 pg/g ww in Emperor penguins from Coullman Is. and Cape Washington, respectively, and pg/g ww in South Polar skua [47]. In the research of Chiuchiolo et al. (2004), PBDEs were measured in juvenile and adult krill samples collected in Antarctic Peninsula. Average concentrations of BDEs are 568±209, 622±253, and 128±50.5 ng/g lw for BDE-47, BDE-99, and BDE-100, respectively in juvenile krill. Average concentrations of BDEs in adult krill are 2.0±0.5, 2.5±0.6, and 0.5±0.1 ng/g lw for BDE-47, BDE-99, and BDE-100, respectively. [49]. After two years, Corsolini et al. (2006) investigated PBDEs in krill (Euphausia superb), fish (emerald rockcod, Trematomus bernacchii) and eggs of Adélie penguin (Pygoscelis adeliae) in the Ross Sea with the concentrations of 5.6±1.12, 5.81±2.32, 4.57±0.17 and 3.06±3.27 ng/g lw respectively. [48]. Corsolini et al. (2007) collected blood samples for analysis of POPs 12

20 in three species of penguin breeding sympatrically at King George Island, Antarctic Peninsula. PBDEs were 291±477, 108±105 and 117±108 pg/g ww in Adelie, Chinstrap and Gentoo penguins, respectively [50]. N. Borghesi et al. (2008) reported the accumulation of PBDEs in the tissues of two species of Antarctic fish (Chionodraco hamatus and Trematomus bernacchii). The concentrations of PBDEs ranged from pg/g lw in C. hamatus muscle to pg/g lw in T. bernacchii liver [52]. Schiavone et al. (2009) analyzed four blubber samples of Antarctic fur seal (Arctocephalus gazelle) pups from Livingston Island to determine levels of PBDEs. Only BDE-47 and BDE-66 were found with values 10±18 ng/g lw and 0.02±0.02 ng/g lw [53]. In the same year, G.T. Yogui et al. investigated concentrations of PBDEs in seabird eggs with the average concentration of 6.78 ng/g lw (range: ng/g lw), 8.12 ng/g lw (range: ng/g lw) and 146 ng/g lw (range: ng/g lw) for chinstrap penguin, gentoo penguin and south polar skua respectively [54]. Another research of N. Borghesi et al. in 2009 evaluated the levels of PBDEs in four species of Antarctic fish: The average concentration of PBDEs in these samples ranged from 0.09 ng/g lw to 0.44 ng/g lw [55]. In 2012, Cipro et al. analyzed seal samples collected from King George Island. Only one sample (fat from L. weddellii) was detected concentration of BDE-99 which is 2.04 ng/g lw [56]. N.B. Lana et al. (2014) investigated levels of PBDEs in tissues such as muscle, liver, gonads and gills of three Antarctic notothenioid species. The ΣPBDE levels ranged from 1.2 to 114 ng/g lw [57]. The study of Colabuono et al. (2015) assessed levels of PBDEs in the eggs and embryos of Antarctic birds. The PBDE congeners were detected only in the eggs of C. antarctica (PBDE 47 and 153) and S. vittata (PBDE 47) with the range of ng/g ww [58]. In the same year, other several researches presented the occurrence of PBDEs. H. Wolschke et al. detected the occurrence PBDEs in samples of an Antarctic rock cod (Trematomus bernacchii), a young gentoo penguin (Pygoscelis papua), and a brown skua (Stercorarius antarcticus) collected from King George Island. The mean concentration of ΣPBDEs is 681 pg/g dw. [59]. Dorneles et al. documented the levels and profiles of PBDEs and MeO-PBDEs in humpback whales that feed in nearshore Antarctic Peninsula waters. The ΣPBDE concentrations varied between 0.15 and 50.8 ng/g lw. [71]. Average concentration of total PBDEs in various biota samples from Antarctic environment is shown in table 5. The figure 7 shows the site of all the scientific stations that have been mentioned before. 13

21 Map resource: Fig. 7 Antarctica map and locations of sampling in former researches 14

22 Table 4 Average concentration of total PBDEs in marine mammal samples from Arctic environment Species Location Year Tissue n PBDEs Reference Ringed seal (ng/g ww) Pangnirtung, Canada 2006 Blubber Muir et al. (2007) Resolute Bay, Canada 2006 Blubber Muir et al. (2007) Sachs Harbour, Canada 2006 Blubber Muir et al. (2007) Arviat, Canada 2006 Blubber Muir et al. (2007) Holman Island, Canada 2006 Blubber Muir et al. (2007) Holman Island, Canada 2002 Blubber 8 5 Ikonomou et al. (2005) Holman Island, Canada 2003 Blubber Ikonomou et al. (2005) Svalbard, Norway 2003 Blubber Sørmo et al. (2006) Ittoqqortoormiit, East Greenland 2004 Blubber Rigét et al. (2006) Hooper Bay and Little Diomede, Alaska 2003 Blubber Rigét et al. (2006) Bearded seal (ng/g ww) Hooper Bay and Little Diomede, Alaska 2003 Blubber Rigét et al. (2006) Spotted seal (ng/g ww) Hooper Bay and Little Diomede, Alaska 2003 Blubber Rigét et al. (2006) Ribbon seal (ng/g ww) Hooper Bay and Little Diomede, Alaska 2003 Blubber Rigét et al. (2006) 15

23 Species Location Year Tissue n PBDEs Reference Beluga Western Hudson Bay, Canada Liver McKinney et al. (2006) Svalbard, Norwegian Arctic Blubber 4 72 Wolkers et al. (2006) Sanikiluaq, E. Hudson Bay, Canada Blubber 35 Tomy et al. (2007) Pangnirtung, Baffin Island, Canada 2005 Blubber Tomy (2007) Pangnirtung, Baffin Island, Canada 2006 Blubber 5 22 Tomy (2007) Pangnirtung, Baffin Island, Canada 2005 Blubber Tomy et al. (2008) Hendrikson Island, Canada 2006 Blubber 8 18 Tomy et al. (2008) Hendrikson Island, Canada 2005 Blubber Tomy et al. (2008) Hendrikson Island, Canada 2006 Blubber Tomy et al. (2008) Igloolik, Canada 2005 Blubber 5 54 Tomy et al. (2008) Resolute, Canada 2003 Blubber 6 36 Tomy et al. (2008) Minke whale West Greenland 2004 Blubber 3 63 Sonne et al. (2008) 16

24 Table 5 Average concentration of total PBDEs in various biota samples from Antarctic environment Species Location Year Tissue n PBDEs (ng/g lw) Reference Adélie penguin: Pygoscelis adeliae Edmonson Point 2001/02 blood (pg/g) Corsolini et al. (2004) South Polar skua: Cataracta maccormicki Edmonson Point 2001/02 blood (pg/g) Corsolini et al. (2004) Emperor penguin: Aptenodytes forsteri Cape Washington and Coullman Island 2001/02 blood (pg/g) Corsolini et al. (2004) Krill (Euphausia superba) Ross Sea 2000,2001/02 whole body 2 pools 5.6 Corsolini et al. (2006) Emerald Rockcod (Trematomus bernacchii) Terra Nova Bay 2000,2001/02 whole body muscle Corsolini et al. (2006) Penguins (Pygoscelis adeliae) Edmonson Point rookery 1995/96 eggs Corsolini et al. (2006) Adelie penguins (Pygoscelis adeliae) Lenie Field Station 2004 blood (pg/g ww) Corsolini et al. (2007) Chinstrap penguins (Pygoscelis Antarctica) Lenie Field Station 2004 blood (pg/g ww) Corsolini et al. (2007) Gentoo penguins (Pygoscelis papua) Lenie Field Station 2004 blood (pg/g ww) Corsolini et al. (2007) Chionodraco hamatus Ross Sea 2005 muscle liver (pg/g ww) 205 (pg/g ww) Borghesi et al. (2008) Trematomus bernacchii Ross Sea 2005 Muscle liver (pg/g ww) 790 (pg/g ww) Borghesi et al. (2008) Antarctic fur seal pup (Arctocephalus gazella) Livingston Island 2004 blubber 4 11 Schiavone et al. (2009) chinstrap penguin King George Island 2004/05 eggs Yogui et al. (2009) 17

25 Species Location Year Tissue n PBDEs (ng/g lw) Reference south polar skua King George Island 2004/05 eggs Yogui et al. (2009) gentoo penguin King George Island 2005/06 eggs Yogui et al. (2009) Antarctic fish (Chionodraco hamatus) Ross Sea 2005 muscle ww Borghesi et al. (2009) Antarctic fish (Chaempsocephalus gunnari) Ross Sea 2001/02 muscle ww Borghesi et al. (2009) Antarctic fish (Gymnoscopelus nicholsi) Ross Sea 2001/02 muscle ww Borghesi et al. (2009) Antarctic fish (Trematomus eulepidotus) Ross Sea 2001/02 muscle ww Borghesi et al. (2009) Weddell seal (Leptonychotes weddellii) King George Island 2004/2005 fat Cipro et al. (2012) Fish (Trematomus newnesi) King George Island muscle liver Fish (Notothenia coriiceps) King George Island muscle liver Fish (Notothenia rossii) King George Island muscle liver Lana et al. (2014) Lana et al. (2014) Lana et al. (2014) Antarctic rock cod (Trematomus bernacchii), Chines Great Wall Station 2010/11 n.d pg/g d.w. Wolschke et al. (2015) gentoo penguin (Pygoscelis papua) Chines Great Wall Station 2010/11 n.d pg/g d.w. Wolschke et al. (2015) brown skua (Stercorarius antarcticus) Chines Great Wall Station 2010/11 n.d. 1 <LOD-6460 pg/g d.w. Wolschke et al. (2015) 18

26 4.2. Presence of dechloranes in remote environment Presence of Dechloranes in Arctic There are many former researches indicated the presence of DP and its related compounds in the environment. But few of them showed available data in remote regions. Möller et al. (2010) reported the occurrence of DP in marine boundary layer air as well as surface seawater from East Greenland Sea toward Antarctica through Atlantic. The concentrations of DP ranged from 0.05 to 4.2 pg/m 3 in the atmosphere and from below limit of detection to 1.3 pg/l in seawater [136]. Möller et al. (2011) reported the DP concentration in air samples and seawater samples from East China Sea to the high Arctic. DP was detected in all air samples mainly in the particulate phase from 0.01 to 1.4 pg/m 3. Seawater concentrations ranged from to 0.4 pg/l [61]. Shen et al. (2012) presented that Dec 602 and its monohydro-dec 602 derivative were measured in approximately equal concentrations, ranging from 25 to 300 pg/g lw [137] Presence of Dechloranes in Antarctic Möller et al. (2012) reported the atmospheric concentration of DP in this area but no previous study has reported the occurrence of dechloranes in the other Antarctic matrices before Kim et al. (2015) reported levels of DP and Dec 602 in avian tissue samples from King George Island, Antarctica. The DP concentrations were ng/g lw in the penguin tissues and ng/g lw in the skua tissues. The levels of Dec 602 were and ng/g lw in the penguin and skua tissues, respectively [110]. Roscales et al. (2016) reported the concentration of syn-dp and anti-dp in plasma of southern giant petrels (Macronectes giganteus) breeding on Livingston Island, with the value of 93 and 86 ng/g lw respectively [111]. 19

27 CHAPTER 2 OBJECTIVES AND METHODS 1. Objectives POPs are chemicals resistant to environmental degradation and can therefore remain intact for exceptionally long periods of time. They can further become widely distributed throughout the environment and accumulate in the fatty tissue of living organisms. In the past few years, increasing concerns have been expressed about growing worldwide contamination by FRs. Some of them, such as PBDEs and HBCD, have already been banned, and as a result, new compounds are being used today. Several studies have shown that these new FRs are also persistent and bioaccumulative. However, there are few data on whether they are also capable of being transported over long distances. The main objective of this master is to provide new data on the transport over long distances of different FRs. Although there are very few human impacts (only from scientific stations) because of the geographic isolation and extreme climate of Antarctic Circle, researches have demonstrated that POPs have reached this isolated continent and ocean area unfortunately. In this study, we will analyse a total of 34 samples of 2 species of seals (Mirounga leonina and Arctocephalus gazella) collected in Livingston Island and Penguin Island (Antarctica). The samples correspond to different tissues: muscle, nervous system (NS), fat and fur. Identification and quantification will be carried out by GC-MS-MS. The work includes analysis of classical FRs already banned (PBDEs) as well as emerging FRs (HBB, PBEB, DBDPE and dechloranes) which appeared as a substitute for the above. Specific aims of this study are: The presence of emerging FRs in these samples can prove the transport capacity of long range with risks to the environment and living beings. The levels of FRs found will be used to assess the degree of exposure of these seals in this area, and compared with other studies published in areas of the Antarctic. With regard to the different tissues analyzed, pollution levels in fur samples are evaluated and correlated with those of other fatty tissues which are normally used for the analysis of these pollutants. A discussion of the possibility of using fur samples as a matrix in biomonitoring will be presented, which are always much more accessible and available. With fat and NS available samples of the same individual, we will check the ability 20

28 of these pollutants to cross the blood brain barrier (BBB). These data are particularly interesting in the case of contaminants that can cause neurological effects. 2. Study Area* Samples that are analyzed in this study were taken from 2 islands of South Shetland Islands: Livingston Island (Hannah Point, Elephant Point and Byers Peninsula) and Penguin Island. The location of these two islands is showed in figure 8. Map resource: Wikipedia Fig. 8 location of Livingston and Penguin Island *Study area information resource: Wikipedia 2.1. Livingston Island Livingston Island is an Antarctic island in the South Shetland Islands, Western Antarctica lying between Greenwich Island and Snow Islands. This island was the first land discovered south of 60 south latitude in 1819, and the name Livingston. Hannah Point is a point on the south coast of Livingston Island in the South Shetland Islands, Antarctica. It forms the east side of the entrance to Walker Bay and the west side of the entrance to South Bay. Surmounted by Ustra Peak to the north, with Liverpool Beach extending between the peak and the tip of Hannah Point. Among the birds that make their home here are the gentoo and macaroni penguins as well as kelp gulls. Southern giant petrels nest here as do blue-eyed shags, skuas, andsnowy sheathbills. Southern elephant seals and Antarctic fur seals are among the larger life forms observed at the point. Elephant Point is a small predominantly ice-free promontory projecting 2 km into Bransfield Strait at the south extremity of the west half of Livingston Island in the South Shetland Islands, Antarctica. The point forms the southwest side of the 21

29 entrance to Kavarna Cove, and is surmounted by Rotch Dome on the north. The area was visited by early 19th century sealers. Byers Peninsula is a mainly ice-free peninsula forming the west end of Livingston Island in the South Shetland Islands of Antarctica. It occupies 60 km 2 [1] and includes the small freshwater Basalt Lake. The peninsula has been designated an Antarctic Specially Protected Area (ASPA 126) for, as well as its birdlife, several additional scientific and historical values. Map resource: Wikipedia Fig. 9 Topographic map of Livingston Island and sampling locations 2.2. Penguin Island Penguin Island is one of the smaller of the South Shetland Islands of Antarctica. The island, which is ice-free and oval shaped, 1.4 km wide by 1.7 km long, lies close off the south coast of the much larger King George Island, and marks the eastern side of the entrance to King George Bay. It has a shoreline of low cliffs, with a beach on the north coast providing access. The island has been identified as an Important Bird Area (IBA) by Bird Life International because it supports a wide range of seabirds including a breeding colony of over 600 pairs of southern giant petrels. 22

30 3. Sampling Samples were obtained by a group of scientific researchers of Federal University of Rio de Janeiro, Brazil in 2014 and Figures below show the Authorization for activities for scientific purposes of the university and some processes of sampling. Fig.10 Authorization for activities for scientific purposes of UFRJ Fig. 11 Some sampling processes The study contains 31 samples of 2 species of seals: southern elephant seal (Mirounga leonine) and Antarctic fur seal (Arctocephalus gazella) collected in Livingston Island and Penguin Island (Antarctica). The samples correspond to different tissues: muscle, NS, fat and fur. In table 6, codes, types of samples, weight of samples and sampling positions are presented. Southern elephant seals (Mirounga leonine) are pups which they fed in Antarctic and Antarctic fur seal (Arctocephalus gazella) are adults which they fed in sub-antarctic seawater. Photos of these two seals are shown in figure 12 and figure

31 Fig. 12 Southern elephant seals (Mirounga leonine) Fig. 13 Antarctic fur seal (Arctocephalus gazella) Table 6 codes, types, weight and locations of samples Marine Mammal Samples - Antarctic, South Shetland Islands AEM Southern elephant seal Mirounga leonina ALB Antarctic fur seal Arctocephalus gazella Samples weight (g) Code Fins (muscle) NS Fat Fur Elephant seals from Livingston Island - Hannah Point ILH-AEM

32 Elephant seals from Livingston Island - Byers Peninsula ANEM / ILB AEM ANEM / ILB AEM ANEM / ILB AEM ANEM / ILB AEM ANEM / ILB AEM ANEM / ILB AEM Elephant seals from Livingston Island - Elephant Point ANEM / ILE AEM ANEM / ILE AEM ANEM / ILE AEM ANEM / ILE AEM ANEM / ILE AEM ILE AEM Antarctic fur seals from Penguin Island ANEM / IP ALM ANEM / IP ALM ANEM / IP ALM ANEM / IP ALM ANEM / IP ALM Materials and methods 4.1. Solvents, patterns, material and equipment Solvents The solvents (ethyl acetate, acetonitrile, dichloromethane and hexane) purchased from Sigma-Aldrich and Merck. Analytical standards: Standards of EFR (HBB, DBDPE, PBEB), of MeO-PBDEs (5-MeO-BDE-47, 6-MeO- BDE-47, 4'-MeO-BDE-49, 2'-MeO-BDE-68, 5'-MeO-BDE-99, 5'-MeO-BDE-100, BDE-4'-MeO-4'-MeO-101 and BDE-103) and 13 C-PBDEs (BDE-28, BDE- 47, BDE-99, BDE-100, BDE-153, BDE-154, BDE-183 and BDE-209) were purchased from Wellington Laboratories Inc. (Guelph, ON, Canada). 25

33 Native PBDEs, HBCDs (α, β and γ isomers) and d18-hbcd (α, β and γ isomers), syn- DP, anti-dp and 13 C-syn-DP were purchased from Cambridge Isotope Laboratories Inc.. (Andover, MA, USA). Dec 602 (95%), Dec 603 (98%) and Dec 604 (98%) were purchased from Toronto Research Chemical Inc. (Toronto, ON, Canada). The material and equipment: Hamilton syringes from 10 to 1000 ml scintillation vials diatomaceous earth Agilent Technologies 25 ml centrifuge tubes vials of accelerated solvent extraction (ASE) cartridge solid phase extraction (SPE): Basic alumina (5 g in 25 ml of Interchar) and C18 (2 g in 15 ml of Isolute) gas chromatography vials Baker ultrasonic shaker Centrifuge Eppendorf Centrifuge 5810 R nitrogen evaporators: Caliper and reactive TurboVap LV-Vap III Pierce gas chromatograph: Agilent Technologies 7890 GC System injector: Injector Series 7683B Agilent Technologies DB-5ms capillary column (15 m 0.25 mm, 0.1 microns thick film) mass spectrometer: 7000B GC / MS Triple Quad Agilent Technologies liquid chromatograph: HP 1100 Binary Pump with Agilent Technologies Symmetry C18 column ( mm, 5 microns particle size) with precolumn C18 ( mm) mass spectrometer: hybrid triple quadrupole - linear ion trap (QqLIT) MSD Sciex 4000QTRAPTM Applied Biosystem electrospray ionization (ESI) Photos of GC MS/MS and LC-MS/MS are shown in Figure 13 (5 and 6) Sample preparation For the extraction and purification of samples, the methodologies used were similar to those previously optimized [ ]. The lyophilized sample is weighted (Figure 14 (1)) and spiked with isotopically labeled internal standards: 20 µl of 13 C-PBDEs ( 13 C-BDE-28, 13 C-BDE-47, 13 C-BDE-99, 26

34 13 C-BDE-100, 13 C-BDE-154, 13 C-BDE-153, 13 C-BDE-183, and 13 C-BDE-209) to 0.2 ng/g and 20 µl of 13 C-syn-DP to 0.2 ng/g. And blank sample is carried out spiking1g diatomaceous earth. After a day in the refrigerator, pressurized liquid extraction (PLE) is made (Figure 14 (2)). For the process of PLE, a cell of 11 ml is used and diatomaceous earth is used to fill the dead volume. A mixture of hexane:dichloromethane (1:1) is used as extraction solvent, with a temperature of 100 C and a pressure of 1500 psi and two static cycles of 10 mins. Extracts were concentrated to incipient dryness. After extraction, the lipid content is determined gravimetrically. Then, extract was redissolved in hexane and treated with concentrated sulfuric acid to remove organic matter (Figure 14 (3)). The remaining organic phase is purified by SPE cartridge with neutral alumina conditioned with 20 ml of hexane and eluted with 20 ml of mixture of hexane: dichloromethane (1:2) (Figure 14 (4)). The extract is evaporated to dryness with nitrogen and reconstituted with 40 ul of toluene for instrumental analysis. The proces of sample preparation is shown in figure Instrumental analysis Gas chromatography coupled to tandem mass spectrometry (GC-MS-MS) is utilized for the analysis of FRs, except for HBCD, which was analysed by liquid chromatography coupled to tandem mass spectrometry (LC-MS-MS). The instrumental conditions and elution program are based on previous works [ ]: injection volume 1 ml injector temperature: 280 C Column: DB-5ms carrier gas: 1 ml/min temperature gradient: 0-2 min at 140 C, C 2-19 min, min at 310 C ionization source temperature: 300 C reactant gas: ammonia at torr Retention times and the two selected transitions (SRM1 and SRM2) for each PBDE congeners, MeO PBDE congeners, PBEB, HBB, DBDPE and dechloranes are shown in table 7. 27

35 Table 7 Retention times, two transitions (SRM 1 and SRM 2) of each compounds analyzed. compounds Rt (min.) SRM 1 (m/z) SRM 2(m/z) PBDEs tri-bde > >248 tetra-bde > >328 penta-bde > >404 penta-bde > >404 hexa-bde > >484 hexa-bde > >484 hepta-bde > >564 deca-bde > >280 MeO-PBDEs 2-MBDE > >358 6-MBDE > >358 5-MBDE > >358 4-MBDE > >358 5-MBDE > >436 4-MBDE > >436 5-MBDE > >436 4-MBDE > >436 Emerging BFRs HBB > >310 PBEB > >406 PBEB > > C-PBDEs 13 C-BDE > > C-BDE > > C-BDE > > C-BDE > > C-BDE > > C-BDE > > C-BDE > > C-BDE > >286 Dechloranes Dec >35 614>35 Dec >35 638>37 Dec >79 460>79 syn-dp >35 654>37 anti-dp >35 654>37 13 C-syn-DP 13 C-syn-DP >35 664>37 HBCD is analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS-MS). The instrumental conditions and elution program are based on previous work [117]: injection volume: 4 ml Column: Symmetry C18 with precolumn eluents: water: methanol (3: 1 v / v) (A) and methanol (B) 0.25 ml/min 28

36 gradient elution: an initial composition of 100% A was ramped to 10% in 17 mins and the initial conditions were reached again in 3 mins and maintained for an additionall 15 mins. ionization source temperature: 350 C Retention times and the two selected transitions (SRM1 and SRM2) of α-hbcd, β- HBCD and γ-hbcd are shown in table 8. Table 8 Retention times, two transitions (SRM 1 and SRM 2) of each compounds analyzed. compounds Rt (min.) SRM 1 (m/z) SRM 2(m/z) HBCD α-hbcd >79 639>81 β-hbcd >79 639>81 γ-hbcd >79 639>81 d 18-HBCD d 18-α-HBCD >79 656>81 d 18- β-hbcd >79 656>81 d 18- γ-hbcd >79 656>81 Data obtained from instrumental analysis was inputted to software Masshunter for quantifying. Examples of quantifying process are shown in Figures below. Figure 15 and 16 showed, as an example, some chromatograms of the real sample analysis. Figure 15 showed the two selected transitions for the detection of MeO-BDE-68 in the sample ILB01fins. As shown, a peak is observed in both transitions, at the same retention time as the standard substance, and the area ratio between the two transitions was similar to that observed with standards. These three characteristics are the conditions that allow us to confirm that the chromatographic peak is our analyte, the MeO-BDE-68. We have the same for figure 16 with BDE-47 in the same sample. In this case, besides the 2 transitions of the compound, the two transitions corresponding to the internal standard ( 13 C-BDE-47) were also shown. This is the same compound but isotopically labeled, and is used as standard for quantification. 29

37 : weighting; 2: PLE extraction; 3: acid treatment; 4: SPE purification; 5: GC-MS/MS; 6: LC-MS/MS Fig. 14 Photos of sample preparation and Instrumental analysis 30

38 Fig. 15 Chromatogram of 2-MBDE-68 in sample ILB01fins 31

39 13 C-BDE-47 BDE-47 Fig. 16 Chromatogram of 13 C-BDE-47 and BDE-47 in sample ILB01fins 32

40 4.4. Analytical parameters Limit of detection (LOD) were determined for each compound as the minimum amount of analyte that gave a signal to noise ratio (S/N) of 3, and the limit of quantification (LOQ) were determined as the minimum amount of analyte that gave a S/N of 10. As for the recovery, Table 9 show the values previously obtained during the different analytical method developments in the IDAEA-CSIC [142,143]. Table 9 Recovery values (%), LOD and LOQ (ng/g lw) for all studied analytes. Analyte R(%) LOD LOQ Analyte R(%) LOD LOQ BDE MBDE BDE MBDE BDE MBDE BDE MBDE BDE MBDE BDE Dec BDE Dec HBB Dec PBEB syn-dp DBDPE anti-dp MBDE α-hbcd MBDE β-hbcd MBDE γ-hbcd R: recovery Recoveries ranged from 56 to 87%, 70 to 77%, 71 to 76 % and 95 to 105% for PBDEs, MeO-PBDEs, emerging BFRs and HBCDs, respectively. And they were between the acceptable range in analytical methods working with isotopic dilution quantification (40-120%). Moreover, LODs are in the range between low pg/g lw (HBCD) and low ng/g lw (BDE-209 and DBDPE). As expected, LODs increased as bromination degree increase, being the highest LODs those corresponding to decabrominated compounds, BDE-209 and DBDPE. 33

41 CHAPTER 3 RESULTS AND DISCUSSION In the analytical process, 8 PBDE congeners, 8 MeO-BDE congeners, 3 emerging BFRs (PBEB, HBB and DBDPE) and 5 dechloranes (syn- and anti-dp and Dec 602, 603 and 604) were monitored in all 31 samples. And α-, β- and γ-hbcd were analyzed in 10 samples. In this chapter, all results are reported and discussed. 1 PBDEs, MeO-PBDEs and emerging BFRs in samples All samples were monitored through instrumental analysis of PBDEs, MeO-PBDEs and emerging BFRs (PBEB, HBB, DBDPE) by GC-MS-MS. Detected concentrations of these organic compounds and lipid content of samples are reported in table 10. Complete Antarctic seal samples data sets for PBDEs, MeO-PBDEs, PBEB, HBB, DBDPE are shown in tables 11 and 12. Only PBDEs were detected, whereas emerging BFRs were not detected in any sample. MeO-PBDEs were detected in some samples but all with values below LOQ. Among the different BDE congeners, only tri-bde-28, tetra-bde-47 and penta-bde-99 were detected with values higher than LOQ, but BDE-99 was detected only in one fins sample at concentration level of 1.11 ng/g lw. The average concentration of ΣPBDEs for M. leonina was 1.26 ± 1.21 (range: nd 2.89 ng/g lw) and 2.09 ± 2.64 (range: nd 5.13 ng/g lw) ng/g lw in fins and fat samples, respectively. PBDEs in fur samples of M. leonina were lower than LOQ. And PBDEs were not found in any NS samples. And for A. gazella, the average concentration of ΣPBDEs was 1.82 ± 0.94 ng/g lw (range: nd 2.74 ng/g lw) in fat samples. No presence of NS samples was shown neither for A. gazella. In the case of MeO-PBDEs, 2-MBDE-68 were detected in fins, fat, and fur samples of M. leonina and 2-MBDE-68 and 5-MBDE-100 were found in two fat samples of A. gazella with values lower than LOQ. There was no presence of MeO- PBDEs in NS samples of both seals. Table 10 Lipid content (%) and concentrations (ng/g lw) of PBDEs in two species of seals collected in South Shetland Islands. Values are mean ± standard deviation. Species M. leonina A. gazella fins NS fat fur fat NS n Lipid (%) 26.5± ± ± ± ± ±22.3 BFRs (ng/g lw) BDE ±0.09 nd 2.43 nd 0.47±0.30 nd BDE ±0.58 nd 1.28±1.24 nd 1.19±0.57 nd BDE nd nd nd nd PBDE 1.26±1.21 nd 2.09±2.64 nd 1.82±0.94 nd 2-MBDE-68 nq nd nq nq nq nd 5-MBDE-100 nd nd nd nd nq nd 34

42 Table 11 Lipid content (%) and concentration of PBDEs, PBEB, HBB, DBDPE and MeO-PBDEs (ng/g lw) in tissues of elephant seals from three sampling locations in Livingston Island * sample code ILH01 ILB01 ILB02 ILB03 ILB04 ILB05 ILB06 ILE01 ILE02 ILE03 ILE04 ILE05 ILE06 tissues NS fins NS fur Fur fins fat fur fins fur fins fat fur fat fur Fur fat fur fur fur fur fins sample weight (g) % lipid BFRs (ng/g lw) BDE-28 nd 0.31 nd nq nd nq 0.15 nd nd nd nd nd nq nd nd nd nd nd nd nd BDE-47 nd 1.45 nd nq nq nq 0.24 nq nq nd nq nd 0.74 nd nd nd nd 1.10 BDE-100 nd nq nd nd nd nd nd nq nq nd nd nd nd nd nd nd nd nd nd nd nd nd BDE-99 nd nq nd nd nd nd nd 3.48 nq nq nd nd nd nd nd nd nd nd nd nd nd nd 1.11 BDE-154 nd nq nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd BDE-153 nd nq nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd BDE-183 nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd BDE-209 nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd PBDEs PBEB nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd HBB nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd DBDPE nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd 2-MBDE-68 nd nq nd nd nd nq nq nq nq nq nq nq nq nd nd nq nd nd nd nd nq 6-MBDE-47 nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd 5-MBDE-47 nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd 4-MBDE-99 nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd 5-MBDE-100 nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd 4-MBDE-100 nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd 5-MBDE-99 nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd 4-MBDE-101 nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd MeO-PBDEs nd: not detected; nq: <LOQ 35

43 Table 12 Lipid content (%) and concentration of PBDEs, PBEB, HBB, DBDPE and MeO- PBDEs (ng/g lw) in tissues of Antarctic fur seals from Penguin Island * 1 sample code IP01 IP02 IP03 IP04 IP05 tissues fat NS fat NS fat NS fat NS fat sample weight (g) % lipid BFRs (ng/g lw) BDE nd 0.25 nd 0.89 nd nd nd 0.24 BDE-47 nd nd 0.93 nd 1.85 nd nd nd 0.80 BDE-100 nd nd nq nd nd nd nd nd nq BDE-99 nd nd nq nd nd nd nd nd nq BDE-154 nd nd nq nd nd nd nd nd nq BDE-153 nd nd nq nd nd nd nd nd nq BDE-183 nd nd nd nd nd nd nd nd nd BDE-209 nd nd nd nd nd nd nd nd nd PBDEs PBEB nd nd nd nd nd nd nd nd nd HBB nd nd nd nd nd nd nd nd nd DBDPE nd nd nd nd nd nd nd nd nd 2-MBDE-68 nd nd nq nd nd nd nd nd nq 6-MBDE-47 nd nd nd nd nd nd nd nd nd 5-MBDE-47 nd nd nd nd nd nd nd nd nd 4-MBDE-99 nd nd nd nd nd nd nd nd nd 5-MBDE-100 nd nd nq nd nd nd nd nd nq 4-MBDE-100 nd nd nd nd nd nd nd nd nd 5-MBDE-99 nd nd nd nd nd nd nd nd nd 4-MBDE-101 nd nd nd nd nd nd nd nd nd MeO-PBDEs nd: not detected; nq: <LOQ *sampling locations: 1Penguin Island 2Hannah Point 3Byers Peninsula 4Elephant Point 36

44 concentration (ng/g lipid) The distribution of PBDE homolog groups in seal tissues is shown in figure 17. As showing in the figure, there is no sample detected to contain hexa-, hepta- and deca- BDEs. Levels of tri-bde-28 are higher than those of tetra-bde-47 in fat samples of M. leonine. In contrast, tetra-bde-47 are predominant in fat samples of A. gazelle. There is a stepped increase of PBDEs concentrations in fins samples of M. leonine. As BDE-47, -99 and -100 are the predominant congeners in commercial mixtures of PBDEs, these are the congeners that have been detected with a greater abundance in biota samples in areas which are not remote. Here BDE-47 and -99 are also detected, but surprisingly the levels of BDE-28 were also relatively high in some samples. And this can be due to the lower degree of bromination of the compound has, more volatile it will be and more easily it can be transported over long distances. This would explain the presence of BDE-28 levels in these samples. Goutte et al. (2013) detected PBDEs in 9 of the 30 analyzed biological samples (starfish, Antarctic yellowbelly rock cod, Antarctic krill, egg of snow petrel, and bald notothen) collected from Pointe Géologie Archipelago, Adélie Land, Antarctica, and BDE-47 was predominant in two upper trophic level species, the snow petrel and the Antarctic yellowbelly rockcod [138]. Corsolini et al. (2006) reported that BDE-47 was the dominant congener in various Antarctic organisms from the same area, such as Antarctic krill (Euphausia superbia) and emerald rockcod (T. bernacchii), and in three penguin species (Pygoscelis adeliae, Pygoscelis antarctica and Pygoscelis papua) from the Antarctic Peninsula [48]. These previous results can prove that BDE-47 is the predominant PBDEs congener in the food web of seal, and in present study, fins samples of M. leonine and fat samples of both seal were all detected with high levels of BDE-47 relatively. 3 fins (M. leonina) fat (M. leonina) fat (A. gazella) tri tetra penta hexa hepta deca Homolog Series Fig. 17 The distribution of PBDE homolog groups in seal tissues 37