Brominated natural products at different trophic levels in ...196895/FULLTEXT01.pdf · Comparison...

Brominated natural products at different trophic levels in the Baltic Sea

Identification of polybrominated dioxins, hydroxylated and methoxylated diphenyl ethers

Anna Malmvärn

Department of Environmental Chemistry

Stockholm University 2007

- ii -

- iii -

Doctoral Thesis 2007 Department of Environmental Chemistry Stockholm University SE-106 91 Stockholm Sweden

Abstract

Over time, the Baltic Sea has been contaminated by increasing discharges of pollutants from human activities. Persistent organic pollutants (POPs) have caused toxic effects in wildlife and excess of nutrients have led to eutrophication. Furthermore, there are indications that certain polyhalogenated compounds similar in structure to man-made POPs are produced by the biota present in this sea. In the late 1990’s both methoxylated polybrominated diphenyl ethers (MeO-PBDEs) and hydroxylated polybrominated diphenyl ethers (OH-PBDEs) were identified in fish and seals living in this environment. OH-PBDEs can originate from metabolism of polybrominated diphenyl ethers (PBDEs), but both OH- and MeO-PBDEs are also known to be natural products in marine environments. Another group of POPs, the polybrominated dibenzo-p-dioxins (PBDDs), are not produced commercially, but are known to be by-products of chemical industry and of the combustion of, e.g., brominated flame retardants (BFRs). In contrast to the OH- and MeO-PBDEs, PBDDs have not previously been shown to be natural products, although certain related compounds have been indicated to have a natural origin. This thesis describes the identification of several brominated compounds in algae, blue mussels and fish living in the brackish water of the Baltic Sea. Several of these have not previously shown to be present in biota. Accordingly, OH-PBDEs, and MeO-PBDEs, as well as mixed brominated chlorinated hydroxylated diphenyl ethers were detected in Baltic macroalgae (Ceramium tenuicorne) and blue mussels (Mytilus edulis). Furthermore, 1,3,7- and 1,3,8-triBDD were identified in these same organisms for the first time. The concentrations of these compounds in the blue mussels were remarkably high (160 ng/g fat) compared to the level of, e.g., the persistent PCB congener 2,2’,4,4’,5,5’-hexachlorobiphenyl (CB-153) (50 ng/g). In addition, we found indications for the presence of another set of PBDDs in these algae and mussels. PBDDs, OH-PBDEs and MeO-PBDEs were also detected in cyanobacteria (Aphanizomenon flos-aquae). Moreover, PBDDs were present in fish, mussels, shrimp and crabs from different regions of the Baltic Sea and from the west coast of Sweden, but not in organisms from freshwater environments. The levels of these compounds in Baltic fish generally exceeded those of their chlorinated analogues. The origin of the PBDDs identified is somewhat unclear, but the high levels present in blue mussels and the pattern of congeners observed indicate natural production. The presence of PBDDs, OH-PBDEs and MeO-PBDEs in fish and shellfish constitutes a potential risk to both humans and wildlife and requires further investigation. © Anna Malmvärn ISBN 91-7155-366-5 US-AB universitetsservice, 2007

- iv -

Table of contents

Abstract ...............................................................................................................iii

Table of contents................................................................................................. iv

Abbreviations......................................................................................................vi

List of papers......................................................................................................vii

1 The Baltic Sea: A general introduction................................................. 1

2 Background considerations .................................................................... 7

2.1 Natural halogenated products................................................................ 7 2.1.1 The role of haloperoxidases in formation of halogenated compounds10

2.2 Hydroxylated polybrominated diphenyl ethers (OH-PBDEs) ............ 11 2.2.1 Chemical structures and physicochemical properties .........................11 2.2.2 Occurrence in the environment ...........................................................13 2.2.3 Biological effects of OH-PBDEs ........................................................14

2.3 Methoxylated polybrominated diphenyl ethers (MeO-PBDEs) ......... 15 2.3.1 Chemical structures and physicochemical properties .........................15 2.3.2 Occurrence in the environment ...........................................................15 2.3.3 Biological effects of MeO-PBDEs......................................................16

2.4 Polybrominated dibenzo-p-dioxins (PBDDs) ..................................... 16 2.4.1 Chemical structures and physicochemical properties .........................16 2.4.2 Occurrence in the environment ...........................................................16 2.4.3 Biological effects of PBDDs ...............................................................17

3 Aims of the present thesis .....................................................................20

4 Analytical approaches...........................................................................21

4.1 Samples and sampling procedures ...................................................... 21

4.2 Extraction methods.............................................................................. 24

4.3 Clean-up and separation of different classes of substances................ 27 4.3.1 Determination of extractable material.................................................27 4.3.2 Derivatization ......................................................................................27 4.3.3 Elimination of matrix and lipids..........................................................29 4.3.4 Separation of different classes of substances ......................................29

4.4 Instrumental analysis........................................................................... 30

4.5 Quality aspects on the analyses........................................................... 32 4.5.1 Identification and quantification .........................................................32 4.5.2 Recovery..............................................................................................34

- v -

4.5.3 Analytical procedures.......................................................................... 34

5 Additional results .................................................................................. 35

6 General discussion................................................................................. 39

6.1 The presence of OH-PBDEs and MeO-PBDEs in the Baltic Sea ...... 39 6.1.1 Sources of OH-PBDEs and MeO-PBDEs........................................... 39

6.2 The occurrence of PBDDs in Baltic biota .......................................... 41 6.2.1 Distribution and levels of PBDDs in the Swedish environment......... 41 6.2.2 The origin of PBDDs .......................................................................... 42

6.3 OH-PBDEs, MeO-PBDEs and PBDDs in different matrices............. 43

6.4 Impact on the Baltic ecosystem .......................................................... 46

7 Future perspectives ............................................................................... 48

8 Acknowledgements................................................................................ 49

9 References .............................................................................................. 52

- vi -

Abbreviations

AHH aryl hydrocarbon hydroxylase AHR aryl hydrocarbon receptor BDE brominated diphenyl ether BFR brominated flame retardant DCM dichloromethane DDE 2,2-bis(4-chlorophenyl)-1,1-dichloroethene DDT 2,2-bis(4-chlorophenyl)-1,1,1-trichloroethane ECD electron capture detection ECNI electron capture negative ionization EI electron ionization EOM extractable organic matter EROD ethoxyresorufin O-deethylase GC gas chromatography HBCDD hexabromocyclododecane HPC halogenated phenolic compound HRMS high resolution mass spectrometry LRMS low resolution mass spectrometry MeO-PBDE methoxylated polybrominated diphenyl ether MTBE methyl tert-butyl ether OH-PBDE hydroxylated polybrominated diphenyl ether PBDE polybrominated diphenyl ether PBDD polybrominated dibenzo-p-dioxin PBDF polybrominated dibenzofuran PCB polychlorinated biphenyl PCDD polychlorinated dibenzo-p-dioxin PCDF polychlorinated dibenzofuran PFK perfluorokerosene REP relative potency RRT relative retention time SIM selected ion monitoring TEF toxic equivalents factor TEQ toxic equivalents

- vii -

List of papers

This thesis is based on the following publications, which will be referred to in the text by their respective Roman numerals. The two published articles are reproduced here with the permission of the publisher. Certain unpublished results are also presented.

I. Hydroxylated and Methoxylated Brominated Diphenyl Ethers in the Red Algae Ceramium tenuicorne and Blue Mussels from the Baltic Sea. Anna Malmvärn, Göran Marsh, Lena Kautsky, Maria Athanasiadou, Åke Bergman and Lillemor Asplund. (2005) Environ. Sci. Technol. 39, 2990-2997. Key results: The red macroalga C. tenuicorne and blue mussels from the Baltic Sea were analysed for MeO- and OH-PBDEs. Seven OH-PBDEs and four MeO-PBDEs were identified in both of these organisms. In addition, monochlorinated OH-tetraBDEs were found in alga and mussels.

II. Identification of Polybrominated Dibenzo-p-dioxins in Blue Mussels (Mytilus edulis) from the Baltic Sea. Anna Malmvärn, Yngve Zebühr, Sören Jensen, Lena Kautsky, Erik Greyerz, Takeshi Nakano and Lillemor Asplund. (2005) Environ. Sci. Technol. 39, 8235-8242. Key results: Several PBDDs were detected in blue mussels (Mytilus edulis) from the Baltic Sea. Two coeluting triBDDs (1,3,7- and 1,3,8-triBDD) were identified by comparison of their RRTs and fragmentation patterns with those of authentic reference standards, as well as by accurate mass determination. The levels of the two triBDDs were estimated to 160 ng/g fat.

III. Hydroxylated and methoxylated polybrominated diphenyl ethers and polybrominated dibenzo-p-dioxins in a red alga and cyanobacteria from the Baltic Sea. Anna Malmvärn, Yngve Zebühr, Lena Kautsky, Åke Bergman and Lillemor Asplund. Manuscript. Key results: TriBDDs were identified and quantified in red alga from the Baltic Sea. Comparison of the levels of the MeO- and OH-PBDEs, identified in Paper I, to those of PCB and PBDE supported the conclusion that the former are natural products. Cyanobacteria was analysed for MeO-, OH-PBDEs and PBDDs. Several of the studied analytes were detected in the cyanobacteria.

IV. Brominated dibenzo-p-dioxins - A new class of marine toxins? Peter Haglund, Anna Malmvärn, Sture Bergek, Anders Bignert, Lena Kautsky, Takeshi Nakano, Karin Wiberg, Lillemor Asplund. Environ. Sci. Technol, In press. Key results: The levels of PBDDs in fish and mussels from several areas in the Baltic Sea, as well as in fish, mussels, shrimp and crabs from the west coast of Sweden were measured. The levels of these compounds in fish from the Baltic Sea generally exceeded those of their chlorinated analogous. No PBDDs were detected in fish living in freshwater.

- viii -

The Baltic Sea: A general introduction

- 1 -

1 The Baltic Sea: A general introduction

The Baltic Sea is a unique, semi-enclosed brackish body of water connected with the North Sea area of the Atlantic Ocean only via the narrow and shallow Danish Straits. The exchange of water between the Baltic and North Seas is therefore limited. Water, rich in nutrients and environmental contaminants, flows into the Baltic from a large drainage area stretching far inland into the surrounding countries, including several urbanized and industrial areas.

The Baltic Sea is divided into three main areas: the Baltic Proper, the Bothnian Sea and the Bothnian Bay (Figure 1.1). With increasing distance from the Atlantic, its salinity decreases from 30 to 3 practical salinity units (psu), as a consequence of dilution by freshwater from rivers and land run-off. This brackish water is the home of a variety of marine and freshwater species, all living under the physiological stress of salinity that is lower or higher than that of their native habitat. In evolutionary terms the Baltic Sea is young; few species, most of marine origin, have been able to adapt to its brackish water and, consequently, the biodiversity of its fauna is low and declines as the salinity decreases.

Figure 1.1 Map of the Baltic Sea showing the three main areas.

BothnianBay

BothnianSea

Baltic

Proper

BothnianBay

BothnianSea

Baltic

Proper


- 2 -

During the last half century, human activities have led to elevated levels of inorganic nitrogen and phosphorous in the Baltic, causing eutrophication of this entire sea, which was originally oligotrophic [1-3]. These high levels of nutrients favour the survival and reproduction of certain species at the expense of others, thereby altering an ecosystem that already contained a relatively small number of species. For example, the growth of phytoplankton is favoured by such high levels of nutrients, which, in turn, decreases the penetration of light into the water, one reason why the distribution of the slowly growing brown seaweed Fucus vesiculosus (Figure 1.2) in the Baltic has been reduced [4]. Moreover, the growth of filamentous alga species such as Pilayella littoralis, Ceramium tenuicorne, Cladophora glomerata and Polysiphonia fucoides, in contrast to F. vesiculosus, benefits from high concentrations of nutrients [5]. Furthermore exudates from P. fucoides [6] and from P. littoralis [7], both appear to exert a negative effect on the reproduction of F. vesiculosus. Fucus vesiculosus is a key contributor to the survival of fish fry, functioning as a nursery and hiding-place, as well as a feeding-place for numerous species. In addition to such repercussions, the huge quantities of P. fucoides produced have caused devastation of beaches, particular along the coast of the island Öland (Figure 1.3).

In addition to such changes in species composition, nutrient enrichment may enhance the formation of biomass and the degradation of large amounts of biomass is a major cause of the oxygen depletion characteristic of the Baltic Sea. Oxygen deficiency leads to the reduction of iron, which in its reduced form is not able to bind phosphate, thus leading to release of phosphate. In the presence of an excess of phosphorous, the supply of nitrogen becomes limiting for the growth of algae, with the exception of cyanobacteria, the only group of “algae” capable of utilizing gaseous nitrogen dissolved in water and thus not dependent on inorganic forms of nitrogen, such as nitrate [8]. Under such conditions, undisturbed by competition from other algae, the cyanobacteria are able to form massive blooms, one of which occurred in the summer of 2005 (Figure 1.4). However, analysis of sediment has revealed that cyanobacteria blooms have been a recurrent event in the ecosystem of the Baltic Sea for at least the last seven thousand years [9], although such blooms are believed to occur more frequently today.

In addition to the repercussions of eutrophication, another serious threat to the biodiversity of the Baltic Sea is over-fishing. This has severely reduced the size of several populations of fish including cod and herring.


- 3 -

Figure 1.2 Part of a belt of the brown seaweed Fucus vesiculosus growing submerged in the Baltic Proper.

Figure 1.3 A beach on the east coast of the island Öland in 2001. The algae covering this beach consist mainly of the red filamentous alga Polysiphonia fucoides.


- 4 -

Figure 1.4 The bloom of cyanobacteria (Nodularia spumigena) that occurred in the Baltic Proper in the summer of 2005.

The Baltic Sea is also known to be heavily contaminated by persistent organic pollutants (POPs), pesticides and industrial chemicals. As early as the end of the 1960´s, this environment was found to be severely polluted by DDT, PCBs and several other organohalogens [10,11]. As a result of their lipophilic properties and chemical stability, many of these compounds are bioaccumulated and biomagnified in food webs, resulting in high concentrations and toxic effects, particularly in species at high trophic levels, e.g., marine mammals (seal, mink and otter) and birds of prey (cf. below).

In the Baltic grey seal (Halichoerus grypus), ringed seal (Phoca hispida) and harbour seal (Phoca vitulina) reproductive failure involving uterine lesions, sterility, and skeletal deformities has occurred [12-14]. These effects are most probably due to high concentrations of organochlorines [12-14]. Moreover, reproduction by the white-tailed sea eagle (Haliaeetus albicilla), another top predator in the aquatic food web of the Baltic Sea, has been severely disrupted


- 5 -

by environmental contaminants. In the early 1960’s a few well-studied pairs on the coast of Sweden failed to breed over a period of several years [15]. By the 1970’s the average brood size had declined by more than 75% [16] and the sea eagle almost became extinct. This decline was brought about by a dramatic reduction in the thickness of the eggshell which was strongly correlated with the levels of contamination by 4,4’-DDE, the major metabolite of 4,4’-DDT, which is the primary component in commercial DDT preparations. Moreover, there are indications that the presence of PCBs exerts negative effects on the reproduction of these eagles [17].

In Sweden the use of DDT was restricted in 1970 and phased out 1975, while the use of PCBs was banned in 1972. These actions have led to significant decreases in the levels of DDT, 4,4’-DDE and PCBs in the Baltic environment and accompanying recovery of both the white-tailed sea eagle [17,18] and of seals [19]. However, uterine tumours and intestinal ulcers are still frequently observed in seals, and the average brood of some populations of the sea eagle has not yet regained its original size [17,20]. Furthermore, fatty fish in the Baltic Sea, i.e., herring and salmon are still contaminated to a significant degree, even though the concentrations of PCB and DDT in herring have been declining since 1980 [21,22]. The remaining relatively high concentrations of POPs and, in particular, of PCDDs/Fs and co-planar PCBs in fish have resulted in recommendations that humans limit their consumption of fatty fish from the Baltic Sea (www.slv.se).

A number of other environmental contaminants are also present in Baltic Sea biota and the levels of other POPs in addition to PCB, DDT and PCDD/F are also being followed by the Swedish Monitoring Program [21]. Two such examples are the persistent and bioaccumulated flame retardants, polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecane (HBCDD), which can today be detected in any Baltic biota [21,23-25]. The levels of PBDEs in guillemot eggs is decreasing, whereas the level of HBCDD shows the opposite trend [21,25].

Another contaminant in the Baltic environment, bis(4-chlorophenyl)sulfone (BCPS), was first detected by Olsson and Bergman in 1995 [26]. BCPS has been detected in fish, mammals and birds [26-28], with the highest level being present in guillemot [29]. An additional, more recently reported group of substances are the perfluorinated compounds (PFCs) [30], used primarily as surfactants. Perflurooctane sulfonate (PFOS), one of these compounds, is present in guillemot eggs collected at Stora Karlsö, at levels that has increased from 1970 to 1995 [31]. The influence of the PFCs on species in the Baltic Sea is not yet known.


- 6 -

During the past few decades reproduction failure has been reported in several species of fish including perch and pike in the Baltic Proper [32]. One of the most serious disorders observed is the M74 syndrome in salmon (Salmo salar), which causes early mortality [33]. This syndrome is associated with a deficiency in thiamine (vitamin B1) and oxidative stress [34], but the actual cause has still not been clarified. Its symptoms appear in newly hatched fry during the period when the yolk sac is being resorbed for nutrition, before the fry begin to feed on external sources [33].

Among numerous hypotheses concerning the cause of the M74 syndrome are i: lowered thiamine levels in fish that the salmon prey on, ii: ecological changes in the Baltic food chains, iii: pollutants, iv: microbial infections, v: and/or genetic changes in the salmon population [35-37]. A recent study revealed no correlation between the levels of common environmental contaminants (e.g., DDT, PCB and PBDE) in the salmon and the production of fry with M74 [38]. Several methoxylated polybrominated diphenyl ethers (MeO-PBDEs) and hydroxylated polybrominated diphenyl ethers (OH-PBDEs) of unknown origin were also detected in the salmon [38].

Reproduction of the Baltic cod (Gadus morhua) is also impaired, which is believed to be due to the low levels of oxygen in the deepwater spawning grounds of this species. As a consequence of this poor reproduction, in combination with heavy fishing and predation by sprat and herring on cod eggs and larvae, the cod population is decreasing continuously [39]. The cod is a key species in the Baltic environment and its depletion may trigger a cascade of disturbances in this ecosystem.

Furthermore, an unusually high rate of mortality among herring (Clupea harengus) eggs in the Baltic has been observed, both in the field and in connection with laboratory studies [40-42]. Although the underlying cause remains unclear, it has been proposed that toxic substances exuded by filamentous algae (Pilayella littoralis [40] and Furcellaria lumbricalis [41]) may be involved [40]. Thus, certain natural compounds may pose an additional threat to fish populations and, thereby, overall biodiversity. The elevated production of such compounds by the filamentous algae whose growth is promoted by the nutrient enrichment associated with eutrophication may thus exert further negative effects on the food web.

Background considerations

- 7 -

2 Background considerations

Halogenated compounds in the environment may originate from both biogenic and anthropogenic sources. This chapter is aimed to give a brief overview of some halogenated compounds, relevant to the scope of this thesis.

2.1 Natural halogenated products

More than 3800 halogenated compounds, most containing chlorine and/or bromine, but a few with iodine and/or fluorine, are known to be produced by living organisms or through natural abiotic processes involving volcanic activity, other geothermal processes or forest fires [43]. The single largest source of biogenic organohalogens are seaweeds, sponges, corals, tunicates, bacteria and other marine life in our oceans [43], which synthesize a wide range of compounds, from simple halocarbons to complex phenols, terpenes, pyrroles and indoles [43-51]. A few examples of such compounds are depicted in Figure 2.1. Several reviews on the occurrence and identities of naturally occurring halogenated compounds are available for further reading [50-54].

At least 50 simple bromophenols (consisting of one aromatic ring) of which the most widely distributed appears to be 2,3-dibromo-4,5-dihydroxybenzyl alcohol (lanosol) (Figure 2.2) [55] are produced naturally, mainly by marine

Figure 2.1 Examples of a few selected halogenated natural compounds. A: 2,2’,3,3’-tetrabromo-4,4’,5,5’-tetrahydroxydiphenylmethane; B: 1-hydroxy-3,6,8-triBDD; C: 1,1’-dimethyl-3,3’,4,4’-tetrabromo-5,5’dichloro-2,2’-bipyrrole; D: 6,6’-dibrom-1,1’-(2,2’)biindolyliden-3,3’-dione (also known as Tyrian Purple); E: bis(2,3-dibromo-4,5-dihydroxybenzyl)ether.

Br Br

HO

Br Br

OH

OH OH

N

N

BrBr

Cl

CH3

CH3 Br Br

Cl

NH

OHN

OBr

Br

O

O

OH

Br

Br

Br

O

BrBr

Br Br

OH

HO

OH

OH

A C

D E

B


- 8 -

plants and animals [44]. Lanosol is common in several species of red algae [54,56-60], including a number that grow along the west coast of Sweden [57]. 2,3,2’,3’-Tetrabromo-4,5,4’,5’-tetrahydroxydiphenylmethane (Figure 2.1) has also been identified in these red algae [61].

Mono-, di-, and tribromophenols (Figure 2.2) are present in a large number of red, green and brown marine algae [62-64], as well as in marine sponges [65], and variations of the concentrations of these compounds occur in several species of algae [62,63,66]. 2,4,6-Tribromophenol (Figure 2.2) is known to have both anthropogenic and natural origin and has been isolated from algae at levels as high as 1900 ng/g fresh weight (f.w.) [62]. Brominated phenols appear to be more common in algae than their methylated counterparts, i.e., the anisoles, (e.g., 2,4-dibromoanisole and 2,4,6-tribromoanisole (Figure 2.2), which were recently detected for the first time in the marine alga Polysiphonia sphaerocarpa [64]). In contrast to the brominated phenols, the anisoles are, in general, not produced or used industrially in technical quantities. Two of them, 2,4- and 2,6-dibromoanisole, have been indicated to be of biogenic origin [67,68].

In addition to the simple phenols, algae are known to release large amounts of volatile halocarbons, including bromoform, chloromethane, bromomethane and iodomethane [44], as well as more complex substances such as the biologically active 2’-MeO-2,3’,4,5’-tetraBDE [69], OH-PBDEs [70], bis(2,3-dibromo-4,5-dihydroxybenzyl)ether [56,60] and polybrominated diphenyl methanes [56,61]. Moreover, marine sponges, and in particular Dysidea, contain a series of brominated compounds, such as MeO-PBDEs [71,72], OH-PBDEs [73-80], di-MeO-PBDE and OH-MeO-PBDEs [81], as well as hydroxylated or methoxylated brominated dibenzo-p-dioxins (OH- and MeO-PBDD) [82,83]. In a recent study 2‘-OH-BDE68 was found to account for 12% of the dry weight of a sponge [84]. It is believed that the primary source of many of these compounds is the filamentous cyanobacterium (Oscillatoria spongeliae) that lives in symbiosis with the sponges [84,85].


- 9 -

Figure 2.2 Examples of monocyclic halogenated phenols and anisoles which have been isolated from natural sources.

OHBr

Br

Br

OHBr Br

OHBr

Br

2,4,6-tribromophenol

2,6-dibromophenol 2,4-dibromophenol

OHBr

OH

2-bromophenol 4-bromophenol

OH

HOOH

Br

2,3-dibromo-4,5-dihydroxybenzyl- alcohol (lanosol)

OH

HO OH

Br

Br2,4-dibromo-1,3,5-benzentriol

OHBr

BrHO

2,3-dibromo-1,4-benzenediol

BrHO OH

OH OHBrCl

OH2-bromo-6-chloro-1,4-benzendiol2-bromo-1,3,5-benzentriol

OBr

Br2,4-dibromoanisole

OBr

Br

Br

2,4,6-tribromoanisole

CH3 CH3

Br

Br


- 10 -

2.1.1 The role of haloperoxidases in formation of halogenated compounds Marine waters contain relatively high concentrations of chloride ions, but bromide, iodide and fluoride ions are also present. If the availability of halide ions was a key factor in the formation of halogenated substances, then chlorinated compounds would be most common; but, in fact, most of the organohalogens that occur naturally in the marine environment are brominated. This apparent discrepancy may be explained by the role played by haloperoxidases in the halogenation of organic substrates [45]. The oxidation of a halide (i.e., chloride, bromide, or iodide) by hydrogen peroxide, catalyzed by these enzymes, results in halogenation, and since bromide and iodide have lower oxidation potentials, than chloride, the former are more readily oxidized via this route [86].

By tradition, the nomenclature for the haloperoxidases is based on the most electronegative halide which is able to be oxidized by hydrogen peroxide, and catalyzed by the enzyme. Accordingly, chloroperoxidase catalyzes the oxidation of chloride, bromide, and iodide; bromoperoxidase catalyzes the oxidation of bromide and iodide; while iodoperoxidase only catalyses the oxidation of iodide by hydrogen peroxide. Many species of red, green and brown algae, as well as a variety of bacteria and invertebrates, contain bromoperoxidase [45,87]. However, the presence of bromoperoxidase in algae is not always correlated with the presence of halogenated natural products, indicating the existence of a broader biological function(s), for these enzymes [87].

In addition to bromide, the bromination reaction catalyzed by bromoperoxidase, requires suitable precursors and hydrogen peroxide. In marine algae the formation of halogenated compounds is dependent on photosynthesis as a source of hydrogen peroxide [88]. Since hydrogen peroxide can be toxic to algae, it has been proposed that the formation of halogenated compounds by haloperoxidase may provide a mechanism for means of scavenging hydrogen peroxide before it reaches toxic concentrations in the algae [88].

Org-H + X- + H2O2 + H+ X-Org + 2H2OOrg-H + X- + H2O2 + H+ X-Org + 2H2O


- 11 -

2.2 Hydroxylated polybrominated diphenyl ethers (OH-PBDEs)

2.2.1 Chemical structures and physicochemical properties The general structure of OH-PBDEs, also known as brominated phenoxyphenols, is depicted in Figure 2.3. The abbreviations applied for the OH-PBDEs are derived from the patterns of halogenation, following the system of the International Union of Pure and Applied Chemistry (IUPAC) for numbering positions in polychlorinated biphenyls (PCBs) [89,90], which is applicable for PBDEs as well. The abbreviation system used herein is the same as previously presented for OH-PCBs [91].

There is not much information available regarding the physicochemical properties of OH-PBDEs. Their solubility in water is low, and decreases with increasing number of bromine (ACD/LABs softwareTM) [92]. The calculated log Kow values for the protonated OH-PBDEs identified in Paper I, together with the corresponding pKa values, as generated by the ACD computer model, are presented in Table 2.1. As is the case for, e.g., OH-PCBs, the OH-PBDEs are not likely to accumulate in lipids [93]. On the other hand, OH-PBDEs may be retained in the blood in the same manner as OH-PCBs, which involve binding to transthyretin (TTR), a protein that normally binds and transports thyroxine [94]. The stability of OH-PBDEs is influenced by the presence of the hydroxy group, which renders them more prone to undergo chemical reactions than either the corresponding unsubstituted PBDEs or their methylated counterparts, the MeO-PBDEs.

Table 2.1 Calculated a log Kow and pKa values for various OH-PBDEs

Compound log Kow pKa

2’-OH-BDE68 7.2 ± 0.6 6.6 ± 0.2

6-OH-BDE47 6.8 ± 0.2 6.8 ± 0.2

6-OH-BDE90 8.2 ± 0.7 5.7 ± 0.2

6-OH-BDE99 8.4 ± 0.7 5.2 ± 0.2

6-OH-BDE85 8.3 ± 0.7 6.2 ± 0.2

2-OH-BDE123 8.3 ± 0.7 5.7 ± 0.2

6-OH-BDE137 9.3 ± 0.8 4.7 ± 0.2 a employing ACD/LABs TM software


- 12 -

Figure 2.3 General chemical structures and abbreviations of the compounds of primary interest in the work presented in this thesis.

O

Brx

OMeO

Brx

OH

O

Brx

OH

Brx

O

BrxO

BrxO

O

Brx / Cly

OHO

Brx / Cly

OMe

Methoxylated polybrominateddiphenyl ethers(MeO-PBDEs)

Hydroxylated polybrominateddiphenyl ethers(OH-PBDEs)

Methoxylated polybrominated/chlorinateddiphenyl ethers(MeO-PBCDEs)

Polybrominated dibenzo-p-dioxins(PBDDs)

Polybrominated dibenzofurans(PBDFs)

Polybrominated diphenyl ethers(PBDEs)

Polybrominated phenols(PBPs)

Hydroxylated polybrominated/chlorinateddiphenyl ethers(OH-PBCDEs)


- 13 -

2.2.2 Occurrence in the environment Although certain OH-PBDE congeners are known to be metabolites of PBDEs [95,96], some naturally occurring OH-PBDEs have also been isolated and their structures determined by NMR, (see further below). To date, OH-PBDEs have never been associated with any commercial production.

As metabolites OH-PBDEs have been identified in several studies after exposure to different PBDE congeners as reviewed by Hakk et al. [96]. For instance, BDE-47 was shown to form OH-PBDE metabolites in mice, rat [95,97], and pike [98]. A number of OH-PBDE metabolites have been identified in rats exposed to BDE-99 [99], BDE-100 [100], BDE-209 [101], or a mixture of PBDE congeners (i.e., BDE-47, BDE-99, BDE-100, BDE-153, BDE-154, BDE-183 and BDE-209) [102]. In the latter study, Malmberg and co-workers [102] identified four OH-PBDEs (4-OH-BDE42, 3-OH-BDE47, 4’-OH-BDE49 and 6-OH-BDE47) and detected an additional sixteen hydroxylated and two dihydroxylated PBDE metabolites in rat blood plasma.

Several OH-PBDE congeners are found as natural products, mainly in marine species. 6-OH-BDE47 (one of the metabolites of PBDE mentioned above) has also been isolated from tunicates [103,104], and marine sponges [74-77]. Moreover, marine sponges contain a number of OH-PBDEs, i.e., 2’-OH-BDE68 [75-78], 6-OH-BDE90 [105], 6-OH-BDE99 [73], 6-OH-BDE85 [74,75,79], 2-OH-BDE123 [79] and 6-OH-BDE137 [78,80]. Several OH-PBDE congeners have also been identified, by comparison with authentic reference standards, in wildlife, e.g., in salmon [38,90], common carp and largemouth bass [106], glaucous gull and polar bear [107], as well as in the bald eagle [108]. Further, 6-OH-BDE47 has been identified in human plasma [109], and, recently, a total of eighteen OH-PBDE congeners were reported in human plasma from children working and living at a garbage dump in Nicaragua [110].

The origin of OH-PBDEs in environmental samples is often far from clear, and a critical issue is to decide, which OH-PBDEs are metabolites of anthropogenic compounds and, which are natural products. In general, the OH-PBDEs identified as metabolites have the hydroxy group in meta- or para-position relative to the diphenyl ether oxygen, while a hydroxy group in an ortho-position is most common for OH-PBDEs of natural origin. Furthermore, the non-hydroxylated ring of OH-PBDEs isolated as natural products is commonly substituted with bromine at the 2- and/or 4- positions. However, there are exceptions, e.g., 6-OH-BDE47, a metabolite of BDE-47 [95,98]. Another exception is the 4’-OH-2,3’,4-triBDE, recently identified in red alga [70].


- 14 -

2.2.3 Biological effects of OH-PBDEs The structure of OH-PBDEs resembles that of the thyroid hormone thyroxine (T4) (Figure 2.4) and in vitro studies have revealed that OH-PBDEs can bind to human TTR [111], with 6-OH-BDE47 demonstrating higher binding affinity [112] than the PBDEs investigated [111,112]. Malmberg and co-workers [92] have reported that OH-PBDEs with the hydroxy group in the meta- or para-position binds to transthyretin with an affinity 3.6-3.9 folder greater than that of T4 itself; while 6-OH-BDE47, with its hydroxy group in the ortho-position, exhibits a lower binding affinity than T4. Binding of hydroxylated organohalogens to TTR in vivo may result in facilitated transport of these compounds across the placenta to the foetal compartment, thereby leading to a reduction in thyroid hormone levels in both the mother and foetus, with consequences for foetal brain development, as observed following prenatal exposure to hydroxylated PCB [113,114].

Figure 2.4 Structure of the thyroid hormone, thyroxine (T4).

Further, two OH-PBDEs, with their hydroxy group in the para-position have been shown to possess estrogenic activity [115]; whereas, 6-OH-BDE47, with its hydroxy group in the ortho-position, shows no such activity relative to estradiol [112]. In a recent study, by Cantón and co-workers [116], the steroidogenic enzyme aromatase (CYP19) activity (which mediates the conversion of androgens to estrogens), was shown to be inhibited by 6-OH-BDE47 in a human adrenocarcinoma H295R cell line, which may be due to the significant increase in cytotoxicity observed for the 6-OH-BDE47. However, the corresponding methoxylated congener, 6-MeO-BDE47, for which no cytotoxicity was observed, also inhibited the aromatase activity, but to a lesser extent.

In addition, certain OH-PBDEs exert antibacterial activity [78,79,117].

OI

HO

I

I

I

NH2

OH

O


- 15 -

2.3 Methoxylated polybrominated diphenyl ethers (MeO-PBDEs)

2.3.1 Chemical structures and physicochemical properties As is the case for the OH-PBDEs, the information presently available concerning the MeO-PBDEs (Figure 2.3) (also referred to as brominated phenoxyanisoles in the literature) is highly limited. In this thesis the system of abbreviation employed for MeO-PBDEs is the same as that for the OH-PBDEs (see above). MeO-PBDEs are, to the best of my knowledge, not produced commercially, but they may be formed naturally by O-bio-methylation of the corresponding halogenated phenols.

MeO-PBDEs are neutral, lipophilic, non-volatile and biplanar compounds. The octanol-water partition coefficients (log Kow) for 2’-MeO-BDE68 and 6-MeO-BDE47 have been estimated to be approximately 6.85 [118], indicating a pronounced enrichment in lipid-rich compartments and bioaccumulation in biota [118,119]. Similar to PBDEs, MeO-PBDEs are chemically non-reactive. Although microbial dealkylation of methylated chlorophenols occurs under anaerobic conditions in sediment [120-122], similar dealkylation of MeO-PBDEs has not yet been reported.

2.3.2 Occurrence in the environment MeO-PBDEs are known to be natural products. 2’-MeO-BDE68 and 6-MeO-BDE47 have been isolated from marine sponges, their structures determined by NMR and confirmed to be natural products [71,72]; and 2’-MeO-BDE68 was isolated from green alga as early as 1985 [69]. MeO-PBDEs were first detected in seals and fish living in the Baltic Sea by Haglund and co-workers [123] and, subsequently, several of these compounds were found in various Baltic biota, including salmon (Salmo salar) [38,90,124], herring (Clupea harengus) [24,125], the white-tailed sea eagle (Haliaeetus albicilla) [126] and guillemot (Uria aalga) [124]. Moreover, MeO-PBDEs are present in herring caught off the west coast of Sweden [125], as well as in pike (Esox lucius) living in a Swedish lake [24].

Apart from the MeO-PBDEs in the Baltic environment they have been detected in largemouth bass from the Detroit River [106]; whales, dolphins, dugong, seals and green turtles from Queensland, Australia [119,127]; human milk from the Faeroe Islands [128]; beluga and pilot whales from the Faeroe Islands and Svalbard [129,130]; whales and dolphins from the Mediterranean Sea [131]; two Canadian populations of beluga whales [132]; polar cod [130], glaucous gulls and polar bears from Svalbard [107]; and in California sea lions [133]. 2’-MeO-BDE68 and 6-MeO-BDE47 were recently identified in True´s beaked whale living in the North Atlantic Ocean and a natural origin


- 16 -

of these two congeners was established by measurement of the 14C content [118].

2.3.3 Biological effects of MeO-PBDEs At present, exceedingly little is known about the biological effects of MeO-PBDEs. 2’-MeO-BDE68 exhibits, anti-bacterial and anti-inflammatory activity [69]. Moreover, aromatase activity is inhibited by 6-MeO-BDE47 [116], but to an extent only 50% of the inhibition exerted by the corresponding phenol.

2.4 Polybrominated dibenzo-p-dioxins (PBDDs)

2.4.1 Chemical structures and physicochemical properties PBDDs are co-planar aromatic compounds (Figure 2.3), with eight positions where halogens can be substituted (Figure 2.5). Altogether, there are 75 possible PBDD congeners and a number of these of relevance in the present context are shown in Figure 2.6.

Figure 2.5 General structure of and numbering system for dibenzo-p-dioxins. There is much less experimental data concerning the physical and chemical properties of PBDDs than in the case of the polychlorinated dibenzo-p-dioxins (PCDDs). In comparison to their chlorinated analogues, PBDDs have higher molecular weights (raging from 263 to 815 for the fully brominated compound), higher melting points and lower vapor pressures and are also less water soluble [134]. As a consequence of their bromine substituents these compounds are highly lipophilic, exhibiting log Kow values from 3-8 (modelled values), and are, therefore, largely found dissolved in lipids or bound to particles. In the presence of indoor light or sunlight, PBDDs undergo photolysis more rapidly than PCDDs [135,136].

2.4.2 Occurrence in the environment PBDDs are not manufactured (except for scientific purposes), but it is well established that they are formed as by-products in connection with the manufacture of brominated flame retardants (BFRs) [134], as well as during

O

O

12

346

7

89


- 17 -

the combustion of products containing such flame retardants [134,137,138]. Although experimental investigations indicate that polybrominated dibenzofuranes (PBDFs) can be formed by photolysis of decaBDE [139,140], to date photolytical formation of PBDDs has not been demonstrated.

At present, little information concerning the occurrence of PBDDs in the general environment is available, and in most investigations the samples are collected in areas directly linked to automobile exhausts [141] and/or combustion [134]. However, a few studies have detected PBDDs in biological samples, i.e., in fish and mussels [142], in human adipose tissue [143] and in human milk [144]. Recently, PBDD congeners detected in freshwater sediment collected in Sweden were suggested to be degradation products of airborne PBDEs [145]. Otherwise, little information exist regarding the presence of PBDDs in uncontaminated areas, either in Sweden or elsewhere.

Although PBDDs/PBDFs are not known to occur as natural products [134], derivatives of PBDDs have been isolated from marine sponges [82,83] and a non-halogenated dibenzo-p-dioxin from the ascidian Aplidiopsis ocellata [146]. Moreover, a derivative of PBDF has been identified in red alga [70].

2.4.3 Biological effects of PBDDs In order to compare the toxicity of different dioxins, the concept of toxic equivalents, which involves relating the toxicity of individual dioxin congeners to the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was developed. In this context toxicity is expressed by applying toxic equivalent factor (TEF) [147,148], with TCDD, the most toxic congener, being given a TEF value of 1. The total toxic equivalents (TEQ) concentration is obtained by multiplying the concentration of each congener by its TEF and than summing all of these values.

The TEF concept is based on the assumption that PCDDs, PCDFs and dioxin-like PCBs all exert their toxicity via a common mechanism, which involves binding to the aryl hydrocarbon (Ah) receptor, an intracellular protein, as well as on the assumption that these toxic effects are additive [149]. Among other things, up-regulation of enzymes, carcinogenicity and reproductive toxicity needs to be included to express the toxicity of these compounds. When the toxic potency of a compound relative to TCDD has been determined in a single in vivo or in vitro study only, this value is referred to as the relative potency (REP).

In contrast to the PCDDs, the TEF concept has not yet been applied to the PBDDs, due to the limited amounts of information concerning the biological effects of these latter compounds. In general, in the limited number of experimental studies that has focused on the toxicity of PBDD, these


- 18 -

substances have been found to exert all of the toxic effects observed with PCDD/PCDF, i.e., lethality, wasting syndrome, thymic antrophy, teratogenesis, reproductive effects, chloracne, immunotoxicity, enzyme induction, decreases in T4 and vitamin A, and increased hepatic porphyrins, are also observed with PBDD/PBDF [134]. The PBDD congener that has received most attention is 2,3,7,8-TBDD, since it is substituted with bromines in the four lateral positions, and is therefore expected to be the most toxic congener. In fact, TBDD has been suggested to be even more potent than TCDD in causing early mortality in fish [150].

With respect to binding to the Ah-receptor, the relative binding affinity of TBDD is only half that of TCDD. The corresponding affinity exhibited by 2,3,7-triBDD is substantially greater than that of the chlorinated counterparts and is, in fact, close to the value of TCDD. However, 2,3,7-triBDD appears to be less potent than TBDD as an inducer of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities [151]. The toxicities of the 1,3,7-triBDD and 1,3,8-triBDD, have not yet been tested and at the moment there are no commercial standards available for these two congeners. It has been demonstrated that addition of bromine in the non-lateral position or removal of a lateral halogen reduces the activity [152].

Overall, the limited information presently available on PBDDs and PBDFs supports the hypothesis that they exert biological effects similar to those caused by their chlorinated counterparts [151]. Moreover, on basis of mechanistic considerations, it has been proposed that the TEF/TEQ concept would be applied to PBDDs as well [148], but this is not yet the case.


- 19 -

Figure 2.6 Structures of the PBDDs analysed in composite biota samples (Paper IV).

O

O

O

O

Br

Br

Br

Br

Br Br

1,3,7-Tribromodibenzo-p-dioxin 1,3,8-Tribromodibenzo-p-dioxin

O

O

Br

Br

1,7-Dibromodibenzo-p-dioxinO

O

BrBr

1,8-Dibromodibenzo-p-dioxin

O

O

Br


O

O

Br


Br

Br

O

O

Br

Br

Br

2,3,7-Tribromodibenzo-p-dioxin

O

O

Br

Br

Br

Br

2,3,7,8-Tetrabromodibenzo-p-dioxin

O

O

Br

Br


Br

BrO

OBr


Br

Br

Br

Aims of the present thesis

- 20 -

3 Aims of the present thesis

The overall objective of this thesis has been to investigate the occurrence of OH-PBDEs, MeO-PBDEs and PBDDs in biota from the Baltic Sea and attempt to determine the origin of these compounds.

More specifically, the aims have been:

1. to investigate whether filamentous macroalgae from the Baltic Sea could be a producer of OH-PBDEs and MeO-PBDEs (Paper I),

2. to investigate whether OH-PBDEs and MeO-PBDEs are present in mussels living in close vicinity of these algae (Paper I),

3. to identify the OH-PBDEs and MeO-PBDEs present in alga and mussels (Paper I), as well as to estimate their concentrations in the alga (Paper III),

4. to structurally identify the triBDDs present in blue mussels and filamentous alga and estimate their concentrations (Papers II and III),

5. to look for the possible presence of OH-PBDEs, MeO-PBDEs and PBDDs in cyanobacteria (Paper III),

6. to study the distributions of PBDDs and measure their concentrations in biota from Baltic and North Seas (Paper IV).

Analytical approaches

- 21 -

4 Analytical approaches

The primary goal of this chapter is to present and discuss the analytical procedures, from sample extraction to instrumental analysis, applied in Papers I-IV. The extraction and clean-up procedures employed in Papers I and III are illustrated in Figure 4.1, and the approach utilized in Paper II is depicted in Figure 4.2. The experimental details are described in the individual papers.

4.1 Samples and sampling procedures

Most of the samples investigated in this thesis were collected in the Baltic Sea, with the exception of certain of the samples analyzed in Paper IV, which came from the west coast of Sweden and from freshwater lakes. Samples were collected from the west coast primarily in order to characterize the distribution of PBDDs in a wider geographical context.

Due to the increasing eutrophication of the water, blooms of cyanobacteria in the Baltic Sea seem to have become more frequent. These blooms in the pelagic Baltic Proper contain three genera of cyanobacteria, i.e., Aphanizomenon, the toxic Nodularia, and Anabaena. Nodularia spumigena (Figure 4.3, right) and Aphanizomenon flos-aquae (Figure 4.3, left) dominate,

Figure 4.3 The two most common species of cyanobacteria in the Baltic Sea: Aphanizomenon flos-aquae (left) and Nodularia spumigena (right).

Camilla Bollner/azote.seCamilla Bollner/azote.se

Camilla Bollner/azote.seCamilla Bollner/azote.seCamilla Bollner/azote.seCamilla Bollner/azote.se


- 22 -

while the several species of Anabaena present, constitute only a minor portion of the total biomass of the bloom. Aphanizomenon is present in the Baltic throughout the year, whereas Nodularia only appears during the summer months [8]. In general, Aphanizomenon sp. is more common in the northern and N. spumigena in the southern area of the Baltic Proper [153], which may reflect the fact that the optimal temperature and salinity for growth of N. spumigena are higher.

The cyanobacteria analyzed here were collected during the bloom that occurred in July of 2003 and consisted mainly of Aphanizomenon flos-aquae (Paper III). The collection utilizing a string bag was performed in the vicinity of Askö Island, (N58o49’, E17o37’), which lies in the southern part of the Stockholm archipelago, i.e., in the north-eastern Baltic Proper. It was not possible to separate cyanobacteria from zooplankton with this sampling method. However, the cyanobacteria were estimated to constitute the major (>90%) proportion of the sample.

Figure 4.4 The red alga Ceramium tenuicorne.

The red alga Ceramium tenuicorne (Kütz.) Waern (Figure 4.4), a common fast-growing filamentous macroalgae, is favoured by the increased amount of nutrients in the Baltic Proper. The C. tenuicorne algae, analyzed in Papers I and III, were collected by diving at the end of June of 2000 at a depth of 3-4 meters and close to Askö Island. Following collection, the algae were compressed in order to remove most of the water present; frozen in brown

Camilla Bollner/azote.seCamilla Bollner/azote.se


- 23 -

glass jars, stored at -20°C and later thawed and cut into pieces 0.5-1 cm in size with a pair of scissors prior to extraction and further analysis.

Blue mussels (Mytilus edulis), a key species in the littoral food web, accounts for more than 90 % of the animal biomass in the Baltic Sea [154]. The average sizes of mussels (M. edulis) along the west coast of Sweden is approximately 10 cm, while the size of the same species in the Baltic Sea, is seldom more than 3 cm, due to the physiological stress caused by the lower salinity. Mussels are commonly used for monitoring levels of contaminants, e.g., PCBs and PBDEs [21].

The blue mussels (M. edulis) analyzed in Paper I was collected at the same site and depth as the alga and cyanobacteria (see above). For the studies documented in Paper II, blue mussels were collected in September of 1999 at a depth of 1-2.5 meter along the east coast of Sweden close to Oxlesund (N57o33’6’’, E16o42’0’’) in the Baltic Proper. The mussels were suspended in a string bag overnight in order to eliminate excrement and the entire mussel tissue then stored at -20°C until being analyzed in September of 2003.

The collection of fish, mussels, shrimps and crabs for the investigation described in Paper IV was coordinated by the Swedish Environmental Protection Agency, the National Food Inspectorate and the Swedish Museum of Natural History. These samples were either collected from background areas for purposes of environmental monitoring or from commercial fishing vessels or fish markets for purposes of food control. The mussels from the west coast of Sweden were collected (N57o60’, E9o0’) for food control in 2001. Both the collection and preparation were performed by the National Food Inspectorate in accordance with the guidelines for monitoring the levels of dioxins. Shrimps and crabs were collected at the same site (N58o36’, E11o05’) and the muscle tissue from crabs and the whole shrimps were analysed. Eels (Anguilla anguilla) were also collected for purposes of food control from several locations along the east coast of Sweden, as well as from freshwater lakes (for further details, see Paper IV).

Perch (Perca fluviatilis), a littoral fish, were collected along the east coast of Sweden and from freshwater lakes for purposes of environmental monitoring. Herring (Clupea harengus), a pelagic fish were collected at three different locations, i.e., in the Bothnian Bay and Baltic Proper as well as along the Swedish west coast, again for environmental monitoring. These fish were collected in early autumn according to the schedule of the Swedish Environmental Monitoring Programme. Tissue taken from the middle dorsal muscle layer, of 3-18 fish (see Paper IV for details) was homogenized and used for the analyses. The collection and preparation of these samples were performed by personnel at the Swedish Museum of Natural History.


- 24 -

The blue mussels used for charting the trend in PBDD levels during the period of 1995-2003 (Paper IV) were collected at Kvädöfjärden (N58o02’, E16o46’) using the routine procedures of the Swedish Environmental Monitoring Programme [21].

4.2 Extraction methods

Since the main purpose of the work presented in this thesis was to identify OH-PBDEs, MeO-PBDEs and PBDDs present in biota of the Baltic Sea, extraction methods used routinely at the laboratories were applied initially. It was important to avoid artefacts, including debromination, and these methods are mild, involving no heating or high pressure. They have been utilized successfully to extract lipophilic, neutral and/or phenolic compounds from various matrices, including mussels, fish (www.quasimeme.org) and blood [155], with the exception of the extraction method applied in Paper IV, which is adaptable only for neutral compounds. It is important in connection with comprehensive environmental analysis to extract both lipids and analytes (neutral and phenolic substances) quantitatively. This is usually performed with mixtures of polar and unpolar solvents.

For extraction of the cyanobacteria a liquid-liquid extraction method was applied, which originally was developed for the extraction of phenolic and neutral organohalogens from plasma [155]. The general extraction and clean-up procedure utilized with the cyanobacteria is illustrated in Figure 4.1. Briefly, hydrochloric acid and 2-propanol were added to a concentrated suspension of cyanobacteria prior to the addition of n-hexane:methyl tert-butyl ether (MTBE) for extraction (Paper III). The main reasons for initially choosing this method were the ability to extract phenolic compounds, as well as the possibility to practically handle a matrix like cyanobacteria during the extraction procedure.

The method used for extraction of both filamentous algae and mussels (Papers I-III), originally developed by Jensen and co-workers [10,156], involves homogenization of the matrices; extraction with acetone:n-hexane, to achieve dehydration and denaturation of proteins; subsequent extraction with n-hexane:MTBE; and, finally, partitioning with an acidic aqueous solution, containing sodium chloride. The purpose of this last step was to protonate phenolic compounds and, thereby, promotes their partitioning into the organic phase.

The fish, mussels (from the west coast) and shellfish examined in Paper IV were only analysed for their content of PBDDs, applying another extraction method [157]. In this case the tissues were homogenized with an


- 25 -

Figure 4.1 The general approaches applied for analysis of OH-PBDEs, MeO-PBDEs and PBDDs in algae, mussels and cyanobacteria (Papers I and III).

excess of sodium sulfate to dehydrate the samples. The homogenates were extracted with acetone:n-hexane and further with n-hexane:diethyl ether.

Besides the extraction methods applied in this thesis, several alternative methods have been described in the literature, but the most of these were developed to obtain neutral compounds such as MeO-PBDEs, and are therefore not applicable for extraction of phenolic compounds. Some examples of such alternative approaches include; microwave-assisted extraction (MAE) [127,158], super-critical fluid extraction (SFE) [131] and accelerated solvent extraction (ASE) [133,159,160].

Determination of extractable material

Separation of Neutral and Phenolic compounds

Phenolic compoundsNeutral compounds

Derivatization

GC-LRMS (ECNI)

MeO-PBDE, PBDD

GC-LRMS (ECNI)

OH-PBDE

Clean upSiO2:H2SO4 column

SiO2 column


SiO2 column

Algae & Mussels Cyanobacteria

ExtractionExtraction

Determination of extractable material

Separation of Neutral and Phenolic compoundsSeparation of Neutral and Phenolic compounds

Phenolic compoundsNeutral compounds

Derivatization

GC-LRMS (ECNI)

MeO-PBDE, PBDD

GC-LRMS (ECNI)

OH-PBDE


SiO2 column


SiO2 column

Algae & Mussels Cyanobacteria

ExtractionExtraction


- 26 -

In MAE a mixture of sample and solvent are exposed to microwaves and the efficiency of the extraction depends both on the nature of the sample and of the solvent. MAE is rapid, but the energy input must be optimised for each individual type of sample [161]. For solvents with low dipolar moments, relatively high levels of energy are required for effective extraction, which may result in dehalogenation of brominated compounds. Therefore, polar solvents or solvent mixtures are usually employed.

ASE, which involves the use of conventional solvents under enhanced temperature and pressure [162,163], has been applied successfully for extraction of organohalogens in a number of studies [133,159,160,164]. With this approach, Saito and co-workers [164] extracted neutral brominated compounds, such as PBDEs, with high recoveries. In contrast, low recoveries of phenolic compounds, e.g., pentachlorophenol (PCP) and OH-PCB, were achieved when a sodium sulfate column was utilized to dehydrate the primary extract. The recoveries of phenolic substances were improved by gently shaking the ASE extract with an acidic aqueous solution [156], but the overall recovery of OH-PCBs was still lower than that of most of the neutral compounds investigated [164].

In addition, several methods for isolating similar types of compounds have been developed in the area of natural product research. For example, MeO-PBDEs and similar halogenated phenolic substances have been isolated from algae by leaching with 95% ethanol for a month, subsequent reduction of the solvent volume and, finally partitioning of the algal extract between water and chloroform [69]. In addition, Pedersén first allowed algal material to stand in aqueous solutions of different pH and then extracted these solutions with ethyl acetate [61]. Furthermore, extraction with chloroform and partitioning with n-hexane [82,83] or successive extraction with acetone and methanol [78] have also been utilized. Unfortunately, many of these approaches have been used primarily for identification and their usefulness for quantitative purposes cannot easily be assessed.

An effective and reproducible method for extraction of lipids is required for selection of an optimal extraction method. The procedures developed by Bligh and Dyer [165] and by Folch [166] are classical in this connection. In addition, the original Jensen method [156] used in Papers I-III is known to provide satisfactory lipid yields when extracting samples with a fat content greater than 1%. However, in the case of lean fish with a fat content of less than 1% and, containing a relatively large proportion of polar phospholipids, application of the original Jensen method results in inadequate yields of lipids.


- 27 -

Therefore the method was recently modified by replacing acetone with 2-propanol and n-hexane with diethyl ether in the initial step [167]. The modified method gave lipid recoveries comparable with the Bligh and Dyer and the Folch method. Since blue mussels are rich in polar phospholipids the modified Jensen method will be preferable for extraction of blue mussels in the future. How efficient extractable organic matter (EOM) is isolated from algae and cyanobacteria with the methods applied has not been investigated.

4.3 Clean-up and separation of different classes of substances

The clean-up procedure has to be suited for elimination of co-extracted material, but without degradation or loss of the compounds to be analyzed. Environmental samples often contain a large number of halogenated substances, and the concentrations of these may range over at least six orders of magnitude. Therefore, it is essential to separate substances into classes prior to instrumental analysis, especially when analysing minor compounds. Moreover, in order to allow characterization of unknown compounds, efficient separation into chemical classes and removal of lipids are required. The clean-up approaches utilized in Papers I-III are illustrated in Figure 4.1 and Figure 4.2.

4.3.1 Determination of extractable material All determinations of biological material (dry, fresh or extractable) were done gravimetrically. The weights of extractable lipids and/or organic matter, as well as fresh material, were measured for all of the samples, with the exception of cyanobacteria. The dry weights of mussels and filamentous algae were determined as presented in Papers II and III.

4.3.2 Derivatization In order to improve the chromatography and, thereby, detection of compounds present at low levels, the phenolic compounds are derivatized. Methylation with diazomethane [168] provides stable derivatives and is the method used for derivatization of halogenated phenolic compounds (HPCs) in this thesis. The capability of diazomethane to methylate OH-PBDEs was investigated by derivatization of standards of known concentrations, and the result showed complete methylation (unpublished). Laboratory use of diazomethane requires a special permit due to the cancer risks of this compound. The use of diazomethane has been approved by the Swedish work environment authority.


- 28 -

Figure 4.2 The general procedure, as applied in Paper II, for extraction, isolation and analysis of PBDDs.

Acetylation and silylation were investigated as alternative derivatization methods. However, the acetyl and silyl derivatives obtained proved to be unstable during clean-up and were therefore not applied in the present work (unpublished). Another disadvantage associated with the use of these derivatization reagents is that the derivatives formed have molecular weights, higher than those formed with diazomethane, resulting in long retention times on GC columns.

Lipid Determination

Lipid RemovalSulfuric acid (conc.)

Polarity separationSilicagel column

Separation on number of aromatic ringsAminopropyl column

Two aromatic ringsOne aromatic ring

Isolation of planar compoundsPYE column

Coplanar compoundsLess planar compounds

Removal of bleeding from the PYE columnMultilayer column

GC-HRMS (SIM)

Three aromatic rings

Mussels

Extraction

Lipid DeterminationLipid Determination

Lipid RemovalSulfuric acid (conc.)Lipid Removal

Sulfuric acid (conc.)

Polarity separationSilicagel column



Two aromatic ringsTwo aromatic ringsOne aromatic ringOne aromatic ring



Coplanar compoundsCoplanar compoundsLess planar compoundsLess planar compounds



GC-HRMS (SIM)GC-HRMS (SIM)

Three aromatic ringsThree aromatic rings

Mussels

Extraction

MusselsMussels

Extraction


- 29 -

4.3.3 Elimination of matrix and lipids In Papers I-III concentrated sulfuric acid was utilized to eliminate most of the lipids and/or matrix. This is a destructive, but also an effective method in which the carbonyl groups of glycerides form charged complexes with the sulfuric acid. Accordingly, the lipids will thereby stay in the sulfuric acid phase while, other hydrophobic compounds are recovered in the organic phase. Indeed, not all analytes are resistant to treatment with concentrated sulfuric acid, a limitation which must be taken into consideration before application of the method.

Alternatively, non-destructive removal of lipids can be achieved by gel permeation chromatography (GPC) [169] on a cross-linked polystyrene gel (BioBeads), as demonstrated in a number of studies [38,159,160]. The general separation principle is based on the size of the molecule where molecules with a large volume are excluded from the pores in the beads and are, therefore, less well retained than small molecules. Other factors of importance for the retention of the analytes are choice of mobile phase and degree of planarity and polarity of the analytes [169]. GPC is useful for isolation of, e.g., phenolic compounds without any requirement of derivatization. When this technique was applied in attempt to remove irrelevant material from algae extracts, it proved to be difficult to elute the algal matrix from the column and, thereby, recondition the column (unpublished). Accordingly, this approach could not be employed. An additional non-destructive method for purification of algal samples, involving acetonitrile partitioning with n-hexane was also examined, but, unfortunately the matrix and substances to be analyzed partitioned in a similar manner.

Filtration through columns of silica gel, acid-impregnated silica gel or multilayer columns (Papers II and IV) was used as a last step in the purification of the samples in this thesis.

4.3.4 Separation of different classes of substances As already mention, GPC can be applied to separate different classes of substances on the basis of size. For example, high resolution GPC has successfully been used for separation of, e.g., PCBs from polychlorinated naphthlenes (PCNs) [170], while polychlorinated alkanes can be separated from PCBs by normal GPC [171].

Another approach to the separation of different chemical classes of compounds is adsorption chromatography, which can be performed either as simple open-column chromatography or by high-performance liquid chromatography (HPLC). With this approach different phases and/or solvents


- 30 -

must be utilized in order to optimize the separation. In addition, simple partitioning between different solvents can also be used for the separation of classes of substances [155,172,173]. Adsorption chromatography using silica gel was performed for separation of substances of different characteristics in Papers II and III. In these studies PBDDs were separated from the slightly more polar MeO-PBDEs.

Isolation of tricyclic aromatic compounds, including PBDDs, from aliphatic, monocyclic substances and compounds with three or more aromatic rings can be achieved utilizing an aminopropyl column [174]. However, since the aminopropyl column does not separate different types of compounds containing two aromatic rings, e.g., PCBs and dioxins, additional steps are required for the isolation of PBDDs.

The classical methodology for isolation of coplanar compounds from other substance is based on the shape-selectivity of activated carbon [175]. Carbon column chromatography was applied for isolating PBDDs as described in Paper IV. The non-dioxin like compounds are eluted with n-hexane, followed by n-hexane:dichloromethane. The column elution direction is reversed before eluting the PBDDs and other dioxin like chemicals with toluene.

2-(1-Pyrenyl)-ethyldimethylsilylated silica (PYE) HPLC column, another alternative for separation of coplanar chemicals from non-planar [176,177], was applied in Paper II to isolate PBDDs. The first fraction is obtained by applying n-hexane as the mobile phase in forward direction, while the second fraction (containing the PBDDs) is eluted from the column with DCM in the reverse direction. As a final step prior to analysis by GC-MS, a multilayer column has to be introduced to remove bleeding from the PYE column. One advantage of a PYE column, over a column containing activated carbon, is that in the former non-aromatic solvents can be used as mobile phases, allowing the chromatographic process to be monitored by UV spectroscopy.

Partitioning with potassium hydroxide is useful for separating phenolic from neutral compounds [155]. This method was used for isolation of HPCs as described in Papers I and III.

4.4 Instrumental analysis

Gas chromatography (GC) coupled to an electron capture detector (ECD) is widely used for environmental analysis due to its sensitivity and selectivity towards halogenated compounds. In Paper III GC-ECD was used for quantification of the MeO- and OH-PBDEs present in red algal samples. The detection of compounds by GC-ECD relies on comparison of retention times in the sample relative to retention times of authentic reference standards and


- 31 -

is therefore dependent of no co-eluting interferences. Accordingly, it is not possible to identify unknown compounds with GC-ECD alone.

Mass spectrometry (MS) provides more information and selectivity and is in many cases highly sensitive. Since both chlorine and bromine occurs in two forms in nature, (i.e., 35Cl/37Cl and 79Br/81Br, respectively), MS analysis of compounds containing these elements gives rise to typical patterns of isotope distribution. These patterns reveal both the presence and numbers of chlorine and bromine atoms in a compound.

In this thesis, GC was utilized in combination with two types of MS instruments, i.e., low resolution (LR) quadrupole and high resolution (HR) magnetic sector systems, for identification of OH-PBDEs, MeO-PBDEs and PBDDs in Papers I-IV, as well as for quantification of PBDDs in Papers II-IV. Two different ionization techniques were applied in the papers.

Electron ionization (EI), a reproducible technique that is to a large extent independent of the instrument used, generally provides detailed structural information and unique fragmentation patterns for each compound or group of compounds. There are disadvantages with the EI technique such as high background levels in full scan mode, and if the compounds are easily fragmentized, structural data may be lost. HRMS in the combination with EI using single ion monitoring (SIM), shows low detection limits compared to LRMS (EI, SIM). This is due to the high resolution provided by these instruments, which give higher selectivity, reduces the chemical noise and thus increases the signal-to-noise ratio.

Electron capture negative ionization (ECNI) is a sensitive technique for electronegative compounds which makes it to a useful tool for analysis of halogenated compounds. It is also a “soft” ionization technique in which a buffer gas such as ammonia or, most commonly, methane is bombarded with electrons to create low energy electrons (thermal electrons). The analyte capture the thermal electrons to generate negative ions through two primary processes, as follows:

Associative electron capture AB + e- (thermal) AB- •

Dissociative electron capture AB + e- (thermal) A- + B•

Associative electron capture provides the molecular weight information of the compound, while dissociative electron capture can yield information about fragment ions. Which process that occurs is dependent on the chemical structure of the compound of interest, as well as the MS system, and the MS


- 32 -

conditions. Brominated compounds such as MeO-PBDE primarily undergo dissociative electron capture, forming free bromide ions, while chlorinated compounds such as PCBs (with more than 3 or 4 chorine atoms) undergo mainly associative electron capture. The reason is that bromide ions have better property to act as a leaving group and that chlorinated compounds can, in general, stabilize the molecular ion. However, compounds containing several bromine atoms will give rise to both bromide ions and molecular ion.

PBDEs have traditionally been determined with ECNI, by monitoring m/z 79 and 81 [178], which provides excellent selectivity towards brominated compounds and very low limits of detection [179,180]. However, although this approach provides the retention time of the compound, no structural information can be obtained.

4.5 Quality aspects on the analyses

Quality assurance for the identifications, quantifications and analytical procedures applied in this thesis are described briefly below. It should be stressed that our main interest was the identification of compounds and, therefore, the quality assurance focused on this aspect.

4.5.1 Identification and quantification To analyze the compounds on several GC columns is an important step in the procedure of identification of compounds. By applying several GC-columns with different separation properties the risks for co-elution of analytes will be reduced. The observation of identical retention time for a compound and its reference standard on columns with different polarity renders the identification more reliable, as does the structural information provided by the fragmentation patterns detected by MS. The general behaviour of the substance during the analytical procedure and accurate determination of the mass provide additional information supporting the tentative identification of an “unknown” compound.

The identification of the OH-PBDEs and MeO-PBDEs present in the alga and mussels (Paper I) was achieved by comparisons of the MS spectra (ECNI and/or EI) of the compound with the spectra of authentic reference standards, together with comparisons of relative retention times (RRTs) of the compounds and RRTs of authentic reference standards on two GC columns with different polarity.

The triBDDs (Paper II) were identified by comparison of the MS (EI and ECNI) fragmentation patterns, and RRTs on three different columns with those of reference standards. Finally, the identity was verified by accurate determination of the mass of the triBDD.


- 33 -

However, the question as to whether PBDDs might be formed artificially during clean-up or analysis had to be addressed. For instance, the partitioning procedure employed in Paper I involved the use of diluted potassium hydroxide, which may hypothetically, result in formation of PBDDs. However, when authentic OH-PBDE standards were treated with diluted potassium hydroxide for several hours [181], no formation of PBDDs was detected.

Another potential route of PBDD formation during analysis involves ring closure of 2-OH-PBDEs in the injector and/or the GC column, as has been observed for polychlorinated predioxins [182,183]. This issue was addressed by utilizing fractionation on a silica gel column, to remove phenols and MeO-PBDEs, prior to gas chromatographic analysis (Papers II and III). In addition, a standard of OH-PBDE was injected into the GC-MS system to look for possible formation of PBDDs. In neither case were any PBDDs detected.

A separate study was performed to determine any potential artificial formation of OH-PBDEs via dimerization of simple phenols in the algal sample. Part of the study included extraction by addition of ascorbic acid to prohibit any oxidation (Paper I).

The PBDFs indicated in the alga and mussels (Papers II and III) need further verification, due to the lack of standards and mass interference by other compounds, such as PCBs and PBDEs. With the method employed the possibility that residues of these compounds may still be present in the samples can not be excluded. One way to avoid interference by the chlorinated compounds would be to use ECNI and monitoring m/z 79 and 81. However, this approach will not solve the problem of possible interference by brominated substances such as PBDEs. To overcome this difficulty, comparison with authentic reference standards, as well as very high MS resolution (20000-30000) or accurate mass determinations of PBDFs are required [184].

In the studies where quantification was performed the surrogate standard was chosen to be structurally as similar as possible to the analytes. Quantification of the MeO-PBDEs and OH-PBDEs, present in the red algae (Paper III) was performed by GC-ECD using single point authentic external reference standards. The linearity, of the GC-ECD instrument was investigated by running standards at multiple concentrations in parallel with the samples. The concentrations of the external standards employed were found to be within the linear range of the GC system.

The concentrations of PBDDs, in Papers II-IV, were estimated by analyzing the samples on GC-HRMS (EI, SIM), monitoring two ions, one quantification ion, and one qualifier ion (the two most intense ions of the molecular ion


- 34 -

isotope distribution clusters) for every substance. The limits of detections (LODs) for the di-, tri- and tetraPBDDs were defined as 3 times the levels of the noise of the analytes in the different samples analysed in Paper IV (for further details Paper IV).

4.5.2 Recovery A recovery study was performed in Paper III by measuring the recovery of the surrogate standard (SS) in relation to a recovery standard (RS). The mean recovery of the SS in neutral fractions was 97 % (n=6), with a range of 78-103%. The mean recovery of the SS in the corresponding phenolic fractions was 98%, with a range of 95-101%.

4.5.3 Analytical procedures Possible contamination originating from the solvents, laboratory equipment and/or instruments, was controlled by running procedure solvent blanks in parallel to the samples. Since brominated compounds are known to undergo debromination when exposed to indoor light or sunlight, all samples were kept in the dark or covered with aluminium foil throughout the entire procedure.

The method used in Paper II for isolation of PBDDs in mussels was validated utilizing PBDD standards. The extraction method applied for extraction of the cyanobacteria (Paper III) needs to be evaluated with recovery standards before using this method for quantification purposes. However, in the present study no quantitative data were reported (Paper III).

Although the original Jensen method, applied in Papers I-III has been evaluated with respect to analysis of organohalogens in biological matrices such as fish and mussels (www.quasimeme.org), no extensive effort has been made to evaluate and optimize this method for extraction of OH-PBDEs, MeO-PBDEs and PBDDs from algae.

Additional results

- 35 -

5 Additional results

This chapter is intended to add some unpublished data that are related to this thesis. These data are aimed to contribute to the understanding of polybrominated compounds present in Baltic Sea biota.

Polysiphonia fucoides is one of the algal species in the Baltic Proper, which is favoured by increased levels of nutrients, and is now present in huge quantities along the coasts of the island of Öland (cf. chapter 1, Figure 1.3). Preliminary results from analyses reveal that P. fucoides contains a large number of brominated phenolic compounds as visualized in the GC-MS chromatogram (Figure 5.1). This chromatogram shows that the phenolic fraction of an extract of P. fucoides also contains a compound with six bromine atoms and a molecular ion at m/z 734. Furthermore, a number of isomers and homologues of bis(polybromo-dihydroxybenzyl) ethers appeared to be present in the extract, but it was not possible to verify the identity of these compounds due to the lack of authentic reference standards. In addition, the triBDD identified in C. tenuicorne (Paper III) was also found in P. fucoides, although in lower concentration (unpublished).

Figure 5.1 A full-scan GC-MS (ECNI) reconstructed ion chromatogram (RIC) of the phenolic fraction isolated from P. fucoides. The peaks marked with an asterix (*) do not yield the m/z 79, 81 ions.

10.0 15.0 20.0 25.0 30.0 35.0

*

*

*

* * ** *

m/z 734

Tribromophenol

10.0 15.0 20.0 25.0 30.0 35.010.0 15.0 20.0 25.0 30.0 35.0

*

*

*

* * ** *

m/z 734

Tribromophenol

Additional results

- 36 -

The P. fucoides algae analyzed here were collected over a period of three years (2000-2002) and clear variations in the level of the compound corresponding to m/z 734 were observed (data not shown). However, since the samples were taken at different times each year, it is not clear whether these variations are annual or seasonal. Of interest in this context is the observation that the concentration of bromophenol in the green alga (Ulva lactuca) exhibits seasonal variations, being relatively high in late summer and low during the rest of the year [66].

Moreover, when P. fucoides starts to decompose, intense red leakages occur, visually traceable for several hundred meters out from the shore where the alga has ended up. There are certain indications that exudates from P. fucoides have negative effects on the reproduction of Fucus vesiculosus [6], as well as disturb the behaviour of fish (Popatoshistus microps) [185]. When Del Barga and co-workers [186] recently exposed rainbow trout (Oncorhynchus mykiss) to exudates from P. fucoides, significant induction of single-strand breaks (SSB) in DNA occurred, with a frequency comparable to that induced by exposure to 20 mg benzo[a]pyrene per kg. It is not yet known which compound(s) that cause this genotoxic effect observed in the trout, but analysis of plasma (by monitoring m/z 79, 81) indicated the presence of a larger number of brominated compounds in exposed than in unexposed (control) fish (unpublished). The difference was most pronounced in the earlier region of the chromatogram, where the simple brominated phenols elute.

Analyses of the P. fucoides exudates were performed in the same manner as the plasma of trout (cf. above). Comparisons between peaks in the exudates and peaks present in the exposed fish showed that several peaks were only present in the exposed fish and not detectable in the algal exudates (unpublished). Possibly, compounds in the algal exudates are transformed or degraded in the aquaria and/or metabolized by the fish, giving rise to a larger number of compounds in the fish. Further analytical efforts are required to find out the composition of chemicals in the algal exudates. Even if several brominated compounds are present in the algal exudates, the substances responsible for the SSB in the trout may not necessarily be halogenated, and therefore also non-halogenated compounds need to be assessed.

The brown filamentous alga Pilayella littoralis was examined in another pilot study (unpublished), and both OH-PBDEs and MeO-PBDEs were detected in the alga, but at lower concentrations compared to the concentrations in C. tenuicorne.

Additional results

- 37 -

Figure 5.2 GC-MS (ECNI) chromatogram of bromide ions (m/z 79, 81). The upper chromatogram shows the methylated phenolic fraction and the lower shows the corresponding neutral fraction of the flounder.

In an investigation of blue mussels exposed to P. littoralis in the field, the “Pilayella mussels” were found to contain all of the OH-PBDEs and MeO-PBDEs (unpublished), which where identified earlier in mussels collected in the vicinity of the red alga C. tenuicorne (Paper I). Exudates from the P. littoralis exert toxic effects at early stages of Fucus vesiculosus [7] and, in addition the survival of juvenile Gammarus spp has been shown to decrease

6-O

H-B

DE9

9

6-O

H-B

DE9

0

2´-O

H-B

DE6

8

6-O

H-B

DE4

7

I.S.

Phenolic fraction

21:40 23:20 25:00 26:40 28:20

6-M

eO-B

DE9

0

6-M

eO-B

DE4

7

2´-M

eO-B

DE6

8

I.S.

Neutral fraction

6-O

H-B

DE9

9

6-O

H-B

DE9

0

2´-O

H-B

DE6

8

6-O

H-B

DE4

7

I.S.

Phenolic fraction

6-O

H-B

DE9

9

6-O

H-B

DE9

0

2´-O

H-B

DE6

8

6-O

H-B

DE4

7

I.S.

6-O

H-B

DE9

9

6-O

H-B

DE9

0

2´-O

H-B

DE6

8

6-O

H-B

DE4

7

I.S.

Phenolic fraction

21:40 23:20 25:00 26:40 28:20

6-M

eO-B

DE9

0

6-M

eO-B

DE4

7

2´-M

eO-B

DE6

8

I.S.

21:40 23:20 25:00 26:40 28:2021:40 23:20 25:00 26:40 28:20

6-M

eO-B

DE9

0

6-M

eO-B

DE4

7

2´-M

eO-B

DE6

8

I.S.

Neutral fraction

Additional results

- 38 -

when exposed to P. littoralis (unpublished). Moreover, the high mortality among eggs of the Baltic herring, observed both in the field and laboratory, has been proposed to be a consequence of exposure to exudates from P. littoralis [40-42].

Flounder (Platichthys flesus) in the Baltic Sea feed mainly on blue mussels (Mytilus edulis), which commonly live in the vicinity of macroalgae and, thereby, the flounder represent a high trophic level organism in a relatively straight food web. Since flounders are used for human consumption they also represent a species in the human food web. In the present study the flounders were collected close to Öland, where P. fucoides is the dominant algal species.

The phenolic fraction isolated from flounder plasma contained four OH-PBDEs (2’-OH-BDE68, 6-OH-BDE47, 6-OH-BDE90 and 6-OH-BDE99) and in the corresponding fraction containing neutral compounds, 2’-MeO-BDE68, 6-MeO-BDE47 and 6-MeO-BDE90 were detected by comparison of their RRTs with authentic reference standards by monitoring the bromide ions (m/z 79, 81) with GC-MS (ECNI) (Figure 5.2). The GC-MS analysis was performed applying only one column requesting further confirmation of the structures suggested above (unpublished). However, it is worth noting that the pattern of both the OH-PBDEs and MeO-PBDEs in flounder plasma resemble the patterns of OH-PBDEs and MeO-PBDEs in salmon plasma [90] (Paper I).

General discussion

- 39 -

6 General discussion

This chapter discuss the main findings of this thesis, as presented in Papers I-IV and in chapter 5.

6.1 The presence of OH-PBDEs and MeO-PBDEs in the Baltic Sea

In the studies presented in Paper I, several OH-PBDEs and MeO-PBDEs were identified in the red alga (Ceramium tenuicorne) and blue mussels (Mytilus edulis) collected from the Baltic Sea. One of the compounds, 6-MeO-BDE137, is, to the best of my knowledge, reported for the first time in Paper I. OH- and MeO-PBDEs were also detected in cyanobacteria (Aphanizomenon flos-aquae) collected from the same area (Paper III).

In connection with a PCB inventory study [187], presented in part in Paper II, MeO-PBDEs were found in mussels living at a number of different locations along the east coast of Sweden. The presence of OH-PBDEs and MeO-PBDEs in several species of filamentous macroalgae (unpublished and Paper I), cyanobacteria (Paper III) and mussels (Paper I), as well as in flounders (see chapter 5), confirms that these compounds are widely distributed in the Baltic Sea environment.

6.1.1 Sources of OH-PBDEs and MeO-PBDEs As far as we know there are no primary anthropogenic sources of OH-PBDEs and MeO-PBDEs, i.e., none of these classes of compounds are formed as by-products or produced commercially. A potential source of traces of OH-PBDEs may be reactions between OH radicals and PBDEs in the atmosphere [188], in a manner similar to the formation of OH-PCBs from PCBs [189]. However, it is highly unlikely that such a minor source can explain the presence of OH-PBDEs and MeO-PBDEs in the biota examined in this thesis. In contrast, it is well known that both of these types of compounds are isolated as natural products in marine sponges [71-73,77,78,105], ascisidians [103,104] and algae [69]. Table 6.1 lists the OH- and MeO-PBDEs shown by others to be natural products, together with the OH- and MeO-PBDEs identified in the present thesis.

In addition, OH-PBDEs are formed by metabolism of PBDEs [95-100,102]. In general, the hydroxy group of OH-PBDEs of metabolic origin is located meta or para to the oxygen of the diphenyl ether. In contrast, all of the OH-PBDEs that we identified in red alga and blue mussels (Table 6.1) have their hydroxy group in one of the ortho-positions. The MeO-PBDEs identified (2’-MeO-BDE68, 6-MeO-BDE47, 6-MeO-BDE85, and 6-MeO-BDE137) are

General discussion

- 40 -

substituted with the methoxy group at this same position (Paper I), which seem to be a general structural feature of OH-PBDEs and MeO-PBDEs of natural origin. Two of these (2’-MeO-BDE68 and 6-MeO-BDE47) have been shown unambiguously to originate from the biosphere, by analyzing their content of the 14C isotope [118]. The origins of natural and anthropogenic OH-PBDEs have been discussed in detail by Marsh [190].

Table 6.1 Overview of the presence of OH-PBDEs and MeO-PBDEs reported to be natural products, together with the corresponding congeners identified in red alga and blue mussels (Paper I).

Compound Species

2’-OH-BDE68 a) Marine sponge [75-78], b) Red alga, blue mussels. 6-OH-BDE47 a) Marine sponge [74-77], a) Tunicates [103,104], b) Red alga, blue

mussels. 6-OH-BDE90 a) Marine sponge [105], b) Red alga, blue mussels. 6-OH-BDE99 a) Marine sponge [73], b) Red alga, blue mussels. 6-OH-BDE85 a) Marine sponge [74,75,79],b) Red alga, blue mussels. 2-OH-BDE123 a) Marine sponge [79], b) Red alga, blue mussels. 6-OH-BDE137 a) Marine sponge [78,80], b) Red alga, blue mussels. 2’- MeO-BDE68 a) Marine sponge [71,83], a) Green alga [69] b) Red alga, blue

mussels, c) Trues beaked whale [118] 6-MeO-BDE47 a) Marine sponge [72], b) Red alga, blue mussels c) Trues beaked

whale [118]

6-MeO-BDE85 b) Red alga, blue mussels. 6-MeO-BDE137 b) Red alga, blue mussels. a) Isolated, structure identified by NMR, and confirmed to be natural products. b) Identified in Paper I by comparison to authentic reference standards. c) Identified as natural products on the basis of the content of 14C.

As shown in Paper III, the concentrations of OH-PBDEs in the same algal samples were two orders of magnitude higher than the concentrations of the PBDEs. This is considered to be an argument for their natural origin since metabolites are rarely present at higher concentrations than their precursors in any biota. An exception is of course DDE being a metabolite of DDT. Further, the levels of OH-PBDEs in the phenolic fraction isolated from the red alga were considerably higher than the MeO-PBDEs in the neutral fraction (Paper III). Although the reason for this is not obvious, one plausible explanation is that OH-PBDEs are the primary products formed by the algae, while MeO-PBDEs may be formed as secondary products, by biomethylation of the corresponding OH-PBDE congeners. Bacteria and fungi can O-

General discussion

- 41 -

methylate brominated and chlorinated phenols [191,192], but it is not yet known whether marine algae can perform this type of methylation, although certain marine arctic macroalgae can methylate mercury and lead [193]. It is also worth mentioning that a similar relationship between the concentrations of simple bromophenols (2,4-dibromophenol and 2,4,6-tribromophenol) and the corresponding anisoles (2,4-dibromoanisole and 2,4,6-tribromanisole) has previously been shown in the red alga Polysiphonia sphaerocarpa collected along the east coast of Australia [64].

Further, a few mixed brominated chlorinated hydroxylated/methoxylated diphenyl ethers were also indicated both in alga and mussels (Paper I). However, due to the lack of authentic reference standards these compounds could not be structurally unambiguously identified. However, the occurrence of such compounds also supports natural production, since mixed diphenyl ethers are not known to be manufactured.

6.2 The occurrence of PBDDs in Baltic biota

Two triBDDs, 1,3,7-triBDD and 1,3,8-triBDD, were identified in blue mussels (M. edulis) collected south of Stockholm in the Baltic Proper (Paper II), as well as in the red alga Ceramium tenuicorne (Papers III and IV). In addition, several other PBDDs were detected, but not structurally identified, in blue mussels, algae and cyanobacteria (Papers II and III). As far as we know, PBDDs have not been identified previously in biota from the Baltic Sea, even though they have been analyzed for [194]. PBDFs also appeared to be present in mussels and alga (Papers II and III), but due to the uncertainties according their identities (see section 4.5.1) they will not be further discussed here.

6.2.1 Distribution and levels of PBDDs in the Swedish environment Following identification of 1,3,7- and 1,3,8-triBDD in blue mussels (Paper II) and of diBDDs and triBDDs in perch from Kvädöfjärden in the Baltic Proper [195], we wanted to investigate the distributions and levels of these compounds in the Swedish ecosystem. Accordingly, fish, mussels and shellfish from several locations in the Baltic Sea (Paper IV), as well as samples from the west coast of Sweden were analyzed for PBDDs (Paper IV). The 1,3,7- and 1,3,8-triBDDs were identified in perch (Perca fluviatilis) and herring (Clupea harengus) from the Bothnian Bay; in perch from one of the sampling locations in the Bothnian Sea, and in all species investigated (i.e., eel, perch, blue mussels and herring) from all sampling locations in the Baltic Proper. Several diBDDs (1,3-/2,7-/2,8-/1,7-/1,8-diBDD) were also identified in these samples, with the exception of herring. Furthermore, 2,3,7-triBDD

General discussion

- 42 -

and 1,3,6,8- and 1,3,7,9-tetraBDD were identified in mussels, perch and eel from the Baltic Proper, but not at all sampling locations.

A number of other PBDDs, were also identified in mussels, crab, shrimps and herring collected along the west coast. Thus, 2,3,7-triBDD, 1,3,6,8-tetraBDD and 2,7-/2,8-diBDD were identified in crabs and 1,3,6,8- and 1,3,7,9-tetraBDD and 2,7-/2,8-/1,7-diBDD in the shrimps. All of the congeners found in the blue mussels from the Baltic Proper were also identified in the blue mussels from the west coast of Sweden, but at 200-fold lower concentrations. The sum of the concentrations of the PBDDs (∑PBDDs) (on a fresh weight basis) was 50 to 2000 times lower in fish, shrimps and crab than in the mussels from the Baltic Sea. Additional PBDDs were detected, but not structurally identified in mussels, perch and eels from the Baltic Sea and in mussels, shrimp and crabs from the west coast (Paper IV).

In contrast, no PBDDs were detected freshwater fish (Paper IV). However, PBDD congeners have recently been detected in lake sediment from Sweden [145]. Perhaps the concentrations of any potential PBDDs, in the analysed fish were too low. However, further analyses of other matrices, such as filter feeding, freshwater mussels are required, to address the question about PBDDs in freshwater biota.

The presence of PBDDs in the algae examined (i.e., C. tenuicorne (Paper II) and P. fucoides (unpublished)), fish, mussels, crabs and shrimp (Papers II and IV), cyanobacteria (Paper III), and the findings of triBDD in mussels living along the east coast of Sweden [187] (Paper II), illustrate the ubiquitous distribution of these compounds in the Baltic Sea. Still it is premature to be too conclusive about the general occurrence of PBDDs in the Baltic Sea environment. This will require further analysis of additional species and compartments of the Baltic ecosystem.

6.2.2 The origin of PBDDs As discussed above (section 2.4.2), PBDDs are not produced intentionally, except for scientific purposes. In addition to the sources already mentioned in section 2.4.2, the results as presented in Papers II-IV, indicate that PBDDs may be natural products, which has not been reported in the literature to date. On the other hand, compounds whose structures are very closely related, i.e., OH-PBDDs and MeO-PBDDs, were reported as natural products in marine sponge some time ago [82,83]. In addition, 4,7,9-tribromo-2-methoxydibenzofuran was recently identified in algae from Japan [70].

The absence of detectable levels of PBDDs in freshwater fish (Paper IV) supports the theory of natural origin of these compounds since, if the PBDDs identified herein, originate from combustion, they would most likely be found

General discussion

- 43 -

in freshwater environments as well. However, PBDDs may be present in freshwater fish at very low concentrations, especially in the light of the fact that PBDD congeners have been detected in freshwater sediment [145]. However, the remarkably high concentrations of the two triBDDs in mussels (160 ng/g fat) and algae (where the concentration is in the same range as that of CB-153) are strong indications that these PBDDs are of natural origin.

In addition to these considerations, a PBDD/F peak pattern, which is dominated by two triBDDs, as in the blue mussels (Paper II), is unlikely to be explained by combustion of BFRs or photolysis of PBDEs, since these processes would normally produce many more congeners. The occurrence of PBDDs in cyanobacteria (A. flos-aquae) also indicates natural production. Potential routes for natural formation of PBDDs, based on the reasonable assumption that the precursor molecules are simple phenols or brominated phenols, which are common natural products, are proposed in Paper IV. Another potential pathway for biogenic formation may involve bromination of suitable precursor molecules, as suggested by Flodin and co-workers [196,197]. One such suitable precursor, for the formation of PBDDs, would be the non-halogenated dibenzo-p-dioxin which has been isolated from ascidian [146].

In summary, the findings presented here, together with the additional knowledge currently available in the literature, provide strong support for the conclusion that PBDDs, and in particular the 1,3,7- and 1,3,8-triBDDs, are produced naturally in biota from the east and west coasts of Sweden. This does not exclude that combustion also being a source of PBDDs.

6.3 OH-PBDEs, MeO-PBDEs and PBDDs in different matrices

To improve the understanding of the complexity of OH-PBDEs, MeO-PBDEs and PBDDs in the species studied, comparisons between patterns and concentrations are presented below. For instance, the concentrations of contaminants such as PCB in algae may fluctuate, as a result of rapid population growth. Rapid algal growth presents a kinetic limitation to sorbtion equilibrium [198,199]. Therefor it is difficult to make a comparison between different studies. Another problem is that the very low content of extractable organic matter (EOM) in algae and cyanobacteria, may lead to errors in concentrations of analyzed compounds if the samples are EOM adjusted. Moreover, the large water content of algae and mussels also renders comparisons based on wet weight uncertain, both within one and the same and between different studies. An alternative is to relate the levels of compounds in the alga to organic carbon (OC), which seem to be appropriate for matrices with low EOM content [200].

General discussion

- 44 -

One approach to express concentrations of lipophilic compounds within matrices where the levels fluctuate is to relate the concentrations of the analytes, in this case OH- and MeO-PBDE in the red algae, to concentrations of well known contaminants such as PCBs and PBDEs in the same sample (Paper III). The concentration of CB-153 was shown to be present in three-fold lower concentration than 2’-MeO-BDE68 in the red alga (Paper III) and the BDE-47 concentration was 10 times lower than this MeO-PBDE (Paper III). The concentration of the triBDD identified in the algal sample was estimated to be 45 ng/g lipid weight, which is in the same range as the concentration of CB-153 in the alga (37-66 ng/g lipid weight). The concentrations of the two coeluting triBDDs (1,3,7-triBDD and 1,3,8-triBDD) in the mussels were as high as 160 ng/g lipid weight (Paper II). Comparison of the levels between these matrices, on lipid weight basis, showed around three to four fold higher concentration of triBDD in blue mussels than in the red alga. Hence, this may be taken as an indication that triBDD is accumulated in the mussels.

The similar patterns of the OH-PBDEs present in cyanobacteria and the red alga Ceramium tenuicorne, are visualized in Figure 6.1, where the major OH-PBDEs are indicated by name. In addition, the patterns of MeO-PBDEs in these two species were also similar (Papers I and III). The fact that the patterns of both the OH-PBDEs and MeO-PBDEs in blue mussels (M. edulis) and red alga (C. tenuicorne) resemble one another (Paper I) is not surprising, since the mussels are filter feeding and live intermingled with the algae on rocky-bottoms.

Interestingly, the patterns of OH-PBDE and MeO-PBDE congeners in the salmon (Salmo salar) [90] (Paper I) and flounder (Platichthys flesus) caught off the coast of Öland (see chapter 5, Figure 5.2) resemble each other, even though their ecology differs. The flounder are stationary and feed mainly on mussels, while Baltic salmon are sea-run and a fish predator.

If the patterns of compounds in the phenolic fraction of fish (chapter 5, Figure 5.2 and Paper I) are compared to the pattern in algae and mussels (Paper I), the 6-OH-BDE137, 6-OH-BDE85, 6-OH-BDE123 are absent, and 6-OH-BDE99 is only present at low concentrations in the fish. In the neutral fraction, the relationship is similar with the absence of 6-MeO-BDE137, 6-MeO-BDE85 and 6-MeO-BDE99 in the two fish species. The reason(s) for these differences is not clear. Perhaps, more highly brominated congeners exhibit lower bioavaibility, or certain congeners are retained selectively in the blood of the fish. Still another possibility may be that the OH-PBDE and MeO-PBDE patterns in the salmon and flounder are influenced by debromination of 6-OH-BDE137, 6-OH-BDE85, 2-OH-BDE123, 6-OH-

General discussion

- 45 -

BDE99, 6-MeO-BDE99, 6-MeO-BDE85 and 6-MeO-BDE137, (all of which are present in the algae and mussels (Paper I)) to yield, e.g., 6-OH-BDE47, 2’-OH-BDE68, 6-MeO-BDE47 and 2’-MeO-BDE68. Debromination has been observed to occur in several species of fish, for example, common carp convert BDE-99 to BDE-47 [201] and pike, trout and carp can transform BDE-209 to less highly brominated diphenyl ethers [202-204].

Figure 6.1 GC-MS (ECNI) chromatograms of bromide ions (m/z 79, 81), derived from the methylated phenolic fraction of the red alga Ceramium tenuicorne (upper) and the cyanobacterium Aphanizomenon flos-aquae (lower). The OH-PBDE congeners indicated in the upper chromatogram were identified in Paper I and those in the lower were tentatively identified in Paper III. The differences in retention times in these two chromatograms reflect the fact that the cyanobacteria sample was analyzed on a non-polar column (DB-5MS, 15 m x 0.25 mm i.d. and 0.1 µm thickness), while the red alga sample was analysed on a non-polar column (CP-Sil 8CB, 30 m x 0.25 mm i.d. and 0.25 µm thickness).

S.S.

2’-O

H-B

DE-

68

6-O

H-B

DE-

47

6-O

H-B

DE-

85

6-O

H-B

DE-

137

6-O

H-B

DE-

906-

OH

-BD

E-99

2-O

H-B

DE-

123

Red alga

2’-O

H-B

DE-

686-

OH

-BD

E-47

6-O

H-B

DE-

85

6-O

H-B

DE-

137

6-O

H-B

DE-

906-

OH

-BD

E-99Cyanobacteria

S.S.

2’-O

H-B

DE-

68

6-O

H-B

DE-

47

6-O

H-B

DE-

85

6-O

H-B

DE-

137

6-O

H-B

DE-

906-

OH

-BD

E-99

2-O

H-B

DE-

123

S.S.

2’-O

H-B

DE-

68

6-O

H-B

DE-

47

6-O

H-B

DE-

85

6-O

H-B

DE-

137

6-O

H-B

DE-

906-

OH

-BD

E-99

2-O

H-B

DE-

123

Red alga

2’-O

H-B

DE-

686-

OH

-BD

E-47

6-O

H-B

DE-

85

6-O

H-B

DE-

137

6-O

H-B

DE-

906-

OH

-BD

E-99Cyanobacteria

2’-O

H-B

DE-

686-

OH

-BD

E-47

6-O

H-B

DE-

85

6-O

H-B

DE-

137

6-O

H-B

DE-

906-

OH

-BD

E-99Cyanobacteria

General discussion

- 46 -

6.4 Impact on the Baltic ecosystem

It is important to ask what implications the findings presented in this thesis might have for the well-being of the Baltic Sea and its wildlife. The most important results presented in this thesis are the identification of PBDDs, OH-PBDEs and MeO-PBDEs in cyanobacteria, the red macroalga Ceramium tenuicorne, blue mussels and fish, at relatively high concentrations. Perhaps the most intriguing result is the identification of triBDDs (1,3,7- and 1,3,8-triBDD) at high concentration in blue mussels.

It is interesting to make a rough calculation of the amounts of PBDDs, OH-PBDEs and MeO-PBDEs that might be present in the Baltic Sea. The net annual production of red filamentous algae, consisting primarily of Ceramium tenuicorne and Polysiphonia fucoides, along the Swedish coast in the Baltic Proper has been estimated to be approximately 570 thousand ton dry weight [205]. Assuming that half of the red filamentous algae are C. tenuicorne and provided that the concentrations of triBDD, ∑OH-PBDEs and ∑MeO-PBDEs are the same throughout the entire alga community, the quantities of these chemicals produced each year by this alga can be estimated to be 0.1 kg (100 g), 40 kg and 1 kg, respectively.

In addition, these compounds have been detected in other filamentous macroalgae (unpublished) as well as, cyanobacteria (Paper III). Further, it is not possible to overlook the possibility of other potential producers. Accordingly, these calculations of the amounts of triBDDs, OH-PBDEs and MeO-PBDEs may be underestimations.

The presence of PBDDs in fish and shellfish may well result in human exposure. Indeed, in perch and eel from the Baltic proper, the ∑PBDD concentration exceeds the ∑PCDD concentration (Paper IV). Data concerning the toxicity of PBDDs are scarce. However, Ah-receptor binding, AHH and EROD enzyme induction data are available for some of the identified congeners (2,7-/2,8-diBDD and 2,3,7-triBDD). If TEQs are assessed from these available REP values the Ah-receptor-TEQ of the crabs and eels, and the EROD- and AHH-TEQs of the Kvädöfjärden mussels are close to the maximum concentration of PCDD/F TEQ that is legally permitted and/or considered as acceptable in food. Furthermore, the potential toxicities of 1,3,7- and 1,3,8-triBDD have not been included in this calculation, since these congeners have not undergone toxicity studies. However, these calculations should be used with great caution since there are a lot of uncertainties therein.

On the other hand, dioxins may exert profound negative effects on health and the environment. For instance, dioxins are associated with the occurrence of the blue sac syndrome in salmonids in the Great Lakes [206]. In this context,

General discussion

- 47 -

2,3,7,8-TBDD has been proposed to be even more potent than 2,3,7,8-TCDD in causing this syndrome [150].

At present, information concerning the toxic effects of OH-PBDEs is also very limited (see section 2.2.3). As mentioned above, OH-PBDEs exhibit antibacterial properties and this is a characteristic property of halogenated phenoxyphenols, such as triclosan [207,208]. Certain OH-PBDEs have been reported to exert endocrine effects, e.g., by binding to TTR [92]. 6-OH-BDE47 inhibits the enzyme aromatase activity [116], which mediates the conversion of androgens to estrogens and may thereby affect the sex ratio of new-born fish.

Pentachlorophenol (PCP) and triclosan can act as uncouplers of the oxidative phosphorylation [209,210] and thereby influence the energy metabolism of organisms. It is not known whether OH-PBDEs can exert a similar effect.

As a consequence of the increases in the levels of nutrients in the Baltic Sea during the past century, species such as mussels and fish, which consume and/or live among algae, may be exposed to elevated levels of the compounds analyzed here. In addition to the high loads of anthropogenic contaminants and the low salinity of the Baltic Sea, such exposure may enhance the stress experienced by these species. One such example is the negative effect exerted by exudates from algae on the survival of herring eggs [40]. However, as indicated by the remarks above, it is difficult to draw firm conclusions from the present results and the need for future research is addressed in the next chapter.

Future perspectives

- 48 -

7 Future perspectives

Several of the OH-PBDEs, MeO-PBDEs and PBDDs identified in this thesis are probably natural products that are formed by algae and cyanobacteria. Further research is required to definitively establish the origin. It is important to open up for identification of potential additional sources of OH-PBDEs MeO-PBDEs and PBDDs in the Baltic Sea ecosystem. This will require investigations of additional species in this environment.

The presence and identities of the MeO- and OH-PBDEs and PBDDs detected in the cyanobacteria Aphanizomenon flos-aquae must be verified by analysis of additional samples and accurate mass determination of the mass of individual congeners. Over all, there is a need for in-depth further identification work.

To characterize the distribution and bioaccumulation of MeO-PBDEs and PBDDs in food webs, additional species need to be analysed. Juvenile eiders (Somateria mollissima) are good candidates in this respect, since they are more or less stationary and feed on mussels that live in the vicinity of algae.

Further analysis of sediment samples in areas where filamentous algae are highly represented may be performed. Through analysis of dated sediment cores it may be possible to gain information about occurrence of these substances over time.

One of the most important issues for the future will be to evaluate the toxicities of OH-PBDEs, MeO-PBDEs and PBDDs, beginning with the compounds identified in this thesis. This is important both from a human and environmental point of view.

Finally, more research is required to address the issue; if climate change is a driver for increased production of the naturally formed organobromines shown to occur in the Baltic Sea biota in this thesis.

Acknowledgements

- 49 -

8 Acknowledgements

Jag vill♫ tacka livet som har gett mej så mycket ♪♫

Jag tänkte inte göra som alla andra utan skriva acknowledgements i tid….och nu sitter jag ändå här i sista sekund,

Det hela började med att en före detta gymnasiekompis (Maria Eliasson) var doktorand hos Joe de Pierre och redan då var Miljökemi känt för att vara en toppenbra institution med massor av roliga fester. Hon övertygade mej om att jag skulle stormtrivas och hon skulle bara veta hur rätt hon hade.

Jag vill börja med att tacka Lillemor, som fått mej att känna att jag klarar ”det”, för att du stått ut då jag envist måste överbevisas innan jag ger mej och för alla härliga skratt som bidrar med minnen att den här tiden varit fantastiskt rolig,

Åke, för din fantastiska drivkraft och för att du genom uppmuntran har förmågan att få en bergsbestigning över Mount Everest att framstå som en tur med Bergodalbanan, tyvärr så besitter jag den motsatta egenskapen och oroar mej hellre än gång för mycket än en för lite,

Jana, den mest fantastiska rumskompis och vän man kan önska sej, som när allt känts som värst peppat mej och dessutom med jämna mellan rum fått stå ut med min dessvärre mindre bra sångröst. Ser fram emot ett kommande projekt ihop, det är ju bara den etiska ansökan vi måste slipa lite på. Jag vet att du inte håller med mej, men Jana det hade aldrig gått utan dej!!!

Emelie, min andra underbara rummis, för all hjälp, för dina snilliga små hyss som förgyller tillvaron, för allt från nötblandnig till apelsiner och för att du tappert försökt få mej till ett sundare leverne - jag ska bli bättre gällande den biten framöver,

Ioannis, för alla givande diskussioner om allt mellan himmel och jord, som bidragit till att öka min kunskap på ett antal områden, tänk att kunna slänga sej med datatermer som får datanissarna att höja på ögonbrynen, men också för alla härliga skratt de här åren,

Ulrika, för att du alltid tar dej tid, hur idiotiska frågorna är, för hjälp med allt från referenser till layout (utan dej hade avhandlingen fortfarande varit i bitar), men framför allt för att du förgyller tillvaron ☼ med allt från☺ till − ser fram emot en vin kväll (lör),

Anita, för all hjälp och för din ständiga omtanke, det värmer,

Acknowledgements

- 50 -

Sören, för att du alltid tar dej tid för frågor och diskussioner, för din ofantliga ström av idéer och för att du inspirerar, fick jag bestämma så skulle du få nobelpriset,

Lena Kautsky, för provtagning, granskning av avhandlingen men framförallt för att du bidragit till att öka mitt kunnande på det biologiska området,

Birgit, för att du alltid kommer till min undsättning, för att du lyckas få till att vi träffas allihop, ser fram emot ytterligare en välbehövlig SPA helg,

Per, Hasse och Maggan som från början fick mej att fastna på Miljökemi,

∫Lars Ehrenberg, i mina tankar kommer du och dina underbara historier alltid att finnas kvar∫

Anna Battle, för att du är en fantastik arbetskompis, matte (Torstens & Tysons inlägg) och vän, nu hoppas jag det blir mer tid över för att öka kunskapen på fler plan än det kemiska, en kurs i japanska kanske vore något Hiiiiii,

Maria, hade fler varit som du hade definitivt den här världen varit bättre, Göran för att du alltid har tid att bolla idéer, jag är så glad att du är tillbaka!

Lotta, för ditt underbara skratt, Linda, kommer alltid att tänka på dej så fort jag ser något lilarosa, Hrönn, för att du introducerat mej på Island och hoppas nu att promenaderna kommer att bli fler framöver, Karin Löfstrand för ett intensivt arbete under året som gått och för att det är just du som fortsätter på den här tråden,

Resten av riskgruppen Anna W, Hans, Jeanette, Ulf, Ulla, Hitesh, Ronnie tack vare dej har jag fått öva min knaggliga engelska……

Syntesgänget, Tati, Carl Axel, Anna C, Johan, för att ni alltid ställer upp, Patricia, räddaren i nöden, Lisa, alltid lika omtänksam, Daniel, du är toppen, ditt leende i korridoren kan få en mus att känna sig som en prinssessa,

Nykomlingarna Keri, it is great to have you here, Maria S, Johan Fång och Biplop, jag är övertygad om att ni kommer att stormtrivas,

Alla på institutionen, en extra gång, för hjälp med både layout och granskning av avhandlingen, men framförallt för att ni stöttat mej, jag är lyckligt lottad som har er som jobbarkompisar och handledare, ni är verkligen fantastiska!

Alla före detta medarbetare Britta, Tina, Sara, Anna Sandholm, Stina, Eden, Charlotta, Johanna, Kaj, Karin Nordström och alla andra som jag eventuellt kan ha glömt,

Acknowledgements

- 51 -

Övriga medförfattare Peter Haglund, Yngve Zebür, Erik Greyerz, Takeshi Nakano, Anders Bignert, Karin Wiberg, Sture Bergek, utan er hade det aldrig blivit någon avhandling,

Ulla Eriksson, för att du alltid ställer upp hur mycket du än har att göra, glöm inte −Bahamas väntar, vi behöver bara få ihop en ansökan…..

Kerstin Nylund och Ann-Sofie Kärsrud för all hjälp under åren som gått, Cynthia för språkgranskningar i tid och otid och Anna-Karin för allt från musselextraktioner till GIS och kattvakt,

Alla övriga ITMare, för att ni alltid hjälpt till när jag behövt,

Hillevi, för all hjälp med att få fram artiklar, du är toppen! Torleif, för provtagning, Joe de Pierre för granskning av språket i avhandlingen och alla uppmuntrande ord, nu blir det champagne,

Alla mina vänner, för att ni finns,

Siv världens bästa svärmor, för allt från kattvakt till de godaste matlådorna,

Mina föräldrar, för att ni alltid stöttat och trott på mej, pappa för att du gjort allt du kunnat för att göra mitt liv lättare,

∫Min underbara envisa lilla mamma, jag saknar dej∫

Sist men inte minst vill jag tacka min sambo Christer som bidragit till att det hela faktisk gått vägen. Du besitter alla egenskaper jag söker, tålmodig, förstående, alla de gånger du lagat middag, som blivit kall för att jag ska bara………, för att du tagit hand om allt, hemmet, katter, vänner, svärföräldrar och när allt rasat (vilket det ofta gör om man är tidsoptimist) har du styrt upp allt efter bästa förmåga, som fredagskvällen vid kopiatorn eller semester på Öland för att plocka alger. Tänker inte lova att jag ska bättra mej men,

Jag älskar dej

This thesis was financially supported by the Swedish Research Council FORMAS, the Swedish Environmental Protection Agency, Stockholm Marine Research Centre (SMF) and Carl Tryggers Foundation.

References

- 52 -

9 References

1. Kautsky, L. and Kautsky, N., (2000), The Baltic Sea including the Bothnian Sea and the Bothnian bay, In: Seas in the Millenium - an environmental evaluation, Ed. Sheppard, C.R.C., Elsevier, Amsterdam, 121-133.

2. Elmgren, R., (1989), Man's impact on the ecosystem of the Baltic Sea: energy flows today and at the turn of the century, Ambio, 19, 326-332.

3. Hänninen, J., Vuorinen, I., Helminen, H., Kirkkala, T., and Lehtilä, K., (2000), Trends and gradients in nutrient concentrations and loading in the archipelago sea, northern Baltic, in 1970-1997, Estuarine Coastal and Shelf Science, 50, 153-171.

4. Kautsky, N., Kautsky, H., Kautsky, U., and Waern, M., (1986), Decreased depth penetration of Fucus vesiculosus (L.) since the 1940's indicates eutrophication of the Baltic Sea, Mar.Ecol.Prog.Ser., 28, 1-8.

5. Eriksson, B. K., Johansson, G., and Snoeijs, P., (1998), Long-term changes in the sublittoral zonation of brown algae in trhe southern Bothanian Sea, Eur.J.Phycol., 33, 241-249.

6. Eklund, B., Svensson, A. P, Jonsson, C., and Malm, T., (2005), Toxic effects of decomposing red algae on littoral organisms, Estuarine Coastal and Shelf Science, 62, 621-626.

7. Råberg, S., Berger-Jönsson, R., Björn, A., Graneli, E., and Kautsky, L., (2005), Effects of Pilayella littoralis on Fucus vesiculosus recruitment: implications for community composition, Marine Ecology Progress Series, 289, 131-139.

8. Almesjö, L., (2006), Filamentous, diazotrophic cyanobacteria in the Baltic Sea: abundance and nitrogen fixation, Licentiate Thesis, Department of Systems Ecology, Stockholm University.

9. Bianchi, T. S., Engelhaupt, E., Westman, P., Andrén, T., Rolff, C., and Elmgren, R., (2000), Cyanobacterial blooms in the Baltic Sea: Natural or human-induced?, Limnol.Oceanogr., 45, 716-726.

10. Jensen, S., Johnels, A. G., Olsson, M., and Otterlind, G., (1972), DDT and PCB in herring and cod from the Baltic, the Kattegat and the Skagerrak, Ambio Special Report, 1, 71-85.

11. Jensen, S., Johnels, A. G., Olsson, M., and Otterlind, G., (1969), DDT and PCB in marine animals from Swedish waters, Nature, 224, 247-250.

12. Helle, E., Olsson, M., and Jensen, S., (1976), DDT and PCB levels and reproduction in ringed seal from the Bothnian Bay, Ambio, 5, 188-189.

13. Helle, E., Olsson, M., and Jensen, S., (1976), PCB levels correlated with pathological changes in seal uteri, Ambio, 5, 261-263.

14. Olsson, M., Karlsson, B., and Anland, E., (1994), Diseases and environmental contaminants in seals from the Baltic and the Swedish west coast, Sci.Total Environ., 154, 217-227.

15. Olsson, V., (1963), Berguv och havsörn i fara!, Sveriges Natur, 54, 177-184. 16. Helander, B., (1985), Reproduction of the white-tailed sea eagle, Holarctic

Ecology, 8, 211-227. 17. Helander, B., Olsson, A., Bignert, A., Asplund, L., and Litzén, K., (2002), The role

of DDE, PCB, coplanar PCB and eggshell parameters for reproduction in the white-tailed Sea Eagle (Haliaeetus albicilla) in Sweden, Ambio, 31, 386-403.

References

- 53 -

18. Helander, B., (1998), Toppkonsumenter, Östersjö '97, Stockholms Marina Forskningsråd.

19. Bergman, A., (1999), Health condition of the Baltic grey seal (Halichoerus grypus) during two decades. Gynaecological health improvement but increased prevalence of colonic ulcers, APMIS, 107, 270-282.

20. Helander, B., (2003), The white-tailed sea eagle in Sweden-reproduction, numbers and trends, SEA EAGLE 2000, 57-66.

21. Bignert, A., Nyberg, E., Asplund, L., Eriksson, U., and Wilander, A., (2006), Comments concerning the National Swedish Contaminant Monitoring Programme in Marine Biota, Swedish Museum of Natural History, http://www.nrm.se/download/18.3f4924f310cc92438458000546/Marina_programmet2006.pdf.

22. Bignert, A., Olsson, M., Persson, W., Jensen, S., Zakrisson, S., Litzén, K., Eriksson, U., Häggberg, L., and Alsberg, T., (1998), Temporal trends of organochlorines in Northern Europe, 1967-1995. Relation to global fractionation, leakage from sediments and international measures, Environ.Pollut., 99, 177-198.

23. de Wit, C., (2002), An overview of brominated flame retardants in the environment, Chemosphere, 46, 583-624.

24. Kierkegaard, A., Bignert, A., Sellström, U., Olsson, M., Asplund, L., Jansson, B., and de Wit, C., (2004), Polybrominated diphenyl ethers (PBDEs) and their methoxylated derivatives in pike from Swedish waters with emphasis on temporal trends, 1967-2000, Environ.Pollut., 130, 187-198.

25. Sellström, Ulla, Bignert, Anders, Kierkegaard, Amelie, Häggberg, Lisbeth, de Wit, Cynthia A., Olsson, Mats, and Jansson, Bo, (2003), Temporal trend studies on tetra- and pentabrominated diphenyl ethers and hexabromocyclododecane in guillemot egg from the Baltic Sea, Environ.Sci.Technol., 37, 5496-5501.

26. Olsson, A. and Bergman, Å., (1995), A new persistant contaminant detected in Baltic wildlife: Bis (4-chlorophenyl) sulfone, Ambio, 24, 119-123.

27. Norström, Karin, Olsson, Anders, Olsson, Mats, and Bergman, Åke, (2004), Bis(4-chlorophenyl) sulfone (BCPS) in Swedish marine and fresh water wildlife-a screening study, Environ.Int., 30, 667-674.

28. Olsson, A., (1999), Applications of various analytical chemical methods for exposure studies of halogenated environmental contaminants in the Baltic environment, PhD Thesis, Department of Environmental Chemistry, Stockholm University.

29. Jörundsdóttir, Hrönn, Norström, Karin, Olsson, Mats, Pham-Tuan, Hai, Hühnerfuss, H., Bignert, Anders, and Bergman, Åke, (2006), Temporal trend of bis(4-chlorophenyl) sulfone, methylsulfonyl-DDE and -PCBs in Baltic guillemot (Uria aalge) egg 1971-2001 - A comparison to 4,4'-DDE and PCB trends, Environ.Pollut., 141, 226-237.

30. Kissa, E., (2001), Fluorinated surfactants and repellents, Marcel Dekker, Inc., New York, USA.

31. Holmström, K., Järnberg, U., and Bignert, A., (2005), Temporal trens of PFOS and PFOA in guillemot eggs from the Baltic Sea, 1968-2003, Environ.Sci.Technol., 39, 80-84.

32. Ljungren, L., Sandström, A., Johansson, G., Sundblad, G., and Karås, P., (2005), Rekryteringsproblem hos Östersjöns kustfiskbestånd, Fiskeriverket, http://www.fiskeriverket.se/publikationer/finfo/pdf/2005/finfo05_5.pdf.

References

- 54 -

33. Bengtsson, B.-E., Hill, C., Bergman, Å., Brandt, I., Johansson, N., Magnhagen, C., Södergren, A., and Thulin, J., (1999), Reproductive disturbances in Baltic fish: A synopsis of the FiRe project, Ambio, 28, 2-8.

34. Vuori, K. A. M., Koskinen, H., Krasnov, A., Koivumäki, P., Afanasyev, S., Vuorinen, P. J., and Nikinmaa, M., (2006), Developmental disturbances in early life stage mortality (M74) of Baltic salmon fry as studied by changes in gene expression, BMC Genomics, www.biomedcentral.com/1471-2164/7/56.

35. Bengtsson, B.-E., Hill, C., and Nellbring, S., (1996), Report from the second work-shop on reproduction disturbance in fish, 4534, Swedish Environmental Protection Agency.

36. Norrgren, L., Amcoff, P., Börjesson, H., and Larsson, P.-O., (1998), Reproductive disturbance in Baltic fish, In: Early life stage mortality syndrome, 8-17.

37. Snoeijs, P., (1998), Diatoms and environmental changes in brackish waters, In: The diatoms: Application to the environmental and earth sciences, Eds. Stoermer, E.F. and Smolt, J.P., Cambridge University Press 14, 1-23.

38. Asplund, L. T., Athanasiadou, M., Sjödin, A., Bergman, Å., and Börjesson, H., (1999), Organohalogen substances in muscle, egg and blood from healthy baltic salmon (Salmon salar) and baltic salmon that produced offspring with the M74 syndrome, Ambio, 28, 67-76.

39. Vallin, L., Nissling, A., and Westin, L., (1999), Potential factors influencing reprodutive success of Baltic cod, Gadus morhua: A review, Ambio, 28, 92-98.

40. Aneer, G., (1987), High natural mortality of Baltic herring (Clupea harengus) eggs caused by algal exudates?, Marine Biology, 94, 163-169.

41. Rajasilta, M., Laine, P., and Eklund, J., (2006), Mortality of herring eggs on different algal substrates (Furcellaria spp. and Cladophora spp.) in the Baltic Sea - an experimental study, Hydrobiologia, 554, 127-130.

42. Rajasilta, M., Eklund, J., Hänninen, J., Kurkilahti, H., Kääriä, J., Rannikko, P., and Soikkeli, M., (1993), Spawing of herring (Clupea harengus membras L.) in the archipelago sea, ICES J.Mar.Sci., 50, 233-246.

43. Gribble, G. W., (2003), The diversity of naturally produced organohalogens, Chemosphere, 52, 289-297.

44. Gribble, G.W., (1996), Naturally occuring organohalogen compounds- a comprehensive survey, Springer-Verlag, Wien New York.

45. Neidleman, S.L. and Geigert, J., (1986), Biohalogenation principles, basic roles and applications, Wiely, New York.

46. Gribble, G. W., (1996), The diversity of natural organochlorines in living organisms, Pure Appl.Chem., 68, 1699-1712.

47. Gribble, G. W., (1998), Naturally occurring organohalogen compounds, Acc.Chem.Res., 31, 141-152.

48. Gribble, G. W., (1999), The diversity of naturally occurring organobromine compounds, Chem.Soc.Rev., 28, 335-346.

49. Gribble, G. W., (1999), The natural production of organobromine compounds, Organohalogen compounds, 41, 23-26.

50. Faulkner, D. J., (2001), Marine natural products, Nat.Prod.Rep., 18, 1-49. 51. Faulkner, D. J., (2002), Marine natural products, Nat.Prod.Rep., 19, 1-48. 52. Blunt, J. W., Copp, B. R., Munro, M. H. G, Northcote, P. T., and Prinsep, M. R.,

(2005), Marine natural products, Nat.Prod.Rep., 22, 15-61.

References

- 55 -

53. Gribble, G. W., (2000), The natural production of organobromine compounds, Environ.Sci.Pollut.Res., 7, 37-49.

54. Kladi, Maria, Vagias, Constantinos, and Roussis, Vassilios, (2005), Volatile halogenated metabolites from marine red algae, Photochem.Rev., 3, 337-366.

55. de Carvalho, L. R. and Roque, N. F., (2004), Correlations between primary and secondary metabolites in Ceramiales (Rhodophyta), Biochemical Systematics and Ecology, 32, 337-342.

56. Fan, X., Xu, N.-J., and Shi, J.-G., (2003), Bromophenols from the red alga Rhodomela confervoides, J.Nat.Prod., 66, 455-458.

57. Pedersén, M., Saenger, P., and Fries, N., (1974), Simple brominated phenols in red algae, Phytochemistry, 13, 2273-2279.

58. Kurata, K., Taniguchii, K., Takashima, K., Hayashi, I., and Suzuki, M., (1997), Feeding-deterrent bromophenols from Odonthalia corymbifera, Phytochemistry, 45, 485-487.

59. Kurata, K. and Amiya, T., (1977), Two new bromophenols from the red alga Rhodomela larix, Chem.Lett., 1435-1438.

60. Kurihara, H., Mitani, T., Kawabata, J., and Takahashi, K., (1999), Two New Bromophenols from the Red Alga Odanthalia corymbifera, J.Nat.Prod., 62, 882-884.

61. Pedersén, M., (1978), Bromochlorophenols and brominated diphenylmethane in red algae, Phytochemistry, 17, 291-293.

62. Whitfield, F. B., Helidoniotis, F., Shaw, K. J., and Svoronos, D., (1999), Distribution of bromophenols in species of marine algae from eastern Australia, J.Agric.Food Chem., 47, 2367-2373.

63. Chung, H. Y., Joyce Ma, W. C., Ang, P. O., Kim, J.-S., and Chen, F., (2003), Seasonal variations of bromophenol in brown algae (Padina arborescens, Sargassum siliquastrum, and Lobophora variegata) collected in Hong Kong, J.Agric.Food Chem., 51, 2619-2624.

64. Flodin, C. and Whitfield, F. B., (2000), Brominated anisoles and cresols in the red alga Polysiphonia Sphaerocarpa, Phytochemistry, 53, 77-80.

65. Vetter, Walter and Janussen, Dorte, (2005), Halogenated natural products in five species of antarctic sponges: Compounds with POP-like properties?, Environ.Sci.Technol., 39, 3889-3895.

66. Flodin, Carina, Helidoniotis, Fay, and Whitfield, Frank B., (1999), Seasonal variation in bromophenol content and bromoperoxidase activity in Ulva lactuca, Phytochemistry, 51, 135-138.

67. Führer, U. and Ballschmiter, K., (1998), Bromochloromethoxybenzenes in the marine troposphere of the atlantic ocean: a group of organohalogens with mixed biogenic and anthropogenic orgin, Environ.Sci.Technol., 32, 2208-2214.

68. Ballschmiter, K., (2003), Pattern and sources of naturally produced organohalogens in the environment: biogenic formation of organohalogens, Chemosphere, 52, 313-324.

69. Kuniyoshi, M., Yamada, K., and Higa, T., (1985), A biologically active diphenyl ether from the green alga Cladophora fascicularis, Experientia, 41, 523-524.

70. Kitamura, M., Koyama, T., Nakano, Y., and Uemura, D., (2005), Corallinafuran and corallinaether, novel toxic compounds from crustose coralline red algae, Chem.Lett., 34, 1272-1273.

References

- 56 -

71. Cameron, G. M., Stapleton, B. L., Simonsen, S. M, Brecknell, D. J., and Garson, M. J., (2000), New sesquiterpene and brominated metabolites from the tropical marine sponge Dysidea sp., Tetrahedron, 56, 5247-5252.

72. Anjaneyulu, V., Nageswara Rao, K., Radhika, P., and Muralikrishna, M., (1996), A new tetrabromodiphenyl ether from the sponge Dysidea herbacea of the Indian Ocean, Indian J.Chem., 35B, 89-90.

73. Bowden, B. F., Towerzey, L., and Junk, P. C., (2000), A new brominated diphenyl ether from the marine sponge Dysidea herbacea, Aust.J.Chem., 53, 299-301.

74. Capon, R., Ghisalberti, E. L., Jefferies, P. R., Skelton, B. W., and White, A. H., (1981), Structural studies of halogenated diphenyl ethers from a marine sponge, J.C.S.Perkin Trans.1, 2464-2467.

75. Carté, B. and Faulkner, D. J., (1981), Polybrominated diphenyl ethers from Dysidea herbacea, Dysidea chlorea and Phyllospongia foliascens, Tetrahedron, 37, 2335-2339.

76. Fu, X., Schmitz, F. J., Govindan, M., and Abbas, S. A., (1995), Enzyme inhibitors: New and known polybrominated phenols and diphenyl ethers from four indo-pacific Dysidea sponges, J.Nat.Prod., 58, 1384-1391.

77. Fu, Xiong and Schmitz, F. J., (1996), New brominated diphenyl ether from an unidentified spieces of Dysidea Sponge. 13C NMR data for some brominated diphenyl ethers, J.Nat.Prod., 59, 1102-1103.

78. 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.

79. Sharma, G. M., Vig, B., and Burkholder, P. R., (1969), Antimicrobial substances of marine sponges IV, In: Food drugs from the sea. Procedings 1969, Ed. Youngken, H.W.Jr., Marine Technology Society, Washington, DC, USA, 307-310.

80. Utkina, N. K., Kazantseva, M. V., and Denisenko, V. A., (1987), Brominated diphenylethers from the marine sponge Dysidea fragilis, Chem.Nat.Compd., 1, 508-509.

81. Hattori, T., Konno, A., Adachi, K., and Shizuri, Y., (2001), Four new bioactive bromophenols from the palauan sponge Phyllospongia dendyi, Fisheries Sci., 67, 899-903.

82. Utkina, N. K., Denisenko, V. A., Scholokova, O. V., Virovaya, M. V., Gerasimenko, A. V., Povov, D. Y., Krasokhin, V. B., and Popov, A. M., (2001), Spongiadioxins A and B, Two new polybrominated dibenzo-p-dioxins from an Australian marine sponge Dysidea dendyi, J.Nat.Prod., 64, 151-153.

83. Utkina, N. K., Denisenko, V. A., Virovaya, M. V., Scholokova, O. V., and Prokofeva, N. G, (2002), Two new minor polybrominated dibenzo-p-dioxins from the marine sponge Dysidea dendyi, J.Nat.Prod., 65, 1213-1215.

84. 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, Marine Biology, 119, 1-11.

85. Unson, M. D. and Faulkner, D. J., (1993), Cyanobacterial symbiont biosynthesis of chlorinated metabolites from Dysidea herbacea (Porifera), Experientia, 49, 349-353.

86. Butler, A. and Walker, J. V., (1993), Marine haloperoxidases, Chem.Rev., 93,

References

- 57 -

1937-1944. 87. Moore, C. A. and Okuda, R. K., (1996), Bromoperoxidase activity in 94 species of

marine algae, J.Natural Toxins, 5, 295-305. 88. Pedersén, M., Collén, J., Abrahamsson, K., and Ekdahl, A., (1996), Production of

halocarbons from seaweeds: an oxidative stress reaction?, Scientia Marina, 60, 257-263.

89. Ballschmiter, K., Mennel, A., and Buyten, J., (1993), Long chain alkyl-polysiloxanes as non-polar stationary phases in capillary gas chromatography, Fresenius J.Anal.Chem., 346, 396-402.

90. Marsh, G., Athanasiadou, M., Bergman, Å., and Asplund, L., (2004), Identification of hydroxylated and methoxylated polybrominated diphenyl ethers in Baltic Sea salmon (Salmo salar) blood, Environ.Sci.Technol., 38, 10-18.

91. Norström, K., (2006), Bis(4-chlorophenyl) sulfone and PCB methyl sulfone metabolites - Trends and chirality in the Baltic Sea environment, PhD Thesis, Department of Environmental Chemistry, Stockholm University, Sweden.

92. Malmberg, T., (2004), Identification and characterisation of hydroxylated PCB and PBDE metabolites in blood. Congener specific synthesis and analysis, PhD Thesis, Department of Environmental Chemistry, Stockholm University.

93. Letcher, R. J., Klasson Wehler, E., and Bergman, Å., (2000), Methyl sulfone and hydroxylated metabolites of polychlorinated biphenyls, In: New types of persistent halogenated compounds, Ed. Paasivirta, J., Springer-Verlag, Berlin, 315-359.

94. Brouwer, A., Morse, D. C., Lans, M. C., Schuur, A. G., Murk, A. J., Klasson Wehler, E., Bergman, Å., and Visser, T. J., (1998), Interactions of persistent environmental organohalogens with the thyroid hormone system: mechanisms and possible consequences for animal and human health, Toxicol.Ind.Health, 14, 59-84.

95. Marsh, Göran, Athanasiadou, Maria, Athanassiadis, Ioannis, and Sandholm, Anna, (2006), Identification of hydroxylated metabolites in 2,2',4,4'-tetrabromodiphenyl ether exposed rats, Chemosphere, 63, 690-697.

96. Hakk, H. and Letcher, R. J., (2003), Metabolism in the toxicokinetics and fate of brominated flame retardants - a review, Environ.Int., 29, 801-828.

97. Örn, U. and Klasson Wehler, E., (1998), Metabolism of 2,2',4,4'-tetrabromodiphenyl ether in rat and mouse, Xenobiotica, 28, 199-211.

98. 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[14C]diphenylether in pike (Esox lucius) after dietary exposure, Organohalogen Compounds, 52, 58-61.

99. 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.

100. Hakk, H., Huwe, J., Low, M., Rutherford, D., and Larsen, G., (2004), Tissue disposition, excreation, and metabolism of 2,2',4,4',6-pentabromodiphenyl ether (BDE-100) in male Sprague-Dawley rats, Brominated Flame Retardants, 429-432.

101. Mörck, A., Hakk, H., Örn, U., and Klasson Wehler, E., (2003), Decabromodiphenyl ether in the rat: absorption, distribution, metabolism, and excretion, Drug Metab.Dispos., 31, 900-907.

102. 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,

References

- 58 -

5342-5348. 103. Fu, Xiong, Hossain, M. B., Schmitz, F. J., and van der Helm, D., (1997),

Longithorones, unique prenylated para- and metacyclophane type quinones from the tunicate Aplidium longithotax, J.Org.Chem., 62, 3810-3819.

104. Schumacher, R. W. and Davidson, B. S., (1995), Didemnolines A-D, new N9-substituted β-carbolines from the marine ascidian Didemnum sp., Tetrahedron, 51, 10125-10130.

105. Agrawal, M. S. and Bowden, B. F., (2005), Marine sponge Dysidea herbacea revisited: Another brominated diphenyl ether, Mar.Drugs, 3, 9-14.

106. Valters, Karlis, Li, Hongxia, Alaee, Mehran, D'Sa, Ivy, Marsh, Göran, Bergman, Åke, and Letcher, Robert J., (2005), Polybrominated diphenyl ethers and hydroxylated and methoxylated brominated and chlorinated analogues in the plasma of fish from the Detroit River, Environ.Sci.Technol., 39, 5612-5619.

107. Verreault, Jonathan, Gabrielsen, Geir W., Chu, Shaogang, Muir, Derek C. G., Andersen, Magnus, Hamaed, Ahmad, and Letcher, Robert J., (2005), Flame retardants and methoxylated and hydroxylated polybrominated diphenyl ethers in two norwegian arctic top predators: Glaucous gulls and polar bears, Environ.Sci.Technol., 39, 6021-6028.

108. McKinney, Melissa A., Cesh, Lillian S., Elliott, John E., Williams, Tony D., Garcelon, David K., and Letcher, Robert J., (2006), Brominated flame retardants and halogenated phenolic compounds in North American West Coast bald eaglet (Haliaeetus leucocephalus) Plasma, Environ.Sci.Technol., 40, 6275-6281.

109. Hovander, L., Malmberg, T., Athanasiadou, M., Athanassiadis, I., Rahm, S., Bergman, Å., and Klasson Wehler, E., (2002), Identification of hydroxylated PCB metabolites and other phenolic halogenated pollutants in human blood plasma, Arch.Environ.Contam.Toxicol., 42, 105-117.

110. Bergman, Å. and Athanasiadou, M., (2006), Hydroxylated PBDE metabolites in human blood, Organohalogen compounds, 68, 635-638.

111. Meerts, Ilonka A. T. M., Van Zanden, Jelmer J., Luijks, Edwin A. C., Van Leeuwen-Bol, Ingeborg, Marsh, Göran, Jakobsson, Eva, Bergman, Åke, and Brouwer, Abraham, (2000), Potent competitive interactions of some brominated flame retardants and related compounds with human transthyretin in vitro, Toxicol.Sci., 56, 95-104.

112. Legler, J. and Brouwer, A., (2003), Are brominated flame retardants endocrine disruptors?, Environ.Int., 29, 879-885.

113. Morse, D. C., Plug, A., Wesseling, W., van den Berg, K. J., and Brouwer, A., (1996), Persistent alterations in regional brain glial fibrillary acidic protein and synaptophysin levels following pre- and postnatal polychlorinated biphenyl exposure, Toxicol.Appl.Pharm., 139, 252-261.

114. Morse, D. C., Wehler, E. K., Wesseling, W., Koeman, J. H., and Brouwer, A., (1996), Alterations in rat brain thyroid hormone status following pre- and postnatal exposure to polychlorinated biphenyls (Aroclor 1254), Toxicol.Appl.Pharm., 136, 269-279.

115. Meerts, I. A. T. M., Letcher, R. J., Hoving, S., Marsh, G., Bergman, Å., Lemmen, J. G., van der Burg, B., and Brouwer, A., (2001), In vitro estrogenicity of polybrominated diphenyl ethers, hydroxylated PBDEs, and polybrominated bisphenol A compounds, Environ.Health Perspect., 109, 399-407.

116. Canton, Rocio F., Sanderson, J. Thomas, Letcher, Robert J., Bergman, Åke, and

References

- 59 -

van den Berg, Martin, (2005), Inhibition and induction of aromatase (CYP19) activity by brominated flame retardants in H295R human adrenocortical carcinoma cells, Toxicol.Sci., 88, 447-455.

117. Salva, J. and Faulkner, D. J., (1990), A new brominated diphenyl ether from a philippine Dysidea speicies, J.Nat.Prod., 53, 757-760.

118. Teuten, E. L., Xu, L., and Reddy, C. M., (2005), Two abundant bioaccumulated halogenated compounds are natural products, Science, 307, 917-920.

119. Vetter, W., Stoll, E., Garson, M. J., Fahey, S. J., Gaus, C., and Müller, J. F., (2002), Sponge halogenated natural products found at parts-per-million levels in marine mammals, Environ.Toxicol.Chem., 21, 2014-2019.

120. Allard, A.-S., Remberger, M., Viktor, T., and Neilson, A. H., (1988), Environmental fate of chloroguaiacols and chlorochatechols, Wat.Sci.Tech., 20, 131-141.

121. Neilson, A. H., Allard, A.-S., Lindgren, C., and Remberger, M., (1987), Transformation of chloroguaiacols, chloroveratroles, and chlorocatechols by stable consortia of anaerobic bacteria, Appl.Environ.Microbiol., 53, 2511-2519.

122. Remberger, Mikael, Allard, Ann Sofie, and Neilson, Alasdair H., (1986), Biotransformations of chloroguaiacols, chlorocatechols, and chloroveratroles in sediments, Appl.Environ.Microbiol., 51, 552-558.

123. Haglund, P. S., Zook, D. 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.

124. Sinkkonen, S., Rantalainen, A.-L., Paasivirta, J., and Lahtiperä, M., (2004), Polybrominated methoxy diphenyl ethers (MeO PBDEs) in fish and guillemot of Baltic, Atlantic and Arctic environments, Chemosphere, 56, 767-775.

125. Asplund, L., Bignert, A., and Nylund, K., (2004), Comparison of spatial and temporal trends of methoxylated PBDEs, PBDEs, and hexabromocyclododecane in herring along the Swedish coast, Organohalogen Compounds, 66, 3938-3943.

126. Olsson, A., Ceder, K., Bergman, Å., and Helander, B., (2000), Nestling blood of the white-tailed sea eagle (Haliaeetus albicilla) as an indicator of territorial exposure to organohalogen compounds - An evaluation, Environ.Sci.Technol., 34, 2733-2740.

127. Vetter, W., (2001), A GC/ECNI-MS method for the identification of lipophilic anthropogenic and natural brominated compounds in marine samples, Anal.Chem., 73, 4951-4957.

128. Vetter, W. and Jun, W., (2003), Non-polar halogenated natural products bioaccumulated in marine samples. II. Brominated and mixed halogenated compounds, Chemosphere, 52, 423-431.

129. van Bavel, B., Dam, M., Tysklind, M., and Lindström, G., (2001), Levels of polybrominated diphenyl ethers in marine mammals, Organohalogen Compounds, 52, 99-103.

130. Wolkers, Hans, van Bavel, Bert, Derocher, Andrew E., Wiig, Oeystein, Kovacs, Kit M., Lydersen, Christian, and Lindström, Gunilla, (2004), Congener-Specific Accumulation and Food Chain Transfer of Polybrominated Diphenyl Ethers in Two Arctic Food Chains, Environ.Sci.Technol., 38, 1667-1674.

131. Pettersson, A., van Bavel, B., Engwall, M., and Jimenez, B., (2004), Polybrominated diphenylethers and methoxylated tetrabromodiphenylethers in

References

- 60 -

cetaceans from the Mediterranean Sea, Arch.Environ.Contam.Toxicol., 47, 542-550.

132. McKinney, Melissa A., De Guise, Sylvain, Martineau, Daniel, Beland, Pierre, Lebeuf, Michel, and Letcher, Robert J., (2006), Organohalogen contaminants and metabolites in beluga whale (Delphinapterus leucas) liver from two Canadian populations, Environ.Toxicol.Chem., 25, 1246-1257.

133. Stapleton, H. M., Dodder, N. G., Kucklick, J. R., Reddy, C. M., Schantz, M. M., Becker, P. R., Gulland, F., Porter, B. J., and Wise, S. A., (2006), Determination of HBCD, PBDEs and MeO-BDEs in California sea lions (Zalophus californianus) stranded between 1993 and 2003, Marine Pollution Bulletin, 52, 522-531.

134. WHO/IPCS, (1998), Polybrominated dibenzo-p-dioxins and dibenzofurans, Environmental Health Criteria (205) ISBN 94-4-1572051.

135. Chatkittikunwong, W. and Creaser, C. S., (1994), Stability of bromo- and bromochloro-dibenzo-p-dioxins under laboratory and environmental conditions, Chemosphere, 29, 547-557.

136. Buser, Hans-Rudolf, (1988), Rapid photolytic decomposition of brominated and brominated/chlorinated dibenzodioxins and dibenzofurans, Chemosphere, 17, 889-903.

137. Buser, H. R., (1986), Polybrominated dibenzofurans and dibenzo-p-dioxins: thermal reaction products of polybrominated diphenyl ether flame retardants, Environ.Sci.Technol., 20, 404-408.

138. Sakai, S., Watanabe, J., Honda, Y., Takatsuki, H., Aoki, I., Futamatsu, M., and Shiozaki, K., (2001), Combustion of brominated flame retardants and behavior of its byproducts, Chemosphere, 42, 519-531.

139. Eriksson, Johan, Green, Nicholas, Marsh, Göran, and Bergman, Åke, (2004), Photochemical decomposition of 15 polybrominated diphenyl ether congeners in methanol/water, Environ.Sci.Technol., 38, 3119-3125.

140. Söderström, Gunilla, Sellström, Ulla, de Wit, Cynthia A., and Tysklind, Mats, (2004), Photolytic debromination of decabromodiphenyl ether (BDE 209), Environ.Sci.Technol., 38, 127-132.

141. Haglund, P. S., Egebäck, K., and Jansson, B., (1988), Analysis of polybrominated dioxins and furans in vehicle exhaust, Chemosphere, 17, 2129-2140.

142. de Jong, A. P. J. M, van der Heeft, E., Marsman, J. A., and Liem, A. K. D., (1992), Investigation on the occurrence of polyhalogenated (Br/Cl) dibenzodioxins and dibenzofurans in cow's milk and fish tissue, Chemosphere, 25, 1551-1557.

143. Choi, J., Fujimaki, S., Kitamura, K., Hashimoto, S., Ito, H., Suzuki, N., Sakai, S., and Morita, M., (2003), Polybrominated dibenzo-p-dioxins, dibenzofurans, and diphenyl ethers in Japanese human adipose tissue, Environ.Sci.Technol., 35, 817-821.

144. Kotz, A., Malisch, R., Kypke, K., and Oehme, M., (2005), PBDE, PBDD/F and mixed chlorinated-brominated PXDD/F in pooled human milk samples from different countries, Organohalogens Compounds, 67, 1540-1544.

145. Hagberg, J., Grahn, E., van Bavel, B., and Lindström, G., (2005), Occurrence and levels of PCDD/Fs and PBDD/Fs in two Swedish lake sediments, Organohalogen Compounds, 67, 2030-2032.

146. Wolf, D., Schmitz, F. J., Hossain, M. B., and van der Helm, D., (1999), Aplidioxins A and B, two new dibenzo-p-dioxins from the Ascidian Aplidiopsis ocellata, J.Nat.Prod., 62, 167-169.

References

- 61 -

147. van den Berg, M., Birnbaum, L., Bosveld, B. T. C., Brunström, B., Cook, P., Feeley, M., Giesy, J., Hanberg, A., Hasegawa, R., Kennedy, S. W., Kubiak, T., Larsen, J. C., van Leeuwen, F. X. R., Liem, A. K. D., Nolt, C., Peterson, R. E., Poellinger, L., Safe, S., Schrenk, D., Tillitt, D., Tysklind, M., Younes, M., Waern, F., and Zacharewski, T., (1998), Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife, Environ.Health Perspect., 106, 775-792.

148. van den Berg, M., Birnbaum, L., Denison, M., De Vito, M., Farland, W., Feeley, M., Fiedler, H., Håkanssson, H., Hanberg, A, Haws, L., Rose, M., Safe, S., Schrenk, D., Tohyama, C., Tritscher, A., Tuomisto, J., Tysklind, M., Walker, N., and Peterson, R. E., (2006), The 2005 World Health Organization reevluation of human and mammalian toxic equivalency factors for dioxins and dioxin-like compounds, Toxicol.Sci., 93, 223-241.

149. 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.

150. Hornung, M. W., Zabel, E. W., and Peterson, R. E., (1996), Toxic equivalency factors of polybrominated dibenzo-p-dioxin, dibenzofuran, biphenyl, and polyhalogenated diphenyl ether congeners based on rainbow trout early life stage mortality, Toxicol.Appl.Pharm., 140, 227-234.

151. Birnbaum, L. S., Staskal, D. F., and Diliberto, J. J., (2003), Health effects of polybrominated dibenzo-p-dioxins (PBDDs) and dibenzofurans (PBDFs), Environ.Int., 29, 855-860.

152. Mason, G., Zacharewski, T., Denomme, M. A., Safe, L., and Safe, S., (1987), Polybrominated dibenzo-p-dioxins and related compounds: Quantitative in vivo and in vitro structure activity relationships, Toxicology, 44, 245-255.

153. Stal, L. J., Albertano, P., Bergman, B., von Bröckel, K., Gallon, J. R., Hayes, P. K., Sivonen, K., and Walsby, A. E., (2003), BASIC: Baltic Sea cyanobacteria. An investigation of the structure and dynamics of water blooms of cyanobacteria in the Baltic Sea-responses to a changing environment, Continental Shelf Res., 23, 1695-1714.

154. Kautsky, N., (1981), On the trophic role of blue mussel (Mytilus edulis L.) in a Baltic coastal ecosystem and the fate of the organic matter produced by the mussels, Kieler Meeresforschungen, 5, 454-461.

155. 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.

156. Jensen, S., Reutergårdh, L., and Jansson, B., (1983), Analytical methods for measuring organochlorines and methyl mercury by gas chromatography, FAO Fish.Tech.Pap., 212, 21-33.

157. Wiberg, K., Bergman, A., Olsson, M., Roos, A., Blomkvist, G., and Haglund, P., (2002), Concentrations and enantiomer fractions of organochlorine compounds in Baltic species hit by reproductive impairment, Environ.Toxicol.Chem., 21, 2542-2551.

158. Vetter, W., Scholz, E., Gaus, C., Müller, J. F., and Haynes, D., (2001), Anthropogenic and natural organohalogen compounds in blubber of dolphins and dugongs (Dugong dugon) from Northeastern Australia, Arch.Environ.Contam.Toxicol., 41, 221-231.

159. Kelly, B. C. and Ikonomou, M. G., (2005), PBDEs and their hydroxylated and

References

- 62 -

methoxylated derivatives in marine sediment and biota from Canada´s Eastern Artic, Organohalogen Compounds, 67, 843-846.

160. Melcher, J., Olbrich, D., Marsh, G., Nikiforov, V., Gaus, C., Gaul, S., and Vetter, W., (2005), Tetra- and tribromophenoxyanisoles in marine samples from Oceania, Environ.Sci.Technol., 39, 7784-7789.

161. Vetter, W., Weichbrodt, M., Hummert, K., Glotz, D., and Luckas, B., (1998), Combined microwave-assisted extraction and gel permeation chromatography for the determination of chlorinated hydrocarbons in seal blubber and cod livers, Chemosphere, 37, 2439-2449.

162. Waldebäck, A., (2005), Pressurized fluid extraction. A sustainable technique with added values, PhD Thesis, Department of Chemistry, Uppsala University, Sweden.

163. Björklund, E., Nilsson, T., and Bøwadt, S., (2000), Pressurrised liquid extraction of persistent organic pollutants in environmental analysis, Trends Anal.Chem., 19, 434-445.

164. Saito, Koichi, Sjödin, Andreas, Sandau, Court, Davis, Mark D., Nakazawa, Hiroyuki, Matsuki, Yasuhiko, and Patterson, Donald G., (2004), Development of an accelerated solvent extraction and gel permeation chromatography analytical method for measuring persistent organohalogen compounds in adipose and organ tissue analysis, Chemosphere, 57, 373-381.

165. Bligh, E. G. and Dyer, W. J., (1959), A rapid method of total lipid extraction and purification, Can.J.Biochem.and Physiol., 37, 911-917.

166. Folch, J., Lees, M., and Stanley, G. H. S., (1957), A simple method for the isolation and purification of total lipids from animal tissues, J.Biol.Chem., 226, 497-509.

167. Jensen, S., Häggberg, L., Jörundsdóttir, H., and Odham, G., (2003), A quantitative lipid extraction method for residue analysis of fish involving nonhalogenated solvents, J.Agric.Food Chem., 51, 5607-5611.

168. Vogel, (1980), Vogel's Elementary Practical Organic Chemistry 1: Preparations, Longman Inc., New York.

169. Bicking, M. K. L. and Wilson, R. L., (1991), High performance size exclusion chromatography in environmental method development. 1. The effect of mobile phase and temperature on the retention of dioxins, furans and polychlorinated biphenyls, Chemosphere, 22, 421-435.

170. Haglund, Peter, Jakobsson, Eva, Athanasiadou, Maria, and Bergman, Åke, (1993), Determination of polychlorinated naphthalenes in polychlorinated biphenyl products via capillary gas chromatography-mass spectrometry after separation by gel permeation chromatography, J.Chromatogr., 634, 79-86.

171. Jansson, B., Andersson, R., Asplund, L., Bergman, Å., Litzén, K., Nylund, K., Reutergårdh, L., Sellström, U., Uvemo, U.-B., Wahlberg, C., and Wideqvist, U., (1991), Multiresidue method for the gas chromatographic analysis of some polychlorinated and polybrominated pollutants in biological samples, Fresenius J.Anal.Chem., 340, 439-445.

172. Bergman, Å., Athanasiadou, M., Bergek, S., Haraguchi, K., Jensen, S., and Klasson Wehler, E., (1992), PCB and PCB methyl sulfones in mink treated with PCB and various PCB fractions, Ambio, 21, 570-576.

173. Janák, K., Becker, G., Colmsjö, A., Östman, C., Athanasiadou, M., Valters, K., and Bergman, Å., (1998), Methyl sulfonyl polychlorinated biphenyls and 2,2-bis(4-chlorophenyl)-1,1-dichlorethene in gray seal tissues determined by gas chromatography with electron capture detection and atomic emission detection,

References

- 63 -

Environ.Toxicol.Chem., 17, 1046-1055. 174. Colmsjö, A. L., Zebühr, Y. U., and Östman, C. E., (1987), Group separation of

PCDD:s, PCDF:s, PAC:s and aliphatic compounds on an amino bonded stationary phase for HPLC., Chromatographia, 24, 541-544.

175. Danielsson, Conny, Wiberg, Karin, Korytar, Peter, Bergek, Sture, Brinkman, Udo A. T., and Haglund, Peter, (2005), Trace analysis of polychlorinated dibenzo-p-dioxins, dibenzofurans and WHO polychlorinated biphenyls in food using comprehensive two-dimensional gas chromatography with electron-capture detection, Journal of Chromatography, A, 1086, 61-70.

176. Haglund, P., Asplund, L., Järnberg, U., and Jansson, B., (1990), Isolation of mono- and non-ortho polychlorinated biphenyl from biological samples by electron-donor acceptor high performance liquid chromatography using a 2-(1-pyrenyl)ethyldimethylsilylated silica column, Chemosphere, 20, 887-894.

177. Haglund, P., Asplund, L., Järnberg, U., and Jansson, B., (1990), Isolation of toxic polychlorinated biphenyls by electron donor-acceptor high-performance liquid chromatography on a 2-(1-pyrenyl)ethyldimethylsilylated silica column, J.Chromatogr., 507, 389-398.

178. Jansson, B. and Asplund, L., (1987), Brominated flame retardants - Ubiquitous environmental pollutants?, Chemosphere, 16, 2343-2349.

179. Athanasiadou, M., Marsh, G., Athanassiadis, I., Asplund, L., and Bergman, Å., (2006), Gas chromatography and mass spectrometry of methoxylated polybrominated diphenyl ethers (MeO-PBDEs), J.Mass Spectrom., 41, 790-801.

180. 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.

181. Malmvärn, A., Kautsky, L., Jensen, S., Athanassiadis, I., and Asplund, L., (2003), Detection of a possible tribromodibenzo-p-dioxin in blue mussels (Mytilus edulis) from the Baltic Sea, Organohalogen Compounds, 61, 143-146.

182. Jensen, S. and Renberg, L., (1972), Contaminants in pentachlorophenol: Chlorinated dioxins and predioxins (chlorinated hydroxy-diphenyl ethers), Ambio, 1, 62-65.

183. Rappe, C. and Nilsson, C.-A., (1972), An artifact in the gas chromatographic determination of impurities in pentachlorophenol, J.Chromatogr., 67, 247-253.

184. Söderström, Gunilla, (2003), On the combustion and photolytic degradation of some brominated flame retardants, Department of Chemistry, Environmental Chemistry, Univerity of Umeå, SE-901 87 Umeå, Sweden.

185. Engkvist, R., Malm, T., Svensson, A., Asplund, L., Isaeus, M., Kautsky, L., Greger, M., and Landberg, T., (2001), Makroalgblomningar längs Ölands kuster, effekter på det lokala näringslivet och det marina ekosystemet, Rapport 2001:2.

186. Del Barga, I., Frenzilli, G., Scarcelli, V., Nigro, M., Malmvärn, A., Asplund, L., Förlin, L., and Sturve, J., (2006), Effects of algal extracts (Polysiphonia fucoides) on rainbow trout (Oncorhynchus mykiss): A biomarker approach, Marine Environ.Res., 62, S283-S286.

187. Greyerz, E., Bignert, A., and Olsson, M., (2000), Miljögifter i blåmussla längs svenska Östersjökusten 1999, Analysrapport från RSL, 1.

188. Ueno, D., Pacepavicius, G., Alaee, M., Campbell, L., Letcher, R., Bergman, Å.,

References

- 64 -

Marsh, G., Muir, D., and Brown, S., (2005), Detection of hydroxylated polybrominated diphenyl ethers (OH-PBDEs) in abiotic samples from southern Ontario, Canada, Organohalogen Compounds, 67, 851-853.

189. Darling, Colin, Alaee, Mehran, Campbell, Linda, Pacepavicius, Grazina, Ueno, Daisuke, and Muir, Derek, (9-10-2004), Hydroxylated PCBs in abiotic environmental matrices: Precipitation and surface waters, Organohalogen Compounds, 66, 1497-1502.

190. Marsh, G., (2003), Polybrominated diphenyl ethers and their hydroxy and methoxy derivatives. Congener specific synthesis and analysis, Ph.D. Thesis, Department of Environmental Chemistry, Stockholm University, Sweden.

191. Allard, Ann Sofie, Remberger, Mikael, and Neilson, Alasdair H., (1987), Bacterial O-methylation of halogen-substituted phenols, Appl.Environ.Microbiol., 53, 839-845.

192. Hundt, Kai, Martin, Dierk, Hammer, Elke, Jonas, Ulrike, Kindermann, Markus Karl, and Schauer, Frieder, (2000), Transformation of triclosan by Trametes versicolor and Pycnoporus cinnabarinus, Appl.Environ.Microbiol., 66, 4157-4160.

193. Pongratz, R. and Heumann, K. G., (1998), Production of methylated mercury and lead by polar macroalgae - a significant natural source for atmospheric heavy metals in clean room compartments, Chemosphere, 36, 1935-1946.

194. Wiberg, K., Rappe, C., and Haglund, P., (1992), Analysis of bromo-, chloro- and mixed bromo/chloro-dibenzo-p-dioxins and dibenzofurans in salmon, osprey and human milk, Chemosphere, 24, 1431-1439.

195. Haglund, P., Lindkvist, K., Malmvärn, A., Wiberg, K., Bignert, A., Nakano, T., and Asplund, L., (2005), High levels of potentially biogenic dibromo and tribromo dibenzo-p-dioxins in Swedish fish, Organohalogens Compounds, 67, 1267-1269.

196. Flodin, C. and Whitfield, F. B., (1999), Biosynthesis of bromophenols in marine algae, Wat.Sci.Tech., 40, 53-58.

197. Flodin, C. and Whitfield, F. B., (1999), 4-Hydroxybenzoic acid: a likely precursor of 2,4,6-tribromophenol in Ulva lactuca, Phytochemistry, 51, 249-255.

198. Carlson, D. L. and Swackhamer, D. L., (2006), Results from the US Great Lakes fish monitoring program and effects of lake processes on bioaccumulative contaminants concentrations, J.Great Lakes Res., 32, 370-385.

199. Smith, K. E. C. and McLachlan, M. S., (2006), Concentrations and partitioning of polychlorinated biphenyls in the surface waters of the southern Baltic Sea - Sesonal effects, Environ.Toxicol.Chem., 25, 2569-2575.

200. Skoglund, R. S. and Swackhamer, D. L., (1999), Evidence for the use of organic carbon as sorbing matrix in the modeling of PCB accumulation in phytoplankton, Environ.Sci.Technol., 33, 1516-1519.

201. Stapleton, H. M., Letcher, R., and Baker, J. E., (2004), Debromination of polybrominated diphenyl ether congeners BDE-99 and BDE-183 in the intestinal tract of the common carp (Cyprinus carpio), Environ.Sci.Technol., 38, 1054-1061.

202. Kierkegaard, A., Balk, L., Tjärnlund, U., de Wit, C. A., and Jansson, B., (1999), Dietary uptake and biological effects of decabromodiphenyl ether in Rainbow trout (Oncorhynchus mykiss), Environ.Sci.Technol., 33, 1612-1617.

203. Stapleton, H. M., Alaee, M., Letcher, R. J., and Baker, J. E., (2004), Debromination of the flame retardant decabromodiphenyl ether by juvenile carp (Cyprinus carpio) following dietary exposure, Environ.Sci.Technol., 38, 112-119.

204. Stapleton, Heather M., Brazil, Brian, Holbrook, David R., Mitchelmore, Carys L.,

References

- 65 -

Benedict, Rae, Konstantinov, Alex, and Potter, Dave, (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.

205. Kautsky, U. and Kautsky, H., (1995), Costal productivity in the Baltic Sea, In: Biology and Ecology of Shallow Coastal Waters. Proc. 28th Eur. Mar. Biol. Symp., Eds. Eleftheriou, A., Ansell, A.D., and Smith, C.J., Olsen & Olsen, Fredensborg, Denmark, 31-38.

206. Cook, P. M., Zabel, E. W., and Peterson, R. E., (1997), The TCDD toxicity equivalence approch for characterizing risk for early life-stage mortality in trout, In: Chemically induced alteration in functional development and reproduction of fishes, SETAC Press, Pensacola, FL, 9-27.

207. McMurry, L. M., Oethinger, M., and Levy, S. B., (1998), Triclosan targets lipid synthesis, Nature, 394, 531-532.

208. Heath, R. J., Rubin, R. J., Holland, D. R., Zhang, E., Snow, M. E., and Rock, C. O., (1999), Mechanism of triclosan inhibition of bacterial fatty acid synthesis, The Journal of Biological Chemistry, 274, 11110-11114.

209. Mäenpää, K. A., Penttinen, O.-P., and Kukkonen, J. V. K., (2004), Pentachlorophenol (PCP) bioaccumulation and effect on heat production on salmon eggs at different stages of development, Aquatic Toxicology, 68, 75-85.

210. Newton, Ana Paula Negrelo, Cadena, Silvia Maria, Rocha, Maria Eliane Merlin, Carnieri, Eva Gunilla Skaere, and Martinelli de Oliveira, Maria Benigna., (2005), Effect of triclosan (TRN) on energy-linked functions of rat liver mitochondria, Toxicol.Lett., 160, 49-59.

Brominated natural products at different trophic levels in ...196895/FULLTEXT01.pdf · Comparison...

Documents

Transcript of Brominated natural products at different trophic levels in ...196895/FULLTEXT01.pdf · Comparison...