Identification of brominated organic compounds in aquatic

biota and exploration of bromine isotope analysis for

source apportionment

Maria Unger

Doctoral Thesis

Department of Materials and Environmental Chemistry (MMK)

Stockholm University

Stockholm 2010

2

© Maria Unger, Stockholm 2010

ISBN 978-91-7447-070-3 pp 1-58

Printed in Sweden by US-AB, Stockholm 2010

Distributors: Department of Materials and Environmental

Chemistry (MMK), Stockholm University

Department of Applied Environmental Science (ITM), Stockholm

University

Cover photo by Sven-Erik Nilsson. Smedfjärden/Kvädöfjärden.

Farleden mellan Huvududden på Stora Askö och Huvudskär.

3

What will the winter bring

if not yet another spring

Kamelot, Serenade, Black Halo 2005

4

Forward momentum!

5

Abstract

Brominated organic compounds (BOCs) of both natural and

anthropogenic origin are abundant in the environment. Most

compounds are either clearly natural or clearly anthropogenic

but some are of either mixed or uncertain origin. This thesis

aims to identify some naturally produced BOCs and to develop a

method for analysis of the bromine isotopic composition in BOCs

found in the environment.

Polybrominated dibenzo-p-dioxins (PBDDs) in the Baltic Sea are

believed to be of natural origin although their source is

unknown. Since marine sponges are major producers of

brominated natural products in tropical waters, BOCs were

quantified in a sponge (Ephydatia fluviatilis) from the Baltic Sea

(Paper I). The results showed that the sponge does not seem to

be a major producer of PBDDs in the Baltic Sea. In this study,

mixed brominated/chlorinated dibenzo-p-dioxins were however

discovered for the first time in a background environment

without an apparent anthropogenic source.

The use of nuclear magnetic resonance spectroscopy (NMR) is

unusual in analytical environmental chemistry due to its sample

requirements. Preparative capillary gas chromatography was

used to isolate a sufficient amount of an unidentified BOC from

northern bottlenose whale (Hyperoodon ampullatus) blubber

(Paper II) to enable NMR analysis for identification of the

compound.

The bromine isotopic composition of BOCs may give information

on the origin and environmental fate of these compounds. The

first steps in this process are the development of a method to

determine the bromine isotope ratio in environmentally relevant

BOCs (Paper III) and measuring the bromine isotope ratio of

several standard substances to establish an anthropogenic

endpoint (Paper IV).

6

Table of contents Abstract ............................................................................ 5

Table of contents ................................................................ 6

Abbreviations ..................................................................... 8

List of papers ..................................................................... 9

Statement ....................................................................... 10

Thesis objectives .............................................................. 11

1. Brominated organic compounds in the marine environment 13

1.1 Naturally produced brominated organic compounds ............................................................................ 13

1.2 Natural production of brominated organic compounds

............................................................................ 17

2. Bromine isotopes .......................................................... 20

2.1 Kinetic isotope effect ......................................... 20

2.2 Isotope fractionation processes ........................... 20

3. Samples and purification methods .................................. 23

3.1 Samples .......................................................... 23

3.2 Extraction and lipid removal methods .................. 24

3.3 Chromatographic separation ............................... 27

4. Analysis ....................................................................... 29

4.1 GC-MS ............................................................. 29

4.2 NMR ................................................................ 30

4.3 GC-multiple-collector-ICP-MS ............................. 31

5. Summary of papers....................................................... 33

5.1 Paper I - Identification and quantification of PBDDs and Br/Cl-DDs ........................................................ 33

5.2 Paper II - NMR-identification of a novel brominated organic compound isolated from whale blubber .......... 34

5.3 Paper III - Development of a GC-MC-ICP-MS method

for brominated diaromatic compounds ...................... 34

5.4 Paper IV - Measurements of δ81Br in anthropogenic and natural monoaromatic compounds ...................... 35

7

6. Results and Discussion .................................................. 36

6.1 Identification of natural brominated organic

compounds ............................................................ 36

6.2 Development of CSIA-Br .................................... 39

7. Conclusions .................................................................. 42

8. Future perspectives ....................................................... 43

9. Sammanfattning på svenska .......................................... 44

10. Acknowledgements ..................................................... 45

11. References ................................................................. 48

8

Abbreviations

ACN Acetonitrile

BOCs Brominated organic compounds

Br/Cl-DDs Mixed brominated/chlorinated dibenzo-p-

dioxins

CIS Cooled injection system

CSIA Compound specific isotope analysis

DDT 2,2-bis(4-chlorophenyl)-1,1,1-trichloroethane

ECNI Electron capture negative ionization

EI Electron ionization

GC-MC-ICP-MS Gas chromatography multiple collector

inductively coupled ion plasma mass

spectrometry

GC-MS Gas chromatography – mass spectrometry

HRMS High resolution mass spectrometry

ICP Inductively coupled plasma

KIE Kinetic isotope effect

MBB Monobromobenzene

MeOMe-BDE42 6-Methoxy-5-methyl-2,2’3,4’-tetrabromo

diphenyl ether

MeO-PBDEs Methoxylated polybrominated diphenyl ethers

NMR Nuclear magnetic resonance spectroscopy

OH-PBDEs Hydroxylated polybrominated diphenyl ethers

PBB Polybrominated biphenyl

PBDDs Polybrominated dibenzo-p-dioxins

PBDEs Polybrominated diphenyl ethers

PBDFs Polybrominated dibenzofurans

PCBs Polychlorinated biphenyls

PCDDs Polychlorinated dibenzo-p-dioxins

PCGC Preparative capillary gas chromatograph

R Isotope ratio

RF Radio frequency

SIM Selective ion monitoring

TCDD 2,3,7,8-Tetrachlorodibenzo-p-dioxin

TriBDD s Tribromodibenzo-p-dioxins

9

List of papers

Paper I

Maria Unger, Lillemor Asplund, Peter Haglund, Anna Malmvärn,

Kristina Arnoldsson, Örjan Gustafsson. Polybrominated and

Mixed Brominated/Chlorinated Dibenzo-p-Dioxins in Sponge

(Ephydatia fluviatilis) from the Baltic Sea. Environmental

Science & Technology, 2009, 43, 8245-8250

Paper II

Maria Unger, Lillemor Asplund, Göran Marsh, Örjan Gustafsson.

Characterization of an abundant and novel methyl- and

methoxy-substituted brominated diphenyl ether isolated from

whale blubber. Chemosphere, 2010, 79, 408-413

Paper III

Henry Holmstrand, Maria Unger, Daniel Carrizo, Per Andersson,

Örjan Gustafsson.

Compound-specific bromine isotope analysis of brominated

diphenyl ethers using GC-multiple collector-ICP-MS. Submitted

to Rapid Communications in Mass Spectrometry

Paper IV

Daniel Carrizo, Maria Unger, Henry Holmstrand, Per Andersson,

Örjan Gustafsson, Sean P. Sylva, Christopher M. Reddy.

Compound-specific bromine isotope composition of natural and

industrially-synthesized organobromine substances. Manuscript

Papers I and II are reproduced with permission from American

Chemical Society and Elsevier, respectively.

10

Statement

I, Maria Unger, have contributed with the following to the

papers included in this thesis:

In Paper I, I planned and performed the sampling and a major

part of the purification. I was responsible for data analysis and

interpretation of mass spectra. I had main responsibility for

writing the paper.

In Paper II, I planned the project. I did a major part of the

sample purification and was responsible for the PCGC isolation

of the compound, data analysis and interpretation of mass

spectra. I took part in the NMR spectra interpretation and had

main responsibility for writing the paper.

In Paper III, I took part in planning the set up of the

instrument and experiments. I took part in the measurements

and data evaluation and assisted in writing the paper.

In Paper IV, I took part in planning the set up of the

instrument and experiments. I took part in the measurements,

data evaluation and in writing the paper.

Papers not included in the thesis

Emma L. Teuten, Carl G. Johnson, Manolis Mandalakis, Lillemor

Asplund, Örjan Gustafsson, Maria Unger, Göran Marsh,

Christopher M. Reddy Spectral characterization of two

bioaccumulated methoxylated polybrominated diphenyl ethers.

Chemosphere, 2006, 62, 197-203

Maria Unger and Örjan Gustafsson PAHs in Stockholm window

films: Evaluation of the utility of window film content as

indicator of PAHs in urban air. Atmospheric Environment, 2008,

42, 5550-5557

11

Thesis objectives

The aims of this thesis were to identify natural brominated

compounds in the aquatic environment and to develop a

method for source apportionment of natural and anthropogenic

organobromine compounds based on their stable bromine

isotopic composition.

The specific objectives for the papers were:

Paper I:

Investigate the role of an abundant sponge in the

production of polybrominated dibenzo-p-dioxins (PBDDs)

in the Baltic Sea by identification and quantification of

halogenated dibenzo-p-dioxins in the sponge.

Paper II:

Isolate a specific unidentified organobromine compound

from northern bottlenose whale blubber using

preparative capillary gas chromatography to obtain a

sufficient amount of pure compound for identification

with nuclear magnetic resonance spectroscopy (NMR).

Paper III:

Develop a method for online compound specific isotope

analysis of stable bromine isotopes (δ81Br) in

polybrominated diphenyl ethers (PBDEs) using a gas

chromatograph coupled to an inductively coupled plasma

and a multiple collector mass spectrometer (GC-MC-ICP-

MS).

Paper IV:

Evaluate the variation in δ81Br in anthropogenic

brominated organic monocyclic aromatic compounds to

establish an anthropogenic endpoint for bromine isotope

analysis of environmentally occurring brominated organic

compounds (BOCs).

12

13

1. Brominated organic compounds in the

marine environment

There are thousands of halogenated compounds of both natural

and anthropogenic origin in the environment. The most well-

known environmentally occurring organohalogen compounds

are DDT, polychlorinated biphenyls (PCBs) and polychlorinated

dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs). During

the last decades other anthropogenic halogenated substances

such as brominated flame retardants (BFRs) (de Wit et al.,

2006; Law et al., 2006) and polyfluorinated compounds (Giesy

et al., 2001) have received growing attention as environmental

contaminants. However, it has also been recognised that a

majority of the halogenated organic compounds in the aquatic

environment may be of natural origin.

Figure 1.1. Well-known anthropogenic organohalogen environmental

contaminants. Structures of A.) DDT, B) a PCDD, C.) a PCB and D.) a

PCDF.

1.1 Naturally produced brominated organic compounds

Nearly 5000 naturally produced halogenated organic compounds

(Gribble et al., 1999) have been identified, from simple gaseous

methyl halides to much larger and sometimes very complex

A.

D.C.

B.

14

structures (Gribble, 1998; Gribble, 1999; Faulkner, 2000;

Faulkner, 2001).

Some of the brominated organic compounds (BOCs) that have

been identified as natural products are brominated phenols

(Whitfield et al., 1999; Fan et al., 2003) methoxylated and

hydroxylated polybromodiphenyl ethers (MeO-PBDEs and OH-

PBDEs) (Fu et al., 1995; Cameron et al., 2000), brominated

bipyrrols (Tittlemier et al., 1999; Teuten et al., 2006c; Teuten

et al., 2007), dimethoxylated polybrominated biphenyls (diMeO-

PBBs) (Marsh et al., 2005; Teuten et al., 2007; Vetter et al.,

2008) and methoxylated and hydroxylated polybrominated

dibenzo-p-dioxins (MeO-PBDDs and OH-PBDDs) (Utkina et al.,

2001; Utkina et al., 2002). (Figure 1.2).

These compounds are mostly detected at low levels but are

sometimes present in high amounts in the producing organisms

(Unson et al., 1994). While some natural BOCs may, for

example, serve as pigments (Saenger et al., 1976), feeding

deterrents (Kurata et al., 1997), or have antibacterial effects (

Sharma et al., 1969), the biological role of the majority is still

unknown.

Figure 1.2. Naturally produced BOCs. A) 2,4,6-tribromophenol, B) a

diMeO-PBB C) 1,1’-dimethyl-2,2’-hexabromobipyrrol and D) a OH-

PBDD

Brominated phenols

Brominated phenols of both natural (Fu et al., 1995; Fielman et

al., 1999; Teuten et al., 2006b) and anthropogenic (World

Health Organization, 2005) origin are common in the

environment. For instance, 2,4,6-tribromophenol is produced by

red, brown and green alga (Fielman et al., 1999; Flodin et al.,

1999; Whitfield et al., 1999; Flodin et al., 2000; Chung et al.,

HH

A. B. C. D.

15

2003) and polychaeta (Fielman et al., 1999) but is also

anthropogenically produced and used as a flame retardant and

for wood preservation (World Health Organization, 2005).

OH-PBDEs and MeO-PBDEs

Marine organisms, such as algae, sponges and turnicates,

produce OH-PBDEs and MeO-PBDEs (Figure 1.3) (Fu et al.,

1995; Gribble, 1998; Gribble, 1999; Gribble et al., 1999; Utkina

et al., 2001). These compounds are found worldwide at different

trophic levels of the marine food web (Haglund et al., 1997;

Marsh et al., 2004a; Melcher et al., 2005; Malmvärn et al.,

2008; Pena-Abaurrea et al., 2009).

Figure 1.3. Structure of A) 6-OH-2,2’,4,4’-tetrabromodiphenyl ether

(OH-BDE47) and B) 2’-MeO-2,3’,4,5’-tetrabromodiphenyl ether (MeO-

BDE68)

OH-PBDEs are produced naturally, but there are also formed

through the metabolism of anthropogenic PBDEs (Marsh et al.,

2004b; Malmberg et al., 2005). Studies show that metabolised

PBDEs tend to have the OH-group in meta- or para-position to

the diphenyl ether bond (Marsh et al., 2004b; Malmberg et al.,

2005) whereas naturally produced OH-PBDEs tend to be

substituted with OH in ortho-position. OH-PBDEs with meta- and

para-substitution have been found to dominate in human serum

at sites known to be heavily contaminated with PBDEs

(Athanasiadou et al., 2008) whereas ortho-substitution

dominates for OH-PBDEs and MeO-PBDEs found in the ambient

environment (Marsh et al., 2004a; Malmvärn et al., 2008). The

HA. B.

16

biogenic synthesis of MeO-PBDEs is not clear but bacterial O-

methylation of phenolic compounds (Allard et al., 1987)

presents the possibility that OH-PBDEs are precursors to the

MeO-PBDEs.

Based on the substitution pattern it is thus likely that the OH-

and MeO-PBDEs found in the environment predominantly are of

natural origin. This theory has been strengthened by

measurements of a contemporary 14C signal of two MeO-PBDEs

isolated from whale blubber from the western North Atlantic

(Teuten et al., 2005) and by their presence in pre-industrial

whale oil (Teuten et al., 2007).

PBDDs and Br/Cl-DDs

Polybrominated dibenzo-p-dioxins (PBDDs) and furans (PBDFs)

(figure 1.4) are formed during combustion of materials

containing PBDEs (Buser, 1986; Thoma et al., 1987; World

Health Organization, 1998). If chlorine is present during the

combustion process, mixed brominated/chlorinated dibenzo-p-

dioxins (Br/Cl-DDs) can also be formed (Weber et al., 2003).

These compounds have been found in combustion processes

(Sovocool et al., 1988; Sovocool et al., 1989) and in sediments

from an industrial area in Japan (Takahashi et al., 2006).

The few available studies on the toxicity of PBDDs and Br/Cl-

DDs mainly focus on the binding affinity to the aryl hydrocarbon

receptor. Their results indicate a similar response as for the

more thoroughly studied PCDDs (Birnbaum et al., 2003; Van

den Berg et al., 2006; Olsman et al., 2007).

Figure 1.4. Examples of A.) a PBDD and B.) a PBDF

A. B.

17

High concentrations of mainly tribromodibenzo-p-dioxins

(triBDDs) have been reported from blue mussels (Malmvärn et

al., 2005; Haglund et al., 2007), alga (Malmvärn et al., 2008)

and sponge (Unger et al., 2009) from the Baltic Sea. Lower

concentrations have also been reported from several species of

fish and shellfish from the Baltic Sea and Swedish west coast

(Haglund et al., 2007). Although no natural sources have been

identified for these compounds, the congener pattern and lack

of clear anthropogenic source have led to the conclusion that

these PBDDs probably are of natural origin.

1.2 Natural production of brominated organic compounds

Simply described, the natural synthesis of BOCs consists of two

steps; first the hydrocarbon skeleton is formed and thereafter

bromination occurs (Nielson, 2003) Larger and more complex

natural BOCs can also be formed from smaller BOCs.

Formation of the hydrocarbon skeleton

There are three main biosynthesis pathways for organic

compounds starting with mevalonic, acetic and shikimic acid,

respectively (Figure 1.5). Generally, mevalonic acid is the basis

for terpenes and steroids, shikimic acid for aromatic compounds

and acetic acid for fatty acids and some aromatic compounds

(Nielson, 2003). Acetic acid and shikimic acid also combine with

simple nitrogen containing compounds to form amino acids.

Figure 1.5. The three starting points for biosynthesis of hydrocarbon

compounds. A) Mevalonic acid, B) Acetic acid and C) Shikimic acid.

COOH

OH

OH

HO

O

OH

OH

OHO

OH

A CB

18

Halogenation

Although chlorine is about 500 times more abundant than

bromine in sea water, the naturally produced BOCs are almost

as abundant as the natural organochlorine compounds (Gribble

et al., 1999). The reason for this is probably that bromine is the

more readily oxidized species in the enzymatic halogenation

process. Haloperoxidases, enzymes that oxidize halogens

through reactions with hydrogen peroxide, are common in

marine organisms (Butler et al., 1993; Moore et al., 1996). The

general formula for the haloperoxidase reaction is (Nielson,

2003):

𝑂𝑟𝑔𝐻 + 𝐻+ + 𝐻2𝑂2 + 𝐵𝑟− → 𝑂𝑟𝑔𝐵𝑟 + 2 𝐻2𝑂

The bromination process is a radical reaction with several steps

involving the relevant enzyme. The actual bromination of the

organic compound is with the oxidized electrophilic Br+ or Br.

formed from reaction of Br- with H2O2. The presence of oxidized

Br in the halogenation process can be seen in the common

bromination pattern in natural products, where Br is found in

positions where electron density is highest in a radical reaction

such as ortho- and para-positions in phenolic compounds

(Nielson, 2003).

19

Formation of larger structures

Larger brominated structures, such as OH-PBDEs and OH-PBBs,

can be formed from the brominated phenols (Figure 1.5).

Figure 1.5. Formation of OH-PBDE and OH-PBB from brominated

phenols by an enzymatically catalysed free radical reaction. The figure

is modified from schemes presented by Nielson (2003).

H.

.

H

+

H HH

20

2. Bromine isotopes

Bromine has two naturally occurring stable isotopes, 79Br

(50.7%) and 81Br (49.3%). The isotopic composition of Br is

usually expressed as the isotope ratio (R) of the heavier-over-

lighter isotope, i.e. R= 81Br/79Br. This is used to define a δ-value

which is the per mil deviation of the R in a sample compared to

R in a reference standard according to equation 1.

𝛿81𝐵𝑟 = 𝑅𝑆𝑎𝑚𝑝𝑙𝑒 − 𝑅𝑅𝑒𝑓𝑒𝑟𝑒𝑛𝑐𝑒

𝑅𝑟𝑒𝑓𝑒𝑟𝑒𝑛𝑐𝑒 × 1000

Equation 1.

The reference standard for stable Br isotopes is the naturally

occurring isotope ratio in seawater. This ratio is constant since

the residence time for Br in the ocean (Broecker and Peng,

1982) is substantially longer than the time for global ocean

mixing (130 million years vs. 1500 years) (Brownlow, 1996).

2.1 Kinetic isotope effect

The mass difference between the two bromine isotopes, 79Br

and 81Br, induce a slight difference in bond energy for the

heavier and the lighter isotope. Thus, the chemical bond

strength is isotope specific. The heavier isotope forms a slightly

shorter and stronger bond that requires somewhat higher

energy to break, causing a slower reaction rate during the

scission of C-Br bonds during dehalogenation reactions. This

leads to a kinetic isotope effect (KIE), which is the difference in

reaction rates for the two isotopes.

2.2 Isotope fractionation processes

Due to the KIE, formation and degradation of BOCs might result

in bromine isotope fractionation, leading to different bromine

isotopic ratios in different compounds. Figure 2.1 shows an

overview of organobromine compounds in the marine

21

environment and illustrate processes that may affect their

isotopic composition.

Figure 2.1. Overview of the processes that affect BOCs in the

environment. Due to the KIE these processes may change the isotopic

composition of the BOCs.

A KIE during the synthesis of halogenated compounds may

create a specific isotope composition for different synthesis

paths. The δ37Cl for anthropogenic abiotically synthesized

organochlorine compounds has been shown to range between

-5.10‰ and +1.22‰ (Drenzek et al., 2002). In contrast, a

study with haloperoxidase showed δ37Cl values of -11‰ and

-12‰ for the enzymatic chlorination of organic compounds

(Reddy et al., 2002). This difference in δ37Cl potentially allows

for differentiation of natural and anthropogenic compounds

based on their chlorine isotopic composition.

A similar KIE might be observed for BOCs but, due to the

smaller relative mass difference, the range in isotopic

composition is expected to be smaller for the heavier bromine

atoms than for chlorine. One estimate suggests that the

difference in KIE for enzymatic and abiotic bromination would

yield a δ81Br difference of 4‰ (Sylva et al., 2007). Based on

the results of stable chlorine isotope analysis, one of the aims

for this thesis was to develop a compound specific isotope

analysis (CSIA) method for Br to investigate the possibility of

Photo degradation

Anthropogenic production

Natural production

Metabolism

Bacterial degradation

22

using δ81Br to distinguish between natural (presumably

enzymatical bromination) and anthropogenic (presumably

abiotic bromination) sources of BOCs in the environment.

Since the 79Br-carbon bond is slightly easier to break than the 81Br-carbon bond, the dehalogenation processes that occur in

nature may cause isotope fractionation. The isotope

fractionation should result in an enrichment of 81Br in the

remaining non-degraded fraction compared to the starting

material. Determination of the Br isotopic composition of

environmental contaminants might thus tell something of the

degree of degradation of the organobromine contaminants

found in nature.

This approach is similar to studies of stable chlorine isotopes,

which have been used to assess the extent of degradation of

DDT in the Baltic Sea (Holmstrand et al., 2007).

23

3. Samples and purification methods

In this thesis, BOCs have been studied in four aquatic species

An overview of the sample treatment is given in figure 3.1.

Figure 3.1. Schematic presentation of the sample treatment leading to

identification, quantification and bromine isotope analysis of BOCs in

this thesis.

3.1 Samples

A common freshwater sponge (Ephydatia fluviatilis) and blue

mussels (Mytilus edulis) were sampled at the same site in the

south-western Baltic Sea and analysed for PBDDs (Paper I). The

brackish water of the Baltic Sea enables freshwater and marine

species to coexist, albeit under some stress due to the too high

or too low salinity, respectively.

Blubber from a northern bottlenose whale (Hyperoodon

ampullatus) was used to isolate several µg of lipophilic BOCs

(Papers II and III).This species dwells in the northern Atlantic

Sample extraction(organic solvent)

Lipid removal(Sulfuric acid)

Chromatographic separation

Identification / quantification

GC-MS GC-MC-ICP-MSNMR

24

and the young male in this study was found dead on the

Swedish west coast. In Paper IV, dibromophenol isolated from a

marine benthic worm (Saccoglossus bromophenolosus) was

analysed.

3.2 Extraction and lipid removal methods

Extraction and purification of target compounds are

prerequisites for accurate quantification and identification. By

removing the sample matrix the limits of detection and

quantification are lowered.

Small scale sample extraction (Jensen method)

The Jensen extraction method (Jensen et al., 1983) was used to

extract halogenated dibenzo-p-dioxins from sponge and blue

mussel samples (Paper I). This method has been developed to

quantitatively extract the lipids, and hence lipophilic

compounds, from a complex sample matrix. Briefly, the samples

are homogenized in acetone/n-hexane followed by extraction

with n-hexane/diethyl ether. Partitioning of the organic solvent

phase with a slightly acidic 0.9 % NaCl water phase removes

water and water soluble compounds from the extract.

The Jensen method is a standard method for extraction of

organohalogen compounds, including BOCs, in our laboratory

and has previously been used successfully in the extraction of

PBDDs. Other possible extraction methods are Soxhlet

extraction or accelerated solvent extraction (ASE). However,

these methods use heat and/or pressure which could cause

debromination of the target BOCs or cyclisation of OH-PBDEs in

the sample, forming PBDDs. It is possible that either of these

methods could be suitable, though neither have been evaluated

for quantitative extraction of PBDDs from biological material.

Large scale lipid extraction (Wallenberg perforator)

A sample of 10 kg whale blubber tissue was used to isolate

several µg of BOCs (Paper II). Blubber tissue consists of about

95% liquid lipids that can be isolated from the blubber tissue by

grinding followed by centrifuging. The lipids are thus separated

from the rest of the tissue. The 10 kg whale blubber sample was

25

reduced to approximately 9.5 kg pure lipids by this initial

treatment. A custom manufactured Wallenberg perforator as

designed by professor Sören Jensen (Jensen et al., 1992) was

used to partition the lipophilic compounds from the lipids to an

acetonitrile (ACN) phase (Figure 3.2). Aromatic compounds

partition into the ACN phase through interactions of their π-

electrons with the nitrile group.

Figure 3.2. The Wallenberg perforator (Jensen et al., 1992). Arrows

indicate the flow direction for ACN from the cooler through the liquid

lipids and back to the heated collector bottle.

The Wallenberg perforator uses refluxing ACN, allowing it to

pass repeatedly through the lipids. Briefly, ACN is heated in the

collector bottle (Figure 3.2) causing the ACN vapour to rise into

the cooler. Liquid ACN flows through a system of glass tubes

from the cooler down to the bottom of a flat-bottomed flask

Liquid lipids

Collector bottle

Heating

Cooling

26

filled with liquid lipids. Continuous stirring cause the ACN to

spiral through the lipids as it, due to the lower density of ACN

compared to the lipids, rise to the top of the flask. The

halogenated compounds are extracted with the ACN during this

process and are transported back to the collector bottle. The

ACN continues to cycle through the system, while the BOCs,

having higher boiling points, accumulate in the collector bottle.

The extraction is stopped when the amount of ACN having

passed through the lipids is the equivalent of 30 times the lipid

volume to ensure that more than 95% of the BOCs are

extracted. Although some lipids are also dissolved in the ACN

and follow the analytes to the collector bottle, the method

efficiently removes about 90% of the lipids using a relatively

small amount of solvent. A second extraction round remove an

additional 70% of the remaining lipids.

Removal of lipid residues

Removal of lipid residues was done with sulfuric acid in both

Papers I and II. This method was not considered for the entire

large blubber lipid sample because of the enormous volume of

sulfuric acid that would have been required. Sulfuric acid

treatment is a destructive method and many compounds react

with the acid and are destroyed or partition into the sulfuric acid

phase because of the Lewis acid properties.

An example of a non-destructive method for the removal of the

lipid residues is gel permeation chromatography which

separates the analytes from the lipid molecules by size. This

method is time consuming, and was deemed unnecessary since

all target compounds were known to survive the sulfuric acid

treatment.

27

3.3 Chromatographic separation

Purification by class separation reduces the risk of compounds

co-eluting during analysis, which is a prerequisite for both

proper identification and quantification.

Column chromatography

Before instrumental analysis, the analytes were fractionated

according to polarity on a silica gel column. This removed the

ubiquitous non-polar PCBs from the slightly polar target MeO-

PBDEs and PBDDs, which were also separated into different

fractions. The more polar OH-PBDEs are retained on the column

and thus removed from the sample. This minimizes the risk of

PBDDs forming from OH-PBDEs in the gas chromatography (GC)

injector, GC column or in the mass spectrometric (MS) ion

source during analysis.

In Paper I the PBDD fraction was further purified on a column

prepared with active carbon dispersed on Celite (Haglund et al.,

2007), separating compounds based on their planarity to isolate

the planar dibenzo-p-dioxins and dioxin-like compounds from

unplanar substances.

Preparative capillary gas chromatography

The NMR method used for identification of an unknown BOC

(Paper II) required a much larger amount of pure compound

than normally used in analytical work with environmental

contaminants. Preparative capillary gas chromatography (PCGC)

(Figure 3.3) is a method that can be used to isolate large

amounts of single compounds from a sample extract. Different

versions of the technique have been used since the 1970s (e.g.,

(Badings et al., 1975; Roeraade et al., 1986). Environmental

applications of the PCGC technique in more recent years include

the isolation of single organic compounds for offline CSIA of

δ14C (Eglinton et al., 1996; Eglinton et al., 1997; Mandalakis et

al., 2004; Teuten et al., 2005; Kumata et al., 2006; Zencak et

al., 2007; Sheesley et al., 2009) and δ37Cl (Holmstrand et al.,

2006a; Holmstrand et al., 2006b; Holmstrand et al., 2007). The

isolation of environmentally occurring BOCs for structural

elucidation with NMR (Teuten et al., 2006a, Unger et al., 2010

28

(Paper II)) and of organic contaminants in ground water for test

of genotoxicity (Meinert et al., 2010) have also reported.

Figure 3.3. Overview of the PCGC process with schematic picture of the

PCGC instrument. A whale blubber sample was injected and a single

BOC was isolated in 99% purity. Mass chromatograms were scanned

between mass-to-charge ratios (m/z) 50-700.

The PCGC technique was optimized for semi-volatile compounds

in environmental samples by Mandalakis et al. (2003). A sample

is repeatedly injected into the GC where it is separated and the

individual compounds are directed into different cooled quartz

traps at the GC outlet. A cooled injection system (CIS) and a

megabore column are used to enable larger sample amounts

and minimize the number of injections needed. The retention

times are carefully monitored and the trap times are adjusted to

correlate with changes in retention time over the sampling

period of normally a few days. This results in sample purities of

>95%.

Effluent splitter

1%

Autosampler

Cooling

Fraction collectorDetector

99%

Megabore column

CIS

22 24 26 2822 24 26 28

29

4. Analysis

Three different instrumental analysis methods have been used

for identification (Papers I and II), quantification (Paper I) and

bromine isotope analysis (Papers III and IV) of BOCs in this

thesis. These analysis techniques are briefly described below.

4.1 GC-MS

The most common method for the analysis of environmental

contaminants is gas chromatography–mass spectrometry (GC-

MS). The method is also applied for the analysis of naturally

produced BOCs. Identification of compounds with GC-MS is

most reliable when authentic reference standards are used to

compare both the GC retention times and mass spectra of the

target compounds. Many novel compounds in environmental

samples still lack standards, limiting identification to the

interpretation of mass spectra fragmentation patterns. In this

context, high-resolution MS is particularly useful for determining

the accurate mass of the analysed compounds. Based on

accurate mass, it is possible to calculate the empirical formula

of the analysed compounds. This technique was used for

identification of Br/Cl-DDs (Paper I) and as part of the

identification of 6-methoxy-5-methyl-2,2’3,4’-tetrabromo

diphenyl ether (MeOMe-BDE42) (Paper II).

Two MS ionization techniques have been used in the analysis of

BOCs in this thesis. Electron ionization (EI) is a reproducible

technique that generally provides detailed structural information

through characteristic fragmentation patterns for different

groups of compounds. Electron capture negative ionization

(ECNI) is a softer ionization method where a buffer gas, in this

case ammonia, is bombarded with electrons to create low

energy thermal electrons. These are captured by the analytes

and negative ions are formed. A high temperature in the ion

source typically causes BOCs to form mainly Br- ions, resulting

in a high concentration of Br- in the detector. Thus, by using

selective ion monitoring (SIM) to detect only the Br-, this

technique is highly sensitive and permits quantification of small

amounts of compounds. This method was used for

30

quantification of MeO-PBDEs (Paper I) and for estimating the

concentration of MeOMe-BDE42 (Paper II). The PBDDs and

Br/Cl-DDs in Paper I were quantified with high resolution MS

using EI and SIM of appropriate ions.

4.2 NMR

Nuclear magnetic resonance spectroscopy (NMR) is a routine

method for structural elucidations in organic chemistry.

However, since NMR requires nearly mg amounts of pure

compound the technique is rarely used in analytical

environmental chemistry.

The 1H NMR spectra provide information on the position of all

protons in a molecule, both in terms of what kind of carbon

structure they are attached to (aliphatic, aromatic, alcohol

etcetera) and where they are located in relation to other

substituents in the molecule, as the chemical shift of each

proton is affected by its surroundings. Protons attached to

adjacent carbons also affect each other’s signal, resulting in

signal splitting (Figure 4.1).

The amount of sample required for 13C NMR is larger than for 1H

NMR because of differences in the intrinsic magnetic properties

of the 1H and 13C nuclei, and the low natural abundance of 13C

(1.1% of total carbon). When the sample amount is too small

for regular 13C NMR, some 13C NMR shifts may be obtained by

using heteronuclear single quantum coherence (HSQC). This

technique is based on 1H-13C interactions and gives 13C shifts for

carbons directly attached to protons by showing the cross-peaks

in a two-dimensional spectrum even though the intensity of the 13C spectrum is too low to be seen. Figure 4.1 shows the HSQC

NMR spectrum used for identification of MeMeO-BDE42 in Paper

II.

31

Figure 4.1. Two-dimensional HSQC NMR spectrum of MeMeO-BDE42. 1H-spectrum is shown on top with enlargement of the area between

6.2 - 7.8 ppm to show signal splitting. 13C-shifts are shown on the y-

axis.

4.3 GC-multiple-collector-ICP-MS

Inductively coupled plasma (ICP) is an efficient ion source that

in connection with a MS equipped with multiple ion beam

collectors can be used to determine isotope ratios for a wide

range of elements. The plasma is created by Ar gas in an

electromagnetic field generated by a radio frequency (RF)

current in the RF-coil (Figure 4.2).

Inorganic or polar organic compounds in aqueous solution are

aspirated into the plasma and ionized. The ions are transferred

into the MS, where they are separated based on their mass-to-

charge ratio and detected. The continuous flow of sample to the

plasma yields a continuous ion signal to the detector. The signal

can be scanned repeatedly permitting a large number of

measurements of the isotope ratio.

7.5 5.0

160

80

0

2.5

1H- shift (ppm)

13C

-shift

(ppm

)

32

Figure 4.2. Schematic drawing of the GC-MC-ICP-MS instrument used

for bromine isotope measurements of BOCs in Papers III and IV.

Besides aqueous solutions, it is possible to use other sample

introductory systems, including GC. The direct connection of a

GC to the plasma and MS (Figure. 4.2) was explored in Papers

III and IV. This on-line system simplifies the purification process

needed before analysis by separating and directly introducing a

specific compound from a complex sample matrix into the ICP

ion source. The sample is manually injected into the megabore

coloumn of the GC where the compounds are separated,

allowing single compounds to elute through the heated

transferline into the plasma where they are ionized.

In contrast to an aqueous solution, which is continuously

aspired into the ICP ion source, each compound generates a

transient signal as they elute from the column. The peak width

is only a few seconds and probably not isotopically homogenous

(Krupp et al., 2005; Holmstrand et al., 2006b). The entire

signal thus needs to be integrated in order to obtain the isotope

ratio of the compound.

Manual injection

Megabore column

Hexapole

collision cell

Multiple collector

Faraday cups

Ambient pressure High vacuum

Ar

Transfer line

ICP torch

RF-coil

MS inlet cones

Plasma gas (Ar)Auxillary gas (Ar)

Sample

Mass

analyser

33

5. Summary of papers

5.1 Paper I - Identification and quantification of PBDDs

and Br/Cl-DDs

In Paper I the common fresh water sponge Ephydatia fluviatilis

was collected from Borgholm harbour in the south-western

Baltic Sea and analysed for halogenated dibenzo-p-dioxins. High

concentrations of PBDDs, especially 1,3,7- and 1,3,8-TriBDD,

were found. As there are no known anthropogenic sources for

PBDDs in the Baltic area and the bromine substitution pattern of

the detected PBDDs are in agreement with what could be

expected for naturally produced compounds, these findings

strengthen the idea that PBDDs are produced naturally in the

Baltic Sea.

Mixed brominated/chlorinated dioxins were also found in the

sponge. To our knowledge this was the first time Br/Cl-DDs

were reported from a background environment with no apparent

anthropogenic sources. This led to the conclusion that Br/Cl-

DDs are also probably natural products in this region. It is worth

noting that the concentrations of Br/Cl-DDs, though lower than

PBDDs, were higher than PCDDs, although PCDD concentrations

are considered to be a problem in the Baltic Sea.

Figure 5.1. Examples of the compounds identified in Paper I A.) 1,3,7-

triBDD, B.) 1,3,8-triBDD, C.) 1,2,4,7-tetraBDD, D.) a Br2Cl-DD and E.)

a Br3Cl-DD.

A. B. C.

D. E.

34

5.2 Paper II - NMR-identification of a novel brominated

organic compound isolated from whale blubber

In Paper II an unknown BOC, present in large amounts in

northern bottlenose whale (Hyperoodon ampullatus) blubber,

was identified. The combination of GC retention times, mass

spectra and NMR enabled us to determine the structure of a

PBDE with both MeO- and Me-substitution (Figure 5.2). The use

of NMR for identification of unknown compounds is unusual in

analytical environmental chemistry as it requires several µg of

pure compound. In this case it was possible to isolate about 100

µg of pure compound by using a PCGC. To our knowledge this

was only the second time PCGC was

used to isolate compounds for

identification with NMR.

Figure 5.2. MeOMe-BDE42 isolated from

whale blubber and identified in Paper II.

5.3 Paper III - Development of a GC-MC-ICP-MS method

for brominated diaromatic compounds

The bromine isotopic composition of BOCs found in the

environment could potentially reveal information of the origin

and fate of these compounds. Paper III describes the first

attempts to develop a method for CSIA of bromine in PBDEs

and MeO-PBDEs with an on-line GC-MC-ICP-MS system. Three

different transferlines were tested and evaluated for their

efficiency in conducting the analytes from the GC to the ICP.

Standard bracketing with monobromobenzene (MBB) with

known δ81Br was used to calculate δ81Br for three PBDEs,

2,2’,4,4’-tetrabromodiphenyl ether (BDE-47), 2,2’4,4’,5-

pentabromodiphenyl ether (BDE-99) and 2,2’4,4’,6-

pentabromodiphenyl ether (BDE-100) (figure 5.3). An attempt

was also made to compare the δ81Br of the structurally similar

BDE-47 and 6-methoxy-2,2’,4,4’-tetrabromo diphenyl ether

(MeO-BDE47).

35

Figure 5.3. The three PBDEs analysed in Paper III. A) BDE-47 B) BDE-

99 and C) BDE-100.

5.4 Paper IV - Measurements of δ81Br in anthropogenic

and natural monoaromatic compounds

In Paper IV δ81Br values were determined for four industrially

produced monoaromatic brominated compounds and a

brominated naphtalene (Figure 5.4) using a method developed

in Paper III. This was done to test the variation in bromine

isotopic composition among different industrial products. A

naturally produced compound, isolated from the marine worm

Saccoglossus bromophenolosus and structurally identical to one

of the anthropogenic compounds, was also analysed. No

statistical difference could be seen between the anthropogenic

and the natural product. More research is needed before any

final conclusions can be drawn in this area.

Figure 5.4. BOCs analysed in Paper IV. A) 2,4-dibromoanisole, B) 2,4-

dibromophenol, C) 4-bromophenol, D) 1,4-dibromophenol, E) 2,4,6-

tribromophenol, F) 1,3,5-tribromobenzene and G) 1-bromonaphtalene.

A. C.B.

H

H

HA.

G.F.E.

B. C. D.

36

6. Results and Discussion

The dual aims of this thesis were the identification of natural

BOCs and the development of a method for compound specific

bromine isotope analysis. The following section is thus split in

two, discussing first the identification of BOCs (Papers I and II)

and thereafter bromine isotope analysis (Papers III and IV).

6.1 Identification of natural brominated organic

compounds

Since the first report of PBDDs in Baltic Sea biota was published

in 2005 (Malmvärn et al., 2005) additional studies have been

published (Haglund et al., 2007; Malmvärn et al., 2008; Unger

et al., 2009 (Paper I); Haglund, 2010) attempting to identify a

natural source and the biosynthetic pathways of these

compounds. There are several lines of evidence that these

compounds are of natural origin. There is no known commercial

or other intentional production of PBDDs. There are however

unintentional anthropogenic formation of PBDFs and PBDDs via

combustion of bromine containing material e.g. PBDEs (Thoma

et al., 1987). In this context, PBDFs are present in far higher

concentrations than PBDDs. In analysed matrices from the

Baltic Sea, the levels of PBDDs greatly exceeded the

concentrations of PBDFs (Haglund et al., 2007; Malmvärn et al.,

2008; Unger et al., 2009 (Paper I); Haglund, 2010). A general

pollution of PBDDs due to combustion ought to also result in

similar concentrations in lakes as in the Baltic Sea, which is not

reported (Haglund et al., 2007). The congener pattern that is

seen in the Baltic Sea samples, where triBDD is clearly

dominant, also indicate natural production since combustion of

PBDEs yield a larger variety of PBDD congeners.

The concentrations of triBDDs found in the sponge (Ephydatia

fluviatilis) were similar to the levels reported in blue mussels.

Both sponges and blue mussels are filter feeders and their

respective PBDD concentrations probably reflect a similar

uptake process. This indicates that sponges are not major

producers of PBDDs in the Baltic Sea.

37

Several single congener PBDDs were identified and quantified in

ambient biota for the first time as reported in Paper I.

Identification and quantification of individual PBDD congeners is

a step towards a better understanding of the biosynthesis of

these compounds. It has been shown that the most abundant

PBDDs found in Baltic Sea biota can be formed through either

enzymatic coupling of abundant bromophenols, condensations

of common OH-PBDEs or, for some triBDDs, debromination of

higher brominated dibenzo-p-dioxins (Haglund, 2010).

Identification and quantification of single congeners is also a

base for future risk assessment since different congeners most

certainly have different toxic effects. A less toxic compound can

have a larger effect than a more toxic compound if the

concentration is high enough. The triBDDs found in high

concentration in Baltic Sea biota were present at 25 000 times

higher concentration than the toxic 2,3,7,8-tetrachloro dibenzo-

p-dioxin (TCDD) in the sponge (Paper I). Several other PBDD

and Br/Cl-DD congeners were also more abundant that the

PCDD congeners the sponge (Paper I). The triBDDs appear to

be relatively rapidly excreted by fish (Haglund et al., 2010) and

are thus less likely to cause toxic effects, but more work is

required to confirm this speculation.

The findings of Br/Cl-DDs in sponge (Paper I) was the first time

these compounds were reported in a background environment.

Previous reports have been from sites associated with

combustion (Sovocool et al., 1988; Sovocool et al., 1989;

Weber et al., 2003; Haglund et al., 2007). Accordingly, the very

high levels of these compounds in the sponge, by far exceeding

those of PCDDs, are a strong indication a natural production of

Br/Cl-DDs in the Baltic Sea.

It seems clear that PBDDs and even mixed Br/Cl-DDs are

produced naturally in concentrations that exceed the

anthropogenic input of these compounds to the Baltic Sea.

Since PBDDs in the Baltic Sea are likely formed from OH-PBDEs

or brominated phenols, the question of natural or anthropogenic

origin should perhaps be expanded to also include the origin of

these precursor compounds.

38

The use of NMR vs. GC-MS for identification of BOCs in the

environment

The use of NMR for identification of bioaccumulated BOCs is

limited by the requirement of near mg amounts of pure

compound for analysis. Its main use for environmentally

occurring BOCs has thus been for natural products isolated from

the producing organisms (Unson et al., 1994; Cameron et al.,

2000; Takahashi et al., 2002; Utkina et al., 2002).

Although NMR gives more information on the chemical structure

than GC-MS, the ability of GC-MS to detect compounds in pg

concentrations in sample extracts (as for PBDDs in Paper I)

makes it a more useful method for most analytical

environmental chemistry purposes. The different requirements

for sample amount and purity make it possible to analyse

samples with GC-MS that are impossible with NMR, due to the

presence of other compounds in the sample or the small

amounts of analyte available. Isolating a single compound from

a more complex environmental extract using a PCGC, alleviates

the problem with pure compounds for NMR analysis. To isolate

up to mg amounts of an analyte for NMR, either a sample with

very high concentrations of the compound of interest or a very

large sample (as in Paper II) is needed.

Paper II describes the identification of a novel BOC with a

combination of NMR and GC-MS. A diMeO-PBB with the same

molecular weight as the unknown compound has been reported

(Marsh et al., 2005; Teuten et al., 2007; Vetter et al., 2008).

The mass spectra of our compound indicated a MeOMe-BDE

structure, as opposed to a diMeO-PBB structure, and the 1H

NMR spectra unequivocally showed the presence of one

methoxyl group and one methyl group, both placed on an

aromatic ring. The sample amount was too low for 13C NMR.

Still, with the somewhat limited information given by 1H NMR

and HSQC, it was possible to limit the possibilities to three

structures. One of these compounds were then synthesised and

the final identification of MeOMe-BDE42 was with GC retention

times.

39

6.2 Development of CSIA-Br

A range of isotope systems are used for studies of transport and

fate of compounds and elements in the environment. This

includes the ratios of 15N/14N to study food webs (Broman et al.,

1992; Tittlemier et al., 2002), 14C/12C to separate between

biogenic and fossil fuel combustion products (Mandalakis et al.,

2004; Sheesley et al., 2009) and to determine the natural or

anthropogenic origin of BOCs in the environment (Teuten et al.,

2005), and 13C/12C for studies of degradation of organic

pollutants (Schmidt et al., 2004; Zwank et al., 2005;

Holmstrand et al., 2007). Similarly, δ37Cl has been used to

study the degradation of DDT in the Baltic Sea (Holmstrand et

al., 2007) and for source apportionment of high levels of PCDDs

in clay (Holmstrand et al., 2006a). The possibility that accurate

measurements of the bromine isotopic composition of BOCs in

the environment may be used for determining the sources and

fates of these compounds was the reason for the method

development described in Papers III and IV.

Standard bracketing

Isotope ratio determination using MC-ICP-MS suffers from

variations in the absolute isotope ratios due to instrument drift

and mass bias. Corrections for these effects can be obtained

with different techniques, including the standard-sample-

bracketing method. This method relies on a substance with

known isotopic composition that is analysed between the

samples. Here, MBB with a known isotope ratio was co-injected

with all samples and used as the isotopic anchor point (Papers

III and IV). A linear drift in the measured isotope ratios was

assumed between the two MBB measurements immediately

preceding and following the sample peaks. A theoretical 81Br/79Br ratio for MBB was calculated for the time of elution for

the PBDEs. This value used to calculate δ81Br values for the

PBDEs (Paper III). The difference in δ81Br for the three PBDEs

remained constant within all measurement series, although the

absolute values varied between series. MBB and PBDEs are two

different types of compounds however, and it is likely that the

ionization yields in the plasma differ. This could enhance any

40

variation caused by slight differences in the physical setup of

the instrument before each measurement campaign and explain

the larger difference between data series than within each

measurement series

This idea was tested with measurements of structurally similar

MeO-PBDE and PBDE isolated from whale blubber (Paper III).

BDE-47 was used as a standard for MeO-BDE47 and the

precision between two measurement series was similar to that

previously found within one series of PBDEs. This result

supported the idea that a structurally similar reference standard

gives a better precision. This was further explored with

measurements of four anthropogenic monocyclic aromatic BOCs

and a brominated naphthalene using MBB as reference standard

(Paper IV). All of these compounds are structurally similar to

MBB and have similar GC retention times, which place the

measurement of the standard closer in time to the analyte than

for PBDEs. The precision for these measurements was 0.8‰.

The internal instrument drift and the compound-specific

ionization yields apparently call for a reference standard with

similar ionization efficiency in the plasma that can be measured

close in time to the analyte.

Transfer lines

The higher boiling points for PBDEs compared to monocyclic

BOCs require a transfer line between the GC and the ICP ion

source with enough heating capacity to keep the analytes from

condensing. Thus the transfer line needed to be heated all the

way from the GC to the plasma with no cold spots on the way.

Three different transfer lines were tested and evaluated. A

detailed schematic drawing of all three transfer lines is

presented in Paper III (Figure 1 in Paper III).

The first transfer line (T1 in Paper III) was electrically heated

between the GC and the quartz torch of the ICP ion source. It

was assumed that the heated gas from the GC and the heat

from the plasma would be sufficient to carry the PBDEs through

the torch to the plasma. The results showed that the standard

MBB passed through but no PBDE signals could be detected. A

version of this transfer line was however used during later

analysis of monocyclic aromatic BOCs (Paper IV).

41

The second transfer line (T2 in Paper III) consisted of a thin

stainless steel capillary that was fed into the torch and

electrically heated through its entire length. The steel capillary

ended 20 mm from the plasma, any further and the intense

heat from the plasma would have melted the steel. Although

this system gave good response for PBDE the precision between

measurement series was lower than expected and the precision

for MBB went from 0.4‰ for T1 to 2‰. We hypothesized that

this was an effect of electrostatic interference from the electric

heating of the transfer line causing instability in the plasma.

The third transfer line (T3 in Paper III) was constructed to keep

the sample constantly heated all the way from the GC to the

plasma while eliminating the risk of electrostatic effects. Instead

of heating the transfer line itself, the Ar gas carrying the sample

into the plasma was heated in the GC oven. The sensitivities for

both MBB and PBDEs were the same as for T2 but unfortunately

the precision did not improve. The variations in isotope ratios

might be caused by the heating of the gas affecting the stability

of the plasma.

Evaluation of δ81Br for enzymatic and anthropogenic

bromination

The δ81Br of both industrially synthesised and naturally

produced dibromophenol were found to have a difference of

1.4±0.9‰ (Paper IV). The δ81Br for all the anthropogenic

compounds in this study ranged from -4.3‰ to -0.45‰. The

observed difference between enzymatic and anthropogenic

bromination was therefore not statistically significant. The

difference in δ81Br for presumably natural MeO-BDE47 and

anthropogenic BDE-47, both isolated from whale blubber (Paper

III), was even lower at 0.3±0.7‰.

With the existing methods and results it is not possible to

discern a difference in the bromine isotopic composition for

anthropogenic and biogenic BOCs.

42

7. Conclusions

Based on their distribution in algae, blue mussels and

sponge, it is possible that PBDDs in the Baltic Sea do not

derive from a single producing organism, but rather

reflect a general production process active in several

organisms (Paper I).

Mixed Br/Cl-DDs are produced in the Baltic Sea (Paper

I).

PCGC is a useful tool to facilitate the identification of

unknown compounds in the environment. By allowing a

large amount of pure compound to be isolated NMR

analysis is enabled (Paper II).

Reference standards with ionization yields similar to the

analytes alleviate the problem of internal instrument drift

during isotope measurements with GC-MC-ICP-MS

(Papers III and IV).

With the existing methods and results it is not possible

to discern a difference in the bromine isotopic

composition for anthropogenic and biogenic BOCs

(Papers III and IV).

43

8. Future perspectives

Although there are strong indications that PBDDs and Br/Cl-DDs

are naturally produced in the Baltic Sea the question is not fully

resolved. Studies of PBDDs, Br/Cl-DDs, OH-PBDEs and

brominated phenols in sediments from the pre-industrial era up

to now could bring clarity to this issue. CSIA of 14C, or possibly

Br isotopes, could be used for this purpose.

Information on the effects of PBDDs and mixed Br/Cl-DDs is still

very limited. More studies are needed in order to evaluate the

potential effects these compounds have on the environment.

NMR has a clear advantage over GC-MS for identification of

unknown compounds although the requirement of near mg

amounts of pure compound is daunting to an analytical

environmental chemist. Large scale extraction methods such as

the Wallenberg perforator and PCGC for isolation of the

compound make NMR a possible choice for the identification of

unknown organic compounds in the environment.

Source apportionment of BOCs is increasingly interesting as

more are discovered in the environment. The attempts here to

use bromine isotopes should be further investigated. It is

necessary to repeat the experiment done for organochlorine

compounds with chloroperoxidase also for BOCs, using a range

of enzymes, to establish a natural production endpoint or range.

Until there is a relatively simple, unquestionable way of

separating anthropogenic and naturally produced compounds in

the environment, the discussion of the relative importance of

the different sources to the environment will continue.

44

9. Sammanfattning på svenska Bromerade organiska föreningar (här förkortat BOCs, från

engelskans brominated organic compounds) är vanliga i miljön.

Många har kända källor och är antingen naturliga eller

antropogena, dvs. industriellt tillverkade. För en del ämnen

finns dock både naturliga och antropogena källor medan andra

är av okänt ursprung. Den här avhandlingen syftar till att dels

identifiera ett antal naturligt producerade BOCs och dels till att

utveckla en metod för att mäta sammansättningen av

bromisotoper i BOCs i miljön.

Polybromerade dibenzo-p-dioxiner (PBDDs) i Östersjön har

bedömts vara naturligt producerade, även om man ännu inte

har lyckats identifiera någon specifik naturlig producent.

Eftersom många BOCs i tropiska vatten är producerade av

marina svampdjur, analyserades förekomsten av BOCs i en

vanligt förekommande tvättsvampsart (Ephydatia fluviatilis)

från Östersjön (Paper I). Resultaten visade att den här

tvättsvampen troligen inte är någon betydande källa till PBDDs i

Östersjön. Utöver PBDDs fann vi också dibenzo-p-dioxiner med

både klor och brom i tvättsvampen. Detta var första gången

sådana föreningar rapporterades från en bakgrundsmiljö, dvs.

utan någon antropogen källa i närheten.

Kärnmagnetisk resonansspektroskopi (NMR) används sällan i

analytisk miljökemi eftersom metoden kräver en stor mängd ren

substans. I Paper II användes en preparativ

kapillärgaskromatograf (PCGC) för att isolera ca 100 µg av en

tidigare oidentifierad BOC från späck från en nordlig näbbval

(Hyperoodon ampullatus). Detta var en tillräcklig mängd för att

kunna använda NMR för att identifiera föreningen.

Bromisotopsammansättningen (δ81Br) i en BOC påverkas av

olika processer och skulle därför kunna ge information om dess

ursprung och öde i miljön. De första stegen mot en sådan

applikation är utvecklingen av en metod för att bestämma

bromisotopkvoten i de BOCs man finner i miljön (Paper III)

samt att definiera var på δ81Br-skalan de antropogena

respektive naturliga ämnena befinner sig. I Paper IV

analyserades därför bromisotopkvoten i ett antal antropogena

BOCs i ett första försök att definiera det antropogena området

på skalan.

45

10. Acknowledgements När jag började på ITM var jag 23 år och hade förlorat min

mamma ett knappt år tidigare. Att säga att jag var ung och lite

vilsen är nog inte helt fel. Under mina år här jag har inte bara

lärt mig mer om laborerande, analyser och skrivande utan

också blivit många erfarenheter rikare och utvecklats som

person. Jag har blivit vuxen. Tack alla ni som varit med mig på

den resan!

Först vill jag tacka mina handledare. Tack Örjan för din

förmåga att entusiasmera och se möjligheter där jag ser

svårigheter. Tack Lillemor för allt ditt stöd och för att du alltid

tagit dig tid att lyssna på mig, vare sig det gällt arbete eller

privatliv. Tack Per för att du alltid funnits där i bakgrunden,

trevlig julsushi och för all hjälp under skrivandet av själva

avhandlingen.

Ett stort tack också till Åke för stöd då jag behövt det och för

alla kommentarer och tips under avhandlingsskrivandet.

Henry, du förtjänar egentligen ett helt eget kapitel! Din lugna

personlighet och speciella humor kan göra vilken dålig dag som

helst mycket bättre. Utan dig har jag svårt att se hur jag skulle

gått i mål med det här, inte minst med tanke på att pek 3 och 4

hade varit oändligt mycket svårare (jag säger inte omöjligt, för

inget är omöjligt!) att nå iland med utan dig.

Tack Karin, för att du tar hand om oss alla och för att du är så

bra på att organisera de trevliga små påhitt som skapar

sammanhållning.

Bodil, tack för alla tips och råd om extraktioner av jätteprover!

Och tack för att du är en underbar människa som både lyssnar

och säger rätt saker.

Tack till mina rumskompisar på gamla ITM. Marie och Cissi,

som tog hand om mig när jag var ny. Tack Marie för alla långa

både vetenskapliga och ovetenskapliga samtal. Nu skiter vi i det

här och går hem! Och tack Cissi för att du ibland kan prata

nästan lika mycket som jag. Vi fick inte mycket gjort vissa

förmiddagar inte!

46

Labbsällskapet Kerstin, Hanna, Jorien, Ulla E, Dennis och

Anna M, tack för både tips och råd och för mer eller mindre

urspårade (freestylade?) diskussioner under många och långa

timmar på lab. Tack Yngve för din oerhörda kunskap om allt

som rör masspektrometri och ditt intresse och din entusiasm för

nya oidentifierade substanser!

Tusen tack Marsha för trevligt sällskap och för att du tagit bort

säkert 90 % av alla however, also och thus i den här avhandlingen!

All current and previous post docs in the MPG group, Manolis,

Kuma, Bart, Laura, Rebecca (I seriously miss our morning

coffees!), Daniel, Brett, Christoph and August. Thank you

Zdenek for taking more time than you really had to help me

with everything from handling the PCGC to structuring excel

sheets.

De “nya” doktoranderna i MPG gruppen, Axel, Elena, Charline

och Emma. Ni är ett härligt gäng! Thank you Elena for

introducing me to the wondrous combination of condensed milk,

lots of nuts and simple pie!

Tack till doktorandgänget på Miljökemi, Anna S, Linda, Johan

F, Hans, Maria S och alla andra både nuvarande och tidigare

doktorander för trevliga fester, seminarier, fikan och en och

annan lunch. Tack Karin L, min räddare i nöden vid flera

tillfällen, för hjälp med kiselgel, standarder, provletande,

referenser och allt vad det må vara!

Tack Anita och Birgit (och Ulrika) för att ni alltid har haft tid

när jag ringt eller kommit förbi och pratat en stund.

Tack till mina medförfattare för långa och givande diskussioner i

samband med peken. Tack Göran för att du visade mig vilken

information det faktiskt går att få fram ur ett NMR spektrum och

tack Peter för snabba svar om allt som rör dioxiner.

Tack också till alla andra nuvarande och tidigare kollegor på ITM

som hjälpt till att göra min tid som doktorand så bra som den

varit. Det känns lite sorgligt att lämna er nu men jag tror att jag

är redo att möta världen utanför universitetet!

47

Även livet utanför jobbet påverkas av de fram- och, i mitt fall

frekventa, motgångar man drabbas av på jobbet och det är

vännerna som får ta smällen.

Så, tack hela gänget för att ni, trots att ni egentligen inte fattat

vad det är jag pysslar med (hmm, typ kemi va?) ändå snällt

lyssnat på mig när jag beklagat mig över trasslande instrument

och svårhanterade prover.

Nahal, tack för att du finns. Du gjorde mitt liv lättare när det

var svårt.

Tack Lisa för alla luncher, med eller utan promenad. Hur hade

det gått för oss om vi inte hade haft vår timme i veckan att ösa

ur oss allt för någon som faktiskt förstår vad det är man pratar

om?

Ett tack också till mina vänner i Hudik, jag saknar er alla! Cia

och Tommy (och barnen) för sköna slöa kvällar, med eller utan

bastu(!) och för en fullkomligt underbar skidsemester! Gerald

och Katarina för härliga fikastunder och roliga samtal. Jag

kommer snart och hälsar på igen! Tack Martin, för att du

visade mig ett annat liv.

Tack familjen Nilsson för att ni omedelbart släppte in mig i er

gemenskap. Jag ser fram emot många fler besök i Motala och i

skärgården!

Jonas, tack för att du hittade mig! Tänk vad lite kyckling kan göra…

Till slut kommer jag till de som nog egentligen är viktigast. Ett

stort tack till hela min underbara familj! Jag inser att jag är

oerhört lyckligt lottad som har en familj som förstår vad jag

håller på med. Mina systrar Anna och Jenny och deras

respektive Joav och Pascal. Världens bästa syskonbarn,

Adrian och Elsa! Tack pappa för allt. Och mamma, älskade

mamma! Jag önskar att du kunde varit här. Vi saknar dig, nu

och för alltid.

Wow, tre sidor…. Nåväl, hur många känner ni som pratar mer än jag?

This thesis was financially supported by MISTRA Idéstöd, the

Swedish Research Council FORMAS and the Stockholm marine

research Center (SMF)

48

11. References

Allard,A.S., Remberger,M., Neilson,A.H., Bacterial O-Methylation of

Halogen-Substituted Phenols. Applied and Environmental

Microbiology 1987. 53, 839-845.

Athanasiadou,M., Cuadra,S.N., Marsh,G., Bergman,Å., Jakobsson,K.,

Polybrominated diphenyl ethers (PBDEs) and bioaccumulative

hydroxylated PBDE metabolites in young humans from Managua,

Nicaragua. Environmental Health Perspectives 2008. 116, 400-408.

Badings,H.T., Vanderpol,J.J.G., Wassink,J.G., Wide-Bore Glass

Capillary Columns for Gas-Chromatography - Their Preparation and

Application. Chromatographia 1975. 8, 440-448.

Birnbaum,L.S., Staskal,D.F., Diliberto,J.J., Health effects of

polybrominated dibenzo-p-dioxins (PBDDs) and dibenzofurans

(PBDFs). Environment International 2003. 29, 855-860.

Broecker, W.S. and Peng, T.H., Tracers in the sea. 1982, Eldigio Press

Lamont Doherty Geological Observatory

Broman,D., Näf,C., Rolff,C., Zebuhr,Y., Fry,B., Hobbie,J., Using Ratios

of Stable Nitrogen Isotopes to Estimate Bioaccumulation and Flux of

Polychlorinated Dibenzo-P-Dioxins (Pcdds) and Dibenzofurans (Pcdfs)

in 2 Food-Chains from the Northern Baltic. Environmental Toxicology

and Chemistry 1992. 11, 331-345.

Brownlow,A.H, Geochemistry. Second Edition ed 1996, New Jersey:

Prentice Hall

Buser,H.R., Polybrominated Dibenzofurans and Dibenzo-Para-Dioxins -

Thermal-Reaction Products of Polybrominated Diphenyl Ether Flame

Retardants. Environmental Science & Technology 1986. 20, 404-408.

Butler,A., Walker,J.V., Marine Haloperoxidases. Chemical Reviews

1993. 93, 1937-1944.

49

Cameron,G.M., Stapleton,B.L., Simonsen,S.M., Brecknell,D.J.,

Garson,M.J., New Sesquiterpene and Brominated Metabolites from

the Tropical Marine Sponge Dysidea sp. Tetrahedron 2000. 56, 5247-

5252.

Chung,H.Y., Ma,W.C.J., Ang,P.O., Kim,J.S., Chen,F., Seasonal

variations of bromophenols in brown algae (Padina arborescens,

Sargassum siliquastrum, and Lobophora variegata) collected in Hong

Kong. Journal of Agricultural and Food Chemistry 2003. 51, 2619-

2624.

de Wit,C.A., Alaee,M., Muir,D.C.G., Levels and trends of brominated

flame retardants in the Arctic. Chemosphere 2006. 64, 209-233.

Drenzek,N.J., Tarr,C.H., Eglinton,T.I., Heraty,L.J., Sturchio,N.C.,

Shiner,V.J., Reddy,C.M., Stable chlorine and carbon isotopic

compositions of selected semi-volatile organochlorine compounds.

Organic Geochemistry 2002. 33, 437-444.

Eglinton,T.I., Aluwihare,L.I., Bauer,J.E., Druffel,E.R.M., McNichol,A.P.,

Gas chromatographic isolation of individual compounds from complex

matrices for radiocarbon dating. Analytical Chemistry 1996. 68, 904-

912.

Eglinton,T.I., BenitezNelson,B.C., Pearson,A., McNichol,A.P.,

Bauer,J.E., Druffel,E.R.M., Variability in radiocarbon ages of individual

organic compounds from marine sediments. Science 1997. 277, 796-

799.

Fan,X., Xu,N.J., Shi,J.G., Bromophenols from the red alga Rhodomela

confervoides. Journal of Natural Products 2003. 66, 455-458.

Faulkner,D.J., Marine Natural Products. Natural Product Reports 2000.

17, 7-55.

Faulkner,D.J., Marine natural products. Natural Product Reports 2001.

18, 1-49.

50

Fielman,K.T., Woodin,S.A., Walla,M.D., Lincoln,D.E., Widespread

occurrence of natural halogenated organics among temperate marine

infauna. Marine Ecology-Progress Series 1999. 181, 1-12.

Flodin,C., Helidoniotis,F., Whitfield,F.B., Seasonal variation in

bromophenol content and bromoperoxidase activity in Ulva lactuca.

Phytochemistry 1999. 51, 135-138.

Flodin,C., Whitfield,F.B., Brominated anisoles and cresols in the red

alga Polysiphonia sphaerocarpa. Phytochemistry 2000. 53, 77-80.

Fu,X.O., Schmitz,E.J., Govindan,M., Abbas,S.A., Hanson,K.M.,

Horton,P.A., Crews,P., Enzyme-Inhibitors - New and Known

Polybrominated Phenols and Diphenyl Ethers from 4 Indo-Pacific

Dysidea Sponges. Journal of Natural Products-Lloydia 1995. 58,

1384-1391.

Giesy,J.P., Kannan,K., Global distribution of perfluorooctane sulfonate

in wildlife. Environmental Science & Technology 2001. 35, 1339-

1342.

Gribble,G.W., Naturally occurring organohalogen compounds. Accounts

of Chemical Research 1998. 31, 141-152.

Gribble,G.W., The diversity of naturally occurring organobromine

compounds. Chemical Society Reviews 1999. 28, 335-346.

Gribble,G.W., Blank,D.H., Jasinski,J.P., Synthesis and identification of

two halogenated bipyrroles present in seabird eggs. Chemical

Communications 1999. 2195-2196.

Haglund,P., On the identity and formation routes of environmentally

abundant tri- and tetrabromodibenzo-p-dioxins. Chemosphere 2010.

78, 724-730.

51

Haglund,P., Löfstrand,K., Malmvärn,A., Bignert,A., Asplund,L.,

Temporal Variations of Polybrominated Dibenzo-p-Dioxin and

Methoxylated Diphenyl Ether Concentrations in Fish Revealing Large

Differences in Exposure and Metabolic Stability. Environmental

Science & Technology 2010. 44, 2466-2473.

Haglund,P., Malmvärn,A., Bergek,S., Bignert,A., Kautsky,L., Nakano,T.,

Wiberg,K., Asplund,L., Brominated Dibenzo-p-Dioxins: A New Class of

Marine Toxins? Environmental Science & Technology 2007. 41, 3069-

3074.

Haglund,P.S., Zook,D.R., Buser,H.R., Hu,J.W., Identification and

quantification of polybrominated diphenyl ethers and methoxy-

polybrominated diphenyl ethers in Baltic biota. Environmental Science

& Technology 1997. 31, 3281-3287.

Holmstrand,H., Gadomski,D., Mandalakis,M., Tysklind,M., Irvine,R.,

Andersson,P., Gustafsson,Ö., Origin of PCDDs in ball clay assessed

with compound-specific chlorine isotope analysis and radiocarbon

dating. Environmental Science & Technology 2006a. 40, 3730-3735.

Holmstrand,H., Mandalakis,M., Zencak,Z., Andersson,P.,

Gustafsson,Ö., First compound-specific chlorine-isotope analysis of

environmentally-bioaccumulated organochlorines indicates a

degradation-relatable kinetic isotope effect for DDT. Chemosphere

2007. 69, 1533-1539.

Holmstrand,H., Mandalakis,M., Zencak,Z., Gustafsson,Ö.,

Andersson,P., Chlorine isotope fractionation of a semi-volatile

organochlorine compound during preparative megabore-column

capillary gas chromatography. Journal of Chromatography A 2006b.

1103, 133-138.

Jensen,S., Athanasiadou,M., Bergman,Å., A Technique for Separation

of Xenobiotics From Large Amounts of Lipids. Organohalogen

Compounds 1992. 8, 79-80.

Jensen,S., Reutergårdh,L., Jansson,B., Analytical methods for

measuring organochlorines and methyl mercury by gas

chromatography. FAO Fish Technical Paper 1983. 212, 21-33.

52

Krupp,E.A., Donard,O.F.X., Isotope ratios on transient signals with GC-

MC-ICP-MS. International Journal of Mass Spectrometry 2005. 242,

233-242.

Kumata,H., Uchida,M., Sakuma,E., Uchida,T., Fujiwara,K., Tsuzuki,M.,

Yoneda,M., Shibata,Y., Compound class specific C-14 analysis of

polycyclic aromatic hydrocarbons associated with PM10 and PM1.1

aerosols from residential areas of suburban Tokyo. Environmental

Science & Technology 2006. 40, 3474-3480.

Kurata,K., Taniguchii,K., Takashima,K., Hayashi,I., Suzuki,M., Feeding-

deterrent bromophenols from Odonthalia corymbifera.

Phytochemistry 1997. 45, 485-487.

Law,R.J., Allchin,C.R., de Boer,J., Covaci,A., Herzke,D., Lepom,P.,

Morris,S., Tronczynski,J., de Wit,C.A., Levels and trends of

brominated flame retardants in the European environment.

Chemosphere 2006. 64, 187-208.

Malmberg,T., Athanasiadou,M., Marsh,G., Brandt,I., Bergmant,A.,

Identification of hydroxylated polybrominated diphenyl ether

metabolites in blood plasma from polybrominated diphenyl ether

exposed rats. Environmental Science & Technology 2005. 39, 5342-

5348.

Malmvärn,A., Zebuhr,Y., Jensen,S., Kautsky,L., Greyerz,E., Nakano,T.,

Asplund,L., Identification of polybrominated dibenzo-p-dioxins in blue

mussels (Mytillus edulis) from the Baltic Sea. Environmental Science

& Technology 2005. 39, 8235-8242.

Malmvärn,A., Zebuhr,Y., Kautsky,L., Bergman,Å., Asplund,L.,

Hydroxylated and methoxylated polybrominated diphenyl ethers and

polybrominated dibenzo-p-dioxins in red algae and cyanobacteria

living in the Baltic Sea. Chemosphere 2008. 72, 910-916.

Mandalakis,M., Gustafsson,Ö., Optimization of a preparative capillary

gas chromatography-mass spectrometry system for the isolation and

harvesting of individual polycyclic aromatic hydrocarbons. Journal of

Chromatography A 2003. 996, 163-172.

53

Mandalakis,M., Gustafsson,Ö., Reddy,C.M., Li,X., Radiocarbon

apportionment of fossil versus biofuel combustion sources of

polycyclic aromatic hydrocarbons in the Stockholm metropolitan area.

Environmental Science & Technology 2004. 38, 5344-5349.

Marsh,G., Athanasiadou,M., Athanassiadis,I., Bergman,Å., Endo,T.,

Haraguchi,K., Identification, Quantification and Synthesis of a Novel

Dimethoxylated Polybrominated Biphenyl in Marine Mammals Caught

Off the Coast of Japan. Environmental Science & Technology 2005.

39, 8684-8690.

Marsh,G., Athanasiadou,M., Bergman,Å., Asplund,L., Identification of

hydroxylated and methoxylated polybrominated diphenyl ethers in

Baltic Sea salmon (Salmo salar) blood. Environmental Science &

Technology 2004a. 38, 10-18.

Marsh,G., Athanasiadou,M., Bergman,Å., Asplund,L., Identification of

hydroxylated and methoxylated polybrominated diphenyl ethers in

Baltic Sea salmon (Salmo salar) blood. Environmental Science &

Technology 2004b. 38, 10-18.

Meinert,C., Schymanski,E., Kuster,E., Kuhne,R., Schuurmann,G.,

Brack,W., Application of preparative capillary gas chromatography

(pcGC), automated structure generation and mutagenicity prediction

to improve effect-directed analysis of genotoxicants in a

contaminated groundwater. Environmental Science and Pollution

Research 2010. 17, 885-897.

Melcher,J., Olbrich,D., Marsh,G., Nikiforov,V., Gaus,C., Gaul,S.,

Vetter,W., Tetra- and tribromopbenoxyanisoles in marine samples

from Oceania. Environmental Science & Technology 2005. 39, 7784-

7789.

Moore,C.A., Okuda,R.K., Bromoperoxidase activity in 94 species of

marine algae. Journal of Natural Toxins 1996. 5, 295-305.

Nielson, A,H., Organic bromine and iodine compounds. In: The

handbook of environmental chemistry vol 3 R, 2003, Springer-Verlag

Berlin Heidelberg

54

Olsman,H., Engwall,M., Kammann,U., Klempt,M., Otte,J., van Bavel,B.,

Hollert,H., Relative differences in aryl hydrocarbon receptor-mediated

response for 18 polybrominated and mixed halogenated dibenzo-p-

dioxins and -furans in cell lines from four different species.

Environmental Toxicology and Chemistry 2007. 26, 2448-2454.

Pena-Abaurrea,M., Weijs,L., Ramos,L., Borghesi,N., Corsolini,S.,

Neels,H., Blust,R., Covaci,A., Anthropogenic and naturally-produced

organobrominated compounds in bluefin tuna from the Mediterranean

Sea. Chemosphere 2009. 76, 1477-1482.

Reddy,C.M., Xu,L., Drenzek,N.J., Sturchio,N.C., Heraty,L.J., Kimblin,C.,

Butler,A., A chlorine isotope effect for enzyme-catalyzed chlorination.

Journal of the American Chemical Society 2002. 124, 14526-14527.

Roeraade,J., Blomberg,S., Pietersma,H.D.J., High-Resolution

Preparative Gas-Chromatography .1. A Microprocessor-Controlled

System for Automated Fraction Collection. Journal of

Chromatography 1986. 356, 271-284.

Saenger,P., Pedersen,M., Rowan,K.S., Bromo-Compounds of Red Alga

Lenormandia-Prolifera. Phytochemistry 1976. 15, 1957-1958.

Schmidt,T.C., Zwank,L., Elsner,M., Berg,M., Meckenstock,R.U.,

Haderlein,S.B., Compound-specific stable isotope analysis of organic

contaminants in natural environments: a critical review of the state of

the art, prospects, and future challenges. Analytical and Bioanalytical

Chemistry 2004. 378, 283-300.

Sharma, G.M., Vig,B., Burkholder,P.R., Antimicrobial substances of

marine sponges IV. In: Food drugs from the sea. Proceednings 1969.

Ed. Youngken,H.W.Jr, Marine Technology Society, Washington, DC,

USA.

Sheesley,R.J., Krusa,M., Krecl,P., Johansson,C., Gustafsson,Ö., Source

apportionment of elevated wintertime PAHs by compound-specific

radiocarbon analysis. Atmospheric Chemistry and Physics 2009. 9,

3347-3356.

55

Sovocool,G.W., Donnelly,J.R., Munslow,W.D., Vonnahme,T.L.,

Nunn,N.J., Tondeur,Y., Mitchum,R.K., Analysis of Municipal

Incinerator Fly-Ash for Bromo-Dioxins and Bromochloro-Dioxins,

Dibenzofurans, and Related-Compounds. Chemosphere 1989. 18,

193-200.

Sovocool,G.W., Mitchum,R.K., Tondeur,Y., Munslow,W.D.,

Vonnahme,T.L., Donnelly,J.R., Bromo-Polynuclear and Bromochloro-

Polynuclear Aromatic-Hydrocarbons, Dioxins and Dibenzofurans in

Municipal Incinerator Fly-Ash. Biomedical and Environmental Mass

Spectrometry 1988. 15, 669-676.

Sylva,S.P., Ball,L., Nelson,R.K., Reddy,C.M., Compound-specific Br-

81/Br-79 analysis by capillary gas chromatography/multicollector

inductively coupled plasma mass spectrometry. Rapid

Communications in Mass Spectrometry 2007. 21, 3301-3305.

Takahashi,S., Sakai,S., Watanabe,I., An intercalibration study on

organobromine compounds: Results on polybrominated

diphenylethers and related dioxin-like compounds. Chemosphere

2006. 64, 234-244.

Takahashi,Y., Daitoh,M., Suzuki,M., Abe,T., Masuda,M., Halogenated

metabolites from the new Okinawan red alga Laurencia

yonaguniensis. Journal of Natural Products 2002. 65, 395-398.

Teuten,E.L., Johnson,C.G., Mandalakis,M., Asplund,L., Gustafsson,Ö.,

Unger,M., Marsh,G., Reddy,C.M., Spectral characterization of two

bioaccumulated methoxylated polybrominated diphenyl ethers.

Chemosphere 2006a. 62, 197-203.

Teuten,E.L., King,G.M., Reddy,C.M., Natural C-14 in Saccoglossus

bromophenolosus compared to C-14 in surrounding sediments.

Marine Ecology-Progress Series 2006b. 324, 167-172.

Teuten,E.L., Pedler,B.E., Hangsterfer,A.N., Reddy,C.M., Identification

of highly brominated analogues of Q1 in marine mammals.

Environmental Pollution 2006c. 144, 336-344.

56

Teuten,E.L., Reddy,C.M., Halogenated organic compounds in archived

whale oil: A pre-industrial record. Environmental Pollution 2007.

145, 668-671.

Teuten,E.L., Xu,L., Reddy,C.M., Two abundant bioaccumulated

halogenated compounds are natural products. Science 2005. 307,

917-920.

Thoma,H., Hauschulz,G., Knorr,E., Hutzinger,O., Polybrominated

Dibenzofurans (Pbdf) and Dibenzodioxins (Pbdd) from the Pyrolysis of

Neat Brominated Diphenylethers, Biphenyls and Plastic Mixtures of

These Compounds. Chemosphere 1987. 16, 277-285.

Tittlemier,S.A., Fisk,A.T., Hobson,K.A., Norstrom,R.J., Examination of

the bioaccumulation of halogenated dimethyl bipyrroles in an Arctic

marine food web using stable nitrogen isotope analysis.

Environmental Pollution 2002. 116, 85-93.

Tittlemier,S.A., Simon,M., Jarman,W.M., Elliott,J.E., Norstrom,R.J.,

Identification of a novel C10H6N2Br4Cl2 heterocyclic compound in

seabird eggs. A bioaccumulating marine natural product?

Environmental Science & Technology 1999. 33, 26-33.

Unger, M., Asplund, L., Marsh, G., Gustafsson, Ö., Characterization of

an abundant novel methyl- and methoxy-substituted brominated

diphenyl ether isolated from whale blubber. Chemosphere 2010 79,

408-413.

Unger,M., Asplund,L., Haglund,P., Malmvärn,A., Arnoldsson,K.,

Gustafsson,Ö., Polybrominated and Mixed Brominated/Chlorinated

Dibenzo-p-Dioxins in Sponge (Ephydatia fluviatilis) from the Baltic

Sea. Environmental Science & Technology 2009. 43, 8245-8250.

Unson,M.D., Holland,N.D., Faulkner,D.J., 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 1994. 119, 1-11.

57

Utkina,N.K., Denisenko,V.A., Scholokova,O.V., Virovaya,M.V.,

Gerasimenko,A.V., Popov,D.Y., Krasokhin,V.B., Popov,A.M.,

Spongiadioxins A and B, two new polybrominated dibenzo-p-dioxins

from an Australian marine sponge Dysidea dendyi. Journal of Natural

Products 2001. 64, 151-153.

Utkina,N.K., Denisenko,V.A., Virovaya,M.V., Scholokova,O.V.,

Prokof'eva,N.G., Two new minor polybrominated dibenzo-p-dioxins

from the marine sponge Dysidea dendyi. Journal of Natural Products

2002. 65, 1213-1215.

Van den Berg,M., Birnbaum,L.S., Denison,M., De Vito,M., Farland,W.,

Feeley,M., Fiedler,H., Hakansson,H., Hanberg,A., Haws,L., Rose,M.,

Safe,S., Schrenk,D., Tohyama,C., Tritscher,A., Tuomisto,J.,

Tysklind,M., Walker,N., Peterson,R.E., The 2005 World Health

Organization reevaluation of human and mammalian toxic

equivalency factors for dioxins and dioxin-like compounds.

Toxicological Sciences 2006. 93, 223-241.

Vetter,W., Turek,C., Marsh,G., Gaus,C., Identification and

quantification of new polybrominated dimethoxybiphenyls (PBDMBs)

in marine mammals from Australia. Chemosphere 2008. 73, 580-

586.

Weber,R., Kuch,B., Relevance of BFRs and thermal conditions on the

formation pathways of brominated and brominated-chlorinated

dibenzodioxins and dibenzofurans. Environment International 2003.

29, 699-710.

Whitfield,F.B., Helidoniotis,F., Shaw,K.J., Svoronos,D., Distribution of

bromophenols in species of marine algae from eastern Australia.

Journal of Agricultural and Food Chemistry 1999. 47, 2367-2373.

World Health Organization, Environmental Health Criteria 205:

Polybrominated dibenzo-p-dioxins and dibenzofurans. 1998.

World Health Organization, Concise international chemical assessment

document 66: 2,4,6-tribromophenol and other simple brominated

phenols, 2005

58

Zencak,Z., Klanova,J., Holoubek,I., Gustafsson,Ö., Source

apportionment of atmospheric PAHs in the western balkans by

natural abundance radiocarbon analysis. Environmental Science &

Technology 2007. 41, 3850-3855.

Zwank,L., Berg,M., Elsner,M., Schmidt,T.C., Schwarzenbach,R.P.,

Haderlein,S.B., New evaluation scheme for two-dimensional isotope

analysis to decipher biodegradation processes: Application to

groundwater contamination by MTBE. Environmental Science &

Technology 2005. 39, 1018-1029.

No regrets.