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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
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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
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