Post on 24-Apr-2020
Levels of organohalogen compounds in White-tailed sea
eagles (Haliaeetus albicilla) in relation to reproduction
impairment in the Bothnian Sea
Ulrika Nordlöf
Doctoral Thesis
Department of Applied Environmental Science
Stockholm University
2012
ii
Doctoral Thesis 2012
Department of Applied Environmental Science (ITM)
Stockholm University
SE-106 91 Stockholm
Sweden
© Ulrika Nordlöf, Stockholm 2012
ISBN 978-91-7447-478-7, pp. ii-ix, 1-45.
Printed in Sweden by US-AB, Stockholm 2012
Distributor: Department of Applied Environmental Science
Cover picture: Göran Nyrẻn
Illustrations inside: My and Gustav Nordlöf
iii
Till My och Gustav
”Ingenting är omöjligt, det tar bara lite längre tid ”
iv
Abstract
The white-tailed sea eagle (Haliaeetus albicilla) is in the top of the
marine food chain and its existence in Sweden has been threatened
several times. The species succeeded to recover from the deep decline in
the 1970s when the exposure to persistent organic pollutants influenced
the reproduction negatively. The species is today spread over the country
but is still seen as a near threatened species. Even though a recovery has
occurred there is still some reproduction problems seen in the region of
the south Bothnian Sea. The work presented in this thesis has focused on
expanding the knowledge of bromine and chlorine containing compounds
in the white-tailed sea eagles and to correlate the levels found with the
reproduction impairment in the region of south Bothnian Sea. Eggs and
blood from nestlings collected from different subpopulations, from the
Arctic (Lapland) in the north to the Baltic Proper in the south of Sweden,
have been studied for polychlorinated dibenzo-p-dioxins (PCDD),
dibenzofurans (PCDF), non-ortho-substituted polychlorinated biphenyl
(PCB), brominated flame retardants, methoxylated- (MeO-), and
hydroxylated- (OH)-polybrominated diphenyl ethers (PBDEs). Some of
the investigated compounds have been compared with levels found in a
unique white-tailed sea eagle egg collected in the Baltic Proper in 1941.
The pattern and levels of PCDD and PCDF in 1941 were similar and in
the same range as found today around the same area. Furthermore, the
human activity has raised the PCB levels in the white-tailed sea eagles
with over 120 times over the last 60 years but these levels are still much
lower than in the 1970s. In conclusion none of the investigated
compounds in this thesis could be correlated to the reduced reproduction
seen in the south Bothnian Sea but the levels of PCDD, PCDF, non-
ortho-PCB and OH-PBDE are high in the populations inhabiting the
Baltic Sea and are in the same range as found to cause different biological
responses in other avian species worldwide.
v
Swedish summary Havsörnen (Haliaeetus albicilla) är högst upp i den marina näringskedjan
och dess existens i Sverige har varit hotat vid ett flertal tillfällen. Arten
återhämtade sig från den djupa nedgången under 1970-talet då den nästan
utrotades till följd av att den utsattes för höga halter av miljöföroreningar
som påverkade reproduktionen negativt. I dag är havsörnen spridd över
hela landet men den anses fortfarande som en nära hotad art och även fast
en återhämtning har skett så finns det fortfarande reproduktions problem i
området kring södra Bottenhavet.
Arbetet som presenteras i denna avhandling har fokuserat på att
öka kunskapen om bromerade och klorerade substansers förekomst i
havsörn och att undersöka halterna av dessa föreningar i förhållande till
med den reproduktions störning som setts i södra Bottenhavet. Ägg och
blod från havsörns ungar har samlats in från olika svenska
subpopulationer, från Lappland i norr till Egentliga Östersjön i söder.
Dessa matriser har analyserats för polyklorerade-dibenzo-p-dioxiner
(PCDD), polyklorerade dibenzofuraner (PCDF), non-ortho-substituerade
polyklorerade bifenyler (PCB), bromerade flamskyddsmedel,
metoxylerade- (MeO-), och hydroxylerade- (OH)- polyklorerade difenyl
etrar (PBDE). Vissa av de undersökta ämnena har jämförts med halterna i
ett unikt havsörnsägg som hittats vid Dalarö utanför Stockholm 1941. De
mönster och halter av PCDD/PCDF som detta gamla ägg innehöll liknar
de som hittats i dagens havsörnsägg. Denna jämförelse visar också på hur
människans användning har ökat PCB halterna i havsörn med över 120
gånger under de senaste 60 åren men dessa halter är ändå mycket lägre än
de halter som uppmättes i havsörnsägg under 1970-talet. Slutsatsen som
kan dras av denna studie är att ingen av de undersökta föreningarnas
koncentrationer kan korreleras till den sämre reproduktionen i södra
Bottenhavet men att nivåerna av PCDD/PCDF, non-ortho-PCB och OH-
PBDEs ändå är speciellt höga i de populationer som finns längs
Östersjöns kust. Dessa halter är i samma storleksordning som har orsakat
olika biologiska responser i andra fågelarter runt om i världen.
vi
Table of contents Abstract ............................................................................................ iv
Swedish summary ............................................................................. v
Table of contents .............................................................................. vi
List of papers .................................................................................... vii
Statement .......................................................................................... viii
Abbreviations ................................................................................... ix
Chapter 1: Introduction ................................................................. 1
The white-tailed sea eagle (Haliaeetus albicilla) ............................. 2
Objectives .................................................................................. 3
Chapter 2: Environmental contaminants ..................................... 4
Polychlorinated biphenyls (PCBs) ................................................. 4
Polychlorinated dibenzo-p-dioxin (PCDD) & dibenzofurans (PCDF) .. 5
Polybrominated diphenyl ethers (PBDEs) ......................................... 5
Hydroxylated polybrominated diphenyl ethers (OH-PBDEs) ............ 6
Methoxylated polybrominated diphenyl ethers (MeO-PBDEs) .......... 6
Hexabromocyclododecane (HBCD) .................................................. 7
Other organohalogen compounds found in the WTSE ....................... 7
Toxicology ....................................................................................... 9
Chapter 3: Samples ........................................................................ 12
Egg samples ...................................................................................... 12
Blood/plasma samples ...................................................................... 13
Reproduction .................................................................................... 13
Chapter 4: Analytical methods and analysis .................................. 14
Extraction ......................................................................................... 14
Sample clean-up ............................................................................... 14
Lipid removal ................................................................................ 16
Class separation of PCDD/Fs and non-ortho PCBs .............................. 16
Separation of phenolic substances from neutrals ................................. 16
Screening study; further clean-up ..................................................... 17 Analysis, detection and identification ................................................ 17
Quality assurance and quality control (QA/QC) ................................ 18
Presentation of data........................................................................... 19
Chapter 5: Additional results ............................................................ 20
Screening of white-tailed sea eagle eggs ........................................... 20
Chapter 6: Summary and discussion of papers ............................. 21
Paper I; Brominated flame retardants and MeO-PBDEs in WTSE eggs .. 21
Paper II; Organohalogen substances in a 60 year old WTSE egg .......... 21
Paper III; Dioxin and dioxin-like substances in WTSE egg samples ....... 22
Paper IV; PBDEs, MeO-PBDEs and OH-PBDEs in nestling blood ....... 22
General discussion ............................................................................ 23
Chapter 7: Concluding remarks and future perspectives ............ 29
Chapter 8: Acknowledgement ........................................................... 30
Chapter 9: References ......................................................................... 32
vii
List of papers This thesis is based on the following publications, which will be referred
to in the text by their roman numerals I-IV. Papers I and II, are reprinted
with the kind permission of the publishers of respective journal:
Paper I Levels of brominated flame retardants and methoxylated
polybrominated diphenyl ethers in eggs of white-tailed sea
eagles breeding in different regions of Sweden.
Ulrika Nordlöf, Björn Helander, Anders Bignert and Lillemor Asplund.
Science of the Total Environment, 2010. 409; 238-246.
Paper II Comparison of organohalogen substances in a white-tailed
sea eagle egg laid in 1941 with five eggs from 1996-2000. Ulrika Nordlöf, Björn Helander, Ulla Eriksson, Yngve Zebühr and
Lillemor Asplund.
Chemosphere, 2011.xxxx-xxx, In press.
Paper III Polychlorinated dibenzo-p-dioxins, polychlorinated dibenzo-
furans and non-ortho PCBs in eggs of white-tailed sea eagles
collected along the Swedish coast in the Baltic Sea. Ulrika Nordlöf, Björn Helander, Anders Bignert, Yngve Zebühr and
Lillemor Asplund.
Submitted manuscript to Science of the Total Environment, 2012.
Paper IV Polybrominated diphenyl ethers and hydroxylated/
methoxylated analogues in plasma from white-tailed sea
eagle (Haliaeetus albicilla) nestlings from four different
regions of Sweden Ulrika Nordlöf, Björn Helander, Kerstin Nylund, Anders Bignert and
Lillemor Asplund
Manuscript.
viii
Statement I have contributed with the following to the papers included in this thesis:
In paper I, I was involved in the planning and did most of the chemical
analysis, responsible for the data analysis and writing the paper.
In paper II, I was responsible for planning, did most of the chemical- and
data analysis and was responsible for writing the paper.
In paper III, I was involved in planning, was involved in chemical
analysis, did most of the data evaluation and shared the responsibility for
writing the paper.
Paper IV, I was responsible for planning the project, did all of the
chemical analysis, did most of the data evaluation and responsible for
writing the paper.
All samples included in this thesis were collected and provided by Björn
Helander at the Swedish Museum of Natural History, Department of
Contaminant Research. The collection was done with authorization from
the Swedish Environmental Protective Agency (EPA). Anders Bignert
from the same department has made valuable contributions with the
statistical evaluations in three of the four papers (I, III and IV). All other
coauthors have made valuable contributions to the planning, chemical
analysis, data analysis/evaluation and writing of the papers.
ix
Abbreviations Ah-receptor Aryl hydrocarbon receptor
BP Baltic Proper
Brood size The number of nestlings
p,p’-DDT 1, 1, 1-trichloro-2, 2-bis (4-chlorophenyl)ethane
p,p’-DDE 1,1-dichloro- 2, 2-bis (4-chlorophenyl)ethylene
ECNI Electron capture negative ionisation
EI Electron ionisation
GC-MS Gas chromatography – mass spectrometry
GC-ECD Gas chromatography-Electron capture detector
GrL Greenland
H2SO4 Sulfuric acid
HBCD Hexabromocyclododecane
HRMS High resolution mass spectrometry
Inl Inland fresh water lakes
Lap Lapland
LOEL Low-observed effect level
MeO-PBDEs Methoxylated polybrominated diphenyl ethers
MeSO2-PCBs Methylsulfonyl-polychlorinated biphenyls
MTBE Methyl-tert-butyl ether
NT Near threatened
OH-PBDEs Hydroxylated polybrominated diphenyl ethers
PBBs Polybrominated biphenyls
PCBs Polychlorinated biphenyls
PCNs Polychlorinated naphthalenes
PBDEs Polybrominated diphenyl ethers
PCDDs Polychlorinated dibenzo-p-dioxins
PCDFs Polychlorinated dibenzofurans
POP Persistent organic pollutants
Productivity Mean number of fledglings per checked
territorial pair
sBS south Bothnian Sea
SIM Single ion monitoring
Successful breeding Territory or nest, attended by a mated pair of
eagle producing fledged young
TEF Toxic Equivalent Factor
TEQ Toxicity Equivalent
Territory An area with one or more nests, used by a pair
of eagles (often under many years)
T3 Thyronine
T4 Thyroxine
TTR Transthyretin
Unsuccessful breeding An occupied territory not producing fledged
young, including non-breeding, hatching failure
and loss of broods
WTSE White-tailed sea eagle
1
1 Introduction
The Baltic Sea has received increasing loads of nutrients and
contaminants, turning it into one of the most contaminated marine
“brackish” environments in the world. The limited exchange of saline
water comes from the North Sea via Kattegat and Denmark. Inflowing
fresh waters from rivers in countries surrounding the Baltic Sea is
polluted with both nutrients and environmental contaminants originating
from industrialized and urbanized areas as well as from atmospheric
deposition [1]. Over the last 70 years, since World War II, a large number
of chemicals have been released into the environment. Several of these
compounds are resistant to chemical and biological degradation, they
bioaccumulate and will persist in the ecosystem. In Sweden, monitoring
of organohalogen compounds has been carried out continuously since the
late 1960s with annual sampling of fish, birds, eggs, and mammals. The
material is stored in the Swedish environmental specimen bank and used
for control of national environmental protective measures as well as
retrospective studies of contaminants [2].
In the early 19th century the white-tailed sea eagle (Haliaeetus
albicilla) was breeding in Sweden from north to south, in freshwater
environments, in the archipelago of the Baltic Sea as well as along the
Atlantic coast. However, due to hard and intense hunting of the species
for many years in the beginning of the last century, the white-tailed sea
eagle (WTSE) in Sweden was close to extinction. This resulted in a law
to protect the species in 1924 [3] whereafter the species recovered. A
second threat was during the 1960-1970s due to high levels of
anthropogenic pollutants, especially organochlorine chemicals (e.g., 1, 1,
1-trichloro-2, 2-bis (4-chlorophenyl)ethane (p,p’-DDT), polychlorinated
biphenyls (PCB)) as well as methyl mercury (CH3Hg). The major DDT
metabolite, 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (p,p’-DDE) was
the chemical primarily responsible for causing eggshell thinning, leading
to low reproductive success and a decline in the WTSE population as
well as populations of other raptors as a result [4-11]. After the Swedish
ban of these chemicals in the early 1970s and the initiation of a
conservatory program designed to save the WTSE, the productivity
(mean brood size per occupied territory for a given period) and the
population have increased. In the beginning of the 1970s there were
approximately only 50 WTSE pairs in Sweden, nesting primarily on the
southeast coast, whereas today there are more than 500 pairs breeding
throughout the country [12]. However, even though their reproduction
seems to have recovered from the deep decline in the 1970s, there are
2
significant signs of continuing reproduction problems in the region of the
south Bothnian Sea (Baltic coast), where the productivity is lower than in
the country as a whole [12-14]. The underlying reason for this is still
unclear.
The white-tailed sea eagle (Haliaeetus albicilla) The WTSE is a long-lived bird with a low reproduction rate. This is
compensated for by many reproduction events under its lifetime. They
first reproduce at about 5 years of age and the normal clutch size is one or
two, but sometimes up to three eggs. Mated sea eagle pairs show strong
site fidelity and typically use the same territory for breeding year after
year, so a strong influence from local and regional contamination could
be expected. Differences in the food composition and patterns of
migration of the WTSE will also be reflected in the levels of
contaminants in their eggs [6]. Territorial adult eagles in the coastal areas
and in and around southern and central inland freshwater lakes are
essentially stationary all year round and thus produce their eggs from
locally acquired lipids and proteins. In contrast, as food becomes largely
unavailable to WTSEs up north in Lapland when waters freeze during
winter, the territorial adults breeding there move to the south of Sweden
or to the Atlantic coast of Norway in late autumn and often spend the
winter in the same places from year to year (Helander unpubl.). Most of
these birds leave the wintering areas no later than during February and
return to their breeding sites in Lapland during March [15].
3
Objectives A large number of organohalogen compounds have been identified in the
Baltic Sea ecosystem. Strong effects have been demonstrated from DDT
and PCB on the reproduction of white-tailed sea eagles breeding in the
Baltic Sea but effects from other organohalogen contaminants have so far
only been investigated to a limited extent. The sea eagles show
comparatively high residue concentrations of a number of compounds.
Despite a reduction in DDT and PCB concentrations which today are
below estimated critical levels and the improvement in the sea eagle
population, the nestling brood size is still below the background (pre-
1954) level in the south Bothnian Sea.
The major hypothesis of this thesis is that the low brood size in the south
Bothnian Sea is caused by high levels of organohalogen compounds. The
objectives of this study were:
to study if the concentrations of brominated substances,
polychlorinated dibenzo-p-dioxins and -furans as well as non-ortho-
PCBs correlate with a lower productivity and a reduction in brood
size (paper I, III and IV)
to compare different concentration levels of known organohalogen
compounds before 1950 with the levels of today (paper II)
to screen for other organohalogen compounds in WTSE eggs from
different regions in Sweden (Additional results)
4
2 Environmental contaminants
This thesis concentrates on anthropogenic organohalogen contaminants as
well as their metabolites and some naturally produced halogenated
organic compounds, some of them are bio-accumulative and end up in
high concentrations in top predators such as the white-tailed sea eagles.
Some of the compounds found as well as their toxicology are briefly
described in this chapter.
Polychlorinated biphenyls (PCBs) PCBs have been produced in many countries
under various trade-names e.g., Aroclor
(USA), Clophen (Germany), Kanechlor
(Japan). Due to their physical-chemical
properties (e.g., low electrical conductivity,
high thermal conductivity and resistance to
thermal degradation), PCBs were used in a
multitude of industrial applications such as
dielectric fluids in electrical equipment, plasticizers in paints, sealants,
lubricants and self-copying paper [16]. There are theoretically 209
possible PCB congeners containing 1-10 chlorine atoms. These are
numbered according to the system originally suggested by Ballschmiter
and co-workers [17]. The commercial products are mixtures of PCB
congeners with different degrees of chlorination. PCBs can be divided
into three groups according to the number of chlorine atoms in the ortho-
position (Figure 1) e.g., non-ortho-PCBs, mono-ortho-PCBs and ortho-
PCBs. The non-ortho-PCBs have a planar configuration due to their lack
of chlorine in the ortho-position. PCBs have low vapour pressures and
low water solubilities. Their octanol/water partition coefficients (log
KOW), range from 4 to 8 [18]. In general, the water solubility decreases
and lipophilicity increases with increasing number of chlorines in the
PCB molecule. PCBs were first identified in WTSEs as environmental
pollutants in 1966 [19]. Due to their persistence and lipophilicity, PCBs
are found widespread throughout the ecosystem and many congeners
bioaccumulate and biomagnify in the food chains. In the early 1970s the
open usage of PCB in Sweden was restricted by law and in 1995 there
was a complete ban.
Cl x Cly5
ortho meta
para
6
3
4
56
4
3 2 2
Figure 1: General structure of Polychlorinated biphenyl (PCB)
´ ´
´
´´
5
Polychlorinated-dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) PCDDs and PCDFs (Figure 2), often referred to as dioxins, are two
related substance groups of organic, tricyclic and planar compounds with
high chemical stability. The group consists of 210 theoretical congeners
comprising 75 PCDDs and 135 PCDFs. Seventeen of these (7 PCDDs
and 10 PCDFs) are substituted with chlorine in the 2,3,7,8-positions, and
are therefore of major toxicological concern [20] (described under the
toxicology section). PCDD/Fs are not manufactured as such but are
formed as contaminants during combustion processes (e.g., municipal
waste incineration), or are formed unintentionally as by-products, in
chemical products or in industrial processes e.g., metallurgic industry
(production/processing of iron, steel and other metals) [21], pulp/paper
mills, chloralkali industry [22]. Due to their high lipophilicity (log KOW 6-
8) [23] PCDD/Fs will bioaccumulate and biomagnify in the adipose
tissues of mammals, birds and other wildlife leading to elevated
concentrations at higher trophic levels. The non-ortho PCBs are coplanar
and are often analysed together with the PCDD/Fs as they have similar
toxicological activities.
Polybrominated diphenyl ethers (PBDEs) PBDEs belong to the large family of
brominated flame retardants (BFRs). They
have been used since the beginning of the
1970s as additives in plastics, textiles,
polyurethane foams, furniture and
electronic devices [24-26]. Theoretically
there are 209 possible congeners, all non
planar with 1-10 bromine atoms in the
molecule (Figure 3) and are numbered according to the same system as
for PCBs [17]. PBDEs have been manufactured as three major
commercial products: Penta-, Octa- and DecaBDE, containing mainly
congeners with 3-6, 6-10 and 9-10 bromine atoms, respectively [24,27].
PBDEs are physically mixed into the polymers to make them more fire
resistant [25] and can more easily be released to the surroundings than
covalently bound BFRs. PBDEs are lipophilic with log KOWs from 6 to 10
Cly
O
O
1
2
3
46
7
8
9
Clx O6
8
7
9
4
21
Clx Cly
Figure 2: General structures of a) Polychlorinated dibenzo-p-dioxin (PCDD) and b) dibenzofuran (PCDF)
a) b)
O
BrxBry
Figure 3: General structure of Polybrominated diphenyl ether (PBDE)
2
3
4
5
6
2
5
4
3
6´
´
´
´
´
6
Figure 5: Structure of 6-MeO-BDE47
OMe
Br
Br
O
Br
Br
OH
Br
Br
O
Br
Br
Figure 4: Structure of 6-OH-BDE47
[24], [28,29]. They are relatively stable in the environment although
several studies have reported debromination of e.g., BDE-209 to lower
brominated congeners by biodegradation or photolysis [30-33]. PBDEs
which are accumulated in the fatty tissues of organisms, have been shown
to biomagnify [34], and are found in many different environmental
compartments, e.g., sediments, sewage sludge and biota, including
humans [35,36].
Hydroxylated polybrominated diphenyl ethers (OH-PBDEs) OH-PBDEs have low water solubility.
Modeled values of logKOW for the
protonated form of OH-PBDEs range
between 6.6 to 10 and the corresponding
acid dissociation constant (pKa) values
range between 4.5 to 7.0 [37]. These
substances are not expected to accumulate
in the lipids but more likely retained in the blood (proteinophilic) in the
same manner as OH-PCBs [38]. OH-PBDEs (Figure 4) have been
identified in marine wildlife, both as a product of PBDE metabolism [40-
41] and as naturally products [39] by e.g., algae, marine sponges and
cyanobacteria [42-45]. Naturally formed OH-PBDE is generally ortho-
substituted relative to the diphenyl ether bond whereas biotransformation
of PBDEs results in meta- and para-substitution [40,41,46-50].
Demethylation of naturally produced MeO-PBDEs has also been
suggested as a source for OH-PBDEs in biota [51]. Methoxylated polybrominated diphenyl ethers (MeO-PBDEs)
MeO-PBDEs are more lipophilic than the
corresponding OH-PBDEs and are found in
the lipid rich tissues within the organisms
and bioaccumulate to some extent in biota
[52]. Research on MeO-PBDEs
demonstrates that most of these substances
are substituted with a MeO-group in the
ortho-position (Figure 5) to the diphenyl ether bond and are naturally
produced by different species in the marine environment [39,45,53-55].
In the Baltic Sea, MeO-PBDEs have been measured in red algae
(Ceramium tenuicorne) and blue mussels (Mytilus edulis), all substituted
with the MeO-group in the ortho-position and were proposed to be of
natural origin [45]. It has been suggested that MeO-PBDEs could be
formed by O-methylation of hydroxylated diphenyl ethers (OH-PBDEs)
by intestinal micro-flora or via microbial methylation in sediments
7
[56,57]. This presents the possibility that OH-PBDEs are precursors to
the MeO-PBDEs. In particular meta- and para-substituted MeO-PBDEs
could be the result of in vivo methylation of OH-PBDEs [58]. Recently
MeO-PBDE formation has been reported in rainbow trout after exposure
to decabromodiphenyl ether [59]. There is little known about the
biological effects of MeO-PBDEs but some congeners have been shown
to be antibacterial and anti-inflammatory [60].
Hexabromocyclododecane (HBCD) HBCD (Figure 6) is a commonly used flame
retardant in expanded and extruded
polystyrene for thermal insulation foams and
in the back-coating of textiles [61-63]. HBCD
ranks third in global flame retardant
production [64]. Technical HBCD products
consist of (mainly) three diasteromers: α-, β-
and γ-HBCD, of which γ-HBCD is present to
the largest extent. In biological samples however, α-HBCD is the most
abundant diastereomer [62,65,66]. Monitoring efforts worldwide indicate
that avian species are subject to HBCD bioaccumulation [67-72]. Other organohalogen compounds found in the WTSEs Polybrominated biphenyls (PBBs) PBBs are structural analogues to PCBs, but
with bromine instead of chlorine
substitution (Figure 7). They are
chemically stable and resistant to
breakdown by acids, bases, heat, reducing
agents, and oxidizing agents, and have
been used as additive flame retardants in
polymers for numerous applications. These
properties also make them persistent in the environment [73,74]. In 1973,
the primary technical PBB product used, FireMaster®BP-6 (containing
mainly BB-153), was inadvertently incorporated in animal feed that was
distributed throughout parts of Michigan, USA [75]. This led to the
poisoning of both animals and humans and shortly thereafter the
production of these chemicals ceased.
Br
Br
Br
Br
Br
Br
Figure 6: General structure of Hexabromocyclododecane (HBCD)
O
BrxBry
Figure 3: General structure of Polybrominated diphenyl ether (PBDE)
2
3
4
5
6
2
5
4
3
6
´
´
´
´
´
8
Figure 11: General structure of Polychlorinated naphtalene (PCN)
ClyClx
18
7
6
2
3
45
Cl
Cl
Cl
Cl
Cl
Cl
Figure 10: Structure of Hexachlorobenzene (HCB)
Cl
Cl
Cl Cl
Cl
Cl
Figure 9: General structure of Hexachlorocyclohexane (HCH)
1, 1, 1-trichloro-2, 2-bis (4-chlorophenyl)ethane (DDT) DDT (Figure 8) has been used as an insecticide since 1945, especially for
the control of malaria mosquitos. Several studies have linked the use of
DDT and its metabolite DDE to the eggshell thinning in predatory birds
that caused the decline in reproduction in the 1960s [4-7].
Hexachlorocyclohexane (HCH) HCH (Figure 9) is a broad spectrum
organochlorine insecticide used throughout the
world, which is comprised of several isomers.
The insecticidal properties of the γ-isomer, also
known as Lindane, were disclosed in 1942. In
biota the α- and β-isomers are generally the
most abundant [76].
Hexachlorobenzene (HCB) HCB (Figure 10) has been registered as a
fungicide for use on seed grains (e.g., wheat,
oats and rye). HCB also occurs as an industrial
by-product in the manufacturing of several
chlorine containing products [77]. It has been
measured world-wide in air, water and biota. It
is very persistent in the environment and has a
high bioaccumulation potential [78].
Polychlorinated naphthalenes (PCNs) The PCNs (Figure 11) constitute a class of
dicyclic, planar and persistent organic
pollutants structurally similar to PCDD/Fs
and non-ortho PCBs. PCNs have been
commercially produced and used in a variety
of applications similar to those of PCBs, but
are also produced in combustion processes
and are present as impurities in PCB mixtures. PCNs are toxic and have a
high bioaccumulative potential in the environment [79,80].
ClCl Cl
ClCl
a)
ClCl
ClCl
b)
Figure 8: General structure of a) DDT and b) DDE
9
Hydroxylated-PCBs (OH-PCBs) and Methylsulfonyl-PCBs (MeSO2-PCBs) OH-PCBs and MeSO2-PCBs (Figure 12) are formed as metabolites of
PCBs, as described under “Toxicology” below. Residues of OH–PCBs
congener have mainly been reported from blood, liver and egg matrices
[81] while MeSO2-PCBs are found in eggs but also in different tissues,
especially in the organism’s adipose and liver tissues [82].
Toxicology The toxic response is dependent on several factors including dose,
species, age and the sex of the animal as well as route and duration of
exposure. Metabolic biotransformation is an important factor in
determining the bioaccumulation, biomagnification, pharmacokinetics
and toxicities of organic contaminants in wildlife, including birds.
Metabolism occurs in two steps, phase I and II biotransformation, which
produce more water-soluble and easily excreted compounds. Organic
contaminants are subject to phase I oxidative metabolism (e.g., the
addition of an OH-group via an epoxide intermediate or directly to the
parent compound) that is mediated by the cytochrome P450 (CYP)
enzyme system and dependent on the structure of the compounds and
CYP metabolic capacity of the species [83]. Phase II enzymes conjugate
the phase I metabolites with endogenous molecules (e.g., via the
mercapturic acid pathway with endogenous glutathione (GHS)) which by
extension leads to the formation of MeSO2-PCBs[84]. In experimental
animals, metabolism of non-ortho-PCBs containing vicinal hydrogen
(H)-atoms at the ortho-meta carbons was shown to be mediated via
CYP1A enzymes. For other PCBs, with vicinal H-atoms at the meta-para
carbons, metabolism was mediated by CYP2B- and CYP3A-like iso-
enzymes [85].
Figure 12: General structures of a) Hydroxylated polychlorinated biphenyl (OH-PCB) and b) Methylsulfonyl polychlorinated biphenyl (MeSO2-PCB)
Cl x Cl y5
2
6
3
4
56
4
3 2
OH
Cl x Cl y
MeSO2
´ ´
´
´´
10
Dioxins and dioxin-like compounds The toxic effects of dioxins and dioxin-like compounds observed in avian
species include endocrine disruption, reduced egg production and
hatching success, developmental abnormalities, immunotoxicity and
mortality [86,87].
PCDD/Fs and other planar compounds such as non-ortho PCBs and PCN
have been shown to cause their toxic effects via mechanisms where the
molecules bind to the cytosolic aryl-hydrocarbon (Ah) receptor [20,88].
The Ah-receptor is present in the cytoplasm of almost all vertebrate cells
and a structurally diverse range of chemicals can bind to and/or activate
the Ah-receptor dependent gene expression, leading to a variety of
biological and toxic effects [89,90]. The sensitivity to PCDD/Fs varies
substantially among species. For example, guinea pigs (Cavia porcellus)
are approximately 1000 times more sensitive to 2,3,7,8-TCDD than
hamsters (Mesocricetus auratus) [91,92]. Bald eagles (Haliaeetus
leucocephalus), herring gulls (Larus argentatus), common terns (Sterna
hirundo) and American kestrels (Falco sparverius) are up to 500 times
less sensitive to the embryotoxic effects of “dioxin”-like compounds than
domestic chickens (Gallus gallus) [93,94], and in general, fish-eating
birds seem to be less sensitive to PCDD/Fs than domestic chickens
[94,95].
The different toxicity of various PCDD/Fs and other dioxin-
like compounds led to the introduction of the concept of toxic equivalent
factors (TEFs). In this system the most toxic congener (2,3,7,8-TCDD) is
assigned a TEF value of one and all other congeners/compounds are
assigned TEF values related to how toxic they are in comparison to
TCDD. An estimate of the total toxic potency of a mixture can be
obtained by multiplying the individual TEF values for each PCDD, PCDF
and non-ortho PCB congener by its concentration in the sample, and
adding these together. This sum is called the total toxic equivalent (TEQ)
of the sample and is an estimation of the toxic risk that is caused by being
exposed to the mixture of dioxin-like compounds. The TEF approach was
developed as an administrative tool and allows quantitative analytical
data for individual congeners to be converted into a single TEQ-value.
This tool has limitations due to a number of simplifications and it is
presumed that the toxic effects are additive, not synergistic or
antagonistic [96,97].
Brominated flame retardants Flame retardants such as PBDE and HBCD have been shown to possess a
wide spectrum of toxicity in laboratory animals, including birds
[32,35,98-107]. The effects include cancer, developmental
11
neurobehavioral effects, reproductive disorders and disruption of the
immune system. Both HBCD and various PBDE congeners have also
been shown to reduce plasma thyroxine (T4) levels in avian species
[101,108].
OH-PBDEs The structure of OH-PBDEs is similar to that of the thyroid hormones T4
(Figure 13) and thyronine (T3) and have an impact on thyroid
homeostasis [109]. The thyroid hormone transport system consists of a
complex of two proteins; transthyretin (TTR) which contains two binding
pockets for thyroid hormones and a retinol binding protein which
contains a binding site for retinol (Vitamin A) [38,110]. It has been
shown that OH-PBDE mimics T4 and competes with the hormone which
is displaced from its binding sites on TTR. Some OH-PBDEs have been
reported to have a higher affinity to TTR than both T3 and T4, and non-
hydroxylated PBDEs [111]. The T3 and T4 hormones are involved in
several processes of avian physiology, including regulation of
metabolism, growth, development of the central nervous system, cell
differentiation, and reproduction [112,113]. Thus, disruption of thyroid
hormone homeostasis by HBCD, PBDEs or OH-PBDEs in avian species
could be critical.
Mitochondrial oxidative phosphorylation (OXPHOS), a general
process for energy production in most cells and species [115] has been
reported to be disturbed by certain OH-PBDEs in low levels [114]. This
disturbance will result in a low adenosine triphosphate (ATP) production,
leading to energy depletion which may cause wasting syndrome with
severe body weight loss and hyperthermia [114]. Several other effects of
both PBDEs and OH-PBDEs have been reported including disruption of
the endocrine system causing both estrogenic and anti-estrogenic effects
[116,117].
Figure 13: Structure of the thyroid hormone, thyroxine T4
NH2
O
I
I
O
OH
I
I
OH
12
3 Samples A variety of appropriate matrices for analysis of environmental
contaminants in biota can be chosen depending on the characteristics of
the chemicals of interest, e.g., eggs, different tissues, organs or blood. In
this thesis, eggs and blood from WTSE were used. Eggs are often used to
study contaminant burden in female birds and maternal transfer to the
embryos [118-123]. Blood or plasma can be used to analyse substances
that are more hydrophilic or bound to proteins, such as phenolic
metabolites (e.g., OH-PCBs and OH-PBDEs) and can also give
information on the internal exposure in the animal.
Egg samples Unhatched eggs from the nests of
WTSE have been collected since
the mid-1960s. The collection
was initiated in order to find out
why the WTSE population
decreased so dramatically and has
been done with permission from
the Swedish EPA. The egg
samples presented in paper I
were sampled from four different
geographical areas in Sweden: 1)
freshwater habitats in the arctic
zone of Lapland (Lap), 2) inland
freshwater lakes in central and
southern Sweden (Inl), 3) the
southern coast of south Bothnian Sea (sBS), and 4) the coast of the Baltic
Proper (BP) (Figure 14). In paper II, both the egg from 1941 and the
more recent eggs were from the same geographical region along the coast
of the Baltic Proper (4). In paper III, the eggs were from the two coastal
areas 3 and 4. These were compared with eggs from western Greenland
(not indicated in the map). Additional results from a screening study of
WTSE eggs collected from all four areas are presented in Chapter 5.
All eggs in this study were unhatchable i.e., dead. Over the
study period the WTSE has been classified as endangered (EN),
vulnerable (VU) and at the present time as a near threatened species (NT)
and collection of fresh samples was not allowed. The dead eggs may not
represent random fresh eggs from the various populations, which may
3
Figure 14: Map over the Scandinavian countries withthe sampling areas marked 1 to 4.
13
introduce a potential bias. Some of the eggs were rotted and putrefied
when collected but the influence of this has earlier been investigated and
concluded to have no effects on the residues concentrations when
expressed on a lipid weight basis [7,124] The eggs used in this thesis
were extracted (see chapter 4) in the year of collection and the extracted
lipids (dissolved in n-hexane) have since been stored in sealed ampoules
in the dark.
Blood samples The monitoring of WTSE reproduction is part of the national Swedish
Environmental Monitoring Programme on Contaminants in Biota [14],
and blood samples from young eagle nestlings (4-8 weeks of age) are
taken annually in connection with nest visits to determine reproductive
outcome and for banding of the nestlings. Blood is taken from the
brachial vein of the wing, kept cold on ice in the field and subsequently
centrifuged to obtain serum, which was stored frozen (-80 C) until
analysis. The blood samples presented in paper IV were collected in the
same geographical areas as the egg samples in paper I (Figure 14).
Reproduction Up to the beginning of the 1950s the breeding success was about 72%
along the Swedish east coast (Baltic Sea) with a mean nestling brood size
of 1.84 per successful breeding attempt and a productivity of
approximately 1.3 chicks per mated pair and year. These data are today
seen as normal production and can be used for reference values for the
coast [13,125].
The different egg samples included in this thesis (paper I and
III) have been evaluated for their reproduction data and correlated to the
measured residue concentrations. To test for the possibility of a smaller
egg clutch size to explain the smaller nestling brood size in the sBS, the
frequency distribution of successful nests containing dead eggs together
with the young was investigated during 1993-2006 and during 2000-2009
[12-14] and are described more in detail in paper III.
14
4 Analytical methods and analysis
Extraction After homogenization, extraction is the first step in a sample clean-up
procedure. Neutral organohalogen compounds (like most of those studied
in this thesis) are primarily lipophilic and extracted by nonpolar solvents.
The eggs in this thesis (paper I-III and screening) were extracted
according to Jensen et al. [127]. In short, the samples are homogenized
with acetone/n-hexane, and then the lipids (and lipophilic substances) are
extracted with a mixture of n-hexane and diethyl ether. The combined
extract is then washed with an acidic water solution. This method extracts
lipids and lipophilic contaminants with good recovery and is suitable for
organisms/tissues with relatively high lipid content, while for lean
organisms/tissues (<1% lipids), a modified version has been developed
[128]. Other alternative extraction methods have been described in the
literature for biological samples, e.g., Bligh and Dyer [129], Folch [130],
Soxhlet extraction [131] and accelerated solvent extraction (ASE) [132].
The plasma samples in paper IV were extracted according to
Hovander et al. [133]. This method extracts both neutral and phenolic
halogenated substances from the serum. In short, the serum proteins were
denatured with iso-propanol and acidified with hydrochloric acid. The
neutral compounds (PCBs, PBDEs and MeO-PBDEs) and the phenolic
water-soluble substances (OH-PCBs and OH-PBDEs) were extracted
with a mixture of n-hexane/methyl-tert-butyl ether (MTBE). After the
extraction, the organic solvents were evaporated and the lipid content
determined gravimetrically.
Sample clean-up Before the instrumental analysis, lipids and other co-extracted material
must be removed. The clean-up methodologies used in paper I –IV and
the screening are briefly described below and illustrated in Figure 15.
15
Extractio
n, L
ipid
determ
inatio
nE
ggs; Jensen
et al. 19
83
Plasm
a; Ho
van
der et al. 2
00
2
Pa
pe
r II, IIIP
CD
D/F
s, no
n-o
rtho
PC
Bs
GC
-MS
An
alysis
1.
Pa
pe
r I, II1
. PB
DE
s, MeO
-PB
DE
s2
. PC
Bs, D
DT
, HC
H, H
CB
Co
nc.
H2 SO
4
GC
-EC
DA
naly
sis
2.
KO
H
Partitio
nin
g
Pa
pe
r IVP
BD
Es, M
eO-P
BD
Es, O
H-P
BD
Es
GC
-LR
MS
An
alysis
Neu
tralP
hen
olic
Co
nc. H
2 SO4
Deriv
atization
H2 SO
4 :SiO2
Co
lum
n
SiO2
Co
lum
n
Co
nc.
H2 SO
4
Mu
ltilayer SiO
2co
lum
n
Am
ino
pro
py
l colu
mn
Separatio
n acco
rdin
g to n
um
ber o
f arom
atic rings
PY
E co
lum
nIso
lation
of
plan
ar com
po
un
ds
On
earo
matic
ring
Tw
oa
rom
atic
ring
s
Th
ree or m
ore
arom
atic rings
Less p
lanar
com
po
un
ds
Co
pla
na
r com
po
un
ds
PC
DD
/Fs, n
on
-orth
o P
CB
SiO2
colu
mn
GC
-LR
MS
An
alysis
Fig
ure
15
: Schem
atic presen
tation
of th
e extraction
and
clean-u
p m
etho
ds u
sed in
pap
er I-IV an
d screen
ing, resp
ectively.
Scre
en
ing
e.g., PC
B, M
eSO2 -P
CB
, OH
-PC
B
AC
NP
artition
ing
KO
H
Partitio
nin
g
PC
B, M
eSO2 -P
CB
O
H-P
CB
Deriv
atization
Co
nc. H
2 SO4
H2 SO
4 :SiO2
Co
lum
n
PC
BW
ater/Hx
MeSO
2 -PC
B
90
% H
2 SO4 :SiO
2
Co
lum
n
GC
-LR
MS
An
alysis
GC
-LR
MS
An
alysis
16
Lipid removal The name lipid comes from the greek word “lipos” meaning fat and the
chemical definition of a lipid is a substance that is soluble in nonpolar
solvents. There are several different types of lipids, all with different
functions in organisms. Triglycerides are used for the storage of energy
and glycerol-phospholipids are the key components in the lipid bilayer of
cells as well as being involved in metabolism and cell signaling [134].
The relative proportions of the different lipids vary between tissues and
species.
There are many methods for lipid reduction/removal, both non-
destructive (e.g., liquid/liquid partitioning with acetonitrile [135] or gel
permeation chromatography (GPC)) [136] and destructive treatment with
concentrated sulfuric acid. The choice of method depends on the question
to be answered. Non-destructive methods are essential if the attempt is to
identify unknown substances as these allow a wider range of substances
to be analyzed. Treatment with concentrated sulfuric acid has the benefit
of being simple and rapid but can destroy or partition the compounds of
interest into the acid. In papers I-IV, concentrated sulfuric acid was used
to remove the lipids. In the additional study in Chapter 5 where results
from a screening-study of WTSE eggs are presented, the non-destructive
partitioning with acetonitrile was used.
For PBDEs, HBCD, PBBs and MeO-PBDEs analysed in paper
I; and for HCB, HCH, p,p´-DDT, p,p´-DDE, p,p´-DDD and ortho-
substituted PCBs in paper II, no further clean-up after sulfuric acid
treatment was necessary.
Class separation of PCDD/Fs and non-ortho PCBs The clean-up procedure for PCDDs/Fs and non-ortho PCBs included an
aminopropyl silica column followed by a 2-(1-pyrenyl)
ethyldimethylsilylated silica (PYE) column and is described in detail in
Ishaq et al. (2003) and Bandh et al. (1996) [137,138] and in the
supporting information (SI) in paper II. In environmental samples,
PCDD/Fs and non-ortho PCBs are most often present in much lower
concentrations than many other lipophilic compounds e.g., ortho-
substituted PCBs. A more extensive clean-up is therefore necessary to
obtain high quality analysis.
Separation of phenolic substances from neutrals In paper IV, neutral and phenolic compounds were separated using
liquid-liquid partitioning of the sample extract in n-hexane with a
potassium hydroxide ethanol/water solution [133]. The de-protonated
17
phenolic substances will partition into the alkaline water phase while the
neutral compounds stay in the organic solvent. After acidification of the
water phase, the phenolic compounds were re-extracted with n-
hexane/diethyl ether and methylated with diazomethane [133].
Screening study; further clean-up of egg samples Half of the lipid extract was used for further clean-up. For lipid reduction
ACN was used. ACN dissolve the aromatic analytes to a higher extent
than lipids and the extracted lipids were treated three times with ACN
resulting in approximately 70% lipid reduction.
The extract was further separated into a neutral and a phenolic
fraction by the use of an alkaline solution of potassium hydroxide (KOH).
The phenolic fraction was derivatized with diazomethane [133] while the
neutral fraction was further treated with sulfuric acid which separates
methyl sulfone metabolites (e.g., MeSO2-PCB) from compounds without
this functional group. Methyl sulfone containing compounds partitioned
into the acid and was further re-extracted by n-hexane after adding water
to the acid. The methyl sulfone fraction was subjected to further clean-up
on a silica gel column impregnated with sulphuric acid (90%).
The remaining neutral fraction was fractionated on a silica gel
column, collecting sex fractions using n-hexane; n-hexane:diethyl ether.
Analysis, detection and identification Gas chromatography/mass spectrometry (GC/MS) is a powerful
analytical technique for environmental trace analysis of organohalogen
compounds [139], and was used for most analyses in this thesis.
Substances containing chlorine (35
Cl/37
Cl) and/or bromine (79
Br/81
Br),
give rise to typical isotopic distribution patterns that give information on
the number of chlorine/bromine atoms in the molecule. In general MS is a
sensitive and selective detector with a large linear range that makes it
suitable for quantitative work [140]. Commonly used ionization
techniques are electron ionization (EI) which is a “hard” technique and
electron capture negative ionization (ECNI), which is a softer technique.
EI produces positively charged molecular ions and fragments that provide
structural information for the compounds while ECNI in general gives
less fragmentation of the analytes than EI and often a stronger molecular
ion signal. In ECNI brominated substances forms bromide ions and is
and monitoring m/z -79 and -81 can be utilized as a selective and
sensitive method for analyzing brominated compounds [141].
18
In this thesis, HBCD, PBB, PBDE, OH-, and MeO-PBDE were
analysed with a low resolution (quadrupole) MS run in the ECNI mode
measuring bromine ions (paper I, II and IV) and for the analysis of
compounds in the screening study, full scan was used. For the PCDD/F
and non-ortho PCB analyses in paper II and III, a high resolution
(magnetic sector) instrument was used in EI mode.
The quantitative analyses of DDT, ortho-substituted PCBs,
HCH and HCB in paper II were conducted with a GC coupled to an
electron capture detector (ECD). This detector is selective and sensitive
to electronegative compounds.
The choice of appropriate surrogate standards for trace analysis
can be delicate if 13
C-labelled standards are not an option. In WTSE eggs
there are a huge number of peaks in the chromatograms (known and
unknown) that can interfere with the chosen surrogates. PBDE analysis in
ECNI mode, monitoring the bromide ions, is a very sensitive method but
excludes the use of 13
C-labelled surrogates for analytes with few
bromines. Dechlorane®603 was used as surrogate standard in paper I and
II and as volumetric standard in paper IV and has been shown to give
good results in intercalibration exercises [142] although it is not optimal
[143]. However, no trace of Dechlorane®603 was found when a large
aliquot of WTSE egg samples was analyzed before it was added as
surrogate standard. 13
C-labelled surrogates were also not an option for the
ECD-analyses in paper II which were conducted according to the same
procedure as for an accredited PCB method used in our laboratory. For
the analyses of PCDD/Fs and non-ortho-PCBs in paper II and III, 13
C-
labelled standards were used.
Quality assurance / Quality control The objectives of the analytical work are to obtain reliable results of a
defined quality. In this thesis, internal quality control criteria were
followed to guarantee consistent analytical results, including the use of
method blanks, laboratory reference material (paper I and II), duplicate
injections of reference standard solutions between six to nine levels for
every set of samples analyzed and recovery standards (paper III and IV).
The limit of detection (LOD) for individual compounds was set to a
signal-to-noise (S/N) ratio of 3 whereas the limit of quantification (LOQ)
was S/N = 5.
19
Presentation of data There are several ways to present concentrations, e.g., on a molar-, dry-,
wet- or lipid weight basis. The use of lipid normalized data makes it
possible to compare concentrations in different tissues that have different
lipid contents and also to compare concentrations in different species in
the food chain. As most of the examined substances are highly lipophilic
with a biomagnification potential through the food chain, lipid weight
normalization is preferable for the eggs. For plasma the concentrations
are presented both on wet and lipid weight basis as most of the results in
the literature are presented on a wet weight basis.
20
5 Additional results In this chapter unpublished results from a screening study of WTSE eggs
are presented as well as some compounds found in the nestling plasma.
WTSEs contain a cocktail of organohalogen compounds and the results
of some possible compounds are listed in Table 1 but which needs to be
confirmed using authentic standards. These results will contribute to the
understanding of the complexity of WTSE contamination.
The GC/MS chromatograms in Figure 16 (neutral fraction, third fraction
on a SiO2 column) indicates occurrence of a large number of compounds
of which only a few have been quantified but much more is left to
identify.
m/z: 79.00, 81.00
100
100
TIC
Figure 16: GC/MS (ECNI) chromatograms of a white-tailed sea eagle egg sample;a) SIM m/z 79, 81 b) Full scan
Indicated compounds Abbreviation Egg Blood serum
Neutral fraction Clordane x
Methoxylated polychlorinated diphenyl ethers MeO-PCDE x
Polychlorinated diphenyl ethers PCDE x x
Polychlorinated terphenyls PCT x x
Phenolic fraction Hydroxylated polychlorinated biphenyls OH-PCB x x
Hydroxylated polybrominated diphenyl ethers OH-PBDE x
Hydroxylated polychlorinated diphenyl ethers OH-PCDE x x
Sulfone fraction Bis (4-chlorophenyl) sulfone BPCS x
Methylsulfonyl polychlorinated biphenyls MeSO2-PCB x
Table 1: Indicated compounds (other than quantified in this thesis) in white-tailed sea eagle eggs and blood serum from nestlings
21
6 Summary and discussion of papers
Paper I; Brominated flame retardants and MeO-PBDE in WTSE eggs This study included the analysis of 44 unhatched WTSE eggs
representing four different subpopulations in Sweden: Lapland around the
inland fresh water lakes in the northern (Arctic) parts, inland fresh water
lakes in the central/south parts, and two subpopulations from the Baltic
Sea: south Bothnian Sea and the Baltic Proper. The aim was to measure
the concentrations of a number of brominated compounds (e.g., PBDEs,
PBBs, HBCD and MeO-PBDEs) in the eggs and to study correlations
between these and reduced reproductive success observed in the south
Bothnian Sea subpopulation. High levels of PBDE were found in the
populations inhabiting the Baltic Sea regions but no significant
differences (p>0.05) were observed in analyte concentrations between
these two populations. Differences were instead observed between the
Lapland population and the central/south fresh water population as well
as between these and the two coastal populations. The presence of MeO-
PBDEs in all of the inland samples indicates that there is an as-yet-
unidentified source of these compounds in the freshwater ecosystem. The
impaired reproduction in the south Bothnian Sea population could not be
explained by the results from this study as the contaminant concentrations
there were equal to those in the Baltic Proper population, which have
normal reproduction.
Paper II; Organohalogen substances in a 60-year-old WTSE egg sample In paper II, the concentrations of a number of organohalogen compounds
determined in a WTSE egg collected on the Baltic Sea coast in 1941 were
compared to those in more recent eggs (1996-2001) collected from the
same geographical area. The concentrations of PCDD/Fs, MeO-PBDEs
and HCB were similar in the old and the new eggs, whereas the PCB
concentrations (sum of 7) were more than 120 times higher in recent
years. HCH, DDT and its metabolites and PBDEs were all detected in the
recent eggs but none of these were detected in the old egg. This illustrates
the exposure of WTSEs to a wider range of bioavailable chemicals in
recent time.
22
Paper III; Dioxin-like substances in WTSE egg samples In paper III, WTSE eggs from two populations on the Baltic Sea (south
Bothnian Sea and Baltic Proper) were analyzed for 2,3,7,8-substituted
PCDD/Fs and dioxin-like non-ortho PCBs in an attempt to correlate
possible differences in concentrations to the reproductive impairment
observed in the south Bothnian Sea population. A collection of WTSE
eggs from Greenland were included as a reference for contamination
levels in WTSEs outside the Baltic Sea region. High concentrations of
PCDD/Fs and non-ortho PCBs were seen. No significant difference in
total TEQ concentrations was found between the two Baltic Sea
populations. In eggs from all three areas, 2,3,4,7,8-PCDF was the most
abundant PCDD/F congener. The non-ortho PCBs (mainly CB-126)
contributed most to the total TEQ values (calculated based on WHO-TEF
for birds [144]) in eggs from the Baltic Sea. Thus, the reproductive
impairment observed in the south Bothnian Sea could not be linked to the
compounds analyzed in this study. However, TEQ values are still high
enough to be suspected of causing biological response in both
populations.
Paper IV; PBDEs, MeO-PBDEs and OH-PBDEs in blood from nestlings In this paper, blood serum from 58 WTSE nestlings (4-8 weeks of age),
from 4 different subpopulations (see Paper I above) were analyzed for
PBDEs, MeO-PBDEs and OH-PBDEs. The hypothesis was that the lower
brood size observed in the population from sBS could be explained by the
levels of these substances in the blood. ƩPBDE concentrations in the sBS
nestlings were 2-fold higher and significantly separated (p<0.05) from
those in the other populations. ƩMeO-PBDE and ƩOH-PBDE
concentrations were not significantly different between the two
populations inhabiting the Baltic Sea. In Lap and the Inl populations, the
concentrations of MeO-PBDE and OH-PBDE were several times lower
compared to the coastal populations and most of the samples were below
limit of quantification. Based on the literature, the PBDE concentrations
found in the Baltic populations are probably not of the magnitude that
cause harm to WTSEs. However, ∑OH-PBDE concentrations found in
both the coastal populations are in the same range that has been found to
be associated with changes in T3 in bald eagles [113].
23
General Discussion The annual monitoring of sea eagle nests along the Baltic coast have
showed that the recovery from the deep decline in the 1970s of the
WTSEs is lower in sBS compared to eagles higher up and further down
on the Baltic coast, implying an impairment. It has also been statistically
verified that the WTSEs in the region of sBS have more unhatched eggs
in their nests (paper III, [12-14]). The hypothesis in this thesis was that
the concentrations of the investigated compounds could be correlated to
the lower reproduction outcome in WTSE from the region of the south
Bothnian Sea. However, the reproductive impairment observed therein
could not be linked to the concentrations of the organohalogen
compounds analyzed in this study.
Concentration changes in relation to biological responses In paper II, the unique egg sample from 1941 contained several known
persistent organic pollutants (POPs) such as PCBs, HCB, PCDD and
PCDF, but also naturally produced MeO-PBDEs were found.
In 1941 the commercial use of PCBs had been ongoing for
about 10 years so the detection of PCBs was not surprising. Sixty years
later the average Ʃ7PCB level (240 µg/g lipid) found in the eagle
population was on average more than 120 times higher. This is below the
critical levels for reproduction disturbance seen in the WTSE during the
1960s to 1980s when the levels for ƩPCBs exceeded 1000 µg/g lipid.
These concentrations were two times higher than the estimated lowest
observable effect level (LOEL) in WTSE, indicating that it was a cause of
embryo mortality [7].
The old sample did not contain any detectable concentrations of
DDE (major DDT metabolite) while today the WTSEs have levels that
have risen to on average 100 µg/g lipid. This is close to the indicative
LOEL value (120 µg/g) for depressed productivity in WTSE [7] but
about seven times lower than the mean DDE concentration in the 1965-
1978 in sea eagle eggs from nests along the Baltic coast [122].
In 1941 the levels of ∑17PCDD/Fs were comparatively high
(2 900 pg/g lipid) and are on average in the same range as found today
(approx. 4 000 pg/g lipid) as presented in paper II and III. These results
were unexpected but after consideration not surprising. In Sweden,
especially along the Baltic coast, there has been a long historical input of
these chemicals from several different industries known to produce
dioxins as by-products [21,145,146] but also the atmospheric long range
transport of these compounds has to be considered [145] and might have
had an input to the Baltic Sea already in the 1940s. Some of the industries
24
started their production already in the mid-1800s but since the early
1990s the industries have been changing their production methods to
reduce the dioxin emissions. Looking back it would have been interesting
to have included some eggs from the time before the change in the
industrial processes to assess any possible variation in levels “before and
after”. However, there are no signs of decreased levels during the
sampling period (1991-2005) as presented in paper III. When recent
PCDD/F and non-ortho PCB concentrations in WTSE eggs from the
Baltic coast are converted to total TEQ, the WTSEs have on average two
times higher total TEQ than the LOEL (12 600 pg TEQ/g) for hepatic
CYP1A induction reported in laboratory studies on North America bald
eagles (Haliaeetus leucocephalus), a species related to the European
WTSE.
In paper I, high levels of PBDEs were reported in recently
collected WTSE eggs from the Baltic Sea (average ∑5PBDE of 4 200
ng/g lipid or 210 ng/g wet weight (w.w.)) while the inland regions had
lower levels (average 720 to 1 500 ng/g lipid). The highest average levels
found in the WTSEs are about five times lower than the productivity
threshold levels indicated for ospreys from North America (>1 000 ng/g
w.w.) [147]. In kestrels (Falco sparverius) a negative influence on
reproduction was seen at egg levels averaging 470 ng/g w.w. (Σ12PBDEs)
[104], which correspond to about two times higher levels than reported
for the two populations of WTSEs from the Baltic coast.
MeO-PBDEs were found in the old egg sample in
concentrations almost identical with the concentrations from the Baltic
coast of today (120 ng/g lipid) which is presented in paper I and II. The
findings of MeO-PBDEs in the old sample are thus consistent with the
hypothesis that these are natural products produced by marine organisms.
These results are therefore consistent with the literature [43,45,148-150].
Thus, MeO-PBDEs are probably not transformation products of PBDE
precursors, as PBDEs were not in commercial use in the 1940s.
In paper IV, blood serum were analyzed the highest
concentrations of ∑PBDE were found in sBS (111 ng/g lipid) which was
approximately 2-fold higher than in the other populations. In bald eagles
no impact on circulating thyroid hormone levels were seen when the
plasma concentrations of ∑PBDEs found were 5 ng/g w.w. (500 ng/g
lipid) [113]. However, high concentrations of ∑OH-PBDEs were found
in both Baltic Sea populations (approximately 210 ng/g lipid) being in the
same range (10-210 ng/g lipid) as found to correlate with an increase in
circulating T3 in bald eagles [113].
25
Congener patterns in eggs and blood The differences in congener pattern between different species may be
caused by differences in the emissions from various sources but the
patterns will also be influenced by differences in the uptake, metabolism
and excretion rates of the different congeners in the different species
and/or in their food webs as well as their migration patterns. Differences
in congener patterns, as well as possible differences between species in
their response to specific congeners, are important to take into account in
population studies when comparing and interpreting effects and
estimating threshold levels of certain compounds/congeners. Birds
exhibit species-specific feeding strategies. Some feed exclusively in
freshwater or marine systems, with fish comprising the majority of their
diet. Others consume primarily terrestrial-based food, such as passerines,
terrestrial mammals and/or insects.
The most abundant PBDE congener in the WTSE eggs reported
in paper I, was BDE47. Congeners were decreasing in the order BDE47
>99 >100 >153 >154. The dominance of BDE47 is consistent with that
found in other marine birds worldwide reviewed by Chen at al. [34],
whereas many terrestrial bird species have a different pattern with higher
contribution from the more highly brominated congeners such as
BDE209. The WTSE did not contain any detectable levels of BDE209 (in
the lipid amount analysed) while this congener have been found in
peregrine falcons (Falco peregrinus) breeding in Sweden [151] as well as
in America [152] but also in other terrestrial birds [34]. The level of
BDE47 in WTSE was several-fold higher along the Swedish coast than in
the inland areas. For the WTSE in Lapland the congener pattern was
slightly different with less dominance of BDE47. In paper IV, the PBDE
pattern in the blood serum was similar (Figure 17) to that in eggs from
the same collection area, indicating maternal transfer of contaminants
from female to the egg and then to the nestling, but the contaminants are
also given to the nestlings in the food and concentrations may be related
to sources in the surrounding environments. In paper IV, 6-MeO-BDE47 and 6-OH-BDE47 were the most
abundant congeners, which was 3-12 and 30-40 times greater,
respectively, along the coast compared to the both inland regions. This is
in agreement with the high levels of these compounds in the Baltic Sea.
26
In the eggs investigated for PCDD/Fs in paper II and III, the
2,3,4,7,8-PeCDF congener clearly dominated the furan pattern in all three
sampling regions (sBS, BP and GrL), which is similar to several other
bird studies reported worldwide [153-158]. This furan congener is formed
as a major impurity in technical PCB mixtures but is also formed in
combustion processes [159-161]. The major PCDD congener in WTSE
from all areas was 1,2,3,7,8-PeCDD while 2,3,7,8-TCDD in sBS were
higher than in the two other populations (BP and GrL) and comprised
approximately 12% of the total PCDD/Fs while the share of this congener
in BP was 5% and GrL 4%. This difference might be due to a higher
frequency of industries in this region. Of non-ortho-PCBs, CB-126 was
highest in all regions and contributed most to total TEQ-values.
Ratio between blood and eggs The average concentrations of ∑PBDE in serum were comparatively low
compared to the eggs with ratios serum/egg (calculated on wet weight) of
0.009, 0.005, 0.005 and 0.003 in the different populations; Lap, Inl, sBS
and BP, respectively. This is of the same magnitude as has been
calculated in plasma/egg for ∑PCB in bald eagles from the Great Lakes
[162]. It should be kept in mind though that the eggs and blood does not
come from the same nests or from the same year of collection.
0%
20%
40%
60%
80%
100%
Lap
(Eg
g)
Lap
(Se
rum
)
Inl (
Egg)
Inl (
Seru
m)
sBS
(Egg
)
sBS
(Se
rum
)
BP
(Eg
g)
BP
(Se
rum
)
BDE154
BDE153
BDE100
BDE99
BDE47
Figure 17: Concentration proportions of individual congeners BDE47, -99, -100, -153 and -154 to∑PBDEs. First bar of each set are values of PBDE in eggs and the second bar in serum from eacharea; Lap, Inl, sBS and BP.
27
However, the eggs have much higher levels of the contaminants than the
blood. Wild birds lay only a few eggs in each breeding season and the
egg-yolk is a major route for excretion of lipophilic compounds by the
female bird and the egg offers an enclosed environment for the embryo
under development. Thereafter, the concentrations in the young birds are
primarily influenced by intake through the diet, as well as the rapid
changes in the bird´s size. The much lower residues in the blood might
partly be due to a very efficient metabolism but also due to mass dilution
from hatching to fledging.
The occurrence of MeO-PBDEs in the interior parts of Sweden Most literature concerning MeO-PBDEs in wildlife report results from
the marine environment suggesting that these compounds mostly are a
result of accumulation via natural sources e.g., via formation in aquatic
sponges and algae, while MeO-PBDEs in freshwater ecosystems claimed
as unlikely [163]. However, in the WTSEs, MeO-PBDEs were found in
all eggs and some of the blood samples from the population inhabiting
the fresh water lakes (Inl). Some of these adult eagles can have the
opportunity to find their food in the marine waters (paper I) while most
of them do not. The findings in paper I and IV, are in an agreement with
earlier reported results in fish from this area [164] and when study the
content of MeO-PBDEs in different fish species from different lakes,
these compounds were found in 10 of 32 fresh water lakes in Sweden but
only a few lakes contained high levels and were not specific associated to
eutrophic lakes [165]. This indicates that there are unidentified sources of
these compounds in the freshwater ecosystem as well but highest
concentrations are still found in the marine environment.
Occurrence of OH-PBDE in the Baltic Sea The WTSE populations in the coastal areas had approximately 13 times
higher concentrations of ∑OH-PBDEs than the Lap population but the
difference between the coast and Inl were up to 44 times with higher
concentrations along the coast. OH-PBDEs have been shown to be
present in high levels in several species in the Baltic Sea such as primary
producers; cyanobacteria (Nodularia spumigena and Aphanizomenon-
flos) and algae (Ceramium tenuicorne) [44,45] but also in blue mussels
(Mytilus edulis L.) [166], Baltic Sea salmon (Salmo salar) [39] and ringed
seals (Phoca hispida) [167]. Routti et al. [167] reported ∑OH-PBDE
concentrations in ringed seals 19 times higher than in the same species
from Svalbard (0.36 and 0.019 ng/g w.w. or 55 and 2.8 ng/g lipid,
28
respectively) OH-PBDEs are retained in the blood but they do not seem
to biomagnify through the food chains [168]. In the WTSE from the
coastal populations ∑PBDE were two times lower in sBS than ∑OH-
PBDE whereas in BP the same relationship was 3.7 times lower.
A wider perspective of brominated compounds in the Baltic Sea Today the human activities impact on the Baltic Sea ecosystems resulting
in rapid and large-scale environmental changes. The biota in the Baltic
Sea is exposed to high levels of anthropogenic contaminants (e.g.,
nutrients, heavy metals, chemicals) as well as toxins produced by
different organisms. In addition, stress in the form of decreased salinity
and increased temperature of the Baltic ecosystem, is expected as an
effect of global warming [169]. The ban of DDT and PCB in the end of
the 1970s led to a recovery of the WTSE populations as well as for many
other species inhabiting the Baltic Sea. Although there has been a
reduced input of these chemicals there is still concern about disruption of
the ecosystems due to eutrophication caused by the human activity, which
seems harder to reduce. Global warming might lead to increased
occurrence of algal blooms and favor the growth of filamentous algae
which might produce more of the naturally produced brominated
compounds such as MeO-PBDEs and OH-PBDEs. OH-PBDEs in turn are
known to interact as a thyroid hormone disrupting agent in higher
species, which could lead to increased biological responses such as
disturbed reproduction, metabolism and growth.
Additional results As indicated in the screening study on eggs and additional results on
blood serum, there are a large number of organohalogen compounds in
the WTSEs. In paper II the levels of PCB are currently still high (in µg/g
lipid range). The PCB concentrations in the eggs reflect the exposure of
PCB from the environment while the MeSO2-PCB reflects the
metabolism of PCBs in the birds. Also the nestling serum contained
metabolic residues such as OH-PCBs which are retained in the blood and
some of these congeners have earlier been measured in WTSEs [119].
The indicated level of OH-PCB in the nestling serum might also be a
potential risk as PCBs still are present in high concentrations in the
environment. Future studies should focus on effects of both OH-PBDEs
and OH-PCBs on endocrine disruption in WTSEs.
29
7 Concluding remarks and future perspectives The WTSE is a powerful example of the dramatic effects that certain
environmental contaminants (e.g., DDT and PCB) can have on
reproduction. The sea eagle population in Sweden has recovered in
tandem with reductions in environmental concentrations of these
contaminants, except for the impairments observed in the area of the
south Bothnian Sea. The hypothesis in this thesis was that higher
concentrations of the investigated chemicals would be correlated to the
lower reproductive success of WTSE in the region of the south Bothnian
Sea.
However, based on the results in the papers included in this
thesis, no clear-cut associations were seen between the levels of the
investigated compounds and the reproductive impairment observed in the
south Bothnian Sea. Instead these studies have made a valuable
contribution to understanding how a species at the top of the food chain is
contaminated with anthropogenic as well as naturally produced
organohalogen compounds at high levels.
During this work, several questions have emerged which need
to be studied in the future. Even though no correlations with productivity
were seen, the levels of total TEQ for the dioxin-like compounds
(PCDD/Fs and non-ortho-PCB) and OH-PBDE concentrations are high in
the populations inhabiting the Baltic Sea region and are in the same range
as found in other bird species that show biological responses or
toxicological effects. Therefore it is important to include biomarker
studies in the WTSE as well and also continue yearly collection and
analysis of nestling blood and eggs. As seen in the additional results, OH-
PCBs are present in eggs and in blood from nestlings while MeSO2-PCB
is present in the eggs. These results need to be verified and specific
compounds need to be identified and quantified. PCB, DDT and its
metabolites are still found in these birds in high levels and might cause
negative effects on the species as well. All together there is a huge
number of unidentified and identified compounds in this top predator, of
which only a few have been quantified. It would be interesting to
investigate if the combined effects of this chemical cocktail results in
adverse effects in the WTSE. Further, with many new contaminants
entering the environment and continued reproductive impairment still
visible in some WTSE populations, there is clearly still a need for
continued studies of this predatory species.
30
8 Acknowledgement
Det känns lite overkligt att jag tagit mig hela den långa vägen ända hit,
men FY vad det var krångligt! Tiden har trots en del avbrott medvetet
(Gustav) och omedvetet valda (VAB, VAB, VAB,…, och lite annat smått
o gott) gått otroligt snabbt och till största delen varit både lärorikt och en
bra period i livet. Men detta till trots kan jag inte förneka att det även
varit jobbigt med en hel del frustration och negativ stress. Men jag
överlevde ju det också!
Ett stort tack till mina handledare. Lillemor, en person med
stor förståelse för att forskningen inte alltid kommer på första plats, inte
en gång du klagat på att jag satt min familj främst. Björn som vi faktiskt
har att tacka att vi i huvudtaget har några havsörnar i Sverige, det har
varit roligt att få känna ditt enorma engagemang! Yngve för att du alltid
är så positiv och för att du delat med dig av dina kunskaper om
dioxinanalyser.
Det finns några speciella personer som jag vill tacka för att ni
gjort tiden på ITM lite extra rolig; Mina underbara gruppmedlemmar Ulla
och Kerstin, TACK, för all kunskap Ni delat med er av och all hjälp
genom åren! Lotta en person med stort hjärta utan dig hade detta varit så
mycket svårare. Tack för alla frukoststunder som lätt blev för låååånga
men helt nödvändiga! Katarina som tog emot mig på ITM de första
dagarna och som sedan blivit en kär vän, utan dig hade detta inte varit
möjligt. TACK, till Cindy och Ulla S, som peppat, kommit med goda
råd och hjälpt till på slutet, vilket bra team ni är! Ett stort tack också till
Henriette, som alltid har haft tid att lyssna och genom ditt lugn tagit ner
mig på jorden då jag ville lyfta eller sjunka av olika anledningar! Anna-
Lena för ditt lugn och påhejande de sena kvällar vi delat tillsammans.
Ulrika F, som var fantastisk då det var som tyngst – du har en fin
egenskap av att bry dig om andra. Johanna, vi har haft mycket att skratta
åt genom åren tillsammans med Anna-Karin och Anna M, Ni har alla
betytt mycket och har alltid tagit er tid för att lyssna och jag ser fram
emot nästa påhitt på stan. Shahid som är en härlig rumskompis med både
lugn och humor. Ann-Sofie för din värme, påhejande och att du alltid
bryr sig om! Maggan och Amelie för att ni alltid har skrattet nära till
hands, det blir så mycket lättare att jobba då.
Ett stort tack också till, Kerstin och Hanna, som tog emot
mig på Mx och hjälpte mig med dioxin-uppreningen utan att blinka, och
som lärde mig lyssna på P1 (fast det var bra trist i början), Kasia som
varit till stor hjälp med allt diskande och trevliga diskussioner på tidiga
morgnar när jag var helt ensam i korridoren.
31
Och till sist ett varmt tack till Michael som hjälp till på slutet med
diskussioner och stöd samtidigt som du gav mig möjligheten att bli färdig
(at last!) trots att det drog ut lite på tiden, men fyra år går ju så väldigt
fort!
Jag vill också passa på att tacka alla andra doktorander och
arbetskamrater som funnits till hands under dessa år, Ni har kommit med
kloka råd då problem uppstått, men också bara för trevligt sällskap vid
lunch och fika, TACK!
På museet finns det några personer som jag vill tacka lite extra
och det är Anders som alltid med ett leende tagit sig tid för diskussion
och statistik och Mats som preppat prover på bästa sätt, TACK!
Till de bästa väninnorna Kicki och Helene som alltid har tid för mig och
som fått mig att tänka på annat, tack för att Ni finns! Yasar min gulliga
studiekamrat för peppning genom åren, är så glad att vi hittade varandra!
Janne som hela tiden funnits vid min sida och med din erfarenhet
kunnat lotsa mig igenom detta, det kan inte ha varit lätt. Inte ett ord om
att jag varit frånvarande (det brukar ju inte riktigt vara jag som är det), vi
har ju trots allt våra fantastiska barn My och Gustav som krävt sitt och
som nu ska få 500% uppmärksamhet, jag älskar er! Även ett stort tack till
Mamma och Pappa som alltid ställt upp med vad nu än kan ha varit, Ni
är bäst i mina ögon!
The Swedish Environment Protection Board within the National
Environment Monitoring Program is acknowledged for their permission
to use eggs and blood samples. The Swedish Research Council for
Environment, Agricultural Sciences and Spatial Planning (FORMAS)
provided financial support for this project.
Blott de tama fåglarna längtar. De vilda flyger……..!
32
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