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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