Microsoft word - thesis-5.doc
APPENDIX 1
Levels and effects of persistent organic pollutants (POPs) in seabirds
Retinoids and α-tocopherol – potential biomarkers of POPs in birds?
Kari Mette Murvoll
Doctoral thesis for the degree of Philosophiae Doctor (PhD)
Norwegian University of Science and Technology
Faculty of Natural Sciences and Technology
Department of Biology
Trondheim 2006
CONTENTS
LIST OF PAPERS . 5
SUMMARY . 7
1. INTRODUCTION. 9
1.1 Organochlorines (OCs). 9
1.1.1 Polychlorinated biphenyls (PCBs) . 10 1.1.2 Organochlorine pesticides (OCPs) . 11
1.2 Brominated flame retardants (BFRs). 14
1.2.1 Polybrominated diphenyl ethers (PBDEs). 15 1.2.2 Hexabromocyclododecane (HBCD). 16
1.3 Toxic mechanisms of persistent organic pollutants (POPs). 16
1.4 Persistent organic pollutants in seabirds. 17
1.5 Environmental monitoring and biomarkers. 19
1.6 Vitamins . 21
1.7 Vitamins as biomarkers of POPs . 22
2. OBJECTIVES . 24
3. METHODS . 26
4. DISCUSSION . 29
4.1 The choice of seabird species. 29
4.2 Levels of POPs in hatchlings of the present study. 29
4.2.1 Levels of POPs . 29 4.2.2 Levels of POPs in comparison to other studies . 34 4.2.3 Pattern of POPs . 36
4.2.3.1 PCBs. 36 4.2.3.2 OCPs. 38 4.2.3.3 PBDEs and HBCD. 39
4.3 Responses of morphological variables to exposure to POPs. 41
4.4 Responses of vitamins to exposure to POPs. 42
4.4.1 Normal temporal variation in vitamin levels. 42 4.4.2 Vitamin responses . 42
4.4.2.1 Responses of α-tocopherol to POPs. 42 4.4.2.2 Responses of retinol to POPs. 46
4.4.3 Vitamins as potential biomarkers . 47
4.5 Possible linkages to higher level effects . 48
4.6 Seabirds as bioindicator species. 50
5. CONCLUDING REMARKS AND FUTURE PERSPECTIVES. 52
6. REFERENCES. 55
List of papers
1.
Murvoll, K.M., Jenssen, B.M. and Skaare, J.U. 2005. Effects of pentabrominated diphenyl
ether (PBDE-99) on vitamin status in domestic duck (
Anas platyrhynchos) hatchlings.
J
Toxicol Environ Health A 68: 515-533.
2.
Murvoll, K.M., Skaare, J.U., Anderssen, E. and Jenssen, B.M. 2006. Exposure and effects
of persistent organic pollutants in European shag (
Phalacrocorax aristotelis) hatchlings from
the coast of Norway.
Environ Toxicol Chem (Volume 25). In press.
3.
Murvoll, K.M., Skaare, J.U., Moe, B., Anderssen, E. and Jenssen, B.M. (Accepted).
Spatial trends and associated biological responses of organochlorines and brominated flame
retardants in hatchlings of North-Atlantic kittiwakes (
Rissa tridactyla).
Environ Toxicol Chem.
4.
Murvoll, K.M., Skaare, J.U., Jensen, H. and Jenssen, B.M. (Submitted). Associations
between persistent organic pollutants and vitamin status in Brünnich's guillemot and common
eider hatchlings.
In the present thesis, levels of polychlorinated biphenyls (PCBs), some chosen organochlorine
pesticides (OCPs), polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecane
(HBCD) were analyzed by gas chromatography in the yolk sac of newly hatched chicks of
European shag (
Phalacrocorax aristotelis), kittiwake (
Rissa tridactyla), Brünnich's guillemot
(
Uria lomvia) and common eider (
Somateria mollissima) from the Norwegian coast and
Svalbard. Levels of vitamin A (retinol), retinyl palmitate and vitamin E (α-tocopherol) were
measured in plasma and liver of the hatchlings using high-performance liquid
chromatography (HPLC). Using statistics, possible significant relationships between levels of
the persistent organic pollutants (POPs) and vitamin levels were examined. Hence, the study
aimed to elucidate retinoids and tocopherol as potential biomarkers of POP exposure. An
exposure study on domestic duck (
Anas platyrhynchos) eggs was also conducted to assess the
effects of 2,2',4,4',5-pentabromodiphenyl ether (PBDE-99) on vitamin levels under controlled
laboratory conditions.
There were significant differences in POP levels between the bird species included in the
present study. In general, kittiwake hatchlings had higher levels of POPs than the other
species, followed by shag, Brünnich's guillemot and common eider hatchlings. Levels of
organochlorine compounds in the hatchlings seemed to be higher than reported in sea bird
eggs from the Canadian Arctic but lower than reported in eggs of other seabirds from the
Netherlands, the Baltic, the Great Lakes and Japan. In contrast to this, the levels of PBDEs
and HBCD seemed to be high in some of the species (kittiwakes, shags) relative to a
Negative relationships were revealed between POPs and morphology in Brünnich's guillemot
hatchlings, indicating that this species may be more responsive with respect to effects of POPs
on morphological variables than the other species included in the present study. The
importance of considering possible confounding impacts of lipid content when studying
effects of POPs on morphological variables was emphasized in shag hatchlings.
The study revealed negative correlations between POPs and liver tocopherol levels in
domestic duck and shag hatchlings. In Brünnich's guillemot hatchlings, liver tocopherol
levels also were negatively associated with POPs, but the relationships were less strong when
the effect of body mass on tocopherol levels was accounted for. In kittiwake and common
eider hatchlings, however, there seemed to be a positive influence by POPs on tocopherol
levels. Thus, the results should encourage further research on the effects of POPs on
tocopherol levels (including oxidized forms of the vitamin).
In shag hatchlings, negative relationships between POPs and plasma retinol levels were
observed, in line with several previous studies on birds. Since retinol was not influenced in
any other species included in the study, tocopherol levels might be more responsive than
retinol levels to POP exposure. Additional studies should, however, be conducted before
certain conclusions are drawn.
Concerning the work needed for further development of vitamins as biomarkers of POP,
effort should be done to characterize confounding factors, such as diet and condition of the
avian mothers. Although there was no obvious link between the observed responses of
vitamins to POP exposure and effects at higher biological levels (i.e. reproduction
disturbances, population decline), the relevance of vitamins as potential biomarkers of POP
exposure should not be repelled.
1. INTRODUCTION
At the beginning of World War II, several pesticides and other chemical agents were under
experimental investigation (Ecobichon 2001). The continuing demand for many new materials
in modern civilization and the concomitant development of the chemical industry resulted in
production of man-made chemicals in large numbers and quantities, greatly contributing to
our convenient and pleasant lifestyle (Tanabe et al 1994). However, many persistent organic
pollutants (POPs), such as polychlorinated biphenyls (PCBs) and organochlorine pesticides
(OCPs), also had undesirable outcomes. The chemicals were found to bioaccumulate in the
food-chain (Reijnders 1980, Tanabe et al 1983) because of their resistance to biodegradation
(recalcitrance) and high lipophilicity (Niimi 1987, Mackay et al 1992a; 1992b, Augistijn-
Beckers et al 1994). In addition, negative effects on wildlife were revealed (Ratcliffe 1967,
Delong et al 1973, Helander et al 1982, Bignert et al 1995). The reports on widespread
environmental contamination of organochlorines (OCs) and the documentation on their toxic
effects lead to restrictions on production and use of the compounds in industrialized countries
(Peterle 1991a, AMAP 1998). In Norway, restrictions on use of PCBs and OCPs were given
in the early 1970s (Ingebrigtsen et al 1984). Today there seems to be a decreasing temporal
trend of PCBs and OCPs in biota (Bignert et al 1998, Braune et al 2001), but the compounds
still exert a potential risk to human and wildlife health because of leakage from sediments and
products (Tanabe 1988, AMAP 1998). Recently, other POPs such as brominated flame
retardants (BFRs) have received much attention due to a reported temporal increase in human
milk and Arctic biota (Meironyté et al 1999, Ikonomou et al 2002). Because of high
production volumes of BFRs and structural resemblance of some of these compounds to well-
known environmental contaminants, such as PCBs, concern has arisen for human and wildlife
health (Darnerud 2003). Hence, due to continued discharges and leakage of POPs, monitoring
of the environment with regard to these pollutants and their potential risk to wildlife is of
great importance.
1.1 Organochlorines (OCs) OCs are organic chemicals with one or more hydrogen atoms on the carbon skeleton
substituted by chlorine atoms (Walker et al 2001). Although they have diverse chemical
structures, the common characteristics for most of them are low water solubilities, high
lipophilicity and resistance to biodegradation (Niimi 1987, Mackay et al 1992a; 1992b,
Augistijn-Beckers et al 1994). These combined characteristics lead to bioaccumulation in
fatty tissues of organisms (Reijnders 1980, Tanabe et al 1983, Barrett et al 1996, Champoux
et al 2002). OCs are grouped in three major classes; industrial chemicals (e.g. PCBs),
pesticides (e.g. dichlorodiphenyltrichloroethane; DDT) and by-products of combustion and
industrial processes (e.g. polychlorinated dibenzo-
p-dioxins; PCDDs). The present thesis
deals with PCBs and some chosen pesticides (in addition to BFRs, see 1.2).
1.1.1 Polychlorinated biphenyls (PCBs) Theoretically there are 209 PCB congeners (i.e. many variants or configurations
of a common chemical structure), and approximately 120 of these are present in commercial
products such as Aroclor 1254, Aroclor 1260 and Chlopen A60 (Walker et al 2001).
Ballschmiter and Zell (1980) proposed a simple numbering system of the PCB congeners,
giving each congener a number from one to 209. This system was later adapted by IUPAC
(International Union of Pure and Applied Chemistry) (www.iupac.org).
Figure 1: General molecular structure and Cl-substitution positions of PCBs.
PCBs were made commercially available in about 1930 (Peterle 1991a). PCBs were used as
hydraulic fluids, coolant-insulation fluids in transformers and plasticizers in paints due to their
physical properties of chemical stability and low volatility (Walker et al 2001). In 1966, the
Swedish scientist Sören Jensen discovered the presence of high PCB levels in environmental
samples when analysing for the insecticide DDT (Jensen 1966). The further discovery of
widespread environmental contamination (Risebrough et al 1976, Reijnders 1980, Tanabe et
al 1983) and the subsequently reported negative impact on wildlife by PCBs (Delong et al
1973, Helander et al 1982) lead to restrictions on the production and use of the compounds
(Peterle 1991a, AMAP 1998). In Norway, restrictions on the use of PCBs were imposed in
1971, and a ban was introduced in 1980 (Ingebrigtsen et al 1984). Today PCBs are globally
banned in accordance with the Stockholm Convention of 17 May 2004 (www.pops.int). Some
of the documented effects of exposure to PCBs on human health and wildlife are impaired
immunity (DeSwart et al 1996), neurotoxicity (Stewart et al 2003), carcinogenicity
(Cajaraville et al 2003), and hormonal and reproductive effects (Helander et al 1982, van den
Berg et al 1994, Bustnes et al 2001).
1.1.2 Organochlorine pesticides (OCPs) The OCPs are a relatively large group of chemicals. The chemicals have been used for the
control of agricultural pests and diseases (Walker et al 2001).
p,p'-dichlorodiphenyldichloroethylene (
p,p'-DDE) is the most stable metabolite of DDT
(Peterle 1991a). The insecticidal properties of DDT were discovered by Paul Müller of the
firm Ciba-Geigy in 1939. DDT was used for vector control during the Second World War,
and thereafter it was widely used for control of agricultural pests, diseases (e.g. malaria
mosquitoes) and insects (Walker et al 2001). The use of DDT has been restricted in several
industrial countries for decades (Goldberg 1991), but has frequently been used in pest control
programs in developing countries (Forget 1991). In Norway, the use of DDT was banned in
1988 (AMAP 1998). According to the Stockholm Convention, the global production and use
of DDT is now limited to controlling disease vectors such as malaria mosquitoes
(www.pops.int). Thus, some new use of DDT will also in future years lead to environmental
releases. The metabolites
p,p'-DDE and
o,p'-dichlorodiphenyldichloroethane (
o,p'-DDD) are
primarily found at higher trophic levels. DDT and its metabolites are reported to affect
reproduction, especially in birds due to egg-shell thinning (Ratcliffe 1967, Longcore and
Stendell 1977, King et al 2003), to impair immunity (Wong et al 1992, Misumi et al 2005)
and to influence hormonal systems (WHO 1989, Mayne et al 2004).
Figure 2: p,p'-DDE.
Hexachlorobenzene (HCB) is a by-product in the production of several chlorinated
compounds. It had a limited use in the 1960s as a fungicide (AMAP 1998). The chemical
bioaccumulates due to high lipophilicity and long half-life in biota (Niimi 1987, Augistijn-
Beckers et al 1994). The worldwide production and use of the compound is now limited to
narrowly prescribed purposes in accordance with the Stockholm Convention (www.pops.int).
Several effects of exposure to HCB are reported, such as reproductive and developmental
effects (Boersma et al 1986, Helberg et al 2005), interruption of the immune system (Bleavins
et al 1983, Bustnes et al 2004) and tumour promotion (Stewart et al 1989, Randi et al 2003).
Figure 3: Hexachlorobenzene (HCB).
Oxychlordane is a toxic metabolite of the chlorinated pesticide chlordane, a mixture of at least
120 compounds. The most important components are
cis-chlordane,
trans-chlordane and
trans-nonachlor (Dearth and Hites 1991). Chlordane is very persistent (Augistijn-Beckers et
al 1994), and reproductive impacts (Lundholm 1988, Bustnes et al 2005), immunosuppression
(Spyker-Cranmer et al 1982, Bustnes et al 2004) and cancer (WHO 1984) are among the
documented toxic effects. According to the Stockholm Convention, the production and use of
chlordane compounds are now limited to prescribed purposes (www.pops.int).
Figure 4: Two important components of chlordane,
cis-chordane and
trans-chlordane. Oxychlordane is a toxic
metabolite of the chlordane mixture.
Mirex is an insecticide and fire retardant, used mainly in the USA and Canada. It has never
been used in Norway (AMAP 1998). Mirex is extremely persistent in soils and sediment in
addition to being lipophilic (Augistijn-Beckers et al 1994). Its present in the Arctic at low
levels is consistent with its volatility and persistence, and today the production and use of
mirex is limited to narrowly prescribed purposes in accordance to the Stockholm Convention
(www.pops.int). Documented effects on vertebrates include reproductive impact (Naber and
Ware 1965, Dai et al 2001) and reduced immune function (Wong et al 1992).
Figure 5: Mirex.
β-Hexachlorohexane (β-HCH) is an isomer of technical HCH, of which γ-HCH (lindane) is
the most biologically active insecticidal isomer. β-HCH has been banned for use in the USA
and most other circumpolar countries since the late 1970s (AMAP 1998). HCH is much less
bioaccumulative than other OCs because of its relatively low lipophilicity and short half-life
in biota (Niimi 1987). β-HCH is documented to be estrogenic (Van Velsen 1986, Steinmetz et
al 1996) and to affect the immune system (Cornacoff et al 1988).
Figure 6: β-HCH.
1.2 Brominated flame retardants (BFRs) Flame retardants are materials added or applied to a material to increase the fire resistance of
that product. In order to meet fire safety regulations, flame retardants are applied to
combustible materials, such as plastics, wood paper and textiles (de Wit 2002, Alaee et al
2003). Halogens are very effective in capturing free radicals that are produced during
combustion processes and hence removing the capability of the flame to propagate. Due to
different stability among the halogens, chlorinated and brominated compounds, and not
fluorinated and iodinated compounds, are used as flame retardants. However, with higher
trapping efficiency and lower decomposing temperature, brominated compounds have
become more popular than their organochlorine counterparts as flame retardants (Alaee et al
2003). The BFRs are divided into three subgroups from the incorporation of brominated
compounds into the polymers; monomers, reactive and additive. Brominated monomers are
incorporated into polymers containing both brominated and nonbrominated monomers.
Reactive flame retardants, such as tetrabromobisphenol A (TBBPA), are chemically bonded
to plastics. Additive flame retardants, such as polybrominated diphenyl ethers (PBDEs) and
hexabromocyclododecane (HBCD), are blended with the polymers and are more likely to
leach out of the products (Alaee et al 2003). Most used BFRs are polybrominated biphenyls
(PBBs), TBBPA and its derivatives, PBDEs and HBCD (Darnerud 2003). The present thesis
deals with PBDEs and HBCD (in addition to OCs, see 1.1).
1.2.1 Polybrominated diphenyl ethers (PBDEs) There are theoretically 209 PBDE congeners. Diphenyl ether molecules contain ten hydrogen
atoms, which can be substituted by bromine. The structure of PBDEs is corresponding to that
of PCBs, and the individual PBDE congeners are numbered according to the IUPAC system,
based on the position of the halogen atoms on the carbon skeleton (de Wit 2002). The
commercial PBDE products predominately consist of so-called penta-, octa- and
decabromodiphenyl ether products (Darnerud 2003). The Penta-BDE (Bromkal 70) consists
of mainly tetra- and pentabrominated congeners (i.e. PBDE-47 and PBDE-99, respectively),
whereas the Octa-BDE contains mainly hepta-, octa- and nonabrominated diphenyl ethers (e.g.
PBDE-183 [hepta] and PBDE-203 [octa]). Commercially produced Deca-BDE contains
mainly the decabrominated diphenyl ether PBDE-209 (Alaee et al 2003).
Figure 7: General molecular structure of PBDEs.
The PBDEs have been used in plastic components in electronic equipment, in textiles, in cars
and in building materials (Darnerud 2003). In the environment, PBDEs were first discovered
in Sweden in fish samples taken downstream from several textile industries (de Wit 2002).
During recent years, the compounds have been detected in sediments, birds, seals and human
blood serum (Allchin et al 1999, Sjödin et al 1999, Ikonomou et al 2002, Herzke et al 2003,
Vorkamp et al 2004). Although the knowledge about the effects of PBDEs in wildlife and
man is scarce (Darnerud 2003), several critical effects of the compounds have been reported
in rodents, such as effects on neurobehavioral development (Eriksson et al 2001), as well as
effects on thyroid hormone homeostasis (Darnerud and Sinjari 1996) and on vitamin A status
(Hallgren et al 2001). The use of the commercial mixtures Penta-BDE and Octa-BDE was
prohibited in all applications for the EU Market from 15 August 2004 (www.bsef-
site.com/regulation), and the Penta-BDE is also voluntarily withdrawn from the Japanese
market (Alaee et al 2003). In addition, California, Maine and Michigan of the USA have
prohibited the use of Penta-BDE from 2006-2008 (www.bsef-site.com/regulation). However,
due to the recalcitrance of the compounds, they will persist in the environment for decades. In
addition, no global ban on the production and use of the compounds is yet adopted.
1.2.2 Hexabromocyclododecane (HBCD) HBCD has been used for about 20 years in foams and expanded polystyrene (de Wit 2002),
which is largely used in insulation panels and blocks for building constructions (Darnerud
2003). The few available studies indicate that HBCD has low water solubility and a high
bioaccumulation potential (de Wit 2002). The compound has been detected in various
environmental compartments and biota (de Wit 2002), but there is a lack of studies of high
quality with regard to toxic effects (Darnerud 2003). However, behavioural effects may be a
sensitive endpoint for HBCD (Eriksson et al 2002), although also other physiological effects
Figure 8: HBCD.
1.3 Toxic mechanisms of persistent organic pollutants (POPs) The mechanisms of toxic action of the POPs are diverse and all of them still not fully solved
(Darnerud et al 2001). However, the toxicity of PCBs and OCPs has been extensively studied
(Parkinson 2001). For the PCBs, a considerable amount of evidence supports a certain
mechanism of toxic action of the non-
ortho (e.g. PCB-77, -126, -169) and mono-
ortho (e.g.
PCB-105, -118, -156, -157) congeners (Brouwer 1991). These compounds have a planar
configuration and elicit toxicity through the nuclear aryl hydrocarbon receptor (AhR), of
which 2,3,7,8-tetrachlorodibenzo-
p-dioxin (TCDD) has the highest affinity (dioxin-like
toxicity) (Safe 1994). Binding to the Ah-receptor mediates the induction of cytochrome P450
(CYP) 1A enzymes in the liver, which are constituents of the most important enzyme system
involved in xenobiotic biotransformation (Safe 1994). Di-
ortho PCB congeners (e.g. PCB-
128, -137, -138, -153) do not as easily induce CYP 1A enzymes as the more planar congeners,
but are able to induce CYP 2B enzymes via another nuclear receptor (Boon et al 1992,
Parkinson 2001). Also OCPs induce CYP 2B enzymes (Parkinson 2001). Inducers of the CYP
enzymes will affect the metabolism and toxicokinetics of other contaminants and may lead to
increased formation of reactive intermediates, giving increased toxicity (additive or
synergistic effects), or increased detoxification (antagonistic effects). The CYP inducers will
also be substrates for the enzymes and thus be metabolized to degradation products (Boon et
al 1992). Hydroxy- and methylsulfonyl PCB metabolites formed by actions of detoxifying
enzymes have been shown to cause a variety of toxic effects, including reduced vitamin A and
thyroid hormone levels (Brouwer et al 1998). Metabolites of DDT are reported to elicit
various toxic effects, such as impaired reproduction (Ratcliffe 1967, Longcore and Stendell
1977, King et al 2003), suppressed immunity (Wong et al 1992
, Misumi et al 2005) and
endocrine disruption (WHO 1989, Mayne et al 2004).
The knowledge about the toxicology of PBDEs and HBCD is scarce (Darnerud 2003).
However, many of the adverse effects of these brominated compounds resemble those of
PCBs, which may indicate similar toxic mechanisms. Actually, tetra-, penta- and
hexabrominated diphenyl ethers have been found to induce the CYP enzymes (von Meyerinck
et al 1990, Pettersson et al 2001). In cultured chick embryo livers, the most potent CYP 1A
inducer of the tested congeners was PBDE-99 (Pettersson et al 2001).
1.4 Persistent organic pollutants in seabirds Highly lipophilic and persistent organic compounds such as POPs are released into the sea,
where they bioaccumulate in marine organisms (Macdonald and Bewers 1996). The POP
bioaccumulation depends on the uptake and elimination ability of the organism, and the
compounds' physico-chemical properties (Walker et al 2001). Although diet and trophic
position is the dominant factor influencing concentrations of hydrophobic and recalcitrant
compounds in seabirds (Borgå et al 2004), several other factors will also be of importance
when considering accumulation of POPs, e.g. the organisms' age (Donaldson et al 1997),
condition and reproductive status (Henriksen et al 1996) and migration pattern (Buckman et al
Uptake of organic compounds in seabirds takes place through the food chain
(biomagnification) (Walker et al 2001). The xenobiotic metabolism activity is related to the
organism's metabolic rate, which generally increases from marine invertebrates to vertebrates
(Livingstone et al 1992) and further from fish (poikilotherms) to seabirds and mammals
(homeotherms). However, many seabirds have shown low ability to metabolize contaminants
via CYP enzymes (Walker 1992). The reason why fish-eating birds are deficient in CYP
enzymes could be that there has been no requirement for sophisticated enzymic detoxication.
In contrast, many herbivore terrestrial birds have evolved systems to detoxify lipophilic
xenobiotics in their food, which contains a wide range of lipophilic compounds that are
normal constituents of plants, but are not produced by animals (Walker 1992). Thus, when
considering the problem of bioaccumulation in marine ecosystems, particular attention should
be paid to fish-eating birds because they are top-predators and because they seem to have low
metabolic capacity towards lipophilic contaminants. Several studies on fish-eating birds have
also shown negative relationships between high levels of OC pollution and reproductive and
developmental parameters, such as malformations of chicks, reduced eggshell thickness and
reduced hatching and breeding success (Fox et al 1991, Bignert et al 1995, Dirksen et al 1995).
Although fish-eating birds in general show relatively low ability to metabolize contaminants,
between-species differences in metabolic ability exist among seabirds (Borgå et al 2001; 2005,
Fisk et al 2001a; 2001b). In addition, the classifications of birds with reference to
development patterns could possibly influence metabolism of contaminants in embryos and
hatchlings. The traditional classifications of development patterns of birds recognize several
categories arranged along a gradient (altricial-precocial) according to a combination of
morphological, physiological and behavioural characteristics of the neonates (Starck and
Rickleffs 1998). The precocial extreme of the spectrum includes species whose young are
totally independent of their parents, and in some species the chicks can fly from the first day
of postnatal life. Semi-precocial species have chicks with relatively less developed locomotor
activity, stronger nest attendance, and complete dependence of the parents for food. Species
whose chicks remain in nest for much or all of their development are altricial. Fully altricial
hatchlings hatch with closed eyes en exhibit little motor activity other than begging and no
visible feathers (Starck and Rickleffs 1998). Precocial hatchlings are covered with down and
are able to respond effectively to heat and cold in contrast to altricial hatchlings, which have
little ability to regulate body temperature at ambient temperatures below 35ºC or above 40ºC
(Dawson and Whittow 2000). Hence, when studying levels and effects of POPs in embryos or
hatchlings of seabirds, it could be of relevance to focus at different species according to the
altricial-precocial spectrum. Their different metabolic ability could influence the levels and
pattern of, and responses to, the compounds.
Furthermore, when studying levels and effects of POPs it could be of interest to include
populations of the same species from different geographical locations. Populations of seabirds
situated close to sources of production and/or widespread use of POPs are expected to show
higher contaminant levels than remote populations far from the sources. Thus, when studying
different populations of birds, this could give opportunities of providing information on
spatial trends of POPs and on possible differences within the same species in responses to
1.5 Environmental monitoring and biomarkers Traditionally, monitoring of environmental pollution has focused to a large extent on
chemical measurements of well-known contaminants in sediments, water or living organisms.
However, chemical data alone is insufficient to reliably assess the potential biological
responses of the complex mixture of contaminants in the natural environment (Peakall and
McBee 2001). There are cases where chemicals may interact in a way that results in an
increase (synergism) or decrease (antagonism) in their overall effect compared with the sum
of the effects of the individual components (Peterle 1991b). Unknown substances could also
lead to a discrepancy between the actual and predicted risk of the contaminants. In addition, a
great variety of organisms and different environmental and biological processes have to be
considered (Fent 2001). Thus, to provide more detailed information on the possible effects of
contaminants,
biomarkers have been introduced as helpful tools in environmental monitoring
(Huggett et al 1992, Walker et al 2001). Some inconsistency, however, applies to the
definition of biomarker (Walker et al 2001). The most common use of the biomarker term
refers to biochemical, physiological or histological changes as well as aberrations in
organisms that can be used to estimate either exposure to chemicals or resultant effects
(Huggett et al 1992). Peakall (1994) defines a biomarker as "a biological response to a
chemical or chemicals that gives a measure of exposure and sometimes, also, of toxic effects"
whereas Walker et al (2001) defines biomarker as "any biological response to an
environmental chemical at the individual level or below demonstrating a departure from the
normal status". Changes at organizational levels above that of the individual, i.e. population,
community and ecosystem, are termed bioindicators (Walker et al 2001).
A number of classifications of biomarkers have been proposed. The most widely used is
division into "biomarkers of exposure" and "biomarkers of effect". Biomarkers of exposure
are those that indicate exposure of the organism to chemicals, but give no information on the
degree of adverse effects that this changes causes. Biomarkers of effect are those which
demonstrate an adverse effect of the organism (Walker et al 2001). However, Peakall and
McBee (2001) argue that this classification is misleading. All biomarkers indicate exposure
and demonstrate an effect of some kind.
The idea of measuring a biological parameter as an indicator of the well-being of an organism
is very old. In ancient China, physicians were able to assess the health of their patients by
tasting urine, by smelling faeces, etc. Modern medicine has refined this approach, and
measurements of heart rate, body temperature, blood counts, enzyme levels etc. are now
commonly used to monitor the patient's health (Depledge 1994). Due to the complexity of
ecosystems, the development of biomarkers in ecotoxicology has to deal with many
challenges that medical toxicology more easily solves. Nevertheless, the parallel to medical
toxicology should only encourage the further work on biomarkers for environmental
The specificity of biomarkers to chemical contaminants varies greatly. Both specific and non-
specific (general) biomarkers have their place in environmental assessment (Peakall and
McBee 2001). Further, some biomarkers can be applied throughout the animal kingdom, for
example the inhibition of acetylcholinesterase, whereas the induction of vitellogenin is
confined to those vertebrates that lay eggs. Some biomarkers respond instantaneously while
others need years to develop. The response may also be transient or irreversible (Huggett et al
1992). Hence, a number of aspects need to be considered when evaluating biomarkers. In
addition to the factors already mentioned, it will also be of importance that the biomarker is
sensitive compared to other endpoints, such as mortality or reproductive impairment, and the
responses should be distinguished from natural stressors. A biomarker will also be more
useful if there is a clear linkage to effects at higher levels of organization (Peakall and McBee
Seabirds have been used in several environmental monitoring studies. Fish-eating birds may
be well suited for the assessment of effects of POPs due to their wide distribution (Fox 1993).
They also bioaccumulate relatively high levels of POPs due to their higher trophic levels and
due to their limited abilities to metabolize anthropogenic compounds (Walker 1992). In birds
induction of CYP 1A enzymes (Sanderson et al 1994, Henriksen et al 2000), retinol
homeostasis (Spear and Moon 1986, Boily et al 1994, Kuzyk et al 2003), thyroid function
(van den Berg et al 1994, Verreault et al 2004) and various malformations (Fox et al 1991)
have been studied as responses to POP exposure and thus as potential biomarkers of POPs.
However, still there is a need of more studies to establish the application of biomarkers in
environmental monitoring programmes.
1.6 Vitamins Vitamin A (retinol) and vitamin E (tocopherol) are water-insoluble and lipophilic vitamins
(Combs 1992, Traber et al 1993), which are found in plants as carotinoids (pro-vitamin A),
tocopherols and tocotrienols (Combs 1992, Sheppard et al 1993). The most biologically active
form of vitamin E is α-tocopherol (Sheppard et al 1993).
The liver stores 50-80 % of retinol in vertebrates as retinyl esters, of which retinyl palmitate is
the most important (Blomhoff 1994, Karadas et al 2005). Mobilization of retinol from the
liver occurs by hydrolysis of retinyl esters. However, the transport of retinol in plasma is
dependent on specific transport molecules, retinol-binding proteins (RBPs), which have been
shown to be partly evolutionary conserved across the avian and mammalian species (Soprano
and Blaner 1994). In plasma, retinol-RBP binds to transthyretin (TTR), the transport protein
of thyroid hormones. The RBP-TTR complex is vital to prevent filtration of retinol-RBP
through the kidneys (Combs 1992). In contrast, there is no evidence of a specific vitamin E
plasma carrier protein. Tocopherol is transported in the blood within plasma lipoproteins and
erythrocytes, which has two important consequences: One is the protection by tocopherol
from free-radical attack on fatty acids and lipids. The other is that tocopherol concentrations
do not entirely depend on dietary intake (Traber et al 1993). Following absorption of
tocopherol from the intestine, tocopherol is incorporated in chylomicrons (mammals) or
portomicrons (birds) (Traber et al 1993, Surai 1999). Avian portomicrons (large lipoproteins)
are transported directly via the portal system to the liver prior to plasma entry (Surai 1999). In
contrast, mammalian chylomicrons are catabolized to chylomicron remnants and then taken
up by the liver. Once in the liver, α-tocopherol is secreted into the bloodstream within
lipoproteins (Traber et al 1993).
Retinol is essential for normal vision, reproduction, cellular immune function and the
maintenance of differentiated epithelia and mucous secretions in higher animals (Fox 1993).
Deficiency in retinol also influences growth and development of tissues and organs of
embryos and young animals (Spear et al 1986). In birds, nutritional excess or deficiency is
associated with changes in several reproductive parameters, such as secondary sexual
characteristics, spermatogenesis, egg-laying, egg size, embryo survival and hatching success
(Boily et al 1994). In the avian embryo, antioxidant defence based on natural antioxidants
such as vitamin A and vitamin E is responsible for the restriction and prevention of oxidative
chain formation and propagation (Surai 1999).
Tocopherol is essential for normal neurological structure and function (Traber et al 1993). As
a part of the antioxidant defences, tocopherol is also important in reducing the negative effects
of cellular oxidative stress (Saito 1990). In avian embryos, vitamin E is considered to play a
particular important role in the antioxidant defence being actively accumulated in embryonic
tissues (Surai 1999).
1.7 Vitamins as biomarkers of POPs Several studies report associations between retinoid status and OC compounds in rodents
(Brouwer et al 1983), seals (Brouwer et al 1989, Jenssen et al 2003), polar bears (Skaare et al
2001) and birds (Spear and Moon 1986, van den Berg et al 1994, Murvoll et al 1999,
Champoux et al 2002, Kuzyk et al 2003, Martinovic et al 2003). Thus, retinoid status has been
proposed as a promising biomarker for exposure to such compounds (Simms and Ross 2000).
Further, in rats and mice, exposure to PBDE mixtures have been shown to cause a reduction
in hepatic retinol levels (Hallgren et al 2001). Studies also document that α-tocopherol is
influenced by PCBs (Palace et al 1996, Twaroski et al 2001). It is therefore possible that
alterations in tocopherol levels could be a useful biomarker of exposure to POP compounds.
Proposed mechanisms for the reducing effect of OCs on retinoid stores are reviewed by Chen
et al (1992) and include (1) increased glomular filtration due to conformational changes by
hydroxy-metabolites in transport molecules for retinol in plasma (TTR-RBP), (2) loss or
transformation of stellate storage cells in the liver and (3) increased metabolism of several
retinol forms by induced phase I and phase II enzymes. Alternatively, increased utilization of
retinol as an antioxidant may also deplete retinol stores (Palace et al 1996).
The influence on α-tocopherol by PCBs is believed to be caused by the cellular oxidative
stress initiated by the substances, which cellular antioxidant defences, such as tocopherol, try
to counteract (Saito 1990). It is assumed that the oxidative stress induced by specific
environmental contaminants is due to the interaction of these compounds with the aryl
hydrocarbon (Ah) receptor and activation of the CYP 1A enzymes (Toborek et al 1995) which
lead to formation of reactive oxygen species (ROS). Some PBDEs have also been shown to
induce CYP 1A enzymes (von Meyerinck et al 1990, Pettersson et al 2001), and these
compounds may therefore also initiate the formation of ROS and oxidative stress. Thus,
tocopherol might be a potential biomarker of the oxidative stress exerted by POPs.
2. OBJECTIVES
The aim of the present thesis was to provide up-to-date information on levels of PCBs, some
OCPs, PBDEs and HBCD in different seabirds along the coast of Norway and from Svalbard,
and to contribute to the development of vitamins as potential biomarkers of POP exposure.
Several knowledge gaps make the use of biomarkers in environmental monitoring limited.
However, in the future, biomarkers would possibly make monitoring of species and
ecosystems more comprehensive and nuanced.
To accomplish the aim, the following was done:
1. Concentrations of POPs
The concentrations of 23 PCB congeners, 5 OCPs, 6 PBDEs and HBCD were analyzed in
the yolk sac of newly hatched chicks of four seabird species; European shag
(
Phalacrocorax aristotelis), kittiwake (
Rissa tridactyla), Brünnich's guillemot (
Uria
lomvia) and common eider (
Somateria mollissima). Samples from shag hatchlings were
taken at Sklinna (65°12' N, 11°00' E), an island at the coast of Norway, while samples
from kittiwake hatchlings were taken at Runde (62°24' N, 5°36' E), another island on the
coast of Norway, and from Kongsfjorden (78°55' N, 12°30' E) close to Ny-Ålesund at
Svalbard. Samples of Brünnich's guillemot and common eider hatchlings were taken from
Kongsfjorden (see Fig. 9).
2. Responses in vitamin levels
An exposure study on domestic duck (
Anas platyrhynchos) eggs was performed using
injections of PBDE-99 to study possible effects of this congener on vitamin levels under
controlled laboratory conditions. Vitamin levels (retinol, retinyl palmitate and α-
tocopherol) were measured in liver and plasma of newly hatched chicks of domestic ducks,
shags, kittiwakes, Brünnich's guillemot and common eider. Possible relationships
between POP levels and vitamins were statistically examined. In both field and lab studies
the age of the hatchlings were standardized (< 12 hrs) to reduce confounding factors. In
kittiwakes, also sex of the hatchlings was determined using PCR techniques, allowing a
correction for sex variation in the kittiwake data.
Figure 9: A map indicating where Runde, Sklinna and Ny-Ålesund are situated.
3. METHODS
3.1 Sampling At hatching, body mass, tarsus length and head size were recorded before blood samples (1-2
ml) of anaestisized chicks were taken by heart-puncture using hypodermic needles. A small
fraction of the collected blood was used to measure hematocrit, and the remaining blood was
centrifuged. The plasma was transferred to cryo vials and kept frozen at -40ºC. To prevent
photodegradation of retinol and tocopherol, the plasma-filled vials were covered with
aluminium foil. The yolk sac and liver were removed and weighed, before packed in
aluminium foil and stored at -40ºC. All samples were taken within 12 hrs after hatching to
reduce the risk of confounding due to normal temporal variations in vitamin levels during the
first 24 hours after hatchling. The sampling was approved by Norwegian Animal Research
Authority (Oslo, Norway).
The yolk sac of the hatchlings was the most appropriate matrix for the POP analysis (see 3.2)
due to the available amounts. Plasma and liver samples from the hatchlings were of small
quantities, but could be used for the analysis of vitamin levels (see 3.3). Hence, POP levels
and vitamin levels were measured in different matrices. The statistical analysis of possible
relationships between the POPs (predictor variables) and vitamins (response variables) could
thus be confounded. Nevertheless, Henriksen et al (1996) found that lipid weight PCB
concentrations were highly significantly correlated between tissues (liver vs. fat vs. brain) in
adult kittiwakes, and from this it was assumed that the levels of POPs in yolk sacs of
hatchlings probably correlated with levels of POPs in plasma and liver.
3.1.1 Exposure study In the laboratory study, the yolk of eggs of domestic duck was injected by PBDE-99 (0.05.
0.5 and 5 µg/ml) (
Paper I). After the injection, the eggs were sealed with paraffin and placed
in an incubator. The eggs hatched after 27-28 days of incubation.
3.1.2 Field studies In the field studies, eggs of kittiwakes, Brünnich's guillemot and common eider were taken
from the nests and transferred to a field laboratory where the eggs were put in incubators
(
Paper III, IV). The eggs hatched during 1-25 days. European shag hatchlings were collected
from their nests within 12 hours after hatching (
Paper II). The eggs/hatchlings were not
artificially exposed to POPs, and hence the POP levels measured represented natural levels.
3.2 Analysis of POPs The analyses of POPs were carried out at the Environmental Toxicology Laboratory at the
Norwegian School of Veterinary Science as described in
Paper I-II. In short, the organic
compounds were extracted with acetone and cyclohexane, concentrated by evaporation and
lipids removed from the extracts with sulphuric acid. The extracts were then injected to a gas-
chromatograph (GC) with a MS- (brominated compounds) and EC- (chlorinated compounds)
detector (
Paper I-II).
The laboratory is accredited according the requirements of NS-EN ISO/IEC 17025:2000 for
the relevant analytical methods. The laboratory's accredited analytical quality for the
chlorinated and brominated compounds has been approved in several international
intercalibration tests.
The 23 PCB congeners analyzed represented both lower- and higher chlorinated PCBs. Some
PCBs previously shown to contribute very little to ∑PCBs in seabirds (i.e. PCB-31, -87, -136,
-110, -151, -132, -141, -199, -206, -209) (Murvoll 1996) were not included. In addition, the
choice of PCB congeners was based on the configuration of the compounds to include the
most persistent ones (i.e. PCB-153, -138, -128, -180, -170) (Boon et al 1997, see 4.2.3.1) and
inducers of the Ah receptor (e.g. PCB-105, -118, -156, -157) (Boon et al 1992, see 1.3). The
chosen OCPs were included to represent the most abundant OCPs in marine biota (AMAP
1998). Furthermore, the 6 PBDE congeners analyzed represented both lower- and higher
brominated PBDEs. The chosen PBDEs were also believed to be the most abundant ones
based on information from available studies on seabirds in 2002 (Lundstedt-Enkel et al 2001).
3.3 Analysis of vitamins The analyses of vitamins were carried out at Department of Biology, Norwegian University of
Science and Technology (NTNU) as described in
Paper I. In short, the vitamins were
extracted with hexane, concentrated by evaporation under pure N2 and dissolved in methanol (mobile phase). Before evaporation, liver samples also had to be sonicated to break the cells
and to facilitate the extraction of vitamins. The extracts were then injected to a high-
performance liquid chromatograph (HPLC) with a fluorescence detector (
Paper I).
The vitamins were chosen due to the previously documented negative relationships between
retinol and OCs in birds and between tocopherol and PCBs in vertebrates (see 1.7). Plasma
and liver are most frequently used as matrices for analysis of these lipid-soluble vitamins. In
addition to retinol, levels of retinyl palmitate were measured due to the possible mobilization
of retinol from retinyl palmitate in the liver in response to POP exposure (Murk et al 1991,
Murk et al 1994).
3.4 Statistical analysis To investigate possible relationships between POP concentrations and vitamin levels,
univariate (
Paper I, IV) and multivariate (
Paper II, III) regression tests were used. In
addition to possible relationships between POP levels and vitamins, it was also statistically
explored if there were any correlations between POPs and morphological variables.
4. DISCUSSION
4.1 The choice of seabird species
The seabird species (i.e. shag, kittiwake, Brünnich's guillemot, common eider) included in the
present study were chosen since they represented different trophic levels and belonged to
different developmental categories along the altricial-precocial spectrum for birds. Common
eider, feeding on benthic organisms (Dahl et al 2003), occupies a lower trophic position
compared to Brünnich's guillemot, primarily feeding on crustaceans and fish (Borgå et al
2001), and kittiwakes and shags, feeding predominately on fish (Barrett et al 1990, Borgå et al
2001). According to the altricial-precocial spectrum, shag represented the altricial species,
kittiwake and Brünnich's guillemot represented the semi-precocial species and common eider
(and domestic duck) represented the precocial species (Starck and Rickleffs 1998).
The species of the present thesis have also been used in previous studies on POP levels
(Barrett et al 1996, Murvoll et al 1999, Franson et al 2004), which could be interesting in the
light of temporal trends. Moreover, samples from kittiwake hatchlings from both the coast of
Norway and from Svalbard were of special usefulness in the comparisons of POP levels
between Norway and Svalbard (Arctic) (i.e. spatial differences) due to the reduction of
confounding factors when using the same species in accordance to the same methodology and
analytical procedures. Also possible differences in responses to POP exposure within a
species could be revealed when studying kittiwakes from the two populations. Furthermore,
ecological monitoring of some of the colonies from where the hatchling samples were taken
provided opportunities of linking observed effects at lower biological levels to those observed
at higher biological levels.
4.2 Levels of POPs in hatchlings of the present study
4.2.1 Levels of POPs The concentrations of POPs in different seabirds vary due to several factors. Diet and trophic
position is the dominant factor influencing concentrations of hydrophobic and recalcitrant
compounds (Borgå et al 2004). However, differing metabolizing capacity between species is
also of importance (Borlakoglu et al 1990, Fisk et al 2001a), in addition to age, (Donaldson et
al 1997), condition and reproductive status (Henriksen et al 1996) and migration pattern
(Buckman et al 2004). Newly hatched chicks reflect the avian mothers' contamination,
although some elimination could happen as the metabolizing capacity increases during
embryonic development (Lorenzen et al 1997, Hoffman et al 1998).
In general, kittiwake hatchlings had significant higher levels of POPs in comparison to
European shag, Brünnich's guillemot and common eider hatchlings. Further, all POP levels
were significantly higher in shag hatchlings compared to Brünnich's guillemot and common
eider hatchlings (p<0.00 for all POPs). There were also significant differences in POP levels
between Brünnich's guillemot and common eider hatchlings (
Paper IV). Figure 10 A-C
shows the POP levels of the seabird hatchlings of the present thesis.
As stated above, several factors influence differences in POP levels between different species
and different locations. Kittiwakes were higher contaminated by most POPs compared to
other species, despite that they occupy a fairly similar intermediate trophic position as
European shags (Nils Røv pers comm.) and Brünnich's guillemots (AMAP 1998, Hop et al
2002). In addition to the influence of minor discrepancies in trophic positions, the higher POP
concentrations in kittiwakes could be due to the fact that kittiwakes migrate widely and may
thus ingest contaminants outside the study area (Barrett et al 1996,
Paper III). Differences in
metabolizing capacities between the species could also affect POP levels. However,
kittiwakes are believed to have a higher metabolic activity and excretion efficiency than
Brünnich's guillemots (Borgå et al 2001), which make it reasonable to assume that higher rate
of food intake (Ellis and Gabrielsen 2002) and diet are more important than elimination
ability in explaining high POP levels of kittiwakes. Higher levels of POPs in kittiwakes than
in Brünnich's guillemots from the Arctic environment is in accordance to previous studies
(Borgå et al 2001, Hop et al 2002).
Some exceptions from the general trend of kittiwakes having the highest POP levels were
found: The kittiwakes from Runde and shags from Sklinna seemed to have ∑PCBs and HCB
levels of same magnitude (∑PCBs: p=0.39, HCB: p=0.47), whereas the levels of
p,p'-DDE
and HBCD were higher in shag hatchlings than in kittiwake hatchlings from both Runde and
Kongsfjorden (p<0.04). The PCBs have been prohibited in most industrial countries for
decades, and local sources and input are at present minimized, although some leakage from
Figure 10:
A. Concentrations of ∑PCBs in kittiwake (
Rissa tridactyla) hatchlings from Runde (
n=19) and Kongsfjorden (
n=18), in shag
(
Phalacrocorax aristotelis) hatchlings from Sklinna (
n=30), in Brünnich's guillemot (
Uria lomvia) hatchlings from
Kongsfjorden (
n=9) and in common eider (
Somateria mollissima)
hatchlings from Kongsfjorden (
n=14). In shag hatchlings,
12 individuals had not detectable levels of PCB-52. In common eider hatchlings, no individuals had detectable levels of PCB-
52, -47 and -114. One common eider hatchling had not detectable levels of PCB-157.
B. Concentrations of ∑PBDEs and HBCD in kittiwake (
Rissa tridactyla) hatchlings from Runde (
n=19) and Kongsfjorden
(
n=18), in shag (
Phalacrocorax aristotelis) hatchlings from Sklinna (
n=30), in Brünnich's guillemot (
Uria lomvia) hatchlings
from Kongsfjorden (
n=9) and in common eider (
Somateria mollissima) hatchlings from Kongsfjorden (
n=14). In shag
hatchlings, PBDE-28 was not detected in two individuals. In common eider hatchlings, PBDE-28 was not detected in any
individuals, and PBDE-47, -99, -100, -153, -154 were detected in three to eleven hatchlings. HBCD was detected in only on
hatchling of common eider.
C. Concentrations of OCPs in kittiwake (
Rissa tridactyla) hatchlings from Runde (
n=19) and Kongsfjorden (
n=18), in shag
(
Phalacrocorax aristotelis) hatchlings from Sklinna (
n=30), in Brünnich's guillemot (
Uria lomvia) hatchlings from
Kongsfjorden (
n=9) and in common eider (
Somateria mollissima) hatchlings from Kongsfjorden (
n=14).
products and sediments still lead to environmental releases (AMAP 1998). Hence, migration
of kittiwakes to potential higher contaminated area may not be of same importance as earlier
with regard to accumulation of PCB levels. Thus, it is possible that the levels of PCBs in the
hatchlings now to a greater extent reflect local contamination on the Norwegian coast. Since
shags from Sklinna actually reside not far from Runde during winter time (Røv 1991), this
could explain the fairly similar levels of PCBs to kittiwakes from Runde. The higher levels of
p,p'-DDE in shag hatchlings compared to kittiwake hatchlings may also be explained by the
assumption that OC levels in birds along the coast of Norway reflect the local contamination.
Hence, differences in food choice, in addition to between-species differences in elimination
properties, are probable causes of the observed higher levels of
p,p'-DDE in shag hatchlings.
With respect to the high levels of HBCD in shag hatchlings, this could be due to local sources,
although no survey with regard to BFRs is yet conducted nearby Sklinna where shags breed.
However, a national survey of brominated compounds in sediments and mussels has revealed
very high levels of both HBCD and PBDEs in Åsefjorden, approximately 40 km from Runde
(Fjeld et al 2004,
Paper III). The high levels are believed to be due to local discharges. Since
shags from Sklinna reside not far from Runde during winter time (Røv 1991), it may be
speculated that the local source in Åsefjorden contribute to the high HBCD levels observed in
the shag hatchlings. In addition, species-dependent differences in uptake and/or metabolism of
this congener and different food choice will influence the levels of HBCD.
It should be noted that shags are lean bird species, having a very low lipid content (6.84 %)
compared to the other species (kittiwakes: 17.5-19.8 %, Brünnich's guillemots: 28.1 %,
common eiders: 20.9 %). This will make lipid weight concentrations relatively high compared
to wet weight concentrations in shags. Being an altricial species (Starck and Rickleffs 1998),
shag hatchlings may also metabolize POPs slower than semi-precocial and precocial species.
This could be of relevance in explaining the relatively high levels of POPs in shag hatchlings
when taking into consideration that the adult shag mothers reside along the coast of Norway
during the year (Røv 1991).
Kittiwake hatchlings from Kongsfjorden had significantly higher levels of ∑PCB and OCPs
than those from Runde (
Paper III). A possible explanation to this could be that the expected
decrease of PCB and OCP concentrations in biota, following the regulatory restrictions on the
use of these compounds, is slower in Arctic regions than in more temperate regions because
the Arctic region serves as sinks for some POPs (Wania and Mackay 1993). This could
probably also be of importance in explaining the similar levels of
p,p'-DDE in Brünnich's
guillemot hatchlings to kittiwake hatchlings from Runde (p=0.84), being significantly higher
contaminated by POPs in general. Although not discussed in
Paper III because of high
degree of uncertainty, another cause for the differences observed between kittiwakes from
Kongsfjorden and Runde could be related to energy expenditure of the adult female prior to
egg-laying in the breeding season or during the winter. Values of field metabolic rates (FMR)
of chick rearing kittiwakes in Kongsfjorden (Fyhn et al 2001) and on Runde (Gangås 1994)
indicate higher energy expenditure in kittiwakes from Kongsfjorden. Thus a higher rate of
food intake and bioaccumulation of PCBs and OCPs could be expected in Arctic kittiwakes.
This contrasts, however, to the conclusion by Golet et al (2000) that energy expenditure in
breeding kittiwakes is relatively invariant between populations from different environmental
conditions. It is very difficult to assess possible differences in energy expenditure or food
intake during winter between the two study populations of kittiwakes because specific
wintering areas are not known. However, there is a tendency for birds breeding in northern
colonies to migrate further north and east than those breeding in southern colonies (Anker-
Nilssen et al 2000). If this applies to the populations in question, on one hand it could imply
higher winter energy expenditure in the kittiwakes from Kongsfjorden (due to higher costs of
thermoregulation), but on the other hand it could imply that kittiwakes from Runde feed on
more contaminated food during winter. Hence, different POP accumulation because of
possible disparities in energy expenditure and food intake between the two populations of
kittiwakes can not yet be concluded.
In contrast to what was found for OC compounds, kittiwake hatchlings from Runde had
higher levels of HBCD than those from Kongsfjorden (
Paper III). It is possible that this
could be explained by a local source on the coast (Åsefjorden) close to Runde, identified in a
national survey of brominated compounds (Fjeld et al 2004,
Paper III).
The shag hatchlings had higher levels of POPs than Brünnich's guillemot and common eider.
None of these species migrate widely, but reside along the coast of Norway and in Nordic
waters during the years (Røv 1991, Anker-Nilssen 2000, Borgå et al 2005). Only minor
influence of migration on the POP burdens in these species should thus be expected due to
their resident behaviour. From the species' breeding sites, it could probably also be expected
that shags from the coast of Norway had higher levels of POPs than Brünnich's guillemots
and common eiders from the remote Arctic. In addition, common eider feeds on benthic
organisms (Dahl et al 2003,
Paper IV) and occupies a lower trophic position compared to
Brünnich's guillemot, primarily feeding on crustaceans and fish (Borgå et al 2001), and shag,
feeding on predominately on fish (Barrett et al 1990). In addition, common eider hatchlings
are precocial (Starck and Rickleffs 1998) and thus have a higher metabolic rate, indicating a
potential of faster elimination of POPs compared to semi-precocial Brünnich's guillemot
hatchlings and altricial shag hatchlings. POP level differences between shag and Brünnich's
guillemot hatchlings could also be explained by different food choice and disparities with
regard to metabolic activity. Being a semi-precocial species (Starck and Rickleffs 1998),
Brünnich's guillemot hatchlings might metabolize POPs faster than altricial shag hatchlings
4.2.2 Levels of POPs in comparison to other studies It should be noted that comparison of contamination levels between studies often is difficult
due to differences in matrix analyzed and in the analytical procedures. Nevertheless,
comparisons could give valuable information on spatial and temporal trends of POP levels. In
comparison with previous studies on PCB and OCP levels, kittiwake and shag hatchlings
herein had similar, or somewhat higher, levels than reported earlier along the Norwegian coast
and at Svalbard (Barrett et al 1996,
Paper II-III). Hatchlings of Brünnich's guillemots had
lower and corresponding levels of PCB and OCPs, respectively, compared to earlier reports
(Barrett et al 1996,
Paper IV). Based on other studies of temporal trends of PCBs in seabirds
(Bignert et al 1998) the PCB concentrations from 1990s until early 2000s should be expected
to be decreasing. As discussed in
Paper III, the lack of an obvious decline in OC levels from
the 1990s in some of the species included in the present study, despite the regulatory
restrictions on the use of the compounds, could be that the expected decline was most evident
during the 1980s and early 1990s, and that PCB and OCP levels will fluctuate around these
relatively low levels in years to come (Barrett et al 1996).
The levels of most OCs were higher in both kittiwake and Brünnich's guillemot hatchlings
than reported in eggs of the same species from the Canadian Arctic (
Paper III-IV). The same
applied to common eider hatchlings and eggs (Franson et al 2004,
Paper IV). The shag
hatchlings seemed to have relatively low levels of OC compounds in comparison with
Phalacrocoracidae species from other European waters, the Great Lakes and Japan (
Paper II,
Kumar et al 2005).
As shown in Table 1, the PBDE levels of the species included in the present study were low
compared to the reported levels in eggs of Great Lakes herring gull (
Larus argentatus)
(Norstrom et al 2002), in eggs of great blue herons (
Ardea herodias) and double-crested
cormorants (
Phalacrocorax auritus) from British Colombia (Elliott et al 2005) and in eggs of
common cormorant (
Phalacrocorax carbo) from Japan (Watanabe et al 2004). However, the
PBDE levels in kittiwake and shag hatchlings were high compared to levels of brominated
compounds reported in eggs of guillemot (
Uria aalge) from the Baltic (Sellström et al 2003),
in eggs of black guillemot (
Cepphus grylle) from the Greenland (Vorkamp et al 2004) and in
eggs of fulmars (
Fulmarus glacialis) from the Faroe Islands (Fängström et al 2005).
Moreover, the HBCD levels were very high in shag hatchlings compared to all other available
studies on HBCD levels in seabirds (
Paper II). In a European scale, the relatively high levels
of BFRs seem to be in contrast to the situation for the OC levels, as the reported PCB levels in
guillemots from the Baltic are much higher than in birds from the Norwegian coast, e.g. shags
(Bignert et al 1998). In addition to migration of kittiwakes to potential higher contaminated
areas, the most obvious explanation of the high BFR levels is linked to local sources (i.e.
Åsefjorden). However, as mentioned above, to our knowledge no survey is conducted nearby
Sklinna where shags breed, and possible local sources remain to be substantiated. Since shags
from Sklinna reside not far from Runde during winter time (Røv 1991), the local source in
Åsefjorden (Fjeld et al 2004), close to Runde, may contribute to the high BFR levels observed
also in the shag hatchlings. Another possible link between the high levels of BFRs in shags
and the local source in Åsefjorden could be the Norwegian spring-spawning herring (
Clupea
harengus). This herring spawn at the coastal banks off Møre where Åsefjorden is situated, and
the juveniles migrate northward to the Barents Sea to feed. A minor part of the juveniles also
feed in the coastal areas from Trøndelag to Finnmark, while the adult stock mainly migrates
westward to feed (Sissener and Bjørndal 2005). Hence, herring in the Møre area could
accumulate BFRs and transfer the pollution to shags, breeding at the coast of Trøndelag
(Sklinna), where the herring migrates to feed.
Table 1: Levels of polybrominated diphenyl ethers (PBDEs) in different species of seabirds. ww; wet weight
concentrations, lw; lipid weight concentrations.
Species Matrix
Location
∑PBDEs ww ∑PBDEs lw PBDE-47
References
0.71 ng/g lw Present thesis
Sellström et al 2003
Double-crested Egg
Footnotes: 1=∑PBDE
6, 2=∑PBDE9, =∑PBDE7, = ∑PBDE20, = ∑PBDE18, 6= ∑PBDE5
4.2.3 Pattern of POPs
4.2.3.1 PCBs In all species included in the study, PCB-153 was the most abundant congener, which could
be expected from the persistency towards biodegradation of this congener (Boon et al 1997).
The pattern of PCBs in shag (
Paper II) seemed to be in accordance to earlier reports. Also the
PCB pattern observed in both populations of kittiwakes (PCB-153 > PCB-138 > PCB-180 >
PCB-118), Brünnich's guillemot (PCB-153 > PCB-138 > PCB-118 > PCB-99) and common
eider (PCB-153 > PCB-138 > PCB-180 > PCB-118) hatchlings was in fairly good accordance
with the pattern reported in previous studies from Norway and Svalbard (Savinova et al 1995,
Henriksen et al 1996, Hop et al 2002, Borgå et al 2005) and from Great Britain (Malcolm et al
Figure 11: Relative patterns of polychlorinated biphenyls in kittiwake (
Rissa tridactyla) hatchlings from Runde (
n=19)
and Kongsfjorden (
n=18), in shag (
Phalacrocorax aristotelis) hatchlings from Sklinna (
n=30), in Brünnich's guillemot (
Uria
lomvia) hatchlings from Kongsfjorden (
n=9) and in common eider (
Somateria mollissima) hatchlings from Kongsfjorden
(
n=14). In shag hatchlings, 12 individuals had not detectable levels of PCB-52. In common eider hatchlings, no individuals
had detectable levels of PCB-52, -47 and -114. One common eider hatchling had not detectable levels of PCB-157. The
trichlorinated biphenyl was PCB-28, the tetrachlorinated biphenyls were PCB-52, -47, -74 and -66, the pentabrominated
biphenyls were PCB-101, -99, -118, -114 and -105, the hexabrominated biphenyls were PCB-149, -153,
-137, -138, -128, -156 and -157, the heptabrominated biphenyls were PCB-187, -183, -180, -170 and -189, the
octabrominated biphenyl was PCB-194.
As shown in Figure 11, the lower chlorinated biphenyl congeners (tri- to penta-) were more
abundant in Brünnich's guillemot and common eider hatchlings compared to kittiwake and
shag hatchlings. An increase of higher chlorinated congeners with trophic position of seabirds
has also been reported by others (Borgå et al 2001). The penta-, hexa- and heptachlorinated
congeners were the dominated PCB congeners in all species. These congeners were also in
higher proportion than other congeners in the commercial mixtures of PCBs and have the
greatest bioaccumulation potential (McFarland and Clarke 1989). Tri- and tetrachlorinated
biphenyl congeners are more readily metabolized, while higher chlorinated congeners are
more tightly bound to sediments and particles and therefore less bioavailable. Once
accumulated in organisms, however, high-chlorinated congeners of PCBs tend to biomagnify
due to their relcalcitrant nature (Philips and Rainbow 1993).
The higher proportions of hexa- to octachlorinated biphenyl congeners in kittiwakes than in
Brünnich's guillemots is in accordance with previous studies (Borgå et al 2001). Several of
these higher chlorinated congeners are persistent (e.g. PCB-153, -138, -180) towards
metabolizing enzymes according to a classification by Boon et al (1997) based on the position
of the chlorine atoms on the biphenyl ring. The most persistent congeners are without vicinal
hydrogen atoms (e.g. PCB-153, -180) or have vicinal hydrogen atoms exclusively in the
ortho- and
meta-positions in combination with two or more
ortho-chloro substitutions (e.g.
(PCB-128, -138, -170) (Boon et al 1997). The higher scores of persistent congeners in
kittiwakes suggest that kittiwakes have higher contaminant metabolic activity than Brünnich's
guillemots (Borgå et al 2001). From this, data from the present study indicate that shags and
kittiwakes have a corresponding metabolic activity towards PCBs, although species-specific
differences with regard to elimination of certain congeners most likely exist. The rate of
metabolism may also differ between shag and kittiwake hatchlings due to their different
classification according to the altricial-precocial spectrum.
Common eiders had a somewhat deviating PCB pattern, as the proportion of hepta- and
octachlorinated congeners were somewhat greater than in kittiwakes and shags, at the same
time as the lower chlorinated congeners were more abundant relative to what was found in
kittiwakes and shags. The special pattern may reflect the lower trophic position of the eiders,
as they are benthic feeders (Dahl et al 2003). Levels of lower chlorinated biphenyls are
expected to be higher in animals at lower trophic levels as these congeners are more readily
metabolized and thus are not biomagnified to the same extent as higher chlorinated congeners
through the food-chain (Philips and Rainbow 1993). The somewhat higher proportion of
hepta- and octachlorinated congeners could reflect a relatively high metabolic activity,
possibly linked to the precocial nature of the common eider hatchlings.
4.2.3.2 OCPs The patterns of OCPs were fairly similar between the different species included in the present
study (Table 2). The patterns seemed to correspond well to earlier reports on OCPs in
kittiwakes (Barrett et al 1996, Braune et al 2001), shags (Murvoll 1996), Brünnich's
guillemots (Hop et al 2002) and common eiders (Savinova et al 1995). β-HCH is the most
biodegradable compound of the OCPs herein, and on this background kittiwake and common
eider hatchlings seemed to be more able to metabolize OCPs than shag and Brünnich's
guillemot hatchlings. The trophic position, food choice and distances to pollution sources will
however also influence the pattern of OCPs. The species' classification according to the
altricial-precocial pattern for birds (metabolic activity) possibly also influences OCP pattern.
Table 2: Gross pattern of organochlorine pesticides in kittiwake (
Rissa tridactyla) hatchlings from Runde (
n=19) and
Kongsfjorden (
n=18), in shag (
Phalacrocorax aristotelis) hatchlings from Sklinna (
n=30), in Brünnich's guillemot (
Uria
lomvia) hatchlings from Kongsfjorden (
n=9) and in common eider (
Somateria mollissima) hatchlings from Kongsfjorden
Gross pattern of organochlorine pesticides
p,p'-DDE > HCB > oxychlordane > mirex > β-HCH
Kittiwake Kongsfjorden
p,p'-DDE > HCB > oxychlordane > mirex > β-HCH
Eur shag Sklinna
p,p'-DDE > HCB > oxychlordane > β-HCH > mirex
Br guillemot Kongsfjorden
p,p'-DDE > HCB > oxychlordane > β-HCH > mirex
Eider Kongsfjorden
p,p'-DDE > HCB > oxychlordane > mirex > β-HCH
4.2.3.3 PBDEs and HBCD The patterns of PBDEs were dominated by PBDE-47. This corresponds to previous reports
that PBDE-47 is one of the most abundant congeners of PBDEs in wildlife and humans
(Darnerud et al 2001, Guvenius and Norén
2001). PBDE-47 is found in high concentrations in
biota due to the fact that PBDE-47 together with PBDE-99 constitutes more than 70 % of the
commercial mixture Penta-BDE (Sjödin et al 1998), which has been one of the most used
commercial products. However, PBDE-47 was not the most abundant compound of the
brominated flame retardants in shag and common eider hatchlings (Table 3). In these species
HBCD was the most abundant BFR. HBCD is also one of the most used BFRs.
Table 3: Pattern of polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecane (HBCD) in
kittiwake (
Rissa tridactyla) hatchlings from Runde (
n=19) and Kongsfjorden (
n=18), in shag (
Phalacrocorax aristotelis)
hatchlings from Sklinna (
n=30), in Brünnich's guillemot (
Uria lomvia) hatchlings from Kongsfjorden (
n=9) and in common
eider (
Somateria mollissima) hatchlings from Kongsfjorden (
n=14). In shag hatchlings, PBDE-28 was not detected in two
individuals. In common eider hatchlings, PBDE-28 was not detected in any individuals, and PBDE-47, -99, -100, -153, -154
were detected in three to eleven hatchlings. HBCD was detected in only on hatchling of common eider.
Pattern of PBDEs and HBCD
PBDE-47 > HBCD > PBDE-99 > PBDE-100
Kittiwake Kongsfjorden
PBDE-47 > HBCD > PBDE-99 > PBDE-100
Eur shag Sklinna
HBCD > PBDE-100 >
PBDE-47 > PBDE-153
Br guillemot Kongsfjorden
PBDE-47 > HBCD > PBDE-99 > PBDE-100
Eider Kongsfjorden
HBCD > PBDE-153 >
PBDE-47 > PBDE-99
It should be noted that very low levels of all the PBDE congeners were detected in common
eider hatchlings, and all the congeners were detected only in some (or none) of the specimens.
Further, HBCD was detected only in one individual. Thus, too much weight should not be
attached to the observed pattern of PBDEs in common eider hatchlings.
As shown in Figure 12, the PBDE pattern of shag hatchlings deviated much from the
corresponding pattern of kittiwake and Brünnich's guillemot hatchlings. In shags, PBDE-100
(pentabrominated) and PBDE-153 (hexabrominated) constituted relatively more of ∑PBDE.
Furthermore, in shags HBCD was more abundant than any of the PBDE congeners. The
reasons why the shag hatchlings have a diverging pattern of PBDEs may be due to local
discharges or differences in diet and metabolism (
Paper II).
Figure 12: Relative patterns of polybrominated dipenyl ethers in kittiwake (
Rissa tridactyla) hatchlings from Runde (
n=19)
and Kongsfjorden (
n=18), in shag (
Phalacrocorax aristotelis) hatchlings from Sklinna (
n=30), in Brünnich's guillemot (
Uria
lomvia) hatchlings from Kongsfjorden (
n=9) and in common eider (
Somateria mollissima) hatchlings from Kongsfjorden
(
n=14). In shag hatchlings, PBDE-28 was not detected in two individuals. In common eider hatchlings, PBDE-28 was not
detected in any individuals, and PBDE-47, -99, -100, -153, -154 were detected in three to eleven hatchlings.
The PBDE pattern in shags seemed to deviate some from what has been reported in cormorant
(
Phalacrocorax carbo) livers from England and Wales (PBDE-47 > PBDE-100 > PBDE-99)
(Allchin et al 2000, Law et al 2002), which could indicate different distances from the sources
of PBDEs. In addition,
Phalacrocoracidae species could differ in their species-specific
metabolism and in food-choice. The PBDE pattern of kittiwake hatchlings was similar to that
reported in herring gull (
Larus argentatus) eggs from the Great Lakes (Norstrom et al 2002),
and this may reflects that gull species have similar capacities to metabolize PBDEs.
Brünnich's guillemot hatchlings showed a PBDE pattern deviating somewhat from the pattern
reported in guillemot eggs from the Baltic proper (PBDE-47 > PBDE-100 > PBDE-99)
(Lundstedt-Enkel et al 2001), possibly reflecting differences between alcid species with
regard to metabolism of brominated compounds, although several other factors also will
influence the pattern observed (e.g. local sources, diet).
The pattern of PBDEs was in general dominated by lower (tetra-) brominated compounds in
contrast to the pattern of PCBs, dominated by higher (hexa-) chlorinated compounds (Fig. 2).
These differences most likely reflect the newer input of PBDEs into the environment due to
continuing production and use of these compounds in most of the world, whereas the use of
PCBs have been restricted for decades in most countries. Thus, the most persistent congeners
of PCBs will be most available in the environment and in biota.
4.3 Responses of morphological variables to exposure to POPs No relationships were found between POPs and morphological variables in the experimentally
exposed domestic duck, the free-living kittiwake and common eider hatchlings (
Paper I,
Paper III-IV). In shag hatchlings, head length and tarsus length correlated negatively to
several PCBs, PBDEs, HBCD and some OCPs. However, when the variation explained by
yolk lipid content was removed from the statistical model, no correlations between POPs and
morphology were found (
Paper II), and this finding underlines the importance of considering
possible confounding impacts of lipid content when studying effects of POPs on
morphological variables. Lipids not only accumulate pollutants, but fat reserves are also an
important pool of energy, which influences body mass and body condition (O'Connor 1976,
Blem 1990). Thus, size parameters, such as hatching mass, head size, tarsus length etc. could
be influenced by the amount of lipids allocated into the yolk by the avian mothers.
In Brünnich's guillemot hatchlings, POPs seemed to have a negative influence on morphology
(
Paper IV). In this species, no correlations were found between lipids and morphology, i.e.
lipid content was not confounding the relationships between POPs and morphology. Several
studies on birds have reported effects of PCBs on morphological parameters related to growth
and size (Hoffman et al 1986, van den Berg et al 1994, Champoux et al 2002). The negative
relationships between POPs and growth parameters in birds may be related to the relatively
well documented effects of these compounds on thyroid hormones and retinol in birds (Spear
and Moon 1986, van den Berg et al 1994). Both thyroid hormones and retinol are important
for normal growth and development in birds (Spear et al 1986). In the present study no effect
on retinol was revealed in Brünnich's guillemot hatchlings. Thyroid hormone status could,
however, still be influenced. Dilution of contaminants with growth could also be an
explanation to the negative relationships between POPs and morphological variables
(Champoux et al 2002), as this would result in lower POP levels in larger animals.
The lack of associations between POPs and morphology in most of the species included in the
present study may indicate that levels of chlorinated or brominated contaminants were below
those that induce effects associated with growth in the respective species. Brünnich's
guillemot might be a more responsive species regarding effects of POPs on morphological
4.4 Responses of vitamins to exposure to POPs
4.4.1 Normal temporal variation in vitamin levels In order to reduce the risk of confounding from normal temporal variations in vitamin levels
in hatchlings, a study on normal temporal variation in domestic duck hatchlings were
conducted (
Paper I). The results showed no significant differences in hepatic tocopherol and
retinyl palmitate levels during the first 24 hrs after hatching. However, hepatic retinol levels
were significantly reduced between 3 and 24 hrs after hatching (
Paper I). Nevertheless, the
choice of taking samples from the hatchling within 12 hrs after hatching seemed to be a
reasonable approach to reduce the risk of confounding from normal temporal variations in
vitamin levels. Although similar studies on temporal variation were not conducted in the field
species, the results from the laboratory experiment were considered normative. Thus it was
assumed that the vitamin levels of hatchlings of wild species also showed no significant
differences with respect to vitamin status within the first 12 hrs after hatching.
4.4.2 Vitamin responses
4.4.2.1 Responses of α-tocopherol to POPs In domestic duck and shag hatchlings, negative relationships were revealed between POPs
and liver tocopherol levels (
Paper I, II). In addition, negative associations between some
OCPs and liver tocopherol levels were found in Brünnich's guillemot hatchlings, although the
relationships seemed to be influenced by body mass, which was related to POPs and liver
tocopherol levels in a negative and positive manner, respectively
(Paper IV). Reduced levels
of hepatic tocopherol levels have also been reported in minks, rats and fish species exposed to
PCBs (Palace et al 1996, Käkelä et al 1999, Twaroski et al 2001). However, to my knowledge,
the present study is the first to reveal negative relationships between POPs and tocopherol
levels in birds. Further, to my knowledge, no previous studies have reported on correlations
between OCPs (
Paper IV), or PBDEs (
Paper I, II), and liver tocopherol levels. The results
thus may indicate that tocopherol is a sensitive response to various POPs in birds. Further, the
results emphasize the need of considering possible impact of morphological variables (size)
when studying the effect of POPs on vitamins.
Tocopherol levels are believed to be reduced by exposure to PCBs because of the cellular
oxidative stress initiated by the substances (Saito 1990), which tocopherol tries to counteract.
The oxidative stress is probably linked to the interaction of PCBs with the Ah-receptor and
the activation of CYP 1A enzymes (Toborek et al 1995). The CYP 1A enzymes catalyze
monooxygenation reactions in which one atom of molecular oxygen is incorporated into a
substrate, and if the catalytic cycle is interrupted, oxygen is released as superoxide anion (O -
or hydrogen peroxide (H2O2) (Parkinson 2001), which both are reactive oxygen species (ROS) (Pitot and Dragan 2001). Some PBDEs have also been shown to induce CYP 1A enzymes (e.g.
PBDE-99, correlating negatively to liver tocopherol levels in domestic duck hatchlings,
Paper I) (von Meyerinck et al 1990, Chen et al 2001, Pettersson et al 2001). The
configuration of PBDE-28, which correlated negatively to liver tocopherol levels in shag
hatchlings (
Paper II), indicates a potential for inducing CYP 1A enzymes (
Paper II).
However, an
in vitro study using a rat hepatoma cell line indicated that PBDE-28 was not able
to activate the Ah-receptor (Meerts et al 1998). The congener was shown to be a 2,3,7,8-
tetrachlorodibenzo-p-dioxin (TCDD) antagonist (Meerts et al 1998), although the observed
antagonism may be due to competition between PBDE-28 and TCDD at the Ah-receptor level
(de Wit 2002). Nevertheless, all CYP enzymes catalyze monooxygenation reactions
(Parkinson 2001), and thus induction of other CYP enzymes than CYP1A may also result in
oxidative stress. In a study on neurotoxicity of PCBs in rats' brain, non-dioxinlike PCBs lead
to increase of ROS and induction of cell death (Mariussen et al 2002), which indicates that not
only the Ah-receptor (dioxin receptor) is important for generating oxidative stress in
organisms. Also OCPs induce CYP 2B enzymes (Parkinson 2001) and hence potentially
oxidative stress, which may explain the negative associations between OCPs and liver
tocopherol levels in Brünnich's guillemot hatchlings (
Paper IV). Studies on endosulfan and
dieldrin, which are OCPs not included in the present study, have also shown induction of
oxidative stress (Stevenson et al 1995, Sohn et al 2004). Furthermore, in Brünnich's guillemot
hatchlings, ∑PCBs were not found to affect the levels of tocopherol, despite the potential of
several of the congeners included in ∑PCBs (e.g. PCB-118, -114, -105, -156, -157, -189)
(Boon et al 1992) to induce CYP 1A enzymes. It is therefore possible that the ability to induce
CYP 1A enzymes of Brünnich's guillemots is lower than that of CYP 2B enzymes. Actually,
in a study on PCB pattern and biotransformation in seabirds, Brünnich's guillemots showed a
limited induction of CYP 1A enzymes in response to PCB exposure (Borgå et al 2005). At the
same time, previous studies have suggested elevated biotransformation of OCPs in Brünnich's
guillemots compared to that of other alcid species (Fisk et al 2001b), indicating a higher
activity of CYP 2B enzymes.
In kittiwake hatchlings, a positive influence of PCBs on plasma and liver tocopherol levels
were indicated (
Paper III). Further, in common eider hatchlings positive relationships
between some POPs and tocopherol levels also were revealed (
Paper IV). Positive
relationships between POPs and tocopherol levels contradict the reduced hepatic levels of
tocopherol in response to POP exposure in other studies and in the other birds of the present
study. However, some studies have also documented positive relationships between POPs and
tocopherol levels (Saito 1990, Palace and Brown 1994, Nyman et al 2003). Increased hepatic
tocopherol levels have been linked to PCB-induced fatty liver (Saito 1990), which can be a
toxicological endpoint from exposure to PCBs (Chu et al 1996; 1998). No certain conclusions
regarding fatty liver can, however, be drawn since no histological examination was conducted
of the kittiwake and common eider livers (
Paper III-IV). In common eider hatchlings, the
levels of POPs were probably too low to induce fatty liver. It could, however, be that the
dose-response relationship connected to the effect of POPs on tocopherol is J-shaped
(hormesis) (Calabrese and Baldwin 2003), which means a lower incidence of oxidative stress
at low POP doses followed by an increasing incidence of oxidative stress at higher POP levels.
A J-shaped dose-response curve should on the other hand lead to more oxidative stress, and
depleted tocopherol levels, in kittiwakes. Nevertheless, between-species differences could
exist for the dose-response relationships.
In addition to possible different dose-response relationships, a study on rat (Wistor strain) has
shown that dietary PCBs can increase tissue tocopherol and the absorption of tocopherol from
the intestine (Katayama et al 1991). The increase in tocoherol levels in response to PCB
exposure was probably due to a simultaneously increase in the fraction of high density
lipoprotein (HDL) cholesterol (Katayama et al 1991). Tocopherol is transported in the blood
within plasma lipoproteins and erythrocytes, and thus high correlations exist between
tocopherol concentrations and total lipids or cholesterol (Traber et al 1993). Hence, due to a
PCB-induced increase in the fraction of HDL cholesterol, increased tocopherol levels may
result from PCB exposure. However, since tocopherol is gained from the diet (Sheppard et al
1993), the elevation of tocopherol levels is most likely limited by the tocopherol contents in
the diet. In this respect it should be noted that common eiders feed on benthic organisms
(Dahl et al 2003). It is therefore possible that diet and nutritional contents (i.e. vitamins) of
food items of common eiders influence in a different manner the levels of tocopherol in
hatchlings and thereby the relationships between POPs and tocopherol (
Paper IV).
The (possible) positive influence by PCBs on tocopherol levels in kittiwake and common
eider hatchlings could also reflect other mechanisms of toxic action than in the other species.
Brouwer (1991) found great variations among fish-eating birds with regard to their ability to
induce CYP 1A enzymes and to produce OH-metabolites of PCBs (Ah-responsive and Ah-
non-responsive species). Similar species-dependent differences probably exist for other CYP
enzymes and possible subsequent toxic actions. According to Brouwer (1991), herring gull
was regarded as an Ah-non-responsive species and thereby relatively resistant to some toxic
actions of PCBs. In contrast, cormorants seemed to be Ah-responsive, while common eider
responded by induction of CYP 1A levels but apparently did not form OH-metabolites (Ah-
responsive without formation of some toxic metabolites). On this background, kittiwakes,
being a gull species, may be less responsive to some toxic effects by POPs than other species
such as cormorants and possibly also shags (
Phalacrocoracidae species). Borgå et al (2005),
however, documented CYP 1A activities in kittiwakes, indicative of Ah-responsiveness,
although the CYP 1A activities seemed to be low. Common eiders, forming no OH-
metabolites, may also be less responsive compared to other species, or other mechanisms of
toxic actions initiate different responses in this species.
It should also be mentioned that tocopherol exists in several oxidized forms (due to the
reactions with ROS) (Twaroski et al 2001). Some oxidized forms can be reduced back to
tocopherol by other antioxidants, such as ascorbic acid (Katayama et al 1991). Thus, redox
reaction may also influence (confound) levels of tocopherol levels, especially when the
oxidative stress is low.
The influence on tocopherol levels in the different bird species were caused by different POPs,
e.g. PBDE-99 (
Paper I), PBDE-28 (
Paper II), PCB-74, -66, -118, -153, -105, -138 (
Paper
III) and OCPs (
Paper IV). This may be linked to the bird species' different concentrations of
POPs, to the disparities in metabolic activity and functionality, and to possible differences in
toxic mechanisms and diet.
4.4.2.2 Responses of retinol to POPs In shag hatchlings, several negative correlations were revealed between POPs and plasma
retinol levels (
Paper II). These negative relationships contradicted the borderline significant
positive correlation between PCBs and plasma retinol levels found in shag hatchlings from
Sklinna in a previous study (Murvoll et al 1999). The differences between the studies could be
caused by between-year variations in dietary levels of retinoids or differences in prey species.
It is also possible that recent increase of other pollutants than PCBs, such as PBDEs and
HBCD, have influenced on the vitamin homeostasis in a synergistic manner (
Paper II).
Temporal trends of PBDEs in double-crested cormorants from British Colombia (Elliott et al
2005) and in guillemots from the Baltic (Sellström et al 2003) show a peak during 1990-1995,
followed by decreasing levels. However, Great Lake herring gulls show a continuing increase
in PBDE levels in the period 1981-2000 (Norstrom et al 2002). Thus, an increase of BFRs in
shags from Sklinna from 1995 to 2002 could be real, although not documented due to lack of
Kittiwake hatchlings, having higher levels of POPs in general than shag hatchlings, showed
no signs of interrupted retinol levels in connection with POP exposure. It could be linked to
possible differences in metabolic ability and functionality. As stated above, Brouwer (1991)
found cormorants to be Ah-responsive and to develop effects related to OH-metabolites of
PCBs, in contrast to herring gull being Ah-non-responsive. Reduction of retinol levels by
PCBs has been associated with the formation of OH-metabolites (Brouwer and van den Berg
1986, Brouwer et al 1990), although other toxic mechanisms for retinol reduction also are
proposed (Chen et al 1992). The OH-metabolites have high affinity for TTR, the transport
molecule of thyroid hormones in plasma (Brouwer and van den Berg 1986). In addition,
retinol-RBP binds to TTR. The RBP-TTR complex is vital to prevent filtration of retinol-RBP
through the kidneys (Combs 1992). Binding of OH-metabolites to TTR results in
conformational changes and in destabilization of RBP-TTR complex (Brouwer et al 1990).
Hence, glomerular filtration of retinol-RBP will increase, leading to reduced levels of retinol.
The European shag, being a
Phalacrocoracidae species and thereby possibly Ah-responsive,
may respond to POP exposure by forming OH-metabolites which is linked to reduced levels
of retinol. Thus, because of possible differences in the xenobiotic elimination (e.g. Ah-
responsiveness),
Phalacrocoracidae species might show responses in retinol levels to POP
exposure whereas gull species may not.
4.4.3 Vitamins as potential biomarkers In the present study, tocopherol levels seemed to be affected by POPs in all species included,
although more studies are needed to clarify the effects of POPs on tocopherol in the different
species. Nevertheless, the results indicate a potential of tocopherol as a biomarker for
exposure to organic compounds. Retinol levels were only influenced in shag hatchlings,
which may imply that higher levels of POPs are needed to elicit this response.
The age of the hatchlings (study objects) was standardized to reduce the risk of confounding
due to normal temporal variations in vitamin levels. Possible temporal variations in vitamin
levels due to reproductive status etc. of the avian mothers were avoided when using hatchlings.
In kittiwakes, being differentiated into males and females, vitamin levels were corrected for
sex variation using linear regression residuals. Thus, the present study minimized the number
of possible confounding factors. However, still there is a lack of knowledge regarding vitamin
levels in the diet and how diet of the avian mothers affects vitamin levels in hatchlings. Also
other factors may influence the vitamin concentrations (e.g. the physiology and condition of
the avian mothers). Also POP levels could be influenced by the condition of the avian
mothers (i.e. age, lipid stores, food choice). Limited resources made it impossible to take
samples of the avian mothers and their diet in the present study. Hence, further (and more
extensive) studies should be conducted to investigate closer tocopherol and retinol levels as
possible biomarkers of POPs in birds.
In addition, the confounding by redox cycling could be diminished by measuring also other
forms of tocopherol. Twaroski et al (2001) proposed that tocopheryl quinone, an irreversible
oxidized form of tocopherol (Michal 1999), could be a more sensitive marker of oxidative
stress than tocopherol. In a study on rats given PCBs, tocopheryl quinone levels were elevated
in both short-time and long-time exposure groups and in both gender (due to increased
oxidative stress), whereas tocopherol levels were reduced only in short-time exposure groups
and in males. Actually, efforts were made to detect tocopheryl quinone in the samples of the
present study without succeeding. Due to limited resources these efforts could not be pursued.
4.5 Possible linkages to higher level effects Reduced plasma retinol levels may indicate that growth and development in hatchlings could
be affected, as vitamin A is important for these processes in birds (Spear and Moon 1986).
Tocopherol is essential for normal neurological structure and function (Traber et al 1993). As
a part of the antioxidant defences, tocopherol is also important in reducing the negative effects
of cellular oxidative stress (Saito 1990). Many toxic, carcinogenic and pathological processes
are believed to be partly due to oxidative stress occurring through the generation of reactive
oxygen species (ROS), which react with lipids, proteins and DNA (Smith et al 1995). Thus, if
the cellular antioxidant defences are depleted, this may have consequences such as mutagenic
damage and carcinogenicity.
The population of kittiwakes at Runde has in later years declined significantly. Further
research is needed to completely assess the cause and effect relationship of this decline,
although changes in food access are proposed as possible explanations (Lorentsen 2003).
However, if POP levels are linked to the observed reduction in population size, vitamin levels
may not to be useful biomarkers to reveal such disturbance. Levels of vitamins in the
kittiwake hatchlings seemed to be only minor influenced by POPs, although further studies
should be conducted to provide more data on the issue.
The shag population from Sklinna at present shows no signs of detrimental effects at higher
biological levels, i.e. reproduction disturbances or population decline (Nils Røv pers comm.,
Lorentsen 2003). Hence, the negative relationships between POPs and vitamin levels
observed in shag hatchlings does not seem to be linked to effects at higher biological levels.
The PCB contamination in the shag hatchlings is also much lower than what has been
documented to reduce hatching success and reproduction in
Phalacrocoracidae species
(Tillitt et al 1992, Dirksen et al 1995). Still vitamin levels indicate a potential as useful "early
warners" of POP exposure by responding to the pollutants, and it could be possible that higher
POP levels both relate negatively to retinol and tocopherol levels and show link to effects at
higher biological levels. It should be mentioned that the shag population at Runde has
declined to one fourth in 2003 compared to 1975, although the size of the population has been
relatively stable in the last ten years (Lorentsen 2003). Possible differences between these
populations of shags with regard to POP levels and effects could exist. Several environmental
factors may also interact (food access variation, habitat destruction, climate changes, pollutant
levels), making the situation for each population unique and complex. It could therefore have
been interesting to study also other populations of shags with regard to POP levels and
The populations of the bird species from Kongsfjorden are reported to be reasonably stable
(kittiwake, Brünnich's guillemot, common eider) (Anker-Nilssen et al 2000), despite great
between-year variations with regard to the number of breeding birds (www.miljo.npolar.no).
Recent data also suggest a negative trend for both kittiwake and Brünnich's guillemot
populations from Kongsfjorden (www.miljo.npolar.no/mosj/MOSJ, H. Strøm unpubl results).
It is difficult to assess whether the observed responses in vitamin levels could be linked to the
negative trends in the development of the sea bird populations. On the other hand, the
population of common eider shows a small increase (3-4 %) since the 1980s
(www.miljo.npolar.no/mosj/MOSJ, G.W. Gabrielsen pers comm.). However, it should be
noted that this species was significantly reduced until the early 1990s due to harvesting of
eggs and down, which has been prohibited on Svalbard since 1963 (www.miljo.npolar.no/
mosj/MOSJ). Both Brünnich's guillemots and common eiders are believed to be vulnerable
species towards anthropogenic disturbance such as over-fishing (Brünnich's guillemot) and
increased exploitation of benthic organisms (common eider), and oil spill (Anker-Nilssen et al
2000, www.miljo.npolar.no/mosj/MOSJ). These species also seemed to be responsive to POP
exposure with regard to tocopherol levels, although the response in Brünnich's guillemot
hatchlings was influenced by body mass. It should also be kept in mind that the levels of
POPs in common eiders were very low. The kittiwake seemed to be a less responsive species
towards POPs with regard to influence of vitamins, possibly due to between-species
differences in the metabolic functionality (i.e. Ah-responsiveness, toxic mechanisms, etc.).
Still other physiological variables may be interrupted in kittiwakes. In the Arctic, having low
temperatures, extreme seasonal variations in light and lack of nutrients, it is possible that the
environmental conditions impose an additional stress to the organisms living there, making
them more vulnerable to anthropogenic pollutants such as POPs compared to organisms living
in more temperate or tropical biomes. The relatively high POP levels (in an Arctic scale)
observed in kittiwake hatchlings from Kongsfjorden could thus be of ecotoxicological interest
due to the special environmental conditions and in light of potential additive effects between
chemicals of old origin and of new (and still unknown) sources.
Although the responses in vitamin levels to POP exposure in the seabird species herein can
not clearly be linked to higher levels effects, the responses could be regarded as "early
warners" of effects of POPs in the studied seabird populations, including populations from the
remote Arctic. In light of the precautionary principle of the Rio Declaration of 1992
(www.unep.org), the continuing use and release of brominated flame retardants in several
parts of the world could be worrying and should be considered seriously by supranational
organizations (e.g. United Nations).
4.6 Seabirds as bioindicator species Seabird bioindicator species represent top predators and are thus relevant in environmental
biomonitoring of POP exposure. Hatchlings of seabirds are appropriate bioindicators due to
the opportunities of control of important factors such as age and development. On the other
hand, sampling from hatchlings requires more resources than sampling of eggs. However,
eggs will not give the advantage of several matrices for analysis of both POPs and biological
parameters (i.e. biomarkers) as do hatchlings.
The results from the present study indicate that shag is a responsive species with regard to
POP exposure, and thereby have a potential as a bioindicator species. Shags breed along the
outer part of the whole Norwegian coastline in addition to the coast of several European
countries such as Iceland, the Faeroes, Russia, Great Britain, Ireland, France, Spain, Italy and
Greece (Snow and Perrins 1998). Thus, shags could be monitored in several countries. In
some colonies, shags can be difficult to get because they breed in-between cliffs. However,
because they breed on the ground, samples of eggs, hatchlings or adults are relatively easy to
collect in most locations. Brünnich's guillemots also seemed to be responsive to POP
exposure, although the relationships between OCPs and liver tocopherol levels were less
strong when the variation explained by body mass was accounted for. The species could be
useful as an indicator species of the Arctic region, as Brünnich's guillemots breed only in the
Arctic region (Snow and Perrins 1998). It is also regarded as a potential indicator of the
productivity of the Arctic ecosystem by The Norwegian Polar Institute (www.miljo.npolar.no/
mosj/MOSJ). Common eider hatchlings responded to POP exposure by elevated levels of
liver tocopherol. Common eiders breed in the Arctic region and along the coast of the Nordic
countries, Russia, Great Britain, Ireland, the Netherlands, and Germany (Snow and Perrins
1998). Still common eiders accumulate very low levels of POPs and are probably influenced
by most POPs to a minor degree.
Kittiwakes accumulate rather high levels of POPs and thus show a potential as an indicator
species. However, vitamin levels seem not to be useful biomarkers of POP exposure and
effects in kittiwakes, although further studies need to be conducted before certain conclusions
are drawn. Other physiological variables may be influenced and could be possible biomarkers.
Kittiwakes breed mainly on low- and high-arctic coasts (i.e. Spitsbergen, the coast of Norway,
Iceland, the Faeroes, Russia), but also to a minor extent in several other countries (Great
Britain, Ireland, Germany, France, Spain) (Snow and Perrins 1998), allowing widespread
monitoring. The kittiwakes breed in steep cliffs and mountains, and sampling may be
challenging. Due to migration kittiwakes will not reflect POP levels from breeding regions
only, and hence the species is not as useful as more resident species regarding monitoring of
regional pollutant levels. Nevertheless, possible responses of kittiwakes to POP exposure
could be of importance in the assessment of effects at higher biological levels, including
The Norwegian and Svalbard coast are regarded relatively clean due to the pristine
environment with low population density and industrial activity. In a broader sense, POP
levels and biomarker responses in Norwegian seabirds could contribute with data from the
lower exposure range in the environment. However, in a European scale, PBDE and HBCD
levels were relatively high in some of the species included in the present study, implying the
difficulties in making general conclusions. The present study thus implies the importance of
follow-up surveys, especially with respect to BFRs.
5. CONCLUDING REMARKS AND FUTURE PERSPECTIVES
This study provided up-to-date data on POP concentrations and vitamin levels in seabirds
from the coast of Norway and from Kongsfjorden, Svalbard. In short, levels of OC
compounds in the hatchlings seemed to be higher than reported in sea bird eggs from the
Canadian Arctic but lower than reported in eggs of other seabirds from the Netherlands, the
Baltic, the Great Lakes and Japan. In contrast to this, the levels of PBDEs and HBCD seemed
to be high in some of the species (kittiwakes, shags) relative to a European scale. With regard
to national temporal trends, the study gave no indications on a further decline in OC levels
compared to the 1990s (Barrett et al 1996), probably due to fluctuations around relatively low
levels following regulatory restrictions on the use of OC compounds decades ago. Due to the
lack of temporal data regarding BFRs, and the relatively high levels documented herein, it
would be recommended to be attentive in this respect in years to come.
There were significant differences in POP levels between the sea bird species included in the
study. In general, kittiwake hatchlings had higher levels of POPs than the other species,
followed by shag, Brünnich's guillemot and common eider hatchlings. The differences could
be explained by trophic position, diet and rate of food intake (i.e. metabolic activity), in
addition to migration pattern, to breeding sites (Norway vs. Svalbard) and possibly also to the
species' different classification in accordance to the altricial-precocial spectrum for birds.
Negative relationships between morphological variables and POP levels were revealed in
Brünnich's guillemot hatchlings. This species is probably more responsive with regard to the
effects of POPs on morphological variables than the other species included in the present
study. In shag hatchlings, the importance of considering possible confounding impacts of lipid
content when studying effects of POPs on morphological variables, was emphasized.
The study revealed interesting relationships between POPs and liver tocopherol levels (
Paper
I-IV). In domestic duck, a negative relationship between PBDE-99 and liver tocopherol levels
was revealed under controlled laboratory conditions. Further, in free-living shag hatchlings,
negative relationships between POPs and liver tocopherol levels were found. In addition, liver
tocopherol levels were negatively associated with POPs in Brünnich's guillemot hatchlings,
although the relationships seemed to be influenced by variation in body mass. However, in
kittiwake hatchlings, there seemed to be a possible positive influence by POPs on tocopherol
levels. In common eider hatchlings, positive relationships between POPs and tocopherol
levels also were revealed. Despite the apparently opposite effects of POPs on tocopherol
levels in different bird species, an influence of liver tocopherol was a consistent trend in these
species, representing different trophic positions and types of birds according to the precocial-
altricial spectrum. The species' responses probably reflect differences with regard to dose-
response relationships, toxic mechanisms and trophic position (diet). The results should
encourage further research on the effects of POPs on tocopherol levels.
In shag hatchlings, a negative relationship between POPs and plasma retinol levels were
observed (
Paper II), in line with several previous studies on birds (Greichus and Hannon
1973, Spear and Moon 1986, Champoux et al 2002). Since the same correlation was not found
in any other species included in the study, tocopherol levels might be more responsive than
retinol levels to POP exposure. Further studies should, however, be conducted before firm
conclusions are drawn.
Concerning the work needed for further development of vitamins as biomarkers of POP, it
should be kept in mind that still there is a lack of knowledge with regard to the natural
physiological events that can alter vitamin concentrations. Hence, efforts should be made to
characterize confounding factors, such as diet and condition of the avian mothers, for the
further validation of retinol and tocopherol as biomarkers. In addition to tocopherol levels,
measurements of the oxidized form tocopheryl quinone could be useful.
Although the observed responses of vitamins to POP exposure were difficult to link to effects
at higher biological levels, the relevance of vitamins as potential biomarkers of POP exposure
should not be repelled. Further studies should be conducted on birds to investigate closer the
vitamins as possible biomarkers. Other possible physiological variables may also have
potential as biomarkers. To ease the work and limit the need of resources, a method for
analyzing vitamins in yolks of eggs (in addition to POPs) should be encouraged.
Relatively high levels of BFRs, possibly increasing, were documented in the present study.
The knowledge of the distribution and effects of other chemicals than POPs, such as
perfluorooctane sulfonate (PFOS), is however scarce. PFOS has also been shown to increase
oxidative damage in fish (Oakes et al 2005), and due to the potential synergistic action of
chemicals, severe oxidative damage could occur earlier than expected from the concentrations
of one group of chemicals only. Associations between vitamin levels and POPs could thus be
early warners of negative impact on biological systems (i.e. populations or ecosystems),
although not yet demonstrated.
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The Stromal Chloroplast Deg7 Protease Participates inthe Repair of Photosystem II after Photoinhibitionin Arabidopsis1[W][OA] Xuwu Sun, Tingjiao Fu, Ning Chen, Jinkui Guo, Jinfang Ma, Meijuan Zou, Congming Lu, and Lixin Zhang* Photosynthesis Research Center, Key Laboratory of Photobiology, Institute of Botany, Chinese Academy ofSciences, Beijing 100093, China Light is the ultimate source of energy for photosynthesis; however, excessive light leads to photooxidative damage and hencereduced photosynthetic efficiency, especially when combined with other abiotic stresses. Although the photosystem II (PSII)reaction center D1 protein is the primary target of photooxidative damage, other PSII core proteins are also damaged anddegraded. However, it is still largely unknown whether degradation of D1 and other PSII proteins involves previouslyuncharacterized proteases. Here, we show that Deg7 is peripherally associated with the stromal side of the thylakoidmembranes and that Deg7 interacts directly with PSII. Our results show that Deg7 is involved in the primary cleavage ofphotodamaged D1, D2, CP47, and CP43 and that this activity is essential for its function in PSII repair. The double mutants deg5deg7 and deg8 deg7 showed no obvious phenotypic differences under normal growth conditions, but additive effects wereobserved under high light. These results suggest that Deg proteases on both the stromal and luminal sides of the thylakoidmembranes are important for the efficient PSII repair in Arabidopsis (Arabidopsis thaliana).
Induction of Diploid Eggs With Colchicine During Embryo Sac Development in Populus By J. WANG1),2), X. Y. KANG1),2),*), D. L. LI2), H. W. CHEN2) and P. D. ZHANG1),2) (Received 22nd August 2009) induction of 2n pollen, due to easy screening by their Diploid (2n) eggs were induced by treating developing size. JOHNSSON and EKLUNDH (1940) first induced 2n
