Comparative evaluation of the impacts of anthropogenic pollutants on the health status of key species in the Gulf of Bothnia (Halichoerus
grypus and Pusa hispida botnica)
INAUGURAL DOCTORAL THESIS
in partial fulfilment of the requirements of the degree of Doctor of Natural Sciences
- Doctor rerum naturalium – (Dr. rer. nat.)
submitted by
Britta Schmidt (née. Müller) Glandorf
Büsum 2020
University of Veterinary Medicine Hannover, Foundation
Prof. Dr. Maren von Köckritz-Blickwede Institute for Physiological Chemistry,
University of Veterinary Medicine Hannover, Foundation First Supervisors: Prof. Prof. h. c. Dr. Ursula Siebert
Institute for Terrestrial and Aquatic Wildlife Research, University of Veterinary Medicine Hannover, Foundation Prof. Dr. Maren von Köckritz-Blickwede
Institute for Physiological Chemistry,
University of Veterinary Medicine Hannover, Foundation
Second Supervisor: Prof. Dr. Marion Schmicke
Institute for Agricultural and Food Sciences, Martin-Luther-University, Halle-Wittenberg
Date of oral examination: 11.11.2020
Amling, M., Siebert, U. (2020). Variation in skull bone mineral density of ringed seals (Phoca hispida) from the Gulf of Bothnia and West Greenland between 1829 and 2019.
Environmental International Special Issue 143, 105968.
https://doi.org/10.1016/j.envint.2020.105968
Schmidt, B., Sonne, C., Nachtsheim, D., Wohlsein, P., Persson, S., Dietz, R., Siebert, U.
(2020). Liver histopathology of Baltic grey seals (Halichoerus grypus) over three decades. Environmental International Special Issue 145, 106110.
https://doi.org/10.1016/j.envint.2020.106110
Parts of this study are in preparation for a publication in a peer-reviewed journal:
Schmidt, B., Hollenbach, J., Mühlfeld, C., Pfarrer, C., Persson, S., Kauhala, K., Siebert, U.
(in prep.). Number of primordial follicles in juvenile ringed seals (Phoca hispida) from the Gulf of Bothnia and West Greenland.
Results of this study were presented on the following conferences:
Schmidt, B., Sonne, C., Nachtsheim, D., Dietz, R., Persson, S., Oheim, R., Rolvien, T., Amling, M., Siebert, U. (2020). Variation in skull bone mineral density of ringed seals (Phoca hispida) from the Gulf of Bothnia and West Greenland between 1829 and 2019.
In: Poster presentation, abstract book. World Marine Mammal Conference, Barcelona, Spain, 7th – 12th of December 2019.
Results of this study were presented on the following meetings:
Schmidt, B., Sonne, C., Nachtsheim, D., Dietz, R., Persson, S., Oheim, R., Rolvien, T., Amling, M., Siebert, U. (2020). Variation in skull bone mineral density of ringed seals (Phoca hispida) from the Gulf of Bothnia and West Greenland between 1829 and 2019.
In: Oral talk, BaltHealth Project Meeting, Turku, Finland, 2nd – 4th of March 2019.
For Patrick & My Parents
with love and gratefulness
List of Abbreviations ...11
Introduction ...15
Chapter 1 Variation in skull bone mineral density of ringed seals (Phoca hispida) from the Gulf of Bothnia and West Greenland between 1829 and 2019 ...23
Abstract ...28
1 Introduction ...29
2 Materials and Methods ...30
2.1 Samples ...30
2.2 Identification of sex ...33
2.3 Bone mineral density (BMD) ...36
2.4 Statistical analyses ...37
3 Results ...39
3.1 Gulf of Bothnia ...39
3.1.1 Sex ...39
3.1.2 BMD and sex differences ...39
3.1.3 Time trends ...40
3.1.4 BMD vs. contaminants ...41
3.2 Greenland ...41
3.2.1 BMD and age group/sex differences ...41
3.2.2 Time trends ...42
3.3 Greenland vs. Gulf of Bothnia ...42
3.3.1 BMD and country differences ...42
3.3.2 Time trends ...43
4 Discussion ...44
4.1 Identification of sex ...44
4.2 Bone mineral density of skulls from ringed seals ...44
4.3 Period differences and time trends in skull BMD ...45
4.4 Contaminants and effects ...45
Conclusions...46
Acknowledgement ... 47
Appendix ... 48
Chapter 2 Liver histopathology of Baltic grey seals (Halichoerus grypus) over three decades ... 51
Abstract ... 56
1 Introduction ... 57
2 Materials and Methods ... 58
2.1 Samples ... 58
2.2 Age determination ... 59
2.3 Histology ... 60
2.4 PCB and DDT analyses ... 61
2.5 Statistical analyses ... 62
3 Results ... 62
3.1 Liver changes vs. contaminants ... 68
4 Discussion ... 70
4.1 Temporal trends... 71
4.2 Contaminant exposure ... 71
Conclusions ... 72
Acknowledgement ... 72
Appendix ... 74
Chapter 3 Number of primordial follicles in juvenile ringed seals (Phoca hispida) from the Gulf of Bothnia and West Greenland ... 81
Abstract ... 86
1 Introduction ... 87
2 Materials and Methods ... 88
2.1 Samples ... 88
2.2 Histology preparation ... 89
2.3 Stereology - Total primordial follicle number ... 90
2.4 Statistical analyses ... 92
3 Results ... 93
3.1 Different follicle and ovary volumes (V(ovary), Vv(follicle/ovary), V(follicle/ovary), V (follicle)) ... 97
3.4 Number of primordial follicle and differences between countries ...99
4 Discussion ... 100
4.1 Identification of primordial follicle number ... 100
4.2 Country differences with regard to primordial follicle ... 102
4.3 Volume differences ... 102
Conclusion ... 103
Acknowledgement ... 103
Appendix ... 105
Overall Discussion... 109
Summary ... 117
Zusammenfassung ... 119
Bibliography ... 121
List of figures ... 141
List of tables... 144
Acknowledgement ... 147
AIC Akaike information criterion
BMD Bone mineral density in (g/cm2)
CBL Condylobasal length
C-M2 Length from the anterior of the alveole of
the canine, to the posterior of the alveole of the 2nd molar
CWN Glomerular capillary wall nodules
CWT Glomerular capillary wall thickening
CYP Hemoprotein cytochrome P
DDT Dichlorodiphenyl trichloroethane
DXA Dual-energy x-ray absorptiometry
FFH Flora-Fauna-Habitat Directive
GAM Generalized additive model
GC Gas chromatography
GLM Binomial generalized linear model
HE Hematoxylin-Eosin
HELCOM Helsinki-Commission
IUCN International Union for Conservation of
Nature
I1-M2 Length from the anterior of the alveole of
the 1st incisor, to the posterior of the alveole of the 2nd molar
LM Linear regression analysis/model
LUKE Natural Research Institute in Finland
OCs Organochlorines
OHCs Organohalogen contaminants
OPF Maximal distance between the
opistocranion and the postorbital process of the frontal bone
P4-M2 Length from the anterior margin of the 4th
premolar to the posterior margin of the 2nd molar
PBB Polybrominated bipehnyls
PCB Polychlorinated biphenyls
POH Postorbital height
POPs Persistent Organic Pollutants
SMNH Swedish Museum of Natural History
„Just because you can’t see something, it doesn’t mean it isn’t there”
- Willard Wigan
Introduction
The most common marine mammals in the Baltic Sea are grey seals (Halichoerus grypus) and ringed seals (Phoca hispida/ Pusa hispida), which can be mainly found in the Gulf of Bothnia. These two species are the only larger predators in the local ecosystem. The harbour porpoises (Phocoena phocoena) and harbour seals (Phoca vitulina) can also be found in the Baltic Sea with lower population numbers. Ringed seals occur in the northern hemisphere and a subspecies is located in the Baltic Sea, which is called Pusa hispida botnica (HÄRKÖNEN AND HEIDE-JØRGENSEN 1990; KINGSLEY AND BYERS 1998; REEVES; RIEDMAN 1990). Being the seal species that is most strongly ice-associated, ringed seals inhabit arctic regions (KELLY ET AL.2010). In the Baltic Sea, they occur in the Gulf of Finland, the Gulf of Riga and the Gulf of Bothnia, where the greatest concentration of ringed seals can be found (HÄRKÖNEN 1992). Grey seals occur throughout the North Atlantic and are also found in the Baltic Sea. Three different stocks are known: the Western Atlantic stock, the Eastern Atlantic stock and the Baltic stock, which is considered as the sub-species Halichoerus grypus balticus (SMITH 1966). Based on the Helsinki Commission (HELCOM), the Baltic Sea can be divided in 17 areas (Figure 1, HELCOM2018A). In the present study, the areas Bothnian Bay, The Quark and Bothnian Sea are summarised as “Gulf of Bothnia”.
The grey seals inhabit the whole area including the Gulf of Bothnia, Gulf of Finland and the Baltic Proper (BERGMAN ET AL.1981;HARDING ET AL.2007).
Figure 1. The Baltic Sea is divided in 17 sub areas. Bothnian Bay, The Quark and Bothnian Sea are summarised as Gulf of Bothnia in this study (HELCOM2018A).
Ringed and grey seals represent the key species of the Gulf of Bothnia. Key species are individuals, which structure and stabilize the ecosystem by their activities and abundances (PAINE 1969). Both species are also K-selected species due to longevity, long generation times and low number of pups born every year (KLOTZ AND KÜHN 2002). K-selected species are supposed to have a large effect on structuring their ecosystems (APOLLONIO 2002;RAY
1981) and are easily vulnerable to direct (e.g. toxic substances) and indirect effects (e.g.
changes in food chain). Therefore, substantial changes in population dynamics of ringed and grey seals may have cascading effects on the whole ecosystem (HAMILTON ET AL.2015;
LUQUE ET AL 2014;MOUQUET ET AL 2013).
In the beginning of the 20th century, a large number of ringed and grey seals could be found in the Baltic. In the 1940s, the number of both seals slowly decreased due to the annual hunt in large parts of Sweden and Finland (ALMKVIST ET AL.1980;HÄRKÖNEN ET AL.2008).
Nevertheless, the population numbers stabilised until 1965. This year was followed by a marked decrease in the Baltic Sea seal populations (ALMKVIST ET AL.1980;HARDING AND
HÄRKÖNEN 1999). Unlike the former decreases, it was not caused by hunting, because in some Baltic areas hunting was already restricted (ALMKVIST ET AL. 1980). Instead, the reproduction capacity massively decreased since the late 1960s (KOKKO ET AL.1999) mainly caused by high levels of organochlorines in the marine environment (BERGMAN AND OLSSON
1986;HELLE 1980;HELLE ET AL.1976A,1985). It is known that the Baltic Sea is one of the marine areas most heavily used by humans worldwide and one of the most contaminated seas in the world (FONSELIUS 1972). The Baltic Sea is surrounded by nine countries inhabiting around 85 million people (KELLY ET AL.2010). Therefore, it is not surprising that the impact of eutrophication and anthropogenic contaminants, such as heavy metals, is high (DIETZ ET AL. 1990, 96; HANSEN 1981; JANSSON 2003; MUIR ET AL. 1992; SMITH AND
AMSTRONG 1978). Furthermore, different contaminants like the compounds of persistent organic pollutants (POPs) can be found in unusually high concentrations (NYMAN ET AL. 2002). POPs are organic compounds that have been produced for agricultural purposes, such as dichlorodiphenyltrichloroethane (DDT), and have been used as plasticizer, like polychlorinated biphenyls (PCBs). PCB as well as DDT are the most present contaminants in the Baltic Sea (HELCOM2010A). They are very persistent in the environment and show also a high chemical stability (NYMAN ET AL.2002). PCBs and DDTs, as well as other POPs, are lipophilic, which is the reason why they accumulate in the blubber layers of marine mammals after ingestion (BOON ET AL.1992;REEVES 1998;TANABE ET AL.1988). Through the 1960s and 1970s PCB and DDT were widely used. Therefore, the concentrations of those contaminants in the water did not decrease until the late 1980s (KELLY ET AL.2010).
The decrease of PCB was based on its restriction in different countries of the Baltic Sea.
First of all, in Sweden and Finland a legal protection for seals in Swedish and Finnish waters was decreed in 1974, followed by the ban of PCB use in 1978 (HELANDER AND BIGNERT
1992). Another important measure was the Stockholm Convention from 2001, in which PCB and DDT were banned in the whole Baltic Sea. The reason for the ban was the different negative impacts on birds, marine mammals and fish. Since seals are top predators, and it is known that different contaminant concentrations increase towards higher trophic levels in the marine ecosystem (FANT ET AL.2001), high concentrations of POPs accumulate in their bodies. Such accumulation leads to toxic effects, which can be seen in the reproductive system, the endocrine system as well as the hormonal and vitamin D status (BROUWER ET AL.1989;DE SWART 1995;REIJNDERS 1986;ROSS 1995). Despite the ban of PCB and DDT, pollutant levels are still high enough (JONSSON ET AL.1996) to cause multiple chronic organ
lesions, for example in the female reproductive organs, intestines, adrenals, livers and kidneys (BERGMAN 2007; HARDING AND HÄRKÖNEN 1999; HELLE ET AL. 1976B, 1980;
HYVARINEN AND SIPILA 1984). Moreover, severe lesions were present in the skeleton (LINDT ET AL.2003;PERTOLDI ET AL.2018).
Today it is a challenge to analyse the health status of marine mammal populations, especially considering anthropogenic influences, and find measures to preserve species like ringed or grey seals. Because the access to free living individuals is limited and it is often difficult to find the individuals due to very low individuals’ numbers. Marine mammals are known to be very mobile, which is another explanation, why it is a challenge to examine the health status (SJÖBERG ET AL.1995). The ban on the usage of toxic substances, as described above is one effective measure; another step is the enlisting of endangered species in official preservation lists (HELANDER AND BIGNERT 1992). The International Union for Conservation of Nature (IUCN) lists the ringed seal as vulnerable species since 1994, as well as in the EU Habitats Directive in Appendix II and V. The grey seal is listed as least concern in IUCN since 2016 but also listed in the EU Habitats Directive (Appendix II and V).
Until today, the ringed seal population recovered and increased to 10,000 individuals in the Baltic Sea, out of which the highest abundance is found in the Gulf of Bothnia (HELCOM 2018B). The grey seal population increased to 30,000 individuals in the whole Baltic Sea (HELCOM2018B).
To effectively preserve seals, it is important to identify reasons for population decreases and gather detailed information about negative influences, especially anthropogenic, on the seal species. Suited for such examinations are vulnerable body parts of ringed and grey seals regarding pollutants such as bones and liver. Multiple studies deal with organochlorines and their effects on the immune, endocrine and skeletal systems of aquatic mammals (BEINEKE ET AL.2005;DAS ET AL.2006; JEPSON ET AL.2016; LIND ET AL.2003;
ROUTTI ET AL.2008). In different marine mammals (e.g. otters (Lutra lutra), grey seals or polar bears (Ursus maritimus)) a relationship between contaminants and bone lesions was found (ROOS ET AL.2010;ROUTTI ET AL.2008;SONNE ET AL.2004). Two different bone cell types, which are under normal circumstances ideally balanced, can be affected by contaminants. Osteoblasts, which are one of the bone cell types, are responsible for the formation, whereas bone resorption is performed by osteoclasts, which represents the other bone cell type. Furthermore, it is known that contaminants like PCB and DDT can affect endocrine homeostasis, which can then end in bone changes (HAAVE ET AL.2003;LETCHER ET AL.2010;SONNE ET AL.2004). PCB and DDT can bind at sexual steroid receptors and can change the reactions of the normal hormone cascade. Furthermore, PCB and DDT are
also known to affect liver tissue of Baltic seals (BERGMAN ET AL.2001). Many studies show liver changes, which can be a result of PCB and DDT burden (BERGMAN ET AL.2001;SONNE ET AL.2005,2007B,2010). In polar bears, increasing numbers of lipid granulomas can be associated with mercury (SONNE ET AL.2007B). The study of SONNE ET AL. (2010) show that mercury and organochlorine can be co-factors in the development of mononuclear cell infiltrations in pilot whales (Globicephala melas). The different lesions show that the liver can be easily disturbed by different contaminants. One possible explanation can be the main functions of the liver, which are filtration of blood, exposition of hormones and rebuilding of steroid or thyroid hormones (RAMADORI ET AL.2008), as well as protecting the individuals from toxic contaminants (GRANT 1991). The mechanisms of contaminant filtration of the blood can explain the high concentrations of contaminants, as well as different contaminants occurring in the liver.
Another of the above-mentioned aspects affected by pollutants are the female reproduction organs and more generally the reproduction, which is an important research area for the observation of population dynamics and the overall health status of a species (ROOS ET AL. 2012). In addition to contaminants, also other environmental influences can affect the reproduction, such as photoperiod and nutritional status (ATKINSON 1997). The reproduction and more specific the reproduction cycle reacts very sensitive to environmental changes, because it is very complex (AMOROSO ET AL.1965). The reproduction of seals can be divided into mating, gestation, parturition and lactation (ATKINSON 1997). Gestation includes the embryonic diapause (BOYD 1991), which can be defined as the phase, where the blastocyst stays inactive in the lumen of the uterus (BONNER 1989;RENFREE 1993;RENFREE AND SHAW
2000;POMEROY 2011). Therefore, the diapause is among other factors an important feature for the timing of the birth (BOYD 1991). In most seal species, the parturition takes place in the time period when the pup has the best chance of survival (GALES ET AL.1997). Food availability, climate, body conditions of the females and other environmental factors have an influence on the time of the parturition (GALES ET AL.1997). It is well known, that lower food availability and a consequently worse body condition is the main factor to delay the parturition or even end the gestation in an abortion (BOURDON AND BRINKS 1982;MARSHALL AND HAMMOND 1926;SMITH 1987). However, not only blubber reserves can have a negative effect on the reproduction success, also environmental changes and contaminants do.
Contaminants, such as PCB and DDT, have an impact on the health status and reproductive system of seals (ICES 2003). The consequences of PCB and DDT depend on their concentration levels (BERGMAN AND OLSSON 1985). High concentrations lead to reproductive failure (JEPSON ET AL. 2016) and reproduction disturbances e.g. uterus
stenosis and occlusions, which can especially still be found in Baltic ringed seals (BERGMAN AND OLSON 1985). It is known, that PCB and DDT can also have hormonal impacts, because they function as hormonal trigger, which is why they intensify or inhibit the hormonal function of the reproduction receptors (LEISEWITZ AND KAMRADT 1996).
Information about contaminant effects on bones, liver and ovaries in ringed and grey seals from the Baltic Sea are rare. All existing studies were performed for specific years or shorter time periods or were investigated in other mammals (e.g. polar bears, whale species, sledge dogs) (BERGMAN ET AL.2001;LAW ET AL.1991;LI ET AL.2003;LIND ET AL.2003;MORTENSEN ET AL.1992;RAWSON ET AL.1993;SONNE ET AL.2004,2013). Thus, the examination of the effects of pollutants in the Baltic ringed and grey seals over a long time-span until today fills an important research gap and will allow an early detection of possible environmental disturbances. Furthermore, the studies carried out in the context of this dissertation help to understand the effects of contaminants on the Baltic ringed and grey seals. The methodology of comparing it to a reference population in Greenlandic waters, which was presumably exposed to lower contaminant concentrations from the beginning of the 1960’s onwards (AMAP1998;HOLDEN 1970), can aid identifying explanatory factors.
This study covers three important and until now insufficiently investigated topics of the Baltic ringed and grey seals:
1. the variation in bone mineral density (BMD) of ringed seals over time (chapter 1) 2. the examination in liver lesions in Baltic grey seal population over three decades
(chapter 2)
3. the determination of the primordial follicles in ringed seal ovaries (chapter 3)
In chapter 1 and 3, the Baltic ringed seals are compared to a reference population in Greenlandic waters. These two examinations of the thesis complement existing studies and enlarge the scientific knowledge about the Baltic ringed seals. In addition to the above- mentioned topics, this thesis also examined liver lesions in the Baltic grey seal population over three decades (chapter 2).
The variation in BMD of ringed seals is examined between 1829 and 2019 (chapter 1). This time-span includes different phases of pollutant usage: before the anthropogenic pollution, impact begins, during high pollution concentrations in the Baltic Sea, which was overlapped by a drastic decrease in population numbers, and after the extensive use of pollutants, when concentrations of different pollutions were still present and alternatives were used. This long-time analysis is relevant for management decisions relating to long-term effects of anthropogenic impacts.
In addition to chapter 1), this thesis also covers liver lesions in the Baltic grey seal population over three decades (chapter 2). As a result, liver changes were determined and the possible effects of contaminant concentrations over the examined timespan are determined. This timeframe starts directly after the heavy decline of the Baltic grey seal population in the 1970’s following the introduction of PCBs and DDTs. It also includes the assessment of current contaminant levels and its effects on the seal livers in the Gulf of Bothnia.
The number of primordial follicles in ringed seal ovaries is examined between 2018 and 2019 and is a topic not yet investigated and firstly published in this thesis (chapter 3). This analysis is important to identify an explanation for the slow recovery of the ringed seals in the Baltic and is therefore a relevant and important tool for management decisions.
The knowledge gained from this doctoral thesis enables early recognition of negative changes in reproduction and the overall health status of two key species in the Gulf of Bothnia due to anthropogenic impacts. As a result, management plans can be developed more accurately and be adapted, if necessary.
Chapter 1
Variation in skull bone mineral density of ringed seals
(Phoca hispida) from the Gulf of Bothnia and West Greenland
between 1829 and 2019
Published:
Schmidt, B., Sonne, C., Nachtsheim, D., Dietz, R., Persson, S., Oheim, R., Rolvien, T., Amling, M., Siebert, U. (2020). Variation in skull bone mineral density of ringed seals (Phoca hispida) from the Gulf of Bothnia and West Greenland between 1829 and 2019. Environmental International Special Issue 143, 105968
Variation in skull bone mineral density of ringed seals (Phoca hispida) from the Gulf of Bothnia and West Greenland between 1829 and 2019
Britta Schmidta, Christian Sonneb, Dominik Nachtsheima, Rune Dietzb, Ralf Oheimc, Tim Rolvienc,d, Sara Perssone, Michael Amlingc, Ursula Sieberta,b*
aInstitute for Terrestrial and Aquatic Wildlife Research, University of Veterinary Medicine Hannover, Foundation, Werftstr. 6, 25761 D-Büsum, Germany
bMarine Mammal Research, Department of Bioscience, Aarhus University, Frederiksborgvej 399, P.O. Box 358, DK-4000 Roskilde, Denmark
cDepartment of Osteology and Biomechanics, University Medical Center Hamburg- Eppendorf, Lottestr. 59, 22529 D-Hamburg, Germany
dDepartment of Orthopedics, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 D-Hamburg, Germany
eDepartment of Environmental Research and Monitoring, Swedish Museum of Natural History, P.O. Box 50007, SE-104 05 Stockholm, Sweden
*Address correspondence to U. Siebert, Institute for Terrestrial and Aquatic Wildlife Research, University of Veterinary Medicine Hannover, Foundation, Werftstr. 6, 25761 D- Büsum, Germany. Telephone: +49511-856-8158. E-mail: ursula.siebert@tiho-hannover.de
Abstract
Bone is remodelled constantly through a balance of bone formation and resorption. This process can be affected by various factors such as hormones, vitamins, nutrients and environmental factors, which can create an imbalance resulting in systemic or local bone alteration. The aim of the present study was to analyse the changes in bone mineral density (BMD) over time in skulls of ringed seals (Phoca hispida) from the Baltic and Greenland using museum samples. Overall, 303 skulls (102 Male, 89 Female, 112 unknown) were used for bone investigations and were divided into three periods according to collection year: before 1958 (N=167), between 1958 and 1989 (N=40) and after 1994 up to 2019 (N=96). All skulls were examined by dual-energy x-ray absorptiometry to obtain the BMD.
Skull BMD of the Baltic seals was positively correlated with the historical polychlorinated biphenyls (PCB) contamination having potential effects on the constitution of bones. BMD fluctuated between the three study periods (LM: p-value<0.001, F-value=47.5) with the lowest BMD found between 1897 and 1957, in the Gulf of Bothnia, where the highest peak of contaminant concentration was in the second period. BMD levels increased with increasing PCB concentration (LM: p<0.001). The Greenland population showed significant lower BMD levels in the pollution and post-pollution period than the Baltic population (LM:
p<0.001). It also revealed a higher BMD in males than in females (LM: p=0.03). In conclusion, the variations between 1829 and 2019 in the Baltic Sea and Greenland may to a certain extent reflect normal fluctuations; however, this study revealed several factors affecting BMD, including sex and PCB levels.
Keywords: Ringed Seal, Skull, Arctic, PCB, Long-term study
1 Introduction
The continuous renewal of bone (i.e., bone remodelling) is mediated mainly by two different bone cell types. While osteoblasts are responsible for the formation, bone resorption is performed by osteoclasts. The process of bone remodelling is influenced by matrix- embedded osteocytes, which are terminally differentiated osteoblasts (BONEWALD 2002). It not only serves for the renewal of bone in response to skeletal loading, but also for the maintenance of mineral homeostasis (i.e., calcium, phosphate) (ROLVIEN ET AL. 2017).
While the remodelling process is ideally balanced, it is influenced by multiple environmental factors including hormones, vitamins, nutrients and organochlorines (HILL 1998;JOHANSSON ET AL.2002;LEDER ET AL.2001;ROOS ET AL.2010;SARAZIN ET AL.2000;SONNE ET AL.2004).
Environmental contaminants in particular may affect this process, resulting in an imbalance that leads to a decrease in bone mass and pathological changes including porosity, alveolar deterioration and decreased overall bone density (BERGMAN ET AL.1992;LIND ET AL.2003;
SONNE ET AL.2004).
Some of the most widely used chemicals over the past 80 years are organochlorines, which include polychlorinated biphenyls (PCB) and chlorinated pesticides such as dichlorodiphenyltrichloroethane (DDT). PCBs and DDTs are found in all fresh, brackish and marine waters around the world (OGATA ET AL.2009). These substances were regulated and banned in most parts of the world during the 70’ and 80’s (AMAP1998), but due to their persistence and tendency to bioaccumulate, they can still be found in ecosystems worldwide today. For the Baltic Sea, the Stockholm Convention banned the usage of PCB in 2001 while Sweden already restricted the use of PCB in 1978. Before the ban, a decrease in the seal populations of both ringed (Phoca hispida) and grey seals (Halichoerus grypus) was observed in the Baltic (HELLE 1980,HARDING AND HÄRKÖNEN 1999).
The reduction of the population size was due to intensive hunting during the first part of the century but later the exposure to organochlorine contaminants, affected the seals’
reproductive system and led to infertility (BERGMAN 1999; BERGMAN AND OLSSON 1986;
HELLE 1980;REIJNDERS 1986;HARDING AND HÄRKÖNEN 1999). Between 2003 and 2016, the growth rate of the ringed seal populations in the Baltic Sea was less than the intrinsic capacity (the biotic potential or maximal growth rate for a species) (HELCOM2018B).
Multiple publications report on organochlorines and their effects on the immune, endocrine, reproduction and skeletal systems of aquatic mammals (BEINEKE ET AL.2005;BERGMAN ET AL.1992;DAS ET AL.2006;JEPSON ET AL.2016;LIND ET AL.2003;ROUTTI ET AL.2008). In Eurasian otters (Lutra lutra) from Sweden, PCBs have been shown to be correlated positively with cortical bone variables including cortical area, cortical mineral content and
cortical thickness (ROOS ET AL.2010). In subadult Eastern Greenland polar bears (Ursus maritimus), a negative correlation between skull bone mineral density (BMD) and PCB and chlordane burden was found (SONNE ET AL.2004). Moreover, a study of ROUTTI ET AL. (2008) showed a relationship between contaminants and endocrine homeostasis and also demonstrated that the bone lesions of Baltic grey seals could be a combination of contaminant-mediated vitamin D and thyroid disruption. The study showed that seals in the Baltic Sea are also exposed to higher concentrations of PCB and DDT than in Canada because the concentrations in the environment are much higher in the former region (ROUTTI ET AL.2008).
Furthermore, it was found that the mandible of Baltic male grey seals had the lowest bone density between 1986 and 1997 compared to the time between 1850 and 1955 (LIND ET AL. 2003). Grey seals, as well as ringed seals are top predators in the marine ecosystem and accumulate high concentrations of environmental contaminants such as PCBs in the internal organs, tissues and blubber. As a top predator, the ringed seal is important for the native ecosystem and is supposed to have a significant effect on structuring its ecosystem (APOLLONIO 2002, RAY 1981; KLOTZ AND KÜHN 2002; MOORE 2008; SERGIO ET AL. 2005).
Therefore, the health status of the ringed seals is used for the evaluation of the overall health and status in a way that this phocid is utilized as a sentinel species (GULLAND AND
HALL 2005).
As a result of the contaminant influence and the slow recovery of the ringed seal population, it has been listed as vulnerable in the HELCOM Red List and has the IUCN Criteria A3c, which means that these seals are under special protection. Against this background it is important to know how they are affected by pollutants in order to be able to take effective actions (HELCOM2013). The present study aims to document the historical event of large- scale environmental pollution by investigating the BMD of ringed seal skulls from the Baltic Sea over the period 1897 to 2018.
2 Materials and Methods 2.1 Samples
The samples from Sweden originate from ringed seals that were found dead (N = 11), bycaught (N = 10) or hunted by locals (N = 14), for 68 individuals the cause of death is unknown. The Greenland samples were hunted by locals. The animals were shot according to the existing hunting law in the respective countries. A sample of 103 ringed seal skulls from the Gulf of Bothnia and 200 ringed seal skulls from west Greenland were examined
(Figure 2; Table 1). The 103 ringed seal skulls from the Gulf of Bothnia (scattered along the northern Swedish east coast) were provided by the Swedish Museum of Natural History (SMNH) in Stockholm, while the skulls from Greenland (Qaanaaq and Qeqertarsuaq) were provided by the Aarhus University and Zoological Museum in Copenhagen, Denmark. The skulls were collected in the period between 1897 and 2018 (Gulf of Bothnia) and the skulls from Greenland between 1829 and 2019. At the SMNH; there are only records of exact treatment method for the individual skulls from recent years. Upon ocular inspection, the skulls from 1897 to 1922 were found to be probably macerated and/or boiled carefully. From 1933 until the 1980s the skulls were boiled with or without adding a cleaning agent. From the end of the 1980s it became more common to use dermestid beetles, and from 2008 and onwards all the skulls were treated with this method. ROOS ET AL.(2010) found no significant differences in bone mineral density between the boiling method at SMNH and cleaning by dermestid beetles. However, too harsh treatment with boiling and chemicals can result in markedly rugged surfaces on the bone. Skulls that had been treated this harshly were excluded from the study. The Zoological Museum in Copenhagen exposed the skulls to different temperatures to clean them. Occasionally, soap was also used to clean the bones.
After preparation, the skulls were stored dry at room temperature at the museums.
Figure 2. Study areas in the Baltic Sea and West Greenland. Samples were predominantly collected in the Gulf of Bothnia and in the locations marked with a triangle at the west coast of Greenland.
The age of the animals was determined by the growth layers counted in the cementum of the canine teeth collected from the lower jaw. First, a decalcification took place, followed by
sectioning and staining with toluidine blue as described in STIRLING ET AL. (1977) and DIETZ ET AL.(1991). 37 ringed seal skulls from the Gulf of Bothnia had already been determined by the SMNH in the same manner. All individuals were separated into three sex and age classes based on age determination: adult males (≥ 5 years old), adult females (≥ 4 years old) and juveniles (remaining individuals) (ATKINSON 1997;KELLY ET AL.2010).
Additionally, the skulls were divided into three groups, according to year of collection. The first group included the skulls from 1829 to 1957, the second from 1958 to 1989 and the last group from 1994 to 2019. These periods represent different contaminant levels in the seals. The first period describes a period with an absence up to very low PCB levels (Supplementary Material, Figure 10) in the environment prior to the industrial production and can be considered as baseline. In this period, 167 skulls were available, where 58 individuals originate from the Gulf of Bothnia and 109 individuals from Greenland (Table 1).
The second period covers the most intense production phase of PCBs (Supplementary Material, Figure 10) and the highest peak of PCBs in the Baltic and includes 40 skulls (Gulf of Bothnia: 26, Greenland: 14, Table 1). In the last period, which represents the decreasing PCB concentrations (AMAP 1998, Supplementary Material, Figure 10) in the Baltic, 96 skulls were available (Gulf of Bothnia: 19, Greenland: 77, Table 1).
Table 1. Number of ringed seal (Phoca hispida) skulls from the Gulf of Bothnia in the Baltic Sea and from West Greenland in the Greenland Sea used in analyses of bone mineral density in the present study.
Gulf of Bothnia West Greenland
Period 1 1897-1933
Period 2 1960-1989
Period 3 2007-2018
Period 1 1829-1957
Period 2 1958-1987
Period 3 1994-2019
Total 58 26 19 109 14 77
Adult 26 25 15 47 2 17
Juvenile 28 0 2 4 1 18
Un. Age group
4 1 2 58 11 42
Known sex
1 26 18 66 5 75
Males 1 11 8 44 1 37
Adult 0 11 6 28 0 12
Juvenile 0 0 1 1 1 7
Un. Age group
0 0 1 16 0 18
Females 0 15 10 22 4 38
Adult 0 14 9 10 1 5
Juvenile 0 0 1 3 0 11
Un. Age group
0 1 0 9 3 22
Un.: unknown
2.2 Identification of sex
The samples came from 114 individuals of the Gulf of Bothnia, of which 59 were of known sex (24 males, 33 females, 2 juvenile males). In order to determine the sex of the 55 individuals with unknown sex, six skull lengths were measured (Table 2, Figure 3). Each distance was measured on both sides of the skulls, using a vernier caliper (Covetrus Inc., 7 Custom House St. Portland ME, 04101). The skulls were measured by two persons and the mean value was used in the calculations. The skull length measurements from individuals with known sex were then tested for differences between the sexes. The aim of the analysis was to predict the sex of individuals with unknown sex by identifying significant sexual differences in ringed seal skull lengths.
Table 2. Definition of the skull lengths of the Baltic ringed seals and Greenland ringed seals (based on BECHSHØFT ET AL.2008).
Length Definition
OPF Maximal distance between the opistocranion and the postorbital process of the frontal bone.
CBL Condylobasal length.
P4-M2 Length from the anterior margin of the 4th premolar to the posterior margin of the 2nd molar.
C-M2 Length from the anterior of the alveole of the canine, to the posterior of the alveole of the 2nd molar.
I1-M2 Length from the anterior of the alveole of the 1st incisor, to the posterior of the alveole of the 2nd molar.
POH Postorbital height.
Figure 3. The six lengths measured on the ringed seal skulls of the Gulf of Bothnia in the Baltic Sea and West Greenland. 1 = OPF, 2 = POH (upper picture), 3 = CBL, 4 = P4-M2, 5
= C-M2, 6 = I1-M2 (bottom picture).
2.3 Bone mineral density (BMD)
Bone mineral density (BMD) was examined for 103 skulls from the Gulf of Bothnia (25 females, 20 males, 58 unknown) and 200 skulls from Greenland’s west coast (64 females, 82 males, 54 unknown), after all skulls which were damaged or not complete were excluded.
Dual-energy X-ray absorptiometry (DXA, Lunar Prodigy iDXA; GE Healthcare; Madison, WI, USA) was used for this, which is a non-invasive and commonly used method for studying BMD (DOWTHWAITE ET AL.2011). In humans, it serves for the detection of osteoporosis. The skulls were scanned with a speed of 5.65 mm/sec and a resolution of 0.5 x 0.61 mm (BOUDOUSQ ET AL.2005). Macerated skulls are difficult to scan, therefore a water tank, which replaced skin and tissue, stands above the samples during the scan process (Figure 4).
After scanning, the results were analysed by the enCORE- software (v15 – GE HealthCare Lunar, Buckinghamshire, UK), which produced an image of the skulls and calculated the BMD in grams per square centimetre (g/cm2). In the analysis the software automatically identifies bone tissue, based on the different X-ray absorption coefficients of bone and other tissue. Nevertheless, this process is not always reliable and the scanner does not necessarily identify the whole skull as bone tissue. This can be manually corrected as the process is supervised.
Figure 4. Experimental set-up of the skull scanning process. The X-ray arm of the DXA scanner is positioned above the water tank, underneath which the macerated skulls are placed.
2.4 Statistical analyses
The statistical analyses were performed with R (version 3.4.3, R Core Team 2014) using the packages “jtools” (LONG 2019), “sm” (BOWMAN AND AZZALINI 2018) and “mgcv” (WOOD
2011).
A binomial generalized linear model (GLM) was used to test for differences in skull length measurements (OPF, POH, CBL, P4-M2, C-M2, I1-M2) between sexes. The aim of the analysis was to subsequently differentiate the sex of seals with unknown sex. This model was only tested with the age group “adult”, as other age groups were underrepresented, and both sides of the skull were analysed separately. The most parsimonious model was selected in a step-wise procedure, as recommended by ZUUR ET AL. (2009). Each explanatory variable (i.e. each skull length measurement) was subsequently dropped from the full model and then this reduced model was compared to the full model by applying likelihood ratio tests using the R function ANOVA. Only significant model terms were
retained. For both sides, the most parsimonious model included only OPF (pleft=0.015, pright=0.04).
The BMD was tested for normality by applying the Shapiro-Wilk test. Level of significance was set at p ≤ 0.05 and a trend was considered between 0.05 < p ≤ 0.10 based on SONNE ET AL. (2004).
The next step was a linear regression analysis (LM), where the relationship of BMD versus period (before, during, after) and also the relationship of BMD versus sex (female, male, unknown) were tested for the skulls from the Baltic Sea and Greenland Sea. For the comparison of both areas first the relationship of BMD versus both areas were tested. To test for period differences an ANCOVA was used. BMD was the dependent variable and period, as well as both areas were independent class variables.
To investigate the effect of organochlorines, particularly PCB, on BMD, reliable estimates of PCB values in ringed seals were needed. As PCB concentrations could only be measured in 17 of the available skull samples (Supplementary Material, Table 3), we modelled a total PCB content based on PCB measurements in blubber samples of ringed seals from the Gulf of Bothnia, Bothnian Bay, Bothnian Sea and Gulf of Finland in 1968 and between 1973 and 2015 (HARDING ET AL., IN PREP.) using a generalized additive model (GAM; R package mgcv). In 1968, 2 individuals and between 1973 and 2015, 337 individuals (121 females, 55 males, 161 unknown; 24 foetus, 69 juveniles, 84 adults and 160 unknown) were analysed. PCB and their 37 congeners (18, 33, 47, 49, 51, 52, 60, 66, 70, 74, 77, 80, 99, 101, 105, 110, 114, 118, 122, 123, 126, 128, 138, 141, 153, 156, 157, 167, 169, 170, 180, 183, 187, 189, 194, 206 ,209) were determined by a gas chromatography and a mass spectrometer. We introduced artificial samples from 1897 to 1905 and assumed a PCB content of 0 as a baseline for the model. For some field work campaigns, the original measurements were not available but only median total PCB values and the corresponding sample sizes were reported. As those sample sizes were usually high, the median values were considered robust. Therefore, the values were repeated as often as the sample size and as such entered in the analysis to make the GAM more robust to outliers from measurements of single individuals. The coefficient of determination was high (R2 = 81.7%) indicating a good model fit. The model output was then used to predict total PCB content of ringed seals from 1897 to 2018 (Supplementary Material, Figure 10). The model values were subsequently assigned to the skull samples from the Gulf of Bothnia based on sampling year. The relationship between BMD and PCB levels was then tested with a linear regression model. After that, the relationship between PCB levels and sex was tested in a separate linear regression model.
3 Results
3.1 Gulf of Bothnia 3.1.1 Sex
The GLMs revealed that only OPF on both sides seems to be significantly different between sexes (pleft=0.02, pright=0.04). Although there is a significant difference, the difference is relatively small and thus biologically not meaningful: mean OPF right females = 8.11 ± 0.36 cm and males = 8.38 ± 0.39 cm, mean OPF left females = 8.16 ± 0.34 cm and males = 8.38
± 0.39 cm (Figure 5). Thus, it is not possible to reliably distinguish sexes only on skull morphometric measurements and to identify the sex of individuals of unknown sex in this way.
Figure 5. Density curves of both OPF sides from Baltic ringed seals males and females.
OPF of males and females overlap strongly, which makes it impossible to reliably distinguish sexes only based on skull morphometric measurements.
3.1.2 BMD and sex differences
BMD was analysed in 103 skulls from the Gulf of Bothnia divided into 25 females (including 1 juvenile), 20 males and 58 unknown individuals representing the period 1897 – 2018. The
exact composition of each time period group can be found in Table 1. Both sexes had significantly higher BMD (mean: female = 0.83 ± 0.16 g/cm2, male = 0.91 ± 0.30 g/cm2, unknown = 0.62 ± 0.10 g/cm2) than the unknown category (LM, punknown vs males < 0.001, punknown vs females < 0.001) (Figure 6). No statistically significant difference could be seen between BMD of males and females.
Figure 6. BMD in Baltic ringed seals during the period 1897 to 2018. It is shown that BMD differs between the periods.
3.1.3 Time trends
Regarding samples from the Bothnian Bay, 58, 26 and 19 skulls were used for the first, second and third periods, respectively (Table 1). The BMD levels of the individuals from the pre-pollution period (1897-1933) were significantly lower than from the pollution period (1960-1989) and from the post-pollution period (2007-2018) (ppollution < 0.001, ppost-pollution <
0.001; mean: pre-pollution period = 0.62 ± 0.10 g/cm2, pollution period = 0.91 ± 0.14 g/cm2, post-pollution period = 0.81 ± 0.32 g/cm2) (Figure 6).
3.1.4 BMD vs. contaminants
BMD was analysed against modelled PCB concentrations in ringed seals from the Gulf of Bothnia (Table 1). The relationship between BMD and PCB concentrations was significant (LM, p < 0.001) (Figure 7). BMD increased with increasing PCB. Males on average have a higher PCB concentration than females between 1897 and 2018 (mean: males = 55672.71
± 46694.47 ng/g, females = 25094.83 ± 30006.85 ng/g).
Figure 7. BMD in Baltic ringed seals as a function of modelled PCB concentrations for the examined ringed seals from 1897 to 2018. It can be seen that the BMD increased with an increasing PCB concentration.
3.2 Greenland
3.2.1 BMD and age group/sex differences
BMD was analysed in 225 skulls divided into 64 females, 82 males and 79 unknown individuals. In contrast to the Gulf of Bothnia the Greenland population showed a significantly higher BMD in males than in females (LM, p = 0.04; mean: females = 0.66 ± 0.24 g/cm2, males = 0.76 ± 0.35 g/cm2). Furthermore the BMD was higher in adult individuals than in juvenile individuals (LM, p = 0.02; mean: adult = 0.73 ± 0.39g/cm2,
juvenile = 0.57 ± 0.1 g/cm2), as well as the group of individuals of unknown sex (LM, p = 0.04; mean: unknown = 0.71 ± 0.23g/cm2, juvenile = 0.57 ± 0.1 g/cm2) (Figure 8).
3.2.2 Time trends
During the whole study period, none of the tested variables were significant. The BMD showed no significant variation over the time span from 1829 to 2019 (Figure 8).
Figure 8. BMD in Greenland ringed seals during the period 1829 to 2019. Males had higher BMD than females.
3.3 Greenland vs. Gulf of Bothnia 3.3.1 BMD and country differences
The BMD varies between sexes and sea regions. In the Gulf of Bothnia, the BMD of the females varies between 0.453 g/cm2 and 1.12 g/cm2 (median: 0.74 g/cm2) and the BMD of the males varies between 0.574 g/cm2 and 1.987 g/cm2 (median: 0.72 g/cm2). Around Greenland the BMD range of the females varies between 0.252 g/cm2 and 1.127 g/cm2 (median: 0.64 g/cm2) and of the males between 0.334 g/cm2 and 2.774 g/cm2 (median: 0.72 g/cm2). No significant difference between the BMD levels of the Greenland population and the Gulf of Bothnia could be shown. The gender composition for the Gulf of Bothnia was for the pre-pollution period one male and 57 individuals with unknown sex. For the pollution
period it was 11 males and 15 females and for the post-pollution period eight males, ten females and one unknown. The gender composition for the Greenland individuals were for the pre-pollution period 44 males, 22 females, 43 unknowns; and for the pollution period one male, four females and nine unknowns; and for the post-pollution period 37 males, 38 females and two unknowns.
3.3.2 Time trends
For the first period, prior to PCB production, 167 skulls were available, in the second period, 40 skulls were examined and for the third period, after the highest peak of contaminant concentration, 96 skulls were available (Table 1). The Greenland population showed a significantly higher BMD in the pre-pollution period than the population in the Gulf of Bothnia (LM, p < 0.001, Figure 9). The population of the Gulf of Bothnia showed significantly higher BMD values in the pollution and post-pollution period than the population of Greenland (LM, ppollution < 0.001, ppost-pollution = 0.005, Figure 9)
Figure 9. BMD of the investigated ringed seals of both areas between 1829 and 2019.
4 Discussion
4.1 Identification of sex
Analysis of the possibility to identify sex based on skull measurements was performed on adult Baltic ringed seals. The test of both skull sides showed a significant difference between sex and OPF (maximal distance between the opistocranion and the postorbital process of the frontal bone), but the difference was relatively small and thus biologically not meaningful in ringed seals. The sex classification based on skull length was also tested in polar bear studies (BECHSHØFT ET AL. 2008), where significant differences between the sexes were detected indicating a marked sexual dimorphism. In contrast to that, female and male ringed seals showed no obvious sexual dimorphism, neither in body length nor in head form. Another study examined the age and sex classification of the skulls of the Mediterranean monk seal (Monachus monachus) (BROMBIN ET AL.2009) using a method which is based on landmarks. With this method, it was possible to detect differences between age classes, but there was no apparent sexual dimorphism similar to ringed seals in this study. Thus, it is not possible to distinguish the sex of ringed seals via skull morphometric measurements and genetic analyses are needed to reliably confirm the sex.
4.2 Bone mineral density of skulls from ringed seals
The skulls from Greenlandic ringed seals showed that males had a significantly higher BMD than females, which can be explained by the effect of the reproduction periods on the females’ body. Females lose BMD during their pregnancy and lactation (KALKWARF ET AL. 1997). Ringed seals give birth to one pup and the suckling period lasts up to 7 weeks (ATKINSON 1997). In this time, females mobilise much of their own calcium and phosphate for the pup (CROSS ET AL.1995;KREBS ET AL.1997;RAMSAY AND STIRLING 1988). Due to requirements for calcium during gestation, the bone density is lower compared to males.
The same result was found in Greenland polar bears and in humans (CROSS ET AL.1995;
SONNE ET AL.2004). The variation of BMD in females seen in this study could also be due to the influence of sexual steroids on the skeletal morphology and physiology. The oestrogen level is important for the bone mineral absorption and the skeletal growth in pups, juveniles and adults (LIND ET AL.2004;DING ET AL.2008;SAGGESE ET AL.1997;YILMAZ ET AL.2005). An inhibition of the oestrogen secretion can lead to a BMD decrease (SAGGESE ET AL.1997).
4.3 Period differences and time trends in skull BMD
Individuals from the Baltic Sea had a significantly higher skull BMD from the period between 1957 and 1990 compared with those from the pre-pollution period. The same can be seen with individuals from the post-pollution period. These results suggest that there could be a linkage between increased BMD and increased contaminant loads. The BMD of males and females increase in the pollution period. The differences between the sampling areas illustrate possible effects from PCB on bones and indirect effects through sterility that cause female BMD not to decrease. The contaminant concentrations in ringed seals around Greenland are almost 100-fold lower than in the Gulf of Bothnia (SKAARE 1996,LETCHER ET AL. 2010, HARDING ET AL. UNPUBLISHED). The PCB concentrations, during the pollution period, were very high in the tissue from ringed seals of the Gulf of Bothnia (BJOURLID ET AL.2018) and reached levels, which can affect immunological parameters. It is known, that environmental contaminants suppress some mechanisms of the immune system (DEAN ET AL. 1982), because the immune system is also influenced by sex steroids, such as oestrogen and progesterone, and stress hormones (ROONEY ET AL. 2003). Such suppressions in turn could be indirectly linked to the present variation in skull BMD (LIND ET AL.2004;ROUTTI ET AL.2008), as a suppressed immune system cannot protect the organism from harmful impacts (ROONEY ET AL.2003).Many other studies show a decrease in skull BMD during the period of high levels of pollution in different species, like grey seals, polar bears and Eurasian otters (Lutra lutra) (BERGMAN ET AL.1992;ROOS ET AL.2010; SONNE ET AL. 2004). In all studies, whether the BMD decreased, increased, the changes were probably caused by hormonal disturbances, which are possibly induced by PCB (ROUTTI ET AL.2008). An inhibition of the oestrogen level can affect the BMD (SAGGESE ET AL.1997) and the disruption of the thyroid homeostasis can also affect the bone homeostasis (ROUTTI ET AL.2008). The contaminants may suppress the circulating vitamin D levels (ROUTTI ET AL.2008), which affects the circulating calcium and phosphate levels.
4.4 Contaminants and effects
Different studies showed that PCB can potentially affect endocrine homeostasis, which can lead to changes in bones (HAAVE ET AL.2003;LETCHER ET AL.2010; SONNE ET AL.2004).
These results may suggest endocrine-related effects (LIND ET AL.2003,SONNE ET AL.2004), because PCB and DDT can bind at sexual steroid receptors and intensify or inhibit the reactions of the normal hormone cascade. This is supported by a study on harbour seals (Phoca vitulina) between 1981 and 2014, where healthy harbours seal skulls were compared to skulls with rather pathological changes and the healthy harbour seal skulls
showed an increase in bone density (PERTOLDI ET AL.2018). However, higher BMD levels do not necessarily indicate better bone quality or stronger bones (LIND ET AL.2000,2004;
PERTOLDI ET AL.2018).Despite higher BMD levels the bones may become brittle (LIND ET AL. 2000) if the increase in BMD is due to PCB contamination. For instance, PCB impaired bone strength and bone composition in rats (LIND ET AL. 2000). A study of LIND ET AL.(2004) demonstrated that the cortical part of the bone of female juvenile American alligators (Alligator mississippiensis) becomes larger, but loses in mass and the trabecular bone becomes smaller, but wins in mass. As a higher amount of the bones consisted of trabecular bone structures, the overall BMD rose (LIND ET AL.2004). Nevertheless, these changes lead to a porous bone structure as a result of differences in the estrogenic hormone balance. As similar results were found in this study, the ringed seals could be exposed to estrogenic compounds resulting in the increase of BMD, because such changes are characteristic of oestrogens (BREEN ET AL.1998). The same effects could be seen in terrestrial vertebrates like mice (BREEN ET AL.1998). Nonetheless, many publications show a loss of BMD related to an increase in contaminants (LIND ET AL.2000, 2003;ROOS ET AL.2010;SONNE ET AL. 2004), which could be a result of exposure to anti-estrogenic compounds (BERGMAN ET AL. 1992; LIND ET AL. 2004). A decrease in BMD leads to osteoporosis, which can have manifestations, such as alveolar bone loss (BERGMAN ET AL.1992). Different hypothesis can be proposed. First, as mentioned before, steroid hormones play an important role for the skeleton, but the bone parts can give different responses (HILL 1998; YILMAZ ET AL. 2005), as well as different species and their bones can give varying responses. Second, the thyroid function has been shown to be influenced by PCB and, therefore, can lead to changes in BMD. That is supported by several studies investigating the thyroid function, which showed changes in the BMD (SØRMO ET AL.2005;ROUTTI ET AL.2008).
In the present study, a positive correlation between PCB concentrations and BMD was observed. The Baltic ringed seals were exposed to higher PCB concentrations for a longer time compared with those from Greenland, which leads to stronger effects on the health status of Baltic ringed seal. This underlines the changes in the BMD of the Baltic ringed seals compared to those from Greenlandic waters.
Conclusions
Skull BMD seemingly increased with increasing contaminant concentrations in the Baltic ringed seals, which can be explained by sterility of females. The increasing BMD can be an indirect effect from samples including a large proportion of female seals affected by sterility.
Sterile females are expected to have higher BMD since they do not loose minerals during lactation as fertile females do. But this also happens, when contaminants lead to changes in the reproduction system and as a consequence, the females end up having a lower number of gestations. BMD levels during the pollution period (1958 – 1989) and the post- pollution period (1994 – 2019) were significantly higher than those found during the pre- pollution period (1829 – 1957), where the seals were not exposed to contaminants. The Baltic ringed seals show a significant higher BMD levels than Greenland ringed seals during the pollution and post-pollution period. Our large sample from Greenland ringed seal give a good baseline for natural variation in BMD in ringed seals, we could also show that males have higher BMD than in females and adults higher than juveniles. Changes in the BMD can be a proof for long-term damages.
Acknowledgement
We are grateful to the Swedish hunters and fishermen for their contribution to the Swedish environmental specimen bank at the Swedish Museum of Natural History, from which the skulls were provided as a loan. We are also grateful to the local hunter in West Greenland, who sent us samples of hunted seals, and the Natural History Museum of Denmark for skull maceration and preparation, as well as providing the samples for us. Thank you to Karin Harding who provided the contaminant data, to Carolin Philipp for designing the maps and to all helpers for taking samples during the field trips. We also thank the anonymous reviewers for their constructive and helpful comments, which improved our manuscript substantially. This study was financed by BONUS BaltHealth. The BaltHealth project has received funding from BONUS (Art. 185), funded jointly by the EU, Innovation Fund Denkmark (grants 6180-00001B and 6180-00002B), Forschungszentrum Jülich GmbH, German Federal Ministry of Education and Research (grant number FKZ 03F0767A), Academy of Finland (decision #311966) and Swedish Foundation for Strategic Environmental Research (MISTRA).
Appendix
Supplementary Material
Figure 10. Predicted total PCB content of ringed seals of the Gulf of Bothnia between 1897 and 2018. The blue area represents the distribution of the modelled PCB concentrations.
The dots show the exact values of 17 examined Baltic ringed seals (blue dots are males, red dots are females, black dot is unknown)
Table 3. Exact PCB concentration (ng/g lipid weight) of 17 Baltic ringed seals, which are examined in this study. Between 1976 and 2018 different ages and sexes were examined for 8 different PCB congeners. YearSex AgeAge class CB28CB52 CB101CB118CB153CB138+ 163 CB180 2008male 7 adult 17450 1600790 47000 28000 20000 2011male 4 juvenile4.5-1347220 140 19001300630 2011female8 adult 5.1-1537170 120 20001300840 2015female8 adult 15160 660 420 730044002600 2018femaleunknown. adult <5.2 44140 9224001400780 2018femaleunknownadult 4.5-14218450760 440 450 2018unknown unknownunknown<4.3 1989901000590 560 2018male unknownunknown5.5-1747170 190 20001200810 2018femaleunknownadult 3.6-11257861860 460 530 2018male unknownadult <5.3 44260 140 340021001200 2018male unknownadult 4.9-1565240 150 10000 57004000 1976female12adult 25300 1500560 600041002100 1977male 9 adult 75550013000 510050000 35000 16000 1980female12adult 19310 2300690 16000 10000 7600 1981female10adult 18290 1300510 550036001700 1983male 8 adult 7518008100170065000 43000 21000 1978female10adult 59180013000 450058000 38000 20000 “range” means value between LOD and LOQ, “<” means value below LOD
Chapter 2
Liver histopathology of Baltic grey seals (Halichoerus grypus)
over three decades
Published:
Schmidt, B., Sonne, C., Nachtsheim, D., Wohlsein, P., Persson, S., Dietz, R., Siebert, U.
(2020). Liver histopathology of Baltic grey seals (Halichoerus grypus) over three decades. Environmental International Special Issue 145, 106110
Liver histopathology of Baltic grey seals (Halichoerus grypus) over three decades Britta Schmidt1, Christian Sonne2, Dominik Nachtsheim1, Peter Wohlsein3, Sara Persson4, Rune Dietz2, Ursula Siebert1,2,*
1Institute for Terrestrial and Aquatic Wildlife Research, University of Veterinary Medicine Hannover, Foundation. Werftstraße 6, D-25761 Büsum, Germany
2Marine Mammal Research, Department of Bioscience, Aarhus University, Frederiksborgvej 399, P.O. Box 358, DK-4000 Roskilde, Denmark
3Department of Pathology, University of Veterinary Medicine, Bünteweg 17, D-30559 Hannover, Germany
4Department of Environmental Research and Monitoring, Swedish Museum of Natural History, P.O. Box 50007, SE-104 05 Stockholm, Sweden
* Address correspondence to U. Siebert, Institute for Terrestrial and Aquatic Wildlife Research, University of Veterinary Medicine Hannover, Werftstr. 6, D-25761 Büsum, Germany. Telephone: +49511-856-8158. E-mail: ursula.siebert@tiho-hannover.de.
Abstract
The liver plays an important role in the metabolism and elimination of endogenic and exogenic lipid-soluble compounds. Multiple studies have shown that polychlorinated biphenyls (PCB) and dichlorodiphenyltrichloroethane (DDT) lead to morphological changes in liver cells. The aim of the present study was therefore to analyse liver changes over time in Baltic grey seals (Halichoerus grypus) and to correlate these with historical PCB and DDT contaminations. A total of 191 liver samples were collected between 1981 and 2015 in the Gulf of Bothnia and northern Baltic Proper. Six histological features were evaluated, including portal mononuclear cell infiltration, random mononuclear cell infiltration, lipid granulomas, hepatocellular fat vacuoles, hepatic stellate cells and mild multifocal bile duct hyperplasia accompanied by portal fibrosis. Three of the six lesions showed a significant correlation with age. Furthermore, a positive correlation between portal mononuclear cell infiltration and mild multifocal bile duct hyperplasia was found. Additionally, lipid granulomas were significantly correlated with hepatic stellate cells. More importantly, hepatic stellate cells and mild multifocal bile duct hyperplasia were correlated with adipose tissue (blubber) concentrations of ƩPCB, measured in a subsample (N = 34) of all individuals. No correlation with lesions and ƩDDT concentrations were found. These results show that age is an important factor for the development of these liver lesions, but PCBs burden may be an influencing factor. This is in agreement with previous studies of marine mammals in the Baltic Sea as well as in the Arctic. We therefore conclude that not only age of the animals, but also exposure to PCBs should be taken into account when understanding and evaluating the current health status of Baltic grey seals.
Keywords: Baltic Grey Seal; Liver; Histopathology; PCB; DDT
