Anthropogenic environmental changes can serve as drivers for evolutionary responses in wild populations. To predict the long-term impact of anthropogenic changes on populations, it is crucial to understand the genetic effects caused by these disturbances. The Baltic Sea is considered to be one of the world’s most contaminated seas, and the increase of anthropogenic chemical pollution is a major threat to its ecosystems. This thesis assesses the impact of harbors and sewage treatment plants on physiological traits and genetic structure of resident populations of blue mussels at replicated sites in the Baltic Sea. The initial evaluation of the overall genetic pattern in blue mussel populations in the Swedish West Coast, the Baltic Proper and the Bothnian Sea found genetic differentiation between the three water basins and a low genetic differentiation within each basin, especially within the Baltic Proper. Despite the low genetic differentiation among blue mussels within the Baltic Proper, a parallel genetic differentiation associated with sewage treatment plant effluents was found in this basin. This included genomic regions with a high degree of differentiation between reference sites and sites affected by sewage plants effluent. This genetic differentiation is suggested to be due to post-dispersal selection acting in each generation. In contrast, no parallel genetic differentiation was associated with harbors. We identified five genomic regions in blue mussels, showing strong signs of selection, shared among three out of four replicated reference sites and sites affected by sewage effluents in the Baltic Proper i.e. Askö, Tvärminne and Karlskrona. An initial characterization of these genomic regions revealed functions related to immune and endocrine responses, oxidative stress and shell formation. Our results indicate that selection caused by sewage effluents involves multiple loci. The same genomic regions are found across different locations in the Baltic Proper but there are also unique genomic regions at each location. No genotoxic or histopathological effects were found among blue mussels from sewage effluent-affected areas but a higher frequency of histological abnormalities in the digestive gland were observed in mussels from harbors. / Evolutionära anpassningar till miljöstörningar i marina ekosystem: genetisk ekotoxikologi i Östersjön

The Baltic Sea is the most polluted sea in the world. Its hydrological conditions and ongoing eutrophication are a high threat for marine biodiversity and ecosystems. Additionally, eutrophication has negative effects on the wellbeing of countries and their societies in the Baltic Sea Region (BSR). Actions to mitigate eutrophication in the Baltic Sea have been implemented through on-land measures in the last 40 years. Although the improvement in the marine environment is notable, it happens very slowly. In order to combat eutrophication, there is a need for a combination of on-land and in-situ measures. In this study, blue mussel farming practices are presented as one of the in-situ measures to combat eutrophication in the Baltic Sea. Blue mussel farming has been implemented in Sweden since the 1980s and has potential to not only mitigate the amounts of nutrients that accumulate in the sea but also brings a circular approach to resource use. In this study, stakeholders from four different sectors that are closely related to blue mussel farming practices and Baltic Sea issues have been interviewed with the aim of making a comprehensive analysis of stakeholder perceptions of blue mussel farming practices in the BSR. Interviewed stakeholders represent four different sectors - academia, entrepreneurs, municipalities and NGOs. A comprehensive analysis of stakeholders’ perceptions on blue mussel farming practises from environmental, social and economic perspective is presented. All interviewed stakeholders are actors in Sweden and represent Swedish perspective on blue mussel farming activities. Potential causes for different perceptions across sectors are discussed.

Hydroxylated polybrominated diphenyl ethers (OH-PBDEs) are naturally produced in aquatic ecosystems e.g. by algae. Many OH-PBDEs have been observed to be highly bioactive and to cause adverse effects through several pathways, e.g. via disrupting oxidative phosphorylation (OXPHOS). The levels of some OH-PBDEs have increased in Baltic biota over the past decades. This may be associated with the nutrient enrichment of the Baltic Sea, which has favored growth of some of the OH-PBDE producers. Ceramium tenuicorne has been suggested to be a producer of OH-PBDEs in the Baltic Sea, which is supported by the results presented in this thesis. The levels of OH-PBDEs were observed to fluctuate greatly in C. tenuicorne over the summer season, and to correlate with the levels of pigments in the algae. However, the observed congener pattern in C. tenuicorne questioned theories regarding the mechanism of their biosynthesis. The results indicate a much more selective pathway for biosynthesis than previously suggested for the production of OH-PBDEs. One of the most abundant OH-PBDEs in C. tenuicorne, 6-OH-BDE137, has previously been observed to be toxic to bacteria, fungi, and crustaceans. Furthermore, Baltic gammarids seemed to change their feeding preferences towards less grazing on C. tenuicorne during the production peek of OH-PBDEs in the alga. This suggests that OH-PBDEs may serve as allelochemical defense agents for C. tenuicorne. The transport and fate of OH-PBDEs through a Baltic food chain was also studied, including C. tenuicorne, Gammarus spp., three-spined stickleback (Gasterosteus aculeatus), and perch (Perca fluviatilis). A small portion of the OH-PBDEs were observed to be methylated in the alga, or by associated bacteria. The methylated OH-PBDEs biomagnified in the food chain up to perch, in which they were converted back to the OH-PBDEs via demethylation. The OH-PBDEs and their methylated counterparts were also partially debrominated in the food chain, which resulted in high concentration of 6-OH-BDE47 in the perch. This congener is the most toxic OH-PBDE with regards to OXPHOS disruption. Another biotransformation of OH-PBDEs was identified in Baltic Sea blue mussels (Mytilus edulis). High concentrations of OH-PBDEs were conjugated with lipophilic moieties, e.g. fatty acids. This increases the residence time of the OH-PBDEs in the mussels. Mussels have been suggested to conjugate steroids with fatty acids as a means to regulate hormone levels. The conjugation of OH-PBDEs to fatty acids may occur due to intrusion into this pathway. Methods were developed to include quantification of conjugated OH-PBDEs in the analysis of mussels. OH-PBDEs were also quantified in blood from Baltic Sea grey seals (Halichoerus grypus). Seals originating from the Baltic proper were observed to be more highly exposed to 6-OH-BDE47 than seals from the Gulf of Bothnia. However, the levels of OH-PBDEs were generally low. A major effort was invested into securing these results, including development of a new analytical method. Blood obtained from dead seals is a difficult matrix for quantification of OH-PBDEs, and previous attempts using an established method yielded unsatisfactory results. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

This thesis focuses on toxicity and occurrence of hydroxylated polybrominated diphenyl ethers (OH-PBDEs) in Baltic Sea biota. The aims were to assess OH-PBDEs potency for disruption of oxidative phosphorylation (OXPHOS) and determine their and related compounds exposure in Baltic blue mussel, herring and long-tailed duck. A method for analysis of OH-PBDEs in herring and long-tailed duck plasma was also evaluated. Relevant OH-PBDEs were tested in vitro for OXPHOS disruption, using a classic rat mitochondrial respiration assay and a cell mitochondrial membrane potential assay. All compounds were found to disrupt OXPHOS either by protonophoric uncoupling and/or via inhibition of the electron transport chain. 6-OH-BDE47 and 6-OH-BDE85, were identified as particularly potent OXPHOS disruptors. Strong synergism was observed when OH-PBDEs were tested as a mixture corresponding to what is present in Baltic blue mussels. Baltic blue mussel is main feed for several species of mussel feeding sea ducks which have decreased dramatically in numbers. To assess long-tailed ducks exposure to brominated substances, liver tissue from long-tailed ducks wintering in the Baltic Sea and blue mussels were analysed. The result confirms that long-tailed duck are exposed to OH-PBDEs via their diet. However, low concentrations were found in the duck livers, which suggest low retention of these compounds despite daily intake. How the nutritional value of blue mussels as feed for sea ducks are affected by OH-PBDE exposure still needs further studies. Other species of sea ducks foraging on Baltic blue mussels during summer months can also be more exposed due to seasonal variation in primary production. Herring sampled in the Baltic Proper and Bothnian Sea, were found to contain OH-PBDEs and high levels of their methylated counterpart, MeO-PBDEs. As demethylation of MeO-PBDEs is known to occur in fish, MeO-PBDEs may pose as additional source for more toxic OH-PBDEs in herring and their roe. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.</p>

Denna studien har gjorts på blåmusslor, Mytilus edulis trossulus, för att undersöka effekter av det antidepressiva läkemedlet Citalopram på blåmusslans fysiologiska funktioner. Blåmusslan är en vanligt förekommande art i Östersjön där den fungerar som en nyckelart som; bidrar till den biologiska mångfalden, är långt ner i näringskedjan och är anpassningsbar. Dessa egenskaper gör den även lämpad som testorganism i denna studie. Citalopram är ett av Sveriges vanligaste läkemedel för att behandla psykisk ohälsa och den verkar genom att blockera återupptaget av serotonin i hjärnan och ingår i läkemedelsgruppen serotoninhämmare (SSRI). Serotonin påverkar flera olika funktioner i kroppen såsom rörelseförmåga, sinnesstämningar och mättnadskänsla. Läkemedel inklusive Citalopram återfinns i sjöar och vattendrag, detta för att läkemedel inte renas bort i våra reningsverk och för att det idag inte finns några krav på läkemedelsrening i svenska avloppsreningsverk. Läkemedel tillverkas för att vara aktiva vid låga koncentrationer vilket gör att de även har en negativ effekt på marina organismer. I denna studie har vi analyserat effekter av Citalopram på blåmusslans fysiologiska funktioner genom tre analyser, filtrering, rörelse och produktion av byssustrådar i tre olika koncentrationer (0 ng/l, 20 ng/l och 200 ng/l). Analyserna utfördes under två tidsperioder, exponering av läkemedel under 96 timmar samt remediering utan läkemedel under 72 timmar. Studien visar en signifikant skillnad i filtrering (P-värde = 0.04416) mellan grupperna under exponeringen där fler blåmusslor som var exponerade för 200 ng/l visade en lägre filtreringsaktivitet (fler individer var helt stängda). Under remedieringen observerades en signifikant högre mortalitet bland blåmusslor som exponerats för 200 ng/l (P-värde = 0.01347). Blåmusslor exponerade för 200 ng/l Citalopram visade även en lägre produktion av byssustrådar (P-värde= 0.02702). I den här studien kan vi se att läkemedlet Citalopram har en negativ påverkan på flera av blåmusslans fysiologiska funktioner. / The aim of this study was to investigate the effects of the antidepressant drug Citalopram on blue mussels, Mytilus edulis trossulus, in the Baltic Sea. The blue mussel is a common species that is considered a key species in the Baltic Sea as it contributes to biodiversity, and is adaptable which makes it suitable as a test organism in this study. Selective serotonin reuptake inhibitors (SSRIs) are the most commonly prescribed antidepressants and a common SSRI substance is Citalopram. This substance is one of Sweden's most common drugs for treating mental illness. Serotonin affects several different functions in the body such as movement, mood and saturation. Many pharmaceuticals including Citalopram are found in lakes and streams, this is because pharmaceuticals are not purified in our wastewater treatment plants and there is no requirements for filtration of pharmaceuticals in Swedish wastewater treatment plants as of today. Pharmaceuticals are manufactured to be active even at low concentrations, a negative side effect of this is that it is also active in the environment causing negative effects on marine organisms. In this study, we analyzed the effects of Citalopram on the physiological functions of the blue mussel by three analyzes; filtration, movement and production of byssus threads using three different concentrations (0 ng /l, 20 ng/l och 200 ng/l) of Citalopram. The analyzes were carried out over two time periods, exposure of drugs for 96 hours and remediation without drugs for 72 hours. The study shows a significant difference in filtration (P-value = 0.04416) between the groups during exposure, where more blue mussels that were exposed to 200 ng/l showed a lower filtration activity (more individuals completely closed). During the remediation, a significantly higher mortality was observed among blue mussels exposed to 200 ng/l (P value = 0.01347). Blue mussels exposed to 200 ng/l Citalopram also showed lower production of byssus threads (P value = 0.02702). In this study we can see that the drug Citalopram has a negative effect on several of the blue mussel's physiological functions.

L’épigénétique se rapporte à un ensemble de mécanismes qui modifient l'expression des gènes sans modifier la séquence nucléotidique sous-jacente, produisant une large gamme de variations phénotypiques et répondant aux fluctuations de l'environnement externe ou interne. La méthylation de l'ADN est le processus épigénétique le plus étudié dans les génomes nucléaires des mammifères. Il existe toutefois des lacunes significatives dans la littérature concernant la méthylation de l'ADN mitochondrial (ADNmt), surtout chez les invertébrés. Les bivalves constituent un modèle particulièrement intéressant pour étudier l’épigénétique mitochondriale ou « mitoépigénétique » puisqu’ils possèdent un système de transmission uniparentale double (DUI) de leur mitochondrie, par lequel les mâles héritent des ADNmt paternel (ou mâle ; M) et maternel (ou femelle ; F). La présente étude avait pour objectif de confirmer l’existence de méthylation dans l’ADNmt de la moule bleue (Mytilus edulis), une espèce à DUI. Notre étude a permis de localiser, par immunofluorescence, des cytosines (5mC) et des adénines (6mA) méthylées, ainsi que des méthyltransférases spécifiques aux adénines et aux cytosines, au sein des mitochondries de cette espèce. Ces résultats sont appuyés par une détection de 5mC et de 6mA dans l’ADNmt par digestions enzymatiques, et confirment la présence de méthylation de l'ADNmt chez M. edulis. Nos résultats en immunofluorescence ont également dévoilé un lien entre la présence de 5mC et le stade de développement des gamètes mâles, c’est-à-dire que les gamètes immatures (spermatides) étaient tous méthylés (au niveau mitochondrial) alors qu’une faible proportion de gamètes matures (spermatozoïdes) présentait ce statut de méthylation. Ceci vient corroborer nos résultats enzymatiques, lesquels ont démontré une plus grande variabilité de méthylation entre les mâles qu’entre les femelles. Enfin, nous avons détecté, par séquençage du méthylome de l’ADNmt, un pic de 5mC en contexte non-CpG conservé entre les ADNmt M et F chez les mâles au sein de la région de contrôle de la réplication, soutenant les résultats d’études antérieures chez les vertébrés. Notre étude est la première à démontrer la présence de méthylation dans l’ADNmt d’une espèce DUI, et met en lumière le rôle potentiel de la méthylation de l'ADN dans leur système de transmission mitochondriale. / Epigenetics refers to a set of mechanisms that modify gene expression without altering the underlying nucleotide sequence, producing a wide range of phenotypic variations, and responding to the external or internal environmental fluctuations. DNA methylation is the most extensively studied epigenetic process in mammalian nuclear genomes. However, there are significant gaps in the literature concerning mitochondrial DNA (mtDNA) methylation, especially in invertebrates. Bivalves are a particularly interesting model for studying mitochondrial epigenetics or “mitoepigenetics”, as they possess a doubly uniparental inheritance (DUI) system of their mitochondria, whereby males inherit both paternal and maternal mtDNAs. The aim of this study was to confirm the existence of methylation in the mtDNA of the blue mussel (Mytilus edulis), a DUI species. Using immunofluorescence, we localized methylated cytosines (5mC) and adenines (6mA), as well as adenine- and cytosine-specific methyltransferases, in mitochondria of this species. These results are supported by the detection of 5mC and 6mA in mtDNA by enzymatic digestions, and confirm the presence of mtDNA methylation in M. edulis. Our immunofluorescence results also revealed a link between the presence of 5mC and the developmental stage of male gametes, i.e. immature gametes (spermatids) were all methylated in their mitochondria while only a small proportion of mature gametes (spermatozoa) showed this methylation status. This corroborates our enzymatic results, which demonstrated greater variability in cytosine methylation status among males than among females. Finally, we detected, by mtDNA methylome sequencing, a 5mC peak in non-CpG context conserved between the paternal and maternal mtDNA in males in the region responsible for control of replication (control region), supporting the results obtained in previous studies on vertebrate. Our study is the first to demonstrate the presence of mtDNA methylation in a DUI species, and highlights the potential role of DNA methylation in their mitochondrial transmission system.