| 1. |
- Castro, Mafalda
(författare)
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Chlorinated Paraffins: improved understanding of their bioaccumulation and toxicity in Daphnia magna
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Chlorinated paraffins (CPs) are industrial chemicals, mainly used as flame retardants, plasticizers and metal cutting fluids. Their production has reached historically high levels in the last decade, with an annual production exceeding one million tonnes. In 2017, short chain chlorinated paraffins (SCCPs) were regulated due to their Persistent, Bioaccumulative and Toxic (PBT) properties, while medium and long chain chlorinated paraffins (MC and LCCPs) were suggested as alternatives. The high hydrophobicity of CPs, which complicates bioaccumulation and aquatic toxicity testing, has hindered proper hazard identification by regulatory authorities. This project was initiated in response to the insufficient understanding of bioaccumulative and toxicological properties of these chemicals, which have even surpassed the environmental levels of legacy Persistent Organic Pollutants (POPs) in certain regions.The research presented in this thesis, contributes to filling these knowledge gaps by adapting methods for reliable bioaccumulation and aquatic toxicity assessment. In Paper I, passive dosing, traditionally used for other highly hydrophobic compounds, was adapted and validated for CPs. SC, MC and LCCPs partitioned from silicone into water and, when the crustacean Daphnia magna was introduced into the test system, the CPs were observed to be effectively taken up by the test organism. This passive-dosing approach was further used in Paper II, to investigate the bioconcentration and bioaccumulation potential in D. magna. All tested CPs were found to bioaccumulate in daphnids, including highly hydrophobic, long chained CP congeners. The two most bioaccumulative CPs in Paper II (CP-52 and Huels70C) were thereafter used in a chronic toxicity study (Paper III) and significantly decreased population growth and disrupted fatty acid metabolism of D. magna. Finally, in Paper IV, liposome-mediated delivery of chemicals to aquatic biota was adapted for the first time for organic contaminants, including CPs. This approach yielded stable body burdens of the tested chemicals in D. magna and allowed for kinetic and toxicity assessments.Overall, two alternative bioaccumulation and aquatic toxicity testing methods were successfully adapted for technically challenging (industrial) chemicals. These methods allowed the determination of endpoints of scientific and regulatory interest, such as the high bioaccumulation and toxicity potential of CPs, but were also used to demonstrate their metabolic disruption potential in small crustaceans.
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| 2. |
- Ribbenstedt, Anton, 1986-
(författare)
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Toxicometabolomics and biotransformation product screening in single zebrafish embryos
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Over the last decade environmental agencies worldwide have escalated their work to phase out animal testing for the purposes of chemical regulation. Meanwhile the number of commercially available chemicals and the requirements for hazard assessments have both increased, creating a large need for substitution of traditional in vivo assays with in vitro tests. One example of this is the replacement of the OECD acute toxicity test of adult fish (test guideline [TG] 203) with zebrafish embryos (TG 236). With new insights into the toxicological properties of chemicals, the demand on these replacement tests is also changing character with a shifted focus towards mechanistic understanding of toxicity. The omics sciences encompass a group of analytical methods which have proven to be very powerful for unveiling of mechanistic information of biochemical processes. Metabolomics is one of the younger members of this family and entails the large-scale analysis of endogenous metabolites and their perturbation in living organisms. The overall objective of this thesis was to develop modifications to the TG236 OECD assay to obtain omic data suitable for use in chemical hazard assessment. To achieve this goal, we started by developing a targeted and non-targeted metabolomics workflow and evaluated the performance of the two types of analysis (Paper I). We also evaluated the efficiency of three signal drift correction approaches, which is an important step in data quality improvement for non-targeted analysis, and reported previously unlisted biochemicals present in NIST reference material. In Paper II we applied the workflow in Paper I to a newly developed, in-plate extraction method for single zebrafish embryos which were exposed to the pharmaceutical and environmental pollutant propranolol. Data processing workflows were developed to overcome challenges arising from the occurrence of the exposure compound and its biotransformation products (or in-source fragments of these) in the final multivariate statistical models, obscuring their outputs and prediction capabilities. Once developed, the workflow allowed us to detect several probable modes-of-action of propranolol in zebrafish, and link them to apical endpoints in the embryos, which were then confirmed through thorough literature searches. The final output from the models was ultimately used to determine a benchmarking dose based on metabolomics endpoints for the first time. In Paper III, the data processing workflow from Paper II was modified to capture propranolol biotransformation products. A total of 7 structures were identified, of which 4 were confirmed with authentic standards, all from the datasets generated in Paper II. In Paper IV we combined the workflows from Papers I, II and III and applied them to the pharmaceutical carbamazepine, which occurs at high concentrations in wastewater treatment plant effluents. Through this approach we determined several modes-of-action for carbamazepine in zebrafish embryos and measured biotransformation products in both embryos and exposure water. Overall, this thesis demonstrated the possibilities of high-throughput chemical mode-of-action determination in single zebrafish embryos using targeted and non-targeted liquid chromatography mass spectrometry, data filtering scripts and multivariate statistics while simultaneously screening for biotransformation products.
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| 3. |
- West, Johannes, 1991-
(författare)
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Degradation Pathways of Dimethylmercury in Natural Waters
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mercury (Hg) is a naturally occurring toxic trace metal whose release into the environment has been (and still is) exacerbated by human activities. The bioavailability of Hg is tied to its chemical speciation, with monomethylmercury (MMHg) being the primary form of Hg that bioaccumulates and biomagnifies. At the top of some food webs, MMHg can reach harmful levels. In addition to MMHg, a second methylated Hg species, dimethylmercury (DMHg), is found in aquatic environments. Dimethylmercury is especially abundant in the oceans, where it could potentially act as an important source of MMHg. The stability of DMHg in natural waters and which biogeochemical processes may be important for its degradation are poorly constrained. This knowledge gap could partially be related to the extreme toxicity and the volatility of DMHg, making it challenging to handle in the laboratory.Through experimental studies, this thesis work has aimed to increase the understanding of DMHg cycling in the aquatic environment. Focusing on the stability and degradation of DMHg, abiotic pathways of DMHg degradation were explored. In Paper I, DMHg degradation via a novel sulfide-mediated pathway was demonstrated for both particulate and dissolved sulfide phases. Key aspects of the process were investigated through the manipulation of pH and DMHg:S ratios. In Paper II, the previously disputed process of aqueous photochemical decomposition of DMHg was confirmed for various natural and artificial water types. This process was studied further in Paper III by investigating the impact of dissolved organic matter and chloride on DMHg photodecomposition rates and the involvement of photochemically produced reactive intermediates. Monomethylmercury was the primary product of both sulfide-mediated decomposition and photodecomposition of DMHg. Rate calculations (Paper I and II) and comparison with MMHg photodecomposition (Paper II and III) confirm that both sulfide-mediated decomposition and aqueous photodecomposition of DMHg could be environmentally significant. Paper IV presents methods and considerations for laboratory work with DMHg, providing insights into both experimental and analytical aspects. This work demonstrates that DMHg can be analyzed together with MMHg and that significant safety risks of working with DMHg can be largely avoided. These insights are important as future work on DMHg is necessary to elucidate its role in aqueous Hg cycling in general and for MMHg cycling in particular.
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| 4. |
- Holliland, Per B., 1984-
(författare)
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Trophic interactions and behaviour : Studies relevant to a Baltic Sea biomanipulation
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The main theme of this thesis is the interactions of animals with the environment and each other. The thesis was written within the framework of a biomanipulation project “Pikeperch in Himmerfjärden”. With the aim to investigate possible trophic pit-falls, give the manipulation the best possible start, and find ways to monitor the progression of the manipulation. In Paper I the diet of the invader cladoceran Cercopagis pengoi is analysed with stable isotopes; conducted prior to stocking. C.pengoi has a preference for large copepods, indicating possible competition with fish. Paper II investigates the behavioural differences between pikeperch fingerlings reared in different environments (pond vs. tank). Results suggest that fish reared in semi-natural ponds are more likely to survive directly after stocking. In Paper III and IV, the diel vertical migrations (DVM) of copepods are in focus. In Paper III the migrations of two copepod species: Acartia spp. and Eurytemora affinis are studied over season and life stage. The amplitude of migration was found to increase with ontogeny for both species, indicating evasion of visual predators. Paper IV examines the varying migratory patterns of adult female E. affinis finding that these animals migrate more actively when feeding conditions deteriorate and growth decreases. The overall conclusions of the thesis are that behavioural, not only direct trophic interactions are key when studying ecosystems.
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| 5. |
- Martella, Giulia, 1993-
(författare)
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DNA adductomics: Method development and integration in biological effect monitoring
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Environmental omics can provide informative biomarkers for identifying pathologies or physiological responses to environmental change. Nucleic acid adductomics, one of the newest omics techniques, is particularly well suited for assessing exposure and effects of environmental contaminants because the technique can capture modifications at the (epi)genome level. However, identifying potential adduct-biomarkers is challenging, especially in non-model organisms. Therefore, the primary aims of this study were to improve the methodology and explore the applicability of nucleic acid adductomics in environmental monitoring of the biological effects of contaminants, using amphipods as sentinel species.From a methodology perspective, this thesis presents a gradual progression in screening and detecting DNA modifications. The mass spectrometry (MS) based approach for identifying candidate DNA adducts utilized the characteristic neutral loss of deoxyribose. In Paper I, the data generated from high resolution MS in Full Scan-Data independent Acquisition mode were analyzed manually using the fragmentation pattern of nucleoside adduct ions. In Paper II, processing time and efficiency were significantly improved by the development of nLossFinder software. Further, TraceFinder software was combined and used to evaluate and quantify detected adducts (Paper III). Another important development was a simultaneous approach for analysis of both DNA and RNA adducts in a single MS run presented in Paper IV. The analysis of amphipod samples based on the existing approaches and methods developed in this thesis showed that reproductive pathologies identified by the microscopic analysis were associated with specific DNA modifications that can be used to classify field-collected individuals according to their health status (Paper I). Moreover, epigenetic marks were the most influential adducts for this classification. Finally, the adduct profile of amphipods differed between the areas with relatively high and low contamination loads based on PAH and trace metal concentrations in the sediment (Paper III). Also, significant correlations were found between DNA adducts and specific contaminants.These method developments and findings uniquely contribute to the field of environmental omics, providing tools for data processing and demonstrating the applicability of nucleic acid adductomics in environmental health research and biological effect monitoring.
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| 6. |
- Ek, Caroline, 1985-
(författare)
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Towards understanding stable isotope signatures in stressed systems
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Stable isotope analysis (SIA) is a valuable tool in ecotoxicology because δ13C and δ15N may provide insights into the trophic transfer of contaminants in a food web. The relationship between a species’ trophic position (TP, determined from δ15N) and internal concentration of biomagnifying contaminants can be established and used for regulatory purposes. However, the exposure of organisms to xenobiotics incurs physiological costs, and the stable isotope signature of a consumer reflects not only diet but also a physiological state. The latter raises questions regarding the interpretation of stable isotope signatures in contaminated areas. Therefore, the aim of this Thesis was to evaluate the behaviour of consumers’ stable isotope signatures in stressed systems, with a primary focus on the effects of environmental contaminants.In paper I, the physiological costs of chemical exposure were found to alter incorporation rates of dietary nitrogen and carbon in a consumer by influencing both growth and metabolic turnover, with resulting changes in isotope signatures relative to a control system. In paper II, the diet-consumer discrimination factors for 15N and 13C were confirmed to increase under chemical exposure mediated via increased metabolic costs. However, the physiological response was low and translated into only minor shifts in the δ13C and δ15N. The predictability of exposure effects on the stable isotope signature was demonstrated in paper III, in which animals exposed to a chemical with a known mode of action presented expected effects on elemental composition, body size, biomarkers of oxidative stress and the stable isotope signatures. Moreover, consumers’ oxidative balance was found to be related to their δ15N values, thus providing evidence of the kinetic isotope effect on the oxidative status. However, despite the alterations in stable isotope signatures observed in laboratory settings (papers I-III), the effect of xenobiotics on the TP estimates was nil or minor in the field-collected animals. Moreover, the TP values were not significantly different between the animals in the contaminated and the reference habitats because of the high overall uncertainties in the TP estimates (paper IV). Also, the TP estimates based on δ15N in bulk material were more similar between the contaminated and the reference systems than TP estimates based on δ15N values in amino acids. Therefore, the latter method appears more sensitive towards xenobiotics (and, possibly, other environmental stressors) and thus less suitable for TP assessment in contaminated areas.This Thesis improved the overall understanding of the applicability of SIA in stressed systems by establishing relationships between various exposure regimes, physiological responses and the stable isotope signatures in consumers. In model species at low trophic levels, the exposure to xenobiotics was found to significantly affect δ13C and δ15N values, which can be expected whenever physiological responses are detected. However, because of the overall high uncertainty in TP estimates, no significant differences between contaminated and control systems were detected, although the estimated TP were consistently higher in the contaminated systems. Future research should focus on higher trophic levels, in which effects of a greater magnitude can be expected. Moreover, the effects in entire food webs should be addressed rather than single prey–consumer relationships as well as other environmental variables that may contribute to the stable isotope variability in and among systems under various environmental pressures.
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| 7. |
- Furuhagen, Sara, 1985-
(författare)
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Application and interpretation of biomarkers in ecotoxicology - from molecular to individual level responses
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The use of biomarkers is considered a promising alternative, or complement, to traditional ecotoxicological assays. Toxic effects are often initially manifested at the molecular or biochemical level, biomarkers are therefore used as sensitive indicators of toxic exposure. Ideally, biomarkers would also indicate reduced fitness and possible later effects at the individual or population levels. However, implementing biomarkers in ecotoxicology is challenging and few biomarkers have an established connection to reduced individual fitness. The aim of this thesis was to increase the value and improve the interpretation of biomarker responses in ecotoxicological studies by examining the impact of confounding factors and the relationship between oxidative biomarkers and reproductive effects in crustaceans.The sensitivity of biomarkers was confirmed in paper I as toxic effects of pharmaceuticals with conserved drug target orthologs were observed at the molecular and biochemical levels both earlier and at lower concentrations than effects on mortality and reproduction. No toxic effects were observed for the pharmaceutical without identified drug target orthologs, thus stressing the importance of considering toxic mechanisms and being aware of the most likely target when evaluating toxic effects also in non-target species. Many xenobiotics and environmental stressors interfere with oxidative processes, making oxidative biomarkers interesting to study in ecotoxicology and stress ecology. Still, feeding rate was identified as a confounding factor for antioxidant capacity (assayed as oxygen radical absorbance capacity, ORAC) and lipid peroxidation in ecotoxicological studies (paper II). However, ORAC normalized to protein was independent of altered feeding rates, hence it can be applied as a suitable exposure biomarker without considering alterations and effects of feeding rate. The connection between reproduction and oxidative stress is dual, as reproduction both can be inhibited by oxidative stress and induce pro-oxidative processes. Further, a positive association was found between ORAC and the occurrence of embryo aberrations in the benthic amphipod Monoporeia affinis (paper III). An association between antioxidant defense and reproduction was also observed for Daphnia magna (paper IV). Threshold values for identification of exposed individuals and prediction of possible later reproductive effects were established for ORAC.This thesis has contributed to diminishing some of the knowledge gaps limiting the use of oxidative biomarkers in ecotoxicology, by contributing to increased understanding of how oxidative biomarkers relate to important life-traits. Moreover, ORAC has been identified as a suitable biomarker of not only exposure, but also reproductive effects. Future research should continue to establish connections between biomarker responses and effects at higher levels, and focus on providing defined threshold values to enable predictions about later effects.
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| 8. |
- Furuhagen, Sara
(författare)
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Integrative approaches in ecotoxicological testing: Implications for biomarker development and application
- 2013
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Ecotoxicology aims to understand toxic effects of chemicals in the environment. Effects can be observed at different levels of the biological organization, from molecular to ecosystem level. Biomarkers on molecular and biochemical levels are used in ecotoxicology as early warning signals of chemical exposure, possible toxic effects and underlying mechanisms. As methods and technologies improve, more biomarkers are being implemented in ecotoxicological studies, due to the general interest in early detection and thus efficient prevention of environmental risks. However, to be of value in ecotoxicological assessment, a connection between biomarker response and effects at higher levels of biological organization should be established. Also, baseline variability for the biomarker in question as well as response to natural fluctuations of environmental factors should be evaluated.The aim of this thesis was to increase value and understanding of biomarkers in ecotoxicological assessment by (1) linking responses across different levels of biological organization, and (2) gaining better understanding of the relative importance of ecological and physiological factors affecting oxidative biomarkers.Paper I is focused on evolutionary conserved drug targets and the toxicity of pharmaceuticals for non-target organisms. The main conclusion from this study is that pharmaceuticals with conserved drug targets in non-target organisms have a higher toxicity than pharmaceuticals for which drug-targets have not been identified in the species. The effects were evaluated using end points at molecular, biochemical and individual levels. Consistent with the expected higher sensitivity of molecular and biochemical end points, the effects on the low-level biomarkers were observed at lower concentrations than at the individual level. Paper II is focused on delineating effects of feeding and toxic exposure on oxidative biomarkers commonly used in ecotoxicology. The results are in agreement with the theory of caloric restriction that links enhanced caloric intake to increased pro-oxidative processes in animals. In our experiments with the cladoceran Daphnia magna, we observed positive effects for both antioxidant capacity and oxidized lipids in response to enhanced feeding rates. This have implications for the use of oxidative stress biomarkers in ecotoxicology as many substances have inhibitory effects on feeding rate and thus, changes in oxidative biomarkers can result from the altered feeding rate rather than other toxic mechanisms. Therefore, possible changes in feeding rate should be assessed when conducting exposure experiments or interpreting field data in studies employing oxidative stress biomarkers. However, it was concluded that the ratio between antioxidative capacity and protein content was independent of feeding rate. Thus, this biomarker is suitable for xenobiotic exposure in D. magna. This thesis have contributed to better understanding of molecular and biochemical biomarkers in ecotoxicological studies in regard to the connections between effects at different biological levels and confounding factors in biomarker response.
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| 9. |
- Gerdes, Zandra, 1989-
(författare)
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Exploring the ecotoxicity of microparticle debris
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The global distribution and anticipated increase of environmental microplastic (MP) pollution are concerning. However, while the impacts of macroplastic litter on wildlife are apparent, we know relatively little about the MP hazard potential. Moreover, the current ecotoxicological methodology is inadequate for solid waste particles and MP hazard assessment because it fails to distinguish particle and chemical effects. This thesis improves our understanding of the particle effects of MP relative to other microparticles.First, a comparative analysis of effect studies on MP and mineral particulates across different biological organisation levels revealed high similarities in responses between these materials (Paper I). At the suborganismal levels, the similarity in the effect concentrations suggests shared particle effect mechanisms. At the higher levels, however, MP induced more severe impacts, possibly due to chemical leaching. Moreover, the highly variable MP effect concentrations motivated exploring the role of polymer properties and ageing status on MP effects; these aspects were addressed in Papers II-III.In Paper II, the possibility of MP acting as a vector of contaminants was evaluated, showing enhanced transport of highly hydrophobic organic contaminants (HOC) at very high HOC and MP concentrations. However, observing it at environmentally relevant contaminant levels would be unlikely. Paper III compared behavioural and physiological responses in benthic amphipods to MP exposure using different polymers (polystyrene and polyethylene terephthalate) and clay as a non-plastic reference particle. The amphipods avoided sediments with high concentrations of the added material regardless of the material type, including aged and virgin MP and clay. Solid waste, including MP, co-occur with various suspended solids in aquatic environments; therefore, the natural solids can serve as reference material when evaluating the MP particle effect. In Paper IV, a novel method for testing MP effects in mixtures with reference particles was proposed. In the exposure experiment with daphnids, the method was used to derive hazard thresholds for the MP contribution to suspended matter conditional on the total suspended solid concentration in the water. Together, these studies add to our understanding of MP-biota interactions and suggest that similarly sized MP and natural particulates share similar particle effects. However, MP might have a higher potential as vectors of chemical contaminants, which needs to be further evaluated in environmentally relevant settings.
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| 10. |
- Motwani, Nisha H., 1982-
(författare)
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Trophic complexity of zooplankton–cyanobacteria interactions in the Baltic Sea : Insights from molecular diet analysis
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Blooms of nitrogen fixing cyanobacteria (NFC) occur in many freshwater and marine systems, including the Baltic Sea. By fixing dissolved nitrogen, they circumvent general summer nitrogen limitation, while also generating a supply of novel bioavailable nitrogen for non-diazotrophic primary producers and ultimately supporting secondary production. Elucidating trophic links between primary consumers and NFC is essential for understanding role of these blooms for secondary production. However, until recently, there was no reliable method to quantify individual prey species for zooplankter feeding in situ. The development of PCR-based methods to detect prey-specific DNA in the diet of consumers, including microscopic animals, allows identification and quantification of trophic linkages in the field.Using molecular diet analysis in combination with egg production measurements, biochemical markers of growth and condition; and stable isotope approach, we explored a possibility to determine (1) whether cyanobacteria are grazed and assimilated by mesozooplankters (Papers I and II), (2) which species/groups are particularly efficient consumers of cyanobacteria (Papers II and III), and (3) how feeding on cyanobacteria affects zooplankton growth and development (Paper I and III). Taken together, these laboratory and field observations, provided evidence that NFC contribute to feeding and reproduction of zooplankton during summer and create a favorable growth environment for the copepod nauplii (Paper I). The favorable growth conditions for juvenile copepods observed during NFC blooms were hypothesized to be mediated by picoplankton that take up bioavailable nitrogen exuded from cyanobacterial cells. This hypothesis found support in Paper II that provided quantitative estimates for the direct picocyanobacteria → mesozooplankton pathway, with highest weight-specific consumption observed in nauplii. Further, using field observations on zooplankton and phytoplankton development during a growth season in the northern Baltic proper, we found that NFC nitrogen is assimilated and transferred to zooplankton via both direct grazing and indirectly through grazing on small-sized phyto- and bacterioplankton (Paper III). Finally, these and other findings emphasizing the importance of NFC for Baltic Sea secondary production during growth season were synthesized to show that diazotrophic nitrogen enters food webs already at bloom initiation (Paper III) and is transferred via multiple pathways to pelagic and benthic food webs and, ultimately, to fish (Paper IV).
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