Nodularin (NODLN) is a pentapeptide produced by the filamentous cyanobacterium Nodularia spumigena that is a bloom-forming species in the Baltic Sea. NODLN is an intracellular hepatotoxin, which can have a negative effect on aquatic life including fish. Toxins are released into the water when cells are lysing, e.g. during a decaying bloom. N. spumigena filaments have previously been shown to have a negative effect on perch egg development and perch larval survival. Coastal fish such as perch (Perca fluviatilis) have suffered from recruitment problems in the Baltic Sea the last decades. However, little is known about the impact of toxic cyanobacteria on juvenile perch. In the autumn of 2007, 1+ perch were exposed, during 29 days to either whole live cells (WC) or a crude extract (CE) of broken N. spumigena cells. Chlorophyll a concentrations in the aquaria were 50 µg L -1. Perch were fed chironomidae larvae twice a day. Unexposed perch either fed (CoF) or without food (Co) served as controls. Length and weight of perch were measured at onset and termination of experiment. NODLN content was measured in N. spumigena filaments, crude extract and perch liver samples using liquid chromatography-mass spectrometry (LC-MS). Total lipids (TL) were extracted and quantified from whole-body lyophilised perch excluding livers. No significant differences for length and weight of perch were found between treatments and fed control. NODLN was detected in the crude extract samples, while no NODLN was detected in the perch livers. Moreover TL determination revealed no significant differences between treatments and fed control. Nodularia spumigena did not affect perch in this experiment, probably due to that the critical period of the first year for the perch was exceeded. Therefore, 1+ perch was not as susceptible to the cyanobacterium as eggs, larvae and younger juveniles of fish found in the literature. Perch liver did not contain NODLN, thus either the toxin was detoxicated with no recorded energetic cost or it was not ingested. The variables studied here did not show any effects of NODLN. However, other chemical methods such as enzymatic activity may disclose effects of NODLN.

<p>Nodularin (NODLN) is a pentapeptide produced by the filamentous cyanobacterium <em>Nodularia spumigena</em> that is a bloom-forming species in the Baltic Sea. NODLN is an intracellular hepatotoxin, which can have a negative effect on aquatic life including fish. Toxins are released into the water when cells are lysing, e.g. during a decaying bloom. <em>N. spumigena </em>filaments have previously been shown to have a negative effect on perch egg development and perch larval survival. Coastal fish such as perch (<em>Perca fluviatilis</em>) have suffered from recruitment problems in the Baltic Sea the last decades. However, little is known about the impact of toxic cyanobacteria on juvenile perch. In the autumn of 2007, 1+ perch were exposed, during 29 days to either whole live cells (WC) or a crude extract (CE) of broken <em>N. spumigena</em> cells. Chlorophyll <em>a </em>concentrations in the aquaria were 50 µg L ^-1. Perch were fed chironomidae larvae twice a day. Unexposed perch either fed (CoF) or without food (Co) served as controls. Length and weight of perch were measured at onset and termination of experiment. NODLN content was measured in <em>N. spumigena </em>filaments,<em> </em>crude extract and perch liver samples using liquid chromatography-mass spectrometry (LC-MS). Total lipids (TL) were extracted and quantified from whole-body lyophilised perch excluding livers. No significant differences for length and weight of perch were found between treatments and fed control. NODLN was detected in the crude extract samples, while no NODLN was detected in the perch livers. Moreover TL determination revealed no significant differences between treatments and fed control. <em>Nodularia spumigena</em> did not affect perch in this experiment, probably due to that the critical period of the first year for the perch was exceeded. Therefore, 1+ perch was not as susceptible to the cyanobacterium as eggs, larvae and younger juveniles of fish found in the literature. Perch liver did not contain NODLN, thus either the toxin was detoxicated with no recorded energetic cost or it was not ingested. The variables studied here did not show any effects of NODLN. However, other chemical methods such as enzymatic activity may disclose effects of NODLN.</p><p> </p>

Cyanobacteria produce a variety of secondary metabolites which possess amongst others antifungal, antibacterial, and antiviral properties. Being primary producers they are also a vital component within the food web. However, certain strains also produce toxic metabolites such as the hepatotoxins microcystin (MC) and nodularin (NOD). Their toxicity in combination with the increasing global occurrence has resulted in a drinking water guideline limit of 1 μg L-1 being issued by the World Health Organisation (WHO). However, these toxins are not only present in water, but can be accumulated by fish and shellfish. Currently, no regulations regarding cyanotoxin contaminated seafood has been established despite similar toxicity to routinely monitored marine toxins such as domoic acid (DA). To facilitate regular monitoring, a high performance liquid chromatography photo diode array (HPLC-PDA) analysis method for the detection of DA was optimised to enable the simultaneous detection of DA and nine cyanotoxins. This method was then utilised to determine cyanotoxin concentration in laboratory cyanobacteria strains. To assess the accumulation and depuration of cyanotoxins in the common blue mussel Mytilus edulis, three feeding trials were performed. During these, mussels were exposed to two cyanobacteria strains, Nodularia spumigena KAC66, Microcystis aeruginosa PCC 7813, both individually and simultaneously. A rapid dose dependent accumulation of cyanotoxins was observed with maximum concentration of 3.4 -17 μg g-1 ww accumulated by M. edulis, which was followed by a much slower depuration observed. During the final feeding trial, with N. spumigena KAC 66 and M. aeruginosa PCC7813, cyanotoxins were still detectable following 27 days of depuration. Mortality in all studies was 7% or less indicating that most mussels were unaffected by the maximum dose of 480 μg L-1 NOD (feeding study 1), 390 μg L-1 MC (feeding study 2), or 130 μg L-1 total cyanotoxins (feeding trial 3), respectively. Mortality in negative control tanks was lower throughout all three feeding trials ( < 1 - 2.6%). Consumption of a typical portion size (20 mussels) would result in ingestion of cyanotoxins at levels significantly higher than the WHO recommended tolerable daily intake (TDI) of 2.4 μg NOD and/or MCs for a 60 kg adult. This value was exceeded not only during the exposure period (maximum levels 270 - 1370 μg cyanotoxins per 20 mussels), but also at the end of the depuration period 39-600 μg cyanotoxins per 20 mussels. These results illustrated that cyanotoxin monitoring of seafood should be considered not only during, but also following bloom events. In an attempt to investigate the cyanotoxin budget of the experimental system, not only mussels, but cyanobacteria cultures, the tank water, and the mussel faeces were also analysed for their cyanotoxin content. Results showed that large quantities of MCs and NOD were unaccounted for during all exposure trials. The combined effect of cyanotoxin metabolism in M. edulis, biotic and/or abiotic degradation, protein binding, and losses during the extraction and analysis were thought to have contributed to the unaccounted cyanotoxin fraction. Mussel flesh was analysed for the presence of glutathione or cysteine conjugates, however, there was no evidence of their occurrence in the samples tested. Due to these discrepancies in the toxin budget of the system, the introduction of correction factors for the analysis of cyanotoxins in M. edulis was suggested in order to protect the general public.

Nodularia spumigena is an estuarine cyanobacteria that produces the toxin nodularin. This toxic cyanobacteria is known to have caused death to domestic and wild animals and is recognised as dangerous to human health. N. spumigena causes harmful algal blooms in many parts of the world including Australia. The toxic solutes of N. spumigena are potentially dangerous when contact is made to contaminated water bodies or is ingested by primary consumers. In Australia blooms of N. spumigena are common in the Gippsland Lakes in South-eastern Victoria and cause socio - economic hardships to the local communities. This PhD investigates the toxic effects of N. spumigena and its solutes to a range of aquatic life. A method known as SPME - HPLC showed promise in environmental monitoring of N. spumigena toxins by measuring nodularin from water samples. Other research presented study into the lethal and sublethal effects of on an extract from N. spumigena to aquatic fauna. Resu lts showed the N. spumigena extract was not lethal to many aquatic fauna although zooplankton from the Gippsland Lakes showed mortality at environmental relevant levels. Biochemical studies focusing on animal detoxification and antioxidation enzymes and DNA integrity showed sublethal effects to the N. spumigena extract. Results presented in this thesis show that an extract of N. spumigena elicited detoxification and antioxidation responses in animals tested. Furthermore, the use of the COMET assay showed increased damage to DNA of animals tested. Results also showed that different organs in animals tested responded differently to the aqueous extract, suggesting mode of uptake maybe important in toxicosis. Further, feeding studies with N. spumigena help elucidate mode of uptake using enzyme response biomarkers. The overall results of this research provided an assessment of the toxic affects of N. spumigena on aquatic fauna with special reference to the Gippsland Lakes, Victoria, Australia.

<p>Summer blooms of filamentous, diazotrophic cyanobacteria are typical of the Baltic Sea Proper, and are dominated by <i>Aphanizomenon </i>sp<i>.</i> and the toxic <i>Nodularia spumigena.</i> Although occurring every summer, the blooms vary greatly in timing and spatial distribution, making monitoring difficult and imprecise. This thesis studies how the spatial variability of Baltic cyanobacterial blooms influences estimates of abundance, vertical and horizontal distribution and N₂-fixation. Implications for sampling and monitoring of cyanobacterial blooms are also discussed.</p><p>The results of the thesis confirm the importance of diazotrophic cyanobacteria in providing N for summer production in the Baltic Proper. It also highlights the large spatial and temporal variation in these blooms and argues that improved spatial coverage and replication could make monitoring data more useful for demonstrating time trends, and for identifying the factors regulating the blooms. The vertical distribution of <i>Aphanizomenon</i> and <i>Nodularia</i> was found to be spatially variable, probably as a combination of species-specific adaptations and ambient weather conditions. Vertical migration in <i>Aphanizomenon</i> was more important towards the end of summer, and is probably regulated by a trade-off between P-availability and light and temperature.</p>

Summer blooms of filamentous, diazotrophic cyanobacteria are typical of the Baltic Sea Proper, and are dominated by Aphanizomenon sp. and the toxic Nodularia spumigena. Although occurring every summer, the blooms vary greatly in timing and spatial distribution, making monitoring difficult and imprecise. This thesis studies how the spatial variability of Baltic cyanobacterial blooms influences estimates of abundance, vertical and horizontal distribution and N2-fixation. Implications for sampling and monitoring of cyanobacterial blooms are also discussed. The results of the thesis confirm the importance of diazotrophic cyanobacteria in providing N for summer production in the Baltic Proper. It also highlights the large spatial and temporal variation in these blooms and argues that improved spatial coverage and replication could make monitoring data more useful for demonstrating time trends, and for identifying the factors regulating the blooms. The vertical distribution of Aphanizomenon and Nodularia was found to be spatially variable, probably as a combination of species-specific adaptations and ambient weather conditions. Vertical migration in Aphanizomenon was more important towards the end of summer, and is probably regulated by a trade-off between P-availability and light and temperature.

In the Himmerfjärden Bay a large excess of nitrogen over phosphorus in the discharge from a large sewage treatment plant (STP) has suppressed growth of diazotrophic cyanobacteria in its inner parts. Implementation of nitrogen removal in the STP in 1997 drastically reduced nitrogen load and triggered growth of diazotrophs, mainly Aphanizomenon sp. This study is part of a long-term series of experiments with the overall aim to test how algal biomass and production in a receiving area can be reduced, without stimulating nitrogen fixation and biomass growth by diazotrophic cyanobacteria. Nitrogen removal was discontinued in the STP during two years (2007-8) and resumed in 2009, and the discharge shifted from 25 to 10 m depth, above the seasonal pycnocline. Cellular 15N showed that N2 was the most important N source for diazotrophic cyanobacteria, and that uptake of combined nitrogen was insignificant. As biomass was declining and at the end of the productive season, we could detect a small, but significant, increase in cellular δ15N at the inner bay stations (H3 and H4). However, this coincided with an increased proportion of Anabaena spp. of the total diazotrophic biomass. This may indicate that Anabaena spp. has a higher uptake of combined nitrogen compared with Aphanizomenon sp. or that declining populations of Aphanizomenon sp. take up combined nitrogen. We also found no evidence of uptake of combined nitrogen during the winter months when nitrogen supply is ample and Aphanizomenon sp. is devoid of heterocysts. During the first half of summer (week 21-27) heterocyst frequencies were higher at the outer stations B1 and H2, compared to the inner bay stations (H4 and H5). The lower frequencies at the inner bay stations are likely due to the reduced growth rate suffered by the Aphanizomenon sp. due to stronger competition for phosphorus by non-diazotrophs at these stations and hence lower need for heterocysts. Towards the end of summer conditions even out along the bay, as the surplus phosphorus from the spring bloom is used up at the outer stations and no heterocyst gradient is present. Heterocyst frequency varied significantly over the summer, with minimum values in the beginning of July, coinciding with the highest water temperatures, and higher frequencies in early and late summer. We suggest this is primarily due to a more efficiently functioning nitrogenase enzyme at high temperatures with a reduced need for “expensive” heterocysts. Spring heterocyst differentiation occurred around 4-6 weeks after depletion of dissolved inorganic nitrogen (DIN) and only when water temperature was 5-9 oC and a pycnocline established. It seems that temperature and light in concert will initiate growth, leading to an internal nitrogen deficiency which starts heterocyst differentiation. / Himmerfjärden eutrophication study

Farmington Bay of Great Salt Lake receives a significant amount of the nutrient-polluted runoff from Salt Lake and Davis Counties, Utah. This nutrient-laden runoff has led to seasonal blooms of blue-green algae, Nodularia spumigena, which produce a toxin called nodularin that has been shown to be toxic to aquatic organisms, birds, and mammals. Nodularia spumigena are the most common algae found in Farmington Bay. This study focused on understanding the physical and chemical factors controlling the growth of Nodularia spumigena in order to improve our knowledge about how nutrients impact algae in the Great Salt Lake. The salinity of the bay ranged from almost fresh water (less than 0.2%) to water twice as salty as the sea (7.0%). Nutrient (nitrogen and phosphorus) levels were high in the bay, and showed patterns of change from south to north. Nodularia spumigena was found in concentrations that greatly exceeded the World Health Organization’s standards for contact recreation. Laboratory studies suggest that nutrients and salinity are significantly correlated with levels of Nodularia spumigena from Farmington Bay. In combination with complex ecosystem interactions, nutrients and salinity in Farmington Bay apparently contribute to the high levels of Nodularia spumigena that we measured.

Due to heavy nutrient load and adverse climate change the occurrence of toxic cyanobacterial blooms have significantly increased during the last decades. Nodularia spumigena is one of the dominant toxic cyanobacteria which produces massive and inherent blooms in brackish water body, the Baltic Sea, particularly in late summer. Nodularia spp. are known to produce nodularins (NOD) and a range of other bioactive peptides such as spumigins and nodulopeptins, all of which have unclear function. In a recent study, three new nodulopeptins with molecular weight of 899, 901 and 917 were characterised from N. spumigena KAC 66. In the present study, N. spumigena KAC 66 was fractionated by reversed phase flash chromatography and their toxicity was determined by their lethality to Daphnia pulex and D. magna along with inhibition of protein phosphatase 1 assay (PP1). All fractions showed lethality to Daphnids and inhibitory activity against PP1, the toxicity was due to additional compounds as NOD and nodulopeptin 901 were only detected in 7 fractions. Pure NOD was lethal to D. pulex and D. magna LC50= 8.4 μg/mL and 5.0 μg/mL, respectively. The newly characterised nodulopeptin 901 was also tested against D. magna (LC50=>100 μg/mL). NOD and nodulopeptin 901 inhibited PP1 with IC50 0.038 μg/mL and 25 μg/mL, respectively. In common with many studies, the maximum amount of NOD was retained within the cells during the seven week growth experiment. In contrast, as much as ~50% of nodulopeptin 901 was detected in the growth media throughout the duration of experiments. To gain further insight on the effects of environmental stress on growth and production of bioactive metabolites in N. spumigena KAC 66, a range of parameters were investigated which included; temperature, salinity, nitrate and phosphorus. In the present study it was investigated that extreme growth conditions have a considerable effect on biomass and toxin levels by N. spumigena KAC 66. The light intensity ranged from 17.35-17.47 μmol/s/m2, 22°C and 11-20 ‰ of salinity were the optimal growth conditions to obtain maximum biomasses, intra and extracellular peptide contents. At 6.5 mg/L nitrate the maximum growth, as indicated by Chl-a and maximum concentrations of intracellular NOD and nodulopeptin 901 were detected found in week 5 and 4, respectively. Temperature had the greatest effect on peptide production. Whilst growth was similar at 22°C, 25°C and 30°C, increase in temperature had a profound effect on NOD production in that an increase from 22°C to 25°C resulted in a 50% decrease in intracellular NOD levels. At 30°C little or no NOD was detected. In contrast, whilst concentrations of nodulopeptin 901 decreased with increasing temperature, they were still detected at consistent levels suggesting they play an important role. The results from phosphate experiment showed Chl-a, cell biomass and peptide production did not show clear dependency on availability of PO-3 4. This is the first study to evaluate the effects of selected environmental parameters on NOD/nodulopeptin 901 production which ultimately may be helpful to explain the distribution, control of natural blooms and toxin levels of N. spumigena in the Baltic Sea and as well as laboratory based experiments. In an attempt further exploit cyanobacterial diversity, 20 strains were isolated from the Dian Lake and 6 from the Dead Sea. The UPLC-PDA-MS analysis of isolates, Microcystis spp. from Dian Lake, China indicated the presence of several peptides namely MC-LR, cyanopeptolin A and aerucyclamides A-D. These new isolates will be examined for biological activity and chemical characterisation in future studies.