Growth, Nitrogen Fixation, and Nodularin Production by Two Baltic Sea Cyanobacteria

In late summer, nitrogen-fixing cyanobacteria Nodularia spumigena and Aphanizomenon flos-aquae form blooms in the open Baltic Sea. N. spumigena has caused several animal poisonings, but Baltic A. flos-aquae is not known to be toxic. In this laboratory study, performed with batch cultures, the influences of environmental conditions on the biomass and nitrogen fixation rate of N. spumigena and A. flos-aquae were compared and the toxin (nodularin) concentration produced by N. spumigena was measured. Several differences in the biomasses and nitrogen fixation rates of N. spumigena and A. flos-aquae were observed. A. flos-aquae preferred lower irradiances, salinities, and temperatures than N. spumigena. The biomass of both species increased with high phosphate concentrations and with accompanying bacteria and decreased with unnaturally high inorganic nitrogen concentrations. Nodularin concentrations in cells and growth media, as well as nitrogen fixation rates, were generally highest under the conditions that promoted growth. Intracellular nodularin concentrations increased with high temperature, high irradiance, and high phosphate concentration and decreased with low and high salinities and high inorganic nitrogen concentrations. Nodularin concentrations in growth media increased with incubation time, indicating that intracellular nodularin was released when cells lysed. The different responses of A. flos-aquae and N. spumigena to changes in salinity, irradiance, and temperature may explain the different spatial and temporal distribution of these species in the Baltic Sea. According to the results, toxic N. spumigena blooms may be expected in late summer in areas of the Baltic Sea with high phosphorus concentrations and moderate salinity.     

Development of Baltic Sea zooplankton in the presence of a toxic cyanobacterium: a mesocosm approach

Cyanobacteria blooms are common in the Baltic Sea and are consideredto be a poor food source and sometimes toxic to zooplankton.Most experiments demonstrating harmful effects have been short-termincubations with monocultures or simple mixtures of food. Inthis study, a mesocosm approach was used to examine zooplanktonresponses over generation timescales. A toxic strain of thecyanobacterium Nodularia spumigena was added to bag enclosuresof ambient water. The initial mesozooplankton concentrationwas either reduced by prescreening the water or enriched withlocally caught zooplankton. Experiments ran for 15 days, longenough to monitor reproductive success and development of thenext mesozooplankton generations. There was no major harmfuleffect on the zooplankton assemblage, even though the concentrationof the toxin nodularin was in the upper range of field observations.The copepod Eurytemora affinis, rotifers Synchaeta spp. andthe cladoceran Bosmina longispina maritima were able to developand reproduce successfully in the presence of N. spumigena.The only species showing impaired recruitment was the copepodAcartia bifilosa. The general lack of population level effectsfrom N. spumigena in this study can be reconciled with previousobservations of adverse effects. Cyanobacteria alone may bepoor food and toxic to zooplankton, but in the mesocosms a richassemblage of microbiota developed, similar to that associatedwith blooms in the field. We suggest that, in the context ofotherwise food-depleted summer situations in the open BalticSea, zooplankton can derive benefit from cyanobacteria bloomassemblages.     

Characterization of the nodularin synthetase gene cluster and proposed theory of the evolution of cyanobacterial hepatotoxins

Nodularia spumigena is a bloom-forming cyanobacterium which produces the hepatotoxin nodularin. The complete gene cluster encoding the enzymatic machinery required for the biosynthesis of nodularin in N. spumigena strain NSOR10 was sequenced and characterized. The 48-kb gene cluster consists of nine open reading frames (ORFs), ndaA to ndaI, which are transcribed from a bidirectional regulatory promoter region and encode nonribosomal peptide synthetase modules, polyketide synthase modules, and tailoring enzymes. The ORFs flanking the nda gene cluster in the genome of N. spumigena strain NSOR10 were identified, and one of them was found to encode a protein with homology to previously characterized transposases. Putative transposases are also associated with the structurally related microcystin synthetase (mcy) gene clusters derived from three cyanobacterial strains, indicating a possible mechanism for the distribution of these biosynthetic gene clusters between various cyanobacterial genera. We propose an alternative hypothesis for hepatotoxin evolution in cyanobacteria based on the results of comparative and phylogenetic analyses of the nda and mcy gene clusters. These analyses suggested that nodularin synthetase evolved from a microcystin synthetase progenitor. The identification of the nodularin biosynthetic gene cluster and evolution of hepatotoxicity in cyanobacteria reported in this study may be valuable for future studies on toxic cyanobacterial bloom formation. In addition, an appreciation of the natural evolution of nonribosomal biosynthetic pathways will be vital for future combinatorial engineering and rational design of novel metabolites and pharmaceuticals.     

Effect of cycles of feed deprivation on growth and food consumption of immature three-spined sticklebacks and European minnows

Individual juvenile three-spined sticklebacks Gasterosteus aculeatus and European minnow Phoxinus phoxinus , from sympatric populations, were subjected to four cycles of 1 week of food deprivation and 2 weeks of ad libitum feeding. Mean specific growth rate during the weeks of deprivation was negative and did not differ between species. The three-spined stickleback showed sufficient growth compensation to recover to the growth trajectory shown by control fish daily fed ad libitum . The compensation was generated by hyperphagia during the re-feeding periods, and in the last two periods of re-feeding, the gross growth efficiencies of deprived three-spined sticklebacks were greater than in control fish. The expression of the compensatory changes in growth and food consumption became clearer over the successive periods of re-feeding. The European minnow developed only a weak compensatory growth response and the mass trajectory of the deprived fish deviated more and more from the control trajectory. During re-feeding periods, there were no significant differences in food consumption or gross growth efficiency between control and deprived European minnows. The differences between the two species are discussed in terms of the possible costs of compensatory growth, the control of growth and differences in feeding biology.     

Feeding and Refuge Use by Small Fish in the Presence of Cyanobacteria Blooms

We studied the effect of cyanobacteria on foraging and refuge use in small fish. We measured pike larval feeding in the presence of cyanobacteria by counting leftover prey. Our results showed that feeding by pike larvae on zooplankton prey decreased significantly in the presence of non-toxic cyanobacteria. The behaviour can be due to lowered vision caused by turbidity or clogging of the gills. Further, we tested whether the three-spined stickleback use toxic cyanobacteria as a refuge against predators in a choice experiment. The choice experiment was performed in a Y-maze fluviarum, where the fish could select between two different environments. Our results support the refuge use hypothesis because the three-spined stickleback clearly preferred toxic cyanobacteria to the chemical predator signal. To conclude, cyanobacteria decrease feeding rates in fish larvae, but may function as important refuge for e.g. sticklebacks, during predation pressure in pelagic algal blooms.     

Reduction of cyanobacterial toxins through coprophagy in Mytilus edulis

An experiment was conducted to follow the fate of the cyanobacterial toxin, nodularin, produced by Nodularia spumigena through ingestion by Mytilus edulis and re-ingestion of faecal material (coprophagy). Mussels were fed with cultures of N. spumigena, and the faeces that were produced were fed to other mussels not previously exposed to N. spumigena. Concentrations of nodularin were measured in the food (N. spumigena), the mussels and in the faeces in order to make a toxin budget. High concentrations of nodularin were found in the mussels and their faeces after 48 h incubation with N. spumigena. When the toxic faeces were fed to new mussels, the toxin content of faeces was reduced from 95 μg nod g⁻¹ dry weight (DW) to 1 μg nod g⁻¹ DW through the process of coprophagy. Hence, when toxic faeces were fed to mussels, the nodularin concentration of the resulting faecal material was reduced by 99%. Pseudofaeces were produced when the mussels were grazing on N. spumigena, but not when grazing on faeces. The pseudofaeces contained high concentrations of nodularin and apparently intact N. spumigena cells. However, these cells were growth-inhibited and their potential contribution to seeding a bloom is probably limited. Our data indicate that a large fraction of ingested nodularin in M. edulis is egested with the faeces, and that the concentration of nodularin in the faeces is reduced when faeces are re-ingested.     

Pattern of hyperphagia in immature three-spined sticklebacks after short-term food deprivation

Juvenile three-spined sticklebacks Gasterosteus aculeatus were fed live enchytraeid worms, and mean daily ad libitum consumption in the absence of periods of deprivation was 11·7% of initial body weight. Then, six groups of five replicate fish were subjected to 1, 3 and 6 days without food. Each period of deprivation was followed by 1 day of ad libitum feeding. The sequential order of deprivations differed between groups. Over the sequence of deprivations and re-feedings, mean cumulative consumption did not differ between groups. Mean daily consumption was 19·8% of initial body weight after a 1-day deprivation, 22·3% after 3 days and 19·5% after 6 days. However, consumption in the 24 h after a given length of deprivation also depended on the prior history of deprivation. Specific growth rate over the experiment did not differ between groups. The results provide evidence for an effective regulation of appetite in growing sticklebacks.     

Enhanced ambient UVB light affects growth,body condition and the investment in innate and adaptive immunity in three-spined sticklebacks (<Emphasis Type="Italic">Gasterosteus aculeatus</Emphasis>)

With ongoing environmental change, ultraviolet-B radiation (UVB) reaching the Earth’s surface has increased over recent decades with consequences for terrestrial and also aquatic ecosystems. Despite evidence for direct physiological and immunological responses of aquatic animals following enhanced UVB exposure, studies investigating indirect impacts of ambient UVB radiation are scarce and mainly used only single doses and/or artificially high amounts of UVB. In the present study, the influence of chronic exposure to elevated UVB levels on growth, body condition and immune function was investigated in three-spined sticklebacks (Gasterosteus aculeatus). Fish were kept outdoors for 68 ± 2 days under two different spectral conditions; one group was exposed to natural solar radiation (UVB-normal), while the other group received additional UVB light for four hours daily (UVB-enhanced). Enhanced UVB radiation was within the range of UVB levels measured at the study site. Fish length and weight were determined at the beginning and end of the experiment to compare growth and body condition between the two treatment groups. At the end of the experiment, the splenosomatic index and the granulocyte-to-lymphocyte ratio were determined as immune parameters. Fish from the UVB-enhanced group showed a reduced growth and body condition as well as a lower splenosomatic index compared to the UVB-normal group. Furthermore, UVB-treated fish had a higher granulocyte-to-lymphocyte ratio representing a relatively higher activation of innate compared to adaptive immunity. Consequently, increased but ecologically relevant levels of ambient UVB negatively affect growth and body condition and have a considerable impact on immunity in three-spined sticklebacks.     

Prey selection by the larvae of three species of littoral fishes on natural zooplankton assemblages

The prey selection of larvae of three common littoral fish species (pike, Esox lucius; roach,Rutilus rutilus; and three-spined stickleback, Gasterosteus aculeatus) was studied experimentally in the laboratory by using natural zooplankton assemblages. Zooplankton prey was offered at four different concentrations to study the functional responses of the planktivores. The diets of pike and sticklebacks were formed mainly of copepod juveniles and adults, which dominated the prey communities, although sticklebacks ate also cladocerans. The diet of roach larvae consisted of rotifers, cladocerans and copepods, without prey selection, in equal proportions indicating a more omnivorous diet. All fish larvae were able to feed selectively although in sticklebacks prey selection was less pronounced. Pike and roach larvae preferred large prey to smaller prey types. Patterns of prey selection are discussed in the context of size-selection theory and apparent vs. true selection.     

Do random fluctuations in daily ration affect the growth rate of juvenile three-spined sticklebacks?

The effect of random fluctuations in daily ration on the growth performance of individual juvenile three-spined sticklebacks Gasterosteus aculeatus was studied in experiments lasting 21 days at 14°C and a photoperiod of 10L:14D. Two mean ration levels were used: a maintenance ration of 2%, and a high ration of 6% of initial body weight per day. For experimental fish, the daily ration varied randomly about the required mean value with a coefficient of variation of 33%. The controls received a constant daily ration. The experiment was replicated in winter (Dec.-Jan.) and spring (Mar.-Apr.). At a given ration, there was no significant difference between the specific growth rates of fish receiving constant or varying ration. Neither the final dry weight, final body water content nor final lipid content (% dry weight) differed significantly. As expected, ration had a significant effect on growth rate, final dry weight and body lipid and water content. There was a significant difference in mean growth rate between the winter and spring replicates. The growth rates observed in these experiments were compared with those predicted from a previously described empirical growth model developed for sticklebacks fed constant rations. The model underestimated mean growth rates.     

Positive feedbacks between bottom-up and top-down controls promote the formation and toxicity of ecosystem disruptive algal blooms: A modeling study

Harmful algal blooms that disrupt and degrade ecosystems (ecosystem disruptive algal blooms, EDABs) are occurring with greater frequency and severity with eutrophication and other adverse anthropogenic alterations of coastal systems. EDAB events have been hypothesized to be caused by positive feedback interactions involving differential growth of competing algal species, low grazing mortality rates on EDAB species, and resulting decreases in nutrient inputs from grazer-mediated nutrient cycling as the EDAB event progresses. Here we develop a stoichiometric nutrient–phytoplankton–zooplankton (NPZ) model to test a conceptual positive feedback mechanism linked to increased cell toxicity and resultant decreases in grazing mortality rates in EDAB species under nutrient limitation of growth rate. As our model EDAB alga, we chose the slow-growing, toxic dinoflagellate Karenia brevis, whose toxin levels have been shown to increase with nutrient (nitrogen) limitation of specific growth rate. This species was competed with two high-nutrient adapted, faster-growing diatoms (Thalassiosira pseudonana and Thalassiosira weissflogii) using recently published data for relationships among nutrient (ammonium) concentration, carbon normalized ammonium uptake rates, cellular nitrogen:carbon (N:C) ratios, and specific growth rate. The model results support the proposed positive feedback mechanism for EDAB formation and toxicity. In all cases the toxic bloom was preceded by one or more pre-blooms of fast-growing diatoms, which drew dissolved nutrients to low growth rate-limiting levels, and stimulated the population growth of zooplankton grazers. Low specific grazing rates on the toxic, nutrient-limited EDAB species then promoted the population growth of this species, which further decreased grazing rates, grazing-linked nutrient recycling, nutrient concentrations, and algal specific growth rates. The nutrient limitation of growth rate further increased toxin concentrations in the EDAB algae, which further decreased grazing-linked nutrient recycling rates and nutrient concentrations, and caused an even greater nutrient limitation of growth rate and even higher toxin levels in the EDAB algae. This chain of interactions represented a positive feedback that resulted in the formation of a high-biomass toxic bloom, with low, nutrient-limited specific growth rates and associated high cellular C:N and toxin:C ratios. Together the elevated C:N and toxin:C ratios in the EDAB algae resulted in very high bloom toxicity. The positive feedbacks and resulting bloom formation and toxicity were increased by long water residence times, which increased the relative importance of grazing-linked nutrient recycling to the overall supply of limiting nutrient (N).     

Nutritional state influences shoaling preference for familiars

Preferences for grouping with familiar individuals are shown in many animal species, including the three-spined stickleback (Gasterosteus aculeatus). Shoaling with familiars is advantageous because of more precise anti-predator behaviours or more stable dominance hierarchies. Additionally, associations with familiar individuals facilitate the evolution of altruistic behaviour. Thus, in situations of increased competition one might expect an increased preference for familiar fish. We gave single juvenile sticklebacks of different nutritional state the choice between shoals composed either of familiar or unfamiliar individuals. Satiated fish preferred to shoal with familiar individuals. A comparative analysis of 8 stickleback studies with 15 different tests using familiars showed that all tests gave similar results, i.e. sticklebacks of all age classes preferred to shoal with familiars in a non-sexual context. In contrast, hungry test fish did not prefer to shoal with familiar fish, but even showed a preference for the unfamiliar group. Because sticklebacks use early-life familiarity to recognize kin, the results suggest the avoidance of competition with relatives. To our knowledge, this is the first study showing an impact of nutritional state on social interactions with familiar individuals.     

Nutritional importance of detritivory in the growth and condition of gizzard shad in an Ohio reservoir

Synopsis Gizzard shad,Dorosoma cepedianum, in Acton Lake, Ohio, ingested foods of varying nutritional quality during the 1981–1983 growing seasons. Adult (ages 2–4) fish fed on a mixed diet (ORG>30%; C:N<7:1) of zooplankton and organic detritus in early summer, and on detrital materials (ORG<16%; C:N>11:1) during the remainder of the growing season. Age 0 (<35 mm standard length) fish ingested only detrital materials. The nutritional quality (ORG = 10 – 20%; C:N<11:1) of these foods displayed little seasonal variation, but was higher than that of organic detritus taken by adult fish in late summer and autumn. Growth and condition of gizzard shad were poor when the diet consisted of detrital materials; however, age 4 fish (1983) grew rapidly and condition improved when zooplankton were consumed. These results suggest that ingestion of poor-quality detritus can reduce the growth and condition of gizzard shad in Acton Lake, whereas the seasonal inclusion of high-quality zooplankton in the diet can result in rapid growth and improved condition.     

Diversity of toxic and nontoxic nodularia isolates (cyanobacteria) and filaments from the Baltic Sea

Cyanobacteria of the genus Nodularia form toxic blooms in brackish waters worldwide. In addition, Nodularia spp. are found in benthic, periphytic, and soil habitats. The majority of the planktic isolates produce a pentapeptide hepatotoxin nodularin. We examined the morphologic, toxicologic, and molecular characters of 18 nodularin-producing and nontoxic Nodularia strains to find appropriate markers for distinguishing the toxic strains from the nontoxic ones in field samples. After classical taxonomy, the examined strains were identified as Nodularia sp., Nodularia spumigena, N. baltica, N. harveyana, and N. sphaerocarpa. Morphologic characters were ambiguous in terms of distinguishing between the toxic and the nontoxic strains. DNA sequences from the short 16S-23S rRNA internally transcribed spacer (ITS1-S) and from the phycocyanin operon intergenic spacer and its flanking regions (PC-IGS) were different for the toxic and the nontoxic strains. Phylogenetic analysis of the ITS1-S and PC-IGS sequences from strains identified as N. spumigena, and N. baltica, and N. litorea indicated that the division of the planktic Nodularia into the three species is not supported by the ITS1-S and PC-IGS sequences. However, the ITS1-S and PC-IGS sequences supported the separation of strains designated N. harveyana and N. sphaerocarpa from one another and the planktic strains. HaeIII digestion of PCR amplified PC-IGS regions of all examined 186 Nodularia filaments collected from the Baltic Sea produced a digestion pattern similar to that found in toxic isolates. Our results suggest that only one planktic Nodularia species is present in the Baltic Sea plankton and that it is nodularin producing.     

Laboratory and field studies on diet choice in three-spined sticklebacks, Gasterosteus aculeatus L., in relation to profitability and visual features of prey

Diet choice by three-spined sticklebacks was investigated under both laboratory and field conditions. In the laboratory, sticklebacks did not always choose the more profitable of two prey items; instead, they apparently selected prey according to a set of proximate decision rules based on visual cues provided by the prey. Studies of the diet of sticklebacks in the field suggest that they may use the same set of proximate decision rules to select their food and that in these more complex conditions this may lead them to feed preferentially on the more profitable types of prey. In general, sticklebacks selected zooplanktonic rather than benthic prey (which is less profitable), but the importance of benthos in the diet increased as the density of zooplankton decreased.     

Sedimentation of Nodularia spumigena and distribution of nodularin in the food web during transport of a cyanobacterial bloom from the Baltic Sea to the Kattegat

Nodularia spumigena is a toxic cyanobacteria that blooms in the Baltic Sea every year. In the brackish water of the Baltic Sea, its toxin, nodularin, mainly affects the biota in the surface water due to the natural buoyancy of this species. However, the fate of the toxin is unknown, once the cyanobacteria bloom enters the more saline waters of the Kattegat. In order to investigate this knowledge gap, a bloom of N. spumigena was followed during its passage, carried by surface currents, from the Baltic Sea into the Kattegat area, through the Öresund strait. N. spumigena cells showed an increased cell concentration through the water column during the passage of the bloom (up to 130 10³ cells ml⁻¹), and cells (4.2 10³ cells ml⁻¹) could be found down to 20 m depth, below a pycnocline. Sedimentation trap samples from below the pycnocline (10–12 m depth) also showed an increased sedimentation of N. spumigena filaments during the passage of the bloom. The toxin nodularin was detected both in water samples (0.3–6.0 μg l⁻¹), samples of sedimenting material (a toxin accumulation rate of 20 μg m^-2 day⁻¹), zooplankton (up to 0.1 ng ind.⁻¹ in copepods), blue mussels (70–230 μg kg⁻¹ DW), pelagic and benthic fish (herring (1.0–3.4 μg kg⁻¹ DW in herring muscle or liver) and flounder (1.3-6.2 μg kg⁻¹ DW in muscle, and 11.7-26.3 μg kg⁻¹ DW in liver). A laboratory experiment showed that N. spumigena filaments developed a decreased buoyancy at increased salinities and that they were even sinking with a rate of up to 1,7 m day⁻¹ at the highest salinity (32 PSU). This has implications for the fate of brackish water cyanobacterial blooms, when these reach more saline waters. It can be speculated that a significant part of the blooms content of nodularin will reach benthic organisms in this situation, compared to blooms decaying in brackish water, where most of the bloom is considered to be decomposed in the surface waters.     

Effects of toxic and non-toxic cyanobacteria on grazing, zooplanktivory and survival of the mysid shrimp Mysis mixta

We measured the effect of toxic and non-toxic cyanobacteria strains on grazing, predation rates and survival of the mysid shrimp Mysis mixta by means of laboratory experiments. Juvenile and adult M. mixta fed most actively on the non-toxic strains Aphanizomenon flos-aquae and Nodularia sphaerocarpa as on high quality food, the green flagellate Brachiomonas submarina, whereas grazing on the toxic N. spumigena was significantly lower than on the other strains. The mysids showed normal functional responses; decreasing clearance rates at increasing concentrations of cyanobacteria. In a separate predation experiment, the feeding rates on copepods declined in the presence of aggregated non-toxic cyanobacteria. However, we could not demonstrate increased mortality in a 7-week experiment where mysids were exposed to high concentrations of toxic N. spumigena     

Interspecific resource competition-combined effects of radiation and nutrient limitation on two diazotrophic filamentous cyanobacteria

The cyanobacterial blooms in the Baltic Sea are dominated by diazotrophic cyanobacteria, the potentially toxic species Aphanizomenon sp. and the toxic species Nodularia spumigena. The seasonal succession with peaks of Aphanizomenon sp., followed by peaks of N. spumigena, has been explained by the species-specific niches of the two species. In a three-factorial outdoor experiment, we tested if nutrient and radiation conditions may impact physiological and biochemical responses of N. spumigena and Aphanizomenon sp. in the presence or absence of the other species. The two nutrient treatments were f/2 medium without NO (3) (-) (-N) and f/2 medium without PO (4) (3-) (-P), and the two ambient radiation treatments were photosynthetic active radiation >395?nm (PAR) and PAR + UV-A + UV-B >295?nm. The study showed that Aphanizomenon sp. was not negatively affected by the presence of N. spumigena and that N. spumigena was better adapted to both N and P limitation in interaction with ultraviolet radiation (UVR, 280-400?nm). In the Baltic Sea, these physical conditions are likely to prevail in the surface water during summer. Interestingly, the specific growth rate of N. spumigena was stimulated by the presence of Aphanizomenon sp. We suggest that the seasonal succession, with peaks of Aphanizomenon sp. followed by peaks of N. spumigena, is a result from species-specific preferences of environmental conditions and/or stimulation by Aphanizomenon sp. rather than an allelopathic effect of N. spumigena. The results from our study, together with a predicted stronger stratification due to effects of climate change in the Baltic Sea with increased temperature and increased precipitation and increased UV-B due to ozone losses, reflect a scenario with a continuing future dominance of the toxic N. spumigena.     

Correlation of the content of hepatotoxin nodularin and glycosidic carotenoids, 4-ketomyxol-2′-fucoside and novel 1′-O-methyl-4-ketomyxol-2′-fucoside,in 20 strains of the cyanobacterium Nodularia spumigena

The carotenoids of 19 different strains of Nodularia spumigena and one Nodularia sphaerocarpa from different global locations were investigated. The molecular structure of the diagnostic pigment in N. spumigena of the Baltic Sea, tentatively named ‘4-keto-myxoxanthophyll-like pigment’ in Schlüter, L., Garde, K., Kaas, H., [2004. A 4-keto-myxoxanthophyll-like pigment is a diagnostic pigment for the toxic cyanobacteria Nodularia spumigena in the Baltic Sea. Mar. Ecol. Prog. Ser. 275, 69–78.] was determined to be a 4-ketomyxol-2′-fucoside. In most of the strains an additional carotenoid was found, identified as the novel 1′-O-methyl-4-ketomyxol-2′-fucoside by 2D NMR. This glycosidic carotenoid methyl ether was found to be a more important diagnostic pigment than the 4-ketomyxol-2′-fucoside for the toxic N. spumigena in the Baltic Sea. Out of the 20 strains 15 were found to produce the hepatotoxin nodularin. The content of carotenoids and nodularin was found to increase relative to chlorophyll a at increasing light intensity and at stationary growth, and nodularin was significantly correlated to both 4-ketomyxol-2′-fucoside and 1′-O-methyl-4-ketomyxol-2′-fucoside, and particular to the sum of these two pigments.