Tropical cyanobacteria in German waters: Causes and consequences - Toxic cyanobacteria of tropical origin have spread to water bodies in northern Germany. Here their population size is determined by the onset of germination: the earlier the time of germination, the larger the summer population. Climate-related early increase in water temperatures over the course of the years has promoted their spreading to temperate regions. Toxins known from these species in tropical regions were also found in German lakes. Surprisingly, these toxins are not produced by the invaders but by native species. Thus, the invasion have drawn our attention to an old problem. However, the invaders itself potentially also poses a health risk in German lakes since they produce other – so far unidentified – toxic substances.

Cylindrospermopsis raciborskii, a cyanobacterium of tropical origin, can produce the toxin cylindrospermopsin (CYN). This originally tropical cyanobacterium (bluegreen algae) has now spread to the distant waters of the Berlin area. Cylindrospermopsin has been detected in two lakes in the area, but none of the C. raciborskii strains isolated here so far were found to produce the toxin. The main objectives of the CYLIN project were therefore to analyze the distribution and regulation of C. raciborskii and cylindrospermopsin and to determine which cyanobacteria are producing this toxin in order to establish a basis with which to predict the further course of development of this species and the related health hazards for humans. The CYLIN project was implemented as a three-part program. A screening program was first conducted in 2004 to test regional water bodies for the presence of cylindrospermopsin and potential CYN-producing cyanobacteria in order to obtain an overview of their distribution in the study region. A total of 142 regional water bodies were sampled once each in this qualitative analysis cylindrospermopsin and cyanobacteria. The screening program was followed by a monitoring program designed to generate quantitative data on the concentrations of dissolved CYN, particulate CYN, cyanobacteria and target environmental parameters at 20 selected lakes, which were sampled 3 times each. Last but not least, we investigated the seasonal dynamics of these parameters at two selected lakes in 2004 and 2005. Apart from this we isolated different cyanobacterial strains and conducted chemical and molecular biological analyses of CYN and CYNcoding genes, in order to identify CYN-producing cyanobacteria. The results show that C. raciborskii and CYN are much more widespread than was previously assumed. C. raciborskii was detected in 22 % of the investigated water bodies, and cylindrospermopsin in 52 %. Additionally, two other toxic cyanobacteria of tropical origin were found for the first time in the BerlinBrandenburg region, Anabaena bergii and Aphanizomenon aphanizomenoides. The mean and maximum CYN concentrations were 1 µg L-1 and 12 µg L-1, respectively. Since the particulate CYN fraction did not exceed 0.5 µg L-1, the dissolved CYN fraction was found to be responsible for the high CYN concentrations. The proposed guideline safety value for cylindrospermopsin in drinking water (1 µg L-1) was exceeded 18 times at 8 different lakes. Although Aphanizomenon flos-aquae (Nostocales) has been unequivocally identified as a producer of cylindrospermopsin, the observed cylindrospermopsin concentrations cannot be attributed to this cyanobacterial species alone. Aphanizomenon gracile was also identified as a potential CYN-producing cyanobacterium. Based on the findings of the CYLIN project, we recommend that cylindrospermopsin be included as a risk factor in drinking and bathing water quality assessments. To identify hazard conditions associated with this cyanotoxin, further investigations are needed to identify all cyanobacteria that produce cylindrospermopsin and to elucidate the mechanisms regulating the occurrence of CYN-producing cyanobacteria, CYN synthesis by these organisms, and CYN decomposition in aquatic ecosystems. Our analysis of C. raciborskii population dynamics showed that its germination is temperature-dependent and its population growth light-dependent. Population size was determined by the time of germination, that is, the earlier the time of germination, the bigger the population. Based on these findings, it appears highly likely that the climate-related early rise in water temperatures over the course of the years has promoted the spread of this species to temperate regions. Our hypothesis for the future course of cyanobacterial and cyanotoxin development in German waters is as follows: The combination of trophic decline and global warming works to the general benefit of cyanobacteria of the order Nostocales and leads to a shift in cyanobacterial species and toxin composition. This may ultimately lead to an increase in the incidence of neurotoxins as well as cylindrospermopsin.

Cylindrospermopsis raciborskii, a cyanobacterium of tropical origin, can produce the toxin cylindrospermopsin (CYN). This originally tropical cyanobacterium (blue-green alga) had spread to the waters of the Berlin area. Cylindrospermopsin had been detected in two lakes in the area, but none of the C. raciborskii strains isolated here so far were found to produce the toxin. The main objectives of the CYLIN project were therefore to analyze the distribution and regulation of C. raciborskii and cylindrospermopsin and to determine which cyanobacteria are producing this toxin in order to establish a basis to predict the further course of development of this species and the related health hazards for humans. The CYLIN project was implemented as a three-part program. A screening program was first conducted in 2004 to test regional water bodies for the presence of cylindrospermopsin and potential CYN-producing cyanobacteria in order to obtain an overview of their distribution in the study region. A total of 142 regional water bodies were sampled once each in this qualitative analysis of cylindrospermopsin and cyanobacteria. The screening program was followed by a monitoring program designed to generate quantitative data on the concentrations of dissolved CYN, particulate CYN, cyanobacteria and target environmental parameters at 20 selected lakes, which were sampled 3 times each. Furthermore, we investigated the seasonal dynamics of these parameters at two selected lakes in 2004 and 2005. Apart from this we isolated different cyanobacterial strains and conducted chemical and molecular biological analyses of CYN and CYN-coding genes, in order to identify CYN-producing cyanobacteria. The results show that C. raciborskii and CYN are much more widespread than was previously assumed for the region. C. raciborskii was detected in 22 % of the investigated water bodies, and cylindrospermopsin in 52 %. Additionally, two other toxic cyanobacteria of tropical origin were found for the first time in the Berlin-Brandenburg region, Anabaena bergii and Aphanizomenon aphanizomenoides. The mean and maximum CYN concentrations were 1 µg L-1 and 12 µg L-1, respectively. Since the particulate CYN fraction did not exceed 0.5 µg L-1, the dissolved CYN fraction was found to be responsible for the high CYN concentrations. The proposed guideline value for cylindrospermopsin in drinking water (1 µg L-1; Humpage and Falconer 2003) was exceeded 18 times at 8 different lakes. Although Aphanizomenon flos-aquae (Nostocales) has been unequivocally identified as a producer of cylindrospermopsin, the observed cylindrospermopsin concentrations cannot be attributed to this cyanobacterial species alone. Aphanizomenon gracile was also identified as a potential CYN-producing cyanobacterium. Based on the findings of the CYLIN project, we recommend that cylindrospermopsin be included as in hazard analyisis for drinking and bathing water quality assessments. To identify risk conditions associated with this cyanotoxin, further investigations are needed to identify all cyanobacteria that produce cylindrospermopsin and to elucidate the mechanisms regulating the occurrence of CYN-producing cyanobacteria, CYN synthesis by these organisms, and CYN decomposition in aquatic ecosystems. Our analysis of C. raciborskii population dynamics showed that its germination is temperature-dependent and its population growth light-dependent. Population size was determined by the time of germination, that is, the earlier the time of germination, the bigger the population. Based on these findings, it appears highly likely that the climate-related early rise in water temperatures over the course of the years has promoted the spread of this species to temperate regions. Our hypothesis for the future course of cyanobacterial and cyanotoxin development in German waters is as follows: The combination of trophic decline and global warming works to the general benefit of cyanobacteria of the order Nostocales and leads to a shift in cyanobacterial species and toxin composition. This may ultimately lead to an increase in the incidence of neurotoxins as well as cylindrospermopsin.

The frequent occurrence of the cyanobacterial toxin cylindrospermopsin (CYN) in the (sub)-tropics has been largely associated with cyanobacteria of the order Nostocales of tropical origin, in particular Cylindrospermopsis raciborskii. C. raciborskii is currently observed to spread northwards into temperate climatic zones. In addition, further cyanobacteria of the order Nostocales typically inhabiting water bodies in temperate regions are being identified as CYN-producers. Therefore, data on the distribution of CYN in temperate regions are necessary for a first assessment of potential risks due to CYN in water used for drinking and recreation. A total of 127 lakes situated in the northeastern part of Germany were investigated in 2004 for the presence of the toxin CYN and the phytoplankton composition. The toxin could be detected in half of the lakes (n ¼ 63) and in half of 165 samples (n ¼ 88). Concentrations reached up to 73.2 _g CYN/g DW. CYN thus proved more widely distributed than previously demonstrated. The analyses of phytoplankton data suggest Aphanizomenon sp. and Anabaena sp. as important CYN producers in Germany, and confirm recent findings of Aphanizomenon flos-aquae as CYN-producing species frequently inhabiting water bodies in temperate climatic regions. The data shown here suggest that CYN may be an important cyanobacterial toxin in German water bodies and that further data are needed to assess this.

The cyanobacterial toxin cylindrospermopsin (CYN) is widely distributed in German lakes, but volumetric data for risk assessment are lacking and it is unclear which cyanobacterial species produce CYN in Europe. We therefore analyzed CYN concentration and cyanobacterial composition of 21 German lakes in 2005. CYN was detected in 19 lakes (102 of 115 samples). In total, 45 samples contained particulate CYN only, and 57 contained both dissolved and particulate CYN. The concentrations were 0.002–0.484 mgL-1 for particulate CYN and 0.08–11.75 mgL-1 for dissolved CYN with a maximum of 12.1 mgL-1 total CYN. A drinking-water guideline value of 1 mgL-1 proposed by Humpage and Falconer [2003. Oral toxicity of the cyanobacterial toxin CYN in male Swiss albino mice: determination of no observed adverse effect level for deriving a drinking water guideline value. Environ. Toxicol. 18, 94–103] was exceeded in 18 samples from eight lakes due to high concentrations of dissolved CYN. CYN occurrence in the German lakes could not be ascribed to the three known CYN-producing species Cylindrospermopsis raciborskii, Anabaena bergii and Aphanizomenon flos-aquae, which were detected in some lakes in low abundances. The highest correlation coefficients were observed between particulate CYN and the native Aphanizomenon gracile. It occurred in 98 CYN-positive samples, was the most abundant Nostocales and was the only Nostocales in five samples. This indicates that A. gracile is a potential CYN producer in German lakes.

Cylindrospermopsis raciborskii, an invasive freshwater cyanobacterium, originated from the tropics but has spread to temperate zones over the last few decades. Its northernmost populations in Europe occur in North German lakes. How such dramatic changes in its biogeography are possible and how its population dynamics in the newly invaded habitats are regulated are still unexplained. We therefore conducted a long-term (1993–2005) study of two German lakes to elucidate the mechanisms behind C. raciborskii population dynamics and to identify the abiotic constraints on its development. Our data revealed that pelagic populations of C. raciborskii thrived for three months during the summer, contributing up to 23% of the total cyanobacteria biovolume. Population sizes varied greatly between years without exhibiting any distinct long-term trends. In the annual lifecycle, C. raciborskii filaments emerged in the pelagic habitat when the temperature rose above 15–17 C. At that time, mean photosynthetically active radiation in the mixed water column (Imix) overstepped its maximum. Rates of population net increase were highest at the beginning of the season (0.15– 0.28 day–1), declined continuously over time, and were significantly positively correlated with Imix. This indicates that the onset of the pelagic population is temperaturemediated and that Imix controls its growth. Since Imix peaks before the population onset, the time of germination is of crucial importance for successful development. To test this hypothesis, we designed a model to simulate pelagic population size, starting at different dates in the annual cycle. Moving the population onset forward by 30 days resulted in a doubling of the population size. We therefore conclude that an earlier rise in water temperature associated with climate change has promoted the spread of C. raciborskii to the temperate zone. Earlier warming permits earlier germination, thereby shifting the pelagic populations to a phase with higher Imix, which advances growth and the population establishment.

Cylindrospermopsis raciborskii is a filamentous freshwater cyanobacterium of tropical and subtropical origin that spread out up to northern temperate zone during the last decades. Belonging to the order Nostocales it can differentiate two types of nonvegetative cells: i) heterocysts, in which it can fix molecular nitrogen, and ii) akinetes, which are resting stages, that allow the species to pass-by unfavourable growth conditions in the sediment. In the tropics and subtropics, C. raciborskii is a perennial species, which often occurs in bloom densities, and which is prominent for the synthesis of various toxins including the potent hepatotoxin cylindrospermopsin (CYN) and neurotoxic paralytic shellfish poisoning toxins. In Europe, its northernmost populations were found in North German lakes. Here, it occurs only during the summer months and has not been observed to form mass developments. The highest documented biomass it attained was 23 % of the total phytoplankton biomass. To investigate the spatial occurrence and relative frequency of C. raciborskii, we undertook a systematic survey, sampling 142 water bodies in the lowland region of Northeast Germany from June till September 2004. The cyanobacteria species present were analysed qualitatively and semi-quantitatively. C. raciborskii was a common species. It was present in 39 of the samples, of which 62 % were new findings. Further, we detected two additional thermophilic Nostocales that have not been reported from Germany before: Anabaena bergii and Aphanizomenon aphanizomenoides. These are the most northerly reports of both species so far. A. bergii occurred in 13 and A. aphanizomenoides in 19 of the 142 water bodies sampled. To elucidate the regulation mechanisms of the population dynamics of C. raciborskii and its abiotic boundaries in the newly invaded habitats furthermore long-term data series (1993 - 2005) of two polymictic shallow lakes in the study area were analyzed. Population sizes largely varied between years without any distinct long-term trend. In the annual course, filaments of the species occurred in the pelagial at temperatures above 15 - 17 °C, i.e. akinetes must have been germinated at temperatures below 15 °C. Population growth started at high rates (1.5 - 2.8 d-1) that declined continuously over the season. Growth rates were not significantly correlated with temperature but they were significantly positive correlated with the mean photosynthetic active radiation in the mixed water column (Imix). At the time population starts growing, Imix has exceeded its annual maximum, and growth rate declines with decreasing Imix. The time of akinete germination is therefore of crucial importance for the success of the population: as earlier akinetes germinate the higher the growth rate will be, resulting in a greater population size, a higher number of akinetes produced, and vice versa. Therefore, we conclude that an earlier increase of the water temperature due to global warming is a major cause for the spreading of C. raciborskii into the temperate zone.

Cylindrospermopsin (CYN) is a potent hepatotoxin produced by different cyanobacteria of the order Nostocales, which is widely distributed in freshwaters and considered a hazard to wildlife and humans. In Europe, only Aphanizomenon flos-aquae is identified as CYN producer so far and the seasonal variability of CYN was not studied yet. Therefore, we studied the seasonal dynamics of the particulate and dissolved CYN concentrations in relation to the cyanobacterial occurrence and environmental factors in two German lakes over two years. Total CYN reached maximum concentrations of 0.34 and 1.80 µg L-1 in Melangsee and Langer See, respectively. In both lakes, the dissolved CYN fraction peaked after the particulate fraction, and reached higher values indicating that CYN is poorly decomposed and accumulates in the water. The cyanobacterial community was very diverse in both lakes, including potentially CYN producing species such as Cylindrospermopsis raciborskii, Anabaena bergii and Aphanizomenon flos-aquae. However, these species could be excluded as the major CYN producers. The stronges significant correlation was found for Aphanizomenon gracile and CYN, strongly indicating that the species is the main CYN producer in Langer See. CYN was also correlated with Planktothrix agardhii in Langer See and Pseudanabaena limnetica in Melangsee, but species of the order scillatoriales are not known to produce CYN. In Melangsee the CYN producer could not be identified. Different correlations of CYN with abiotic factors in the two lakes indicate the presences of further undetected CYN producers as well as different regulation mechanisms of their dynamic and the variability of CYN.

The invasive cyanobacterium Cylindrospermopsis raciborskii is increasingly spreading to temperate freshwater habitats world wide and is of major concern due to its ability to produce potent toxins. It is therefore important to understand the mechanisms behind the dispersal of this species. Different hypotheses have been proposed to explain the phylogeography and mechanisms underlying the recent expansion of C. raciborskii into temperate latitudes, but there is still no conclusive evidence whether the obvious ecological success of C. raciborskii is due to selection mechanisms, physiological tolerance, climatic change or radiation after the last ice age. In this study, new isolates of C. raciborskii from Europe and Africa were genetically characterized by sequencing the ITS1, PC-IGS, nifH and rpoC1 genes and compared to corresponding sequences of C. raciborskii available in GenBank in order to test different phylogeographical hypotheses. The strains were also morphologically examined and screened for production of the hepatotoxic cylindrospermopsin (CYN). We clearly demonstrate that there are phylogenetic, morphological and toxicological differences between the isolated strains. The phylogenetic analyses revealed a clustering of the strains due to geographic origin. The ITS1 and nifH genes separated into American, European and Australian-African groups, whereas the PC-IGS and rpoC1

Cylindrospermopsis raciborskii is considered a cyanobacterium of tropical origin and an alien species to temperate waters. However, it has been detected as far north as northern Germany. While previous studies have shown that all isolated German C. raciborskii strains are hepatotoxic, little is known about the spatial occurrence and relative frequency of this species in temperate Germany. The aim of this study was to investigate the spatial distribution and relative frequency of C. raciborskii close to its northernmost distribution limit, to characterise the habitat in which it is most likely to occur in this climatic zone and to search for any other neocyanobacterial species that might be present in German waters but has so far been overlooked. One hundred forty-two water bodies in northeast Germany were sampled from June until September 2004. All cyanobacteria species were analysed qualitatively and semiquantitatively. Besides C. raciborskii, two additional neocyanobacterial species were detected: Anabaena bergii and Aphanizomenon aphanizomenoides. For both taxa, these findings represent their northernmost occurrence and their first report from German waters. Cylindrospermopsis raciborskii was present in 27%, Anabaena bergii in 9% and phanizomenon aphanizomenoides in 7% of the samples. The occurrence of each species was analysed in relation to maximum lake depth, Secchi depth, lake volume and lake surface area. All three species were present in a wide range of habitats, but C. raciborskii and Anabaena bergii occurred significantly more often in shallow, turbid waters than in deep, transparent water bodies. None of the parameters investigated were significantly correlated with the occurrence of Aphanizomenon aphanizomenoides. In conclusion, alien thermophilic cyanobacterial species are much more widely distributed in temperate Germany than previously known. The results are discussed with respect to the possible mechanisms that enable these organisms to expand northwards.