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.

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.

Ziel dieses Projektes ist es, zur Klärung der derzeitigen Verbreitung und Variabilität des toxischen Cyanobakteriums C. raciborskii und des Toxins Cylindrospermopsin (CYN) in Gewässern der Berliner Region beizutragen und eine Grundlage zu schaffen, auf welcher deren weitere Entwicklung und das damit verbundene Risiko für Mensch und Umwelt abgeschätzt werden kann. Die Verbreitung von C. raciborskii und CYN wurde in einem Pre-Screening Programm im Sommer 2004 untersucht. Für 142 Gewässer, die sich hinsichtlich Morphometrie, Trophie und Mixistypus unterscheiden, wurde die Zusammensetzung der Cyanobakterien einmalig qualitativ und semi-quantitativ analysiert sowie der CYN Gehalt des Sestons ermittelt. C. raciborskii wurde in 27,5 % der 142 untersuchten Gewässer nachgewiesen. Ihre relative Häufigkeit wurde überwiegend als vereinzelt (21,1 %) oder häufig (6,3 %) eingeschätzt. Massenentwicklungen der Art traten zum Zeitpunkt der Untersuchung nicht auf. Als typisches Habitat für C. raciborskii wurden flache eutrophe Gewässer mit niedriger Sichttiefe, und geringen Zeu/Zmix Verhältnissen analysiert. Entgegen bisheriger Annahmen ist die Art jedoch nicht auf Flachseen beschränkt, sondern kann auch in tiefen dimiktischen Gewässern Populationen etablieren. Darüber hinaus wurden vier weitere bedeutende Arten ermittelt. Raphidiopsis curvata und R. mediterranea, die in 5 von 142 Gewässern detektiert wurden. Von beiden Arten ist bekannt, dass sie CYN produzieren können. Anabaena bergii wurde vereinzelt bis häufig in 14,1 % der Gewässer nachgewiesen. Für diese Art wurde der gleiche Habitattyp wie für C. raciborskii festgestellt. Aphanizomenon aphanizomenoides wurde vereinzelt bis häufig in 13,4 % der Gewässer nachgewiesen. Bei beiden Arten handelt es sich wie bei C. raciborskii um Neo-Cyanobakterien, die bisher nur aus tropischen bzw. subtropischen Regionen bekannt waren. Beide produzieren toxische Substanzen, die im Fall von A. aphanizomenoides noch nicht näher identifiziert werden konnten. Im Fall von A. bergii handelt es sich bei einem der Toxine um CYN. Zusammenfassend kann für die hier relevanten Arten festgestellt werden, dass sie weiter verbreitet sind als bisher bekannt war. Die Tatsache, dass C. raciborskii bisher in verhältnismäßig wenigen und A. bergii sowie A. aphanizomenoides bisher gar nicht für das Untersuchungsgebiet beschrieben wurden, wird u.a. auf taxonomische Unklarheiten zurückgeführt. Bislang wurden 96 Sestonproben aus 80 Gewässern auf CYN untersucht. In 63 % der Proben, bzw. 61 % der Seen wurde CYN in Konzentrationen zwischen 0,1 und 100 µg/g TG nachgewiesen und ist somit in Deutschland weiter verbreitet als bisher angenommen. Ein erster Vergleich der Cyanobakterienzusammensetzung mit dem CYNVorkommen zeigt, dass CYN in den untersuchten Gewässern nicht nur von C. raciborskii produziert wird, da es auch in Proben gemessen wurde, in denen die Art nicht nachgewiesen wurde. Derzeit werden die beiden oben beschriebenen Arten A. bergi und A. aphanizomenoides als weitere potentielle CYN-Produzenten in Betracht gezogen sowie eine Reihe weiterer Arten der Gattungen Anabaena und Aphanizomenon. Eine entgültige Klärung wird nach Abschluss der chemischen und molekularbiologischen Analysen der isolierten Stämme erwartet.

Three single-filament isolates of Aphanizomenon flos-aquae from two German lakes were found to produce remarkable amounts of the cyanobacterial epatotoxin cylindrospermopsin (CYN). CYN-synthesis of the strains were evidenced both by LC-MS/MS analysis and detection of PCR products of gene fragments which are implicated in the biosynthesis of the toxin. The strains contain CYN in the range of 2.3–6.6 mg gK1 of cellular dry weight. To our knowledge this is the first report of CYN in A. flos-aquae.

L'Erdre, rivière de l'ouest de la France, a subi ces dernières années une prolifération massive de cyanobactéries, avec des répercussions négatives sur les activités touristiques étant donné le danger potentiel pour la santé humaine lié au rejet de toxines. A la demande de l'établissement public territorial "Entente pour le Développement de l'Erdre Navigable", E.D.E.N., un consortium de spécialistes (SETUDE, Anjou Recherche, KWB, UBA, Bi-Eau, BCEOM, Eco-Environnement Ingénierie) a participé à une étude détaillée sur l’équilibre écologique de l’Erdre, la croissance des cyanobactéries ainsi que le relargage de toxines, afin de développer des stratégies pour limiter ce phénomène. Au sein de ce consortium, le Centre de Compétence des Eaux de Berlin (KompetenzZentrum Wasser Berlin gGmbH, KWB) et l'Agence Fédérale de l'Environnement d’Allemagne (Umweltbundesamt, UBA) ont réalisé des travaux de recherche en laboratoire. Le projet a été mené de fin 2002 à début 2004 par les partenaires scientifiques français et allemands. Les expériences en laboratoire réalisées par les deux partenaires à Berlin ont pour but d’identifier l’influence de trois facteurs sur le développement des cyanobactéries et la libération de leurs toxines : l’impact d’une limitation en nutriments (azote, phosphore), l’influence de la vitesse d’écoulement, et le rôle des sédiments. L’étude porte sur la cyanobactérie filamenteuse Planktothrix agardhii et la toxine microcystine, qui prédominent dans l’Erdre.

Cyanobacteria proliferation and the potential health risk related with the release of the associated toxins have lead the local association EDEN to initiate a comprehensive study on cyanobacteria in the river Erdre. Within the consortium in charge of the project, the Berlin Centre of Competence for Water (KWB) realised lab-scale research in cooperation with the German Federal Environmental Agency (UBA), on the species Planktothrix agardhii which predominates in the river Erdre, and the associated toxin microcystin. The objective was to determine the influence of key factors such as nutrients (nitrogen, phosphorus), light, flow velocity and sediments on cyanobacteria growth and competition as well as microcystin release from the Planktothrix population in the river Erdre. Results from the lab-scale cultures proved that nutrient-limited conditions lead to a decrease of cyanobacteria biomass and may favour some genotypes with reduced needs among the Planktothrix population. Given the current state of scientific knowledge, no differences in competition between toxic and non-toxic Planktothrix strains can be established. Nutrient limitation favours microcystin release from cells, however the global decrease of cyanobacteria biomass induces a decrease of the total quantity of released toxin. These results can be applied in a water body where nutrients concentrations are very low (below 50 µg/L for total phosphorus). In the river Erdre, as long as external nutrients inputs remain considerable, light is the limiting factor. Internal nutrient recycling from the sediments is globally negligible in comparison with external inputs. Culture experiments in a flow simulation flume proved that flow velocity had substantial impact neither on Planktothrix growth nor on microcystin release. Only a short transition phase with negative effects was observed. Overwintering of Planktothrix in Erdre-sediments could be proved by the detection of a substantial population using fluorescence analysis. This inoculum should be large enough for initiation of Planktothrix development in the next vegetation period. However, the high adsorption capacities of the analysed sediments from the river Erdre allow to put aside a potential risk of microcystin release from sludge. While providing innovative results on the species Planktothrix agardhii, this project contributes to the comprehensive study initiated by the EDEN association in order to preserve the values associated with environment, health and tourism in the river Erdre.