The occurrence of the cyanobacterial toxins anatoxin-a (ATX) and cylindrospermopsin (CYN) in surface waters has been reported throughout the world. Beside degradation, sorption is an important pathway for toxin elimination if these resources are used for drinking water production via sediment passage. However, to date studies that systematically investigated sorption of these toxins onto sediments are lacking. Therefore, the aim of our work was (i) to determine the adsorption coefficients of ATX and CYN according to the Freundlich and Langmuir model for sediments of various textures and (ii) to derive sorptionrelevant sediment characteristics. We determined sorption parameters in air-dried samples of eight differently textured sediments using batch experiments. Results for both toxins showed best fits with the Langmuir model. Organic C proved to be the main sediment parameter determining CYN sorption. There was no or little CYN sorption on sandy and silty sediments (0e39 mg kg-1), respectively, presumably due to charge repulsion from the negatively charged surfaces. Sorption of ATX (max. sorbent loading ranging from 47 to 656 mg kg-1) was much stronger than that of CYN (max. sorbent loading ranging from 0 to 361 mg kg-1) and predominantly controlled by clay and to a minor degree also by organic C and silt. While ATX sorption to most sediments occurred mainly through cation exchange this mechanism played only a minor role in CYN sorption to organic C. Hence, high mobility for CYN and moderate mobility for ATX during sediment passage has to be expected.

Recent results show that cylindrospermopsin is more frequent and widespread in surface waters than previously assumed. Studies on the fate of CYN in sediments are lacking, but this is important if these resources are used for drinking-water production via sediment passage. Therefore, the aim of our study was to determine a) CYN retention in two sandy sediments as a function of flow rate, CYN concentration, the presence of DOM and the content of fines (1% and 4%, respectively) and b) the influence of sediment preconditioning and DOM composition of the water (aquatic DOM versus DOM released from lysed cells) on CYN degradation. Retention of CYN proved negligible under the investigated conditions. Degradation in virgin sediments showed the highest lag phases (20 days). Preconditioned sediments showed no lag phase. The presence of aquatic DOM yielded highest degradation rates (k1 ¼0.46 and 0.49 day 1) without a lag phase. Readily available organic carbon sources were preferentially metabolized and hence induced a lag phase. Thus, the presence and composition of DOM in the water proved important for both CYN degradation rates in preconditioned sediments and for the lag phase. Cylindrospermopsin degradation took place solely in the sediment and not in the water body.

The neurotoxin anatoxin-a (ATX), has been detected in several northeast German lakes during the last two decades, but no ATX producers have been identified in German water bodies so far. In 2007 and 2008, we analyzed phytoplankton composition and ATX concentration in Lake tolpsee (NE Germany) in order to identify ATX producers. Sixty-one Aphanizomenon spp. strains were isolated, morphologically and phylogenetically characterized, and tested for ATX production potential by liquid chromatography–tandem mass spectrometry (LC–MS/MS). New primers were specifically designed to identify a fragment of a polyketide synthase gene putatively involved in ATX synthesis and tested on all 61 Aphanizomenon spp. strains from L. Stolpsee and 92 non-ATX-producing Aphanizomenon spp., Anabaena spp. and Anabaenopsis spp. strains from German lakes Langersee, Melangsee and Scharmützelsee. As demonstrated by LC–MS/MS, ATX concentrations in L. Stolpsee were undetectable in 2007 and ranged from 0.01 to 0.12 µg l-1 in 2008. Fifty-nine of the 61 strains isolated were classified as Aphanizomenon gracile and two as Aphanizomenon issatschenkoi. One A. issatschenkoi strain was found to produce ATX at concentrations of 2354 ± 273 µg g-1 fresh weight, whereas the other A. issatschenkoi strain and A. gracile strains tested negative. The polyketide synthase gene putatively involved in ATX biosynthesis was found in the ATX-producing A. issatschenkoi strain from L. Stolpsee but not in the non-ATX-producing Aphanizomenon spp., Anabaena spp. and Anabaenopsis spp. strains from lakes Stolpsee, Langersee, Melangsee, and Scharmützelsee. This study is the first confirming A. issatschenkoi as an ATX producer in German water bodies.

Neurotoxic paralytic shellfish poisoning (PSP) toxins, anatoxin-a (ATX), and hepatotoxic cylindrospermopsin (CYN) have been detected in several lakes in northeast Germany during the last 2 decades. They are produced worldwide by members of the nostocalean genera Anabaena, Cylindrospermopsis, and Aphanizomenon. Although no additional sources of PSP toxins and ATX have been identified in German water bodies to date, the observed CYN concentrations cannot be produced solely by Aphanizomenon flos-aquae, the only known CYN producer in Germany. Therefore, we attempted to identify PSP toxin, ATX, and CYN producers by isolating and characterizing 92 Anabaena, Aphanizomenon, and Anabaenopsis strains from five lakes in northeast Germany. In a polyphasic approach, all strains were morphologically and phylogenetically classified and then tested for PSP toxins, ATX, and CYN by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA) and screened for the presence of PSP toxin- and CYN-encoding gene fragments. As demonstrated by ELISA and LC-MS, 14 Aphanizomenon gracile strains from Lakes Melang and Scharmützel produced four PSP toxin variants (gonyautoxin 5 [GTX5], decarbamoylsaxitoxin [dcSTX], saxitoxin [STX], and neosaxitoxin [NEO]). GTX5 was the most prevalent PSP toxin variant among the seven strains from Lake Scharmützel, and NEO was the most prevalent among the seven strains from Lake Melang. The sxtA gene, which is part of the saxitoxin gene cluster, was found in the 14 PSP toxin-producing A. gracile strains and in 11 non-PSP toxin-producing Aphanizomenon issatschenkoi, A. flos-aquae, Anabaena planktonica, and Anabaenopsis elenkinii strains. ATX and CYN were not detected in any of the isolated strains. This study is the first confirming the role of A. gracile as a PSP toxin producer in German water bodies.

In summer 2007 & 2008, 100 water samples were collected from 10 freshwater reservoirs with cyanobacteria issues. Phytoplankton was determined according to the Utermohl method [1]. Intra- and extracellular CYN, ATX-a, STX were analyzed by LC-MS-MS or HPLC-PDA at UBA, and in addition, Veolia tested Abraxis ELISA kits for total CYN and total STX on the 2008 water samples (n=45). Cyanobacterial abundance was comparably low in 2007 & 2008 for all reservoirs, probably because of cooler summer months, with less sunlight, more rain and quickly decreasing fall temperatures (except in reservoir 10, which had low incoming nutrient charges). For instance, average chlorophyll content was 12 µg/L in 2007 and 35 µg/L in 2008 in Western France, when 60-80 µg/L concentrations are usually measured. In spite of these environmental conditions, cyanobacteria were detected in 97% of the samples and cyanotoxins in 55%. WHO level 3 for drinking water (>100 000 cell/mL) was reached for 20-25% of the samples. Among the species observed in the water samples, the following potential CYN, ATX-a, STX producers were observed: Cyanotoxin LC-MS-MS and HPLC-PDA results are given on the right. ELISA results for CYN and STX of the 2008 samples only partially agree with the LC-MS-MS data. This might be due to the differences in extraction procedures of the two methods, cross-reactivity issues of the ELISAs for derivatives, in combination with overall very low concentrations of the toxins.

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.

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.