Occurrence of the cyanobacterial toxin cylindrospermopsin in northeast Germany

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 north-eastern 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 = 63) and in half of 165 samples (n = 88). Concentrations reached up to 73.2 microg 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.     

Seasonal dynamics of cylindrospermopsin and cyanobacteria in two German lakes

Cylindrospermopsin (CYN) is a potent hepatotoxin produced by different cyanobacteria of the order Nostocales. Questions of major concern are: which species produce CYN, which are the seasonal patterns of CYN dynamics and how are they regulated? Therefore, we studied for the first time the seasonal dynamics of particulate and dissolved CYN concentrations, cyanobacterial abundance and environmental factors in two German lakes over 2 years. Total CYN reached maximum concentrations of 0.34 and 1.80 μg L⁻¹ in Melangsee and Langer See, respectively. In both lakes, maxima of the dissolved CYN fraction occurred later in the season than those of the particulate fraction, and it reached higher concentrations. This indicates that CYN is poorly decomposed and accumulates in the water. The cyanobacterial community in both lakes included several potentially CYN-producing species that did not correlate with CYN concentrations. Significant correlations between the particulate CYN concentrations and species biovolume were only found for Aphanizomenon gracile (r_s = 0.803) in Langer See indicating that this species is a CYN producer. Different correlations of CYN with abiotic factors in the two lakes indicate the presence of further undetected CYN producers as well as different regulation mechanisms of their dynamics and the variability of CYN.     

First report on cylindrospermopsin producing Aphanizomenon flos-aquae (Cyanobacteria) isolated from two German lakes.

Three single-filament isolates of Aphanizomenon flos-aquae from two German lakes were found to produce remarkable amounts of the cyanobacterial hepatotoxin 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 g(-1) of cellular dry weight. To our knowledge this is the first report of CYN in A. flos-aquae.     

A comparative study of Florida strains of Cylindrospermopsis and Aphanizomenon for cylindrospermopsin production.

The toxin cylindrospermopsin (CYN) is produced by a variety of cyanobacterial genera. One of these, Cylindrospermopsis raciborskii, is generally assumed to be the source of CYN in lakes and rivers in Florida, USA. However, in this study, none of the eight Florida isolates of this species tested contained the genetic determinants involved in toxin production nor did they produce CYN. We show for the first time that Aphanizomenon ovalisporum isolated from a pond in this state has the genes putatively associated with CYN production. Analysis by liquid chromatography with mass spectrometric detection (LC/MS) revealed that it produced CYN in the range of 7.39-9.33 microg mg(-1) freeze-dried cells. 16S rDNA sequences of this strain showed 99.6% and 99.9% identity to published A. ovalisporum and Anabaena bergii 16S sequences, respectively. These results help to explain the general lack of a defined relationship between the abundance of C. raciborskii in freshwater ecosystems of Florida and observed concentrations of CYN. The latter observation raises the potential that previous reports of CYN may be coincidental with unrecorded presence of another CYN-producing species.     

Accumulation and depuration of the cyanobacterial toxin cylindrospermopsin in the freshwater mussel Anodonta cygnea.

Cylindrospermopsin (CYN) is a toxic alkaloid produced by several genera of freshwater cyanobacteria. This compound has been implicated in outbreaks of human sickness and the death of domestic and wild animals. Given that several of the cyanobacterial genera known to produce CYN are common components of the phytoplankton of freshwaters including aquaculture facilities, we studied the accumulation of CYN in the freshwater mussel (swan mussel) Anodonta cygnea. Anodonta were exposed to CYN-producing cultures of the cyanobacterium Cylindrospermopsis raciborskii for 16 days and were found to accumulate the toxin to concentrations up to 2.52 microg g tissue dry weight(-1). There was considerable variation in the concentrations of CYN detected in different parts of the body. At the end of a 2-week accumulation period the distribution of CYN in the body of Anodonta was as follows: haemolymph (68.1%), viscera (23.3%), foot and gonad (7.7%) and mantle (0.9%). No CYN was detected in the gills or adductor muscle of any animals. Following a 2-week depuration period, approximately 50% of the toxin remained in the tissues. Based on the recently derived guideline value for CYN in human drinking water (1 microg l(-1)) and the concentrations of this compound in animal tissues reported here, there is a clear need for the increased monitoring of this compound in organisms grown for human and animal consumption.     

Occurrence and elimination of cyanobacterial toxins in two Australian drinking water treatment plants.

In Australian freshwaters, Anabaena circinalis, Microcystis spp. and Cylindrospermopsis raciborskii are the dominant toxic cyanobacteria. Many of these surface waters are used as drinking water resources. Therefore, the National Health and Medical Research Council of Australia set a guideline for MC-LR toxicity equivalents of 1.3 microg/l drinking water. However, due to lack of adequate data, no guideline values for paralytic shellfish poisons (PSPs) (e.g. saxitoxins) or cylindrospermopsin (CYN) have been set. In this spot check, the concentration of microcystins (MCs), PSPs and CYN were determined by ADDA-ELISA, cPPA, HPLC-DAD and/or HPLC-MS/MS, respectively, in two water treatment plants in Queensland/Australia and compared to phytoplankton data collected by Queensland Health, Brisbane. Depending on the predominant cyanobacterial species in a bloom, concentrations of up to 8.0, 17.0 and 1.3 microg/l were found for MCs, PSPs and CYN, respectively. However, only traces (<1.0 microg/l) of these toxins were detected in final water (final product of the drinking water treatment plant) and tap water (household sample). Despite the low concentrations of toxins detected in drinking water, a further reduction of cyanobacterial toxins is recommended to guarantee public safety.     

Oral toxicity of the cyanobacterial toxin cylindrospermopsin in male Swiss albino mice: determination of no observed adverse effect level for deriving a drinking water guideline value

The cyanobacterial toxin cylindrospermopsin (CYN) is a frequent contaminant of freshwaters throughout the world, including those that are sources of drinking water. The first cases of human poisoning attributed to this toxin occurred from a treated drinking water supply in Queensland, Australia, in 1979. The toxin causes extensive damage to the liver, kidneys, spleen, heart, and other organs. It is known to be a potent protein synthesis inhibitor, but there is mounting evidence for genotoxicity and that it metabolizes to even more toxic forms. As part of a risk assessment process leading to a guideline for a safe drinking water level for this toxin, we performed a series of experiments to determine a no-observed-adverse-effect level (NOAEL) for this toxin. In the first trial male mice were exposed to CYN-containing cyanobacterial extract in their drinking water (0-657 microg CYN kg(-1) day(-1)) for 10 weeks. In the second trial mice received purified CYN by daily gavage (0-240 microg CYN kg(-1) day(-1)) for 11 weeks. Body and organ weights were recorded; urine, serum, and hematology analyses were performed; and histopathological examination of tissues was carried out. Body weights were significantly increased at low doses (30 and 60 microg kg(-1) day(-1)) and decreased at high doses (432 and 657 microg kg(-1) day(-1)). Liver and kidney weights were significantly increased at doses of 240 microg kg(-1) day(-1) and 60 microg kg(-1) day(-1), respectively. Serum bilirubin levels were significantly increased and bile acids significantly decreased at doses of 216 microg kg day(-1) and greater. Urine total protein was significantly decreased at doses above 60 microg kg(-1) day(-1). The kidney appeared to be the more sensitive organ to this toxin. If it is assumed that increased organ weights and changes in functional capacity are responses to an underlying toxic effect, then the NOAEL based on this data is 30 microg kg(-1) day(-1), which, with standard calculations and uncertainty factors, provides a proposed guideline safety value of 1 microg/L in drinking water.     

Cylindrospermopsin Biodegradation Abilities of Aeromonas sp. Isolated from Rusa?ka Lake

The occurrence of the cyanobacterial toxin cylindrospermopsin (CYN) in freshwater reservoirs is a common phenomenon. However, the biodegradation of this toxin in environmental samples has been observed only occasionally. In this work the biodegradation ability of cylindrospermopsin was investigated based on isolates from lakes with previous cyanotoxin history. Bacterial strains were identified based on the 16S rDNA and rpoD gene comparison. CYN biodegradation was monitored using the HPLC method. The R6 strain identified as Aeromonas sp. was documented as being capable of CYN removal. This biodegradation was dependent on the pH and temperature. Additionally, the stimulation of the growth of the R6 strain in the presence of CYN was indicated. Our discovery supports the hypothesis that (in analogy to the well-known phenomenon of microcystin biodegradation) in lakes dominated by potential CYN-producing cyanobacteria, the processes of microbial utilization of this toxin may occur.     

Cylindrospermopsin occurrence in two German lakes and preliminary assessment of toxicity and toxin production of Cylindrospermopsis raciborskii (Cyanobacteria) isolates.

Cylindrospermopsis raciborskii, a freshwater cyanobacterium of tropical origin, is not only increasingly found in (sub) tropical water bodies, but also in temperate regions. Since this species may produce potent toxins such as cylindrospermopsin (CYN) and paralytic shellfish poisons, its massive occurrence in water bodies used as drinking water sources or for recreation is of major concern. The proliferation of C. raciborskii in German water bodies has been documented for the past decade. We investigated the occurrence of CYN in field populations and isolates of C. raciborskii from two lakes, and assessed the toxicity of culture isolates using the mouse bioassay, primary rat hepatocytes and human derived cell lines. We show for the first time the occurrence of CYN in German water bodies. None of seven isolates of C. raciborskii contained CYN, however, all isolates were toxic to primary rat hepatocytes, human hepatoblastoma (HEP-G2) and human colon adenocarcinoma (CACO-2) cells. Methanolic extracts were more toxic than aqueous extracts. Three isolates tested in the mouse bioassay were toxic at a concentration of 800 mg kg(-1) showing liver and spleen damage and inflammation of the intestine. These results give strong evidence that the German isolates of C. raciborskii contain currently not identified or unknown toxins.     

Hepatotoxic cyanobacterial blooms in the lakes of northern Poland

The lakes of northern Poland are among the recreational sites most valued by Polish and German holiday makers. Given the socioeconomic importance of these lakes, water quality should be maintained at high levels for such intensive recreational purposes. In 2002 studies of species composition, biomass, and toxin production by phytoplankton and the attendant physicochemical variables were performed in order to assess the risk of cyanobacterial blooms in selected northern lakes: Lakes Jeziorak, Jagodne, Szymoneckie, Szymon, Taltowisko, Siecino, and Trzesiecko. The research showed that total phosphorus (0.1 mg P/L) and total nitrogen (1.5 mg N/L) in the studied lakes almost exceeded the permissible limits for eutrophication of water bodies. Most phytoplankton samples were taken in late summer, when cyanobacteria were expected to reach their highest biomass. At the time of sampling most of the lakes were dominated by oscillatorialean and nostocalean species. Average chlorophyll-a concentration was higher than 10 microg/L in almost all the lakes studied, which corresponded with an average microcystin concentration in the range of 4-5 microg/L. The main microcystins in the analyzed samples were dmMC-RR, MC-RR, MC-YR, and MC-LR. The results demonstrated a potential for intensive cyanobacterial blooms to appear during the summer in northern Polish lakes. The levels of cyanobacteria found in the lakes investigated indicated that toxicity had reached the first-alert level according to World Health Organization recommendations. If microcystin-producing cyanobacteria dominate, with a microcystin concentration of 2-4 microg/L, symptoms of toxicity can appear in the swimmers most sensitive to exposure. Analysis of cyanobacterial assemblages in northern Polish lakes also indicated a significant presence of Aphanizomenon species including a Scandinavian species, A. skujae (Skuja) Kom.-Legn. & Cronb. Future investigations of Polish lakes also should assess neurotoxins and study the biology of their producers. This study was the first attempt to evaluate the potential danger of toxic cyanobacterial blooms in the lakes of northern Poland.     

Toxic cyanobacteria and microcystin concentrations in a public water supply reservoir in the Brazilian Amazonia region.

Toxic cyanobacteria in public water supply reservoirs represent a serious health risk as they can release potent cyanotoxins into the water. In the present study we analyzed surface water collected from the Utinga Reservoir, the main source of drinking water for the city of Belem-PA, in order to characterize the reservoir's cyanobacterial biota and to determine the toxicity of these organisms and the concentration of microcystins in raw and treated water. The cyanobacterial biota included potentially toxic genera such as Aphanizomenon, Microcystis, Nostoc, Oscillatoria, Planktothrix and Radiocystis. Mouse bioassays revealed a 43.6% frequency of hepatotoxic strains, including 30.8% Microcystis viridis strains and 12.8% Radiocystis fernandoi strains, with the lowest LD(100) (ip) of 45 and 75 mg kg(-1) body weight, respectively. Subacute hepatotoxicity was observed for Aphanizomenon cf. gracile and Coelomoron pusillum strains. HPLC analysis confirmed the production of microcystins at maximum concentrations of 4.22 microg mg(-1) dry weight for M. viridis and 2.47 microg mg(-1) for R. fernandoi. Microcystins at concentrations of up to 1.25 microg L(-1) detected in raw water by ELISA, together with a cyanobacterial density of 20,000 cells mL(-1), represents the first report of a bloom of cyanobacteria for an Amazonian water body.     

The accumulation of cylindrospermopsin from the cyanobacterium Cylindrospermopsis raciborskii in tissues of the Redclaw crayfish Cherax quadricarinatus.

Redclaw crayfish, Cherax quadricarinatus harvested from an aquaculture pond infested by a bloom of the cyanobacterium Cylindrospermopsis raciborskii (order: Nostocales), were shown to accumulate the toxic alkaloid cylindrospermopsin. Pond water samples collected during the bloom contained 589 microg l(-1) of the toxin (93% in the cyanobacterial cells, 7% in the water). Crayfish from the pond contained cylindrospermopsin at concentrations of 4.3 microg g freeze dried hepatopancreas tissue and 0.9 microg g freeze dried muscle tissue. Trichomes of C. raciborskii were observed in gut contents of crayfish harvested during the cyanobacterial bloom, indicating that the most likely mechanism for accumulation of the toxin was by ingestion of cyanobacterial cells. Crayfish subjected to an extract of harvested bloom material under laboratory conditions for a period of 14 days were also found to accumulate cylindrospermopsin, indicating that this toxin is also absorbed into the tissues by direct uptake of the toxin in solution.     

Occurrence and elimination of cyanobacterial toxins in drinking water treatment plants

Toxin-producing cyanobacteria (blue-green algae) are abundant in surface waters used as drinking water resources. The toxicity of one group of these toxins, the microcystins, and their presence in surface waters used for drinking water production has prompted the World Health Organization (WHO) to publish a provisional guideline value of 1.0 mug microcystin (MC)-LR/l drinking water. To verify the efficiency of two different water treatment systems with respect to reduction of cyanobacterial toxins, the concentrations of MC in water samples from surface waters and their associated water treatment plants in Switzerland and Germany were investigated. Toxin concentrations in samples from drinking water treatment plants ranged from below 1.0 microg MC-LR equiv./l to more than 8.0 microg/l in raw water and were distinctly below 1.0 microg/l after treatment. In addition, data to the worldwide occurrence of cyanobacteria in raw and final water of water works and the corresponding guidelines for cyanobacterial toxins in drinking water worldwide are summarized.     

Toxicity of cylindrospermopsin, and other apparent metabolites from Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum, to the zebrafish (Danio rerio) embryo

Cyanobacteria produce a diverse array of toxic or otherwise bioactive compounds that pose growing threats to human and environmental health. We utilized the zebrafish (Danio rerio) embryo, as a model of vertebrate development, to investigate the inhibition of development pathways (i.e. developmental toxicity) by the cyanobacterial toxin, cylindrospermopsin (CYN), as well as extracts from various isolates of Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum. CYN was toxic only when injected directly into embryos, but not by direct immersion at doses up to 50 μg/ml. Despite the dose dependency of toxicity observed following injection of CYN, no consistent patterns of developmental defects were observed, suggesting that toxic effects of CYN may not target specific developmental pathways. In contrast, direct immersion of embryos in all of the extracts resulted in both increased mortality and reproducible, consistent, developmental dysfunctions. Interestingly, there was no correlation of developmental toxicity observed for these extracts with the presence of CYN or with previously reported toxicity for these strains. These results suggest that CYN is lethal to zebrafish embryos, but apparently inhibits no specific developmental pathways, whereas other apparent metabolites from C. raciborskii and A. ovalisporum seem to reproducibly inhibit development in the zebrafish model. Continued investigation of these apparent, unknown metabolites is needed.     

Microcystins associated with Microcystis dominated blooms in the Southwest wetlands, Western Australia

Potentially toxic cyanobacterial blooms are becoming common in the freshwater wetlands on the Swan Coastal Plain, Western Australia. During summer the dominant bloom-causing species belong to the genera Microcystis and Anabaena and to a lesser extent Aphanizomenon and Nodularia. Although toxic cyanobacteria have been recorded in the Swan-Canning and Peel-Harvey estuaries in Western Australia, very little is known about the blooms in the surrounding freshwater lakes. In this study, a total of 32 natural bloom samples representing 13 lakes were analyzed by HPLC for microcystin (MC)-LR, -RR, and -YR. Twenty-eight samples proved to be toxic. The highest total microcystin concentration ranged from 1645 to 8428.6 microg L(-1), and the lowest concentrations were less than 10 microg L(-1) with some below the detection limit (< 0.05 microg L(-1)). MC-LR (100%) was the predominant microcystin, followed by MC-YR (71.4%) and MC-RR (60.7%). The presence of a Nodularia spumigena bloom in the freshwater Lake Yangebup was associated with the detection of nodularins (1664 microg L(-1)). This is the first study to demonstrate the presence of microcystins and nodularins in urban lakes on the Swan Coastal Plain, Western Australia.     

Immunohistochemical Approach to Study Cylindrospermopsin Distribution in Tilapia (Oreochromis niloticus) under Different Exposure Conditions

Cylindrospermopsin (CYN) is a cytotoxic cyanotoxin produced by several species of freshwater cyanobacteria (i.e., Aphanizomenon ovalisporum). CYN is a tricyclic alkaloid combined with a guanidine moiety. It is well known that CYN inhibits both protein and glutathione synthesis, and also induces genotoxicity and the alteration of different oxidative stress biomarkers. Although the liver and kidney appear to be the main target organs for this toxin based on previous studies, CYN also affects other organs. In the present study, we studied the distribution of CYN in fish (Oreochromis niloticus) under two different exposure scenarios using immunohistochemical (IHC) techniques. In the first method, fish were exposed acutely by intraperitoneal injection or by gavage to 200 µg pure CYN/Kg body weight (bw), and euthanized after 24 h or five days of exposure. In the second method, fish were exposed by immersion to lyophilized A. ovalisporum CYN-producing cells using two concentration levels (10 or 100 µg/L) for two different exposure times (7 or 14 days). The IHC was carried out in liver, kidney, intestine, and gills of fish. Results demonstrated a similar pattern of CYN distribution in both experimental methods. The organ that presented the most immunopositive results was the liver, followed by the kidney, intestine, and gills. Moreover, the immunolabeling signal intensified with increasing time in both assays, confirming the delayed toxicity of CYN, and also with the increment of the dose, as it is shown in the sub-chronic assay. Thus, IHC is shown to be a valuable technique to study CYN distribution in these organisms.     

Uptake and accumulation of dissolved,radiolabeled nodularin in Baltic Sea zooplankton

The mass occurrence of toxic cyanobacteria is a recurrent phenomenon in the Baltic Sea. Grazers may obtain toxins either through ingestion or by direct exposure to dissolved toxins. Despite this, there is little knowledge about the accumulation of cyanobacterial toxins in planktonic organisms present during these blooms. Toxin analyses of tissue samples are complicated to carry out and, because of the small size of microscopic planktonic organisms, often difficult to execute. Therefore, we wanted to use a precise and sensitive method to study toxin uptake and accumulation in zooplankton. We used chemically tritiated nodularin, (3)H-dihydronodularin, to study the uptake of dissolved nodularin, a cyanobacterial hepatotoxin produced by Nodularia spumigena. Cultures of the calanoid copepods Acartia tonsa and Eurytemora affinis, and an oligotrich ciliate Strombidium sulcatum were exposed to (3)H-dihydronodularin in filtered seawater, using naturally occurring concentrations of dissolved nodularin (5 microg L(-1)). All three species took up measurable amounts of radiolabeled nodularin. After 48 h we detected 0.37 +/- 0.22 microg toxin g C(-1) (mean +/- sd) in A. tonsa and 0.60 +/- 0.15 microg toxin g C(-1) in E. affinis, whereas 1.55 +/- 0.50 microg toxin g C(-1) was detected in S. sulcatum after 24 h. The minimum bioconcentration factor (BCF) of (3)H-dihydronodularin was 12 for A. tonsa and 18 for E. affinis. For S. sulcatum our results indicate a maximum BCF of 22. However, because the uptake studies for this species were done in the presence of bacteria, possible particulate transfer cannot be excluded. Nevertheless, our results indicate that dissolved nodularin can be taken up by planktonic organisms. Therefore, the vectorial transport of dissolved toxins to higher trophic levels seems possible, even if some planktonic grazers would avoid feeding on toxic cyanobacteria filaments.     

First occurrence of cylindrospermopsin in freshwater in France

Eleven waterbodies in Western France dominated by cyanobacteria of the genera Aphanizomenon and Anabaena were analyzed in September 2006 for microcystins (MC) and cylindrospermopsin (CYN). CYN was detected for the first time in France in four of them in the presence of Aphanizomenon flos-aquae and in the presence of Anabaena planctonica in the other. The intracellular concentrations of CYN measured by LC-MS/MS ranged between 1.55 and 1.95 microg/L. The occurrence of CYN represents an additional health hazard to MC especially because Aphanizomenon flos-aquae is the third most common species in freshwaters in France.     

Cell-free protein synthesis inhibition assay for the cyanobacterial toxin cylindrospermopsin

The cyanobacterial toxin cylindrospermopsin (CYN) is known to be a potent inhibitor of protein synthesis. This paper describes the use of a rabbit reticulocyte lysate translation system as a protein synthesis inhibition assay for CYN. A dose response curve for protein synthesis inhibition by CYN was constructed and was modeled to a sigmoidal dose response curve with variable slope (R2 = 0.98). In this assay, CYN has an IC50 of 120 nM [95% confidence limits (Cl) = 111-130 nM] with a detection limit in the region of 50 nM in the assay solution. Application of the assay allows quantification of toxin samples within the range 0.5-3.0 microM (200-1200 micrograms/L) CYN. To assess the usefulness of this assay, a range of toxic and nontoxic Cylindrospermopsis raciborskii extracts, including both laboratory strains and environmental samples, were assayed by protein synthesis inhibition. These CYN quantifications were then compared to quantifications obtained by high performance liquid chromatography (HPLC) and HPLC-tandem mass spectrometry (HPLCMS-MS). The results demonstrate that the protein synthesis inhibition assay correlates well with both HPLCMS-MS (r2 = 0.99) and HPLC (r2 = 0.97) quantifications. We conclude that this is an accurate and rapid assay for the measurement of cylindrospermopsin in cyanobacterial extracts.