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.     

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.     

Influence of intracellular toxin concentrations on cylindrospermopsin bioaccumulation in a freshwater gastropod (Melanoides tuberculata).

Scant information is available regarding the bioaccumulation of cylindrospermopsin (CYN) in aquatic organisms, particularly in invertebrates. This study examined toxin bioconcentration and bioaccumulation in the aquatic snail, Melanoides tuberculata, following exposure to freeze-thawed whole cell extracts and a live Cylindrospermopsis raciborskii culture containing CYN. Both bioconcentration and bioaccumulation were evident, but exposure to toxin in the freeze-thawed solutions resulted in minor tissue contamination compared with that resulting from live C. raciborskii exposure. Thus, whilst CYN uptake resulted from both extracellular and intracellular exposures, the availability of intracellular toxin was critical in affecting tissue CYN values. M. tuberculata did not bioconcentrate CYN into the shell. Bioaccumulation of the analog deoxy-CYN was also recorded. Knowledge of intracellular toxin concentrations may be critical in evaluating the bioaccumulation, ecological and human health risks associated with contaminated systems.     

Concentrations of particulate and dissolved cylindrospermopsin in 21 Aphanizomenon-dominated temperate lakes.

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 microg L(-1) for particulate CYN and 0.08-11.75 microg L(-1) for dissolved CYN with a maximum of 12.1 microg L(-1) total CYN. A drinking water guideline value of 1 microg L(-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.     

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.     

Isolation and identification of the cyanotoxin cylindrospermopsin and deoxy-cylindrospermopsin from a Thailand strain of Cylindrospermopsis raciborskii (Cyanobacteria).

A strain of Cylindrospermopsis (Cyanobacteria) isolated from a fishpond in Thailand was examined for its taxonomy based upon morphology and 16S rRNA gene sequence. It was also examined for production of the hepatotoxic cyanotoxin called cylindrospermopsin (CYN) and deoxycylindrospermopsin (deoxy-CYN). The strain (CY-Thai) was identified as C. raciborskii (Woloszynska) Seenaya and Subba Raju based upon morphological examination which was confirmed by 16S rRNA gene sequences and phylogenetic comparisons based upon its 16S rRNA gene. The alkaloid heptatotoxin CYN was confirmed using mouse bioassay, HPLC and HPLC-MS/MS while deoxy-CYN was confirmed using HPLC-MS/MS. The mouse bioassay gave a minimum lethal dose at 250mg dry weight cells/kg body weight within 24h and 125mg/kg at 72h, with signs of poisoning the same as in literature reports for CYN. HPLC chromatographic comparison of the CY-Thai toxin with standard CYN gave the same retention time and an absorbance maximum at 262nm. HPLC-MS/MS confirmed the presence of CYN (M+H 416) and deoxy-CYN (M+H 400). The CYN content in strain CY-Thai was estimated at 1.02mg/g and approximately 1/10 of this amount for deoxy-CYN. This is the first report from Asia of a CYN, deoxy-CYN producing Cylindrospermopsis raciborskii.     

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.     

Effects of Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum (cyanobacteria) ingestion on Daphnia magna midgut and associated diverticula epithelium

This article reports a light and electron microscopy investigation of the effects of Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum ingestion on midgut and associated digestive diverticula of Daphnia magna. Additionally, survivorship and growth effects caused by feeding on cyanobacteria were assessed. Three cyanobacteria were used in the experiments: cylindrospermopsin (CYN)-producing C. raciborskii, CYN-producing A. ovalisporum and non-CYN-producing C. raciborskii. In order to discriminate between the alterations due to the low nutritional value of cyanobacteria and toxic effects, a control group was fed on the chlorophyte Ankistrodesmus falcatus and another control group was not fed. In the chlorophyte fed control, the epithelium lining the midgut and associated diverticula is mainly formed by strongly stained cells with an apical microvilli border. Nevertheless, unstained areas in which cell lyses had occurred were also observed. In the unfed control, the unstained areas became predominant due to an increment of cell lyses. All individuals fed on CYN-producing A. ovalisporum and some of those fed on non-CYN-producing C. raciborskii appear similar to the unfed control. However, some individuals fed on non-CYN-producing C. raciborskii showed similarities with the fed control. In contrast, the midgut and digestive diverticula of D. magna fed on CYN-producing C. raciborskii showed a widespread dissociation of epithelial cells, associated with severe intracellular disorganization, but cell lysis was less evident than in controls. These alterations cannot be attributed to CYN, because those effects were not induced by CYN-producing A. ovalisporum. Therefore, data suggest the production of another unidentified active metabolite by CYN-producing C. raciborskii, responsible for the disruption of cell adhesion in the epithelium of D. magna digestive tract. Data also show that the tested cyanobacteria are inadequate as food to D. magna, due to low nutritional value and toxic content.     

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.     

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.     

The Palm Island mystery disease 20 years on: a review of research on the cyanotoxin cylindrospermopsin

Poisoning of humans resulting from consumption of water affected by the toxic cyanobacterium Cylindrospermopsis raciborskii was first reported almost 20 years ago from Palm Island, northern Queensland, Australia. Since that time a great deal has been learned about this organism and cylindrospermopsin (CYN), the toxin it produces. This article reviews the information now available to us. It summarizes aspects of the chemistry of the toxin-now known to be produced by some cyanobacterial species other than C. raciborskii-and its biosynthesis and chemical synthesis in vitro, as well as its detection and measurement by chemical and biological assay. Some of the factors affecting toxin production by cultured isolates of C. raciborskii are reviewed and the conditions that cause its release from the cells described. The occurrence of CYN in water bodies and the management strategies used to minimize the harmful effects of the toxin are outlined. These include a range of water-treatment practices now in place to remove CYN-producing organisms and/or to neutralize the toxin together with some management procedures that have been tried, with varying degrees of success, to prevent buildup of blooms of the offending organisms. Some of the public-health considerations arising from exposure to water supplies affected by CYN are summarized along with the risk factors and guidance values as they are currently applied. Among the more recent developments described are those that come from the application of molecular techniques for characterizing toxic and nontoxic strains and for exploring the genetic aspects of CYN production.     

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.     

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.     

Studies of the comparative in vitro toxicology of the cyanobacterial metabolite deoxycylindrospermopsin

Cyanobacteria are capable of producing metabolites that are in some cases toxic to humans and other animals. Of these metabolites, the toxin cylindrospermopsin (CYN) is produced by a number of species of cyanobacteria including Cylindrospermopsis raciborskii, and its toxicity has been documented. The CYN analog deoxycylindrospermopsin (deoxyCYN) is commonly produced in varying proportions by the cyanobacteria that produce CYN. The toxicological profile of CYN suggests that it is primarily a hepatotoxin, but with the capacity to damage other organs and tissues. Limited in vivo information is available on the toxicity of deoxyCYN and suggests it to be of low potency. The aim of this research was to determine the comparative toxicology of deoxyCYN using in vitro systems. Using cell viability assays, it was shown that deoxyCYN had inhibitory effects on cell viability and proliferation of a similar magnitude to that of CYN. Morphological changes in deoxyCYN-treated cells were similar to those of CYN. Investigation of protein synthesis inhibition demonstrated that deoxyCYN was of similar potency to CYN. Inhibition of protein synthesis is an acknowledged mechanism of toxicity for CYN, and the results produced here suggest that deoxyCYN operates by similar toxicological mechanisms to CYN and that in vivo animal testing should be undertaken to clarify the potential for risk to humans from this toxin.     

Interaction of the cyanobacterial toxin cylindrospermopsin with the eukaryotic protein synthesis system.

The cyanobacterial toxin cylindrospermospin (CYN) was shown to inhibit the eukaryotic protein synthesis apparatus with similar potency in plant and mammalian cell extracts, IC(50) of 334 nM in wheat germ extract and 110 nM in reticulocyte lysate. [(14)C] CYN binding was assessed in reticulocyte lysates, following separation of ribosomes from free toxin on Sephadex G-50 size exclusion columns. While toxin binding was shown to be concentration-dependent (60-600 nM [(14)C] CYN), it did not correlate with ribosome content. The molar ratio of toxin to ribosomes was 0.02:1 at the binding plateau. Significant binding of [(14)C] CYN was identified in both the ribosome fraction (> 90% total ribosomes) and the first wash fraction eluting from the Sephadex G-50 column, despite the low ribosome content (<10% total ribosomes) in the latter. Bound [(14)C] CYN could be partially displaced by incubation with an excess of unlabelled CYN in both the ribosome and wash fractions, indicating a non-covalent linkage. Molecular weight cut-off filters identified that [(14)C] CYN was associated with high molecular weight material > 100 kDa. These results suggest that CYN's target may not be the ribosome itself, but rather one of the soluble proteins associated with the eukaryotic translation system.     

Nitrogen Limitation Promotes Accumulation and Suppresses Release of Cylindrospermopsins in Cells of Aphanizomenon Sp.

As the biosynthesis of cylindrospermopsin (CYN) is assumed to depend on nitrogen availability, this study investigated the impact of nitrogen availability on intra- and extracellular CYN and deoxy-CYN (D-CYN) contents in three Aphanizomenon strains from temperate waters. Nitrogen deficient (−N) cultures showed a prolonged growth phase and intracellular toxin accumulation by a factor of 2–6. In contrast, cultures with additional nitrate supply (+N) did not accumulate CYN within the cells. Instead, the maximum conceivable CYN release estimated for dead cells (identified by SYTOX^® Green staining) was much lower than the concentrations of dissolved CYN actually observed, suggesting these cultures actively release CYN from intact cells. Furthermore, we found remarkably altered proportions of CYN to D-CYN: as batch cultures grew, the proportion of D-CYN increased by up to 40% in +N medium, whereas D-CYN remained constant or decreased slightly in −N medium. Since +N cultures showed similar toxin patterns as −P cultures with increased extracellular CYNs and higher proportion of D-CYN we conclude that nitrogen limitation may affect the way the cells economize resources, especially the yield from phosphorus pools, and that this has an impact on CYN production and release. For water management, these result imply that nutrient availability not only determines the abundance of potentially CYN-producing cyanobacteria, but also the amount of extracellular CYNs (challenging drinking-water treatment) as well as the ratio of D-CYN to CYN (affecting toxicity).     

New values of molecular extinction coefficient and specific rotation for cyanobacterial toxin cylindrospermopsin.

The molecular extinction coefficient epsilon of cylindrospermopsin (CYN) purified by the anion exchange and the normal-phase HPLC procedures was determined to be 9800 at 262 nm. This epsilon is significantly higher than those (epsilon, 5800-6250) reported previously. In order to determine CYN concentrations in solutions using UV absorption, the epsilon-value of CYN should be corrected from 5800 to 9800. Further, the [alpha](D) value of CYN should be corrected from +12.5 degrees to +17.0 degrees .     

Elucidating the factors influencing the biodegradation of cylindrospermopsin in drinking water sources

The cyanotoxin cylindrospermopsin (CYN) is produced by several species of cyanobacteria and can be persistent in drinking waters supplies, which is of major concern to water authorities because of its potential to severely compromise human health. Consequently, there is a need to fully understand the persistence of CYN in water supplies, in particular, to determine whether this toxin is readily degraded by endemic aquatic organisms. This study provides insights into the environmental factors that can influence the biodegradation of this toxin in Australian drinking water supplies. Biodegradation of CYN was only evident in water supplies that had a history of toxic Cylindrospermopsis raciborskii blooms. In addition, lag periods were evident prior to the onset of biodegradation; however, repeated exposure of the endemic organisms to CYN resulted in substantial decreases in the lag periods. Furthermore, the concentration of CYN was shown to influence biodegradation with a near linear relationship (R(2) of 0.9549) existing between the biodegradation rate and the initial CYN concentration. Temperature was also shown to affect the biodegradation of CYN, which is important since CYN is now being detected in more temperate climates. The presence of copper-based algicides inhibited CYN degradation, which has significant implications since copper-based algicides are commonly used to control cyanobacterial growth in water bodies. The results from this study indicate that the biodegradation of CYN in natural water bodies is a complex process that can be influenced by many environmental factors, some of which include CYN concentration, temperature, and the presence of copper-based algicides.     

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.     

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.