Microcystins are a group of cyclic heptapeptides originating from cyanobacteria. Cyanobacteria also produce a range of peptides and other compounds that can result in complex chromatograms when samples are analyzed by LC-MS. Derivatization with appropriate thiols (e.g., mercaptoethanol) of the olefin in the α,β-unsaturated amide present in most microcystins was shown to simplify analysis of LC-MS chromatograms of sample extracts, making it much easier to identify peaks corresponding to candidate microcystins. Furthermore, interpretation of MS(2) spectra was facilitated by addition of the mass associated with the thiol to the α,β-unsaturated amide of microcystins. Cyanotoxins containing Mdha or Dha reacted readily with thiols, whereas Mser, Ser, Mdhb, and thiol-derivatives of Mdha or Dha did not react under the conditions used. This approach therefore provides a convenient LC-MS method to obtain evidence for the presence of Mdha or Dha and can likely be used to differentiate between the isobaric amino acids Mdha and Dhb in candidate cyanotoxin peaks. When O-(2-mercaptoethyl)-O'-methyl-hexa(ethylene glycol) (MEMHEG) (M(w)t. 356) was used as the thiol, the resulting derivatives eluted in an LC-MS mass window that was largely free of interferences. This approach simplifies detection of candidate microcystin analogues even in the presence of complex mixtures of coeluting components. The method was used for qualitative analysis of a Microcystis aeruginosa culture from Lake Naivasha, Kenya, and the results were verified using precursor-ion scanning and high-resolution mass spectrometry.

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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.

Cyanobacterial mats at hot springs on the shore of the alkaline Lake Bogoria, Kenya, were investigated regarding species community and cyanobacterial toxin content. The hepatotoxins microcystin-LR, -RR, -LF and -YR, and the neurotoxin anatoxin-a were present. The mats were dominated by Phormidium terebriformis, Oscillatoria willei, Spirulina subsalsa and Synechococcus bigranulatus. The concentration of microcystins in mat samples, ranged from 221 to 845 microg microcystin-LR equivalents g(-1) DW of mat. Anatoxin-a concentrations ranged from 10 to 18 microg g(-1) DW of mat. A contribution of the cyanobacterial toxins from the hot spring mats to the mass mortalities of Lesser Flamingos is suggested by: (a), the presence of hot spring cyanobacterial cells and cell fragments, and high concentrations of the cyanobacterial hepato- and neurotoxins in flamingo stomach contents and faecal pellets; (b), observations of neurological signs of bird poisoning at the lake. Cyanobacterial toxins in stomach contents, intestine and fecal pellets were 0.196 microg g(-1) fresh weight (FW) for the microcystins and 4.34 microg g(-1) FW for anatoxin-a. Intoxication with cyanobacterial toxins could occur by uptake of detached cyanobacterial cells from the mats, as the flamingos need to drink fresh or brackish water, and to wash their feathers daily, which they do in the vicinity of the hot springs, where salinity is lower than in the main body of water of the lake.