Application of the neuroblastoma assay for paralytic shellfish poisons to neurotoxic freshwater cyanobacteria: interlaboratory calibration and comparison with other methods of analysis.

Paralytic shellfish poisons (PSPs) are produced by freshwater cyanobacteria and pose a threat to human and animal drinking-water supplies. The wide range of toxin analogues (and the likelihood that further analogues remain to be discovered) means that chromatographic methods are not always reliable indicators of toxicity. Although the mouse bioassay remains the method of choice in the seafood industry, its use is increasingly being questioned on ethical grounds. The cell-based Neuro-2A neuroblastoma toxicity assay is an alternative bioassay validated for testing shellfish extracts, so it was of interest to determine its applicability with the different suite of toxin analogues produced by cyanobacteria. Cyanobacterial bloom samples from Australia, Brazil, and France were assayed using the neuroblastoma assay, liquid chromatography-tandem mass spectrometry (LC-MS/MS), high-performance liquid chromatography with postcolumn derivatization and fluorescence detection, and the Jellett Rapid Test for PSP. To assess interlaboratory variability, the neuroblastoma assay was set up in laboratories in Paris (France) and Adelaide (Australia). Neuroblastoma and chromatographic methods gave comparable results except in the case of the neurotoxic Brazilian samples: LC-MS/MS did not detect the putative new PSPs contained in these samples. Inter- and intralaboratory variability of the neuroblastoma assay was typical of biological assays but no greater than that found for interassay variability between different chromatographic determinations. The batch of Jellett Rapid Tests for PSP used did not yield quantitative results. Overall, the neuroblastoma assay was useful as a screening assay for determination of toxicity caused by saxitoxin neurotoxins in freshwater cyanobacteria, having the advantage of being sensitive to unidentified toxins that currently cannot be quantified by chromatographic means.

Analysis of cyanobacterial-derived saxitoxins using high-performance ion exchange chromatography with chemical oxidation/fluorescence detection.

A single run HPLC method utilizing ion exchange as the separation mode with a novel mobile phase system coupled to chemical postcolumn oxidation and fluorescence detection has been developed and demonstrated to be applicable to the quantitative analysis of paralytic shellfish poisons (PSPs) produced by Australian cyanobacteria (Anabaena circinalis) and other cyanobacteria. Both the cyanobacterial matrix and natural water constituents did not significantly affect the performance of this method. The daily precision of this method was adequate for it to be considered as a routine analytical tool for direct PSP analysis (prePSP concentration is not required) of cyanobacterial extracts and water bodies containing PSPs (C1, C2, GTX2, GTX3, NEO, STX) in the low parts per billion concentration range (10-70 ppb).

Extraction of cyanobacterial endotoxin.

To simplify our efforts in acquiring toxicological information on endotoxins produced by cyanobacteria, a method development study was undertaken to identify relatively hazard-free and efficient procedures for their extraction. One article sourced and two novel methods were evaluated for their ability to extract lipopolysaccharides (LPSs) or endotoxins from cyanobacteria. The Limulus polyphemus amoebocyte lysate (LAL) assay was employed to compare the performance of a novel method utilizing a 1-butanol-water (HBW) solvent system to that of Westphal's (1965) phenol-water system (HPW) for the extraction of endotoxin from various cyanobacteria. The traditional HPW method extracted from 3- to 12-fold more endotoxin from six different cyanobacterial blooms and culture materials than did the novel HBW method. In direct contrast, the novel HBW method extracted ninefold more endotoxin from a non-microcystin producing Microcystis aeruginosa culture as compared to the HPW method. A solvent system utilizing N,N'-dimethylformamide-water (HDW) was compared to both the HPW and HBW methods for the extraction of endotoxin from natural samples of Anabaena circinalis, Microcystis flos-aquae, and a 1:1 mixture of Microcystis aeruginosa/Microcystisflos-aquae. The LAL activities of these extracts showed that the novel HDW method extracted two- and threefold more endotoxin from the Anabaena sample that did the HBW and HPW methods, respectively. The HDW method also extracted approximately 1.5-fold more endotoxin from the Microcystis flos-aquae sample as compared to both the HBW and HPW methods. On the other hand, the HBW method extracted 2- and 14-fold more endotoxin from the Microcystis flos-aquae/Microcystis aeruginosa mixture than did the HPW and HDW methods, respectively. Results of this study demonstrate that significant disparities exist between the physicochemical properties of the cell wall constituents not only of different cyanobacterial species but also of different strains of the same cyanobacterial species, as showing by the varying effectiveness of the solvent systems investigated. Therefore, a sole method cannot be regarded as universal and superior for the extraction of endotoxins from cyanobacteria. Nevertheless, the ability of the novel HBW and HDW methods to utilize easily handled organic solvents that are less hazardous than phenol render them attractive alternatives to the standard HPW method.