Christine Edwards

Extraction and high-performance liquid chromatographic method for the determination of microcystins in raw and treated waters

Increasing concern over the presence of microcystins (cyanobacterial/blue-green algal hepatotoxins) in water supplies has emphasized the need for a suitable analytical method. As many microcystins are known to exist, a method was developed that permits the determination of numerous variants by a single procedure. The method involves filtration to separate cyanobacterial cells from water, allowing intracellular and extracellular toxin levels to be assessed. The cellular components of the samples are extracted repeatedly in methanol, which was found to be the most versatile solvent tested for the extraction of microcystins. The efficiency of this extraction procedure was found to be independent of cell biomass. The filtered water was subjected to trace enrichment using a C18 solid-phase extraction cartridge, followed by identification and determination by photodiode-array high-performance liquid chromatography. The procedure was assessed using four water samples (two raw and two treated) spiked with a mixture of five microcystins and the cyanobacterial hepatotoxin nodularin. Recoveries of all but one microcystin were found to be good when spiked with concentrations as low as 250 ng l-1. The linearity and precision of the experimental procedure were assessed for five microcystins and nodularin. The proposed method permits rapid sample processing and determination of several microcystins.

Identification of anatoxin-A in benthic cyanobacteria (blue-green algae) and in associated dog poisonings at Loch Insh, Scotland

Dog deaths occurred in 1990 and 1991 after the animals drank water containing blooms of benthic cyanobacteria along the shoreline of Loch Insh, Scotland. Signs of poisoning in the affected animals and the high neurotoxicity of bloom extracts in laboratory bioassays indicated acute poisoning due to cyanobacterial neurotoxin(s). The neurotoxic blooms consisted largely of benthic Oscillatoria species which were also observed in the stomach contents of the poisoned dogs. Stomach contents were also neurotoxic in bioassays with the same signs of poisoning as the Oscillatoria blooms. The cyanobacterial alkaloid neurotoxin anatoxin-a was identified in bloom extracts and poisoned dog stomach contents by high-performance liquid chromatography and gas chromatography-mass spectrometry. A species of benthic Oscillatoria has been isolated from the neurotoxic bloom material and shown to produce anatoxin-a in laboratory culture. These findings are the first to associate anatoxin-a toxicoses with benthic, rather than planktonic, cyanobacteria. Procedures for anatoxin-a extraction and identification from the blooms and animal material are also detailed.

Isolation and identification of novel microcystin-degrading bacteria.

ABSTRACT Of 31 freshwater bacterial isolates screened using the Biolog MT2 assay to determine their metabolism of the microcystin LR, 10 were positive. Phylogenetic analysis (16S rRNA) identified them as Arthrobacter spp., Brevibacterium sp., and Rhodococcus sp. This is the first report of microcystin degraders that do not belong to the Proteobacteria.

Biodegradation of microcystins and nodularin in freshwaters.

Microcystin-LR (MC-LR) was readily biodegraded on addition to six different water samples irrespective of their previous exposure to microcystins. Subsequent studies with water from three of these water bodies confirmed the degradation of MC-LR and also demonstrated the biodegradation of MC-LF, nodularin and mixture of microcystins and nodularin. Rates of degradation of MC-LR, MC-LF and NOD in individual water samples ranged from a half-life of 4 to 18d. Analysis by HPLC-PDA-ESI+ and MALDI MS/MS revealed novel intermediate degradation products of MC-LF and nodularin which included demethylation, hydrolysis, decarboxylation and condensation of the parent compound(s). Our study suggests a possible diversity of micro-organisms and/or pathways which has not been previously observed.

Purification of microcystins

Microcystins are an increasingly important group of bioactive compounds produced by a number of mainly planktonic cyanobacteria. They are a family of cyclic heptapeptides that cause both acute and chronic toxicity. Purified microcystins are utilised in a range of research applications including toxicological and biochemical studies, development of detection systems and the investigation of water treatment strategies. The commercial availability of purified microcystins is still relatively limited and for many projects the cost of their purchase prohibitive. The purification of microcystins from both bloom material and laboratory cultures is reviewed including a discussion on extraction, separation, and the determination of purity and yield.

Liquid chromatography-electrospray ionization-mass spectrometry of cyanobacterial toxins

Abstract Cyanobacterial neurotoxins such as anatoxin-a and saxitoxin, peptide hepatotoxins including microcystin variants and nodularin were studied by electrospray ionization-mass spectrometry following direct infusion of samples into the mass spectrometer. This technique was further exemplified when analysing the cyclic hepatotoxic peptides by means of on-line liquid chromatography-mass spectrometry. Peptide toxins were identified in extracts from toxic bloom material and cell cultures by comparison of spectral and chromatographic data to that of authentic materials. When authentic materials were not available, several peptides were tentatively identified by their molecular-weight information provided by this technique.

Laboratory-scale purification of microcystins using flash chromatography and reversed-phase high-performance liquid chromatography

Microcystins were extracted from 7 l (equivalent to 313 g dry weight) of cyanobacterial scum collected from Rutland Water in Leicestershire, UK in 1989. The resulting aqueous extract was rapidly concentrated on a C18 flash chromatography cartridge and microcystins were eluted using a step gradient. Fractions were collected manually and monitored by UV spectrophotometer and analytical HPLC. Fractions containing microcystins of similar polarity were pooled to give three fractions. Simple isocratic methods for separating each fraction were developed on an analytical column and scaled up to a 15 x 7.5 cm I.D. column. Closed-loop recycling was used to maximise yield and purity of two hydrophobic microcystins.

Rapid detection of microcystins in cells and water

Global occurrence and concern about microcystin contamination in water has prompted the development of a range of detection methods for their identification and quantification. However, most protocols are relatively time consuming, expensive and require laboratory expertise. The production of robust and sensitive recombinant antibodies has facilitated the development of a lateral flow immunoassay (ImmunoStrip) which can rapidly detect microcystins and nodularins in the field with minimal equipment or processing. Here we evaluate the sensitivity and cross-reactivity of the commercially produced ImmunoStrip) and apply them to the detection of microcystins in laboratory cultures and natural samples. It was observed that while the ImmunoStrip) are marketed for the detection of 10 microg/l microcystin, all 7 microcystins and nodularin that were tested were detected below 1 microg/l. Furthermore, microcystins and nodularins were successfully detected in a range of laboratory cultures and samples from irrigation ponds.

Rapid separation of triterpenoids from Neem seed extracts

A dichloromethane extract of the triterpenoids from seeds of the Neem tree (Azadirachta indica) can be fractionated rapidly and with economic use of solvents using the Biotage™ flash chromatography system. The fractions can be analysed by thin layer chromatography (TLC), or quantitatively, by supercritical fluid chromatography or high performance liquid chromatography (HPLC), for the determination of 11 tetranortriterpenoids, including the natural insecticide azadirachtin. After a second pass through the Biotage flash column, pure compounds, including azadirachtin and salannin can be obtained directly, although these compounds comprise less than 0.3% of the total mass of the seeds extracted and are present in a complex mixture of similar substances. Copyright

Bioremediation of cyanotoxins.

Cyanobacteria are a diverse group of mainly aquatic microorganisms which occur globally. Eutrophication (nutrient enrichment) of water bodies, often as a result of human activities, results in prolific grow of cyanobacteria that develop into a thick scum or bloom. Many of these blooms are toxic due to the production of hepatotoxins (microcystins and cylindrospermopsin) and/or neurotoxins (saxitoxins and anatoxins) posing a serious health hazard to humans and animals. The presence of these cyanotoxins is of particular concern in drinking water supplies where conventional water treatment often fails to eliminate them. Hence, there is significant interest in water treatment strategies that ensure the removal of cyanotoxins, with the exploitation of microbes being on such possible approach. As naturally occurring compounds it is assumed that these toxins are readily biodegraded. Furthermore, there is no significant evidence of their accumulation in the environment and their relative stable under a wide range of physico-chemical conditions, suggests biodegradation is the main route for their natural removal from the environment. Microcystins, as the most commonly occurring toxins, have been the most widely studied and hence form the main focus here. The review provides an overview of research into the biodegradation of cyanotoxin, including evidence for natural bioremediation, screening and isolation of toxin biodegrading bacteria, genetic and biochemical elucidation of a degradation pathway along with attempts to harness them for bioremediation through bioactive water treatment processes.