Purification, detection and biological effects of cyanobacterial toxins.

McElhiney, Jacqueline

Abstract

Dense growths of cyanobacteria (blue-green algae), known as blooms, commonly occur in fresh and brackish waters throughout the world. Cyanobacterial blooms present a considerable threat to water quality as many species produce toxins. These toxins have been implicated in the poisoning of humans and animals throughout the world, and there is a requirement for simple and effective methods for their detection. This study set out to investigate the detection of several cyanobacterial toxins, including a group of neurotoxins known as the saxitoxins. These toxins are also produced in marine environments by dinoflagellates and bacteria, and can accumulate in edible bivalve shellfish. An alternative assay for the monitoring of these toxins was developed using the desert locust Schistocerca gregaria. The locust bioassay was used in conjunction with an established high-performance liquid chromatography (HPLC) protocol to devise an extraction procedure for saxitoxins from cyanobacterial cells. It was also suitable for screening acid extracts of shellfish flesh for saxitoxins and performed well in a large shellfish monitoring programme, indicating its potential as a replacement to the mouse bioassay. The detection of another group of cyanobacterial toxins called microcystins was also addressed. The routine monitoring of cyanobacterial cultures and natural bloom samples for microcystins relies on HPLC. However, the lack of purified standards has hindered accurate detection and quantification of many microcystin variants. Reversed-phase Flash chromatography was employed for the partial purification of microcystins from a laboratory culture of Microcystis aeruginosa. A technique was then developed to facilitate the separation of two closely eluting hydrophobic variants (microcystin-LW and -LF), using normal-phase flash chromatography. The resulting three-step methodology provided a simple and inexpensive means of extracting and purifying microcystins for use as analytical standards. Purified toxins were also employed to investigate the effects of microcystins on plants. Bioassay methods revealed the inhibitory effects of microcystins on plant growth and development. The accumulation of microcystins in exposed plants was then investigated. A simple plant model lacking roots was exposed to microcystin-LF and extracted using three different solvents to compare toxin recovery. HPLC analysis revealed that the most efficient extraction method was methanol and indicated the presence of additional compounds possibly representing toxin metabolites. The uptake of microcystin-LR was then examined using a larger intact plant. Exposure to the toxin did not inhibit growth for up to 18 days, but had a marked effect on the roots and caused plants to take up less medium than controls. The plants were then extracted in methanol and analysed by HPLC to determine whether toxin had accumulated. However, the co-elution of contaminants prevented the detection of microcystin-LR, highlighting the requirement for alternative clean-up methods for complex biological matrices.

Citation

MCELHINEY, J. 1999. Purification, detection and biological effects of cyanobacterial toxins. Robert Gordon University, PhD thesis.

• Thesis Type: Thesis
• Deposit Date: Sep 23, 2010
• Publicly Available Date: Sep 23, 2010
• Public URL: http://hdl.handle.net/10059/528
• Contract Date: Sep 23, 2010
• Award Date: May 31, 1999