Initial Studies on the Occurrence of Cyanobacteria and Microcystins in Irish Lakes

We report the results of a synoptic survey at 14 sites across the north of Ireland undertaken to determine the occurrence of cyanobacteria and their constituent microcystin cyanotoxins. Seven microcystin toxins were tested for, and five of which were found, with MC-LR, MC-RR, and MC-YR being the most prevalent. Gomphosphaeria spp and Microcystis aeruginosa were the most dominant cyanobacterial species encountered. Together with Aphanizomenon flos-aquae, these were the cyanobacteria associated with the highest microcystin concentrations. The occurrence of several microcystin toxins indicates that there may potentially be more than one cyanobacteria species producing microcystins at many sites. Total microcystin concentrations varied over three orders of magnitude dividing the sites into two groups of high (>1000 ngMC/μgChla, six sites) or low toxicity (<200 ngMC/μgChla, eight sites). © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2010.

Next generation planar waveguide detection of microcystins in freshwater and cyanobacterial extracts, utilising a novel lysis method for portable sample preparation and analysis

The study details the development of a fully validated, rapid and portable sensor based method for the on-site analysis of microcystins in freshwater samples. The process employs a novel lysis method for the mechanical lysis of cyanobacterial cells, with glass beads and a handheld frother in only 10min. The assay utilises an innovative planar waveguide device that, via an evanescent wave excites fluorescent probes, for amplification of signal in a competitive immunoassay, using an anti-microcystin monoclonal with cross-reactivity against the most common, and toxic variants. Validation of the assay showed the limit of detection (LOD) to be 0.78ngmL and the CCß to be 1ngmL. Robustness of the assay was demonstrated by intra- and inter-assay testing. Intra-assay analysis had % C.V.s between 8 and 26% and recoveries between 73 and 101%, with inter-assay analysis demonstrating % C.V.s between 5 and 14% and recoveries between 78 and 91%. Comparison with LC-MS/MS showed a high correlation (R=0.9954) between the calculated concentrations of 5 different Microcystis aeruginosa cultures for total microcystin content. Total microcystin content was ascertained by the individual measurement of free and cell-bound microcystins. Free microcystins can be measured to 1ngmL, and with a 10-fold concentration step in the intracellular microcystin protocol (which brings the sample within the range of the calibration curve), intracellular pools may be determined to 0.1ngmL. This allows the determination of microcystins at and below the World Health Organisation (WHO) guideline value of 1µgL. This sensor represents a major advancement in portable analysis capabilities and has the potential for numerous other applications.

Production of a broad specificity antibody for the development and validation of an optical SPR screening method for free and intracellular microcystins and nodularin in cyanobacteria cultures

A highly sensitive broad specificity monoclonal antibody was produced and characterised for microcystin detection through the development of a rapid surface plasmon resonance (SPR) optical biosensor based immunoassay. The antibody displayed the following cross-reactivity: MC-LR 100%; MC-RR 108%; MC-YR 68%; MC-LA 69%; MC-LW 71%; MC-LF 68%; and Nodularin 94%. Microcystin-LR was covalently attached to a CM5 chip and with the monoclonal antibody was employed in a competitive 4min injection assay to detect total microcystins in water samples below the WHO recommended limit (1µg/L). A 'total microcystin' level was determined by measuring free and intracellular concentrations in cyanobacterial culture samples as this toxin is an endotoxin. Glass bead beating was used to lyse the cells as a rapid extraction procedure. This method was validated according to European Commission Decision 96/23/EC criteria. The method was proven to measure intracellular microcystin levels, the main source of the toxin, which often goes undetected by other analytical procedures and is advantageous in that it can be used for the monitoring of blooms to provide an early warning of toxicity. It was shown to be repeatable and reproducible, with recoveries from spiked samples ranging from 74 to 123%, and had % CVs below 10% for intra-assay analysis and 15% for inter-assay analysis. The detection capability of the assay was calculated as 0.5ng/mL for extracellular toxins and 0.05ng/mL for intracellular microcystins. A comparison of the SPR method with LC-MS/MS was achieved by testing six Microcystis aeruginosa cultures and this study yielded a correlation R(2) value of 0.9989.

Processes influencing surface interaction and photocatalytic destruction of microcystins on titanium dioxide photocatalysts

Microcystins are a family of hepatotoxic peptides produced by freshwater cyanobacteria. Their occurrence in drinking water is of concern since chronic exposure to these toxins causes tumor promotion. It is therefore essential to establish a reliable treatment strategy that will ensure their removal from potable water. We have previously described the rapid destruction of microcystin-LR using TiO₂ photocatalysis, however, since there are at least 70 microcystin variants it is essential that the destruction of a number of microcystins be evaluated. In this study the dark adsorption and destruction of four microcystins was followed over a range of pH. All four microcystins were destroyed although the efficiency of their removal varied. The two more hydrophobic microcystins (-LW and -LF) were found to have high dark adsorption (98 and 91% at pH 4) in contrast to microcystin-RR, which was found to have almost no (only 2-3%) dark adsorption across all pH. 

Physico-chemical treatment methods for the removal of microcystins (cyanobacterial hepatotoxins) from potable waters

The incidence of cyanobacterial blooms in freshwaters, including drinking water reservoirs, has increased over the past few decades due to rising nutrient levels. Microcystins are hepatotoxins released from cyanobacteria and have been responsible for the death of humans as well as domestic and wild animals. Microcystins are chemically very stable and many processes have only limited efficacy in removing them. In this paper we review a range of water treatment methods which have been applied to removing microcystins from potable waters.

Detoxification of microcystins (cyanobacterial hepatotoxins) using TiO2 photocatalytic oxidation

TiO₂ photocatalysis has been used to destroy microcystin-LR in aqueous solution. The destruction of this toxin was monitored by HPLC, and the disappearance was accompanied by the appearance of seven UV detectable compounds. Spectral analysis revealed that some of these compounds retained spectra similar to the parent compound suggesting that the Adda moiety, thought to be responsible for the characteristic spectrum, remained intact whereas the spectra of some of the other products was more radically altered. Six of the seven observed reaction products did not appear to undergo further degradation during prolonged photocatalysis (100 min). The degree to which microcystin-LR was mineralized by photocatalytic oxidation was determined. Results indicated that less than 10% mineralization occurred. Mass spectral analysis of the photocatalyzed microcystin-LR allowed tentative characterization of the reaction process and products. Reduction in toxicity due to the photocatalytic oxidation was evaluated using an invertebrate bioassay, which demonstrated that the disappearance of microcystin-LR was paralleled by a reduction in toxicity. These findings suggest that photocatalytic destruction of microcystins may be a suitable method for the removal of these potentially hazardous compounds from drinking water.

Development of a planar waveguide microarray for the monitoring and early detection of five harmful algal toxins in water and cultures

A novel multiplex microarray has been developed for the detection of five groups of harmful algal and cyanobacterial toxins found in marine, brackish, and freshwater environments including domoic acid (DA), okadaic acid (OA, and analogues), saxitoxin (STX, and analogues), cylindrospermopsin (CYN) and microcystins (MC, and analogues). The sensitivity and specificity were determined and feasibility to be used as a screening tool investigated. Results for algal/cyanobacterial cultures (n = 12) and seawater samples (n = 33) were compared to conventional analytical methods, such as high performance liquid chromatography (HPLC) and liquid chromatography tandem mass spectrometry (LC-MS/MS). Detection limits for the 15 min assay were 0.37, 0.44, 0.05, 0.08, and 0.40 ng/mL for DA, OA, STX, CYN, and MC, respectively. The correlation of data obtained from the microarray compared to conventional analysis for the 12 cultures was r(2) = 0.83. Analysis of seawater samples showed that 82, 82, 70, 82, and 12% of samples were positive (>IC20) compared to 67, 55, 36, 0, and 0% for DA, OA, STX, CYN, and MC, respectively, for conventional analytical methods. The discrepancies in results can be attributed to the enhanced sensitivity and cross-reactivity profiles of the antibodies in the MBio microarray. The feasibility of the microarray as a rapid, easy to use, and highly sensitive screening tool has been illustrated for the five-plex detection of biotoxins. The research demonstrates an early warning screening assay to support national monitoring agencies by providing a faster and more accurate means of identifying and quantifying harmful toxins in water samples.

Multi-detection method for five common microalgal toxins based on the use of microspheres coupled to a flow-cytometry system

Freshwater and brackish microalgal toxins, such as microcystins, cylindrospermopsins, paralytic toxins, anatoxins or other neurotoxins are produced during the overgrowth of certain phytoplankton and benthic cyanobacteria, which includes either prokaryotic or eukaryotic microalgae. Although, further studies are necessary to define the biological role of these toxins, at least some of them are known to be poisonous to humans and wildlife due to their occurrence in these aquatic systems. The World Health Organization (WHO) has established as provisional recommended limit 1 μg of microcystin-LR per liter of drinking water. In this work we present a microsphere-based multi-detection method for five classes of freshwater and brackish toxins: microcystin-LR (MC-LR), cylindrospermopsin (CYN), anatoxin-a (ANA-a), saxitoxin (STX) and domoic acid (DA). Five inhibition assays were developed using different binding proteins and microsphere classes coupled to a flow-cytometry Luminex system. Then, assays were combined in one method for the simultaneous detection of the toxins. The IC₅₀'s using this method were 1.9 ± 0.1 μg L⁻¹ MC-LR, 1.3 ± 0.1 μg L⁻¹ CYN, 61 ± 4 μg L⁻¹ ANA-a, 5.4 ± 0.4 μg L⁻¹ STX and 4.9 ± 0.9 μg L⁻¹ DA. Lyophilized cyanobacterial culture samples were extracted using a simple procedure and analyzed by the Luminex method and by UPLC–IT-TOF-MS. Similar quantification was obtained by both methods for all toxins except for ANA-a, whereby the estimated content was lower when using UPLC–IT-TOF-MS. Therefore, this newly developed multiplexed detection method provides a rapid, simple, semi-quantitative screening tool for the simultaneous detection of five environmentally important freshwater and brackish toxins, in buffer and cyanobacterial extracts.

The Application of Semiconductor Photocatalysis for the Removal of Cyanotoxins from Water and Design Concepts for Solar Photocatalytic Reactors for Large Scale Water Treatment

There has been a significant increase in the occurrence of cyanobacterial blooms in freshwaters over the past few decades due to escalating nutrient levels. These cyanobacteria release a range of toxins, for example microcystins which are chemically very stable. Many cyanotoxins are consequently very difficult to remove from water using existing treatment technologies. Semiconductor photocatalysis, however, has proven to be a very effective process for the removal of these compounds from water. In this chapter we consider the application of this highly versatile and exciting technology for the decomposition of cyanotoxins. Furthermore design concepts for solar photocatalytic reactors that could be utilized for the removal of these toxins are also considered

The degradation of microcystin-LR using doped visible light absorbing photocatalysts

Microcystins are one of the primary hepatotoxic cyanotoxins released from cyanobacteria. The presence of these compounds in water has resulted in the death of both humans and domestic and wild animals. Although microcystins are chemically stable titanium dioxide photocatalysis has proven to be an effective process for the removal of these compounds in water. One problem with this process is that it requires UV light and therefore in order to develop effective commercial reactor units that could be powered by solar light it is necessary to utilize a photocatalyst that is active with visible light. In this paper we report on the application of four visible light absorbing photocatalysts for the destruction of microcystin-LR in water. The rhodium doped material proved to be the most effective material followed by a carbon-modified titania. The commercially available materials were both relatively poor photocatalysts under visible radiation while the platinum doped catalyst also displayed a limited activity for toxin destruction. © 2009 Elsevier Ltd. All rights reserved.

The photocatalytic decomposition of microcystin-LR using selected titanium dioxide materials

Microcystins (cyclic heptapeptides) produced by a number of freshwater cyanobacteria are a potential cause for concern in potable water supplies due to their acute and chronic toxicity. TiO₂ photocatalysis is a promising technology for removal of these toxins from drinking water. It is, however, necessary to have a sufficient knowledge of how the catalyst materials cause the degradation of the toxins through the photocatalytic process. The present study reports microcystin degradation products of the photocatalytic oxidation by using a number of commercial TiO₂ powder (P25, PC50, PC500 and UV100) and granular (KO1, KO3, TiCat-C, TiCat-S) materials, so aiding the mechanistic understanding of this process. Liquid chromatography-mass spectrometry analysis demonstrated that the major destruction pathway of microcystin for all the catalysts tested followed almost the same pathway, indicating the physical properties of the catalysts had little effects on the degradation pathway of microcystin-LR. 

Mechanistic studies of the photocatalytic oxidation of microcystin-LR: An investigation of byproducts of the decomposition process

Microcystins (cyclic heptapeptides) are produced by a number of freshwater cyanobacteria and cause concern in potable water supplies due to their acute and chronic toxicity. The present study reports the structural characterization of the degradation products of the photocatalytic oxidation of microcystin-LR, so aiding the mechanistic understanding of this process. TiO₂ photocatalysis is a promising technology for removal of these toxins from drinking water. However, before it can be adopted in any practical application it is necessary to have a sufficient knowledge of degradation byproducts and their potential toxicity. Liquid chromatography-mass spectrometry analysis demonstrated that the major destruction pathway of microcystin appears to be initiated via three mechanisms: UV irradiation, hydroxyl radical attack, and oxidation. UV irradiation caused geometrical isomerization of microcystin converting the (4E), (6E) of the Adda configuration to (4E), 6(Z) or 4(Z), 6(E). Hydroxyl radical attack on the conjugated diene structure of Adda moiety produced dihyroxylated products. Further oxidation cleaved the hydroxylated 4-5 and/or 6-7 bond of Adda to form aldehyde or ketone peptide residues, which then were oxidized into the corresponding carboxylic acids. Photocatalysis also hydrolyzed the peptide bond on the ring structure of microcystin to form linear structures although this appeared to be a minor pathway.

Mechanistic and toxicity studies of the photocatalytic oxidation of microcystin-LR

Cyanobacterial toxins present in drinking water sources pose a considerable threat to human health. Conventional water treatment systems have proven unreliable for the removal of these toxins and hence new techniques have been investigated. Previous work has shown that TiO₂ photocatalysis effectively destroys microcystin-LR in aqueous solutions, however, a variety of by-products were generated. In this paper, we report a mechanistic study of the photocatalytic destruction of microcystin-LR. In particular, the toxicity by-products of the process have been studied using both brine shrimp and protein phosphatase bioassays.