Kiyomi Tsuji

Stability of microcystins from cyanobacteria: effect of light on decomposition and isomerization.

Microcystins are potent hepatotoxins produced by cyanobacteria. Their geometrical isomers [6(Z)-Adda microcystin] do not essentially show hepatotoxicity and show weaker tumor-promoting activity than their parent toxins. The present study was undertaken to examine stability of microcystins during the analysis and purification and under photolysis conditions in connection with the detoxification. Microcystin LR was very stable because of limited decomposition and isomerization to its geometrical isomer during analysis and purification. While microcystins decomposed very limitedly by exposure with sunlight alone, the addition of pigments extracted from cyanobacteria accelerated their decompositions

Stability of microcystins from cyanobacteria—II. Effect of UV light on decomposition and isomerization

Microcystins are very potent hepatotoxins and strong liver tumor promoters produced by cyanobacteria, and their occurrence has been reported all over the world. They could threaten human health when toxic Microcystis occurs in water supply reservoirs. In this study, we examined the stability of microcystins during photolysis with UV light. The toxins were easily decomposed by UV light at wavelengths around the absorption maxima of the toxins and the decomposition depended on the intensity of the light. The half-life of microcystin LR by 147 microW/cm2 UV irradiation was 10 min, and the toxin was completely decomposed by 2550 microW/cm2 UV after 10 min. When the toxins were irradiated with weaker UV light, isomerization was also observed by a different mechanism from that during photolysis by sunlight and pigment, and several products including three geometrical isomers of the conjugated diene of Adda were detected. Microcystin RR showed almost the same behavior as that of microcystin LR under the same conditions. Since no noxious products were formed in the present study, a water treatment including UV irradiation is very possible for removing microcystins from raw water.

Stability of microcystins from cyanobacteria-IV. Effect of chlorination on decomposition

Microcystins, the cyclic heptapeptide toxins produced by cyanobacteria such as Microcystis, show tumor-promoting activity through inhibition of protein phosphatases 1 and 2A. They potentially threaten human health, and are increasing the world-wide interest in the health risk associated with cyanobacterial toxins. In this study, the effect of chlorination on the decomposition of microcystins-LR and -RR was examined. The toxins were easily decomposed by chlorination with sodium hypochlorite, and the decomposition depended on the free chlorine dose. In this operation, many reaction products were formed, one of which was determined to be dihydroxymicrocystin formed through the chloronium ion at the conjugated diene of Adda [3-amino-9-methoxy-10-phenyl-2,6,8-trimethyl-deca-4(E), 6(E)-dienoic acid], followed by hydrolysis. Other products may be its stereoisomers and/or regioismers. No noxious products were detected from the chlorination process of microcystin-LR. Although these results suggested that chlorination at an adequate chlorine dose is very effective for the removal of microcystin in raw water, preoxidation of the cell itself with chlorine must be avoided, because it frequently causes toxin release from algae and produce trihalomethanes during water treatment.

Analysis of microcystins in sediments using MMPB method

During the course of study on the detoxification of microcystins, the adsorption on sediments in the natural environment was investigated. Because it was very difficult to extract microcystins from sediments using conventional techniques, a physicochemical screening method, the MMPB (2-methyl-3-methoxy-4-phenylbutyric acid) method, including ozonolysis and mass spectrometric detection was developed. This method consisted of the following operations: lyophilized sediments were suspended in methanol and MMPB-d(3) as an internal standard was added to this suspension, which was cooled at -78 degrees C with vigorous stirring and then treated with a stream of ozone/oxygen. After centrifugation, an aliquot of the reaction solution was subjected to EI (electron ionization)-GC/MS analysis after methylation with 14% BF(3)-methanol and liquid-liquid extraction. The established method had a potential for the analysis of microcystins in sediments that are difficult to analyze using conventional methods. Finally, this method was applied to sediment samples collected in Japanese lakes and six of the eleven samples showed positive results. The obtained results clearly indicated that the adsorption on sediments contributes to the detoxification of microcystins under natural conditions.

A clean-up method for analysis of trace amounts of microcystins in lake water.

A clean-up method using high performance liquid chromatography (HPLC) and liquid chromatography/mass spectrometry (LC/MS) was developed to pursue trace amounts of microcystins in lake water. The method consisted of the combined usage of octadecyl silanized (ODS) silica gel and silica gel cartridges. In the first clean-up process, the retention behavior of microcystin RR on ODS silica gel cartridge was carefully observed together with microcystin LR, and 10% water-methanol was chosen as the best solvent system to elute microcystins from the ODS silica gel cartridge. Because many impurities still remained in the desired fraction from the raw water even after the clean-up with ODS silica gel, an additional clean-up process was developed using various cartridges. As a result of extensive experiments, the second clean-up process using silica gel cartridge was established, and the impurities were effectively eliminated. The present method including a tandem cartridge system allowed a precise analysis of microcystins in water samples from three different lakes at a 0.02 ppb level.

Persistence and Decomposition of Hepatotoxic Microcystins Produced by Cyanobacteria in Natural Environment

AbstractMicrocystins, the cyclic heptapeptide toxins produced by cyanobacteria such as Microcystis, show tumor-promoting activity through inhibition of protein phosphatases 1 and 2A. They potentially threaten human health and are increasing the worldwide interest in the health risk asSoc. iated with cyanobacterial toxins. Microcystins are normally considered to be confined within cyanobacterial cells and to enter into the surrounding water after lysis and cell death under field conditions. Five pathways may be considered to contribute to natural routes of detoxification of the microcystins: (1) dilution, (2) adsorption, (3) thermal decomposition aided by temperature and pH, (4) photolysis and (5) biological degradation. In this review, we describe the persistence and decomposition of microcystins under the conditions mentioned above and discuss the fate of the toxins in the natural environment.

Lysis of cyanobacteria with volatile organic compounds

One of bacteria collected from Lake Sagami, Japan, Brevibacillus sp., was found to have a lytic activity of cyanobacteria, but did not produce active compounds. Instead, the co-culturing of Microcystis with the Brevibacillus sp. enhanced the production of two volatile compounds, beta-cyclocitral and 3-methyl-1-butanol, and the former had a characteristic lytic activity. It was confirmed that these volatile compounds were derived from the cyanobacteria themselves. beta-Ionone, geosmin and 2-methylisoborneol derived from cyanobacteria and similar volatile compounds, terpenoids, produced by plants also had a lytic activity. The minimum inhibitory concentration values of the cyanobacterial metabolites were estimated to be higher than those of compounds from plants except for a few compounds. Among them, beta-cyclocitral only produced a characteristic color change of culture broth from green to blue. This color change is similar to the phenomenon observed when a sudden decline in growth of cyanobacteria begins in a natural environment.

Microcystin levels during 1992-95 for Lakes Sagami and Tsukui-Japan.

Toxic cyanobacterial blooms have been frequently observed in Lakes Sagami and Tsukui, Kanagawa Prefecture, Japan, which are used as drinking and recreational water sources. As the first step toward the control and removal of cyanobacterial toxins, the present study evaluated the microcystin level in these lakes. Our established method using HPLC and LC/MS to pursue trace amounts of microcystins was applied to the determination of microcystins within cyanobacteria cells and in water. We could determine precisely the intracellular and extracellular microcystin level in the water environment during 1992-95. Microcystins RR, LR, and YR were detected at 0.02-2.64 micrograms/L in cell-free water and at 0.02-378 micrograms/L in the cells. Although there were many cases in which microcystin concentrations in the cells exceeded the proposed guideline level (1 microgram/L), there was only one example of this happening in cell-free water samples. Because the present monitoring indicated that the amount of microcystins detected in water was much less than that estimated in cells, the release of microcystins from the cells and their stability in lake water were examined in the dark. The resulting toxins persisted at the same concentration level for 14 days and the microcystin concentrations steadily declined, showing that biodegradation using aquatic natural bacterial flora is an effective detoxification process under natural conditions.

Determination of microcystins in lake water using reusable immunoaffinity column.

A reusable immunoaffinity column for purification of microcystins in lake water was prepared by coupling anti-microcystin-LR monoclonal antibodies to immunoaffinity support. Thanks to spherical shape of the immunoaffinity support Formyl-Cellulofine used in this study, applied solutions passed the column smoothly even when used repeatedly. Reusability of the column was examined by determining the recoveries of spiked microcystins-RR, -YR and -LR (100ng each) from lake water. After extraction with a Sep-Pak PS2 cartridge containing styrene-divinylbenzene copolymer, the extract was purified with the immunoaffinity column. The immunoaffinity column was regenerated by washing with Tris-HCl buffer containing bovine serum albumin for repeated uses. Recoveries of spiked microcystins from the first use of the column were 87-88%, and 83-88% from the second and third uses, and the recoveries gradually dropped to 63-77% from the 4-5th uses, the results of which indicated that the column could be used repeatedly for three times. The present method was applied to determine microcystins in water collected from three different lakes in Japan in 1999. In a sample from Lake Suwa, microcystins-RR and -LR were determined by high performance liquid chromatography with photodiode array detection and electrospray ionization-liquid chromatography/mass spectrometry.

Analytical aspects of cyanobacterial volatile organic compounds for investigation of their production behavior.

In order to fully understand the role of volatile organic compounds (VOCs) under natural conditions, an adaptable analytical method was developed as the first step. beta-Ionone, beta-cyclocitral, 2-methyl-1-butanol and 3-methyl-1-butanol were simultaneously analyzed in addition to geosmin and 2-MIB using GC/MS with SPME. The slight modification of a known method allowed the simultaneous detection and quantification of these VOCs. The SIM of the 3-methyl-1-butanol was always accompanied by a shoulder peak, suggesting the presence of two compounds. In order to separate both compounds, the GC/MS conditions were optimized, and the additional peak was identified as 2-methyl-1-butanol by direct comparison of the authentic compound, indicating that the Microcystis strain always produces a mixture of 2-methyl-1-butanol and 3-methyl-1-butanol. Furthermore, it was found that 2-methyl-1-butanol and 3-methyl-1-butanol were predominant in the dissolved fractions. beta-Cyclocitral was easily oxidized to provide the oxidation product, 2,6,6-trimethylcyclohexene-1-carboxylic acid, which causes the blue color formation of cyanobacteria as a consequence of acid stress. The intact acid could be satisfactorily analyzed using the usual GC/MS without derivatization.