Environ. Sci. Technol. 1094, 28, 173-177
Stability of Microcystins from Cyanobacteria: Effect of Light on Decomposition and Isomerization Klyoml Tsuj1,'~tShojl Nalto,t Fumlo Kondo? Naohlsa Ishlkawa,* Marlyo F. Watanabe,s Makoto Suzuk1,ll and Ken-lchl Haradall
Kanagawa Prefectural Public Health Laboratories, 52-2 Nakao-cho Asahi-ku, Yokohama 241, Japan, Aichi Prefectural Institute of Public Health, Tsuji-machi, Kita-ku, Nagoya 462, Japan, Tokyo Metropolitan Research Laboratory of Public Health, Hyakunin-cho, Shinjuku-ku, Tokyo 160, Japan, and Faculty of Pharmacy, Meijo University, Tempaku-ku, Nagoya 468, Japan Microcystins are potent hepatotoxins produced by cyanobacteria. Their geometrical isomers [6(Z)-Adda microcystinl 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. Additionally, the isomerization of microcystins to 6(Z)-Adda microcystins and vice versa occurred under the same conditions. The decomposition and isomerization rates depended upon pigment concentration. The converted microcystin LR from its inactive geometrical isomer showed essentially the same toxicity as that of intact microcystin LR. Photolysis in the presence of pigment may be regarded as one of the detoxification processes for microcystins under field conditions. Introduction Microcystins are potent hepatotoxins produced by cyanobacteriasuch as Microcystis aeruginosa, Microcystis viridis, Nostoc sp., Oscillatoria agardhii, and Anabaena flos-aquae (1-n,and they are structurally monocyclic heptapeptides composed of three D-amino acids, two unusual amino acids, N-methyldehydroalanine, and 3-amino-9-methoxy-l0-phenyl-2,6,8-trimethyldeca-4(E),6(E)dienoic acid (Adda),and two variable L-amino acids (8,9). Over 40 microcystins have been isolated so far (1-7,lO14). Their LD50 (i.p. mouse) were reported to range from 50 to 600 pg/kg (4, 13, 15). Potent hepatotoxicity of microcystins causes hepatocyte necrosis with destruction of sinusoidal endothelium followedby massive intrahepatic hemorrhage and rapid death in mice (16). Recently, it has been reported that microcystins inhibit protein phosphatases 1 and 2A in a manner similar to okadaic acid and have a tumor-promoting activity on rat liver ( I 720).
In the course of isolation of microcystins from natural blooms of Microcystis, we have isolated geometrical isomers of microcystins LR and RR and have determined their structures to be the 4(E),6(Z) isomer of the diene of the Adda portion in microcystins LR and RR, which are tentatively named G(Z)-Adda microcystin LR and 6(Z)Kanagawa Prefectural Public Health Laboratories.
* Aichi Prefectural Institute of Public Health. 8 Tokyo Metropolitan 11 Meijo University.
Research Laboratory of Public Health.
0013-936X/94/0928-0173$04.50/0
0 1993 American Chemical Society
Adda microcystin RR (21),respectively. In Japan, toxic bloom samples usually contain 5-15% of the geometrical isomers. The isolated isomers do not show hepatotoxicity and have weaker tumor-promoting activity than their parent toxins, indicating that the 4(E),6(E)-Adda portion is essential for these biological activities (22-24). Severe outbreaks of toxic cyanobacterial bloom have been observed in water supply reservoirsin many countries. Hughes et al. reported that a toxic substance was detected in the culture filtrate in the early stage of growth (25).We have also observed the release of microcystins into the surrounding culture medium during the decomposition of Microcystis aeruginosa (26). These findings suggest that microcystins are normally confined within the cyanobacterial cells and enter into the surrounding water after lysis and cell death under field conditions. However, the amount of microcystins detected in lake water was at most a few micrograms per liter (27),and the amount was much less than that estimated in the cells. To assess the health implications, it is very important to pursue microcystins under field conditions. To our knowledge, no detoxification study on microcystins has been conducted. Five pathways may be considered to contribute to the natural route of detoxification of microcystins: (1) dilution, (2) adsorption, (3) thermal decomposition aided by temperature and pH, (4)photolysis, and (5) biological degradation. The aims of this study are to examine the stability of microcystins under photolysis conditions. Experimental Section
Materials. Water-Extractable Pigment. Lyophilized cyanobacterial cells (4 g) collected in Lake Kasumigaura, Japan, were extracted with 100 mL of distilled water for 30 min while being stirred. The extract was centrifuged at 1600g, and the supernatant was directly applied to 30 g of reversed-phase silica gel on a column (4 X 40 cm, Chromatorex ODS, Fuji-Davison Chemical Ltd., Kasugai, Japan) to remove toxin. The filtrate was lyophilized, and the dried pigment (0.1 g) was dissolved in 25 mL of water. Solvent-Extractable Pigment. Lyophilized cells (0.25 g) collected in Lake Kasumigaura were extracted with 50 mL of acetone for 30 min while being stirred. The extract was centrifuged at 1600g, and then the supernatant was evaporated to dryness. The residue was dissolved in 20 mL of methanol. Chlorophyll a and 0-carotene (Sigma Chemical Go., St. Louis) were dissolved in methanol at a concentration of 100 mg/L. Chemicals and Glassware. All chemicals used were of analytical grade. All glassware was Pyrex. Toxin Purification. Microcystins LR, RR, YR, and 6(Z)-Adda microcystins LR and RR were isolated from field samples accordingto the method described by Harada Environ. Sci. Technoi., Vol. 28, No. 1, 1994 173
et al. (28). The field samples were collected from water blooms dominated by Microcystis sp. in Lake Suwa, Japan, during the summer season in 1989. Microcystin LR and 6(Z)-Adda microcystin LR were present at a ratio of 85:15 in the toxic fraction. Microcystin RR and 6(Z)-Adda microcystin RR were present in a ratio of 85:15 in the toxic fraction. Stability Test of Microcystin LR during Isolation and Analysis. A solution of microcystin LR in methanol was prepared at a concentration of 20mg/L. One milliliter of the solution in a 10-mL Pyrex test tube with a screw cap was evaporated to dryness with nitrogen, and the residue was dissolved in 2 mL of each of the test solvents. One of the following packing materials (0.1 g), silica gel, ODS silica gel, or TOYOPEARL HW-40F (Tosoh, Tokyo, Japan), was added to the solution. Samples were taken at regular intervals and analyzed by HPLC. Photolysis of Solutions of Microcystins. (1) I n Distilled Water. A solution of microcystin LR (0.007-14 mg/L) with 15% of 6(Z)-Adda microcystin LR was subjected to photolysis using a fluorescent lamp or natural sunlight. Irradiation was continuously performed at 13.9 pE m-2 s-l of fluorescent illumination. Irradiation with sunlight was performed outdoor for 26 days (August 5-31, 1990). Dark controls were employed. Samples were taken at regular intervals and analyzed by HPLC. (2)I n Pigment Solution. A solution of microcystin LR with 15% 6(Z)-Addamicrocystin LR, in one of the various pigment solutions, was exposed to sunlight for 15 days (November 10-24,1990). The solution of microcystin LR or 6(Z)-Addamicrocystin LR in water-extractable pigment or solvent-extractable pigment was exposed to sunlight for 27 days (April 12-May 9,1991). Influence of pigment concentration on isomerization of microcystin LR was examined under sunlight for 29 days (August 8-September 6, 1991). Samples were taken at regular intervals and analyzed by HPLC. Results were expressed to be the mean f the SEM of five individual samples. HPLC determinations were made in triplicate. For frit-fastatom bombardment liquid chromatography/ mass spectrometry (Frit-FAB LC/MS) analysis, microcystin LR (10 mg/L) in water-extractable pigment (0.1 mg/mL) was filtered through a glass GF/C microfiber filter (Whatman, Maidstone, England) after irradiation with sunlight for 60 days and then applied to an ODS cartridge (Baker, Phillipsburgh, NJ). The cartridge was rinsed with 15 mL of water, followed by 15 mL of 20% methanol in water, The desired products were eluted with 20 mL of methanol. The eluate was evaporated, and the residue was used for Frit-FAB LC/MS analysis. Isomerization of G(Z)-Adda Microcystin LR by Irradiation with Sunlight. Purified 6(Z)-Adda microcystin LR (3.6 mg) was dissolved in 50 mL of waterextractable pigment solution (0.5 mg/mL). The solution was exposed to sunlight for 10 days (May 2-12, 1992). After the filtration of the solution, the filtrate was applied to an ODS cartridge (Baker, Phillipsburgh, NJ). The cartridge was rinsed with 20 mL of water, followed by 30 mL of 20% methanol in water. The eluate with 20 mL of methanol was evaporated to give a residue (3 mg). The residue was separated to yield 0.6 mg of microcystin LR and 0.9 mg of 6(Z)-Adda microcystin LR by preparative HPLC using methanok0.05 % trifluoroacetic acid (TFA) (62:38) as the mobile phase. Isomerized microcystin LR was finally purified by using TOYOPEARL HW-40F gel 174
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chromatography (90 X 1.2 cm i.d.), and the obtained microcystin LR (0.25 mg) was used for toxicity assay. HPLC. A high-performance liquid chromatograph with a UV detector was used to separate and quantify microcystins. The system consisted of a Shimadzu (Kyoto, Japan) LC-6A pump coupled to a SPD-6A detector set at 238 nm and a CR-5A integrator. Separation was accomplished under a reversed-phase isocratic condition with a Inertsil ODS (150 X 4.6 mm, GL Sciences, Tokyo, Japan) and a Cosmosil 5C18-P column (250 X 10 mm, Nacalai Tesque, Kyoto, Japan). Three mobile phases, methanol: 0.05 M phosphate buffer (pH 3)(58:42), methanol:0.05 M sodium sulfate (6:4), and methanol:0.05% TFA (6:4) were used. The flow rate was 1mL/min for analysis and 2 mL/ min for preparative separation. Frit-FAB LC/MS. A high-performance liquid chromatograph equipped with a constant-flow pump (LC-6A; Shimadzu) was used. Separation was accomplished under a reversed-phase isocratic condition with Chromatorex ODS (5 pm, 250 X 4.0 mm, Fuji Davison Chemical Ltd., Kasugai, Japan) and a mobile phase of methanol:0.05% TFA (63:37) (containing 0.8% of glycerol). The flow rate was 0.5 mL/min. A mass spectrometer (JMS-AX505W, JEOL Co. Ltd., Tokyo, Japan) was used and connected to a data system JEOL JMA-DA5000. The fast atom beam was operated at 3 kV with xenon gas, and the spectrometer was operated at a 5-kV accelerating potential. The FAB mass spectra were obtained in positive-ion mode by scanning from 40 to 1500 at a cycle time of 6.5 s. A JEOL Frit-FAB probe with a stainless steel frit was connected to a flow splitter. Injected samples were introduced into the ion source at a split ratio of 4500 after a post-column splitting with the flow splitter. Determination of Toxicity. The toxicity of isomerized and intact microcystin LR was tested by intraperitoneal injection using 46 male 5-week-old ddY mice. The dose of isomerized microcystin LR was 40,60,90,135, and 203 pg/kg in saline, and those of intact microcystin LR were 33,50,75, and 113pg/kgof toxin in saline. After injection, the animals were allowed to stay in the cage for 4 h, unless they died. The LDWvalues for both toxins were estimated using the up-and-down method repeatedly three times (29, 30). After death, the mice were autopsied, and the livers were removed for the determination of the liver/ body weight ratio. Statistical Analysis. The statistical comparison of values was accomplishedwith one-wayanalysis of variance. The statistical significance of difference between two means was examined using Scheffe’s method. P < 0.05 was considered significant.
Results and Discussion We have established a total method for analysis and purification of microcystins (28). The method has been used for some studies on microcystins (4, 26). In the present study, HPLC using methanol:0.05 M phosphate buffer (pH 3) and methanok0.05 % TFA as a mobile phase were employed for an examination of the stability. The decomposition ratio was estimated by the comparison of peak areas of toxins at the starting and finishing times of the experiment. The isomerization ratio was similarly obtained by HPLC using the following definition: 6(Z)Adda microcystin/ [microcystin + 6(Z)-Adda microcystin] ( Z / E + 2).
First, the stability of microcystin LR was investigated during analysis and purification. In 5 % acetic acid solution for extraction and mobile phases with silica gel and TOYOPEARL for chromatography, over 90% of the toxin was recovered without decomposition and isomerization to its geometrical isomer after 5 days. Similarly, microcystin LR was stable in HPLC mobile phases. During dissolution of the toxin using an ultrasonic apparatus and centrifugation, no significant loss was observed. These results indicate that microcystin LR is very stable and that 6(Z)-Adda microcystin LR is not formed under these conditions. Sunlight irradiates the earth at wavelengths above 295 nm and is essential in the growth of cyanobacteria. The influence of fluorescent light and natural sunlight on the stability of microcystin LR (0.007-14 mg/L) was observed in distilled water for 26 days with a mixture of microcystin LR and its geometrical isomer (85:15). Over 86% of microcystin LR was found to be present in distilled water after 26 days under these conditions. Although under sunlight irradiation the isomerization ratio was slightly changed at a concentration of microcystin LR (14 mg/L), no significant change was observed in other cases. These results indicate that microcystin LR and its isomer are stable to sunlight and fluorescent light. Cyanobacteria possess several pigments for photosynthesis, including chlorophyll a, @-carotene,mixoxanthophyll, phycocyanins,allophycocyanins,and phycoerythrins (31, 32). The solvent-extractable pigments and waterextractable pigments from cyanobacteria collected in Lake Kasumigaura were used in this experiment. The major components of the former were &carotene, chlorophyll a, and mixoxanthophyll, and the latter were phycocyanins. If cells decompose under field conditions, microcystins would be exposed to sunlight together with co-existing pigments. Figure 1A illustrates the decrease of total microcystin (microcystin LR and its isomer) by irradiation with sunlight for 15 days in the presence of various pigments, indicating that the presence of pigments accelerates the decomposition. Among pigments tested, water-extractable pigments made microcystin LR effectively decompose because of their better stability compared with solvent-extractable pigments and purified chlorophyll a and @-carotene. However, no significant decomposition of microcystin LR occurred in pigment solutions by irradiation with fluorescent light. In order to observe the isomerization, microcystin LR and 6(Z)-Adda microcystin LR were subjected separately to exposure under the same conditions as above. The time courses of the isomerization for both compounds are shown in Figure 1B. Both components were gradually isomerized to the corresponding isomers, and the reactions reached an equilibrium after 18 days in the presence of waterextractable pigments. The isomerization ratios in equilibrium were approximately 0.55. The effect of the concentration of water-extractable pigments on decomposition and isomerization was observed for 29 days. The decomposition rate of microcystin LR increased with increasing pigment concentration as shown in Figure 2A. After 29 days, 5% of microcystin LR only remained in pigment solutions at 5 and 1 mg/mL. Although linear relationships between pigment concentration and isomerization ratio were obtained after 6 and 8 days (Figure 2B), the relationship was not found over 8 days, due to complete decomposition of both micro-
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Flgure 1. (A) Decrease of total microcystin (November 10-24, 1990) and (B) isomerization of microcystin LR and G(Zj-Adda microcystin LR (April 12-May 9, 1991) by irradiation with sunlight in varlous pigment solutions. (A) (+) chlorophyll a (20 mg/L); (0) @-carotene(20 I'nglL); (0)water-extractable pigment (5 mg/mL); (A)solvent-extractable plgment (5 mg/mL). Distilled water without pigment (0). (B) (0) microcystin LR in water-extractable pigment solution; (0)G(Z)-Adda microcystin LR In water-extractable pigment solution; (A)microcystin LR in solvent-extractable pigment solutlon; (A) G(Z)-Adda microcystin LR in solvent-extractable pigment solution. Pigment concentration, 5 mg/mL; bar, standard error.
cystins. Probably decomposition occurred faster than the isomerization, These results indicate that the decomposition and isomerization of microcystin LR occur simultaneously under these conditions, and the former is predominant at higher pigment concentrations. The isomerization was influenced by the presence of pigments in water and gave only one geometrical isomer. The rate of isomerization was dependent on the concentration and the type of pigment. These results suggested that a photosensitized reaction occurs during decomposition and isomerization of microcystin LR. Microcystins RR and YR showed almost the same behavior as that of microcystin LR from photolysis in the presence of pigments with the formation of 6(Z)-Adda microcystins. No significant difference was found for the isomerization rates of microcystins RR, YR, and LR. The toxicity of isomerized and intact microcystin LR was tested, and the LD50 of the intact and isomerized microcystin LR were 67 and 90 pg/kg, respectively. Both microcystin LR showed similar toxicities, indicating that 6(Z)-Adda microcystin LR with no toxicity is converted to toxic microcystin LR by isomerization. The photolysis product of microcystin LR from irradiation with sunlight in the presence of a water-extractable pigment was analyzed using the Frit-FAB LC/MS method, which was developed for rapid separation and identifiEnvlron. Scl. Technol., Vol. 28, No. 1, 1994
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Flgure 2. (A) Decornposltion and (B) isomerization of mlcrocystln LR (10 mg/L) by irradiatlon with sunlight at various concentrations of water extractable pigment (August 8-September 6, 1991). Concentrations of pigment: (0)5 mg/mL; (0)1 mg/mL; (A)0.1 mg/mL; (A)0.01 mg/mL; (0)0.005 mglmL; (a)0 mg/mL. Bar, standard error.
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Figure 3. Frlt-FAB LC/MS analysis of photolysis products of microcystin LR. (A) Total Ion chromatogram and mass chromatograms monitored at mlr 995 and 135; (6)Frlt-FAB LC/MS mass spectra of the products. Frit-FAB LC/MS analysis condltlons were as follows: HPLC, column, chromatorex ODS (5 pm, 250 X 4.0 mm); mobile phase, methanol:0,05% TFA (63:37) (containing 0.8% glycerol); flow rate, 0.5 mL/mln. MS, ion source, Frit-FAB; primary beam, XeD (5 kV); split ratio, 4:500 (using flow splitter).
cation of microcystin in cyanobacteria (33). As shown in Figure 3, three peaks (amajor peakA and two minor peaks, X and Y) are present in addition to that of microcystin LR in the total ion chromatogram (TIC). Peak A can be easily identified to be 6(Z)-Adda microcystin LR by its retention time, mass chromatogram monitored at mlz 995, and mass spectrum. Mass chromatography monitored at 176 Environ. Scl. Technoi., Vol. 28, No. 1, 1994
mlz 135 is a very powerful technique for identification of microcystins (33). Peaks X and Y are detected by this technique, and their molecular weights are 1028,suggesting that they have two hydroxyl groups oxidatively added in the diene group of Adda. They can be key compounds in the elucidation of a mechanism for the isomerization of microcystins by photolysis with pigment.
Acknowledgments
We wish to thank Dr. H. Nakazawa and Mrs. S. Suzuki, National Institute of Public Health, Tokyo, Japan, for useful discussions. Literature Cited Elleman, T. C.; Falconer, I. R.; Jackson, A. R. B.; Runnegar, M. T. Aust. J . Biol. Sci. 1978,31,209. Botes, D. P.;Kruger, H.; Viljoen, C. C. Toxicon 1982,20, 945. Kusumi, T.; Ooi, T.; Watanabe, M. M.; Takahashi, H.; Kakisawa, H. Tetrahedron Lett. 1987,28,4695. Watanabe, M. F.; Oishi, S.; Harada, K.-I.; Matsuura, K.; Kawai, H.; Suzuki, M. Toxicon 1988,26,1017. Harada, K.-I.; Ogawa, K.; Kimura, Y.; Murata, H.; Suzuki, M.; Thorn, P. M.; Evans, W. R.; Carmichael, W. W. Chem. Res. Toxicol. 1991,4,535. Sivonen, K.; Carmichael, W. W.; Namikoshi, M.; Rinehart, K. L.; Dahlem, A. M.; Niemelii, S. I. Appl. Environ. Microbiol. 1990,56, 2650. Meriluoto, J. A.0.;Sandstrom, A.; Eriksson, J. E.; Remaud, G.; Grey Craig, A.; Chattopadhyaya, J. Toxicon 1989,27, 1021. Botes, D. P.;Tuinman, A. A,; Wessels, P. L.; Viljoen, C. C.; Kruger, H.; Williams, D. H.; Santikarn, S.; Smith, R. J.; Hammond, S. J. J . Chem. Soc.,Perkin Trans. 1984,1,2311. Carmichael, W. W.; Beasley, V. R.; Bunner, D. L.; Eloff, J. N.; Falconer, I.; Gorham, P.; Harada, K.-I.; Krishnamurthy, T.; Yu, M.-J.; Moore, R. E.; Rinehart, K.; Runnegar, M.; Skulberg, 0. M.; Watanabe, M. F. Toxicon 1988,26,971. Namikoshi, M.; Sivonen, K.; Evans, W. R.; Carmichael, W. W.; Rouhiainen, L.; Luukkainen, R.; Rinehart, K. L. Chem. Res. Toxicol. 1992,5, 661. Kiviranta, J.; Namikoshi, M.; Sivonen, K.; Evans, W. R.; Carmichael, W. W.; Rinehart, K. L. Toxicon 1992,30,1093. Sivonen, K.; Namikoshi, M.; Evans, W. R.; Fardig, M.; Carmichael, W. W.; Rinehart, K. L. Chem. Res. Toxicol. 1992,5 , 464. Carmichael, W. W. J . Appl. Bacteriol. 1992,72,445. Sivonen, K.; Namikoshi, M.; Evans, W. R.; Gromov, B. V.; Carmichael, W. W.; Rinehart, K. L. Toxicon 1992,30,1481. Carmichael, W. W. In Handbook of Natural Toxins: Vol. 3, Marine Toxins and Venoms; Tu, A. T., Ed., Marcel Dekker, Inc.: New York, 1988;pp 121-147. Falconer, I. R.; Jacson, A. R. B.; Langley, J.; Runnegar, M. T. Aust. J . Biol. Sic. 1981,34,179. Yoshizawa, S.;Matsushima, R.; Watanabe, M. F.; Harada,
K.-I.; Ichihara, A.; Carmichael, W. W.; Fujiki, H. J . Cancer Res. Clin. Oncol. 1990,116,609. (18)Mackintosh, C.;Beattie, K. A.; Klumpp, S.;Cohen,P.; Codd, G. A. FEBS Lett. 1990,264,187. (19)Eriksson, J. E.; Toivola, D.; Meriluoto, J. A. 0.;Karaki, H.; Han, Y.-G.; Hartahorne, D. Biochem. Biophys. Res. Commun. 1990,173,1347. (20) Nishiwaki-Matsushima, R.; Ohta, T.; Nishiwaki, S.; Suganuma, M.; Kohyama, K.; Ishikawa, T.; Carmichael, W. W.; Fujiki, H. J . Cancer Res. Clin. Oncol. 1992,118,420. (21) Harada, K.-I.; Matsuura, K.; Suzuki, M.; Watanabe, M. F.; Oishi, S.; Dahlem, A. M.; Beasley, V. R.; Carmichael, W. W. Toxicon 1990,28,55. (22) Matsushima, R.;Yoshizawa, S.; Watanabe, M. F.; Harada, K.-I.; Furusawa, M.; Carmichael, W. W.; Fujiki, H. Biochem. Biophys. Res. Commun. 1990,171,867. (23)Nishiwaki-Matsushima,R.; Nishiwaki, S.; Ohta, T.; Yoshizawa, S.; Suganuma, M.; Harada, K.-I.; Watanabe, M. F.; Fujiki, H.Jpn. J . Cancer Res. 1991,82,993. (24) Harada, K.-I.; Ogawa, K.; Matauura, K.; Murata, H.; Suzuki, M.; Watanabe, M. F.; Itezono, Y.; Nakayama, N. Chem. Res. Toxicol. 1990,3,473. (25) Hunhes,E. 0.; Gorham, P. R.; Zehnder, A. Can.J . Microbiol. 1958,4,225. (26)Watanabe, M. F.; Tsuji, K.; Watanabe, Y.; Harada, K.-I.; Suzuki, M. Nut. Toxins 1992,1,48. (27)Watanabe, M. M.; Kaya, K.; Takamura, N. J . Phycol. 1992, 28,761. (28) Harada, K.-I.; Matsuura, K.; Suzuki, M.; Oka, H.; Watanabe, M. F.; Oishi, S.; Dahlem, A. M.; Beasley, V. R.; Carmichael, W. W. J . Chromatogr. 1988,448,275. (29)Cochran, W. G.; Cox, G. M. In Expermental Designs; Bradly, R. B., Kendall, D. G., Hunter, J. S., Watson, G. S., Eds.; John Wiley & Sons: New York, 1975;pp 29-31. (30) Dixon, W. J.; Mood, A. M. J . Am. Stat. Assoc. 1948,43,109. (31) Nichols, B. W. In The Biology of Blue-Green Algae; Carr, N. G., Whitton, B. A., Eds.; Blackwell: London, 1973;pp 144-161. (32) Chapman, D. J. In The Biology of Blue-Green Algae; Carr, N. G., Whitton, B. A., Eds.; Blackwell: London, 1973;pp 162-185. (33) Kondo, F.; Ikai, Y.; Oka, H.; Ishikawa, N.; Watanabe, M. F.; Watanabe, M.; Harada, K.-I.; Suzuki, M. Toxicon 1992, 30, 227. Received for review July 6,1993.Revised manuscript received September 13, 1993.Accepted September 23,1993.'
*Abstract published in Advance ACS Abstracts, November 1, 1993.
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