K. Kaya

Graduate School of Environmental Studies　　　　　　　 Tohoku University 　　　　 　

Chemistry and Toxicology of

Cyanobacterial toxins

Causes of WaterQuality Deterioration

Industrial Waste water

Waste Water from Home

１） Pollutants 　　　（ PCDD 、 Endocrine Disruptors 、 Herbicides etc ）

２） Eutrophication　　（ Roading of Nitrogen and phosphorus→ Occurrence of Toxic Cyanobacterial Waterblooms ）

Noctiluca bloom in California

Anabaenopsis sp. bloom in Bedetti Lake, Santo Tome, Santa Fe, Argentina

Toxic Scum of Anabaena sp. in a Drinking Water Reservoir in Finland

Estimation of Asian Water Resource in 21 Century by UNEP

Increases in Population in Asia （ 1/3 of world population), Food Production and Industrial Activities

Increase in Water DemandScarcity of Freshwater ResourceLocalized Torrential Downpour by Global Warmingand Reducing of Forest 　

Scarcity of Freshwater and Eutrophication

N P

Toxic waterblooms are occurred by enrichment of phosphorus and nitrogen in waterbodies(Eutrophication).

Toxins

Toxic Cyanobacterial blooms of Dianchi Lake in Kunming City, Yunnan, PR China

Bang Phra Drinking Water Reservoir (Thailand)

Toxic Blooms of Microcystis aeruginosa in the Reservoir

(1990)

Bloom-forming cyanobacteria)

O. agardhii A. spiroides

M. viridis M. aeruginosa

Cyanobacteria are ProkaryotesComparison of Physiological Functions between Cyanobactera and Higher Plants 　

Cyanobacteria 　　　　　 Higher Plants

Respiration 　 thylakoid 　　　　　　　　　　 mitochondria

Photosynthesis 　 thylakoid(HCO3-) 　 　 chloroplast(CO2) 　

　　　　　　　　 oxygen production oxygen production

Nitrogen source 　　 N2, NO3- NO3

-

Genetic 　　 　 Double Strand DNA 　 DNA-Histon Complex　　　　　　　　　　　　　　　　　　　　　　　 (Chromatin)

movement 　　　 slide 　　　　　　　　　　 　 non

CO2 　 HCO3-

CO32-

84 6 10 12pH

100%

pH Dependency of Soluble Carbonate Ions and cyanobacteria

Optimal pH of cyanobacteria

Toxins Produced by Cyanobacteria

・ Neurotoxins Anatoxin-a, Anatoxin-a(s), Saxitoxin

・ Hepatotoxins microcystin, Nodularin, Cylindrospermopsin

・ Cytotoxins　 Hapalindoles

・ Ichthyotoxins　 Thionsulfolipid

HepatotoxinsOCH3

CH3CH3

HNN CH2

O

HOOC

NH

H3CO NH

HN

O

O

H3C

HN

O

CH3

CH3

HN

COOH

CH3

CH3

O

O

NHH2N

HN

microcystin-LR

OCH3

CH3 CH3

HNN CH

O

COOH

H3CO

CH3 CH3

NH

COOH

O

NHHN

H3C

OO

NHH2N

HN

N NH HN NH

O3SO

H3CNHH

H HO

O

OH

cylindrospermopsin

+

-

nodularin

-

Neurotoxins

N

N

NH

HN

H2NOCO

H2N

HO OH

NH2

RH

saxitoxin : R = Hneosaxitoxin : R = OH

HN N

NH2

N(CH3)2

O

P OCH3O

O

anatoxin-a(s)

+-

H2+Cl-

N RO

anatoxin-a : R = CH3

homoanatoxin-a : R = CH2CH3

NH

ClNC

hapalindole E

NH

Cl

NC

O

hapalindolinone A

Cytotoxins

O

OH

OH

OH

CH2SO3H

O

O

O

C

C

R1

R2

S

O

R-O-C-R1

S+ H2O R-OH + R1-C-OH

SR1-C-SH

O

R1-C-SHO

+ H2O R1-C-OHO

+ H2S

Thionsulfolipid from Synechococcus sp.

Ichthyotoxin

Kaya, K., et al.(1993) Biochim. Biophys. Acta, 1169, 39-45.

tautomerism

H3CNH

COOH

NH2

L--methylaminoalanine (BMAA)

ALS/PDC(The amyotrophic lateral sclerosis / 　　　　　　　　　　　　　　 parkinsonism-dementia complex)

The rate of ALS/PDC in Chamorro people in Guam is higher than those of other people. ALS/PDC is related with fruit-bat soup as a domestic food of the Chamorro.

Neurotoxin (chronic)

Biomagnification of cyanobacterial BMAA in Guam.

Satellite photograph of a Trichodesmium bloom by using SeaWiFS imagery for spectral imaging at 443, 490, and 550 nm off the eastern coast of Florida

on October 30, 1998.

HPLC chromatograph of BMAA peak in Chroococcidiopsis indica GT-3-26 (solid line) and BMAA authenticated standard (dashed line) obtained by using fluorescence detection.

Representative chromatogram depicting BMAA in the frontal superior gyrus tissue of a Canadian Alzheimer’s patient

Microcystins, Nodularins

Bioactive Compounds Isolated from Cyanobacteria

Adda unitAhmf unitUreido unitOscillatoric acid unitOtherTricycloAhp unitAmpa unitOther

Ahd unitChoi unitSaa unitFatty acid unitOther

CyclicPep.

CyclicDepsipep.

LinearPep.

CyclicPep.

Peptides

Alkaloids

Macrolides

Lipids

Other

PuwainaphycinsOscillamidesOscillatorinLaxaphycins

MicroviridinsMicropeptins

CryptophycinsMajusculamidesMicroginensAeruginosinsAeruginoguanidines

SpiroidesinRadiosuminAnatoxins, AphantoxinCylindrospermopsinTolytoxins

Thionsulfolipid

Cracin, Fischerellin A

BioactiveCompounds

H3C NH

NHNH

NCH3

CH3

NHN

H3C CH3

OH3C

O

O

O

OO

OH

OH3C

O

O

NH

NH2

HN

Cyanopeptolin A Oscillapeptin G

3-Amino-6-hydroxy-2-piperidone (Ahp)

Ahp-Containing-Cyclic Depsipeptides

N

O

OH

NH

HN

NH

NHNH

N OH

NHN

H3C

OH3C

O

O

O

OO

OH

OH3C

O

O

CH3

H3C CH3

OH

CH3

NH2O

OH

O

OHHO

Micropeptin A, B M. a. T. Okino et al (1993)Micropeptin 90 M. a. NIES-90 K. Ishida et al (1995)Micropeptin T-20 M. a. NIES (T-20) T. Okano et al (1999)

Oscillapeptin O. a. NIES-204 H. J. Shin et al (1995)Oscillapeptin A, B O. a. NIVA CYA 18 T. Sano & K. Kaya (1998)Oscillapeptin C O. a. CCAP 1459/16 T. Sano (1996)Oscillapeptin D O. a. (China) T. Sano et al (1998)

Nostocyclin Nostoc sp. K. Kaya et al (1996)

Aeruginopeptin 95A, 95-B M.a. TAC 95 K. Harada et al (1993)Aeruginopeptin 228-A M.a. 228A K. Harada et al (1993)

Cyanopeptolin A-D M.a. PCC 7806 C. Marchin et al (1993)

Microcystilode A 　　　　　　　　　　　　 M. a. NO-15-1840 　 S. Tsukamoto et al (1993)

Cyanopeptolin S M. a. bloom C. Jakobi et al (1993)Cyanopeptolin SS M.a. bloom J. Weckesser et al (1996)

Anabaenopeptilide 90-A, 90-B A. c. 90 K. Fujii et al (1995) Anabaenopeptilide 202-A, 202-B A. l. 202A2/41 K. Fujii et al (1995)

A90720A Microchaete loktakensis A. Y. Lee et al (1994)

Variants of Ahp-containing cyclic depsipeptideVariants Source Reference

Oscillapeptin G O. a. NIVA CYA 18 T. Sano & K. Kaya (1996)

Comparison of Acute toxicity between Cyanotoxins and Artificial Toxic Chemicals.

Cyanotoxins andToxic chemicals 　　　　　　 LD50(g/kg) 　　　　 Remarks

Palytoxin*1 　　　　　　　 　 0.15 　　　　　　　 mouse2,3,7,8-TCDD *2 0.6 guinea pig (oral adm.)　　　　　　　　　　　　　　　　　　　　　　　　　　　 mouse(274g/kg)Tetrodotoxin*3 8 mouseSarin 　　　　　　　　　　　　 17 　　　　　　　　　 rabbit mouse (170g/kg)Anatoxin-a(s) 20 mouse (IP)Microcystin-LR 100 mouse (IP)Anatoxin-a 200 mouse (IP)Sodium cyanate 　　　　 2200 　 rabbit (IP)

The underlines express artificial toxic chemicals*1 ： sea anemone toxin 、 * ２： the most toxic in PCDD* ３： globefish toxin

OCH3

CH3CH3

HNN CH2

O

HOOC

NH

H3CO NH

HN

O

O

H3C

HN

O

CH3

CH3

HN

COOH

CH3

CH3

O

O

NHH2N

HN

microcystin-LR

HN

H3C

O

N

R2

NH

N

O

NH

H3COCH3

CH3

R1

COOH

CH2

COOH

CH3

O

OO

XZ

1

23

4

5

67

* L-aminoisobutyric acid

1023

966

980

980

923

1028

1001

959

1019

1037

1044

994

909

Aba*

Arg

Tyr

Ala

Met

Arg

Arg

Arg

Arg

Arg

Arg

Arg

Arg

Leu

Leu

Leu

Leu

Phe

Leu

Tyr

Tyr

Leu

Arg

Tyr

AlaLeu

Z

[D-Asp3,Dha7]microcystin LR

[Dha7]microcystin LR

[D-Asp3]microcystin LR

microcystin LAba

microcystin FR

microcystin LY

microcystin YA

microcystin YM

microcystin RR

microcystin YR

microcystin LR

microcystin LA

[D-Asp3]microcystinRR

H

H

H

CH3

CH3CH3

CH3

CH3

CH3CH3

CH3

CH3CH3

CH3

CH3CH3

CH3

CH3CH3

H

CH3CH3

H

CH3CH3

CH3

CH3CH3

CH3

CH3CH3

CH3

CH3

CH3CH3

CH3

MWX R1 R2

H

Nomenclature of microcystins

Over 70 microcystin variants has been identified in the world at the present time (2004)

ミクロシスチン生合成遺伝子の構造

10kbp

ミクロシスチン

O

NH2

N

ON

N

O

O

OO

H

H HH

H

H

H

O

H

H

CH2H

mcyE mcyD mcyA mcyB mcyCmcyGmcyJ

mcyI

mcyH

mcyF

Me

Me

Me

Me

MeMe

OMe

COOH

NH

Me

NH

HN

NH

H

NH

COOH

NHMe

Structure of Biosynthesis Gene of Microcystin

mcyE mcyDmcyGmcyJ

mcyI

mcyH

mcyF

PCR-primer for detection of toxin gene

mcyA

About 550 bases

NIES 88

mcy

Dm

cyG

mcy

J

mcy

Dm

cyG

mcy

J

NIES 99Toxic Non-toxic

Toxin gene oftoxic strain

940bp

420bp

PCR Detection of Microcystin Gene

Mouse Liver enlarged by microcystin-RR

Microcystin Shock

Microcystin

Bile Acid Tranport SystemReceptor

HyperphophorylatesdProtein

Cytoskeletal changes

Membrane StructureChanges

ＰＡＦInhibition of ProteinPhosphatase

Arachidonic acid

CyclooxygenasePLA2

TXA2

IL-1

TNF-

ＰＡＦ

Arachidonic acid

Cyclooxygenase

TXA2

Hepatocytes Macrophages

Ca2+ ion

PGI2

PGI2

Enclosure A

Enclosure B

Enclosure C

Dianch Lake Side

Cancer Promotion by Microcystin (MC)

Phosphorylated Protein　　 (Activation)

Function （ Proliferation, Differentiation ）

Protein Kinases Protein Phosphatases

Protein (Inactivation)

Inhibition of ProteinPhosphatase by MC

Hyper-Phospho-rylation

Deformationof Cells

CancerPromotion

TNF-

WHO Guideline for microcystin-LR is 1g/litre (drinking water), 1997

(Falconer, I. R. etal .(1994), Toxicity of the blue-green alga (cyanobacterium)Microcystis aeruginosa in drinking water to growing pigs, as an animal model for human injury and risk assessment. Environ. Toxicol. Water Qual.Intern. J., 9, 131-139. )

Outline of the Experiment

１） Animal ： Pig （ Body weight 60 – 65 kg 、 5 heads/group ）

２） Administration ： Oral (microcystin containing water)

３） Dosage ： 1312, 796, 280 and 0g/kg/day

４） experimental Period ： 8 weeks

Outline of Risk Assessment

Minimum Dose （ 280 g/kg/day ） Liver tissue damage.

Safety Factor

１） Only 1% of lifetime exposure------------------A safety factor of 10 is applied２） Use of pig data as an animal model for human injury -------------------------------------A safety factor of 10 is applied３） Difference of health condition due to age, other causes of liver damage, and other-----A safety factor of 10 is applied

Thus a safety factor 1000 is applied to the lowest doserate.This provide a guideline safe intake for humans of 0.28 g/kg/day, Which should result in no adverse effect as seen by direct liver injury

To apply this to a 60kg adults drinking 2L water/day, a consumption,Of water containing 8.4 g microcystin/L should be safe.

4) For tumor prmortion, additional safety factor of 5 or 10 is required

Thus a conservative estimate for water safety is 0.84 g microcystin/ L or approximately 1 g/L.

Determination Methods for Total Microcystin

1)Molecular biological method i) PCR of Toxin gene

2) Biochemical methods i) Protein Phosphatase Inhibition ii) ELISA

3) Physical methods i) HPLC/UV or MS

4) Chemical i) MMPB metho ii) GSH method

1) Inhibition of Protein Phosphatase 2A 　

2) Enzyme-Linked Immunosorbent Assay (ELISA)

Biochemical Determination 　

Microcystin

Bile Acid Tranport SystemReceptor

HyperphophorylatesdProtein

Cytoskeletal changes

Membrane StructureChanges

ＰＡＦInhibition of ProteinPhosphatase

Arachidonic acid

CyclooxygenasePLA2

TXA2

IL-1

TNF-

ＰＡＦ

Arachidonic acid

Cyclooxygenase

TXA2

Hepatocytes Macrophages

Ｃａイオン

PGI2

PGI2

Inhibitory Activity

Not only microcystinbut also other compounds inhibit

ELISA

Secondary antibody

ＨＲＰ

ＨＲＰ

Substrate

Color

MCLR-BSA

Perimary antibody

microcystin

HPLC Analysis of Unknown Microcystins

- Kaya’s Lab. Method -

Check points

1) Absorption ratio at 239 nm / 280 nm

2) Division of Peak Shape

3) UV Spectrum

55% MeOH pH 3.0, 1ml/min, Mightysil 4.6x150 mm Rt of LR was 25 minUnknown microcystins

LR

Rt, 15.4 min Rt, 28 min

Rt, 30 min Rt, 31 min (shoulder)

HN

H3C

O

N

CH3

NH

N

O

NH

H3COCH3

CH3

CH3

COOH

R

COOH

CH3

O

O

O

XZ

H

COOH

OCH3

CH3

MMPB method for total microcystin determination

Chemical Determination

R:CH2 （ normal microcystin ）

→quantitative addition of GSH (C=CH2 +GSH→CH-CH2-SG)

R:C=CH-CH3 (Dhb-microcystin ）→non-reaction with GSH

Selective Determination

KMnO4+NaIO4

GSH method

NH

CH3 CH3

OCH3

CH3O

Mdha

D-MeAsp

D-Ala D-Glu

CH3

OCH3

HO

OKMnO4 / NaIO4

CH3

OCD3

HO

O

2-methyl-3-methoxy4-phenylbutyric acid (MMPB)

MMPB-d3(Internal standard)

Y

X

ＭＭＰＢ法

Kaya,K. and Sano, T, Anal. Chim. Acta 386 (1999) 107-112

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 min

10000

15000

20000

25000

30000

35000

40000

45000

50000

55000Int.

210.00(1.07)207.00(1.00)

SIM Profile of MMPB by LC/MS

COOH

CH3

OCD3

m/z 207(MMPB)

m/z 210 (MMPB-d3)

MMPB-d3

MMPB Method

HN

H3C

O

N

NH

HN

O

NH

H3COCH3

CH3

COOH

COOH

CH3

O

OO

XZ

HN

H3C

O

N

R

NH

HN

O

NH

H3COCH3

CH3CH3

COOH

CH2

COOH

CH3

O

OO

XZ

1

23

4

5

6 R = CH3 (Mdha) R = H (Dha)

H

CH3

H

1

23

4

5

6

(E) or (Z)-Dhb 7

Microcystin (Normal microcystin)

[Dhb7] microcystin

HN

H3C

O

N

NH

HN

O

NH

H3COCH3

CH3

COOH

COOH

CH3

O

OO

XZ

H

OH

H

1

23

4

5

[L-Ser7] microcystin

6L-Ser 7

HN

H3C

O

N

NH

HN

O

NH

H3COCH3

CH3

COOH

COOH

CH3

O

OO

XZ

HCH3

1

23

4

5

[L-Ala7] microcystin

6 L-Ala 7

7

CH3

CH3

Microcystin groups according to the amino acid at unit 7 　

(GSH)

5%K2CO3

TNBS

0.5M NaHCO3-NaOH(pH9.0)40°C, 90 min.

SO3-O2N

NO2

NO2S-CH2-CH-CO-NH-CH2-COOH

NH-CO-CH2CH2-CH-COOH

NH

NO2O2N

NO2

Calorimetric, TLC

2M HCl/ MeOH90°C, 18hr.

+ Internal Standard*MeOOC-CH2-CH2-CH-COOMe

NH

NO2O2N

NO2

D-AlaX

Y D-Glu N

CH2

O-MAsp

Adda

Mdha or Dha

D-AlaX

Y D-Glu N

O-MAsp

Adda

D-AlaX

Y D-Glu N

O-MAsp

Adda

SG

LC/UV, LC/MS

max 332 nm (=15000) ]*

MeOOC-CH2-CH2-CH2-CH-COOMe

NH

NO2O2N

NO2

Internal Standard(Dimethyl 2-TNBaminoadipate)

NH

NH2

O

(CH3CO)2O

10%CH3COONa

NH

HN

OCH3

O

at 25°C, 1.5 hr at 25°C, 2 hr

N-acetylated peptides

microcystin and other peptides

Acetic anhydrideMicrocystin fraction

2000

1800

1600

1400

1200

1000

800

2000

1800

1600

1400

1200

1000

800

2 4 6 8 10 12 14 Rt (min)

2 4 6 8 10 12 14 Rt (min)

O2N

NO2

NO2

NH CHCOOMe

CH2

CH2

COOMe

m/z 385 [(M-H)- ]

O2N

NO2

NO2

NH CHCOOMe

CH2

CH2

CH2m/z 399 [(M-H)

- ]COOMe(b)

(a)

Inte

ns

ity

Inte

ns

ity

Detection of N-TNB-dimethyl glutamate by LC/MS

D-AlaX

Y D-Glu N

O-MAsp

AddaSG

S-CH2-CH-CO-NH-CH2-COOH

NH-CO-CH2CH2-CH-COOH

NH2D-AlaX

Y D-Glu N

O-MAsp

Adda

Microcystin Fr.

NH

NH2

O

R

D-AlaX

Y D-Glu N

CH2

O-MAsp

Adda

TLC

(Identification of individual variants)

C

O

O

OH

OH

(Total microcystin)

Colorimetry

OVERALL PROCEDURE

GSH

Kaya, K. et al Anal. Chim. Acta 450 (2001) 73-80

RR SampleLR AC-Sample

OCH3

CH3CH3

HNN CH2

O

HOOC

NH

H3CO NH

HN

O

O

H3C

HN

O

CH3

CH3

HN

COOH

CH3

CH3

O

O

NHH2N

HN

microcystin

CH

CH3

Dhb-

H3C

CH3

1

2

34

5

67

L-amino acid

L-amino acid

D-Ala

D-Leu

D-Glu

(Z) and (E)

Adda

OCH3

CH3CH3

HNN CH2

O

HOOC

NH

H3C O NH

HNO

O

H3C

HN

OCH3

CH3

HN

COOH

CH3

CH3

O

O

NHH2N

HN

Dhb-microcystinC

CH3

1

2

34

5

67

L-amino acid

L-amino acid

D-Ala

D-Glu Mdh

HN

H

NHO

a5.6-5.7 ppm

(E)-Dhb-

C

CH3HN

H

NHO

b

6.4-6.5 ppm

(Z)-Dhb-

Sano, T.Beattie, K., Codd, G. A., and Kaya, K. J. Nat. Prod. 61, 851-853 (1998)Sano, T. and Kaya, K. Tetrahedron 54, 463-470 (1998)

normal

E-Dhb

Z-Dhb

HN

CH3

Ha

NHb

Ha

HN

Hb

CH3

O

O

O

[Asp3, (E)-Dhb7]microcystin RR O. agardhii

[Asp3, (E)-Dhb7]microcystin HtyR O. agardhii

[Asp3, (E)-Dhb7]microcystin HilR P. rubescens

[Asp3, ADMAdda5, (E)-Dhb7]microcystin RR Nostoc sp.

[Asp3, ADMAdda5, (E)-Dhb7]microcystin HtyR Nostoc sp.

[Asp3, ADMAdda5, (E)-Dhb7]microcystin LR Nostoc sp.

[Asp3, (Z)-Dhb7]microcystin HtyR O. agardhii

[Asp3, (Z)-Dhb7]microcystin LR O. agardhii

Dhb-microcystin

Dhb-microcystin has not been found from Microcystis.

世界地図： http://www.sekaichizu.jp/

Geographical 　 Distribution of Dhb-MC

OCH3

CH3CH3

HNN CH2

O

HOOC

NH

H3CO NH

HN

O

O

H3C

HN

O

CH3

CH3

HN

COOH

CH3

CH3

O

O

NHH2N

HN

microcystin

CH

CH3

Dhb-

H3C

CH3

1

2

34

5

67

L-amino acid

L-amino acid

D-Ala

D-Leu

D-Glu

(Z) and (E)

Adda

D-[D-Leu1] microcystin LR

[D-Leu1]microcystin LR foundfrom Microcystis aeruginosaisolated from Brazil and Canada.

Summary

1)Dhb-microcystins were found from cells of O. agardhii, P. rubescens,and Nostoc sp. isolated from North European countries.

2)[D-Leu1]microcystin was isolated from cells of M. aeruginosa collected from Brazil and Canada, but has not been found any other area.

Problems Are toxin genes in cyanobacteria localized ? Do migratory birds carry cyanobacteria ?

SELECTIVE CONTROL OF TOXIC MICROCYSTIS WATERBLOOMS USING LYSINE AND

MALONIC ACID

Why do we need selective control of toxic cyanobacterial waterblooms?

In Europe, they do not need selective control of toxiccyanobacteria, since they use only drinking.

Therefore, They remove phosphate completely in eutrophicated lake water for control of toxic cyanobacteria.

As the result, there is no phytoplankton in the lake, alsoZooplankton and fish.

In Asia, inland residents have utilized freshwater fish for a major protein source.

Therefore, aquaculture is important, and eutrophication is necessary for growth of phytoplankton, zooplankton and fish, but exclusion of toxic cyanobacteria is necessary for human health and aquaculture.

As the result, we need to develop a method of selective control of toxic cyanobacteria.

As the opposite situation of the European,

Kaya, K. and Sano, T.(1996) Algicidal compounds in yeast extract as a component of microbial culture media. Phycologia, 35(6 Supp.), 117-119

Two algicidal compounds, lysine and malonic acid, were identified from Yeast extract. Lysine was toxic to only Microcystis (cyano-Bacteria, blue-green algae).Cells of Microcystis viridis NIES-102 were completely killed within 48 hr by lysine at the concentration of 1.0 ppm, whereas lysine was non-toxic to Anabaena and Chlorella species. Also, cells of M. viridis were killed by malonic acid at the concentration of 40 ppm.

Why did we select lysine and malonic acid for the control?

We examined effects of lysine and malonic acid on MicrocystisBlooms using enclosures.

Enclosure 10 m10 m

1.3-1.5 m

10 – 20 cm

A B C

Lake Sediments

Enclosure A: ControlEnclosure B: Lysine treatmentEnclosure C: Lysine plus malonic acid treatment

1 m1 m

0.5 m

Sampling Point

Enclosure A

Enclosure B

Enclosure C

Dianchi Lake Side

Macrophytes: Seeds of macrophytes (Myriophllum spicatum and Potamogeton crispus L) and water chestnuts (Trapa sp.) were contained in the lake sediment.

Monitoring:Water pH, DO, Chlorophill-a, Lysine, Malonic acid, Microcystin, Cell numbers of phytoplankton (cyanobacteria, dyatom,eugllena) and zooplankton (cradoceran )

Results were expressed as average of three sampling points with S. D.

Lysine and malonic acid treatments:Lysine was dissolved with water at the concentration of 100g/L, and sprayed with an insecticide sprayer (lysine 10 g/m2).Malonoc acid was sprayed as the same manner as the lysine treatment (malonic acid 10g/m2).

Methods:

１

Ｂ

３

The enclosure surfaces on Day 3 after the treatments

A:　Control

B: lysine

C:　lysine + malonic acid

A CMicrocystis aeruginosa

6

8

10Ｌ

ｙｓ

ｉｎ

ｅ

[ m

g/L]

10 7 140

Days　after　Treatment

2

4

3 5

Enclosure　C

Enclosure　B

Enclosure　A

Fig.1 Decrease in lysine concentration in the enclosures after the treatments. (The zero day means immediately after the treatments, S. D. < 5 %)

HO

O

NH2

NH2 HO

O

NH2

OH

O

+ NH4OH

Degradation

lysine 2-aminoadipic acid

8.0

8.5

9.0

9.5

pH

7.5

20 7 14 21 280

Days　after　Treatment

Enclosure　A

Enclosure　B

Enclosure　C

Fig.2 Changes in pH in the enclosures after the treatments

(S. D. < 5%; *p < 0.05 )

**

60

80

100

120

Ｃｈ

ｌｏ

ｒｏ

ｐｈ

ｙｌ

ｌ　

ａ　

[g

/L]

20 7 14 21 280

20

40

Days　after　Treatment

Enclosure　A　

Enclosure　B

Enclosure　C

Fig.3 Changes in biomass in the enclosures after the treatment of lysine and malonic acid. (S. D. < 20 %)

28

12

16

20

24

Ce

llｓ

　 [

ｘ

10

6/L

]

20 7 14 210

Days　after　Treatment

４

８

Total　phytoplankton

Cyanobacteria

Diatom

Euglena

Enclosure　A　

12

16

20

24

20 7 14 21 280

Days　after　Treatment

４

８

Total　phytoplankton

Cyanobacteria

Diatom

Euglena

Enclosure　B

12

16

20

24

20 7 14 21 280

Days　after　Treatment

４

８

Total　phytoplankton

Cyanobacteria

Diatom

Euglena

Enclosure　C

Fig.4 Changes in phytoplankton compositions in the enclosures after the treatments. (S. D. < 20 %)

Cla

do

cera

n　

[in

div

idu

als

/L]

20 7 14 21 280

Days　after　Treatment

Enclosure　AEnclosure　B

Enclosure　C

100

200

300

400

500

600

700

800

Fig.5 changes in individual number of cladoceran in the enclosures after the treatments. (S. D. < 20 %)

９

12

15

To

tal　

mic

rocy

stin

　[m

g/L

]

20 7 14 21 280

Days　after　Treatment

3

６

Enclosure　A　

Enclosure　B

Enclosure　C

Fig.6 Changes in total microcystyin contents in the enclosures after the treatments. (S. D. < 10 %)

The enclosures surfaces on day 28 after the treatments

A:　Control

B: lysine

C:　lysine + malonic acid

A

B

CMyriophllum spicatum

water chestnut (Trapa sp.)

Microcystis aeruginosa

Conclusion:

The treatment with lysine plus malonic acid is an effective method for the control of toxic Microcystis blooms. The ecological and water qualitative changes derived from the treatment suggested that the incorporation cycles of nitrogen and phosphorus in eutrophicated water were switched from toxic cyanobacteria (Microcystis) to non-toxic macrophytes.

Another Methods for Cyanobacterial Control

Shade [on water surfaces (Shallow Clean®)]

When about 30% of the water surface area of a pond was covered with a shade, cyanobacterial waterblooms were disappeared within 1 month.

What happened to the pond?1) The water temperature just under the shade was 4 ºC lower than that of the open surface.2) Waterblooms were gathered under the shade.3) The cyanobacterial cell growth was suppressed by the shade.4) The water pH was shifted to neutral side by the shade.5) Waterblooms-feeding biofilms were formed on the water-touched surface of the shade.

5 – 50 m

anchor

Shade (Shallow Clean®)

Dry up (of Dam Sediments )

Cyanobacterial (Microcystis) cells are resting on dam sediment at low watertemperature (below 10 ºC) in winter season.When the dam sediment were dried up, the germination rates of the cells on the sediment were dependent on the water content in the sediment.

Water level under normal conditions

S1(31m from WL)

S2(16m from WL)

Water level in the experiment(36m from WL)

(WL)

Water content in the sediment (WCS) and germination rate (GR)

100

0 7 14 21 30Day after dry up

% o

f W

CS

(bar

s)100

75

50

25

0 0

25

50

75

100

% o

f G

R (

clos

ed c

ircl

es)S1

S2

S1

S2

　　　　　　　　　　　　　　　　　　　　

　　　　　　　　　　　　　　　　　　　　　　　　 In Europe: 　 Prof. G. A. Codd (UK) and Dr. H.C. Utkilen (Norway)

　　　　　　　　　 In North America ,and parts of Central America and the Caribbean: 　　　　　 Prof. Wayne Carmichael (USA)

　　　　　　　　　　　 In South America, and parts of the Central America and the Caribbean:

　　　　 Prof. Sandra Azevedo (Brazil)　　　　　　　　　　　　　 In Africa: 　　　　 Dr. William R. Harding (South Africa)　　　　　　　　　　　　　 In Asia (western sector): 　　　　　　　　　　　　　　　　　　　　　　　 Dr. Suvendra Bagchi (India)　　　　　　　　　　　　　 In Asia (eastern sector):

　　　　 Prof. Kunimitsu Kaya (Japan)　　　　　　　　　　　　　 In Australasia and parts of Oceania:　　　　　　　　　　　　　　　　　　　　　　　 M.D. Burch (Australia)

UNESCO-CYANONET Committee Member

WWC

KK

GAC&HCU

MDB

SB

WRH

SA

Thank youfor your attention!