Chemistry and Toxicology of Cyanobacterial toxins

K. Kaya Graduate School of Environmental Studies Tohoku University

Causes of Water Quality Deterioration

Industrial Waste water Waste Water from Home

Pollutants PCDDEndocrine DisruptorsHerbicides etc

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 Asia1/3 of world population), Food Production and Industrial Activities

Increase in Water Demand Scarcity of Freshwater Resource

Localized Torrential Downpour by Global Warming and Reducing of Forest

Scarcity of Freshwater and Eutrophication

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

N P 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 Prokaryotes

Comparison of Physiological Functions between Cyanobactera and Higher Plants

Cyanobacteria Respiration thylakoid

Higher Plants mitochondria chloroplast(CO2) oxygen production NO3DNA-Histon Complex (Chromatin) non

Photosynthesis thylakoid(HCO3-) oxygen production Nitrogen source Genetic movement N2, NO3Double Strand DNA slide

100%

pH Dependency of Soluble Carbonate Ions and cyanobacteria

Optimal pH of cyanobacteria

8 pH

10

12

CO2

HCO3-

CO32-

Toxins Produced by Cyanobacteria

Neurotoxins Anatoxin-a, Anatoxin-a(s), Saxitoxin
Hepatotoxins microcystin, Nodularin, Cylindrospermopsin Cytotoxins Hapalindoles Ichthyotoxins Thionsulfolipid

HOOC

CH3 N O O H3C H N O CH3 H HN N CH2 NH O CH3 CH3

Hepatotoxins

HN OCH3 H3C O NH CH3 CH3 HN H2N N H O

O COOH

microcystin-LR

COOH HN OCH3 H3C O NH CH3 CH3 HN H2N N H O H3C HN O

CH3 CH3 N CH OO NH COOH

-O

SO 3H3C

H N H NH

OH O

NH HN

NH O

cylindrospermopsin

nodularin

Neurotoxins

H2NOCO R N H2N N H H N NH2 N H HO OH

saxitoxin : R = H neosaxitoxin : R = OH
N(CH3)2 HN N O
H2+ClN

NH2

-O

P OCH3 O

anatoxin-a(s)

anatoxin-a : R = CH3 homoanatoxin-a : R = CH2CH3

Cytotoxins

Cl NC Cl NC O N H

N H

hapalindole E

hapalindolinone A

Ichthyotoxin
S CH2 SO 3H O OH OH OH O O C R1 O O C R2

Thionsulfolipid from Synechococcus sp.

S R-O-C-R1 + H 2O O R1-C-SH + H2O S R-OH + R 1-C-OH
tautomerism

O R 1-C-SH

O R1-C-OH + H2S

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

Neurotoxin (chronic)
H3C N H COOH NH2

L--methylaminoalanine (BMAA)
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.

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

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 Alzheimers patient

Bioactive Compounds Isolated from Cyanobacteria

Adda unit Ahmf unit Ureido unit Oscillatoric acid unit Other Tricyclo Ahp unit Ampa unit Other Ahd unit Choi unit Saa unit Fatty acid unit Other Microcystins, Nodularins Puwainaphycins Oscillamides Oscillatorin Laxaphycins Microviridins Micropeptins Cryptophycins Majusculamides Microginens Aeruginosins Aeruginoguanidines Spiroidesin Radiosumin Anatoxins, Aphantoxin Cylindrospermopsin Tolytoxins Thionsulfolipid Cracin, Fischerellin A

Cyclic Pep.

Cyclic Pep.
Peptides Cyclic Depsipep.

Bioactive Compounds

Linear Pep.

Alkaloids
Macrolides Lipids Other

Ahp-Containing-Cyclic Depsipeptides
OH N N H O

3-Amino-6-hydroxy-2-piperidone (Ahp)
NH2 HN NH OH O O O H3C N H O H3C O H3C NH N H O O H3C N H N O CH3
OH

H3C
CH3 O CH3
HO OH H N O O NH2 O O N H O H3C

CH3 O NH N H O O H3C CH3 O N

OH CH O

OH

H3C N H N O

OH

Cyanopeptolin A

Oscillapeptin G

Variants of Ahp-containing cyclic depsipeptide

Variants
A90720A Aeruginopeptin 95A, 95-B Aeruginopeptin 228-A Anabaenopeptilide 90-A, 90-B Anabaenopeptilide 202-A, 202-B Cyanopeptolin A-D Cyanopeptolin S Cyanopeptolin SS Microcystilode A Micropeptin A, B Micropeptin 90 Micropeptin T-20 Nostocyclin Oscillapeptin Oscillapeptin A, B Oscillapeptin C Oscillapeptin D Oscillapeptin G

Source
Microchaete loktakensis M.a. TAC 95 M.a. 228A A. c. 90 A. l. 202A2/41 M.a. PCC 7806 M. a. bloom M.a. bloom M. a. NO-15-1840 M. a. M. a. NIES-90 M. a. NIES (T-20) Nostoc sp. O. a. NIES-204 O. a. NIVA CYA 18 O. a. CCAP 1459/16 O. a. (China) O. a. NIVA CYA 18

Reference
A. Y. Lee et al (1994) K. Harada et al (1993) K. Harada et al (1993) K. Fujii et al (1995) K. Fujii et al (1995) C. Marchin et al (1993) C. Jakobi et al (1993) J. Weckesser et al (1996) S. Tsukamoto et al (1993) T. Okino et al (1993) K. Ishida et al (1995) T. Okano et al (1999) K. Kaya et al (1996) H. J. Shin et al (1995) T. Sano & K. Kaya (1998) T. Sano (1996) T. Sano et al (1998) T. Sano & K. Kaya (1996)

Comparison of Acute toxicity between Cyanotoxins and Artificial Toxic Chemicals.

Cyanotoxins and Toxic chemicals

LD50(mg/kg) 0.15 0.6

8 17 20 100 200 2200

Remarks mouse guinea pig (oral adm.) mouse(274mg/kg) mouse rabbit mouse (170mg/kg) mouse (IP)

Palytoxin*1 2,3,7,8-TCDD *2
Tetrodotoxin*3 Sarin Anatoxin-a(s) Microcystin-LR Anatoxin-a Sodium cyanate

mouse (IP) mouse (IP)

rabbit (IP)

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

HOOC HN OCH3 H3C O NH CH3 CH3 HN H2N N H O

CH3 N O O H3C H N O CH3 H HN N CH2 NH O CH3 CH3

O COOH

microcystin-LR

7 6 COOH 5 R2 N O NH CH3 CH3

X microcystin LA microcystin LR microcystin YR microcystin RR microcystin YM microcystin YA microcystin LY microcystin FR microcystin LAba [D-Asp3]microcystin LR [Dha7]microcystin LR [D-Asp3 ,Dha7]microcystin LR [D-Asp3 ]microcystinRR * L-aminoisobutyric acid Leu Leu Tyr Arg Tyr Tyr Leu Phe Leu Leu Leu Leu Arg Z Ala Arg Arg Arg R1 CH3 CH3 CH3 CH3 CH3 CH3 CH3 H CH3 H H R2 CH3 CH3 MW 909 994

Nomenclature of microcystins

HN H3C

CH2 NH

OCH3

O O H3C H N X 2 1

R1 O

Z 4

COOH 3

CH3 1044 CH3 1037 CH3 1019 CH3 CH3 959 1001

Met CH3 Ala Tyr Arg

CH3 1028 CH3 CH3 H H 923 980 980 966

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

Aba* CH3 Arg Arg Arg Arg

CH3 1023

Structure of Biosynthesis Gene of Microcystin

10kbp

mcy mcy mcy J I H

mcyG

mcy F

mcyE

mcyD

mcyA

mcyB

mcyC

COOH H

Me O N NH CH 2 H O NH Me Me H

N H OMe H Me H O Me H NH H N H H Me H N Me O O Me

O COOH

HN

NH

NH2

PCR-primer for detection of toxin gene

mcy mcy mcy J I H

mcyG

mcy F

mcyE

mcyD

mcyA

About 550 bases

PCR Detection of Microcystin Gene

Toxic
NIES 88

Non-toxic
NIES 99

940bp

Toxin gene of toxic strain

420bp

Mouse Liver enlarged by microcystin-RR

Microcystin

Receptor
IL-1

Bile Acid Tranport System A Enclosure Dianch Lake Side Hepatocytes Enclosure B
Inhibition of Protein Phosphatase
Hyperphophorylatesd Protein

Macrophages

Enclosure C

TNF-a

Arachidonic acid Cyclooxygenase

PGI2

PLA2

Arachidonic acid Cyclooxygenase

Cytoskeletal changes
TXA2

Membrane Structure Changes

PGI2

TXA2

Ca2+ ion

Microcystin Shock

Cancer Promotion by Microcystin (MC) FunctionProliferation, Differentiation

Deformation of Cells

Phosphorylated Protein (Activation)

Inhibition of Protein Phosphatase by MC

HyperPhosphorylation

Protein Kinases

Protein Phosphatases

Cancer Promotion

Protein (Inactivation) TNF-a

WHO Guideline for microcystin-LR is 1mg/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

AnimalPig Body weight 60 65 kg5 heads/group AdministrationOral (microcystin containing water) Dosage1312, 796, 280 and 0 mg/kg/day experimental Period8 weeks

Outline of Risk Assessment

Minimum Dose280 mg/kg/dayLiver tissue damage.

Safety Factor
Only 1% of lifetime exposure------------------A safety factor of 10 is applied Use of pig data as an animal model for human injury -------------------------------------A safety factor of 10 is applied 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 mg/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 mg 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 mg microcystin/ L or approximately 1 mg/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

Biochemical Determination
1) Inhibition of Protein Phosphatase 2A

2) Enzyme-Linked Immunosorbent Assay (ELISA)

Microcystin

Receptor
IL-1

Bile Acid Tranport System Hepatocytes

Inhibition of Protein Phosphatase
Hyperphophorylatesd Protein

Macrophages

TNF-a

Arachidonic acid Inhibitory Activity

Not only microcystin but also other compounds inhibit PGI2

PLA2

Cyclooxygenase

Arachidonic acid Cyclooxygenase TXA2

Cytoskeletal changes

Membrane Structure Changes

TXA2

PGI2

ELISA

Secondary antibody

microcystin

Color

Perimary antibody
MCLR-BSA

Substrate

HPLC Analysis of Unknown Microcystins

- Kayas Lab. Method Check points 1) Absorption ratio at 239 nm / 280 nm 2) Division of Peak Shape 3) UV Spectrum

Unknown microcystins

55% MeOH pH 3.0, 1ml/min, Mightysil 4.6x150 mm

Rt of LR was 25 min

LR

Rt, 15.4 min

Rt, 28 min

Rt, 30 min

Rt, 31 min (shoulder)

Chemical Determination
MMPB method for total microcystin determination GSH method
OCH3 COOH C O O H HN H 3C O O O NH CH3 CH 3 Z CH3 H N X O H 3C NH CH3 N R

CH3

KMnO4+NaIO4

OCH3

R:CH2 normal microcystin quantitative addition of GSH (C=CH2 +GSHCH-CH2-SG)

COOH

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

Selective Determination

D-Ala X D-MeAsp

Mdha

D-Glu O Y N H CH3 OCH3 KMnO4 / NaIO4 HO CH3 CH3 CH3 2-methyl-3-methoxy4-phenyl butyric acid (MMPB) O OCH3

O HO

OCD3

CH3 MMPB-d3 (Internal standard)

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

MMPB Method

SIM Profile of MMPB by LC/MS

m/z 207(MMPB)
OCD3 COOH

MMPB-d3

CH3

m/z 210 (MMPB-d3)

Microcystin groups according to the amino acid at unit 7

7 6 COOH R N O O H3 C CH3 H N X COOH 3 2 NH O 1

L-Ser 7
6 5 OCH3 H3C HN O NH CH3 CH3 4 Z O COOH O O H3C CH3 H N X CO O H 2 3 NH 1 O H N H

5
OCH3 H3 C

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

CH2

HN O NH

OH

CH3 CH3 4

Z O

Microcystin (Normal microcystin)

[L-Ser7] microcystin

6 5 HN OCH3 H3C O NH CH3 CH3 4 Z O COOH O O H3C H N X COOH 2 3 NH 1 O CH3 CH3 H N H

(E) or (Z)-Dhb 7
5 CH3 OCH3 H3C HN

COOH O O

H N

L-Ala 7
CH3 NH 1 O

O
CH 3 H N X H3C

NH
Z 4 O

COOH 2 3

[Dhb ] microcystin

[L-Ala7] microcystin

Microcystin fraction
microcystin and other peptides
O N H NO2 O2N TNBS SO3NO2 Y -MAsp Adda X NH2

Mdha or Dha

Acetic anhydride
(CH 3CO)2O 10%CH3COONa at 25C, 1.5 hr

Adda X

D-Glu D-Ala

N CH2

(GSH) 5%K2CO3 at 25C, 2 hr

Adda X

D-Glu

SG

-MAsp

-MAsp

D-Ala

N-acetylated peptides O H N CH3 N H O D-Glu D-Ala N

S-CH2-CH-CO-NH-CH2-COOH NH-CO-CH2CH2-CH-COOH O O2N NH NO2

Calorimetric, TLC

0.5M NaHCO3-NaOH(pH9.0) 40C, 90 min.

+ Internal Standard* 2M HCl/ MeOH 90C, 18hr.

MeOOC-CH2-CH 2-CH-COOMe NH O2N NO2 LC/UV, LC/MS NO 2 max 332 nm ( =15000) ]

NO2

* Internal Standard(Dimethyl 2-TNBaminoadipate) MeOOC-CH2-CH2-CH2-CH-COOMe NH O2N NO2

NO2

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

OVERALL PROCEDURE
Microcystin Fr.
Y -MAsp Adda D-Glu N CH2 X D-Ala

GSH
O

Y -MAsp

Adda

D-Glu

SG

O N H NH2 R

D-Ala

Y -MAsp

Adda

D-Glu

D-Ala

S-CH2-CH-CO-NH-CH2-COOH NH-CO-CH2CH2-CH-COOH NH2

O OH C O OH

TLC
(Identification of individual variants)

Colorimetry
(Total microcystin)

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

RR

LR

Sample

AC-Sample

(Z) and (E) Dhb6 7 D-Glu

H O OC CH 3

CH C H3
N O O O NH CH 2

5 Adda
HN O C H3 H 3C

H3 C NH
CH 3 CH 3 HN O H N O N H C H3 H HN N O CO OH

1 D-Ala
O

H 3C C H3

D-Leu
C H3 C H3

2 L-amino acid

4 L-amino acid

H 2N

microcystin

Dhb-microcystin

H N

Ha C

5.6-5.7 ppm
C H3

NH

6 7 D -Glu

(E)-DhbM dh
CH2 O NH
1 D -Ala

HO OC
5

CH3
N O CH3 C Hb CH3 CH3L-amino acid
2

HN O CH3 H3C O

6.4-6.5 ppm

NH
CH3 CH3 HN H2N 4 L-a min o acid N H O

H3C H CH3 H HN N N O O CO OH
3

H N

NH

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

Ha N Hb O normal

H N O

Ha CH3

E-Dhb

H N O

CH3 Hb

Z-Dhb

Dhb-microcystin
[Asp3, (E)-Dhb7]microcystin RR [Asp3, (E)-Dhb7]microcystin HtyR [Asp3, (E)-Dhb7]microcystin HilR [Asp3, ADMAdda5, (E)-Dhb7]microcystin RR [Asp3, ADMAdda5, (E)-Dhb7]microcystin HtyR [Asp3, ADMAdda5, (E)-Dhb7]microcystin LR [Asp3, (Z)-Dhb7]microcystin HtyR [Asp3, (Z)-Dhb7]microcystin LR
Dhb-microcystin has not been found from Microcystis.

O. agardhii O. agardhii P. rubescens Nostoc sp. Nostoc sp. Nostoc sp. O. agardhii O. agardhii

Geographical Distribution of Dhb-MC

http://www.sekaichizu.jp/

(Z) and (E) Dhb6 7 D-Glu

H O OC CH 3

CH C H3
N O O O NH CH 2

5 Adda
HN O C H3 H 3C

H3 C NH
CH 3 CH 3 HN O H N O N H C H3 H HN N O CO OH

1 D-Ala
O

H 3C C H3

D-Leu
C H3 C H3

2 L-amino acid

4 L-amino acid

H 2N

[D-Leu1] microcystin LR

microD- stin cy

[D-Leu1]microcystin LR found from Microcystis aeruginosa isolated 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 toxic
cyanobacteria, 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, also

Zooplankton and fish.

As the opposite situation of the European,

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.

Why did we select lysine and malonic acid for the control?
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 (cyanoBacteria, 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.

We examined effects of lysine and malonic acid on Microcystis Blooms using enclosures.
Enclosure
10 m

Sampling Point
10 m

A
1.3-1.5 m 10 20 cm

0.5 m

1m

1m

Lake Sediments

Enclosure A: Control Enclosure B: Lysine treatment Enclosure C: Lysine plus malonic acid treatment

Enclosure A Dianchi Lake Side

Enclosure B Enclosure C

Methods:
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).

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.

The enclosure surfaces on Day 3 after the treatments

A: Control B: lysine C: lysine + malonic acid

Microcystis aeruginosa

10

Enclosure C
Enclosure B
O HO NH2 NH2 HO NH2 O O OH

[ mg/L]

8 6

NH4OH

lysine

2-aminoadipic acid

Degradation

4
2 0 0 1

Enclosure A

7 Days after Treatment

14

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

9.5 9.0 pH 8.5 8.0 7.5 0

Enclosure A

Enclosure B

Enclosure C

14 21 Days after Treatment

28

Fig.2 Changes in pH in the enclosures after the treatments (S. D. < 5%;
*p < 0.05 )

120 100 [mg/L] 80 60

Enclosure A

Enclosure B

40
20 0 0 2

Enclosure C
7 14 21 Days after Treatment

28

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

Enclosure A
24 20 [ 106/L] 16 12 0 Euglena Diatom 0 2 7 14 21 28 Days after Treatment Cyanobacteria Total phytoplankton 24 20 16 12 0

Enclosure B
24 Total phytoplankton 20 16 Cyanobacteria Diatom 12 Euglena

Enclosure C

Total phytoplankton Cyanobacteria Euglena Diatom

Cell

0 0 2 7 14 21 28 0 2 7 14 21 28 Days after Treatment Days after Treatment

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

800 Cladoceran [individuals/L] 700 600 500 400 300 200 Enclosure A Enclosure B

Enclosure C

100
0 0 2 7 14 21 Days after Treatment 28

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

15
12 3 0 0 2

Enclosure A

Total microcystin [mg/L]

Enclosure B

Enclosure C

14 21 Days after Treatment

28

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

water chestnut (Trapa sp.)

C
Microcystis aeruginosa Myriophllum spicatum

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 nontoxic 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 C 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.
Shade (Shallow Clean)

F 5 50 m

anchor

Dry up (of Dam Sediments )

Cyanobacterial (Microcystis) cells are resting on dam sediment at low water temperature (below 10 C) 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 (WL) S2(16m from WL) Water level in the experiment (36m from WL) S1(31m from WL)

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

100 S1 75 % of WCS (bars) 100

S2
50

100

50

25

25

0
0 7 14 21 Day after dry up 30

% of GR (closed circles)

S1

S2

75

UNESCO-CYANONET Committee Member

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)

GAC&HCU

WWC KK
SB SA WRH MDB

T hank y ou for y our attention !