MINISTRY OF EDUCATION AND

VIETNAM ACADEMY OF SCIENCE

TRAINING

AND TECHNOLOGY

GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY

**********************

PHAM THANH NGA

INHIBITORY EFFECT OF EUPATORIUM FORTUNEI
TURCZ. EXTRACTS ON THE GROWTH OF A TOXIC CYANOBACTERIAL SPECIES Microcystis aeruginosa IN
FRESH WATERBODIES

Major: Environmental Engineering
Code: 9.52.03.20

SUMMARY OF DOCTORAL THESIS IN ENVIRONMENTAL
ENGINEERING

Hanoi - 2019


The doctoral thesis was completed at Institute of Environmental Technology (IET),
Graduate University of Science and Technology, Vietnam Academy
of Science and Technology


Among all sorts of microalgae, Microcystis aeruginosa, one of the most common representative species
responsible for the water blooming, can produce hepatotoxins and neurotoxins which may lead to
headache, fever, abdominal pain, nausea, vomiting and even cancer. Therefore, it is of great importance
to inhibit the growth of cyanobacteria, especially M. aeruginosa in eutrophic waters. Basically, there
are three short-term approaches to control harmful algal blooms such as chemical, physical and
biological approaches. Chemical treatments can effectively and rapidly remove algal bloom. However,
some algicidal chemicals can cause secondary pollution of aquatic environment or persistence in the
environment and the inhibitory effects of most chemicals do not selectively target harmful
cyanobacteria; leading to the collapse of aquatic ecosystems. Physical methods like mixing lake water
using an air compressor, pressure devices or ultraviolet irradiation indicate less subsequent secondary
pollution. However, the disadvantages of physical treatments of algal removal are energy intensive and
tend to be low efficiency as well as injury to non-target species. In recent years, biological methods
including using algicidal bacteria have received much more attention as alternatives to chemical agents.
These approaches tend to be environmental friendly and promising methods for controlling toxic
cyanobacteria. However, the efficiency of biological method is influenced by many biotic and abiotic
factors in the environment. For these limitations of the above approaches, the discovery and use of
natural compounds that feature selective toxicity towards phytoplankton communities and are nontoxic
to other aquatic species, have been a significant advance in the management of aquatic ecosystems.
Eupatorium fortunei Turcz, a species of Asteraceae, is a perennial herb used in folk medicine as a
medicinal and has been demonstrated antibacterial activity in various scientific studies. In 2008, Nguyen
Tien Dat and et al carried out the experiments of using plant extracts to inhibit the growth of M.
aeruginosa. The results showed that the extract from E. fortunei indicated the highest inhibitory effect
on the species. This conclusion was confirmed by the publication of Pham Thanh Nga in the following
years. However, these are only preliminary studies investigating the using of the plant extract to control
toxic cyanobacterial bloom.
By wishing to inherit, develop previous research results and solve several reaserch questions
related to the issue, author chose topic: “Inhibitory effect of Eupatorium fortunei Turcz. extracts on
the growth of a toxic cyanobacterial species Microcystis aeruginosa Kützing in fresh waterbodies”
2. RESEARCH PROPOSE OF THE DOCTORAL THESIS
Research to create effective plant extracts from E. fortunei to inhibit growth of Microcystis aeruginosa

6. NEW CONTRIBUTION OF THE DOCTORAL THESIS
Isolation of 02 pure new chemical compounds from Eupatorium fortunei which have not been
published in international scientific journals. Investigation of the biological activity of these compounds to
control M. aeruginosa at the concentrations from 1.0 µg.mL-1 to 50 µg.mL-1. Growth inhibitory effect (IE) was
recorded from 10 to 45% after 72 hours of exposure.
Application of the innovative method to control the growth of toxic microalga (M. aeruginosa) by
using extracts from Eupatorium fortunei Turcz. The experiment was carried out from the laboratory scale in
150- mL flashes with IE of over 90%, then in the 5L aquarium and in the outdoor scale (3 m3) with IE around
of 60 % for evaluating the different efficiency between the theoretical value and practical application. The
ethanol extract proved to be more toxic to M. aeruginosa than to Daphnia magna and Lemna minor.
7. STUCTURE OF THE THESIS
The thesis is organized in the introduction, three chapters and concluding section with 143 pages, 18
tables and 45 figures and graphs. The thesis uses 182 references with more than 40% of the papers published
in the last five years (from 2013 to 2018). Chapter 1 presents an overview about researches related to
eutrophication and the toxic cyanobacterial bloom in aquatic ecosystem and the methods used to control these
problems. Chapter 2 presents research objectives, methods and the design of experiments. Chapter 3 shows the
reaserch results and gives discussion. The chapter 3 will be presented in more detail in the next section.


3

CHAPTER 3. RESULTS AND DISCUSSION
3.1. The process of producing crude extracts, fractions and pure chemical compounds
isolated from E. fortunei Turcz.
Table 3.1. Effeciency of crude extract production in various solvents
Solvent

Gram crude plant extract/100gram
dried materials


Table3.3. Effeciency of isolating 7 chemical compounds from E. fortunei
Compounds
1.
2.
3.
4.
5.
6.
7.

EfD5.1
EfD14.1
EfD1.8
EfD10.1
EfD10.3
EfD4.7
EfD4.8

Mg compound/100 g EtOAc fractions of E.
fortunei.
71.69
20.80
13.34
4.56
3.91
2.61
1.56


4


6

Firuge 3.6.HSQC spectra of EfD4.7
Table3.4.1H NMR and

13

C NMR spectra of EfD4.7 và EfD4.8 compounds

STT

EfD4.7
δH (m, J in Hz)

1
2
3
4
5
6
7
8
9
10

Figure 3.7.HMBC of EfD4.7

6.79 (1H, d, 2.0)
7.20 (1H, d, 7.5)

127.8
131.1
119.0
64.8
149.3
65.8

26.1

5.41 (1H, d, 2.0), 5.20 (1H, d, 2.0)

114.8

Chemical structures of 07 chemical compounds isolated from E. fortunei
1. 7,8,9-trihydroxythymol(EfD4.7)

2. 8,10-didehydro-7,9dihydroxythymol(EfD4.8)

2.

o-Caumaric acid (EfD1.8)


7
3. 8,9,10- Trihydroxythymol
(EfD5.1):

5. 4-(2-hydroxyethyl)benzaldehyde
(EfD10.1):



T6

Time (days)

0.50

B

Control- M.a
E- Eth-500
E- Me-500
E-W-500
CuSO4-5

0.40
0.30
0.20
0.10
0.00

T10

T0

T3

T6

Time (days)

4.00
2.00

Control - M.a
E- Eth-500
E- Me-500
E-W-500
CuSO4-5

6.00

B

4.00
2.00
0.00

0.00
T0

T3

T6

T i me ( d a ys )

T10

T0


T6

5.00
4.00

B

3.00
2.00
1.00

0.20
0.10
0.00

35.00

T3

T6

B

Control- Chlorella
E-Ethanol -50
E-Ethanol-100
E-Ethanol-200
E-Ethanol-500

30.00


T10

10.00

0.00

Cell Density, × 105 TB/mL

T0

Cell Density x 105 TB/mL

0.30

T0

Control- M.a
E-Ethanol 50
E-Ethanol- 100
E-Ethanol-200
E-Ethanol-500

A

Control-Chlorella
E-Ethanol-50
E-Ethanol-100
E-Ethanol- 200
E-Ethanol- 500

growth inhibitory effect at T10 of 88.28% and 69.10%, respectively. The treatment of water crude extract
at 500 μg.mL -1 had inhibitory effect of 52.51% with biomass at T10 of 3.12 ± 0.37 μg.L -1. However, the
treatment at the concentration of 200 μg.mL -1 slightly stimulated growth compared with the control (p

Time (days)

Figure 3.11. Growth of Ch. vulgaris under the
exposure of crude ethanol extract determined by
optical density (A), chlorophyll a content (B) and cell
density (C)


9
The results clearly indicated that ethanol crude extract from E. fortunei at the both 200 and 500 μg.mLconcentration showed effective inhibition on the growth of M. aerguinosa
Table 3.5 shows that the ethanol extracts had selective inhibitory effect between M. auruginosa and
Ch. vulgaris with growth inhibitory values (IE%) on C. vulgaris recorded lower than M. aeruginosa in all three
analytical methods (optical density, chlorophyll a concentration and cell density) (p
E-Ethyl 50
E-Ethyl 100
E-Ethyl 200
E-Ethyl 500

0.50
0.40
0.30

Optical Density (Abs 680 nm)

Optical Density (Abs 680 nm)

3.2.3.

IE (TB)

0.20
0.10

0.60

B

Control - M.a
E-W- 50
E-W- 100
E-W 200
E-W 500



A

Control- M.a
E- Ethyl 50
E- Ethyl 100
E- Ethyl 200
E- Ethyl 500

7.00
6.00
5.00
4.00

Hàm lượng Chlorophyll a , ug/L

Hàm lượng Chlorophyll a , ug/L

Figure 3.12. Growth of M. aeruginosa under the exposure of ethyl acetate (A) and water fractions (B) determined
by optical density

3.00
2.00
1.00
0.00
T0

T3
Time (days)


Figure 3.13. Growth of M. aeruginosa under the exposure of ethyl acetate (A) and water fractions (B)
determined by chlorophyll a content


40.00

40.00

Control-M.a
E-Ethyl -50
E-Ethyl-100
E-Ethyl-200
E-Ethyl -500

35.00
30.00
25.00
20.00

A

Mật độ tế bào x 105Tb/mL

Optical Density x 105 TB/mL

10

15.00
10.00
5.00


Time (days)

T6

T10

Time (days)

Control- Chlorella
E-Ethyl -50
E-Ethyl-100
E-Ethyl-200
E-Ethyl -500

0.40
0.30

Optical Density (Abs 680 nm)

0.50

A

0.20
0.10

B

0.50


T10

Time (days)

Figure 3.15. Growth of Ch.vulgaris under the exposure of ethyl acetate (A) and water fractions (B)
determined by optical density
60.00
50.00
40.00

60.00

A

Control- Chlorella
E- Ethyl -50
E- Ethyl-100
E- Ethyl-200
E- Ethyl -500

Chlorophyll a Concentration ,
ug/L

Chlorophyll a Concentration,
ug/L

Optucal Density, (Abs 680nm)

Figure 3.14. Growth of M. aeruginosa under the exposure of ethyl acetate (A) and water fractions (B)

20.00

10.00

10.00

0.00

0.00
T0

T3

T6

Thời gian (ngày)

T10

T0

T3

T6

Thời gian (ngày)

T10



E-W-100
E-W-200
E-W-500

35.00
30.00
25.00

B

20.00
15.00
10.00
5.00
0.00

T0

T3

T6

T0

T10

Time (days)

T3
Time (days)

(Chla)

(TB)

E-Ethyl 500

93.55

96.16

75.61

76.98

78.40

55.6

E-W-500

58.62

43.46

37.58

32.33

40.16



T48

T72


12

Chlorophyll aConcentration,
µg/mL

3.50

T0

3.00

2.50
2.00
1.50
1.00
0.50
0.00
Control

Cell Density × 105 TB/mL

Figure 18 B. Effect of
plant extracts on the
growth of M.aeruginosa


Table 3.7. Inhibition efficiency
(IE) of extracts from E. fortunei on the growth of M.aeruginosa at the concentrations of 500 µg.mL-1after 72
hours
IE 72h

IE (72h)

IE% (72)

OD

Chla

TB

CuSO4-5

47.4

74.72

35.10

E-Ethanol 500

52.2

67.35



0.25
0.20
0.15
0.10
0.05
0.00
EfD 1.8

EfD 4.8

EfD 4.7

EfD 5.1

EfD 10.1

EfD 10.3

EfD 14.1


Cell Density × 105TB/mL

13
25.0

B
20.0
15.0


Figure 3.20. Transmission electron micrographs (TEM) of Microcystis aeruginosa cells (A) and Ch. vulgaris
(B)
A3

B3

A6

B6

A10

B10


14
C3

C6

C10

Figure 3.21. Transmission electron micrographs (TEM) of M. aeruginosa cells: in the
control (a); incubated with ethanol extract (B), ethyl acetate fraction (B) and water fraction
A3

A6

B3

was fastly increased after 48 hours exposure to the extract. The ethyl acetate fraction was greater toxic to
D.magna than the ethanol extract. At the concentrations of 160 and 120 µg.mL-1 the ethyl acetate fraction killed
D.magna with mortality rate reaching to 100% after 24 and 48 hours, respectively.
Table 3.8. LC50 value of the crude ethanol extract and the ethyl acetate extract fraction after 24 and 48
hours

Mortality rate (%)

Concentration of the ethanol
extract
(µg.mL-1)

Concentration of the ethyl
acetate fraction (µg.mL-1)

24 hours

48 hours

24 hours

48 hours

LC 1

71.4

37.0

7.8


5.4

LC 50

247.8

183.2

47.4

13.6


16
LC 85

431.2

373.4

105.8

43.2

LC 90

491.6

442.0

extract

DO

DO

pH

pH

(T0)

(T48) (T0) (T48)
mg L-

(µg.mL-1)

mg
L-1

0.00

7.77

7.72

7.78

7.42



1

Table 3.10. DO and pH value of D. magna
exposured to the ethyl acetate fraction
from E.fortunei at 0 and after 48 hours.
Concentration
of ethyl
acetate
fraction

DO

DO

pH

pH

(T0)
Mg.
L-1

(T48)
Mg.
L-1

(T0)

(T48


7.70

7.44

6.07

7.57

40.00

7.88

6.88

7.65

7.37

6.83

6.18

6.76

80.00

7.83

6.44


160.00

7.85

7.72

7.29

7.03

500
450
400
350
300
250
200
150
100
50
0

Control-L.minor

A

CuSO4-5
E- Eth-500


500
450
400
350
300
250
200
150
100
50
0

B

E-Ethyl- 500
E-Ethyl- 200
E-Ethyl- 100
E-Ethyl- 50

T0

T1

T2

T3

T4

T5

50.0

40.0

60.0

Control-L.minor
CuSO4-5
E- Eth-500
E-Eth-200

A

30.0
20.0
10.0

Fresh weight (mg)

Fresh Weight (mg)

Figure 3.27. Frond morphological appearance after 5 days of the ethanol extract exposure ethyl acetate
fraction

50.0
40.0
30.0

Control - L.minor
E-Ethyl- 500


18

Pigment Concentration
(mg/gFW)

concentration of 500 µg.mL -1 there was the sighnificant decrease in biomass, of 9,0 ± 1,25 mg, with IE of
77,76%.

A

0.70

Chla

0.60

Chlb

Chl (a+b)

0.50
0.40
0.30
0.20
0.10
0.00

Control - L.minor



E-Ethyl- 200

E-Ethyl- 500

Chlorophyll a Concentration ,
µg/L

Figure 3.29. Pigment concentrations (mg.g-1FW) of L. minor under the treatment of plant extracts
Ethanol crude extract, B. Ethyl acetate fraction
The ethanol extract showed the slight effect on L. minor even at 500 μg.mL-1 with the inhibitory effect of
16 to 25%, whereas ethyl acetate fraction at the concentration of 500 μg.mL -1 proved to be toxic to L.
minor like CuSO4 5 μg.mL-1 with the IE value of 75 to 85% (p
VKL
khác
Green
agla
silic agla
Tảo
lục &
tảo&silic
Phytoplanton
Nhóm
TVN

30.00
25.00

A

20.00
15.00
10.00
5.00
0.00
Control-HK

CuSO4-5

Cell Density × 105 TB/mL

35.00



CuSO4-5

E-Ethanol - 500

E-Ethyl- 500

Figure 3.31. Effect of plant extracts on the growth of phytoplankton in water samples collected from Hoan
Kiem Lake determined by cell density (Laboratory Scale)
T0- the begining (A) and T10- the end (B)

Chlorophyll a,Concentration µg/L

In the control sample, the increase in biomass was observed in all species, especially in Microcystis sps.,
with increasing from (10.91 ± 0.37) x 106 cells.mL-1 at beginning to (21.16 ± 1.27) x106 cells.mL-1 at the last
day of experiment. While biomass of Microcystis sps. in other treatments significantly decreased in comparison
with the control. Cell density of the CuSO4-5 sample was just (11.77 ± 1.24) x 106 cells.mL-1; of the E-Ethanol
500 sample was (13.16 ± 1.12) x106 cells/mL and of the E-Ethyl 500 (11.93 ± 1.14) x106 cells/mL with the IE
values of 44.40; 37.82 và 43.61 %, respectively. However, the ethanol extract showed different inhibitory
effect between Microcsystis spp., green algae, and silic algae indicating lower the IE value, just being of 27.67
%.
3.4.2. Effect of plant extracts on the growth of phytoplankton in water samples collected from Lang
Lake in the laboratory scale.

Control
Ethanol- 500

18.00
16.00



20
20.00

Microcystis
Microcystis
sp sp
VKL khác
Tảo
lục &
tảo silic
Other
cyanobacteria
Nhóm TVN

15.00

A

Green agla & silic agla
10.00
Phytoplanton
5.00
0.00
Control - HL

CuSO4-5

E-Ethanol-500



E-Ethyl-500

Figure 3.33. Effect of plant extracts on the growth of phytoplankton in water samples collected from Lang
Lake determined by cell density (Laboratory scale)
T0- the begining (A) and T10- the end (B)
The IE values of the CuSO4, E-Ethyl 500 and E-Ethanol 500 samples were 58.33; 43.65 và 49.20 %. The
results on Lang Lake’s samples showed that the ethanol extract indicated selective inhibitory effect between
Microcsystis spp.; cyanobacteria (IE from 43.43 to 46.44 %) and green algae; silic algae which indicated lower
the IE value, just of 34.68 % (p>0,05).
Effect of plant extracts on the growth of phytoplankton in water samples collected from Lang
Lake in the outdoor scale.
Chlorophyll a Concentration (µg/L)

3.4.3.

18.00
16.00

Control

CuSO4-5

T1

T3

E- Ethanol- 500

14.00

Other
cyanobacteria
Tảo
lục &
tảo silic
Nhóm TVN
Green agla & silic agla

18.00
16.00
14.00

A

12.00
Phytoplanton

10.00
8.00
6.00
4.00
2.00
0.00

Cell Density × 105TB/mL

Control - HL

CuSO4-5


CuSO4-5

E-Ethanol - 500

Figure 3.35. Effect of plant extracts on the growth of phytoplankton in water samples collected from Lang
Lake determined by cell density (Outdoor scale)
T0- the begining (A) and T10- the end (B)
The ethanol crude extract at the concentration of 500 µg.mL-1 inhibited the growth of Microcystis spp.
with the IE values of 39.92 %. The IE value of phytoplankton was 30.63% while that of other green and silic
algae was just 28.55 %. The results indicated that the extract had selective inhibition effect to toxic
Microcystis spp. more than other species in water samples.


21
3.4.4.

EFFECT OF PLANT EXTRACTS ON ENVIRONMENTAL PARAMETERS

Table.3.11A. Effect of plant extracts on physical parameters in water samples collected from Hoan Kiem Lake (laboratory scale)
Treatment

T (0C)

Control

24.45

CuSO4

24.41

2.17 (1.90 – 2.68)

24.66

0.011(0.010 - 0.012)

17.5 (15.7 - 22.9)

157.5 (113 ÷ 210)

7.03 (6.73 - 7.67)

2.03 (1.14 - 2.50)

24.78

0.011(0.09 - 0.012)

15.3 (14.9 - 16.9)

151.1(107 ÷ 192)

6.72 (6.30 - 6.97)

2.01 (1.16 - 2.34)

(µS/cm)

0.011(0.011 - 0.012)



1.01 (0.96 ÷ 1.15)

0.017 (0.012 - 0.021)

0.127 (0.125 - 0.171)

0.035 (0.019 - 0.033)

1.276 (0.812 - 1.755)

E - Ethanol - 500

1.38 (0.94 ÷ 2.18)

0.028 (0.015 - 0.053)

0.172 (0.150 - 0.272)

0.023 (0.019 - 0.058)

1.827 (1.703 -1.961)

E - Ethyl - 500

1.46 (0.91÷ 1.90)

0.021 (0.025 - 0.056)

0.154 (0.128 - 0.237)

25.21

0.011 (0.011 - 0.012)

22.3 (22.0 - 24.1)

34.6 (47 ÷ 69)

8.82 (7.76 - 9.34)

8.48 (8.43 - 9.05)

CuSO4

25.06

0.011 (0.010 - 0.011)

21.6 (21.1 - 22.0)

33.9 (41÷ 63)

7.61 (6.55 - 8.70)

6.85 (5.9 - 7.37)

E - Ethanol - 500

25.25



(mg/L)

(mg/L)

Control

0.46 (0.36÷ 0.58)

CuSO4

NH4+ (mg/L)

NO2- (mg/L)

Silic (mg/L

0.013 (0.010 - 0.016)

0.096 (0.093 - 0.186)

0.046 (0.013 - 0.063)

2.018 (1.557 - 2.983)

0.38 (0.28÷ 0.43)

0.011 (0.006 - 0.015)

0.99 (0.087 - 0.176)