Nghiên cứu tác dụng ức chế của cao chiết cây mần tưới (eupatorium fortunei turcz ) lên sinh trưởng của vi khuẩn lam độc microcystis aeruginosa kutzing trong các thủy vực nước ngọt tt tiếng anh

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

Supervisors: Prof. Dr. Dang Dinh Kim
Dr. Le Thi Phuong Quynh
Reviewer

1.

Reviewer

2.

Reviewer

3.

This doctoral thesis will be defended at Graduate University of
Science and Technology, Vietnam Academy of Science and Technology at …… on ……
2019

This doctoral thesis can be found at:
Library of the Graduate University of Science and Technology, VAST.


1

INTRODUCTION
1. NECESSITY OF DOCTORAL THESIS
Eutrophication is a widespread problem in aquatic ecosystems around the world due to sewage
and surface run-off. It significantly affects water quality and induces off-flavor problem. Moreover,
cyanobacterial blooms usually break out along with release of cyanotoxins, which cause a series of
adverse effects such as decreasing water quality and biodiversity, and illness in animals and humans.

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

Kützing in the laboratory and outdoor larger scale.
3. TASKS OF THE DOCTORAL THESIS
- Develop the process of producing crude extracts, fractions and pure chemical compounds isolated from E.
fortunei
- Study of inhibitory effect of the crude ethanol extracts from E. fortunei on the growth of M.aeruginosa
and evaluating their ecological safety to non-target aquatic organisms.
- Study of inhibitory effect of the fraction extracts from E. fortunei on the growth of M. aeruginosa and
evaluating their ecological safety to non-target aquatic organisms.
- Study of bioactive properties of chemical compounds isolated from E. fortunei.
- Research on the application of plant extracts to control cyanobacterial bloom in natural water samples (in
the laboratory and outdoor scales)
4. METHODOLOGY OF RESEARCH
The author uses different modern research methods which provide the scientific reliable results and
suitable to Vietnam's conditions. The methods include 1). Methods of plant sample treatment, production of
plant extraction and isolation of pure chemical compounds; 2). Method of identifying the chemical structure
of pure compounds (1H, 13C-NMR, DEPT, HMBC, HR- ESI-MS); 3). Methods of evaluating the growth of
cyanobacterial M. aeruginosa, Ch. vulgaris and phytoplanktons; 4). Method of evaluating the toxicity of plant
extracts to non-target aquatic organisms (Daphnia magna and Lemna minor); 5). Morphology of M.


2
aeruginosa and Ch. vulgaris under the exposure of plant extracts (TEM); 6). Standard methods in water
analysis (physical and chemical parameters).
5. SCIENTIFIC AND PRACTICAL MEANINGs OF THE DOCTORAL THESIS
Water pollution, especially the eutrophication that caused the cyanobacterial bloom including
mainly M. aeruginosa which releases microcystin toxins, has received much attention and research in
recent times. Using plant extracts to control this phenomenon indicates more advantages than other
traditional methods used previously. The results of the doctoral thesis provide a scientific basis of the
feasibility of using plant extracts as a selective inhibitor to the growth of M. aeruginosa in order to control
the toxic cyanobacterial bloom while do not harm to other non-target organisms in aquatic ecosystems.

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

Ethanol

9.17

Methanol

12.75

W (Water)

8.75

Table 3.2. Effeciency of fraction production from crude ethanol extracts of E. fortunei
Fractions

Gram fractions/100 gram crude ethanol
extract (%)

n-hexan

18.97

EtOAc

10.57

W

60.27

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

Figure 3.1. Process of isolating chemical compounds from the ethyl acetate fraction

2 new compounds

Figure 3.2. 7,8,9-trihydroxythymol (EfD4.7)

Figure 3.3. 8,10-didehydro-7,9dihydroxythymol(EfD4.8)


5
EfD4.7 White powders; []D24 = +0,2 (c 0.1, MeOH). The HR-ESI-MS (positive) revealed a peak [M +
Na]+ at m/z 221,0783 (C10H14NaO4). In the 1H NMR spectra of EfD4.7 compound, the presence of aromatic
signalsABX at δH 6,79 (1H, d, J = 2,0 Hz, H-2), 7,20 (1H, d, J = 7,5 Hz, H-5), and 6,81 (1H, dd, J =
7,5, 2,0 Hz, H-6)], one group ethyl at δH 1,58 (3H, s, H-10). 1H NMR (500 MHz, CD3OD) và 13C NMR
(125 MHz, CD3OD) [Table 3.4].

Firuge 3.4.HSQC spectra of EfD4.7

Figure 3.5.HMBC of EfD4.7

Actually, the 1H and 13C-NMR spectra data of EfD4.7 are very similar to that for the 8,9dihydroxythymol compound, except for the appearance of the hydroxymethyl group instead of the methyl
group at C- 7. The data of the HMBC spectra also showed interactions from H-7 (δH 4.52) to C-1, C-2 and C6, from H-9 (δ H 3.76 and 3.65) to C-4, C-8 and C-10, and from H-10 (δH 1.58) to C-4, C-8 and C-9. It can
be concluded that EfD4.7 is 7,8,9-trihydroxythymol, a new compound that was first published.
EfD4.8 is white powder. HR-ESI-MS (positive): m/z181.0864 [M + H]+ (C10H13O2).1H NMR (500 MHz,
CD3OD) và 13C NMR (125 MHz, CD3OD) [Table 3.4]. The 1H và 13C-NMR speactras of EfD4.8 was similar
to that of EfD4.7 compound, excep for the appearance of one methylene group (δC/δH 114,8/5,41 and 5,20)
instead of the methyl group as in the EfD4.7 structure. This is also confirmed by HR-ESI-MS spectra with
chemical formular of C10H13O2. The HMBC speactra was also confirmed the structure of EfD4.8

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)

6.81 (1H, dd, 7.5, 2.0)
4.52 (2H, s)
3.76 (1H, d, 11.0)
3.65 (1H, d, 11.0)
1.58 (3H, s)

EfD4.8
δC

δH (m, J in Hz)
140.1
116.3
156.8
133.5
129.5
120.5
64.9
76.9
72.4

6.82 (1H, d, 2.0)
7.12 (1H, d, 7.5)
6.80 (1H, dd, 7.5, 2.0)
4.55 (2H, s)
4.39 (2H, s)

δC
143.6
115.1
155.8

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):

6. Kaempferol (EfD10.3):

7. 10-Acetoxy-8,9dihydroxythymol (EfD14.1)

0.50

Control - M.a
E- Eth-200
E- Me-200
E-W-200
CuSO4-1

0.40
0.30

A
Optical Density, (λ= 680 nm)

Optical Density (λ= 680 nm)

3.2. Inhibitory effect of plant extracts and pure chemical compounds from E. fortunei on the growth
of M. aeruginosa and Ch. vulgaris
3.2.1. Inhibitory effect of different crude extracts from E. fortunei on the growth of M. aeruginosa

0.20
0.10
0.00
T0

T3

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)

T10

Figure 3.8. Growth of M. aeruginosa under the exposure of crude ethanol extract at the concentration of 200 (A)
and 500 µg.mL- (B) determined by optical density
The ethanol extract of Eupatorium fortunei Turcz at 500 µg/mL with inhibition efficiency of 91.5 %
showed higher potential ability to inhibit the growth of M.aeruginosa than those of the water and methanol
extracts with inhibition efficiencyof 61.7 % and 78.5%, respectively. CuSO4 5 µg/mL significantly inhibited
growth of M. aeruginosa with the IE of 81.7%.

6.00

8.00

A

Control- M.a
E- Eth-200
E- Me-200
E-W-200
CuSO4-1

Chlorophyll a
Concentration , µg/L

Chlorophyll a
Concentration, µg/L

8.00

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

T3

T6

T10

Time (days)

Figure 3.9.Growth of M. aeruginosa under the exposure of crude ethanol extract at the concentration of 200 (A)
and 500 µg.mL- (B) determined by chlorophyll a content


8
In the treatment samples exposed to ethanol and methanol extracts at the concentration of 500

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

Chlorophyll a Concentration ,
àg/L

T0

T3

A

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

25.00
20.00

T10

15.00
10.00
5.00
0.00

0.00

40.00

T3

T6

C

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

30.00
20.00

T0

T10

10.00

0.00

Cell Density, ì 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

0.40

T10

7.00
6.00

Optical Density (λ= 680 nm)

0.50

0.50
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00

Chlorophyll a Concentration,
ug/L

Optical Density (λ= 680 nm)

μg.mL-1 cyanobacteria biomass were lower than that of the control at T3, T6 and T10 (p <0.05) with

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
<0.05).Applications of extraction solvents may have a significant impact on the yield of phenolic compounds
from plant materials. The extract obtained by 96 % ethanol had highest total antioxidant activity as well as
phenolic content compared with those of the methanol and water solvents. It was noted that phenolic
compounds have demonstrated anti-algal inhibitory effect. It may be the reason why the ethanol extract had
shown the most effective cyanobacteria growth inhibition in our study. However, plant extracts at lower
concentration sometimes slightlystimulated the growth of M. aeruginosa.
3.2.2. Inhibitory effect of ethanol crude extracts from E. fortunei on the growth of M. aeruginosa
and Ch. vulgaris

35.00

T3

T6

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

30.00
25.00
20.00

T10

C

15.00
10.00
5.00

0.00
T0

T3

T6

T10

Time (days)

Figure3.10. Growth of M. aeruginosa under the
exposure of crude ethanol extract determined by
optical density (A), chlorophyll a content (B) and cell
density (C)

T0

T3

T6

T10

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 <0.05).
Table.3.5. Inhibition efficiency (IE) of ethanol crude extract from E. fortunei on the growth of M.aeruginosa
and Ch.vulgaris at the concentrations of 200 and 500 µg.mL-1
1

M. aeruginosa

Ch. vulgaris

Concentrations
IE % (OD)

IE % (Chla)

56,10
87,80

61,32

90,13

200 µg/mL
500 µg/mL

IE % (OD)

IE % (Chla)

32,89
70,59

35,89
66,42

51,72
68,6

IE (%) (TB)
41.73
55,61

Inhibitory effect of ethyl acetate and water fractions from E. fortunei ethanol extract on the
growth of M. aeruginosa and Ch. vulgaris

0.60

A

Control- M.a

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

0.50
0.40

0.30
0.20
0.10
0.00

0.00
T0

T3

Time (days)

T6

T0

T10

T3

Time (days)

T6

T10

8.00

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)

T6

T10

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

8.00
7.00
6.00
5.00
4.00
3.00
2.00
1.00
0.00

T0

T3
T6
Time (days)

B

T10

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

0.00
T0

T3

T6

Control- M.a
E-W-50
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

T10

T0

T3

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

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

0.40
0.30

0.20
0.10
0.00

0.00

T0

T3
Time (days)

T6

T0

T10

T3

T6

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)

determined by cell density
The results shown in Figures 3.13 and 3.14 by optical density and chlorophyll a methods both reflect
the same trend. It was clearly demonstrated that the ethyl acetate fraction inhibited more strongly on the
growth of M. auruginosa compared to the water fraction after 10 days of experiment. At the concentrations
of 200 and 500 μg.mL-1, the water fractionation was slightly inhibited M. aeruginosa at the last day of
experiment, measured with the optical values of 0.354 ± 0.015 and 0.199 ± 0.016; with chlorophyll a contents
of 5.76 ± 0.38 and 3.96 ± 0.223 μg / L, respectively. The inhibitory effect on M. aeruginosa growth at 200
μg.mL-1 was 18-20% and 45-60% at 500 μg.mL-1.
In term of ethyl acetate fraction, it indicated high toxicity to M. aeruginosa at the concentrations of
200 and 500 μg.mL-1 after 10 days of exposure. The optical values were 0.102 ± 0.03 and 0.031 ±0.001 và
chlorophyll a contents were 1.78± 0.018 và 0.27 ± 0.019 µg/L, respectively. The inhibitory effect on M.
aeruginosa growth was over 90% at the concentration of 500 µg/mL.

Control- Chlorella
E- W-50
E- W-100
E- W-200

50.00

40.00

B

30.00

30.00

20.00

20.00

10.00

10.00

0.00

0.00
T0

T3

T6

Thời gian (ngày)

T10

T0

T3

T6

Thời gian (ngày)

T10

11

Figure 3.16. Growth of Ch. vulgaris under the exposure of ethyl acetate (A) and water fractions
40.00
35.00
30.00
25.00
20.00
15.00
10.00
5.00
0.00

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

40.00

A

Cell Density x 105Tb/mL

Cell Density x 105 TB/mL

(B) determined by chlorophyll a content
Control- Chlorella
E-W-50

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)

T6

T10

Figure 3.17. Growth of Ch. vulgaris under the exposure of ethyl acetate (A) and water fractions (B)
determined by cell density
Compared with the harmful effect of the extracts on the growth of M. aeruginosa, the extract showed
less toxic to Ch. vulgaris. The sample exposed to water fraction from E. fortunei at 500 μg / mL after 10 days
had the slight lower optical values (0.260 ± 0.013) than that of the control (OD of 0.391 ± 0.0228). The
inhibitory effeciency (IE) of 32.33% by optical density and 40.16% by chlorophyll a concentration. The ethyl
acetate faction showed stronger toxicity than the water fraction to Ch. vulgaris with the IE values of 76.98%
and 78.40%, respectively.
Bảng 3.6. Inhibition efficiency (IE) of ethyl acetate and water fractions from E. fortunei on the growth of
M.aeruginosa at the concentrations of 500 µg.mL-1 after 10 days
M. aeruginosa
Ch. vulgaris
Treatment

IE%
IE % (OD) IE % (Chla)

IE %

IE %

(TB)

IE % (OD)

(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

31.34

The using of plant extracts to control M.aeruginosa bloom after 72 hours of treatment
Control. M.a
Figure 3.18. Effect of
0.28
CuSO4-5
plant extracts on the
E-Ethanol 500
0.24
growth of M.aeruginosa
E-Ethyl 500
after 72 hours treatment
0.20
A. by optical density
0.16

Optical Density (Abs 680nm )

3.2.4.

0.12
0.08
0.04
0.00
T0

T24

Time (hours)

T48

T72


12

Chlorophyll aConcentration,
àg/mL

3.50

T0

3.00

2.50
2.00
1.50
1.00
0.50
0.00
Control

Cell Density ì 105 TB/mL

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

after 72 hours treatment
by Chlorophyll a
concentration

T72

CuSO4-5

25.00

E-Ethanol 500

T0

E-Ethyl-500

T72

Figure 18 C. Effect of
plant extracts on the
growth of M.aeruginosa
after 72 hours treatment
by cell density

20.00
15.00
10.00
5.00
0.00
Control - Ma CuSO4-5 Ethanol-500 Ethyl- 500

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

34.77

E-Ethyl-500

62.8

79.60

37.42

Treatment

Toxicity of chemical compounds isolated from E. fortunei to M. aeruginosa
Optical Density (Abs 680 nm)

3.2.5.

0.30

0 µg/mL

1 µg/mL

10 µg/mL

20 µg/mL

50 µg/mL

A

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

10.0
5.0
0.0
EfD1.8

EfD 4.8

EfD 4.7

EfD 5.1

EfD 10.1

EfD 10.3

EfD 14.1

Figure 3.19. The growth of M. aeruginosa treated by chemical compounds isolated from E. fortunei
after 72 hours by optical density (A) and by cell density (B)
EfD 5.1 showed the highest inhibitory effect to M. aeruginosa by both analyzed methods with the
IE values of 45.6 và 49.0 %, following by 10-acetoxy-8,9-dihydroxythymol (EfD 14.1) and 4-(2hydroxyethyl) benzaldehyde (EfD 10.1); their IE values were 43.1 and 41.6 %; 43.0 % and 39.6 %,
respectively. 8,10-didehydro-7,9-dihydroxytymol (EfD 4.8) had the lower IE values; 39,1% và
41,1%while 7,8,9-trihydroxythymol (EfD 4.7) và aglycone kaempferol (EfD 10.3) slightly inhibited the
growth of M.aeruginosa with IE of 20-25 % at the same concentration after the 72 - hour experiment.
3.2.6.

Effect of the extracts on the ultrastructure of M.aeruginosa và Ch.vulgaris
B

A

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

B6

C3

C6

A10

B10

C10

Figure 3.22. Transmission electron micrographs (TEM) of M. aeruginosa cells: in the control (a); incubated
with ethanol extract (B), ethyl acetate fraction (B) and water fraction (C) at 500 µg.mL-1 after 3 days (3), 6
days (6), and 10 days (10).
3.3.
Safety evaluation of plant extracts to non-target aquatic organisms.
3.3.1. Acute toxicity of the ethanol extract and ethyl acetate fraction from E.fortunei on D.magna


15

Figure 23. Acute toxicity of the ethanol extract from E. fortunei on D. magna after 24 (a) and 48(b) hours

Figure 24. Acute toxicity of the ethyl acetate fraction from E. fortunei on D. magna after 24 (a) and 48 (b)
hours
After 24 hours of ethanol extract's exposure, the mortality percentage of D. magna fluctuated from 0%
(for the control not been exposed to the extract) to 85% (for the sample with adding the extract at 360 µg mL1
) and reached to 100% (for the sample under the treatment of 400 µg mL-1). The mortality rate of D.magna

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

1.8

LC 5

102.8

59.2

13.2

3.2

LC 10

125.0

76.0

17.6

4.4

LC 15

142.4

90.0

21.2

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

128.0

42.4

LC 95

596.8

567.2

169.6

58.6

LC 99

859.2

885.8

287.8

107.4

Table 3.9. DO and pH value of D. magna
exposured to the ethanol extract from E.
fortunei at 0 and after 48 hours.
Concentration
of the ethanol

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

100.00

7.76

7.52

6.87

7.54

200.00

7.82

7.40

6.57

240.00

7.85

7.57

280.00

7.92

320.00
360.00

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

)

(µg.mL-1)
0.00

7.77

7.42

7.77

7.42

10.00

7.87

7.51

7.78

7.49

7.56

20.00

7.85

7.44

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

7.52

7.17

7.86

6.72

6.17

6.55

120.00

7.86

6.92

7.44

7.15

7.86

7.34

6.15

7.14

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

Frond number

Frond Number

There was no significant change in the DO and pH values during the 48 hours of experiment. The DO
and pH of the samples exposed to ethanol crude extract at the concentrations of 0 ÷ 360 mg L-1 fluctuated from
6.83 to 7.92 mg L-1 and from 6.15 to 7.78, respectively, and those exposed to ethyl acetate fraction at the
concentrations of 0 ÷ 160 mg L-1 were 6.44 ÷ 7.88 mg L-1and 7.03 ÷ 7.77, respectively. They were still good
conditions for D. magna growth. D. magna shows good survival, such as 85% survival at the DO of 1.8 mg
L-1and over 90 % at 2.7; 3.7 and 7.6 mg L-1.
3.3.2. Toxicity of the ethanol extract and ethyl acetate fraction from E.fortunei to Lemna minor

E-Eth-200

T0

T1

T2

Time (days)

T3

T4

T5

Control -L.minor

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

Time (days)

Figure 3.25. Influence of E. fortunei extract on the number of L. minor fronds.
A. Ethanol crude extract, B. Ethyl acetate fraction
The ethanol extract at the concentrations 200 and 500 g.mL-1 showed little inhibiting effect on L.
minor in this experiment. However, in L. minor sample exposed to the Ethyl acetate fraction new fronds
developed only in the first – second days and after that they died.


17

A. Control
B. CuSO4

C. E-Ethanol 200

D. E-Ethanol 500
Figure 3.26. Frond morphological appearance after 5 days of the ethanol extract exposure
Control- L.minor

E-Ethyl 500

E-Ethyl 200

E-Ethyl 100

E-Ethyl 50

60.0

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

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

B

20.0
10.0
0.0

0.0

T0

T5

T0

T5

Figure 3.28. Fresh weight (mg) of the duckweed at the beginning (T0) and the end (T5) of the experiment
A. Ethanol crude extract, B. Ethyl acetate fraction
Under the exposure of ethanol crude extract, L.minor still increased biomass through the
experiment. On the last day, the biomass of L. minor in the control and treatments at 200 and 500 g.mL-1 was
about 47.6, 42.5 and 35.6 mg, respectively, which was 2.5 – 3.0 times higher than the one at the beginning
day (15.0±0.1 mg). Fresh weight of the E-Eth200, E-Eth500 and CuSO4-5 samples were 42,5 ±2,08; 35,6
±2,69 và 6,20 ±0,41mg, respectively with the inhibition efficiency of 10,63; 25,18 và 86,93%,
respectively. In term of ethyl acetate fraction from E. fortune, the IE was reported from 5,83 to 10,87%
at the concentration from 50 to 200 µg.mL-1. However, when L. minor exposed to the extract at the higher

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

Pigment Concentration
(mg/gFW)

0.70

CuSO4-5

Chla

E- Eth-500

Chlb

B

E-Eth-200

Chla + b

0.60
0.50
0.40
0.30
0.20
0.10
0.00

Control- L.minor E-Ethyl- 50

E-Ethyl- 100

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 <0.05).
3.4. Application of plant extracts to control cyanobacteria bloom in natural water samples (in the
laboratory and outdoor scales)
3.4.1. Effect of plant extracts on the growth of phytoplankton in water samples collected from Hoan
Kiem Lake in the laboratory scale.
Control - HK
E-Ethanol-500

40.00

CuSO4-5
E-Ethyl-500

35.00
30.00
25.00
20.00
15.00

10.00
5.00

0.00
T0

T3
Time (days)

T6

T10

Figure 3.30. Effect of plant extracts on the growth of phytoplankton in water samples collected from Hoan
Kiem Lake determined by chlorophyll a content (Laboratory scale)
The IE values determined by chlorophyll a concentration was the highest to the E-Ethyl- 500
treatment, 49,91%, following by CuSO4-5 sample (IE of 44,90 %) and the E-Ethanol 500 sample (IE of
34,70 %).


Cell Density x 105 TB/mL

19
35.00

Microcystis
Microcystis
spsp
Other
cyanobacteria

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

E-Ethanol-500

E-Ethyl-500

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

30.00
25.00
20.00
15.00

B

Phytoplanton

10.00
5.00

0.00
Control - HK

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

CuSO4-5
Ethyl - 500

14.00
12.00
10.00

8.00
6.00
4.00
2.00
0.00
T0

T3

T6

T10

Time (days)

Figure 3.32. Effect of plant extracts on the growth of phytoplankton in water samples collected from Lang
Lake determined by chlorophyll a content (Laboratory scale)
The IE values determined by chlorophyll a concentration was the highest to the E -Ethyl- 500
treatment, 58.83 %, following by CuSO 4-5 sample (IE of 54.60 %) and the E-Ethanol 500 sample (IE of
48.42 %).


Cell Density × 105 TB/mL

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

Cell Density ×105 TB/mL

20.00

E-Ethyl-500

Microcystis sp
Microcystis sp
VKL khác
Tảo lục & tảo silic
OtherTVN
cyanobacteria
Nhóm

15.00

B

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

12.00
10.00
8.00
6.00
4.00
2.00
0.00
T0

T6

T10

Time (days)
Figure 3.34. Effect of plant extracts on the growth of phytoplankton in water samples collected from Lang
Lake determined by chlorophyll a content (Outdoor scale)
The IE values determined by chlorophyll a concentration was the highest to the CuSO 4-5 sample
(IE of 51.90 %) and the E-Ethanol 500 sample (IE of 48.39 %).


21

Cell Density x 105 TB/mL

20.00

Microcystis
spsp
Microcystis
VKL khác

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

20.00
18.00
16.00
14.00
12.00
10.00
8.00
6.00
4.00
2.00
0.00

E-Ethanol - 500
Microcystis sp
Microcystis sp
VKL khác
Tảo
lục &
tảo silic
Other
cyanobacteria
Nhóm TVN
Green agla & silic agla

B

Phytoplanton

Control - HL

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

E - Ethanol - 500
E - Ethyl - 500

Conductivity

Salty

Turbidity (NTU)

pH

DO (mg/L)

12.7 (11.5 - 14.6)

46.7 (43 ÷ 57)

10.46 (10.08 - 10.75)

2.36 (1.89 - 2.71)

0.011(0.011 - 0.012)

12.6 (11.2 - 14.4)

44.4 (39 ÷ 48)

10.23 (10.06 - 10.53)

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)

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

Total phosphorus

Total phosphate

(mg/L)

(mg/L)

NH4+ (mg/L)

NO2- (mg/L)

Silic (mg/L

Control

1.13 (0.88 ÷ 1.26)

0.019 (0.015 - 0.038)

0.136 (0.129 - 0.235)

0.026 (0.018 - 0.035)

1.603 (1.296 - 2.404)

CuSO4

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)

0.020 (0.018 - 0.052)

1.792 (1.779 - 1.957)


22
Table.3.12A. Effect of plant extracts on physical parameters in water samples collected from Lang Lake (laboratory scale)
Treatment

Temperature

Conductivity

Salty

0

( C)

(µS/cm)

Turbidity (NTU)

pH

DO (mg/L)

Control

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

0.014 (0.014 - 0.015)

28 (27.9 - 28.3)

118.4 (106 ÷ 204)

6.67 (5.61 - 7.56)

4.74 (3.91 - 6.67)

E - Ethyl - 500

25.37

0.012 (0.011 - 0.012)

23.1 (22.7 - 23.3)

115.6 (104 ÷ 207)

6.63 (6.30 - 6.87)

4.56 (3.23 - 6.01)

Table.3.12B. Effect of plant extracts on chemical parameters in water samples collected from Hoan Kiem Lake (laboratory scale)
Total phosphorus

Total phosphate

Treatment

(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)

0.035 (0.011 - 0.051)

2.02 (1.735 - 2.399)

E - Ethanol - 500

0.52 (0.48 ÷ 0.84)

0.015 (0.013 - 0.025)

0.112 (0.097 - 0.189)

0.037 (0.016 - 0.084)

2.404 (1.990 - 2.343)

E - Ethyl - 500

0.49 (0.42 ÷ 0.77)

0.016 (0.012 - 0.029)

0.105 (0.090 – 0.167)

0.032 (0.258 - 0.0750)

1.945 .465 -1.998)