Summary of doctoral thesis in Environmental engineering: Inhibitory effect of Eupatorium fortunei Turcz. extracts on the growth of a toxic cyanobacterial species Microcystis aeruginosa Kutzing in fresh waterbodies

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Develop the process of producing crude extracts, fractions and pure chemical compounds isolated from E. fortunei; tudy 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.

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Nội dung Text: Summary of doctoral thesis in Environmental engineering: Inhibitory effect of Eupatorium fortunei Turcz. extracts on the growth of a toxic cyanobacterial species Microcystis aeruginosa Kutzing in fresh waterbodies

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 Mg compound/100 g EtOAc fractions of E. Compounds fortunei. 1. EfD5.1 71.69 2. EfD14.1 20.80 3. EfD1.8 13.34 4. EfD10.1 4.56 5. EfD10.3 3.91 6. EfD4.7 2.61 7. EfD4.8 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-7,9- dihydroxythymol(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 8,9- dihydroxythymol 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 C- 6, 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 Figure 3.7.HMBC of EfD4.7 Table3.4.1H NMR and 13 C NMR spectra of EfD4.7 và EfD4.8 compounds STT EfD4.7 EfD4.8 δH (m, J in Hz) δC δH (m, J in Hz) δC 1 140.1 - 143.6 2 6.79 (1H, d, 2.0) 116.3 6.82 (1H, d, 2.0) 115.1 3 - 156.8 - 155.8 4 - 133.5 - 127.8 5 7.20 (1H, d, 7.5) 129.5 7.12 (1H, d, 7.5) 131.1 6 6.81 (1H, dd, 7.5, 2.0) 120.5 6.80 (1H, dd, 7.5, 2.0) 119.0 7 4.52 (2H, s) 64.9 4.55 (2H, s) 64.8 8 - 76.9 - 149.3 9 3.76 (1H, d, 11.0) 72.4 4.39 (2H, s) 65.8 3.65 (1H, d, 11.0) 10 1.58 (3H, s) 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-7,9- 2. o-Caumaric acid (EfD1.8) dihydroxythymol(EfD4.8)
7 3. 8,9,10- Trihydroxythymol 5. 4-(2-hydroxyethyl)benzaldehyde 6. Kaempferol (EfD10.3): (EfD5.1): (EfD10.1): 7. 10-Acetoxy-8,9- dihydroxythymol (EfD14.1) 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 0.50 A Control - M.a B Optical Density (λ= 680 nm) 0.50 Control- M.a Optical Density, (λ= 680 nm) E- Eth-200 E- Eth-500 0.40 E- Me-200 0.40 E- Me-500 E-W-200 E-W-500 0.30 CuSO4-1 CuSO4-5 0.30 0.20 0.20 0.10 0.10 0.00 0.00 T0 T3 T6 T10 T0 T3 T6 T10 Time (days) Time (days) 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%. 8.00 Control- M.a A 8.00 Control - M.a B Concentration , µg/L E- Eth-200 E- Eth-500 Concentration, µg/L E- Me-500 Chlorophyll a E- Me-200 6.00 6.00 Chlorophyll a E-W-200 E-W-500 CuSO4-1 CuSO4-5 4.00 4.00 2.00 2.00 0.00 0.00 T0 T3 T6 T10 T0 T3 T6 T10 T i me ( d a ys ) 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 μg.mL-1 cyanobacteria biomass were lower than that of the control at T3, T6 and T10 (p
9 The results clearly indicated that ethanol crude extract from E. fortunei at the both 200 and 500 μg.mL- 1 concentration 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
10 40.00 40.00 Optical Density x 105 TB/mL Control-M.a Control- M.a 35.00 35.00 E-W-50 B E-Ethyl -50 A Mật độ tế bào x 105Tb/mL 30.00 E-Ethyl-100 30.00 E-W-100 E-Ethyl-200 E-W-200 25.00 25.00 E-W-500 20.00 E-Ethyl -500 20.00 15.00 15.00 10.00 10.00 5.00 5.00 0.00 0.00 T0 T3 T6 T10 T0 T3 T6 T10 Time (days) Time (days) 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. 0.50 B Optical Density (Abs 680 nm) 0.50 Control- Chlorella A Control- Chlorella Optucal Density, (Abs 680nm) E-Ethyl -50 E-W-50 0.40 0.40 E-Ethyl-100 E-W-100 E-Ethyl-200 E-W-200 0.30 E-Ethyl -500 0.30 E-W-500 0.20 0.20 0.10 0.10 0.00 0.00 T0 T3 T6 T10 T0 T3 T6 T10 Time (days) Time (days) Figure 3.15. Growth of Ch.vulgaris under the exposure of ethyl acetate (A) and water fractions (B) determined by optical density Chlorophyll a Concentration, 60.00 A 60.00 Chlorophyll a Concentration , Control- Chlorella Control- Chlorella B 50.00 E- Ethyl -50 50.00 E- W-50 E- Ethyl-100 E- W-100 40.00 E- Ethyl-200 40.00 E- W-200 ug/L E- Ethyl -500 ug/L 30.00 30.00 20.00 20.00 10.00 10.00 0.00 0.00 T0 T3 T6 T10 T0 T3 T6 T10 Thời gian (ngày) Thời gian (ngày)
11 Figure 3.16. Growth of Ch. vulgaris under the exposure of ethyl acetate (A) and water fractions (B) determined by chlorophyll a content 40.00 40.00 Control- Chlorella A Control- Chlorella Cell Density x 105 TB/mL Cell Density x 105Tb/mL E-Ethyl -50 35.00 E-W-50 B 35.00 E-Ethyl-100 30.00 E-W-100 30.00 E-W-200 E-Ethyl-200 25.00 25.00 E-Ethyl -500 E-W-500 20.00 20.00 15.00 15.00 10.00 10.00 5.00 5.00 0.00 0.00 T0 T3 T6 T10 T0 T3 T6 T10 Time (days) Time (days) 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 % IE % IE % (OD) IE % (Chla) (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 3.2.4. The using of plant extracts to control M.aeruginosa bloom after 72 hours of treatment Control. M.a Figure 3.18. Effect of Optical Density (Abs 680nm ) 0.28 CuSO4-5 plant extracts on the 0.24 E-Ethanol 500 growth of M.aeruginosa E-Ethyl 500 0.20 after 72 hours treatment 0.16 A. by optical density 0.12 0.08 0.04 0.00 T0 T24 T48 T72 Time (hours)
12 3.50 Figure 18 B. Effect of T0 T72 plant extracts on the Chlorophyll aConcentration, 3.00 growth of M.aeruginosa 2.50 after 72 hours treatment 2.00 by Chlorophyll a µg/mL 1.50 concentration 1.00 0.50 0.00 Control CuSO4-5 E-Ethanol 500 E-Ethyl-500 Cell Density × 105 TB/mL 25.00 T0 T72 Figure 18 C. Effect of 20.00 plant extracts on the growth of M.aeruginosa 15.00 after 72 hours treatment by cell density 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) Treatment 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 3.2.5. Toxicity of chemical compounds isolated from E. fortunei to M. aeruginosa 0.30 0 µg/mL 1 µg/mL 10 µg/mL 20 µg/mL 50 µg/mL Optical Density (Abs 680 nm) 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
13 25.0 Cell Density × 105TB/mL 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-(2- hydroxyethyl) 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 A B Figure 3.20. Transmission electron micrographs (TEM) of Microcystis aeruginosa cells (A) and Ch. vulgaris (B) A3 A6 A10 B3 B6 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 A10 B3 B6 B10 C3 C6 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 mL- 1 ) 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 Concentration of the ethanol Concentration of the ethyl Mortality rate (%) extract (µg.mL-1) 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 Table 3.10. DO and pH value of D. magna exposured to the ethanol extract from E. exposured to the ethyl acetate fraction fortunei at 0 and after 48 hours. from E.fortunei at 0 and after 48 hours. Concentration DO DO pH pH Concentration DO DO pH pH of the ethanol (T0) (T48) (T0) (T48) of ethyl (T0) (T48) (T0) (T48 extract acetate Mg. Mg. ) mg mg L- fraction L-1 L-1 (µg.mL-1) L-1 1 (µg.mL-1) 0.00 7.77 7.72 7.78 7.42 0.00 7.77 7.42 7.77 7.42 100.00 7.76 7.52 6.87 7.54 10.00 7.87 7.51 7.78 7.49 200.00 7.82 7.40 6.57 7.56 20.00 7.85 7.44 7.70 7.44 240.00 7.85 7.57 6.07 7.57 40.00 7.88 6.88 7.65 7.37 280.00 7.92 6.83 6.18 6.76 80.00 7.83 6.44 7.52 7.17 320.00 7.86 6.72 6.17 6.55 120.00 7.86 6.92 7.44 7.15 360.00 7.86 7.34 6.15 7.14 160.00 7.85 7.72 7.29 7.03 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 500 Control-L.minor A 500 Control -L.minor 450 CuSO4-5 B 450 E-Ethyl- 500 400 Frond Number E- Eth-500 400 E-Ethyl- 200 350 Frond number 300 E-Eth-200 350 E-Ethyl- 100 250 300 E-Ethyl- 50 200 250 200 150 150 100 100 50 50 0 T0 T1 T2 T3 T4 T5 0 T0 T1 T2 T3 T4 T5 Time (days) 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 Figure 3.27. Frond morphological appearance after 5 days of the ethanol extract exposure ethyl acetate fraction 60.0 60.0 Control-L.minor Control - L.minor B A Fresh weight (mg) Fresh Weight (mg) CuSO4-5 50.0 E-Ethyl- 500 50.0 E-Ethyl- 200 E- Eth-500 40.0 E-Ethyl- 100 40.0 E-Eth-200 E-Ethyl- 50 30.0 30.0 20.0 20.0 10.0 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 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 Chlb Chl (a+b) Pigment Concentration 0.60 (mg/gFW) 0.50 0.40 0.30 0.20 0.10 0.00 Control - L.minor CuSO4-5 E- Eth-500 E-Eth-200 B 0.70 Chla Chlb Chla + b 0.60 Pigment Concentration 0.50 (mg/gFW) 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 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