Inhibitory effect of eupatorium fortunei turcz. extracts on the growth of a toxic cyanobacterial species microcystis aeruginosa in fresh waterbodies

.50 T0 T3 T6 T1 0 O p ti c a l D e n si ty (λ = 6 8 0 n m ) T i me ( d a y s ) Control - M.a E- Eth-200 E- Me-200 E-W-200 CuSO4-1 0.00 0.10 0.20 0.30 0.40 0.50 T0 T3 T6 T10 O p ti c a l D e n si ty , (λ = 6 8 0 n m ) Time (days) Control- M.a E- Eth-500 E- Me-500 E-W-500 CuSO4-5 0.00 2.00 4.00 6.00 8.00 T0 T3 T6 T1 0 C h lo ro p h y ll a C o n ce n tr at io n , µ g /L Time (days) Control- M.a E- Eth-200 E- Me-200 E-W-200 CuSO4-1 0.00 2.00 4.00 6.00 8.00 T0 T3 T6 T10 C h lo ro p h y ll a C o n ce n tr at io n , µ g /L Time (days) Control - M.a E- Eth-500 E- Me-500 E-W-500 CuSO4-5 B A A B 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 <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 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) 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) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 T0 T3 T6 T10 O p ti ca l D en si ty (λ = 6 8 0 n m ) Control- M.a E-Ethanol 50 E-Ethanol- 100 E-Ethanol-200 E-Ethanol-500 0.00 0.10 0.20 0.30 0.40 0.50 T0 T3 T6 T10 O p ti ca l D en si ty (λ = 6 8 0 n m ) Control-Chlorella E-Ethanol-50 E-Ethanol-100 E-Ethanol- 200 E-Ethanol- 500 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 T0 T3 T6 T10 C h lo ro p h y ll a C o n ce n tr at io n , µ g /L Control- M.a E-Ethanol 50 E-Ethanol- 100 E-Ethanol-200 E-Ethanol-500 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 T0 T3 T6 T10 C h lo ro p h y ll a C o n ce n tr at io n , u g /L Control- Chlorella E-Ethanol -50 E-Ethanol-100 E-Ethanol-200 E-Ethanol-500 0.00 10.00 20.00 30.00 40.00 T0 T3 T6 T10 C el l D en si ty x 1 0 5 T B /m L Time (days) Control- M.a E-Ethanol-50 E-Ethanol-100 E-Ethanol-200 E-Ethanol-500 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 T0 T3 T6 T10 C el l D en si ty , × 1 0 5 T B /m L Time (days) Control- Chlorella E-Ethanol -50 E-Ethanol-100 E-Ethanol-200 E-Ethanol-500 A B C A B C 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 <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 Concentrations M. aeruginosa Ch. vulgaris IE % (OD) IE % (Chla) IE (TB) IE % (OD) IE % (Chla) IE (%) (TB) 200 µg/mL 56,10 61,32 51,72 32,89 35,89 41.73 500 µg/mL 87,80 90,13 68,6 70,59 66,42 55,61 3.2.3. Inhibitory effect of ethyl acetate and water fractions from E. fortunei ethanol extract on the growth of M. aeruginosa and Ch. vulgaris Figure 3.12. Growth of M. aeruginosa under the exposure of ethyl acetate (A) and water fractions (B) determined by optical density Figure 3.13. Growth of M. aeruginosa under the exposure of ethyl acetate (A) and water fractions (B) determined by chlorophyll a content 0.00 0.10 0.20 0.30 0.40 0.50 0.60 T0 T3 T6 T10 O p ti ca l D en si ty (A b s 6 8 0 n m ) Time (days) Control- M.a E-Ethyl 50 E-Ethyl 100 E-Ethyl 200 E-Ethyl 500 0.00 0.10 0.20 0.30 0.40 0.50 0.60 T0 T3 T6 T10 O p ti ca l D en si ty ( A b s 6 8 0 n m ) Time (days) Control - M.a E-W- 50 E-W- 100 E-W 200 E-W 500 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 T0 T3 T6 T10 H àm l ư ợ n g C h lo ro p h y ll a , u g /L Time (days) Control- M.a E- Ethyl 50 E- Ethyl 100 E- Ethyl 200 E- Ethyl 500 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 T0 T3 T6 T10 H àm l ư ợ n g C h lo ro p h y ll a , u g /L Time (days) Control - M.a E- W 50 E- W 100 E- W 200 E- W 500 A B A B 10 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. Figure 3.15. Growth of Ch.vulgaris under the exposure of ethyl acetate (A) and water fractions (B) determined by optical density 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 T0 T3 T6 T10 O p ti ca l D en si ty x 1 0 5 T B /m L Time (days) Control-M.a E-Ethyl -50 E-Ethyl-100 E-Ethyl-200 E-Ethyl -500 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 T0 T3 T6 T10 M ật đ ộ t ế b ào x 1 0 5 T b /m L Time (days) Control- M.a E-W-50 E-W-100 E-W-200 E-W-500 0.00 0.10 0.20 0.30 0.40 0.50 T0 T3 T6 T10 O p tu ca l D en si ty , (A b s 6 8 0 n m ) Time (days) Control- Chlorella E-Ethyl -50 E-Ethyl-100 E-Ethyl-200 E-Ethyl -500 0.00 0.10 0.20 0.30 0.40 0.50 T0 T3 T6 T10 O p ti ca l D en si ty (A b s 6 8 0 n m ) Time (days) Control- Chlorella E-W-50 E-W-100 E-W-200 E-W-500 0.00 10.00 20.00 30.00 40.00 50.00 60.00 T0 T3 T6 T10 C h lo ro p h y ll a C o n ce n tr at io n , u g /L Thời gian (ngày) Control- Chlorella E- Ethyl -50 E- Ethyl-100 E- Ethyl-200 E- Ethyl -500 0.00 10.00 20.00 30.00 40.00 50.00 60.00 T0 T3 T6 T10 C h lo ro p h y ll a C o n ce n tr at io n , u g /L Thời gian (ngày) Control- Chlorella E- W-50 E- W-100 E- W-200 A B A B A B 11 Figure 3.16. Growth of Ch. vulgaris under the exposure of ethyl acetate (A) and water fractions (B) determined by chlorophyll a content 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 Treatment M. aeruginosa Ch. vulgaris IE % (OD) IE % (Chla) IE% (TB) IE % (OD) IE % (Chla) IE % (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 Figure 3.18. Effect of plant extracts on the growth of M.aeruginosa after 72 hours treatment A. by optical density 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 T0 T3 T6 T10 C el l D en si ty x 1 0 5 T B /m L Time (days) Control- Chlorella E-Ethyl -50 E-Ethyl-100 E-Ethyl-200 E-Ethyl -500 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 T0 T3 T6 T10 C el l D en si ty x 1 0 5 T b /m L Time (days) Control- Chlorella E-W-50 E-W-100 E-W-200 E-W-500 0.00 0.04 0.08 0.12 0.16 0.20 0.24 0.28 T0 T24 T48 T72 O p ti ca l D en si ty (A b s 6 8 0 n m ) Time (hours) Control. M.a CuSO4-5 E-Ethanol 500 E-Ethyl 500 A B 12 Figure 18 B. Effect of plant extracts on the growth of M.aeruginosa after 72 hours treatment by Chlorophyll a concentration Figure 18 C. Effect of plant extracts on the growth of M.aeruginosa after 72 hours treatment by cell density 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 Treatment 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 3.2.5. Toxicity of chemical compounds isolated from E. fortunei to M. aeruginosa 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 Control CuSO4-5 E-Ethanol 500 E-Ethyl-500 C h lo ro p h y ll a C o n ce n tr at io n , µ g /m L T0 T72 0.00 5.00 10.00 15.00 20.00 25.00 Control - Ma CuSO4-5 Ethanol-500 Ethyl- 500 C el l D en si ty × 1 0 5 T B /m L T0 T72 0.00 0.05 0.10 0.15 0.20 0.25 0.30 EfD 1.8 EfD 4.8 EfD 4.7 EfD 5.1 EfD 10.1 EfD 10.3 EfD 14.1 O p ti ca l D en si ty (A b s 6 8 0 n m ) 0 µg/mL 1 µg/mL 10 µg/mL 20 µg/mL 50 µg/mL A 13 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 Figure 3.20. Transmission electron micrographs (TEM) of Microcystis aeruginosa cells (A) and Ch. vulgaris (B) 0.0 5.0 10.0 15.0 20.0 25.0 EfD1.8 EfD 4.8 EfD 4.7 EfD 5.1 EfD 10.1 EfD 10.3 EfD 14.1 C el l D en si ty × 1 0 5 T B /m L B B A A3 A6 B3 B6 B10 A10 14 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 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 C6 C10 C3 A3 A6 A10 B3 B6 B10 C3 C10 C6 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 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 (µg.mL-1) DO (T0) mg L-1 DO (T48) mg L- 1 pH (T0) pH (T48) 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 7.56 240.00 7.85 7.57 6.07 7.57 280.00 7.92 6.83 6.18 6.76 320.00 7.86 6.72 6.17 6.55 360.00 7.86 7.34 6.15 7.14 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 (µg.mL-1) DO (T0) Mg. L-1 DO (T48) Mg. L-1 pH (T0) pH (T48 ) 0.00 7.77 7.42 7.77 7.42 10.00 7.87 7.51 7.78 7.49 20.00 7.85 7.44 7.70 7.44 40.00 7.88 6.88 7.65 7.37 80.00 7.83 6.44 7.52 7.17 120.00 7.86 6.92 7.44 7.15 160.00 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 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. 0 50 100 150 200 250 300 350 400 450 500 T0 T1 T2 T3 T4 T5 F ro n d N u m b er Time (days) Control-L.minor CuSO4-5 E- Eth-500 E-Eth-200 0 50 100 150 200 250 300 350 400 450 500 T0 T1 T2 T3 T4 T5 F ro n d n u m b er Time (days) Control -L.minor E-Ethyl- 500 E-Ethyl- 200 E-Ethyl- 100 E-Ethyl- 50 A B 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 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 0.0 10.0 20.0 30.0 40.0 50.0 60.0 T0 T5 F re sh W ei g h t ( m g ) Control-L.minor CuSO4-5 E- Eth-500 E-Eth-200 0.0 10.0 20.0 30.0 40.0 50.0 60.0 T0 T5 F re sh w ei g h t (m g ) Control - L.minor E-Ethyl- 500 E-Ethyl- 200 E-Ethyl- 100 E-Ethyl- 50 A B 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%. 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. 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 %). 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 Control - L.minor CuSO4-5 E- Eth-500 E-Eth-200 P ig m en t C o n ce n tr at io n (m g /g F W ) Chla Chlb Chl (a+b) 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 Control- L.minor E-Ethyl- 50 E-Ethyl- 100 E-Ethyl- 200 E-Ethyl- 500 P ig m en t C o n ce n tr at io n (m g /g F W ) Chla Chlb Chla + b 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 T0 T3 T6 T10 C h lo ro p h y ll a C o n ce n tr a ti o n , µ g /L Time (days) Control - HK CuSO4-5 E-Ethanol-500 E-Ethyl-500 A B 19 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) 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. 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 CuSO4-5 sample (IE of 54.60 %) and the E-Ethanol 500 sample (IE of 48.42 %). 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 Control - HK CuSO4-5 E-Ethanol - 500 E-Ethyl- 500 C el l D en si ty × 1 0 5 T B /m L Microcystis sp VKL khác Tảo lục & tảo silic Nhóm TVN 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 T0 T3 T6 T10 C h lo ro p h y ll a ,C o n ce n tr at io n µ g /L Time (days) Control CuSO4-5 Ethanol- 500 Ethyl - 500 A B 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 Control-HK CuSO4-5 E-Ethanol-500 E-Ethyl-500 C el l D en si ty x 1 0 5 T B /m L Microcystis sp VKL khác Tảo lục & tảo silic Nhóm TVN Microcystis sp Other cyanobacteria Green agla & silic agla Phytoplanton Microcy tis sp Other cyanoba teria Green agla & silic agla Phytoplanton 20 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). 3.4.3. Effect of plant extracts on the growth of phytoplankton in water samples collected from Lang Lake in the outdoor scale. 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 %). 0.00 5.00 10.00 15.00 20.00 Control - HL CuSO4-5 E-Ethanol-500 E-Ethyl-500 C el l D en si ty × 1 0 5 T B /m L Microcystis sp VKL khác Tảo lục & tảo silic Nhóm TVN 0.00 5.00 10.00 15.00 20.00 Control-HL CuSO4-5 E-Ethanol-500 E-Ethyl-500 C el l D en si ty × 1 0 5 T B /m L Microcystis sp VKL khác Tảo lục & tảo silic Nhóm TVN 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 T0 T1 T3 T6 T10 C h lo ro p h y ll a C o n ce n tr at io n ( µ g /L ) Time (days) Control CuSO4-5 E- Ethanol- 500 A B Microcy tis sp Other cyanobacteria Green agla & silic agla Phytoplanton Microcystis sp Other cyanobacteria Green agla & silic agla Phytoplanton 21 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 val