Journal of Environmental Protection, 2014, 5, 35-41
Published Online January 2014 (
Influences of Cyanobacterial Toxins Microcystins on the
Seedling of Plants
Thanh-Son Dao1*, Thai-Hang Le1, Thanh-Luu Pham2, Lan-Chi Do-Hong3, Phuoc-Dan Nguyen4
1Institute for Environment and Resources, Ho Chi Minh City, Vietnam; 2Graduate School of Life and Environmental Sciences, Uni-
versit y of Tsukuba, Japan; 3Vietnam National University, Ho Chi Minh City, Vietnam; 4University of Technology, Ho Chi Minh City,
Email: *
Received November 26th, 2013; revised December 21st, 2013; accepted J anuary 9th, 2014
Copyright © 2014 Thanh-Son Dao et al. This is an open access article dist ributed under the C reative Commons Attri bution Licens e,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In accor-
dance of the Creative Commons Attribution License all Copyrights © 2014 ar e reserved for SCIRP an d the owner of the intel lectual
property Thanh-Son Dao et al. All Copyright © 2014 are guarded by law and by SCIRP as a guardian.
Cyanobacterial blooms associated by their toxins have been increasing in frequency in fresh water bodies
throug ho ut the world. Among the cy anobacteria l toxins, microcystins (MC) are the most common and cause se-
vere adverse impacts on plants, aquatic organisms and human beings. In this study, the effects of MC (at the
concentrations of 20 and 200 µg·L1) from field water and crude extract of cyanobacterial scum (mainly Micro-
cystis spp.) from the Dau Tieng Reservoir, Vietnam, on the seedlings of three plants, Brassica rapa-chinensi s, B.
narinosa and Nasturtium officinale, were investigated for over a period of 7 days. The results showed that MC
reduced the fresh w eight , roo t and shoo t lengt h of the exposed see dlings. In ad dition, abno rmalities o f leaf shape
and color of B. rapa-chinensis under exposure to MC were observed. The results implied that MC w ere ta ken up
and might be accu mulated i n the seedl ings po ssessing pot ential r isk to co nsumers as seedling s of t hese plants a re
a common food source for Vietna mese. To the best of our knowledge, this is the first report on the effects of MC
on B. rapa-chinensis, B. narinosa and N. off icina le .
Microcystins; Adverse Effects; Fresh Weight; Root and Shoot Length; Abnormalities
1. Introduction
In water bodies, microcystins (MC) have been the most
commonly reported cyanobacterial toxins compared to
other cyanotoxins (e.g. cylindrospermopsin, nodularin,
saxito xins, anatoxi n-a(s), anatoxin-a) [1]. Cyanobacterial
toxins cause a range of adverse effects on aquatic plants,
animals and human beings. In addition, investigations
showed that the toxins can accumulate and adversely
affect on plants at different aspects of enzymatic res-
pons es, photosynthesis, seedlings and growth.
Recently, many studies on bioaccumulation of cyano-
bacterial toxins (e.g. MC) in plants have been conducted
and reported. The uptake and metabolism of MC-LR in
stems, rhizomes and leaves of aquatic macrophytes were
reported with the MC-LR conce ntra tion s be ing hig hest i n
leaves, followed by shoots and lowest in stems [2,3].
Other plants such as broccoli, mustard and duckweed are
able to accumulate MC in their leaves and bark protein,
and whole plant up to 8.7 ng MC g1 fresh weight (FW)
after exposure [4-6]. Furthermore, Microcystis aerugi-
nosa containing MC-LR can also be retained by salad
letture (Lactuca sativa) after spraying with irrigation
water containing the cyanobacterium [7].
Macrophytes and seedlings have been showed to dis-
play the growth inhibitio n wh en irrigated with water co n-
taining toxic cyanobacterial extract [8]. MC inhibit the
growth and development of rape (Brassica napus), rice
(Oryza sativa) [9], mustard (Sinapis alba) [4] and Wolffia
arrhiza [5]. Exposure to MC-LR resulted in a decrease in
germination, roof and leaf length of spinach [10]. MC-
LR, M C-RR and anatoxin-a also reduced the chlorophyll
and carotenoid concentrations in plants [8,11,12] and
*Corresponding author.
Influences of Cyanobacterial Toxins Microcystins on the Seedling of Plants
caused malformation on mustard [4].
Pietsch et al. [13] and Wiegand et al. [14] showed that
the photosynthesis of macrophytes, Vesicularia dubyana
and Ceratophyllum demersum, was inhibited by cyano-
bacterial crude extracts or purified cyanotoxins, MC,
microcin SF608 and anatoxin-a. These authors also rec-
orded the activity alteration of the enzyme glutathione
S-transferase in the macrophytes after toxin exposures.
The activities of antioxidant and biotransformation en-
zymes (superoxide dismutase, peroxidase, catalase, glu-
tathione S-transferase, glutathione peroxidase) from
plants were also significantly changed by cyanobacterial
crude extracts containing MC, pure MC-LR or anatoxin-
a at the concentration from 0.5 - 10 µg·L1 [10,12,14].
MC-LR induced oxidative stress responses in Lepidium
sativum seedlings including lipid peroxidation, change of
tocopherol concentrations and profile, and elevation of
glutathione enz yme acti vitie s [15] . MC-RR decreased the
glutathione level and increased superoxide dismutase and
catalase activities in cells of Arabidopsis thaliana and
tobacco (Nicotiana tabucum) [16,17].
Today, seedlings of many different plants are common
food ingredients especially in Asia. In the field plants
could be irrigated with water containing cyanobacteria
and their toxins. Hence these plants could be affected by
and accumulate cyanotoxins. To our knowledge, there
has been no information on the effects of MC on the
seedlings of Brassica rapa-chinensis, B. narinosa and
Nasturtium off icinale . T he aim o f this stud y is to obser ve
the detrimental effects of MC from field water and cya-
nobacterial crude extract obtained from Dau Tieng Re-
servoir, Vietnam, at the environmentally relevant con-
centrations (20 and 200 µg·L1) on t he see dling s of thr ee
different plants B. rapa-chinen sis, B. narinosa and N.
officinale over 7 days.
2. Materials and Methods
The seeds of B. rapa-chinensis, B. narinosa and N. o ffi-
cinale were purchased from a super market in Hochiminh
City. Two samples: 1) cyanobacterial scum (mainly Mi-
crocystis spp) collected in July 2011; and 2) (raw) field
water sample during cyanobacterial scum (mainly Ana-
baena flos-aquae) from Dau Tieng Reservoir collected in
September 2012, were used for experiments.
2.1. Sample Preparation for Experiments and
Microcystins Analysis
Crude extract from cyanobacterial scum collected in July
2011 was prepared according to Pietsch et al. [13] with
minor modification. Briefly, the dried biomass of scum
on GF/C filters was homogenized, suspended into re-
versed osmosis water, sonicated, frozen at –70˚C over
night and thawed at room temperature. The freeze/thaw
cycle was repeated five times. After the last thawing
cycle, samples were centrifuged at 4500 rpm, 4˚C for 15
min. Supernatant was collected and kept at 70˚C prior
to exp e riments on t he plant se edling s. Field wate r sa mple
collected in September 2012 was filtere d via plankton net
(25 µm mesh size) then centrifuged at 4500 rpm, 4˚C for
15 min and supernatant was collected and stored at
–70˚C prior to exposure to seedlings.
Sub-samples of field water and cyanobacterial crude
extract were centrifuged at 14,000 rpm 4˚C for 15 min
and supernatants were collected for MC analysis by high
performance liquid chromatography (HPLC). HPLC
(Shimadzu, Japan) equipped with a silica based reverse
phase C18 column (Waters SunFire, Ireland) was
maintained at 40˚C. A 0.05 M phosphate buffer (pH 2.5)
in methanol (50/50, v/v), at a flow rate of 0.58 mL min–1,
was used as mobile phase. MC congeners were detected
by the UV detection at 238 nm with a photodiode UV-
visible array detector. The MC variants MC-RR, -YR
and -LR purchased from Wako chemicals company
(Osaka, Japan) were used as standards.
2.2. Exposures of Brassica rapa-chinensis, B.
narinosa and Nasturtium officinale
to Micro cy sti ns
The seedling experiment used 40 seeds of each plant
species (with 3 replicates). The seeds were placed on
tissues paper and watered daily with 5 mL of field water
(containing 20 (DT20) or 200 (DT200) MC µg·L1) or
cyanobacterial crude extract (containing 20 (Sc20) or 200
(Sc200) MC µg·L1). A co ntro l was inc lude d whic h con-
sisted of seeds watered with distilled water only. Expe-
riments were run at 25˚C ± 1˚C, in the dark for the first 2
days. From the third day to the end of incubation (7 days),
the seedlings were placed under light with an inten sity of
around 1500 Lux and a light: dark cycle of 12 h:12 h.
The parameters recorded were fresh weight (FW), shoot
lengt h and roo t length of t he seedli ngs at 2, 4 and 7 da ys
of incubation. The FW was determined using a balance
(Sartorius BP 201S, Germany) and the lengt h was meas-
ured with a ruler of 1 mm spacing.
2.3. Statistical Analysis
Sigmaplot, version 12.0 was used for data analysis. One-
way Analysis of Variance (A NOVA) and Tukey test Post
Hoc were applied to deter mine the sta tistically significant
differences of the FW, shoot and root length of seedlings
after the data were checked for variance homogeneity
(Levenes) and normality (Shapiro-Wilk’s test).
3. Results and Discussion
3.1. Microcystins Concentration in the Field
Water and Cyanobacterial Crude Extr act
The HPLC analysis showed that the cyanobacterial crude
Influences of Cyanobacterial Toxins Microcystins on the Seedling of Plants
extract and field water contained MC-RR, MC-YR and
MC-LR (Figure 1) at the total concentrations of 686.9
µg MC-LReq. g1 dry weight and 1069.2 µg MC-LReq.
L–1, respectively (Table 1). The MC concentration from
cyanobacterial scum sample (collected in July 2011) in
this st udy is in r ange o f pr e vio us r ec or d s fr om the f i eld i n
Vietnam [18]. H owever , the M C co nc e ntr a tio n i n t he r aw
water sample from Dau Tieng Reservoir was much high-
er than those ever reported before from Vietnamese wa-
ters . The hi gh MC conce ntrati ons d uring Microcystis spp
and Anabaena flos-aquae scums proposed a serious risk
to local residents who daily use the water from the re-
servoir for domestic activities. Additionally, this is the
first report of the MC producing cyanobacterium A. flos-
aquae in Vietnam.
Figure 1. HPLC chromatography of MC from control (a),
field water (b) and cya nobacterial s cum sample (c).
Table 1. Microcystins concentrations of the cyanobacterial
s cum (µg·g1 DW) a nd field w at er s ample (µg·L1).
Sam ples MC-RR MC-YR MC-LR Total MC
Cyan oba ct eria
l scum 635 31.7 20.3 686.9
Field water 539.5 30.6 499.1 1069.2
3.2. Effects of Microcystins on the Fresh Weight
of See dl i ngs
After 2, 4 and 7 days of incubation, the FW of all three
plant species from exposures to either field water or
cyanobacterial crude extract was significantly decreased
compared to the control (ANOVA followed by Tukey
test, p < 0.05; Figure 2). Besides, high MC concentration
(DT200 and Sc200) had a stronger impact than the low
toxin one (DT20 and Sc20) on FW of exposed plants
consequently lower seedling FW in treatments with 200
µg M C L1 compared to that with 20 µg MC L1. G ener-
ally, the FW of seedlings was similar when they were
exposed to the same MC concentrations (20 or 200
µg·L1) either from field water or crude extract.
The significantly lower FW of the three seedlings ex-
posed to MC in our study is in line with previous inves-
tigations in which the FW of potato and the germination
of spinach were inhibited by cyanobacterial crude extract
[8,10]. The weight of seedlings of the three plants during
the first days of germination should be involved in the
amount of water they took up. MC induce oxidative
stress response [15] and inhibited ATPase [19] possibly
interfering the metabolism in seedlings during germina-
tion consequently water uptake and seedlings of the ex-
posed seeds and plants. Therefore, from our study it
could be inferred that MC reduce the water uptake ca-
pacity of seeds hence inhibit FW increase of the seedl-
ings. Besides, the detoxification of MC in plant cells to
balance the activities of biotransformation and antioxi-
dant enzymes [10,12,20] would lead to the decrease of
the energy or material for growth of the seedlings which
properly involved in the reduction of FW of seedlings in
the MC exposures compared to the control.
3.3. Effects of Microcystins on the Development
of Root and Shoot
The roo t and sho o t le ng th of the three plant species in the
control treatment alwa ys significantly longer than that in
MC exposures after 2, 4 and 7 days of incubation
(ANOVA followed by Tukey test, p < 0.05; Figures 3
and 4). Additionally, high MC concentration from the
field water and cyanobacterial crude extract had stronger
effects than low MC conce ntration on the root and shoot
development of B. rapachinen sis, B. narmosa and N.
officmale (Figures 3 and 4). The MC from field water or
crude extract at the same concentrations (20 or 200
µg·L1) resulted in the similar root and shoot length of
the seedlings.
Inhib itio n on t he r oo t and sho ot le ngth o f MC -exposed
seedlings in our study is consistent with previous records
of McElhiney et al. [8] and Pflugmacher et al. [10].
Garbers et al. [21] indicated that MC regulated the phy-
tohormone auxin, and MC we re protein phosphatase i nhi-
Influences of Cyanobacterial Toxins Microcystins on the Seedling of Plants
Figure 2. Fresh weight (mg) of the seedlings (mean value ± SD of n = 40) during incubation. Asterisks indicate significant
differenc e between exposures and c ontrol by ANOVA followed by Tukey test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
Figure 3 . Root le ngt h (mm) of t he se edl ing s ( mea n val ue ± SD of n = 40 ) du ring inc ubat ion. As teri sks indi cat e si gnif ic ant dif-
ference between exposures and control by ANOVA foll owed by Tukey test (*, p < 0.05; **, p < 0.01; ** *, p < 0.001).
Influences of Cyanobacterial Toxins Microcystins on the Seedling of Plants
Figure 4. Shoot length (mm) of the seedlings (mean value ± SD of n = 40) during incubation. Asterisks indicate significant
differenc e between exposures and c ontrol by ANOVA followed by Tukey test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
bitors. Therefore, exposure to MC would cause the dis-
order of cell development consequently inhibition of root
and shoot growth of the exposed seedlings. Also, the
water uptake reduction induced by MC as mentioned
above would contribute to the decrease of shoot and root
prolongation. Besides, MC could cause strong alteration
of biotransformation and a ntioxidant e nzyme activ ities in
plants [10,12,20] hence some energy is spent on the MC
detoxification leading to the reduction of hydrocarbon
and nutrient source for root and shoot development.
Summing up the adverse effects MC on plants, the
seedlings exposed to MC would grow slower than those
in the c ontrol as observed i n our experiments .
3.4. Abnormalities of the Seedlings Exposed
to Micro cy sti ns
Among the MC exposures, abnormalities of leaf shape
and color of some seedlings of B. rapa-chinensis were
observed. The two young leaves on some seedlings from
MC exposure were quite difference in size, and the leaf
margin of those seedlings were brown (Figure 5(b))
while young leaves were almost similar in size with
green color in the control incubation (Fig ur e 5(a)).
Gehringer et a l. [22] found that the leaves of Lepidium
sativum exposed to MC were significantly shorter than
control samples which supported the unbalanced size of
two young leaves observed in our study. It is possible
that the brown color on leaf margin of B. rapa-chinensis
in this investigation has been involved in the disappear-
ance or decrease of chlorophyll content at the margin
whi ch was previously reported elsewhere [4]. This phe-
nomenon could also be explained as chlorophyll is inhi-
bited by MC at the concentrations from 5 - 50 µg L1 [8]
which were in the range with or below the MC concen-
trations in our study (20 - 200 µg·L1).
4. Conclusion
Microcystins concentration in water from Dau Tieng
Reservoir was extremely high possessing high risk to
local residents who daily use the water from the reservoir
for their domestic activities. MC from the field water
sample and crude extract of cyanobacteria caused ad-
verse effects on seedling of the tested plants including
signi fica nt d ecr ease s in FW , roo t and shoo t lengt h. T hese
effects could be involved in the alteration of regulated
(protein phosphatase), energetic (ATPase), biotransfor-
mation (glutathione S-transferase) and antioxidant (cata-
lase) enzyme activities caused by MC. Besides, abnor-
malities of leaf shape and brown color from B. rapa-
chinensis seedlings exposed to MC after one week could
Influences of Cyanobacterial Toxins Microcystins on the Seedling of Plants
Figure 5. Normal leaves from control (a) and abnormal
leave s from M C ex posure (b) of Brassica rapa -chinensis at 7
days of incubation. Arrows indicate the difference in size
and the brown c olor at margin of the yo ung leaves.
be attributed to the impact of MC on chlorophyll content.
The results of this study confirm the potent toxicity of
cyanobacterial toxins from Dau Tieng Reservoir on
plants. To our knowledge, this is the first report on the
effects of MC on seedlings of B. rapa-chinensis, B. na-
rinosa and N. officinale.
Ackno wledgements
Thi s stud y is fund ed b y the Vietnam National University
Hochiminh City under the granted projects numbers
B2012-24-01TD and A2013-48-01.
[1] K. Sivonen and G. Jones, “Cyanobacterial Toxins,” In: I.
Chorus andJ. Bartram, Eds., Toxic Cyanobacteria in
Water—A Guide to Their Public Health Consequences,
Monitoring and Management, E & FN Spon, London,
1999, pp . 41-111.
[2] S. Pflugmacher, C. Wiegand, K. A. Beattie, G. A. Codd
and C. E. W. Steinberg, “Uptake of the Cyanobacterial
hepatotoxin Microcystin-LR by Aquatic Macrophytes,”
Journal of Applied Botany, Vol. 72, No. 5-6, 1998, pp.
[3] S. Pflugmacher, C. Wiegand , K. A. Beat tie, E. Krause, C .
E. W. Steinberg and G. A. Codd, “Uptake, Effects and
Metabolism of Cyanobacterial Toxins in the Emergent
Reed Plant Phragmites australis (Cav.) Trin. ex Steud,”
Environmental Toxicology and Chemistry, Vol. 20, No. 4,
2001, pp . 846-852.
[4] K. Kurki-Helasmo and J. Meriluoto, “Microcystin Uptake
Inhibits Growth and Protein Phosphatase Activity in
Mustard (Sinapis alba L.) Seedlings,” Toxicon, Vol. 36,
No. 12, 1998, pp. 1921-1926.
[5] S. M. Mitrovic, O. Allis, A. Furey and K. J. James, “Bio-
accumulation and Harmful Effects of Microcystin-LR in
the Aquatic Plants Lemna minor and Wolffia arrhiza and
the Filamentous Alga Chladophora fracta,” Exotoxicolo-
gy and Environmental Safety, Vol. 61, No. 3, 2005, pp.
[6] S. Jarvenpaa, C. Lundberg-Niinisto, L. Spoof, O. Sjovall,
E. Tyystjarvi and J. Meriluoto, “Effects of Microcystins
on Broccoli and Must ard , and Analysis of Accumulated
Toxin by Liquid Chromatography-Mass Spectrometry,”
Toxicon, Vol. 49, No. 6, 2007, pp. 865-874.
[7] G. A. Codd, J. S. Metcalf and K. A. Beattie, “R etentio n of
Microcystis aeruginosa and Microcystin b y Salad Lett uce
(Lactuca sativa) after Spray Irrigation with Water Con-
taini ng Cyano bact eria, ” To xicon, Vol. 37, No. 8, 1999, pp.
[8] J. McElhiney, LA. Lawton and C. Leifert, “Investigations
into the Inhibitory Effects of Microcystins on Plant
Growt h , and the Toxicity of Plant Tissues Following Ex-
posure,” Toxicon , Vol. 39, No. 9, 2001, pp. 1411-1420.
[9] J. Chen, L. Song, J. Dai and Z. Liu, “ Effect s o f M icro cys -
tins on the Growth and the Activity of Superoxide Dis-
mutase and Peroxidase of Rape (Brassica napus L. ) and
Rice (Oryza sativa L.),” Toxicon, Vol. 43, No. 4, 2004,
pp. 393-400.
[10] S. Pflugmacher, M. Aulhorn and M. Grimm, “Influence
of a Cyanobacterial Crude Extract Containing Microcys-
tin-LR on the Physiology and Antioxidative Defence
Systems o f Differen t Spi nach V ariants, New Phyco logist,
Vol. 175, No. 3, 2007, pp . 482-489.
[11] J. Weiss, H. P. Liebert and W. Braune, “Influence on
Microcystin-RR on Growth and Photosynthetic Capacity
of the Duckweed Lemma minor L.,” Journal of Applied
Botany, Vol. 74, No. 3-4, 2000, pp. 10-105.
[12] M. H. Ha and S. Pflugmacher, “Phytotoxic Effects of the
Cyanobacterial Neurotoxin Anatoxin-a: Morphological,
Physiological and Biochemical Responses in Aquatic
Macrophyte, Ceratophyllum demersum,” Toxicon, Vol.
70, 2013 , pp. 1-8.
[13] C. Pietsch, C. Wiegand, M. V. Ame, A. Nicklisch, D.
Wunderlin and S. Pflugmacher, “The Effects of Cyano-
bacterial Crude Extract on Different Aquatic Organisms:
Evidence for Cyanobacterial Toxin Modulating Factors, ”
Environmental Toxicology, Vol. 16, No. 6, 2 00 1, pp . 535-
[14] C. Wiegand, A. Peuther t, S. Pflug macher and S. Carmeli,
“Effects of Microcin SF608 and Microcystin-LR, Two
Cyanobacterial Compounds Produced by Microcystis sp.,
on Aquatic Organisms,Environmental Toxicology, Vol.
17, No. 4, 2002, pp . 400-406.
[15] J. Stuven and S. Pflugmacher, “Antioxidative Stress Re-
sponse of Lepidium sativum Due to Exposure to Cyano-
bacterial Seco ndar y Metabo li tes,” Toxicon, Vol. 50, No. 1,
2007, pp . 85-93.
Influences of Cyanobacterial Toxins Microcystins on the Seedling of Plants
[16] L. Yin, J. Huang, W. Huang, D. Li and Y. Liu, “Res-
ponses of Antioxidant System in Arabidopsis thaliana
Suspension Cells to the Toxicity of Microcystin-RR,”
Toxicon, Vol. 46, No. 8, 2005, pp. 8 59-864.
[17] L. Yin, J. Huang, W. Huang, D. Li, G. Wang and Y. Liu,
“Micro cystin-RR-Induced Accumulation of Reactive
Oxygen Species and Alteration of Antioxidant Systems in
Tobaco BY-2 Cells,” Toxicon, Vol. 46, No. 5, 2005, pp.
[18] T. S. Dao, T. L. Pham, L. C. Do-Hong and B. T. Bui,
“Occurrence of Toxic Cyanobacteria and Their Toxins
from Freshwater Bodies in VietnamA Short Review,”
Vietnam Journal of Science and Technology, Vol. 50, No.
1C, 2012, pp. 264-269.
[19] A. Mikhailov, A. S. Harmala-Brasken, J. Her man, J. A. O.
Meriluoto and J. E. Eriksson, “Identification of ATP-
Synthetase as a Novel Intracellular Target for Microcys-
tin-LR,” Chemico-Biological Interactions, Vol. 142, No.
3, 2003, pp. 223-237.
[20] S. Pflugmacher, “Promotion of Oxidative Stress in the
Aquatic Macrophyte Ceratophyllum demersum during
Biotransformation of the Cyanobacterial Toxin Microcys-
tin-LR,” Aquatic Toxicology, Vol. 70, No. 3, 2004, pp.
[21] C. Garbers, A. DeLong, J. Deruere, P. Ber nasconi and D.
Soll, “A Mutation in Protein Phosphatase 2A Regulatory
Subumit A Affects Auxin Transport in Arabidopsis,” The
EMBO Journal, Vol. 15, No. 9, 199 6, pp. 2115-2124.
[22] M. M. Gehringer, V. Kewada, N. Coates and T. G.
Downing, “The Use of Lepidiium sativum in a Plant Bio-
assay System for the Detection of Microcystin-LR,” Tox-
icon, Vol. 41, No. 7, 2003, pp. 871-876.