Influences of Cyanobacterial Toxins Microcystins on the Seedling of Plants

doi:10.4236/jep.2014.51005

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Journal of Environmental Protection, 2014, 5, 35-41

Published Online January 2014 (http://www.scirp.org/journal/jep)

http://dx.doi.org/10.4236/jep.2014.51005

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,

Vietnam.

Email: *[email protected]

Received November 26th, 2013; revised December 21st, 2013; accepted J anuary 9th, 2014

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In accor-

ABSTRACT

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·L−1) 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 .

KEYWORDS

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 g−1 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.

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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·L−1 [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·L−1) 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·L−1) or

cyanobacterial crude extract (containing 20 (Sc20) or 200

(Sc200) MC µg·L−1). 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

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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. g−1 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·g−1 DW) a nd field w at er s ample (µg·L−1).

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 L−1 compared to that with 20 µg MC L−1. G ener-

ally, the FW of seedlings was similar when they were

exposed to the same MC concentrations (20 or 200

µg·L−1) 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·L−1) 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-

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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).

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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 L−1 [8]

which were in the range with or below the MC concen-

trations in our study (20 - 200 µg·L−1).

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

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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.

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