toxins
Article
Uptake, Growth, and Pigment Changes in Lemna
minor L. Exposed to Environmental Concentrations
of Cylindrospermopsin
Nelida Cecilia Flores-Rojas 1, Maranda Esterhuizen-Londt 2,3,4,* and Stephan Pflugmacher 2,3,4
1Institute of Ecology, Technische Universität Berlin, Ernst-Reuter-Platz 1, 10587 Berlin, Germany;
2Faculty of Biological and Environmental Sciences, Ecosystems and Environmental Research Programme,
3Korea Institute of Science and Technology Europe (KIST), Joint Laboratory of Applied Ecotoxicology,
Campus 7.1, 66123 Saarbrücken, Germany
4Helsinki Institute of Sustainability Science (HELSUS), University of Helsinki, Fabianinkatu 33,
00014 Helsinki, Finland
*Correspondence: [email protected]; Tel.: +358-50-318-8337
Received: 30 September 2019; Accepted: 5 November 2019; Published: 7 November 2019
Abstract:
Cylindrospermopsin (CYN)-producing cyanobacterial blooms such as Raphidiopsis,
Aphanizomenon,Anabaena,Umezakia, and Lyngbya spp. are occurring more commonly and frequently
worldwide. CYN is an environmentally stable extracellular toxin, which inhibits protein synthesis,
and, therefore, can potentially affect a wide variety of aquatic biota. Submerged and floating
macrophytes, as primary producers in oligotrophic habitats, are at risk of exposure and information
on the effects of CYN exposure at environmentally relevant concentrations is limited. In the present
study, we investigated CYN uptake in the floating macrophyte Lemna minor with exposure to
reported environmental concentrations. The effects were evaluated in terms of bioaccumulation,
relative plant growth, and number of fronds per day. Variations in the concentrations and ratios
of the chlorophylls as stress markers and carotenoids as markers of oxidative stress defense were
measured. With exposure to 25
µ
g/L, L. minor could remove 43% of CYN within 24 h but CYN was
not bioaccumulated. Generally, the pigment concentrations were elevated with exposure to 0.025,
0.25, and 2.5
µ
g/L CYN after 24 h, but normalized quickly thereafter. Changes in relative plant
growth were observed with exposure to 0.25 and 2.5
µ
g/L CYN. Adverse effects were seen with these
environmentally realistic concentrations within 24 h; however, L. minor successfully recovered within
the next 48–96 h.
Keywords: cylindrospermopsin; uptake; pigment contents; relative plant growth; Lemna minor
Key Contribution:
Exposing Lemna minor to environmental concentrations of cylindrospermopsin
caused stress on a physiological level; however, the macrophyte was able to recover within 48 h
of exposure.
1. Introduction
Cylindrospermopsin (CYN) is an alkaloid with a tricyclic guanidine zwitterionic structure attached
to a hydroxymethyluracil [
1
], which makes the toxin highly water-soluble. Two natural variants
of CYN have been identified, namely 7-epicylindrospermopsin and deoxycylindrospermopsin [
2
,
3
].
Nowadays, CYN is recognised as a potent cyanobacterial toxin detected in waterbodies worldwide [
4
]
and more often considered for its toxic consequences. The ever-increasing global occurrence of
Toxins 2019,11, 650; doi:10.3390/toxins11110650 www.mdpi.com/journal/toxins
Toxins 2019,11, 650 2 of 13
massive and prolonged blooms of cylindrospermopsin-producing cyanobacteria, such as Raphidiopsis,
Aphanizomenon,Anabaena,Umezakia, and Lyngbya spp. poses a potential threat to both human health
and the ecosystem balance [
4
,
5
]. Due to its predominantly extracellular availability because of active
excretion, stability under a wide range of conditions, and ability to covalently bind to DNA/RNA [
6
] as
well as to inhibit protein synthesis, CYN has the potential to impact a wide variety of aquatic plant and
animal species [
5
,
7
]. Only a few studies examining the uptake of CYN have focused on aquatic plants
and investigated the subsequent physiological responses and effects on growth associated with CYN
exposure. Submerged and emerged macrophytes, as the primary producers in oligotrophic habitats,
play an important role in stabilising aquatic ecosystems, where the consumers in all trophic levels
depend on them. They are vital in the aquatic carbon cycle, also providing shelter for young animals,
egg-hatching niches and shade to cool the surface waters [
8
]. Their central role thus emphasises the
need to understand how they are affected by hazards in their environment.
Studies focusing on CYN assimilation in aquatic plants have shown low uptake concentrations
and associated bioconcentration factors (BCF). White et al. [
9
] reported a maximum uptake of 15 ng
CYN/g FW in Hydrilla verticillata (BFC 0.045) after seven days with exposure to 400
µ
g/L CYN.
Interestingly, exposure for longer periods (up to 14 days) did not result in bioaccumulation. In Landoltia
punctata (previously Spirodella oligorrhyza), exposed to Cylindrospermopsis raciborskii whole-cell extracts
(containing 0 to 500
µ
g/L CYN), 30 ng CYN/g FW plant material was internalised (BFC 0.038) after six
days of exposure to the highest CYN concentration [
10
]. Santos et al. [
11
] exposed Azolla filiculoides to
crude extracts with three different CYN concentrations (50, 500, and 5000
µ
g/L). After seven days of
exposure, CYN uptake (1.31
±
0.11
µ
g/g plant material fresh weight (FW); BCF 0.40
±
0.04) could only
be quantifiable in plants exposed to the highest CYN concentration.
The aquatic fern A. filiculoides showed a 99.8% growth inhibition after seven days of exposure
to 5000
µ
g/L CYN; although, not with lower exposure concentrations [
11
]. The same study reported
increased chlorophyll, carotenoid, and protein contents. Jambrik et al. [
12
] studied the growth
alterations in Lemna minor and Wolffia arrhiza exposed to a crude extract containing CYN as well
as the purified toxin (ranging from 10 to 20,000
µ
g/L). Both crude extract and pure CYN induced
growth alterations, such as decreased frond numbers and fresh weight in both plants after five days of
treatment. Several studies reported the stimulation of root growth and reduced leaf fresh weight with
exposure to CYN either as a purified toxin or in a crude extract in plants such as H. verticillata [
13
] or
Oryza sativa [14].
CYN exposure has been associated with instigating oxidative stress in plants, as shown in
O. sativa [
14
], and A. filiculoides [
11
]. Few studies investigating the effect of CYN have been conducted
with duckweed species. L. minor exposed to CYN showed increased catalase (CAT) activity within 168 h
of exposure to CYN concentrations of 2.5 and 25
µ
g/L, respectively [
15
]. In the same study, glutathione
S-transferase (GST) and glutathione reductase (GR) activities increased after 24 h with 25
µ
g/L CYN.
However, the highest activities were recorded after 168 h only at the highest CYN concentration [
15
].
The authors suggested that CAT plays a vital role as a first mechanism to control oxidative stress,
while GST and GR play a crucial role during longer exposure times and at high CYN concentrations.
Although research regarding the effects of CYN in aquatic environments has increased in the last
years, the uptake, corresponding effects on growth, and the physiological changes in macrophytes at
environmentally relevant concentrations have not yet been studied extensively. Due to the pivotal role
of macrophytes in the aquatic environment, understanding these effects is required. The information
is moreover necessary to understand how CYN is metabolized and the effects on the plant defence
mechanisms, to further assess the applicability of these plants in phytoremediation technologies for
cyanobacterial toxins such as the Green Liver System®[16].
The aquatic macrophyte L. minor, commonly known as duckweed, is widely used in aquatic
testing [
17
] and has been reported to be a potential scavenger of heavy metals from aquatic environments
and are being applied in wastewater treatment and phytoremediation [
18
]. To further investigate
the application range of L. minor in phytoremediation, the present study investigated its uptake
Toxins 2019,11, 650 3 of 13
ability and the corresponding physiological responses with exposure to pure CYN. The pigment
contents such as chlorophyll aand band carotenoids, as well as the number of fronds, were used as
indicators of changes in the plant’s stress status in response to CYN exposure and uptake. On this
basis, the study assessed the connection between the physiological responses in this macrophyte and if
it can be used for phytoremediation as the inability of an aquatic plant to cope with toxin exposure on
a physiological level may lead to death and re-release of xenobiotics already taken up. Furthermore,
CYN studies to date have been conducted with concentrations far higher than those detected in
the environment [
4
,
19
,
20
]; therefore, in the current study, concentrations resembling environmental
scenarios were used.
2. Results and Discussion
2.1. CYN Uptake
In the negative control plants, no CYN contamination was detected. For the positive control
(Figure 1A), 19% of the CYN in the media was degraded after 24 h, which further increased to 29%
after 168 h. The low natural degradation of CYN previously proven [
7
,
21
–
23
], was thereby confirmed
in the present study. The degradation was assumed to have occurred due to light and temperature
effects or bacterial contamination or possibly protein binding [24].
Toxins 2019, 11, x FOR PEER REVIEW 3 of 14
uptake ability and the corresponding physiological responses with exposure to pure CYN. The
pigment contents such as chlorophyll a and b and carotenoids, as well as the number of fronds, were
used as indicators of changes in the plant’s stress status in response to CYN exposure and uptake.
On this basis, the study assessed the connection between the physiological responses in this
macrophyte and if it can be used for phytoremediation as the inability of an aquatic plant to cope
with toxin exposure on a physiological level may lead to death and re-release of xenobiotics already
taken up. Furthermore, CYN studies to date have been conducted with concentrations far higher than
those detected in the environment [4,19,20]; therefore, in the current study, concentrations resembling
environmental scenarios were used.
2. Results and Discussion
2.1. CYN Uptake
In the negative control plants, no CYN contamination was detected. For the positive control
(Figure 1A), 19% of the CYN in the media was degraded after 24 h, which further increased to 29%
after 168 h. The low natural degradation of CYN previously proven [7,21–23], was thereby confirmed
in the present study. The degradation was assumed to have occurred due to light and temperature
effects or bacterial contamination or possibly protein binding [24].
Figure 1. (A) CYN concentration in the exposure media in the presence (treatment) and absence
(positive control) of L. minor with time (B) CYN concentration taken up by L. minor with time and
corresponding BCFs. Data represent the mean value ± standard deviation (n = 4). Significances
compared to the media control are indicated by the asterisks (* p < 0.05).
The CYN concentrations in the exposure media containing plants were significantly reduced
compared to the positive control, which lacked plants (p < 0.05) within a minute after beginning the
experiment (0.03 h; Figure 1A). At this time, the CYN concentration in the media containing plants
decreased from 21.29 ± 3.1 µg/L to 15.55 ± 2.41 µg/L corresponding to 27% CYN removed. Natural
degradation is not likely to occur this fast [7]; therefore, it is assumed that L. minor was responsible
for the removal. The media CYN concentration in the treatments decreased sharply within the first
24 h to 9.76 ± 3.04 μg/L (p < 0.005), corresponding to a removal percentage of 54%. Taking into account
natural degradation, the percentage of CYN removed by L. minor after 24 h was 43%. After 96 h and
168 h, the CYN concentrations in the exposure media were significantly lower compared to the
control (p < 0.001). However, at 48 h, 96 h, and 168 h, the CYN removal percentages remained similar
among them (p > 0.05). The highest rate of CYN removal corresponds to 861 ng/min (0.03 h), followed
by 13.08 ng/min and 9.29 ng/min after 1 h and 2 h, respectively, while after 24 h, no further uptake
was documented. The results suggest that L. minor could take up CYN most effectively during the
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
0 1 2 24 48 96 168
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
Free CYN BCF
0
5
10
15
20
25
30
00.03 1 2 24 48 96 168
Lemna exposure media
Natural degradation
Exposure time (hours) Exposure time (hours)
CYN exposure media (µg/L)
Free CYN in plant tissue (ng/g FW)
A
**
*
**
B
BCF
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
0 1 2 24 48 96 168
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
Free CYN BCF
0
5
10
15
20
25
30
00.03 1 2 24 48 96 168
Lemna exposure media
Natural degradation
Exposure time (hours) Exposure time (hours)
CYN exposure media (µg/L)
Free CYN in plant tissue (ng/g FW)
A
**
*
**
B
BCF
Figure 1.
(
A
) CYN concentration in the exposure media in the presence (treatment) and absence
(positive control) of L. minor with time (
B
) CYN concentration taken up by L. minor with time and
corresponding BCFs. Data represent the mean value
±
standard deviation (n=4). Significances
compared to the media control are indicated by the asterisks (* p<0.05).
The CYN concentrations in the exposure media containing plants were significantly reduced
compared to the positive control, which lacked plants (p<0.05) within a minute after beginning the
experiment (0.03 h; Figure 1A). At this time, the CYN concentration in the media containing plants
decreased from 21.29
±
3.1
µ
g/L to 15.55
±
2.41
µ
g/L corresponding to 27% CYN removed. Natural
degradation is not likely to occur this fast [
7
]; therefore, it is assumed that L. minor was responsible
for the removal. The media CYN concentration in the treatments decreased sharply within the first
24 h to 9.76
±
3.04
µ
g/L (p<0.005), corresponding to a removal percentage of 54%. Taking into
account natural degradation, the percentage of CYN removed by L. minor after 24 h was 43%. After
96 h and 168 h, the CYN concentrations in the exposure media were significantly lower compared to
the control (p<0.001). However, at 48 h, 96 h, and 168 h, the CYN removal percentages remained
similar among them (
p>0.05
). The highest rate of CYN removal corresponds to 861 ng/min (0.03 h),
followed by 13.08 ng/min and 9.29 ng/min after 1 h and 2 h, respectively, while after 24 h, no further
uptake was documented. The results suggest that L. minor could take up CYN most effectively during
Toxins 2019,11, 650 4 of 13
the first periods of exposure, which is not only consistent with previous studies on CYN uptake in
macrophytes [9], but also other cyanotoxins [25–28].
Free CYN was detected in the plant tissues for each exposure time ranging from 0.82
±
0.03 ng/g
FW to 0.85
±
0.01 ng/g FW (Figure 1B). Free CYN in the plant tissues, and thus the BCFs, did not
show significant changes between the different exposure times (p>0.05); therefore, metabolism or
biotransformation of CYN is unlikely. The highest bioconcentration factor (BCF) was 0.095
±
0.03,
which was achieved after 24 h of exposure (Figure 1B). The low amounts of free CYN in the tissues and
no bioaccumulation (BCF <1) reported in this study are similar to previous studies related to CYN
exposure at higher concentrations [
9
–
11
]. In the current study, the rapid partial removal of CYN within
2 h could be, in part, attributed to the adhesion of CYN on the cell wall because of its zwitterionic
properties together with microbial degradation. White et al. [
9
] discussed the absence of free-CYN
bioconcentration in H. verticillata, leaving open the question of whether CYN can become intracellular
but is transported out of the cell at the same rate at which it enters; or that intracellular toxin could be
enzymatically bound, modified or metabolized within plants preventing detection with quantitative
techniques used. A recent study with crude lysates from different aquatic organisms exposed to 25
µ
g/L
CYN has shown a significant decrease in free CYN percentages. The same study also revealed that the
preparation of the proteins influences the percentage of free CYN detectable and that all CYN could
be accounted for when incubated with amino acids and oxidised or reduced glutathione, suggesting
protein binding [24].
E. densa exposed to 50
µ
g/L CYN displayed 0.05% toxin uptake per exposure organism [
29
]. In the
present study, considering the CYN remaining from the media after the exposures with L. minor,
free CYN in the whole exposed plant biomass represented between 0.15% to 0.30% of CYN removed in
water. The low percentages of CYN in plants suggest that cyanotoxin could be binding to components
of the plant cells. The previous investigations and the current study cannot confirm the association of
CYN to proteins but highlights the need to continue investigating this subject.
2.2. Photosynthetic Pigment Contents
The chlorophyll acontent in L. minor exposed to CYN increased relative to the control after 24 h
with all exposure concentrations (p<0.05) except 25
µ
g/L (Figure 2A); however, this returned to the
same concentration as the control within 48 h (p>0.05). With exposure to 25
µ
g/L, a later (
at 96 h
) but
prolonged elicitation until 168 h was seen (p<0.05). The chlorophyll band total chlorophyll contents
reacted similarly with a sharp increase after 24 h of exposure, followed by normalisation after 48 h
(Figure 2B,C). Enhanced carotenoid content compared to the control was observed after 24 h with
0.025
µ
g/L, 0.25
µ
g/L, and 2.5
µ
g/L CYN exposure concentrations (p<0.05). After 168 h, the carotenoid
concentration remained significantly increased compared to the control for treatments with 0.25
µ
g/L
and 25 µg/L CYN (p<0.05) (Figure 2D).
The chlorophyll ato chlorophyll bratio showed different alterations concerning the four CYN
concentrations used (Figure 3A–D). With exposure to 0.025
µ
g/L and 0.25
µ
g/L CYN, a significant
decrease in the chlorophyll ato bratio was only seen after 168 h (p<0.05) (Figure 3A,B). No significant
effects were observed with exposure to 2.5
µ
g/L (Figure 3C). After 24 h, the chlorophyll ato bratio
decreased with exposure to 25
µ
g/L CYN only (p<0.05), which then returned to the statistically
similar ratio as the control (p>0.05) (Figure 3D). The carotenoids to total chlorophyll ratio decreased
significantly compared to the control after 24 h with exposure to 0.25
µ
g/L, 2.5
µ
g/L, and 25
µ
g/L
(
p<0.05
; Figure 3F–H), but recovered within 48 to 96 h of the disturbance. With exposure to 0.025
µ
g/L,
no significant alterations in the ratio of carotenoids to chlorophyll were observed (Figure 3E).
Toxins 2019,11, 650 5 of 13
Toxins 2019, 11, x FOR PEER REVIEW 5 of 14
Figure 2. The (A) chlorophyll a, (B) chlorophyll b, (C) total chlorophyll, and (D) carotenoid content in
L. minor with exposure to different concentrations of CYN. Data represent mean pigment
concentration ± standard deviation of three independent samples, each determined three times (n =
9). Significance compared to the control is shown by an asterisk (* p < 0.05).
0
100
200
300
400
500
600
700
800
024 48 96 168
Chl a (µg/g FW)
control 0.025 µg/L 0.25 µg/L 2.5 µg/L 25 µg/L
0
50
100
150
200
250
300
350
400
024 48 96 168
Chl b (µg/g FW)
control 0.025 µg/L 0.25 µg/L 2.5 µg/L 25 µg/L
0
200
400
600
800
1000
1200
024 48 96 168
Total Chl (µg/g FW)
control 0.025 µg/L 0.25 µg/L 2.5 µg/L 25 µg/L
0
25
50
75
100
125
150
175
200
024 48 96 168
Total Carotenoids (µg/g FW)
control 0.025 µg/L 0.25 µg/L 2.5 µg/L 25 µg/L
Exposure time (hours)
**
**
*
*
*
*
*
*
*
**
***
**
Chl a(µg/g FW)
Chl b(µg/g FW)
Total Chl (µg/g FW)
Total Carotenoids (µg/g FW)
A
B
C
D
**
0
100
200
300
400
500
600
700
800
024 48 96 168
Chl a (µg/g FW)
control 0.025 µg/L 0.25 µg/L 2.5 µg/L 25 µg/L
0
50
100
150
200
250
300
350
400
024 48 96 168
Chl b (µg/g FW)
control 0.025 µg/L 0.25 µg/L 2.5 µg/L 25 µg/L
0
200
400
600
800
1000
1200
024 48 96 168
Total Chl (µg/g FW)
control 0.025 µg/L 0.25 µg/L 2.5 µg/L 25 µg/L
0
25
50
75
100
125
150
175
200
024 48 96 168
Total Carotenoids (µg/g FW)
control 0.025 µg/L 0.25 µg/L 2.5 µg/L 25 µg/L
Exposure time (hours)
**
**
*
*
*
*
*
*
*
**
***
**
Chl a(µg/g FW)
Chl b(µg/g FW)
Total Chl (µg/g FW)
Total Carotenoids (µg/g FW)
A
B
C
D
**
Figure 2.
The (
A
) chlorophyll a, (
B
) chlorophyll b, (
C
) total chlorophyll, and (
D
) carotenoid content in
L. minor with exposure to different concentrations of CYN. Data represent mean pigment concentration
±
standard deviation of three independent samples, each determined three times (n=9). Significance
compared to the control is shown by an asterisk (* p<0.05).
Toxins 2019,11, 650 6 of 13
Toxins 2019, 11, x FOR PEER REVIEW 7 of 14
Figure 3. The ratio of chlorophyll a to b in L. minor with exposure to (A) 0.025 μg/L, (B) 0.25 μg/L, (C)
2.5 μg/L, and (D) 25 μg/L CYN as well as the ratio of carotenoids to total chlorophyll with exposure
to (E) 0.025 μg/L, (F) 0.25 μg/L, (G) 2.5 μg/L, and (H) 25 μg/L CYN. Significance compared to the
control is shown by an asterisk (* p < 0.05).
1.5
2
2.5
3
3.5
024 48 96 168
Chl a/Chl b
control 25 µg/L
0.12
0.14
0.16
0.18
0.2
0.22
024 48 96 168
Carotenoids/Total Chlorophyll
control 0.025 µg/L
0.12
0.14
0.16
0.18
0.2
0.22
024 48 96 168
Carotenoids/Total Chlorophyll
control 0.25 µg/L
0.12
0.14
0.16
0.18
0.2
0.22
024 48 96 168
Carotenoids/Total Chlorophyll
control 2.5 µg/L
0.12
0.14
0.16
0.18
0.2
0.22
024 48 96 168
Carotenoids/Total Chlorophyll
control 25 µg/L
1.5
2
2.5
3
3.5
024 48 96 168
Chl a/Chl b
control 0.25 µg/L
1.5
2
2.5
3
3.5
024 48 96 168
Chl a/Chl b
control 2.5 µg/L
Exposure time (hours)
Chl a/Chl b
Chl a/Chl b
Carotenoids/Total Chl
Carotenoids/Total Chl
*
*
*
*
**
**
B
CD
EF
GH
1.5
2
2.5
3
3.5
024 48 96 168
control 0.025 µg/L
A
*
1.5
2
2.5
3
3.5
024 48 96 168
Chl a/Chl b
control 25 µg/L
0.12
0.14
0.16
0.18
0.2
0.22
024 48 96 168
Carotenoids/Total Chlorophyll
control 0.025 µg/L
0.12
0.14
0.16
0.18
0.2
0.22
024 48 96 168
Carotenoids/Total Chlorophyll
control 0.25 µg/L
0.12
0.14
0.16
0.18
0.2
0.22
024 48 96 168
Carotenoids/Total Chlorophyll
control 2.5 µg/L
0.12
0.14
0.16
0.18
0.2
0.22
024 48 96 168
Carotenoids/Total Chlorophyll
control 25 µg/L
1.5
2
2.5
3
3.5
024 48 96 168
Chl a/Chl b
control 0.25 µg/L
1.5
2
2.5
3
3.5
024 48 96 168
Chl a/Chl b
control 2.5 µg/L
Exposure time (hours)
Chl a/Chl b
Chl a/Chl b
Carotenoids/Total Chl
Carotenoids/Total Chl
*
*
*
*
**
**
B
CD
EF
GH
1.5
2
2.5
3
3.5
024 48 96 168
control 0.025 µg/L
A
*
Figure 3.
The ratio of chlorophyll ato bin L. minor with exposure to (
A
) 0.025
µ
g/L, (B) 0.25
µ
g/L,
(
C
) 2.5
µ
g/L, and (
D
) 25
µ
g/L CYN as well as the ratio of carotenoids to total chlorophyll with exposure
to (
E
) 0.025
µ
g/L, (
F
) 0.25
µ
g/L, (
G
) 2.5
µ
g/L, and (
H
) 25
µ
g/L CYN. Significance compared to the control
is shown by an asterisk (* p<0.05).
The results suggest that exposure to CYN causes an increase in the concentration of the chlorophyll
aand carotenoids within 24 h at CYN concentrations under 25
µ
g/L. In previous studies, the effects
reported on the chlorophyll aand bcontents with exposure to CYN have been contradictory [
11
,
13
].
Toxins 2019,11, 650 7 of 13
Increased chlorophyll acontent, accompanied by increased carotenoids, may indicate an effect on the
maintenance of the complex chlorophyll-carotenoid binding proteins, which are responsible for the
absorption and conversion of light energy during photosynthesis. The increase of the carotenoids
after 24 h suggest their activity in response to exposure to the xenobiotic CYN in the antioxidative
system to combat excessive ROS formation [
30
]. For the highest CYN exposure concentration (25
µ
g/L),
the stabilisation could only occur later (96 to 168 h), probably after initial damage due to exposure was
overcome by the antioxidative stress enzymes [
15
]. Santos et al. [
11
] also reported increased carotenoid
levels with CYN exposure in A. filiculoides. The changes in the carotenoid contents may be related to
ROS production and plant growth. ROS production and signalling are integrated with the action of
phytohormones in the coordinate regulation of plant growth and stress tolerance [
31
]. Many plant
hormones generate ROS as part of the mechanism that regulates plant growth and development [
31
].
Moreover, the observed changes in the carotenoids to total chlorophyll ratios showed the stabilisation
of the photosynthetic system in L. minor after 96 h for the higher CYN concentrations.
2.3. Plant Growth
In general, changes in the number of fronds with exposure to the different CYN concentrations
were divergent. The number of fronds with exposure to 0.25 and 2.5
µ
g/L CYN were higher compared
to the control (Figure 4B,C), while for 25
µ
g/L CYN, the number of fronds decreased relative to the
control (Figure 4D). With exposure to the lowest concentration, 0.025
µ
g/L CYN, no effects were
observed (Figure 4A).
The L. minor growth evaluated by calculating the relative plant growth (RG) aswell was dissimilar
for the four CYN treatments (Figure 4E). After 4 days, the RG increased significantly only with exposure
to 0.25
µ
g/L CYN (p<0.05) and remained significantly higher until the end of the exposure period.
Interestingly, after 7 days, the RG of the plants grown in the presence of 25
µ
g/L decreased significantly
compared to the control (p<0.05). After the 8th day of exposure, the RG for plants exposed to
0.025
µ
g/L and 2.5
µ
g/L showed a significant increase. Except for plants exposed to 0.25
µ
g/L and
25 µg/L CYN, the RG recovered by the next sampling point.
In two studies by Kinnear et al. [
10
,
13
] with L. punctata and H. verticillata, an increased relative
growth was reported with exposure to CYN. Similarly, Santos et al. [
11
] reported an increase in the
growth of A. filiculoides with exposure to CYN and attributed the observation to it being an approach
to combat the toxicity.
The higher increase in the number of fronds at lower concentrations can be related to hormetic
responses, where low-stress conditions can induce growth. Hormesis has been demonstrated in
L. minor exposed to different pesticides [
32
]. However, this study cannot confirm the hormetic response
in L. minor exposed to CYN, but this assumption could be considered in other investigations.
The decreases of the chlorophyll ato bratio seen at the low exposure concentrations after 168 h may
be explained by the higher increases in the number of fronds at these concentrations. The appearance of
new fronds in L. minor occurs by division from mother fronds. The senescence of the mother fronds was
observed in the experiment; this can affect the change of pigment contents. Additionally, more fronds
at the same surface tend to compete for light.
The increase in the number of fronds below the control at the highest CYN concentration can
be related to changes in the carotenoids to total chlorophyll ratios. Some phytohormones regulating
growth derivate from carotenoid precursors [
33
]. A decrease in the carotenoid contents related to total
chlorophyll can collaborate to delay growth. In this way, L. minor can reduce ROS formation from plant
growth until the enzymatic antioxidant and non-enzymatic antioxidant system regulates oxidative
stress caused by CYN. The stabilisation of the carotenoids to total chlorophyll ratio after 96 h and
168 h may show the regulation of biosynthesis and storage of carotenoids in L. minor, which, therefore,
leads to stabilise growth.
Toxins 2019,11, 650 8 of 13
Toxins 2019, 11, x FOR PEER REVIEW 9 of 14
Figure 4. The growth of L. minor in terms of mean number of fronds (n = 3) with exposure to (A) 0.025
μg/L, (B) 0.25 μg/L, (C) 2.5 μg/L, and (D) 25 μg/L CYN, as well as mean relative plant growth with
exposure to the four CYN concentrations (E). Significance compared to the control is shown by an
asterisk (* p < 0.05).
The higher increase in the number of fronds at lower concentrations can be related to hormetic
responses, where low-stress conditions can induce growth. Hormesis has been demonstrated in L.
minor exposed to different pesticides [32]. However, this study cannot confirm the hormetic response
in L. minor exposed to CYN, but this assumption could be considered in other investigations.
0.00
2.00
4.00
6.00
8.00
10.00
2 3 4 7 8 9 10 11 14
Days
Relative plant growth
0 µg/L 0.025 µg/L 0.25 µg/L 2.5 µg/L 25 µg/L
0
20
40
60
80
100
120
140
160
180
0 2 4 6 8 10 12 14 16
0 µg/L 0.025 µg/L
0
20
40
60
80
100
120
140
160
180
0 2 4 6 8 10 12 14 16
0 µg/L 0.25 µg/L
0
20
40
60
80
100
120
140
160
180
0 2 4 6 8 10 12 14 16
0 µg/L 2.5 µg/L
0
20
40
60
80
100
120
140
160
180
0 2 4 6 8 10 12 14 16
0 µg/L 25 µg/L
Number of fronds
Number of fronds
Number of fronds
Number of fronds
Days Days
Days Days
Relative plant growth
Days
*
*
*
****
*
*
*
*
*
*
*
A B
CD
E
0.00
2.00
4.00
6.00
8.00
10.00
2 3 4 7 8 9 10 11 14
Days
Relative plant growth
0 µg/L 0.025 µg/L 0.25 µg/L 2.5 µg/L 25 µg/L
0
20
40
60
80
100
120
140
160
180
0 2 4 6 8 10 12 14 16
0 µg/L 0.025 µg/L
0
20
40
60
80
100
120
140
160
180
0 2 4 6 8 10 12 14 16
0 µg/L 0.25 µg/L
0
20
40
60
80
100
120
140
160
180
0 2 4 6 8 10 12 14 16
0 µg/L 2.5 µg/L
0
20
40
60
80
100
120
140
160
180
0 2 4 6 8 10 12 14 16
0 µg/L 25 µg/L
Number of fronds
Number of fronds
Number of fronds
Number of fronds
Days Days
Days Days
Relative plant growth
Days
*
*
*
****
*
*
*
*
*
*
*
A B
CD
E
Figure 4.
The growth of L. minor in terms of mean number of fronds (n=3) with exposure to
(
A
) 0.025
µ
g/L, (
B
) 0.25
µ
g/L, (
C
) 2.5
µ
g/L, and (
D
) 25
µ
g/L CYN, as well as mean relative plant growth
with exposure to the four CYN concentrations (
E
). Significance compared to the control is shown by an
asterisk (* p<0.05).
3. Conclusions
In agreement with previous studies in aquatic macrophytes with high concentrations,
CYN exposure
at environmentally relevant concentrations did not result in bioaccumulation.
In response
to exposure, the pigment concentrations were elevated for a short period, probably
to combat the adverse effects of the xenobiotic. Relative plant growth was stimulated; however,
Toxins 2019,11, 650 9 of 13
this was previously proposed to be a response to stress due to CYN exposure. L. minor can tolerate
and even combat adverse effects experienced from exposure to CYN at environmentally reported
concentrations; however, as it does not remove large amounts of CYN from its environment after
the first 24 h of exposure or bioaccumulate CYN continuously, it is not an ideal candidate alone for
sustainable phytoremediation. It is necessary to continue studies on CYN uptake and CYN removal
with other macrophytes to be able to have more tools for designing methods to control CYN in
aquatic environments.
4. Materials and Methods
4.1. Plant Material
L. minor was provided by Wakus (Wakus GmbH Wasserpflanzenkulturen, Germany). The identity
of the macrophyte was verified according to Rothmaler [
34
] and in accordance with the rules given by
the Botanical Society of Britain and Ireland [
35
]. A culture of L. minor was maintained in glass tanks
(60 cm
×
60 cm
×
60 cm) in modified Provasoli’s medium consisting of de-ionized water containing
CaCl
2
(0.20 g/L), NaHCO
3
(0.11 g/L), and sea salt (chloride, sodium, sulfate, potassium, calcium,
carbonate, boron, magnesium, strontium; 0.10 g/L), under cool white fluorescent light (50
µ
E/m
2·
s
irradiation measured with a light meter) with a 14:10 h light:dark photoperiod at 20
±
1
◦
C [
15
]. These
cultivation conditions were also used for all exposures.
4.2. Chemicals
CYN standard (purify >95%) was purchased from Alexis Biochemicals (Lausen, Switzerland) and
dissolved in 70% methanol to obtain a stock solution. All chemicals used in Laboratory experiments
were of analytical-grade quality and were obtained from Sigma-Aldrich, Inc. (Darmstadt, Germany).
4.3. CYN Treatments
Four concentrations of CYN (0.025, 0.25, 2.5, and 25
µ
g/L) were prepared from the stock by
dilution with culture medium. The exposure concentrations were selected according to typical
CYN concentrations previously recorded in aquatic environments [
4
,
19
,
20
]. Before the beginning
of the experiment, the plants were pre-cultured in the exposure vessels to acclimate for seven days.
The experiments were carried out with non-axenic plants under nonsterile conditions. Per treatment,
fronds of L. minor with a total fresh weight (FW) of 2.5
±
0.5 g were exposed to the four specified CYN
concentrations, respectively, in a volume of 150 mL (surface area of 70.8 cm
2
) under the controlled
culture conditions described above for 168 h. Each treatment and its negative control in parallel was
prepared with its independent replicates.
For CYN uptake, only the highest exposure concentration of 25
µ
g/L CYN was used due to the
limitations of the analytical method (limit of quantification was 10 pg on column; see Section 4.4).
The treatment and control replicates (n=4) were sampled after 0.02 h, 1 h, 2 h, 24 h, 48 h, 96 h,
and 168 h
of exposure in order to analyse the uptake of CYN using liquid chromatography–tandem
mass spectrometry (LC–MS/MS, Section 4.4). Assuming that natural degradation of CYN could occur,
an additional
positive control (25
µ
g/L CYN medium without plants; n=4) was set up concomitantly
and sampled at the same exposure times.
The experimental setup was repeated for the analysis of the pigment content and plant growth
with all four exposure concentrations (n=3). Photosynthetic pigment contents were measured at
all concentrations from the samples after 24 h, 48 h, 96 h, and 168 h and analysed as described in
Section 4.5. After exposure, plants were washed twice with 100 mL of de-ionised water to remove any
remaining toxin from the plant surfaces. The plants were shock frozen in liquid nitrogen and stored at
−80 ◦C for further biochemical analysis.
Toxins 2019,11, 650 10 of 13
4.4. Analysis of CYN
Free CYN was extracted from the plant tissue, according to Esterhuizen-Londt et al. [
29
],
with slight modifications. The frozen plant material was ground in liquid nitrogen to a fine powder
and lyophilised (LIO-5P freeze-dryer Kambiˇc Laboratorijska oprema doo, Semiˇc, Slovenija) overnight
(
−
50.3
◦
C; 6.1 mbar). The lyophilised samples (500 mg) were further homogenised mechanically using a
Tissuelyser LT (Qiagen, Hilden, Germany), then suspended in 200
µ
L 95% acetonitrile (ACN) followed
by ultrasonic treatment for 30 min in an ultrasonic water bath (Alpax, Gmbh and Co. KG, Goldach,
Switzerland). The samples were continuously shaken in the dark for 30 min at room temperature using
an overhead Intelli-Mixer RM-2 (Neolab, Heidelberg, Germany). Extracts were then centrifuged at
20,800
×
gfor 15 min at 4
◦
C (Eppendorf Centrifuge 5417 R, Hamburg, Germany). The pellets were
suspended in 300
µ
L of 95% ACN and again, centrifuged as before. Supernatants were combined and
analysed by LC–MS/MS. The concentrations of CYN in the exposure media were also analysed by
LC–MS/MS.
Chromatographic separation of CYN was achieved with a Kinetex HILIC column (2.6
µ
m,
2.1 ×100 mm
) by liquid chromatography (1200 infinity Series, Agilent, Waldbronn, Germany) coupled
to triple quadrupole mass spectrometry (model 6460 Triple Q
TM
, Agilent) with electrospray ionization
(Jet-Stream, Agilent, Santa Clara, CA, USA) according to Esterhuizen-Londt et al. [
29
]. The column
oven temperature was set to 35
◦
C, and an injection volume of 10
µ
L was used for each sample at
a flow rate of 0.5 mL/min. Compound separation was achieved using a gradient elusion starting at
95% ACN (MS grade) for 3 min, which was then decreased to 50% over 4 min with a post time of 3
min, resulting in a retention time of 4.1 min for CYN. For the subsequent MS–MS detection, the MRM
mode (positive mode) was used with a mass transfer of 416 (Q1) to 176 and 194 (Q3) for CYN. The
drying gas temperature and flow settings were 320
◦
C and 12 L/min, respectively and the sheath
gas temperature and flow were set to 380
◦
C and 12 L/min using nitrogen gas. The capillary voltage
applied was 4500 V, and the nozzle voltage was set to 1200 V, together with a nebuliser pressure of
25 psi. The calibrations used for quantification of CYN were linear (R
2
=0.998) between 0.01 and
100 µg/L. The limit of quantification was set at 10 pg on column (S/N≥5).
The bioconcentration factors (BCF) per exposure time were calculated by dividing the CYN
concentration in the plant tissue (ng/g FW) by the corresponding CYN concentration in the media
(ng/mL) [36].
4.5. Photosynthetic Pigment Contents
Chlorophyll and carotenoid contents were measured according to Inskeep and Bloom [
37
] and
Wellburn [
38
], respectively. The plant samples were ground to a fine powder in liquid nitrogen, and
a total of 0.05 g FW were suspended in 5 mL of N,N-dimethylformamide (N,N-DMF) in the dark at
4
◦
C for three days. The extraction solution was centrifuged (Eppendorf Centrifuge 5417 R, Hamburg,
Germany) at 20,800
×
gfor 15 min at 4
◦
C. Spectrophotometric analysis was carried out in the dark in
triplicate at 647 nm, 664.5 nm (for chlorophyll aand brespectively), and 480 nm (for carotenoids) with
1 cm quartz cuvettes.
4.6. Plant Growth Determination
L. minor plants were exposed to four concentrations of CYN (0.025, 0.25, 2.5, and 25
µ
g/L) for
14 days. The exponential growth period was previously assessed (doubling time of frond number
<2.5 days). A total of nine individual colonies, each with two fronds, were inoculated in 100 mL CYN
solution. The number of fronds from each treatment were counted from the second day and within
two weeks. Plant growth was expressed as relative plant growth (RG) according to Equation 1 [
39
] as
follows:
RG =(Nt−N0)/N0
Toxins 2019,11, 650 11 of 13
where N
t
is the number of fronds at day t and N
0
is the number of fronds at the beginning of
the experiment.
4.7. Statistics
All statistical analysis was performed using SPSS software (IBM SPSS Statistics, Version 20,
IBM Corporation, New York, NY, USA). Differences between treatments (toxin concentrations) and
corresponding controls were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s
post-hoc at an alpha level of p=0.05. Before the ANOVA test, normality and homogeneity of variance
among groups were tested by Shapiro–Wilk and Levene’s test, respectively. When necessary, data were
transformed for normalisation to reduce heterogeneity. When the assumptions of homogeneity were
not satisfied, a nonparametric Levene’s test was performed. In this case, significant differences between
treatments and controls were analyzed by the nonparametric Kruskal–Wallis test.
Author Contributions:
Conceptualization, S.P., M.E.-L. and N.C.F.-R.; methodology, S.P., N.C.F.-R., and M.E.-L.,
validation, M.E.-L., analysis, N.C.F.-R. and M.E.-L.; resources, M.E.-L. and S.P.; writing—original draft preparation,
N.C.F.-R.; writing—review and editing, M.E.-L.; supervision, M.E.-L. and S.P.; project administration, M.E.-L. and
S.P.; funding acquisition, S.P.
Funding: This research received no external funding.
Acknowledgments:
The authors thank Sandra Kühn for technical assistance in the laboratory. Open access
funding provided by University of Helsinki.
Conflicts of Interest: The authors declare no conflict of interest.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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