Aquatic Toxicology 155 (2014) 142–150
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Aquatic Toxicology
journal homepage: www.elsevier.com/locate/aquatox
Capacity of the aquatic fern (Salvinia minima Baker) to accumulate
high concentrations of nickel in its tissues, and its effect on plant
physiological processes
Ignacio I. Fuentes, Francisco Espadas-Gil, Carlos Talavera-May, Gabriela Fuentes,
Jorge M. Santamaría ∗
Centro de Investigación Científica de Yucatán, Calle 43 No. 130, Col. Chuburná de Hidalgo, 97200 Mérida, Yucatán, Mexico
a r t i c l e
i n f o
Article history:
Received 20 March 2014
Received in revised form 23 June 2014
Accepted 24 June 2014
Available online 30 June 2014
Keywords:
Nickel uptake and accumulation
Salvinia minima
Heavy metals
Phytoremediation
Physiological processes
a b s t r a c t
An experiment was designed to assess the capacity of Salvinia minima Baker to uptake and accumulate
nickel in its tissues and to evaluate whether or not this uptake can affect its physiology. Our results suggest
that S. minima plants are able to take up high amounts of nickel in its tissues, particularly in roots. In fact,
our results support the idea that S. minima might be considered a hyper-accumulator of nickel, as it is able
to accumulate 16.3 mg g−1 (whole plant DW basis). Our results also showed a two-steps uptake pattern
of nickel, with a fast uptake of nickel at the first 6 to 12 h of being expose to the metal, followed by a slow
take up phase until the end of the experiment at 144 h. S. minima thus, may be considered as a fern useful
in the phytoremediation of residual water bodies contaminated with this metal.
Also from our results, S. minima can tolerate fair concentrations of the metal; however, at concentrations higher than 80 ␮M Ni (1.5 mg g−1 internal nickel concentration), its physiological performance
can be affected. For instance, the integrity of cell membranes was affected as the metal concentration
and exposure time increased. The accumulation of high concentrations of internal nickel did also affect
photosynthesis, the efficiency of PSII, and the concentration of photosynthetic pigments, although at a
lower extent.
© 2014 Published by Elsevier B.V.
1. Introduction
Soil and water contamination with heavy metals has become
a worldwide problem, leading to losses in agricultural yields and
hazardous health effects, as they enter the food chain.
Of the many heavy metals known in nature, Ni is essential as a
trace element for normal plant growth and development, because it
is constituent of some important enzymes such as urease. At toxic
levels, however, it seems to affect a number of biochemical and
physiological processes in plants. Urease contains two Ni ions at the
active site (Ciurli, 2001). However, high concentration of Ni in the
growth medium can lead to toxicity symptoms and reduced growth
Abbreviations: S. minima, Salvinia minima; Ni, nickel; Hoagland, nutrient solution; FQ, phytochelatins; MT, metallothionein; Ci , internal concentration of CO2 ; Pn,
photosynthesis; DW, dry weight; FW, fresh weight; EL, electrolyte leakage; BCF, bioconcentration factor; EDTA, ethylene diamine tetra acetic acid; Fv/Fm, maximum
efficiency of photosystem II; PPFD, photosynthetic photon flux density; ANOVA,
analysis of variance; SD, standard deviation.
∗ Corresponding author. Tel.: +52 01 999 9428330; fax: +52 01 999 9813900.
E-mail addresses: [email protected], [email protected] (J.M. Santamaría).
http://dx.doi.org/10.1016/j.aquatox.2014.06.016
0166-445X/© 2014 Published by Elsevier B.V.
of plants (Seregin and Kozhevnikova, 2006). Toxicity may result
from the binding of metal to sulfhydryl groups involved in the catalytic action or structural integrity of enzymes. Toxic effects of high
concentrations of Ni in growth medium on plants include alteration
in the uptake of essential nutrients, chlorosis, reduced CO2 uptake,
gas exchange disturbances, alterations in water uptake and generation of free radicals and oxygen reactive species that produce
oxidative stress (Seregin and Kozhevnikova, 2006; Ma et al., 2009).
Nickel can also replace Zn or Fe, and other metal ions, in certain
other metalloenzymes of lower plants (Mulrooney and Hausinger,
2003).
Aquatic plants possess an immense potential to remove heavy
metals from wastewater, but not all plants have the same effectiveness for metal removal. They differ in both, the capacity to
accumulate the heavy metal in roots and in the proportion of
metal transferred from roots to aerial parts (Suñe; et al., 2007).
The uptake of heavy metals by aquatic plants is also dependent
on many environmental factors (i.e. temperature, light intensity,
nutrient availability, salinity, presence of other metals, oxygen
level, etc.) (Olguin et al., 2007). Many aquatic ferns have a large
potential for metal removal from wastewaters, specially, in tropical
I.I. Fuentes et al. / Aquatic Toxicology 155 (2014) 142–150
and subtropical regions (Olguin et al., 2007; Suñe et al., 2007).
Floating macrophytes such as Eichhornia crassipes (Mart.) Solms.,
Pistia stratiotes L., and Salvinia herzogii de la Sota, have been studied because of their contaminant removal capacity from water
and their subsequent use in wetlands constructed for wastewater treatment (Delgado et al., 1993; Sen and Bhattacharyya, 1994;
Banerjee and Sarker, 1997; Maine et al., 2001, 2004; Hadad et al.,
2006). In most cases, the research goal was assessing contaminant
removal efficiencies. However, studies of bioaccumulation process
by macrophytes and their toxic effects, would allow us to determine their tolerance and provide basic information related to the
potential use of locally available macrophytes for water depuration
(Cardwell et al., 2002).
The genus Salvinia has about 20 species found in tropical and
temperate regions of the world. They show a high growth rate and
great capacity to survive under adverse environmental conditions
(Oliver, 1993). All Salvinia species can remove heavy metals from
wastewaters and polluted water bodies (Olguin et al., 2007; Dhir
et al., 2009). Heavy metals accumulation in Salvinia is normally
rapid and involves the passive uptake through adsorption of metal
ions, onto the plant surface and/or active uptake into plant cells
(Suñe et al., 2007). Although many studies have shown the ability of Salvinia to remove heavy metals (Olguin et al., 2007; Prado
et al., 2010), there is very limited information on the effect of those
metals on physiological and metabolic parameters involved in the
capacity to accumulate nickel.
The aim of the present work was to analyze the capacity of S.
minima to remove nickel from aqueous solutions and to evaluate
its physiological effects when exposed to Ni at different concentrations and exposure times. The physiological processes studied
were photosynthetic rate, chlorophyll fluorescence (Fv/Fm), photosynthetic pigments concentration and electrolytes leakage as an
estimate of membrane integrity in leaves and roots of S. minima
exposed to different Ni concentrations.
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2.3. Determination of internal Ni concentrations in tissues
At each sampling time, plants from each treatment were washed
with 10 mM EDTA pH 8.0 followed by a rinse with de-ionized water,
to remove external metal ions (Hoffmann et al., 2004). Plants of S.
minima were separated in leaves and roots, and frozen in liquid
nitrogen. Samples were ground and stored at −80 ◦ C until analysis.
Tissues were freeze-dried and 20 mg were mixed with 200 ␮L of
HNO3 (69.9%) and incubated for 3 h at 80 ◦ C in a sand bath in tightly
closed 2 mL reaction plastic tubes. The fully digested biomass was
brought up to 1.8 mL with de-ionized water and strongly agitated
for 5 min. The tubes were centrifuged at 13,000 × g for 15 min.
Before the measurement, the supernatant was filtered (Millipore
0.45 nm), and the supernatant was transferred to a new tube and
stored at −20 ◦ C. The Ni quantification was performed with the ICPOES. The internal Ni concentration was expressed as mg g−1 DW as
in Hoffmann et al. (2004). The bio-concentration factor (BCF) was
calculated as the quotient of the measured concentration of metal
in the plant material (mg Kg−1 DW) per initial metal concentration
in solution (mg L−1 ) (Walker, 1987; Madeira et al., 2003; Hoffmann
et al., 2004).
2.4. Determination of pigment content
Pigments content of control and metal-treated plants were
determined according to the method of Nagata and Yamashita
(1992). One hundred mg of fresh plant tissue were shaken with 5 mL
of acetone–hexane mixture (4:6) for 1 min. The extract was then
centrifuged at 2500 rpm for 5 min. The absorbance of the filtrate
was measured at 505, 645 and 663 nm (UV–vis spectrophotometer, DU-650, Beckman Coulter, USA). Contents of chlorophyll a and
b were calculated according to equations of Nagata and Yamashita
(1992).
2.5. Photosynthesis measurements
2. Materials and methods
2.1. Salvinia minima growing culture
Plants of the aquatic fern S. minima Baker (Salviniaceae) ecotype Yucatán, were cultivated under hydroponics conditions using
a modified Hoagland’s solution (Hoagland and Arnon, 1950;
Hoffmann et al., 2004), at 25 ± 2 ◦ C, in a culture room with a photon flux density 100 ␮mol m−2 s−1 , a relative humidity of 70 ± 5%,
and artificial light photoperiod of 12 h. Plants were cultured under
the mentioned conditions for one month before they were separated in different groups and each group was transferred to aqueous
solutions containing 0, 20, 40, 80, 160 ␮M of NiCl2 (Ni) where they
remained for 144 h. The environmental conditions (temperature,
light, photoperiod, RH) during the metal exposure remained the
same.
2.2. Determination of Ni concentrations in solutions
The quantification of Ni in the solution was made on 2 mL from
each medium in which S. minima plants were exposed to 0, 20, 40,
80, 160 ␮M of NiCl2 for 0, 0.5, 6, 12, 24, 48, 72, 96, 120 and 144 h and
subsequently analyzed in an atomic emission spectrometer inductively coupled to plasma (ICP-OES 400 Perkin Elmer). The detection
of Ni was carried out by readings at 221.647 nm using 1% HNO3 as
blank. A standard solution for nickel ions (100 ppm) was prepared
from analytical grade of NiCl2 (Sigma, USA). The working standards
were prepared by serial dilution of the standard stock solution and
they were used for spectrometer calibration.
The rate of photosynthesis for control and metal-treated plants
was determined with a portable photosynthetic system (LI-COR
Inc., Lincoln, Nebraska, USA), using three samples for treatment
(each with six fronds). Samples were placed in the leaf chamber under 100 ␮mol m−2 s−1 of light (PPFD). The readings were
determined within 60 s of having locked up the leaf chamber. The
measurements were made under the same conditions where the
plants were exposed to the metal.
2.6. Determination of photosynthesis–CO2 (A/Ci ) response curves
and photosynthesis–light (A/Q) response curves
In order to gain further understanding of the effect of nickel
on photosynthesis, non-exposed control plants and those exposed
to the maximum concentration of nickel in the medium (160 ␮M
NiCl2 ), were used, at the end of the 144 h exposure period, for
the construction of both A/Ci and A/Q curves. A portable LI-6400
(LI-COR Inc., Lincoln, Nebraska, USA) was used to measure net
assimilation of CO2 (A) in response to increasing levels of light (Q)
and in response to increasing internal leaf CO2 (Ci ). Light intensity
ranged from 0 to 1600 ␮mol m−2 s−1 automatically adjusted by a
red–blue light-emitting diode (LED) light source, at a constant CO2
concentration of 700 ␮mol mol−1 . On the other hand, Ci concentrations increasing from 0 to 1050 ␮mol mol−1 at a constant light
intensity of 700 ␮mol m−2 s−1 , at 25 ◦ C temperature and at ambient
humidity, with a 2 min acclimation interval between measurements. The functional photosynthetic parameters were calculated
by fitting rectangular hyperbola to the measured points using the
software Photosynthesis Assistant (1.1.2 for Windows by Parsons
and Ogston, Dundee Scientific, UK). Parameters examined included
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Fig. 1. Capacity of S. minima plants to remove nickel from aqueous solutions of NiCl2 , containing different initial concentrations (0, 20, 40, 80 and 160 ␮M NiCl2 ). Internal
nickel concentration found in leaves (A) and roots (B) over time, during an exposure period of 6 days. And the internal concentration of nickel found in roots and leaves (C),
and as a whole-plant (D), when expressed in terms of increasing concentrations of nickel in the medium, at the end of a 6 days exposure period for each initial concentration.
Points are means ± SD of 3 independent experiments. * indicate values differing significantly from the controls (P < 0.05).
apparent quantum efficiency (AQE), carboxylation efficiency (CE),
CO2 compensation point (CO2 ), light compensation point ( L ),
maximum photosynthesis (Amax ), and saturation point (Psat ).
2.7. Chlorophyll fluorescence measurement
Fluorescence emission for control and metal-treated plants was
assessed in fully expanded leaves, with a fluorescence modulated
systems analyzer (FMS2—Hansatech, Norfolk, UK). After 20 min
dark adaptation, each leaf disc was exposed to a saturation pulse
of high light intensity (2500 ␮mol m−2 s−1 ) for 2 s and fluorescence
variables (Fo, Fm, Fv, Fv/Fm) were determined.
2.9. Statistical analysis
Data from all the measured parameters from control and metaltreated plants were analyzed and compared by using a one-way
analysis of variance (ANOVA) followed by post hoc Duncan’s test
(P < 0.05) when necessary. Results were the means of three samples
for treatment, of two independent experiments ± standard deviation (SD). All data analyses were performed using Statgraphic plus
5.1 Software (Statistical graphics Corp., USA).
3. Results
2.8. Electrolyte leakage
3.1. Capacity of Salvinia minima to remove and to accumulate
nickel
Cell membrane stability for control and metal-treated plants
was measured by electrolyte leakage (EL) from leaf tissues using
the method described by Jiang and Huang (2002). The sampled
leaves were cut into discs 2 mm in diameter. The discs were rinsed
three times with distilled water and three discs were incubated in
a test tube containing 6 mL distilled water. The test tubes were agitated on a shaker for about 4 h and conductivity (C1) of the solution
was measured with a conductivity-meter (ORION, model 162). Leaf
discs then were heated in an oven at 80 ◦ C for 1 h, and the conductivity of the solution containing the tissue (C2) was measured after
the tubes had cooled down to room temperature, and they had been
agitated on a shaker for 1 h. The relative ion leakage was calculated
as (C1/C2) × 100.
S. minima plants were able to remove nickel from aqueous
solutions containing different initial Ni concentrations. Ni concentrations in water decreased with time, reaching a plateau after
about 4 days of exposure.
At the same time, S. minima plants accumulated high content of
nickel in both their leaves and roots (Fig. 1A and B) when exposed to
various nickel (NiCl2 ) concentrations. In both tissues, a two-steps
metal uptake was observed; a fast take up of Ni occurred during the
first 12 h, in all the concentrations tested, followed by a slow take
up of Ni later on (Fig. 1A and B.). Tissue accumulation of Ni in both
leaves and root was dose-dependent, the Ni concentration found in
roots and leaves increased with increasing Ni concentrations in the
hydroponic solution. Nickel accumulation was significantly higher
I.I. Fuentes et al. / Aquatic Toxicology 155 (2014) 142–150
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Fig. 2. Visual appearance of leaves of S. minima non-exposed control plants at 0 h, and at the end of a 144 h period ((A) and (C)), when compared to plants before (0 h) and
144 h after being exposed to 160 ␮M of nickel in the medium ((B) and (D)). Bar = 1 mm.
in roots than in leaves for all treatments (Fig. 1C). At the maximum
time of Ni exposure, the capacity of Ni bioaccumulation of Salvinia
roots were 10 mg g−1 DW, while in leaves it was 6 mg g−1 DW. The
maximum nickel accumulation (16.3 mg g−1 DW) was observed in
plants exposed to 160 ␮M nickel (Fig. 1D).
Furthermore, the nickel concentration, expressed as whole
plant basis, were 8.8 mg g−1 DW (BCF = 7429) at 20 ␮M of
Ni; 10.5 mg g−1 DW (BCF = 4496) at 40 ␮M, 14.4 mg g−1 DW
(BCF = 3013) at 80 ␮M and 16.3 mg g−1 DW (BCF = 1713) at 160 ␮M
initial concentration of nickel at the aqueous solution.
These results suggest that S. minima could be considered as a
hyper-accumulator fern for nickel and perhaps a good candidate for
phyto-remediation of water bodies contaminated with this metal.
3.2. Effect of Ni on plant morphology
S. minima plants showed no visual damage when exposed to
NiCl2 concentration of 0, 20, 40, 80 ␮M. However, when exposed
to higher metal concentrations (160 ␮M), a partial necrosis was
observed after being exposed for 144 h (Fig. 2D).
3.3. Effect of Ni on photosynthetic pigments
S. minima control plants showed at the beginning of the experiment a total chlorophyll concentration of nearly 13 ␮g g−1 FW,
that decreased slightly during the first 24 h of the experiment, but
remained steady during the rest of the experiment at values around
of 8 ␮g g−1 FW (Fig. 3A). When plants were exposed to NiCl2 in the
medium, the total chlorophyll concentration was reduced rapidly
during the first 30 min of exposure even at low Ni concentrations
of 20 ␮M, remained steady up to the 72 h exposure, but decreased
further from then onwards, reaching values as low as 2 ␮g g−1 FW,
after 144 h exposure to nickel concentrations of 160 ␮M (Fig. 3A).
Moreover, when expressed as a function of the concentration
of nickel in the medium, pigment contents decreased as the concentration of Ni in the medium increased. A significant reduction
occurred even at low Ni concentration of 20 ␮M when compared
to control plants. From then on the reduction in total chlorophyll concentration decreased almost linearly with increased Ni
concentration in the medium, reaching a significant reduction
(85%) at the end of the 144 h exposure to 160 ␮M Ni (Fig. 3D).
3.4. Effect of Ni on photosynthesis (Pn)
S. minima control plants showed a Pn of 1.6 ␮mol m−2 s−1 during the whole experiment. When plants were exposed to Ni in
the medium, no significant changes in Pn occurred during the first
40 min at any nickel concentration. However, after 1 h exposure,
a significant reduction in Pn occurred in plants exposed to 20, 40
and 60 ␮M of Ni, when compared to control plants, but no significant differences among these three concentrations occurred. When
plants were exposed to the higher Ni concentration, a significant
reduction in Pn occurred after 90 h exposure, reaching the lowest
Pn values at 144 h (Fig. 3B).
When expressed as a function of the concentration of Ni exposed
at the end of a 144 h period, Pn decreased as the concentration
of Ni in the medium increased. Even at low Ni concentration of
20 ␮M, Pn was significantly lower (almost 0) than that of control
plants. From then on, Pn decreased almost linearly with increasing
Ni concentration in the medium, reaching a significant reduction
(−2.7 ␮mol m−2 s−1 ) that means that those plants were no longer
photosynthetic but they were rather experiencing high respiration
rates, at the end of the 144 h exposure to 160 ␮M (Fig. 3E).
3.5. Effects of Ni on chlorophyll fluorescence (Fv/Fm)
S. minima control plants showed a fluorescence of chlorophyll of
PS II (measured as Fv/Fm) of around 0.85 during the whole experiment. On the other hand, plants exposed to nickel in the medium,
showed no significant effects on Fv/Fm during the first 72 h exposure, but from then on Fv/Fm decreased significantly in all metal
concentrations reaching values as low as 0.32, after 144 h exposure
to 160 ␮M of nickel (Fig. 3C).
When expressed in terms of increasing metal concentrations
in the medium, again a significant reduction in Fv/Fm occurred,
when compared to control plants, at nickel concentrations as low
as 20 ␮M. After that point, Fv/Fm values decreased almost linearly
as the concentration of nickel in the medium increased, reaching
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Fig. 3. Total chlorophyll content (A), net photosynthetic rate (B) chlorophyll fluorescence; Fv/Fm (C) of S. minima plants over time, when exposed to various NiCl2 concentrations in the medium (0, 20, 40, 80 and 160 ␮M) during a 6 days exposure period. The same parameters, when expressed in terms of increasing concentrations of nickel in
the medium, at the end of a 6 days exposure period ((D), (E), and (F)). Points are means ± SD of 3 independent experiments. * indicate values differing significantly from the
controls (P < 0.05).
values 62% lower than control plants after being exposed for 144 h
to nickel concentrations of 160 ␮M (Fig. 3F).
measured at non-exposed control plants (1.2 ␮mol m−2 s−1 ), indicating that the exposure to the metal did affect the carboxylation
efficiency of nickel-exposed plants.
3.6. Photosynthesis–CO2 (A/Ci ) response curves
3.7. Photosynthesis–light (Q) response curves
Elevating CO2 concentration in the air suppresses oxygenation reaction of Rubisco and increases photosynthesis of C3
plants. S. minima plants grown without nickel, displayed a typical A/Ci response curve under a constant light intensity of
700 ␮mol m−2 s−1 (Fig. 4A). On the other hand, S. minima plants
exposed to 160 ␮M NiCl2 in the medium, showed a significantly
lower A/Ci response curve. The carboxylation efficiency of Rubisco,
measured as the initial slope of the A–Ci curve (0.08 ␮mol m−2 s−1 ),
was affected when compared to the carboxylation efficiency
S. minima control plants grown without nickel, showed a typical A/Q response curves under non-limiting CO2 (700 ␮mol mol−1 )
(Fig. 4B). The data revealed a slightly lower light–response curve
from nickel-exposed plants (160 ␮M NiCl2 ), when compared to
that from non-exposed control plants. Control plants had a higher
light-saturated photosynthesis (380 ␮mol m−2 s−1 ) than those of
nickel-exposed plants (170 ␮mol m−2 s−1 ). The predicted Amax of
control plants was 16.5 ␮mol m−2 s−1 , that was higher than that for
I.I. Fuentes et al. / Aquatic Toxicology 155 (2014) 142–150
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3.9. Correlations between the internal Ni concentration found in
tissues and its effect on physiological leaf parameters
The internal Ni concentration found in tissues, showed high
correlations with detrimental effects on some physiological parameters evaluated. Fig. 6 shows that some processes were more
sensitive to the metal entering the tissues than others, for instance,
membrane integrity (as measured by EL) was more damaged as the
metal internal concentration increased (r2 = 0.84). Photosynthesis
on the other hand, was slightly affected when the internal nickel
concentration was up to 2 mg g−1 DW, but it rapidly decreased to 0
and even negative values at higher internal nickel concentrations
(Fig. 6B). Similarly, the efficiency of photosystem II (as measured
by the Fv/Fm ratio) was not affected when internal nickel concentrations reached values as high as 2.5 mg g−1 DW, but it rapidly
decreased to values as low as 0.5, at higher internal metal concentrations (Fig. 6D). Interestingly, a low correlation (r2 = 0.35)
was found between the leaf total chlorophyll concentration and
the internal metal concentrations reaching the tissues of exposed
plants (Fig. 6C), suggesting that the presence of the metal interfered in a less direct manner, with the oxidation or biosynthesis of
photosynthetic pigments.
This correlation analysis as a whole, might suggest that metal
stress affected mainly the activities of enzymes associated with
carbon assimilation of S. minima plants, while the light reaction
was less affected. Therefore, inhibition of photosynthetic rate by
metal stress in the present study, was mainly due to stomatal regulation and declined carboxylation efficiency, but less associated
with reduced inactivation of light-reaction processes.
4. Discussion
4.1. Is S. minima an hyper-accumulator of nickel?
Fig. 4. A/Ci response curve (Ci = intercellular CO2 ) (A), light response curve
(PPFD = photosynthetic photon flux density) (B), of S. minima non-exposed control
plants (䊉) or plants subjected to 160 ␮M of NiCl2 for 6 days ().* indicate values
differing significantly from the controls (P < 0.05).
nickel-exposed plants (11.6 ␮mol m−2 s−1 ). However, both treatments showed a similar initial slope in response to light, indicating
that the capacity for electron transport was less affected in S. minima plants exposed to 160 ␮M NiCl2 .
3.8. Effects of Ni on electrolyte leakage
The electrolyte leakage (EL) was determined as a measure of
membrane integrity in leaves and roots from S. minima plants
exposed to different Ni concentrations during a 144 h period. Control plants showed electrolyte leakage values of around 23% for
leaves and 32% for roots during the whole experiment (Fig. 5A and
B).
On the contrary, when plants were exposed to Ni in the medium,
a rapid increase in EL occurred as early as 6 h for all nickel concentrations. The EL remained without further changes up to 120 h,
when increased further at the end of the 144 h exposure period.
Damage in membrane stability was higher in leaves than in roots,
but the changes in EL occurred earlier in roots (Fig. 5A and B).
When expressed in terms of increasing concentrations of nickel
in the medium, in leaves EL values increased from values of 23% in
control plants to values of 54% at 20 ␮M, 59.9% at 40 ␮M, 62% at
80 ␮M and 73% at 160 ␮M, after 144 h of exposure (Fig. 5C).
For roots, EL values increased from 32% in control plants to values of 56% at 20 ␮M, 62% at 40 ␮M, 73% at 80 ␮M and 75% at 160 ␮M
(Fig. 5D).
S. minima have been reported as a fern useful in phytorremediaton of water bodies, in previous reports S. minima plants were
exposed to metals such as Pb, Cd, As (Hoffmann et al., 2004; Olguin
et al., 2007; Estrella et al., 2009) and the fern was capable to accumulate high concentrations of Pb and Cd but not As. No reports
existed however, on the capacity of S. minima to remove nickel
from water solutions, nor on whether it was capable to accumulate
nickel in its tissues. In the best of our knowledge, the present work
represents the first report on the fact that S. minima is an aquatic
plant that can be considered as an hyper-accumulator of nickel.
In fact, the capacity of S. minima to accumulate Ni can be considered higher than that for accumulating other metals assayed before
(Olguin et al., 2007; Estrella et al., 2009).
4.2. The nickel uptake capacity of S. minima is different in roots
and shoots?
In the present work, Ni concentrations in both roots and aerial
parts showed a non-linear relationship with the quantity of Ni
added in the medium. The accumulation of nickel in S. minima
plant tissues occurred especially in roots, after 6 days of exposure.
A similar type of non-linear increase in the uptake of Cu and Cd
was reported previously in water hyacinth (O’Keefe et al., 1984), in
Azolla (Sela et al., 1989) and in Pistia (Satkyala and Kaiser, 1997;
Maine et al., 2001). The heavy metal accumulation occurred more in
roots than in shoots in radish and spinach (Pandey, 2006). Vajpayee
et al. (2001) also reported higher accumulation of heavy metal (Cr)
in roots than in shoots of an aquatic plant (Vallisneria spiralis L.).
Higher metal concentrations in roots, than in leaves exposed to
nickel were observed in the present study, as it has been shown
in other previously reported studies in macrophytes (Sen and
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Fig. 5. Membrane integrity (electrolyte leakage; EL) of S. minima leaves (A) and roots (B) over time, when plants were exposed to various nickel initial concentrations in the
medium (0, 20, 40, 80 and 160 ␮M) during a 6 days exposure period. EL in leaves (A) and roots (B) when expressed in terms of increasing NiCl2 concentration in the medium
at the end of a 6 days exposure period for each initial NiCl2 concentration. Points are means ± SD of 3 independent experiments. * indicate values differing significantly from
the controls (P < 0.05).
Bhattacharyya, 1994; Banerjee and Sarker, 1997; Manios et al.,
2003; Göthberg et al., 2004; Paris et al., 2005). Therefore, a higher
tolerance of roots than that of shoots, together with a decreased
metal translocation to the shoots, might explain the high metal concentration in the roots that might be a common feature to respond
to different metals of the macrophytes studied.
Tissue concentration of Ni in both leaves and roots was dosedependent. The maximum nickel accumulation was observed in the
roots (10 mg g−1 DW) when plants were exposed to 160 ␮M nickel.
Those plants showed low translocation of nickel toward their aerial
parts. It has been suggested that roots of exposed plants may act as
a barrier against heavy metal translocation to upper parts, possibly
as a result of potential tolerance mechanism (Ernst et al., 1992).
It is difficult to ascertain that the concentration of Ni found in
leaves in our experiments was solely the result of translocation of
the metal from roots, as S. minima plants have floating leaves, and
the possibility exists that they may partly take up the metal directly
via the leaves, as they also have direct contact with the medium.
Maine et al. (2004) demonstrated that leaves of S. herzogii directly
absorb Cr from growth solution and this process is the main cause
of the increase of Cr in aerial parts.
4.3. How tolerant is S. minima to nickel exposure?
High concentration of nickel caused a decreased concentration
of photosynthetic pigments and photosynthetic rates. In addition,
the efficiency of PSII was also affected by nickel uptake. It is difficult
to define the exact mechanisms by which nickel affects the physiology of this aquatic plant, but our data on electrolyte leakage
suggests that an excess of nickel, might have caused cell membrane damage and this in turn, might have caused a disruption of
ionic homeostasis, leading to damage to the photosynthetic apparatus. The reduction of photosynthetic pigments concentration by
Ni exposure, is in line with other reports in S. molesta exposed to
50, 100 and 200 mM NaCl (Upadhyay and Panda, 2005). A decline
in chlorophyll content at very high concentrations suggests that
the metal did affect the chlorophyll synthesizing system and the
chlorophyllase activity.
The Fv/Fm ratio may have decreased in nickel exposed plants
as a result of an increased protective non-radiative energy dissipation (non-photochemical quenching) associated with a regulated
decrease in photochemistry and photosynthesis rates, photodamage of PSII centers, or both. The reduced Fv/Fm values for the
Ni-treated S. minima plants, indicate that Ni decreased the capacity for re-oxidizing QA during actinic illumination and increased
the excitation pressure on PSII. A decrease in Fv/Fm is known to be
closely associated with photo-inhibition.
Regarding our data on the effect of nickel on membrane permeability, nickel has been shown to increase membrane permeability
in rice and maize roots (Pavlovkin et al., 2006). Some authors
reported that changes of the membrane proteins or lipidic composition caused by heavy metals, induce structural damage and
alterations in membrane properties (Llamas et al., 2008). Different
concentrations of nickel had a significant effect on sugar leakage
I.I. Fuentes et al. / Aquatic Toxicology 155 (2014) 142–150
149
Fig. 6. Correlations between the internal concentration of nickel measured in the tissues (as a whole plant) when plants were exposed to concentrations from 0 to 160 ␮M
NiCl2 in the medium vs. EL (A), Pn (B), chlorophyll concentration (C) and Fv/Fm (D), measured in leaves of S. minima plants exposed to the corresponding NiCl2 initial
concentration. The r2 for polynomial fits are shown.
from roots of Zea mays seedlings to the culture (Llamas and Sanz,
2008). Our data might indicate that nickel did cause a structural
damage and alterations in S. minima membrane properties, as suggested by Llamas et al. (2008).
The generation of ROS may cause extensive damage to the
membrane by peroxidation of their constituent lipids (Eriyamremu
and Lolodi, 2010). Oxidative stress can seriously disrupt normal metabolism through oxidative damage to lipids, protein and
nucleic acids, leading to changes in the selective permeability of
bio-membranes and thereby to increased membrane leakage and
changes in the activity of membrane bound enzymes (Boominathan
and Doran, 2002). However, in the present study even when S. minima plants may have been suffering from toxic effects due to the
bioaccumulation of Ni, they continued to remove metal from the
aqueous solutions.
4.4. Which mechanisms might have S. minima to tolerate high
internal concentrations of nickel in their tissues?
In previous studies assayed with S. minima in our group, it was
found that when the fern was exposed to Pb, the concentration
of phytochelatins (Estrella et al., 2009) and glutathione (Estrella
et al., 2012) increased in its tissues. In addition, the activity of
enzymes involved in their synthesis (phytochelatin synthase: PCs,
and glutathione synthetase: Gs, respectively) also increased as did
the expression of their respective genes encoding those enzymes.
Phytochelatins are proteins involved in the detoxification of heavy
metals such as Pb, by chelating the metal into cell vacuoles (Estrella
et al., 2009), while glutathione plays an important role in ROS
detoxification mechanism (Estrella et al., 2012). In the present
study, we did not measure phytochelatins or glutathione contents,
but it would be likely that theses mechanisms could also be part of
a S. minima response to nickel, since both phytochelatins and glutathione are involved as part of the plant tolerance mechanism to
deal with heavy metals stresses.
We can then conclude, that S. minima Baker have a high capacity to accumulate nickel in their tissues and can be considered as
a fern useful for the phytoremediation of residual water bodies
contaminated with this metal. It can tolerate fair concentrations
of the metal; although at concentrations higher than 80 ␮M Ni, its
physiological performance can be affected.
Work is now in progress to undertake molecular studies in S.
minima plants exposed to nickel, to better understand the mechanism at gene level, that might be involved in the capacity of this
fern to take up high concentrations of Ni from aqueous solutions,
as well as to understand the mechanisms associated with its great
ability to cope with high levels of nickel in its tissues.
Acknowledgements
We wish to thank Dr. E Sauri (ITM, Yucatán, México) for his kind
support in facilitating the spectrophotometer for the quantification
of nickel. Likewise, we wish to thank Dr. L Pinzon (ITC, Yucatán,
México) for facilitating the portable photosynthesis equipment for
Pn measurements.
150
I.I. Fuentes et al. / Aquatic Toxicology 155 (2014) 142–150
References
Banerjee, G., Sarker, S., 1997. The role of Salvinia rotundifolia in scavenging aquatic
Pb(II) pollution: a case study. Bioprocess Eng. 17, 295–300.
Boominathan, R., Doran, P.M., 2002. Ni-induced oxidative stress in roots of the Ni
hyperaccumulator Alyssum bertolonii. New Phytol. 156, 205–215.
Cardwell, A.J., Hawker, D.W., Greenway, M., 2002. Metal accumulation in aquatic
macrophytes from southeast Queensland, Australia. Chemosphere 48, 653–663.
Ciurli, S., 2001. Electronic structure of the nickel ions in the active site of urease.
Chemistry (Easton) 2001, 99–100.
Delgado, M., Bigeriego, M., Guardiola, E., 1993. Uptake of Zn, Cr and Cr by water
hyacinths. Water Res. 27, 269–272.
Dhir, B., Sharmila, P., Saradhi, P.P., Nasim, S.A., 2009. Physiological and antioxidant
responses of Salvinia natans exposed to chromium-rich wastewater. Ecotoxicol.
Environ. Saf. 72, 1790–1797.
Estrella, G.N., Mendoza, C.D., Moreno, S.R., González, M.D., Zapata, P.O., Hernandez, A.M., Santamaria, J.M., 2009. The pb-hyperaccumulator aquatic fern Salvinia
minima Baker, responds to Pb2+ by increasing phytochelatins via changes in
SmPCS expression and in phytochelatin synthase activity. Aquat. Toxicol. 91,
320–328.
Estrella, G.N., Sauri, D.E., Omar, Z.P., Santamaría, J.M., 2012. Glutathione plays a
role in protecting leaves of Salvinia minima from Pb2+ damage associated with
changes in the expression of SmGS genes and increased activity of GS. Environ.
Exp. Bot. 75, 188–194.
Eriyamremu, G.E., Lolodi, O., 2010. Alterations in lipid peroxidation and some antioxidant enzymes in germinating beans (Vigna unguiculata) and maize (Zea mays)
exposed to nickel. Int. J. Bot. 6, 170–175.
Ernst, W.H.O., Verkleij, J.A.C., Scat, H., 1992. Metal tolerance in plants. Acta Bot. Neerl.
41, 229–248.
Göthberg, A., Greger, M., Holm, K., Bengtsson, B.E., 2004. Influence of nutrient levels
on uptake and effects of mercury, cadmium and lead in water spinach. J. Environ.
Qual. 33, 1247–1255.
Hadad, H., Maine, M.A., Bonetto, C., 2006. Macrophyte growth in a pilotscale
constructed wetland for industrial wastewater treatment. Chemosphere 63,
1744–1753.
Hoagland, D.R., Arnon, D.I., 1950. The water-culture method for growing plants
without soil. Calif. Agric. Exp. Stn., Circ. 347, 1–32.
Hoffmann, T., Kutter, C., Santamaría, J.M., 2004. Capacity of Salvinia minima Baker to
tolerate and accumulate As and Pb. Eng. Life Sci. 4, 61–65.
Jiang, Y., Huang, B., 2002. Protein alterations in tall fescue in response to drought
stress and abscisic acid. Crop Sci. 42, 202–207.
Llamas, A., Sanz, A., 2008. Organ-distinctive changes in respiration rates of rice plants
under nickel stress. Plant Growth Regul. 54, 63–69.
Llamas, A., Ullrich, C.I., Sanz, A., 2008. Ni2+ toxicity in rice: effect on membrane
functionality and plant water content. Plant Physiol. Biochem. 46, 905–910.
Nagata, M., Yamashita, I., 1992. Simple method for simultaneous determination of
chlorophyll and carotenoids in tomato fruit. Nippon Shokuhin Kogyo Gakkaishi
39, 925–928.
Ma, Y., Rajkumar, M., Freitas, H., 2009. Improvement of plant growth and nickel
uptake by nickel resistant plant growth promoting bacteria. J. Hazard. Mater.
166, 1154–1161.
Madeira, P.T., Jacono, C.C., Tipping, P., Van, T.K., Center, T.D., 2003. A genetic survey
of Salvinia minima in the southern United States. Aquat. Bot. 76, 127–139.
Maine, M.A., Duarte, M., Suñé, N., 2001. Cadmium uptake by floating macrophytes.
Water Res. 35, 2629–2634.
Maine, M.A., Suñé, N., Lagger, S.C., 2004. Chromium bioaccumulation: comparison of the capacity of two floating aquatic macrophytes. Water Res. 38, 1494–
1501.
Manios, T., Stentiford, E., Millner, P., 2003. The effect of heavy metals accumulation
on the chlorophyll concentration of Typha latifolia plants, growing in a substrate
containing sewage sludge compost and watered with metaliferus water. Ecol.
Eng. 20, 65–74.
Mulrooney, S.B., Hausinger, R.P., 2003. Nickel uptake and utilization by microorganisms. FEMS Microbiol. Rev. 27, 239–261.
O’Keefe, D.H., Hardy, J.K., Rao, R.A., 1984. Cadmium uptake by water hyacinth: effects
of solution factors. Environ. Pollut. 34, 133–147.
Oliver, J.D., 1993. A review of the biology of giant Salvinia (Salvinia molesta Mitchell).
J. Aquat. Plant Manage. 31, 227–231.
Olguin, E.J., Sanchez-Galvan, G., Perez-Perez, T., 2007. Assessment of the phytoremediation potential of Salvinia minima baker compared to Spirodela polyrrhiza
in high-strength organic wastewater. Water Air Soil Pollut. 181, 135–147.
Paris, C., Hadad, H., Maine, M.A., Suñe, N., 2005. Eficiencia de dos macrofitas flotantes
libres en la absorción de metales pesados. Limnetica 24, 237–244.
Pandey, S.N., 2006. Accumulation of heavy metals (Cd, Cr, Cu, Ni and Zn) in Raphanus
sativus L. and Spinacia oleracea L. plants irrigated with industrial effluent. J.
Environ. Biol. 27, 381–384.
Pavlovkin, J., Luxova, M., Mistrikova, I., Mistrik, I., 2006. Short and long-term effects
of cadmium on transmembrane electric potential (Em) in maize roots. Biol. Plant.
61, 109–114.
Prado, C., Rodriguez-Montelongo, L., Gonzalez, J.A., Pagano, E.A., Hilal, M.,
Prado, F.E., 2010. Uptake of chromium by Salvinia minima: effect on plant
growth, leaf respiration and carbohydrate metabolism. J. Hazard. Mater. 177,
546–553.
Satkyala, G., Kaiser, J., 1997. Studies on the effect of heavy metals Pollution on Pistia
stratiotes L. (water lettuce). Ind. J. Environ. Health 39, 1–7.
Sela, M., Carty, J., Taylor, E., 1989. The accumulation and the effect of heavy metals
on the water fern Azolla filiculoides. New Physiol. 112, 7–12.
Sen, A.K., Bhattacharyya, M., 1994. Studies of uptake and toxic effects of Ni(II) on
Salvinia natans. Water Air Soil Pollut. 78, 141–152.
Seregin, I.V., Kozhevnikova, A.D., 2006. Physiological role of nickel and its toxic
effects on higher plants. Russ. J. Plant Physiol. 53, 257–277.
Suñe, N., Maine, M.A., Sánchez, G., Caffaratti, S., 2007. Cadmium and chromium
removal kinetics from solution by two aquatic macrophytes. Environ. Pollut.
145, 467–473.
Upadhyay, R.K., Panda, S.K., 2005. Salt tolerance of two aquatic macrophytes Pistia
stratiotes and Salvinia molesta. Biol. Plant. 49, 157–159.
Vajpayee, P., Rai, U.N., Ali, M.B., Tripathi, R.D., Yadav, V., Sinha, S., Singh, S.N., 2001.
Chromium—induced changes in Vallisneria spiralis L. and its role in phytoremedation of tannery effluents. Bull. Environ. Toxicol. 67, 246–256.
Walker, C.H., 1987. Kinetic models for predicting bioconcentration factors. Environ.
Pollut. 44, 227–239.