Aquatic Toxicology 189 (2017) 115122

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Aquatic Toxicology
journal homepage: www.elsevier.com/locate/aqtox

Growth and physiological responses of a marine diatom (Phaeodactylum MARK

tricornutum) against two imidazolium-based ionic liquids ([C4mim]BF4 and
[C8mim]BF4)

Xiang-Yuan Denga,b, , Biao Chena, Da Lia, Xiao-Li Hua, Jie Chenga, Kun Gaoa, Chang-Hai Wangb
a
College of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212003, China
b
Jiangsu Provincial Key Laboratory of Marine Biology, Nanjing Agricultural University, Nanjing 210095, China

A R T I C L E I N F O A B S T R A C T

Keywords: Ionic liquids (ILs) have been considered as green substitutes for traditional organic solvents in many existing
Phaeodactylum tricornutum biological and chemical areas. However, they have high solubility and poor biodegradability in water, sug-
1-Butyl-3-methylimidazolium tetrauoroborate gesting that they could become persistent chemical pollutants in aquatic environment. The ability of two widely
1-Octyl-3-methylimidazolium tetrauoroborate used imidazolium-based ILs to aect the growth and physiological characteristics of a marine diatom
Reactive oxygen species (ROS)
(Phaeodactylum tricornutum) was investigated in this study. The diatom was exposed to dierent concentrations
Antioxidant enzymes
of 1-butyl-3-methylimidazolium tetrauoroborate ([C4mim]BF4) and 1-octyl-3-methylimidazolium tetra-
uoroborate ([C8mim]BF4) for 96 h within a batch-culture system. Results showed that [C4mim]BF4 and
[C8mim]BF4 were very stable in seawater during 96 h of exposure, and the compounds signicantly inhibited the
growth of P. tricornutum with 24, 48, 72 and 96 h EC50 values of 30.81, 28.53, 39.92, 45.88 mg L1 and 30.17,
23.36, 28.62, 31.37 mg L1, respectively. In addition, the photosynthetic activity and chlorophyll a synthesis of
P. tricornutum were inhibited by [C4mim]BF4 and [C8mim]BF4, indicating that the structural integrity of
chloroplasts of the diatom may be disrupted or damaged by the two ILs. Compared with that of the controls,
reactive oxygen species (ROS) level was increased by 0.65, 1.17, 1.85, 3.13, 2.94 times and 0.55, 1.77, 2.42,
3.45, 3.47 times in 5, 10, 20, 40 and 60 mg L1 [C4mim]BF4 and [C8mim]BF4 treatments, respectively. The
excessive ROS may cause lipid peroxidation, shortage of metabolic energy and decline of photosynthetic e-
ciency, which may be the main reason for toxicity of the two ILs to marine diatoms. To withstand the damaging
eects of excessive ROS, remarkable physiological and biochemical responses occurred in treatments with the
two ILs to protect the cells of P. tricornutum. Parameters such as soluble protein content, soluble sugar content,
and superoxide dismutase (SOD) and peroxidase (POD) activities of the diatom increased signicantly with
increasing concentrations of the two ILs at 96 h of exposure relative to the controls. These ndings not only
provide strong background for evaluating the ecological risks and toxicity of ILs in marine environment, but also
help to unravel the toxic mechanism of the two ILs to marine diatoms.

1. Introduction
Ionic liquids (ILs) have been considered as a promising green
substitute for traditional organic solvents and areused widely as media
for catalysts, organic syntheses, extractions in electrochemistry appli-
cations, and separation processes (Zhang et al., 2011; Moosavi and
Daneshvar, 2014). However, some previous studies indicated that ILs
could become persistent environmental pollutants because of their high
chemical stability, high solubility and poor biodegradability in water
(Docherty et al., 2007; Pham et al., 2016). Many researchers have
evaluated the toxicity of ILs at dierent trophic and biological levels,
and pointed out that the potential hazards of ILs to environment, par-
ticularly to aquatic environment, cannot be ignored (Pham et al., 2010;
Cvjetko Bubalo et al., 2014b). As we all know, the marine environment
is regarded as an important sink for almost all types of chemical
pollutants, including organochlorine compounds, herbicides, domestic
and municipal wastes, petroleum products, heavy metals, and so forth
(Haynes and Johnson, 2000). Thus, it can be inferred that ILs could
become one kind of marine pollutants once they enter aquatic en-
vironment by accidental leakage or euents.
Recently, a number of studies have focused on the inuence and
toxicity of ILs on marine organisms at physiological, biochemical and

Corresponding author at: College of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, 212003, China.
E-mail address: dengxy2016@foxmail.com (X.-Y. Deng).

http://dx.doi.org/10.1016/j.aquatox.2017.05.016
Received 1 May 2017; Received in revised form 18 May 2017; Accepted 28 May 2017
Available online 31 May 2017
0166-445X/ 2017 Elsevier B.V. All rights reserved.
X.-Y. Deng et al. Aquatic Toxicology 189 (2017) 115122

molecular levels (Deng et al., 2015, 2016, 2017; Tsarpali et al., 2016).
Some toxic mechanisms of ILs have been proposed and summarized as
follows: (1) ILs can interact with cell membrane phospholipid bilayers
and hydrophobic domains of membrane proteins, leading to disruption
of membrane physiological functions, because ILs have similar structure
with detergents, pesticides and antibiotics (Samor et al., 2011; Li et al.,
2012; Nancharaiah et al., 2012); (2) ILs can induce overproduction of
reactive oxygen species (ROS) in the cells, which would cause oxidative
stress and lipid peroxidation, resulting in random target attack and
toxic eects (Wu et al., 2013; Cvjetko Bubalo et al., 2014b; Deng et al.,
2017); (3) hydrolysis of ILs with uorinated anions may result in the
formation of uoride, which is a potent inhibitor of the Na+, K+ and
ATPase, and interferes with processes essential in cell self-maintenance
(Stolte et al., 2006; Fatemi and Izadiyan, 2011). However, knowledge
about toxic mechanisms of ILs to marine microalgae is still incomplete,
due to the great variety of ILs, whose toxic ability may dier depending
on the ions of which they are produced, as well as on the dierent
microalgal species sensitivity (Cvjetko Bubalo et al., 2014b; Tsarpali
et al., 2016).
Microalgae, widely distributed in ponds, lakes, rivers, coastal sea-
water and oceans, are a very diverse group of aquatic photosynthetic
organisms with the ability to convert sunlight to food and energy. They
play an important role in freshwater and marine ecosystem as primary
producers because of their key position in the trophic chain, their
ability to produce high amounts of oxygen, and their participation in
nutrient cycles (Ma et al., 2010; Tsarpali et al., 2016). The ubiquity and
importance of microalgae make them the ideal biological model for
(eco)-toxicological assessments because they have a short life cycle and
can respond quickly to environmental stresses (Pham et al., 2010; Bae
and Park, 2014). Among marine microalgae, diatoms are one of the
most used groups of microalgae in marine bioassays because of their
easy cultivation and signicant sensitivity to metals and chemical
pollutants (Wang and Zheng, 2008; Brandt et al., 2015). Several groups
have studied the eects of ILs on diatoms, such as Cyclotella mene-
ghiniana, Skeletonema marinoi, Phaeodactylum tricornutum and Skeleto-
nema costatum (Lataa et al., 2009a; Samor et al., 2011; Deng et al.,
2015, 2016, 2017). However, the information about potential risk and
toxicity of ILs to diatoms is still limited up to now. In addition, cell wall
plays an important role in transport of substances in and out of the cell
(Samor et al., 2011). Cells of diatoms have a range of features that
make them to diverge greatly from the classical cellular structure of
microalgae and higher plants (Lataa et al., 2009a). The toxic me-
chanisms of ILs to diatoms may be dierent from those of other mi-
croalgal species. Thus, more studies are needed to further assess the
toxicity of ILs to diatoms and to unravel the underlying toxic me-
chanism.
Based on the statistical data about known ILs on the Web of Science,
imidazolium-based ILs are found to be the most widely produced and
used at a large scale (Sun et al., 2017). In the present study, two
commonly used imidazolium-based ILs, 1-butyl-3-methylimidazolium
tetrauoroborate ([C4mim]BF4) and 1-octyl-3-methylimidazolium tet-
rauoroborate ([C8mim]BF4) were selected as the tested ILs because
they are widely used in chemical industry (Gutkowski et al., 2006;
Grishina et al., 2013), and their toxic eects on green microalgae have
been reported by other researchers (Tsarpali and Dailianis, 2015;
Tsarpali et al., 2016). In addition, P. tricornutum was used as a model
organism to investigate the growth and physiological responses of
marine diatoms against the two ILs because it is an important food
resource for marine zooplankton and other ltering organisms in the
marine food chain, and is also used as a standard marine species in
bioassays on the toxicity of chemical pollutants (Wang and Zheng,
2008). The purposes of this study were to evaluate the potential eects
of [C4mim]BF4 and [C8mim]BF4 on growth, photosynthetic activity,
cellular components (soluble protein and sugar contents), antioxidant
enzyme activity, and lipid peroxidation levels of the diatom. It is hoped
that this study could provide more data and a theoretical basis upon
which to unravel the toxic mechanism of ILs to marine diatoms and to
further evaluate the safety of ILs in marine environment.
2. Materials and methods
2.1. Chemical regents
Two imidazolium-based ILs ([C4mim]BF4 and [C8mim]BF4) with a
purity of 99.0% were purchased from Chengjie Chemical Co., Ltd.
(Shanghai, China). The choice of these ILs was based on their popularity
and application in scientic elds, as well as the increased likelihood of
them migrating from industry to the environment (Tsarpali et al.,
2016). All other reagents were of high analytical grade and obtained
from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Stock
solutions of the two ILs were prepared in sterile seawater with f/2
medium (Guillard and Ryther, 1962).
2.2. Marine diatom and cultivation
A marine diatom, P. tricornutum, was obtained from the State Key
Laboratory of Marine Environmental Science (Xiamen, China), and was
grown photoautotrophically in 500 mL Erlenmeyer asks containing
200 mL sterile seawater with f/2 medium. The asks were placed in a
20 1 C incubator (Jiangnan Instrument Factory, Ningbo, China)
with a photoperiod of 12 h light/12 h dark and a light density of
40 mol photons m2 s1. Cool white uorescent lamps were xed
vertically on the two sides of incubator to supply light for the growth of
P. tricornutum. Cells of the diatom were pre-cultivated and induced to
the exponential phase of growth with a cell density of 5.2 107 cells
mL1, which was used for the following experiments.
2.3. Toxicity tests
After pre-cultivation, cells of the diatom were washed twice with
sterile seawater to remove the inuence of other substances, and then
collected for toxicity tests. The tests were conducted in 250 mL asks
containing 100 mL sterile seawater with f/2 medium. Dierent con-
centrations of [C4mim]BF4 and [C8mim]BF4 (0, 5, 10, 20, 40 and
60 mg L1) were injected into the medium according to our pre-
liminary range-nding tests. Cultivation conditions, such as tempera-
ture, light intensity, photoperiod, and other conditions, were main-
tained similar to the ones described above. Three biological replicates
were set up for each treatment and six for the controls. All operations
were carried out aseptically.
2.4. Determination of [C4mim]BF4 and [C8mim]BF4 concentrations
The concentrations of [C4mim]BF4 and [C8mim]BF4 were de-
termined with a high-performance liquid chromatography (HPLC,
Agilent 1260, USA) according to the methods described by Liu et al.
(2016) and Deng et al. (2017). The detailed experimental protocols
have been presented in a previous study (Deng et al., 2017). Each
sample was measured in triplicate, and then the average value was
given as mg L1.
2.5. Growth inhibition analysis
Samples were taken every day during the 96-h exposure, and the
numbers of P. tricornutum cells were determined using a Neubauer he-
mocytometer with an optical microscope (Nikon, YS100, Japan). Cell
density of P. tricornutum (X, cells mL1) was calculated based on the
method of Deng et al. (2017). Each sample was counted at least three
times. Specic growth rate (ri, d1) and growth inhibition percentage
(IR, %) were calculated and analyzed according to a previous study
(Deng et al., 2017).

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X.-Y. Deng et al.
Fig 1. Dynamic variation of [C4mim]BF4 (A) and [C8mim]BF4 (B) concentrations in dierent treatments during the exposure periods. Each point represents the mean of 3 biological
replicates and the error bars represent the standard deviation (SD).
2.6. Measurements of chlorophyll uorescence
Chlorophyll uorescence of photosystem II (PSII) of P. tricornutum
treated with dierent concentrations of [C4mim]BF4 and [C8mim]BF4
was measured with a pulse-amplitude modulation (PAM) uorometer
(AquaPen AP-C100, Photon Systems Instruments, Drasov, Czech
Republic) according to the procedure described by Deng et al. (2017).
In brief, samples of the diatom were kept in the dark for 15 min to
obtain the equilibrium of the PSII oxido-reduction state. Then, max-
imum quantum yield of PSII (Fv/Fm), derived from (Fm F0)/Fm, where
Fm and F0 are the maximum and minimum uorescence of the dark-
adapted cells, was recorded. The eective quantum yield of PSII (PSII),
calculated as (Fm F0)/Fm, where Fm is the maximum light-adapted
uorescence yield and F0 is the lowest uorescence yield at F0 in dark-
adapted cells, was also measured.
2.7. Chlorophyll a, and soluble protein and sugar content determination
Chlorophyll a (Chl a) content of P. tricornutum exposed to dierent
concentrations of [C4mim]BF4 and [C8mim]BF4 was measured and
calculated according to the method of Deng et al. (2015). After 96 h of
exposure, soluble protein and sugar were extracted from the harvested
cells of P. tricornutum according to the method described by Deng et al.
(2017). Soluble protein assay kits and sugar content test kits were ob-
tained from Jiancheng Bioengineering Institute (Nanjing, China). Based
on the manufacturers instructions of these kits, soluble protein and
sugar contents of the diatom were determined and calculated.
2.8. ROS level determination
Reactive oxygen species (ROS) assay kits were purchased from
Jiancheng Bioengineering Institute (Nanjing, China). The ROS level was
determined with a 2,7-dichlorofuorescin diacetate probe, and detected
by a multi-mode microplate reader (Molecular Devices, California,
USA) at the emission and excitation wavelengths of 525 and 485 nm,
respectively.
2.9. MDA content, and SOD and POD activity determination
Malondialdehyde (MDA), and superoxide dismutase (SOD) and
peroxidase (POD) activity assay kits were obtained from Jiancheng
Bioengineering Institute (Nanjing, China). After being exposed to
[C4mim]BF4 and [C8mim]BF4 for 96 h, 40 mL of well-blended cultures
117
Aquatic Toxicology 189 (2017) 115122
in each treatment were taken out and harvested by centrifugation
(6000 g, 10 min, 4 C). The harvested cells of P. tricornutum were
resuspended in 1.5 mL sodium phosphate buer (0.05 mol L1, pH
7.8), and then lysed by sonication (Scientz Biotechnology Co. Ltd.,
Ningbo, China) for 10 min with a repeating duty cycle of 5 s in ice bath.
The cellular homogenate was centrifuged (12,000 g, 10 min, 4 C) to
separate the cell debris, and the liquid supernatant was used to de-
termine the MDA content and enzyme activities of P. tricornutum ac-
cording to manufacturers instructions and the method of Wang and
Zheng (2008).
2.10. Statistical analysis
In this work, EC50 value (the eect concentration of [C4mim]BF4 or
[C8mim]BF4 leading to a 50% growth inhibition of P. tricornutum) was
calculated and analyzed according to the method of Deng et al. (2012)
using SPSS software (version 18.0) for Windows (Statistical Product and
Service Solutions, formerly Statistical Package for the Social Sciences,
distributed by SPSS Inc., Chicago, IL). In addition, the results were
presented as the means standard deviation (SD) of three in-
dependent experiments in each treatment to check the reproducibility
of the data. One-way analysis of variance (ANOVA) followed by Dun-
can's multiple range tests was used to evaluate the statistical sig-
nicance of the data between treatments and controls with SPSS 18.0.
The results were considered statistically signicant if the p-values were
0.05 or less.
3. Results and discussion
3.1. Dynamic variation of [C4mim]BF4 and [C8mim]BF4 concentrations
The residual concentrations of [C4mim]BF4 and [C8mim]BF4 in
seawater with f/2 medium were determined every 24 h, and shown in
Fig. 1. No [C4mim]BF4 and [C8mim]BF4 were detected in the controls
during 96 h of exposure. Residual concentrations of the two ILs changed
no more than 5% in dierent treatments during the whole exposure
period, suggesting that both of them were very stable in seawater with
f/2 medium. Some previous studies indicated that ILs, especially the
imidazolium-based ILs, were not easily biodegraded, and thus were
very stable in the environment. For instance, Liu et al. (2016) reported
that residual concentrations of 1-octyl-3-methylimidazolium chloride
([C8mim]Cl) and [C8mim]BF4 changed no more than 20% in freshwater
during 14 d of exposure. Sun et al. (2017) observed that the
X.-Y. Deng et al. Aquatic Toxicology 189 (2017) 115122

12 12
0 0 e
5 mg L-1 5 mg L-1
e
10 10
Cell density ( 10 6 cells mL-1)

Cell density ( 10 6 cells mL-1)

10 mg L-1 20 mg L-1 10 mg L-1 20 mg L-1 d
d e
d
8 40 mg L-1 60 mg L-1 e 8 40 mg L-1 60 mg L-1 d
d
de c e d
f d c
6 6 d
e c c
d d
d
e c c b
4 c b 4 c b
d cd b
c b b b
b b
2 2 a a
a a a
a a
a
0 0
0 24 48 72 96 0 24 48 72 96
Exposure time (h) Exposure time (h)

A B
Fig. 2. Growth curves of Phaeodactylum tricornutum under dierent [C4mim]BF4 (A) and [C8mim]BF4 (B) concentrations during 96 h of exposure. Each point represents the mean of 3
biological replicates and the error bars represent the standard deviation (SD). Dierent letters indicate signicant dierences at p < 0.05 between the treatments and the controls
according to Duncans multiple range tests.

concentrations of [C8mim]BF4 remained stable in soil during 40 d of
exposure. Our previous results showed that 1-dodecyl-3-methylimida-
zolium tetrauoroborate ([C12mim]BF4) was very stable in seawater
during 96 h of exposure (Deng et al., 2017). Moreover, ILs could keep
their intact properties and become persistent chemical pollutants in
aquatic environment according to the report of Ma et al. (2010). The
ndings in this study could provide more information to prove the
stability and lack of biodegradation of ILs in marine environment.
3.2. Inhibitory eects of two ILs towards the diatom
In this work, P. tricornutum was selected as a model species of
marine diatoms to investigate the potential threats of [C4mim]BF4 and
[C8mim]BF4 to marine environment. As illustrated by the growth
curves in Fig. 2, P. tricornutum showed a normal growth curve in the
controls, but its growth was signicantly inhibited by both [C4mim]BF4
and [C8mim]BF4. Growth rates of P. tricornutum decreased with in-
creasing concentrations of the two ILs. The maximum growth rates of
the diatom were 0.509, 0.470, 0.449, 0.371, 0.251, 0.052 d1 and
0.461, 0.391, 0.420, 0.310, 0.254, 0.043 d1 in 0, 5, 10, 20, 40 and
60 mg L1 [C4mim]BF4 and [C8mim]BF4 treatments, respectively. Si-
milar results were found in our previous studies, growth rates of Ske-
letonema costatum and P. tricornutum were signicantly decreased with
the increase of [C8mim]Br and [C12mim]BF4 concentrations, respec-
tively (Deng et al., 2016, 2017). These ndings suggest that ILs were
toxic to the growth of marine diatoms, and might pose serious potential
threats to marine ecosystems. In addition, EC50 values of [C4mim]BF4
were signicantly higher than those of [C8mim]BF4 at 48, 72 and 96 h
of exposure (Table 1). The higher value of EC50, which is a relatively
sensitive index for evaluating the toxicity of a compound, means lower
toxicity. Thus, it is suggested that the toxicity of [C4mim]BF4 was lower
than that of [C8mim]BF4 to P. tricornutum, which might be attributed to
Table 1
Inhibitory eects of [C4mim]BF4 and [C8mim]BF4 on the growth of Phaeodactylum tri-
cornutum, expressed as median eective concentrations (EC50) standard deviation (SD)
(mg L1) at 24, 48, 72 and 96 h of exposure.
Test EC50 (mg L1)
chemicals
24 h 48 h
[C4mim]BF4 30.81 1.23 28.53 1.41
[C8mim]BF4 30.17 1.30 23.36 1.42
the dierent alkyl chain lengths of the two ILs. Ma et al. (2010) re-
ported that EC50 values of [C4mim]Br, [C6mim]Br, [C8mim]Br,
[C10mim]Br and [C12mim]Br decreased from 22.24, 5.88, 0.34 and
0.1 mg L1 to 0.02 mg L1, respectively, when S. obliquus was exposed
to the ILs for 96 h, notably indicating that toxicity of ILs was mainly
dependent on their alkyl chain length. Pham et al. (2016) observed that
lengthening of the alkyl chain would lead to a concomitant increase in
the toxicity of ILs to a freshwater green microalga, Pseudokirchneriella
subcapitata. Reasons for this phenomenon may be due to the fact that
the increased lipophilic properties of ILs with longer alkyl chains would
increase the membrane permeability and their toxicity. During 96 h of
exposure, the EC50 values decreased rst from 30.81 and 30.17 mg L1
to 28.53 and 23.36 mg L1, and then increased to 45.88 and
31.37 mg L1 in [C4mim]BF4 and [C8mim]BF4 treatments, respectively
(Table 1). It is indicated that the toxicity of [C4mim]BF4 and [C8mim]
BF4 to P. tricornutum increased rst and then decreased with increasing
exposure time, reaching maximum toxicity at 48 h of exposure. The
decrease of toxicity with increase of exposure time probably resulted
from the acclimation of the diatom to ILs (Ertrk and Saan, 2012). This
trend has also been previously discussed by other researchers (Liu et al.,
2015b; Deng et al., 2017), and appears to occur regardless of dier-
ences in the organisms examined and the methodologies employed, and
dierences in the cation and anion combinations of the ILs tested.
Moreover, a recent study (Tsarpali et al., 2016) reported the toxicity of
[C4mim]BF4 and [C8mim]BF4 to a green microalga D. tertiolecta with
24 h, 48 h, 72 h and 96 h IC50 values of > 200, > 200, > 200, >
200 mg L1 and 11.6, 8.0, 23.9, 29.5 mg L1, respectively. The
variability in EC50 or IC50 values among dierent microalgal species
may be caused by the dierences in the inherent physiological status of
dierent species. It was reported that green algae are far less sensitive
to chemical pollutants than diatoms because of the dierent cell wall
structures and cell size (Lataa et al., 2009b). In the case of green algae,
as indigestible cellulose forms the main structural element in the cell
wall. This is far stronger and less sensitive to chemicals than the silica
frustule surrounding diatoms. The dierence in size of microalgal cells
and in production of extracellular organic substances may also lead to
high variability in the sensitivity of dierent algal species to the same
chemical pollutant (Rojkov and Marlek, 1999).
72 h 96 h 3.3. Eects of [C4mim]BF4 and [C8mim]BF4 on chlorophyll uorescence
39.92 1.24 45.88 1.54
28.62 1.31 31.37 1.24 To investigate the photosynthetic status of P. tricornutum under the
stress of [C4mim]BF4 and [C8mim]BF4, the Fv/Fm and the PSII values

118
X.-Y. Deng et al. Aquatic Toxicology 189 (2017) 115122

Fig. 3. Changes of the maximum photochemical eciency of PSII (Fv/Fm) (A and B) and the eective quantum yield of PSII (PSII) (C and D) of Phaeodactylum tricornutum treated with
dierent concentrations of [C4mim]BF4 and [C8mim]BF4, respectively, during 96 h of exposure. Each point represents the mean of 3 biological replicates and the error bars represent the
standard deviation (SD). Dierent letters indicate signicant dierences at p < 0.05 between the treatments and the controls according to Duncans multiple range tests.

were measured (Fig. 3), which, respectively, show the photosynthetic
capacity and eciency of the microalgal cells. As shown in Fig. 3A and
B, Fv/Fm values in the controls were stable throughout the experiments
with mean values of 0.53, indicating that the diatom was in good
physiological state (Maxwell and Johnson, 2000). Compared with that
of the controls, Fv/Fm values were not signicantly aected in [C4mim]
BF4 treatments with a concentration of 40 mg L1. Nevertheless, in
60 mg L1 [C4mim]BF4 treatments, Fv/Fm decreased sharply during
24 h of exposure, and then gradually decreased after 24 h of exposure
(Fig. 3A). The same trend was also observed in [C8mim]BF4 treatments
(Fig. 3B). Similar results were reported in a previous study (Deng et al.,
2017), which showed that Fv/Fm values of P. tricornutum did not change
in treatments with low [C12mim]BF4 concentrations (1 mg L1), but
decreased signicantly in treatments with high concentrations of
[C12mim]BF4 (2.5 mg L1). These ndings were also in line with
those reported by Samor et al. (2011). In general, a decrease of Fv/Fm
values indicated that the ratio of electrons generated in PSII to photons
absorbed by the light-harvesting pigments decreased due to oxidation,
degradation of D1 protein, or severely reduced pigment contents
(Devilla et al., 2005; Li et al., 2014). Moreover, ILs could cause oxi-
dation of electron transport chain or primary electron acceptor of the
PSII, and aect the photosynthesis of microalgae (Samor et al., 2011;
Huang et al., 2013). Eects of [C4mim]BF4 and [C8mim]BF4 on PSII of
P. tricornutum are shown in Fig. 3C and D, respectively. Although there
were no signicant dierences in PSII of P. tricornutum between the
controls and treatments with 5 mg L1 [C4mim]BF4 during 96 h of
exposure (p > 0.05), it was signicantly inhibited by 10, 20, 40 and
60 mg L1 [C4mim]BF4 at all exposure times relative to the controls
(p < 0.05) (Fig. 3C). Similar changes in PSII of P. tricornutum were
also found in [C8mim]BF4 treatments (Fig. 3D). These results are con-
sistent with the previous literature showing that ILs inhibit PSII in
microalgae (Samor et al., 2011; Deng et al., 2017). The marked de-
crease of PSII under the stress of ILs showed an inhibiting eect of ILs
on photosynthetic eciency of the diatom, which may be caused by ILs
stopping the formation of cell assimilatory power (NADPH and ATP) or
aecting the xation and assimilation of carbon in the microalgal cells
(Liu et al., 2015a).
3.4. Eects of [C4mim]BF4 and [C8mim]BF4 on chlorophyll a content
In this work, a reduction of Chl a content of P. tricornutum was
observed in the treatments with [C4mim]BF4 and [C8mim]BF4 (from 5

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X.-Y. Deng et al. Aquatic Toxicology 189 (2017) 115122

0 5 mg L-1 10 mg L-1 0
2.50 2.50 5 mg L-1 10 mg L-1
-1 -1 -1 -1
2.25 20 mg L 40 mg L 60 mg L 2.25 20 mg L 40 mg L -1
60 mg L-1
a a
a a a a a

Content of Chl a ( g 107 cell-1)

Content of Chl a ( g 107 cell-1)

2.00 2.00
a ab
a b b b
1.75 a a
b b c 1.75 b b b b b b
c b b bc
a d b bcd
b c 1.50 c c cd
1.50 d e
d 1.25
1.25

1.00 1.00

0.75 0.75 d
c e d c e
0.50 0.50
f
e
0.25 0.25

0.00 0.00
24 48 72 96 24 48 72 96
Exposure time (h) Exposure time (h)

A B
Fig. 4. Eects of [C4mim]BF4 (A) and [C8mim]BF4 (B) on the mean values (mean SD; number of biological replicates (n) = 3) of chlorophyll a content in Phaeodactylum tricornutum
during 96 h of exposure. Each bar is the mean of 3 biological replicates. Error bar represents the standard deviation (SD). Dierent letters above columns indicate signicant dierences at
p < 0.05 between the treatments and the controls according to Duncans multiple range tests.

to 60 mg L1) during 96 h of exposure (Fig. 4), indicating that the treatments; while an obvious decrease was found in the treatments with
photosynthetic system of the diatom may be partly damaged and its 60 mg L1 of the two ILs. Similar results were obtained in our previous
photosynthetic eciency inhibited by the two ILs. The negative eects studies (Deng et al., 2015, 2016, 2017). The increase of soluble protein
of ILs on the photosynthetic pigments in algae have been reported by content may be attributed to the following reasons: (1) an increase of
many researchers (Ma et al., 2010; Deng et al., 2015, 2017; Liu et al., antioxidant and biotransformation enzymes in the cells because they
2015b). Several mechanisms have been proposed: perhaps the rst one are important components of soluble protein (Kumar et al., 2008); (2)
is that the long alkyl chain of ILs can incorporate into the polar head an elevated and potentially prolonged expression of target genes of the
groups of phospholipid bilayer as described by Cvjetko Bubalo et al. diatom in response to the two ILs (Bajguz and Piotrowska-Niczyporuk,
(2014a). The disruption of phospholipid bilayer of chlorophyll mem- 2014). The decrease of soluble protein content may be due to various
brane, especially by ILs with longer alkyl chains attached to cation core, propagating radicals and ROS in the cells. They would induce oxidative
is an obvious scenario (Kumar et al., 2011; Zhang et al., 2013). Another modications of amino acid side chains, cause ROS-mediated peptide
mechanism could be excessive ROS generation in the cells. ROS would cleavage, reactions of peptides with lipids and carbohydrate oxidation
attack chlorophyll molecule and chloroplast membrane. In plant cells, products, and cause the formation of carbonyl derivatives of proteins
the biosynthesis pathway of Chl a requires the formation of Mg-proto- (Valavanidis et al., 2006).
porphyrin IX from magnesium chelatase and magnesium; the excessive Eects of [C4mim]BF4 and [C8mim]BF4 on the soluble sugar content
ROS would block this process (Walker and Willows, 1997). As a result, of P. tricornutum were investigated in this study. As shown in Table 2,
the microalgal photosynthetic eciency would be inuenced and the soluble sugar content of the diatom increased from 0.27 to 0.41, 0.43,
energy deciency would be aggravated. 0.42, 0.64, 0.78 g 106 cell1 and from 0.25 to 0.44, 0.51, 0.60,
1.04, 1.09 g 106 cell1 at 96 h of exposure in 5, 10, 20, 40 and
60 mg L1 [C4mim]BF4 and [C8mim]BF4 treatments, respectively.
3.5. Eects of [C4mim]BF4 and [C8mim]BF4 on soluble protein and sugar These data were in agreement with those reported in previous studies.
contents An increase of soluble sugar content was found in green algae (Clado-
phora) under the stress of lead (Cao et al., 2015). It was reported that
After 96 h of exposure, soluble protein content of P. tricornutum was synthesis of sugar in microalgae was stimulated by environmental
determined and shown in Table 2. Compared with that of the controls, a stresses, and the algae may use sugar to increase the thickness of cell
remarkable increase of soluble protein content in the diatom was ob- wall or to combine with chemical pollutants (Warr et al., 1985; Kirrolia
served in 5, 10, 20 and 40 mg L1 [C4mim]BF4 and [C8mim]BF4

Table 2
Eects of [C4mim]BF4 and [C8mim]BF4 on the mean values (mean SD; number of biological replicates (n) = 3) of soluble protein and sugar contents, ROS level and MDA content, and
SOD and POD activities in Phaeodactylum tricornutum after 96 h of exposure. Dierent letters indicate signicant dierences at p < 0.05 between the treatments and the controls
according to Duncans multiple range tests.

Test chemicals Concentrations Soluble protein content Soluble sugar content ROS level MDA content SOD activity POD activity
(mg L1) (g 106 cell1) (g 106 cell1) (FU 107 cell1) (nmol 108 cell1) (U mg1 proteins) (U mg1 proteins)

[C4mim]BF4 0 0.78 0.02a 0.27 0.01a 1.26 0.19a 0.38 0.08a 8.33 1.55a 0.75 0.09a
5 1.30 0.10b 0.41 0.04b 2.08 0.03b 0.98 0.15b 18.24 0.95b 1.05 0.07b
10 1.59 0.02c 0.43 0.04b 2.73 0.10c 1.32 0.08c 20.39 1.31b 1.08 0.06b
20 1.62 0.08c 0.42 0.02b 3.59 0.06d 1.55 0.13c 42.20 4.72c 1.62 0.09c
40 1.62 0.04c 0.64 0.04c 5.21 0.08e 1.52 0.10c 51.56 3.66d 2.26 0.07d
60 0.31 0.10d 0.78 0.05d 4.97 0.23f 2.54 0.22d 18.30 9.53b 1.46 0.08e
[C8mim]BF4 0 0.70 0.04a 0.25 0.02a 1.32 0.06a 0.20 0.05a 7.82 0.80a 0.73 0.08a
5 1.57 0.22b 0.44 0.06b 2.05 0.12b 0.53 0.11b 21.03 0.81b 1.32 0.03b
10 1.94 0.14c 0.51 0.03b 3.66 0.08c 0.74 0.05c 24.40 1.87c 1.87 0.07c
20 1.97 0.25c 0.60 0.06b 4.52 0.14d 1.02 0.07d 44.29 4.30d 2.38 0.11d
40 1.70 0.15bc 1.04 0.05c 5.88 0.04e 1.62 0.17e 54.91 4.85e 3.41 0.16e
60 0.68 0.14a 1.09 0.18c 5.90 0.38e 3.54 0.08f 21.96 2.92bc 1.08 0.05f

120
X.-Y. Deng et al.
et al., 2011; Cao et al., 2015). Among dierent solutes accumulating in
response to stress, sugars may play a key role in maintaining the os-
motic regulation in microalgal cells. Thus, the increase of sugar content
in the diatom may be considered as an adaptive measure to protect cells
from the stress of ILs (Roitsch, 1999).
3.6. Eects of [C4mim]BF4 and [C8mim]BF4 on ROS level and MDA
content
In this study, ROS level was measured to conrm whether the
growth inhibition of ILs on microalgae was caused by ROS. As shown in
Table 2, ROS level of P. tricornutum was increased by 0.65, 1.17, 1.85,
3.13, 2.94 times and 0.55, 1.77, 2.42, 3.45, 3.47 times relative to the
controls in 5, 10, 20, 40 and 60 mg L1 [C4mim]BF4 and [C8mim]BF4
treatments, respectively. There are several reports indicating that con-
taminants trigger ROS accumulation in microalgal cells, and the re-
sulting oxidative damage may be a mechanism of toxicity in aquatic
organisms exposed to pollutants (Chen et al., 2014). Thus, it was not
surprising to nd that the ROS level of the diatom was signicantly
increased with increasing concentrations of the two ILs. These results
are consistent with those obtained by Zhang et al. (2013), who observed
that dimethyl thiourea (ROS scavenger) eectively enhanced the anti-
oxidant ability of Lemna minor; and suggested that ROS might be in-
volved in the mechanism of ILs-induced toxicity to L. minor. In our
previous studies (Deng et al., 2016, 2017), a signicant increase of ROS
level was also found in the diatoms under the stress of ILs. According to
the ndings of Asada (1999), ROS are mainly produced through the
transfer of electrons on molecular oxygen (O2) from photosystem I (PSI)
during the photosynthesis process. Thus, an increase of ROS level in the
diatom may be the consequence of the inhibition on photosynthesis as
mentioned above.
Lipid peroxidation is one of the primary manifestations of oxidative
damage in organisms. As the main product of lipid peroxidation, MDA
content can serve as an indicator of the degree of lipid peroxidation and
the severity of cellular damage (Li et al., 2010). In this work, the MDA
content of the diatom cells increased with increasing concentrations of
[C4mim]BF4 and [C8mim]BF4, following a similar trend to that ob-
served for ROS level (Table 2). Similar results were reported by other
researchers (Kumar et al., 2011; Zhang et al., 2013; Deng et al., 2016,
2017), when they studied the toxic eects of ILs on algae, indicating
that, although the ILs and organisms were dierent in these studies, the
toxic mechanism of ILs may be consistent. The increased MDA contents
in the treatments suggested that the integrity of the lipid membrane of
the diatom was seriously damaged by the attacks of excessive ROS
(Kumar et al., 2011; Liu et al., 2016). The contents of Chl a were sig-
nicantly decreased in the diatom treated by the two ILs (Fig. 4), in-
dicating that excessive ROS might reduce the eciency of photo-
synthesis by causing cellular pigments losses. Moreover, the presence of
excessive ROS in the diatom could consume some additional metabolic
energy, which might lead to a shortage of metabolic energy (Zhou et al.,
2013). As a result, the growth of P. tricornutum was inhibited because
the membrane integrity, algal energy metabolism, and photosynthetic
eciency are very important for the proliferation of microalgae.
Therefore, these results obtained in present study conrmed that ILs
induced the diatom to produce excessive ROS, providing an explanation
to the toxic mechanism of ILs to microalgae.
3.7. Eects of [C4mim]BF4 and [C8mim]BF4 on SOD and POD activities
To nd out the ROS-eliminating mechanism, activities of several
antioxidant enzymes including SOD and POD were measured in this
study. Eects of [C4mim]BF4 and [C8mim]BF4 on SOD and POD ac-
tivities of P. tricornutum are presented in Table 2, which show that the
activities of the two antioxidant enzymes increased with increasing the
concentrations of both ILs from 5 to 40 mg L1, and then a sharp de-
crease was observed in 60 mg L1 treatments with the two ILs. Many
121
Aquatic Toxicology 189 (2017) 115122
studies have conrmed that the antioxidant enzyme activities in algae
increased under the stress of ILs (Kumar et al., 2011; Zhang et al., 2013;
Deng et al., 2015, 2016, 2017). The increased SOD activity may be
attributed to the overproduction of superoxide, which is considered to
be a central component of signal transduction, resulting in the activa-
tion of existing enzyme pools or the increased expression of genes that
encode SOD (Mishra et al., 2006). The enhancement of POD activity is
considered to be an adaptive trait against the damage caused by ROS
(Zhang et al., 2013). Thus, the increase of SOD and POD activities may
be due to a protective strategy to eliminate excessive ROS and prevent
oxidative damage to the diatom. In addition, a decrease of SOD and
POD activities observed in 60 mg L1 [C4mim]BF4 and [C8mim]BF4
treatments may be due to ROS levels that were beyond the scavenging
capacity of SOD and POD, leading to inhibition of SOD and POD ac-
tivities. These data are consistent with those obtained by Zhang et al.
(2013), Du et al. (2014) and Deng et al. (2016), where the SOD and
POD activities also increased rst and then decreased with the in-
creasing concentrations of ILs.
4. Conclusion
This study investigated the eects of two imidazolium-based ionic
liquids ([C4mim]BF4 and [C8mim]BF4) on the growth and physiological
characteristics of a marine diatom P. tricornutum for evaluating the
toxicity of ILs to marine environment. The main conclusions were as
follows.
(1) [C4mim]BF4 and [C8mim]BF4 were very poorly biodegraded, and
consequently were very stable in seawater during 96 h of exposure.
(2) [C4mim]BF4 and [C8mim]BF4 signicantly inhibited the growth of
P. tricornutum with 24, 48, 72 and 96 h EC50 values of 30.81, 28.53,
39.92, 45.88 mg L1 and 30.17, 23.36, 28.62, 31.37 mg L1, re-
spectively, indicating that [C8mim]BF4 was more toxic to the
diatom than [C4mim]BF4 and both of them were potential en-
vironmental pollutants.
(3) The photosynthetic activity and Chl a synthesis of P. tricornutum
were inhibited by [C4mim]BF4 and [C8mim]BF4, which suggested
that the structural integrity of chloroplasts in the diatom was dis-
rupted or damaged by the two ILs.
(4) Excessive ROS were produced during the exposure to the two ILs in
the diatom, which could cause lipid peroxidation, shortage of me-
tabolic energy and decline of photosynthetic eciency.
(5) Contents of soluble protein and sugar, and activities of antioxidant
enzymes (SOD and POD) in the diatom were increased under the
stress of the two ILs, which may be viewed as active defense to
protect the cells from injury and poisoning caused by ILs.
(6) Consequently, ILs are potential threats once they are released into
the marine environment accidentally, and their toxicity may mainly
be caused by the ROS overproduction in microalgal cells induced by
ILs. These ndings will be helpful for understanding the ecological
risks and toxicity of ILs in marine environment.
Acknowledgements
This manuscript was supported by the National Natural Science
Foundation of China (no. 31200381) and Jiangsu Provincial Natural
Science Foundation of China (no. BY2015065-10).
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