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International Journal of

Molecular Sciences

Article
Influence of Speciation of Thorium on Toxic Effects to
Green Algae Chlorella pyrenoidosa
Can Peng 1 , Yuhui Ma 2, *, Yayun Ding 2 , Xiao He 2 , Peng Zhang 2 , Tu Lan 2 , Dongqi Wang 2 ,
Zhaohui Zhang 1, * and Zhiyong Zhang 2,3, *
1 School of Public Health, University of South China, Hengyang 421001, China; vintagecancan@icloud.com
2 Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics,
Chinese Academy of Sciences, Beijing 100049, China; dingyy@ihep.ac.cn (Y.D.); hx421@ihep.ac.cn (X.H.);
pengzhang@ihep.ac.cn (P.Z.); lantu@ihep.ac.cn (T.L.); dwang@ihep.ac.cn (D.W.)
3 School of Physical Sciences, University of the Chinese Academy of Sciences, Beijing 100049, China
* Correspondence: mayh@ihep.ac.cn (Y.M.); zhaohuizzh@126.com (Z.Z.); zhangzhy@ihep.ac.cn (Z.Z.);
Tel.: +86-10-8823-3215 (Y.M. & Z.Z.); +86-734-828-1375 (Z.Z.);
Fax: +86-10-8823-5294 (Y.M. & Z.Z.); +86-734-828-1771 (Z.Z.)

Academic Editor: Guido R. M. M. Haenen


Received: 6 March 2017; Accepted: 5 April 2017; Published: 10 April 2017

Abstract: Thorium (Th) is a natural radioactive element present in the environment and has the
potential to be used as a nuclear fuel. Relatively little is known about the influence and toxicity of Th
in the environment. In the present study, the toxicity of Th to the green algae Chlorella pyrenoidosa
(C. pyrenoidosa) was evaluated by algal growth inhibition, biochemical assays and morphologic
observations. In the cultural medium (OECD TG 201), Th(NO3 )4 was transformed to amorphous
precipitation of Th(OH)4 due to hydrolysis. Th was toxic to C. pyrenoidosa, with a 96 h half maximum
effective concentration (EC50 ) of 10.4 µM. Scanning electron microscopy shows that Th-containing
aggregates were attached onto the surface of the algal cells, and transmission electron microscopy
indicates the internalization of nano-sized Th precipitates and ultrastructural alterations of the algal
cells. The heteroagglomeration between Th(OH)4 precipitation and alga cells and enhanced oxidative
stress might play important roles in the toxicity of Th. To our knowledge, this is the first report of
the toxicity of Th to algae with its chemical species in the exposure medium. This finding provides
useful information on understanding the fate and toxicity of Th in the aquatic environment.

Keywords: thorium; speciation; toxicity; green algae; Chlorella pyrenoidosa

1. Introduction
Thorium (Th) is an actinide that occurs naturally as 232 Th with a very long half-life of
1.4 × 1010 years. In the environment, Th exists predominantly in the tetravalent state, and is a trace
constituent in phosphates, simple and multiple oxides, and silicates [1,2]. Th is used for making
ceramics, welding rods, camera and telescope lenses, fire brick, heat resistant paint, and metals used in
the aerospace industry [3]. In recent years, Th has attracted more and more attention because it has the
potential to be used as a cleaner, safer, and more abundant nuclear fuel.
People will always be exposed to small amounts of Th through inhalation, ingestion and skin
penetration, because Th is naturally ubiquitous in air, water, soil and biological materials. Individuals
that work in the mining, milling or thorium industries may be exposed to more Th than usual. There is
evidence that breathing in Th dust increases the risk of lung and pancreatic cancer. Exposure to Th
also causes bone cancer because Th can be stored in bone [4]. In the 1930s and 1940s, ThO2 was used
as a radiographic contrast agent (thorotrast). After intravascular injection, ThO2 was found mainly in
reticuloendothelial systems such as liver, spleen and bone and has been associated with an increased

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Int. J. Mol. Sci. 2017, 18, 795 2 of 10

incidence of liver disease [5]. Recently, studies on the toxicity of Th at cellular and molecular levels
have also been carried out. Oliveira et al. [6] found that Th was not toxic to human lymphocytes
at concentration range from 0 to 1 mM. Allred et al. [7] reported that an iron-binding protein called
siderocalin could bind and transport actinides such as Th, Pu, Am, etc., into cells. It was considered to
be a major advance in understanding the biological chemistry of radioactive actinides.
The aquatic environment is the final sink of all pollutants, but there are currently only a few
published articles on aquatic toxicity of Th. Researchers from Russia investigated the effect of 232 Th to
the fresh water green alga Chlorella pyrenoidosa and found that the 24 h EC50 value of Th was 15.4 µM
and the presence of caffeine significantly increased Th toxicity [8]. However, de Queiroz et al. [9]
reported that the growth of two green microalgae Monoraphidium sp. and Scenedesmus sp. was resistant
to Th. Adverse effects on cell growth were observed only for concentrations higher than 215 µM.
The authors postulated that the presence of the NO3 − in the stock solution of Th could have been used
by the cells as a nutrient source and EDTA presented in ASM-1 medium acted as a complexing agent
for Th, preventing its interaction with the microalgae. The effects that Th has on aquatic invertebrate
and vertebrate species have also been studied. Borgmann et al. [10] evaluated the toxicity of 63 metals
and metalloids including Th to freshwater amphipod Hyalella azteca. Lethal concentration resulting in
50% mortality (LC50 ) of Th was correlated with the hardness of the cultural media. The LC50 s of Th in
tap water was higher than that in soft water. Correa et al. [11] tested the effect of 15 days of waterborne
Th exposure on accumulation, metabolic and oxidative parameters in the bile, gills, liver, muscle, brain,
skin, kidney and blood of the silver catfish (Rhamdia quelen). The concentrations of Th in gills and
skin were the highest among all the organs. CAT and GST activities in the liver and muscle were
altered by Th treatment. A prolonged exposure of 30 days to Th obtained similar results [12]. It is well
known that in the aquatic environment, the most important factor that influences the toxicity of heavy
metals is their chemical species. Ionic Th tend to form hydrolyzed species and/or insoluble residues in
aqueous solutions at pH higher than 3. In most reported studies, the aquatic organisms were treated
with Th(NO3 )4 , but the transformation of Th in the exposure media (modified reconstituted water)
was almost never analyzed. Recently, our group demonstrated that composition of exposed media
significantly influences the Th species and Th was present as particulate ThO2 in the exposure medium
of D. magna. The 24 h and 48 h EC50 of ThO2 were 7.3 and 4.7 µM, respectively [13]. This suggests that
more attention should be paid to the toxicity of Th species that are present in insoluble forms.
Algae, primary producers in the aquatic system, are ubiquitous and have colonized almost every
part of the world. Using green algae (e.g., Scenedesmus obliquus, Chlorella pyrenoidosa, etc.) to investigate
the aquatic toxicology of chemicals is advantageous, since they are easy to culture and sensitive to
pollutants. The objective of this study was to access the toxicity of Th to C. pyrenoidosa on the basis of
the chemical speciation of Th in the exposure medium. The EC50 , chlorophyll a concentrations and
ROS levels of C. pyrenoidosa treated by Th were determined. The morphological changes of algal cells
and uptake of Th were observed by SEM and TEM. This work will provide understanding on the
toxicity of Th to the aquatic environment.

2. Results and Discussion

2.1. Chemical Species of Th in the Media


Th4+ ions tend to form hydrolyzed species and/or insoluble residues in aqueous solutions [14].
Moreover, the formation of colloidal species of this element is known to start at very low pH.
Considering the complexity of OECD medium, the MEDUSA program was used to calculate the
distribution diagrams of different chemical forms of Th in the exposure media. As shown in Figure 1,
the hydroxo-complexes (Th(OH)2 2+ , Th(OH)3+ and ThOH3+ ) aggrandized with the increase of pH
values; as a consequence, the concentration of free Th4+ decreased. Since the pH values of Th-containing
solutions were all above 7.0, according to the distribution diagrams there were no free Th4+ ions in
the exposure media, with all of Th being present as Th(OH)4 . It has been reported that the Ksp value
Int.Int.
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J. Mol. Sci.Sci.
Mol. 2017,
2017,
Sci. 18,18,
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18, 795 33 of
3 of of 10
10 10

free
free ThTh
4+ 4+ ions
ions inin the
the exposure
exposure media,
media, with
with allall
ofofThTh being
being present
present asas Th(OH)
Th(OH) 4. It
4. It hashas been
been reported
reported
forthe
Th(OH) wasfor
only 5.34 × −33.6 [15], which
that
that theKspKsp 4 value
value for Th(OH)
Th(OH) 410
was
4 was only
only 5.34
5.34 × 10× 10 means the
−33.6 [15],
−33.6 solubility
which
[15], which means
means ofthe
the Th(OH) 4 in
solubility
solubility ofthe
of water4 in
Th(OH)
Th(OH) was
4 in

theextremely
the water
water was low.
was extremely
extremely low.
low.

Figure
Figure
Figure Distribution
1. 1.Distribution
Distribution diagram
diagram
diagram ofdifferent
different
of ofdifferent forms
forms
forms of
of of Th
ThTh at
at at the
thethe highest
highest
highest treated
treated
treated concentration
concentration
concentration in
in in
dependence
dependence
dependence on
onon pH
pHpH values.
values.
values.

2.2.
2.2.
2.2. Effects
Effects of of
Effects of Th
Th onon
Th Algal
Algal
on Growth
Growth
Algal Growth
The The
The concentration-
concentration-
concentration- andand
and time-dependent
time-dependent
time-dependent effects
effects
effectsofofThTh
of toto
Th C.C.
to pyrenoidosa
pyrenoidosa
C. pyrenoidosa are
are shown
shown
are shown inin Figure
Figure
in Figure 2. 2.2.
AtAt concentrations
concentrations ofof less
less thanthan 7.27.2
μM, μM,
At concentrations of less than 7.2 µM, Th had no effectTh Th had
had nono effect
effect onon algal
algal growth,
growth, while
while it it showed
showed
algal growth, while it showed significant significant
significant
toxicity
toxicity
toxicity at at
athigher
higher
higher concentrations
concentrations
concentrations at at
ateach
each each exposure
exposure
exposure time.
time. The
The
time. 24-,
24-,
The 48-,
48-,
24-, and
and
48-, 72-,
72-,
and and
and
72-, 96-h
96-h
and ECEC
96-h 50 values
values
50EC ofof
50 values
ThTh to
oftoThC.C.topyrenoidosa
pyrenoidosa are
C. pyrenoidosa are calculated
calculated
are and
calculated and summarized
summarized
and inin
summarized Table
Tablein 1. 1.
The
Table The growth
growth
1. inhibition
inhibition
The growth increased
increased
inhibition with
with
increased
the
the
with increasing
increasing ofof
the increasing exposure
exposure
of exposuretime.
time. This
This
time. result
result
Thiswas was consistent
consistent
result with
with
was consistent other
other
with previous
previous
other reports,
reports,
previous inin which
which
reports, the
in whichthe
toxicity
toxicity ofoflead
the toxicity lead [16],cadmium
of [16],
lead cadmium
[16], cadmium [17]and
[17] and
[17] chlorine
chlorine
and [18]
[18]
chlorine toto
[18] algae
algae
to was
was
algae related
related
was relatedtotothe
tothe incubation
incubation
the incubation time.
time.
time.
After
After
After 9696h hh exposure,
exposure,
exposure, thethe
the inhibition
inhibition
inhibition rates
rates
rates relative
relative toto
to the
the the control
control
control group
group
group of
ofof7.2, 7.2,
7.2, 10.8,
10.8,
10.8, and and
and 14.4
14.4
14.4 µMμM μM
Th Th Th
were
were
were
22.5%, 22.5%,
22.5%,55.1%, 55.1%,
55.1%,
andand and
67.8%, 67.8%,
67.8%, respectively.
respectively.
respectively. The
The 24 The
and 24
2496and and
h EC 96 h EC
9650h values
EC50 values
50 values were respectively
were respectively
were respectively 18.5 and 18.5
18.5 and
10.4 and
µM,
10.4
10.4 μM,
which μM, which
which
was waswas
comparable comparable
comparable toto
to the results thethe results
results
of ofof
Evseeva Evseeva
Evseeva
et et et
al. [8]. al.al. [8].
[8].

Figure 2.
Figure Cell density
2. Cell density of algae cells after exposure to different concentration of Th. Data are showed as
Figure 2. Cell density of of algae
algae cells
cells after
after exposure
exposure to to different
different concentration
concentration of of
Th.Th. Data
Data areare showed
showed
mean value ± ±standard deviations (SD). Significant difference of the experimental value compared
compared
as as mean
mean value
value standard
± standard deviations
deviations (SD).
(SD). Significant
Significant difference
difference of of
thethe experimental
experimental value
value compared
with
with each
each control
control was
was marked
marked with
with “*” (p < 0.05) or “**” (p < 0.01).
with each control was marked with “*”“*”
(p (p < 0.05)
< 0.05) oror “**”
“**” (p (p < 0.01).
< 0.01).
Int. J. Mol. Sci. 2017, 18, 795 4 of 10

Int. J. Mol. Sci. 2017, 18, 795 4 of 10


Table 1. The EC50 values with 95% confidence interval (CI) of Th to C. pyrenoidosa at different
Table 1. time.
exposure The EC50 values with 95% confidence interval (CI) of Th to C. pyrenoidosa at different
exposure time.
Time (h) EC (µM) 95% CI (µM)
Time (h) EC50 50
(μM) 95% CI (μM)
24 24 18.5
18.5 10.9 26.7
10.9 26.7
48 16.7 14.7 21.7
72
48 16.7
11.8
14.7 21.7
10.2 14.1
96 72 11.8
10.4 10.2 14.1
7.9 15.9
96 10.4 7.9 15.9
2.3. Effects of Th on Chlorophyll a Contents
2.3. Effects of Th on Chlorophyll a Contents
Chlorophyll, one kind of photosynthetic pigment, is the basis for photosynthesis of algae cells.
Chlorophyll,
The growth status one kindcan
of cells of photosynthetic
be reflected by pigment,
monitoring is the
thebasis for photosynthesis
intracellular contents ofofchlorophyll.
algae cells.
The growth status of cells can be reflected by monitoring the intracellular contents
The chlorophyll a contents of algal cells after being treated with different concentrations of Th are of chlorophyll. The
chlorophyll a contents of algal cells after being treated with different concentrations
shown in Figure 3. The general trend of chlorophyll changes was similar to that of the growth (Figure 2).of Th are shown in
Figure
With the3.increase
The general trend of chlorophyll
of exposure changes was similar
time and concentrations, to thatof
the contents ofchlorophyll
the growth (Figure 2). cells
a of algae With
the increase of exposure time and concentrations, the contents of chlorophyll
decreased gradually. After 96 h exposure, the content of chlorophyll a in the cells exposed to 10.8 and a of algae cells
decreased
14.4 µM Thgradually.
was reduced After
by 96 h exposure,
34.5% and 58.6% therespectively
content of chlorophyll
compared with a in that
the of
cells
theexposed to 10.8
control group
and 14.4 μM Th was reduced by 34.5% and 58.6% respectively compared with
(p < 0.01). The reduction of chlorophyll contents after exposure to Th indicated the ability of the cellsthat of the control
group
to (p < 0.01).
synthesize The reduction
chlorophyll or theof chlorophyll contents
photosynthetic reactionafter exposure
center to Thwas
complexes indicated the ability
impacted. of
Similar
theeffects
to cells ofto other
synthesize
heavychlorophyll
metals, such or as
thelead
photosynthetic
and cadmium, reaction
on thecenter
green complexes
algae [17], was impacted.
the decreased
Similar to effects of other heavy metals, such as lead and cadmium, on the green
chlorophyll content may result in a decrease in the photosynthetic activity and chlorosis and indicates algae [17], the
decreased chlorophyll content may result in a decrease in the photosynthetic activity
that the ability of the cells to synthesize chlorophyll or the photosynthetic reaction center complexes and chlorosis
and indicates that the ability of the cells to synthesize chlorophyll or the photosynthetic reaction
was impacted after exposure to Th.
center complexes was impacted after exposure to Th.

Figure3.3.Chlorophyll
Figure Chlorophylla acontents
contents
ofof algae
algae cells
cells after
after exposure
exposure to different
to different concentration
concentration of Data
of Th. Th. Data
are
are showed
showed as mean
as mean valuevalue ± standard
± standard deviations
deviations (SD). Significant
(SD). Significant difference
difference of the experimental
of the experimental value
value compared
compared with
with each each control
control was marked
was marked with “*” with
(p <“*” (p <or0.05)
0.05) “**” or
(p “**” (p < 0.01).
< 0.01).

2.4.Morphological
2.4. MorphologicalChanges
Changes
The SEM
The SEM images
images in in Figure
Figure44show
showmorphology
morphology differences
differences between
between thethecontrol
controlandandexposed
exposed
cells. As shown in Figure 4A, a typical untreated cell was intact and enclosed with a
cells. As shown in Figure 4A, a typical untreated cell was intact and enclosed with a rigid cell wall.rigid cell wall.
Afterbeing
After beingincubated
incubatedwith with14.4
14.4µMμMof ofTh
Thfor
for96
96h,
h,however,
however,thethealgae
algaecells
cellswere
wereaggregated
aggregatedtogether
together
and the cells were shrunk and distorted (Figure 4B). Moreover, a number of particle
and the cells were shrunk and distorted (Figure 4B). Moreover, a number of particle aggregates were aggregates were
attached on the surface of the treated cells, indicating the strong agglomeration between
attached on the surface of the treated cells, indicating the strong agglomeration between particles and particles
and cells.
cells. The EDS
The EDS spectrum
spectrum of theofred
thebox
redmarked
box marked in Figure
in Figure 4B shows
4B shows the presence
the presence of Thof(Figure
Th (Figure
4C),
4C), which suggests the attachment of Th on the external surface of algae cells. As
which suggests the attachment of Th on the external surface of algae cells. As mentioned above, Thmentioned above,
was
Th was transformed into Th(OH)4 in the medium. Therefore, the observed precipitation adsorbed to
the surface of alga cells was probably Th(OH)4, which will increase the opportunity contacting with
algal cells, and may thus contribute to the algal toxicity of Th. It has been reported that
Int. J. Mol. Sci. 2017, 18, 795 5 of 10

transformed into Th(OH)4 in the medium. Therefore, the observed precipitation adsorbed to the
surface of alga cells was probably Th(OH)4 , which will increase the opportunity contacting with algal
Int. J. Mol. Sci. 2017, 18, 795 5 of 10
cells, and may thus contribute to the algal toxicity of Th. It has been reported that heteroagglomeration
may lead to direct andmay
heteroagglomeration indirect
leadtoxicity to algae
to direct and through
indirect internalization,
toxicity to algae physical
throughdamage, oxidative
internalization,
stress, and/or shading effects [19,20]. In this study, the heteroagglomeration
physical damage, oxidative stress, and/or shading effects [19,20]. In this study, the between the precipitation
of Th(OH)4 and algaebetween
heteroagglomeration cells could possibly leadoftoTh(OH)
the precipitation a reduction in the light available to cells and
4 and algae cells could possibly lead to a
thus decrease the chlorophyll contents. On the other hand,
reduction in the light available to cells and thus decrease the chlorophyll it might be a mechanism
contents.ofOnegodefence
the other
that the exposed cells aggregated together to decrease the physical contact with
hand, it might be a mechanism of egodefence that the exposed cells aggregated together to decrease Th, as reported by
Zhao et al. [21], who showed that the aggregated algae cells act as a barrier
the physical contact with Th, as reported by Zhao et al. [21], who showed that the aggregated algaeto prevent the direct
damage
cells act as of CuO NPstotoprevent
a barrier the cell the
walldirect
and membrane.
damage of CuO NPs to the cell wall and membrane.

Figure 4. SEM images of C. pyrenoidosa after exposure for 96 h. (A) control algal cell; (B) algal cells
Figure 4. SEM images of C. pyrenoidosa after exposure for 96 h. (A) control algal cell; (B) algal cells
treated with 14.4 μM Th; (C) energy dispersive spectroscopy (EDS) analysis of the area marked by
treated with 14.4 µM Th; (C) energy dispersive spectroscopy (EDS) analysis of the area marked by the
the red box in panel B.
red box in panel B.

2.5. Ultrastructural Alterations


2.5. Ultrastructural Alterations
TEM images in Figure 5 show the ultrastructural differences of algae cells between the control
TEM images in Figure 5 show the ultrastructural differences of algae cells between the control and
and the exposed group. In the control group, all the cells had a well-shaped structure. A clear
the exposed group. In the control group, all the cells had a well-shaped structure. A clear nucleus was
nucleus was observed and the cytoplasm was closely attached to the cell membrane (Figure 5A–C).
observed and the cytoplasm was closely attached to the cell membrane (Figure 5A–C). In contrast, the
In contrast, the algal cells exposed to 14.4 μM Th were deformed with serious plasmolysis (Figure
algal cells exposed to 14.4 µM Th were deformed with serious plasmolysis (Figure 5E,F,H), indicating
5E,F,H), indicating the great algal toxicity of Th. In particular, as shown in Figure 5E, the cytoplasm
the great algal toxicity of Th. In particular, as shown in Figure 5E, the cytoplasm obviously shrunk and
obviously shrunk and the cell nucleus was blurry compared with the untreated cells, which may
the cell nucleus was blurry compared with the untreated cells, which may lead to a dysfunction of
lead to a dysfunction of chloroplasts, such as the limitation of nutrient uptake by algae cells [22]. The
chloroplasts, such as the limitation of nutrient uptake by algae cells [22]. The cell wall/membrane of
cell wall/membrane of the exposed alga cells were irregular and were attached with a number of
the exposed alga cells were irregular and were attached with a number of amorphous precipitations
amorphous precipitations (Figure 5E,F,I, black arrows), and this might result in cell wall and
(Figure 5E,F,I, black arrows), and this might result in cell wall and membrane damage as shown in
membrane damage as shown in Figure 5D. Moreover, Th compounds could also be found inside the
Figure 5D. Moreover, Th compounds could also be found inside the cells (Figure 5G, black arrows).
cells (Figure 5G, black arrows). As shown by SEM and TEM, Th in the form of Th(OH)4 was
As shown by SEM and TEM, Th in the form of Th(OH)4 was nano-sized and internalized by alga cells.
nano-sized and internalized by alga cells. These nano-sized Th(OH)4 deposits may impair normal
These nano-sized Th(OH)4 deposits may impair normal cellular processes.
cellular processes.
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Figure 5. Transmission electron microscopy (TEM) images of ultrathin slices of C. pyrenoidosa after
Figure 5. Transmission
exposure to Th for electron microscopy
96 h. (A–C) control algae(TEM) images
cells; (D–I) algae of ultrathin
cells slices
treated with C.ofpyrenoidosa
14.4ofμM Th. (G,I) after
exposurearetohigher
Th for 96 h. (A–C)ofcontrol
magnification the areasalgae cells;
marked (D–I)
by the algae
black cells
boxes treated
in (H). witharrows
The black 14.4 µM of Th.
denoted the(G,I) are
higher magnification of the areas marked by the black boxes in (H). The black arrows denoted the
precipitation of Th.
precipitation of Th.
2.6. Oxidative Stress
Exposure
2.6. Oxidative Stress of algae cells to Th induced an increase in intracellular ROS levels (Figure 6). At the
first 24 h, the difference of ROS between the control group and 1.8, 3.6, 7.2 and 10.8 μM of Th was
Exposure of algae
insignificant, whilecells to Th
the ROS induced
level an increase
was significantly in intracellular
increased by 169.2% atROS levels
14.4 μM of Th(Figure
compared6). At the
first 24 to
h, the
thecontrol
difference
groupof(pROS between
< 0.05). At 48 htheandcontrol
72 h, nogroup and 1.8, 3.6,relationship
clear does-response 7.2 and 10.8 wasµM of Th was
found,
except while
insignificant, that thethe
ROSROSformation
level wassignificantly increased
significantly at 14.4 μM
increased of Th (p at
by 169.2% < 0.01). At 96ofh,Th
14.4 µM thecompared
ROS to
levels were enhanced approximately 2 and 3 times at exposure of 10.8 and 14.4 M Th, respectively.
the control group (p < 0.05). At 48 h and 72 h, no clear does-response relationship was found, except
Elevated intracellular ROS levels may result in oxidative damage to DNA and other
that themacromolecules
ROS formation significantly increased at 14.4 µM of Th (p < 0.01). At 96 h, the ROS levels
and this may subsequently lead to cell death.
were enhanced The approximately
mechanism of the2 toxicity
and 3 times
of Th at
to exposure of 10.8 is
aquatic organisms andstill14.4 M Th, respectively.
unknown. Common heavy Elevated
intracellular
metal ROS levels
pollutants may
(e.g., Cd,result in etc.)
Pb, Hg, oxidative damage
show high to for
affinity DNA thioland other macromolecules
containing biomolecules (GSHand this
and sulfhydryl
may subsequently proteins)
lead to celland act as a catalyst in Fenton-type reactions, producing oxidative damage
death.
The mechanism of the toxicityinsoluble
[23]. Since Th was present as an of Th to Th(OH) 4 in the exposure medium, the biological behavior
aquatic organisms is still unknown. Common heavy
of Th was probably different from those above mentioned heavy metals. The TEM images show that
metal pollutants (e.g., Cd, Pb, Hg, etc.) show high affinity for thiol containing biomolecules (GSH and
nano-sized Th(OH)4 was deposited both inside and outside the cells. Recent progresses in the
sulfhydryl proteins)
aquatic toxicityand act as a catalyst
of engineered in Fenton-type
nanomaterials reactions,
suggest that insolubleproducing
nano-sizedoxidative
particles damage
might [23].
Since Th was present as an insoluble Th(OH)4 in the exposure medium, the biological behavior
of Th was probably different from those above mentioned heavy metals. The TEM images show
that nano-sized Th(OH)4 was deposited both inside and outside the cells. Recent progresses in
the aquatic toxicity of engineered nanomaterials suggest that insoluble nano-sized particles might
exhibit toxicity to green algae by the following processes: (1) the shading effect may attenuate the
photosynthesis by reducing light transmittance; (2) the heteroagglomeration and physical interaction
Int. J. Mol. Sci. 2017, 18, 795 7 of 10
Int. J. Mol. Sci. 2017, 18, 795 7 of 10

exhibit toxicity to green algae by the following processes: (1) the shading effect may attenuate the
photosynthesis by reducing light transmittance; (2) the heteroagglomeration and physical
may lead to the internalization of nanoparticles, cell membrane disruption and endocyte outflow;
interaction may lead to the internalization of nanoparticles, cell membrane disruption and endocyte
(3) theoutflow;
permeation and
(3) the entry of and
permeation nanoparticles into the cells
entry of nanoparticles intomay induce
the cells maythe elevation
induce of intracellular
the elevation of
ROS levels and the
intracellular membrane
ROS levels andlipid
the peroxidation
membrane lipid [19–21,24–26]. All these processes
peroxidation [19–21,24–26]. might
All these contribute
processes
to the toxic
mighteffects of Th
contribute theC.toxic
to to pyrenoidosa. However,
effects of Th understanding
to C. pyrenoidosa. theunderstanding
However, underlying mechanism requires
the underlying
furthermechanism requires further investigations.
investigations.

Figure 6. ROS generation of algae cells after exposure to different concentration of Th. Data are
Figure 6. ROS generation of algae cells after exposure to different concentration of Th. Data are showed
showed as mean value ± standard deviations (SD). Significant difference of the experimental value
as mean value ± standard deviations (SD). Significant difference of the experimental value compared
compared with each control was marked with “*” (p < 0.05) or “**” (p < 0.01).
with each control was marked with “*” (p < 0.05) or “**” (p < 0.01).
3. Materials and Methods
3. Materials and Methods
3.1. Materials and Chlorella pyrenoidosa Culture
3.1. Materials and used
Th was Chlorella
in thepyrenoidosa Culture
form of Th(NO 3)4∙5H2O with purity over 99%. All chemicals were analytical

grade
wasand were purchased
form offrom Beijing Chemical Plant. Freshwater algae Chlorella pyrenoidosa (C.
Th used in the Th(NO 3 )4 ·5H2 O with purity over 99%. All chemicals were analytical
pyrenoidosa) was obtained from the Institute of Hydrobiology, Chinese Academy of science, Wuhan,
grade and were purchased from Beijing Chemical Plant. Freshwater algae Chlorella pyrenoidosa
China. The algae were cultured in 100 mL conical flasks containing complete Organization for
(C. pyrenoidosa) was obtained from the Institute of Hydrobiology, Chinese Academy of science, Wuhan,
Economic Co-operation and Development (OECD) 201 medium [27]. The flasks were placed on a
China.shaker
The (95
algae were in
± 5 r/min) cultured in 100 mL incubator.
the bed temperature conical flasks containingof complete
The temperature the incubatorOrganization
was set at for
Economic Co-operation and Development (OECD) 201 medium [27]. The flasks were
24 ± 1 °C under illumination of 3000 ± 10% l× light intensity, with a 12-h light and 12-h night daily placed on a
(95 ±During
shakercycle. 5 r/min) in the bed growth
the exponential temperature
phase, incubator. Thewere
the algae cells temperature
calculated of
bythe incubator was set at
a hematocytometer
◦ C the
24 ± 1and cell density
under was monitored
illumination of 3000 ±at 680
10%nm l×(OD 680)intensity,
light with a microplate readerlight
with a 12-h (infinite
andM200
12-h PRO).
night daily
cycle. According
During the to the results of hematocytometer
exponential growth phase, the and algae
OD680 values, therecalculated
cells were was a correlation between the
by a hematocytometer
cell density of C. pyrenoidosa and the optical density (OD680) values. The regression equation was
and the cell density was monitored at 680 nm (OD680 ) with a microplate reader (infinite M200 PRO).
calculated as y (×106 mL−1) = 50.97 OD680 − 0.15 (R2 = 0.99).
According to the results of hematocytometer and OD680 values, there was a correlation between the
cell density C. pyrenoidosa
of Growth
3.2. Algal Assays and the optical density (OD680 ) values. The regression equation was
calculated as y (×10 mL−1 ) = 50.97 OD680 − 0.15 (R2 = 0.99).
6
Following the OECD 201 algal growth-inhibition test guidelines, algae cells in the logarithmic
growth phase were used for all experiments. The cells were collected, washed, and diluted to the
3.2. Algal Growth Assays
initial concentration of 2 × 106 cells L−1 for the exposure of Th. Th(NO3)4 5H2O were added to the
axenic algae
Following 201
the medium
OECD 201toalgal
reachgrowth-inhibition
the final Th concentrations at 0, 1.8, 3.6,
test guidelines, 7.2,cells
algae 10.8, in
and 14.4
the μM.
logarithmic
After the addition of Th, the pH values of the treated algal media were all around
growth phase were used for all experiments. The cells were collected, washed, and diluted to the 7.4. The algae cells
initial in the control group
concentration of 2without Th were
× 106 cells L−1conducted following of
for the exposure theTh.
same procedure.
Th(NO All treatments were
3 )4 5H2 O were added to the
performed in triplicate. The growth of algae cells was examined by monitoring the OD680 values after
axenic algae 201 medium to reach the final Th concentrations at 0, 1.8, 3.6, 7.2, 10.8, and 14.4 µM.
different exposure time (1, 2, 3 and 4 days). Percent inhibition of growth was calculated at each time
After the addition of Th, the pH values of the treated algal media were all around 7.4. The algae cells
in the control group without Th were conducted following the same procedure. All treatments were
performed in triplicate. The growth of algae cells was examined by monitoring the OD680 values after
different exposure time (1, 2, 3 and 4 days). Percent inhibition of growth was calculated at each time for
the estimation of EC50 and 95% confidence interval using Probit analysis in SPSS software (version 18,
IBM, Armonk, NY, USA).
Int. J. Mol. Sci. 2017, 18, 795 8 of 10

3.3. Chlorophyll a Fluorescence Measurements


The fluorescence intensity of chlorophyll a was measured at different exposure times, following
a procedure described by Jeffrey and Humphrey [28]. Four milliliters of ethanol was added to 1 mL
algae suspension of each group to extract the chlorophyll. After 3 h reaction in the dark, the mixing
suspension was measured by a fluorescence spectrophotometer (RF-5301PC). The excitation and
emission wavelengths were 420 and 671 nm, respectively. According to the result of preliminary
experiment, concentrations of chlorophyll a and the fluorescence intensity have a dose relationship
(IF = 179.50c + 5.62, R2 = 0.98).

3.4. Th Speciation in the Cultured Medium


To determine the actual chemical species of Th present in the cultural medium, make equilibrium
diagrams using sophisticated algorithms (MEDUSA program) was used for the construction of
distribution diagrams. The basic parameters, including equilibrium constants that are needed
for the calculation of equilibrium diagrams were in the program database. The program was
written by Ignasi Puigdomenech from the Inorganic Chemistry department of Royal Institute of
Technology, Stockholm, Sweden [29]. The MEDUSA program is a freeware and is available at
http://www.kemi.kth.se/medusa.

3.5. SEM And TEM Observations


After being exposed to 0 or 14.4 M of Th for 96 h, the algae cells were collected by centrifugation
(400× g) for 5 min at 4 ◦ C. Then the supernatant was removed, the collected algae cells were washed
with phosphate-buffed saline (PBS, pH 7.4) for three times. The washed cells were then resuspended
with PBS at certain concentration and placed on sample holder for observation. The morphology
of algae was observed with a scanning electron microscope (SEM, S-4800, Hitachi, Tokyo, Japan),
equipped with the energy dispersive spectroscopy (EDS, EMAX-250, HORIBA, Kyoto, Japan).
The transmission electron microscopy (TEM) observation of algae cells followed a modified
procedure described by Xia et al. [30]. All samples (treated and untreated cells) were postfixed in 1%
osmic acid for 1 h and washed with phosphate butter solution (PBS, pH 7.0) for 3 times. Dehydration
process of the samples was conducted in increasing concentrations of acetone at room temperature.
The samples were permeated and impregnated in resin for 5 h. Ultrathin sections were made and placed
on Cu grids for imaging. The ultrastructure of algae cells were observed with TEM (JEM-1230, JEOL,
Tokyo, Japan).

3.6. Oxidative Stress


ROS generation of algae cells under different treatments was detected using
2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), which is an oxidation-sensitive fluorescent
probe dye. The ROS was measured according to the approach described by Saison et al. [31].
After exposure to Th for 0–96 h, 20 µL of DCFH-DA (100 µM) was added to 180 µL of algae suspension
to reach the final concentration at 10 µM. Then the algal cells were washed three times to remove the
unbound DCFH-DA. DCFH-DA could be transformed into H2 DCF by intracellular esterase if they
enter cells. When intracellular ROS was generated, intracellular H2 DCF could be deacetylated and
then oxidized to the highly fluorescent dichlorofluorescein (DCF). The fluorescence intensity of DCF,
which indicated the extent of ROS generation, was measured using a microplate reader (infinite M200
PRO). The excitation and emission wavelengths were 488 and 525 nm, respectively. The ROS levels in
the treated groups were expressed as percentages relative to the control group.

3.7. Statistical Analysis


Data are reported as mean ± S.D. The significant differences between the control and the
treatments were analyzed by one-way ANOVA with least significant difference (LSD) test or
Int. J. Mol. Sci. 2017, 18, 795 9 of 10

Kruskal-Wallis H ANOVA with Mann–Whitney U test. Analysis was performed using the IBM
SPSS (version 18, IBM). The significant level was set at p < 0.05 (*) or p < 0.01 (**).

4. Conclusions
This study investigated the toxicity of Th to C. pyrenoidosa on the basis of its chemical species in the
cultural medium. Th in the form of Th(OH)4 could inhibit the growth of algae cells, reduce chlorophyll
contents, and enhance the intracellular levels of ROS. Th-containing precipitation was attached on the
surface of algal cells as determined by SEM observation combined with EDS. TEM images showed
the plasmolysis, membrane damage, and ultrastructural changes of the exposed algal cells. Overall,
the direct physical interaction of agglomerations with algal cells and generation of intracellular ROS
were the main reasons for the toxicity of Th to C. pyrenoidosa. This study significantly advances our
understanding of the potential toxicity of Th to aquatic species. In future studies, more attention
should be paid to the toxicity of Th in insoluble forms such as Th(OH)4 .

Acknowledgments: This work was financially supported by National Natural Science Foundation of China
(Grant No. 11375009, 11575208, 11405183, 11675190, 11275215 and 11275218) and the Ministry of Science and
Technology of China (Grant No. 2013CB932703).
Author Contributions: Yuhui Ma and Zhiyong Zhang conceived and designed the experiments; Can Peng
performed the experiments; Yuhui Ma and Can Peng analyzed the data; Yayun Ding, Xiao He, Peng Zhang,
Tu Lan, and Dongqi Wang contributed reagents/materials/analysis tools; Yuhui Ma and Can Peng wrote the
paper; Zhiyong Zhang and Zhaohui Zhang revised the paper.
Conflicts of Interest: The authors declare no conflict of interest.

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