Aquatic Toxicology 158 (2015) 1–13

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

Toxicity of nano-TiO2 on algae and the site of reactive oxygen species

production
Fengmin Li a,∗ , Zhi Liang a , Xiang Zheng b,∗∗ , Wei Zhao a , Miao Wu a , Zhenyu Wang a
a
Key Laboratory of Marine Environmental Science and Ecology, Ministry of Education, College of Environmental Science and Technology,
Ocean University of China, Qingdao 266100, PR China
b
School of Environment and Natural Resources, Renmin University of China, Beijing 100872, China

a r t i c l e i n f o a b s t r a c t

Article history: Given the extensive use of nanomaterials, they may enter aquatic environments and harm the growth of
Received 11 August 2014 algae, which are primary producers in an aquatic ecosystem. Thus, the balance of an aquatic ecosystem
Received in revised form 15 October 2014 may be destroyed. In this study, Karenia brevis and Skeletonema costatum were exposed to nano-TiO2
Accepted 21 October 2014
(anatase, average particle size of 5–10 nm, specific surface area of 210 ± 10 m2 g−1 ) to assess the effects
Available online 3 November 2014
of nano-TiO2 on algae. The findings of transmission electron microscopy-energy dispersive X-ray spec-
troscopy (TEM-EDX) and scanning electron microscopy (SEM) demonstrate aggregation of nano-TiO2 in
Keywords:
the algal suspension. Nano-TiO2 was also found to be inside algal cells. The growth of the two species
Nano
Titanium dioxide
of algae was inhibited under nano-TiO2 exposure. The 72 h EC50 values of nano-TiO2 to K. brevis and S.
Algae costatum were 10.69 and 7.37 mg L−1 , respectively. TEM showed that the cell membrane of K. brevis was
ROS destroyed and its organelles were almost undistinguished under nano-TiO2 exposure. The malondial-
Oxidative stress dehyde (MDA) contents of K. brevis and S. costatum significantly increased compared with those of the
control (p < 0.05). Meanwhile, superoxide dismutase (SOD) and catalase activities (CAT) of K. brevis and S.
costatum changed in different ways. The reactive oxygen species (ROS) levels in both species were signifi-
cantly higher than those of the control (p < 0.05). The site of ROS production and accumulation in K. brevis
and S. costatum under nano-TiO2 exposure was explored with the addition of inhibitors of different elec-
tron transfer chains. This study indicated that nano-TiO2 in algal suspensions inhibited the growth of K.
brevis and S. costatum. This effect was attributed to oxidative stress caused by ROS production inside algal
cells. The levels of anti-oxidative enzymes changed, which destroyed the balance between oxidation and
anti-oxidation. Thus, algae were damaged by ROS accumulation, resulting in lipid oxidation and inhibited
algae growth. The inhibitors of the electron transfer chain showed that the site of ROS production and
accumulation in K. brevis cells was the chloroplast.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction nanotechnologists have expressed concerns about current and

Nanomaterials are increasingly being used in industrial pro-
duction, as well as scientific, biological, and medical research (Nel
et al., 2009; Patra et al., 2012; Shipway et al., 2000; Zhang, 2003).
Nanoscale TiO2 has been widely applied in industrial applications,
including the paint, textile, paper, plastic, sunscreen, cosmetic,
and food industries (Chong et al., 2010; Dastjerdi and Montazer,
2010; Suh et al., 2009; Tong et al., 2012). Given the extensive
use of nanomaterials, several nongovernmental organizations and
∗ Corresponding author. Tel.: +86 053266782780.
∗∗ Corresponding author.
E-mail addresses: lifengmin@ouc.edu.cn (F. Li), zhengxiang7825@sina.com
(X. Zheng).
potential future developments of nanotechnology. The uncertain-
ties about the impact of new nanomaterials on human health
and ecotoxicity require our urgent concern (Manier et al., 2013;
Melegari et al., 2013; Pakrashi et al., 2013; Rodea-Palomares
et al., 2012). Considering the widespread use of nanomaterials,
they may be released in the aquatic environment. The possible
effects of these chemicals to aquatic life, especially among algae,
which are the primary producers in aquatic environments, have
become a large concern. Different toxic effects of nanoparticles
on algae have been studied. Nanoparticles inhibit the growth and
photosynthetic abilities of algae, alter enzymatic activity [e.g.,
malondialdehyde (MDA), superoxide dismutase (SOD), catalase
(CAT), and glutathione (GSH)] in algal cells, and cause DNA dam-
age (Aruoja et al., 2009; Chen et al., 2012; Ma et al., 2013; Melegari
et al., 2013; Wang et al., 2008; Wong et al., 2010). However, the

http://dx.doi.org/10.1016/j.aquatox.2014.10.014
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2 F. Li et al. / Aquatic Toxicology 158 (2015) 1–13
mechanisms of these toxic effects on aquatic organisms are largely
unknown.
The indirect effects of nanoparticles are caused mainly by phys-
ical restraints or release of toxic ions (e.g., metal nanoparticles) or
the production of reactive oxygen species (ROS). Physical restraints
can cause toxicity because of the addition of nanoparticles, such
as the shading effect. The production of nanoparticles on the sur-
face of algal cells might reduce light availability for photosynthesis
(Navarro et al., 2008; Schwab et al., 2011). Aggregation of nanopar-
ticles on the surface of algal cells increased the weight of cells
by two- to three-fold (Huang and Ismat, 2005). Nanoparticles on
the surface of cells might block the cell wall and prevent nutri-
ent molecules from entering cells (Chen et al., 2012; Metzler et al.,
2011). By contrast, Hund-Rinke and Simon (2006) showed that
nanoparticles have no impact on the growth and light availability
of algal cells. Thus, whether the shading effect is the toxic mecha-
nism by which nanoparticles influence algal cells requires further
research. The release of toxic ions of nanoparticles, especially metal
nanoparticles, also causes toxicity toward algal cells (Borm et al.,
2006). Because TiO2 could not release ions in suspension (Wang
et al., 2008), the toxicity of releasing ions was excluded.
The toxicity of nanoparticles to cells may also be attributed to
ROS production. Nanoparticles might produce ROS upon interac-
tion with organisms or agents present in the environment (Navarro
et al., 2008). Nanoparticles, such as TiO2 , produce ROS in aquatic
environments because of their photocatalytic properties (Kus et al.,
2006). The toxicity of nano-TiO2 toward bacteria increases under
ultraviolet (UV) exposure (Adams et al., 2006). Other nanoparticles,
such as fullerol, also generate ROS after UV exposure (Badireddy
et al., 2007). When nanoparticles entered algal cells through the cell
membrane, ROS might be induced to generate in algal cells; thus,
the antioxidant (e.g., ascorbate, glutathion, etc.) and antioxidant
enzymes (such as SOD, CAT, etc.) change following ROS genera-
tion to maintain the pro-oxidation/anti-oxidation balance. Patra
et al. (2012) studied the antifungal effects of zinc oxide nanoparti-
cles, and discovered that these effects were due to ROS generation.
The activities of SOD and CAT increased to eliminate excess ROS.
Lipid peroxidation is an important process involved in the toxic
mechanism of nanoparticles. Chen et al. (2012) suggested that lipid
peroxidation is induced under nano-TiO2 exposure, and found that
the MDA contents initially increased and then decreased after 8 h
of exposure. Chen associated these results to ROS production or
the shading effect, but these findings have not been confirmed.
Therefore, the present study aimed to determine the site of ROS
production.
Three main types of ROS producers exist in plants, namely,
electron transport chains (ETCs) in chloroplasts and mitochon-
dria, some peroxidases and oxidases (e.g., NADPH oxidase, NADH
oxidase, xanthine oxidase, lipoxygenase, glycolate oxidase, amine
oxidase, etc.), and photosensitizers, such as chlorophyll molecules
(Dat et al., 2000; Edreva, 2005; Song et al., 2012). Chloroplast is
one of the main source and site of ROS production (Alberts and
Lewis, 2002; Gill and Tuteja, 2010). If light energy is not com-
pletely consumed, O2 , which has been converted into an electron,
will be a selective receptor of excess light energy that is con-
verted to superoxide free radicals. Many studies showed that plant
cells can produce ROS when exposed to toxic substances. Forti and
Gerola (1977) found inhibited peroxidase activity and photosyn-
thesis rate, as well as low H2 O2 content, when chloroplasts are
isolated from spinach exposed to azide and cyanide. Tripathy and
Mohanty (1980) declared that Zn2+ ions affect the ETC in chloro-
plasts. Photosystem II (PS II) is inhibited under low concentrations
of Zn2+ exposure, whereas photosystem I (PS I) is inhibited under
high concentrations of Zn2+ exposure. Therefore, metal nanopar-
ticles that can release metal ions may also affect the ETC in algal
chloroplasts.
In human and animal cells, the mitochondrion was considered
to be the main locus of ROS production. During aerobic respira-
tion, most electrons transfer to the mitochondrion, which is the
terminal of the ETC, and combine with oxygen to produce water.
However, a small part of electrons leak out of the ETC and combine
with oxygen to produce superoxide free radicals. Hydroxyl radicals
and hydrogen peroxide radicals are produced through specific reac-
tions. Many studies showed that the ETC in the mitochondrion is
affected by toxic substances. Li et al. (2010) suggested that 3-(4-
(benzo[d]thiazol-2-yl)-1-phenyl-1H-pyrazol-3-yl) phenyl acetate
(DPB-5) has a significant cytotoxic effect on Hep G2 cells and
induces cell apoptosis. In the mitochondrion, the GSH content
increases, ROS accumulates, and the membrane potential of the
mitochondrion is destroyed. The addition of rotenone, an inhibitor
of the mitochondrial ETC (Reynafarje et al., 1976), significantly
decreases the ROS content caused by DPB-5. Therefore, the mito-
chondrion is one of the main sites of ROS production.
Lovern et al. (2007) pointed out that certain nanoparticle types
may have impacts on the population and food web dynamics in
aquatic systems. Algae are primary producers that provide energy
and nutrients for other organisms through the food web. Changes
that occur to algal populations under nanoparticle exposure can
alter the entire ecosystem. In the present study, Karenia brevis and
Skeletonema costatum were used as target organisms. Karenia brevis
is a kind of red-tide algae. They were found in many continents such
as Europe, North America, Asian and Australia (Guiry and Guiry,
2014). Marine diatom Skeletonema costatum can provide bait for
fish and shrimp (Hasle and Syvertsen, 1997). They were often found
in China coast (Guo et al., 2014). These organisms are important pri-
mary producers which can affect the balance of ocean ecosystem.
This research aimed to determine the effects of nano-TiO2 on
the growth of two algae species, and observe the changes in ROS
levels and antioxidant enzymes in algal cells under exposure to
nano-TiO2 suspension. Moreover, the mechanisms underlying the
inhibition of algae growth by nano-TiO2 were investigated. The site
of ROS production and accumulation in algal cells under nano-TiO2
exposure was also explored.
2. Materials and methods
2.1. Algae cultures
K. brevis and S. costatum were purchased from the Institute of
Oceanography, Chinese Academy of Sciences. K. brevis and S. costa-
tum were maintained in F/2 medium (Guillard and Ryther, 1962) in
5 L flasks. Cultures were pre-cultured to logarithmic phase before
exposure at 22 ± 1 ◦ C, with cool-white fluorescent light at a photon
intensity of 12,000 lux and a 14:10 h light dark photocycle. Flasks
were shaken by hand twice each day to obtain a homogenous cell
distribution.
2.2. Nano-TiO2
Nano-TiO2 (anatase) was purchased from Wanjingxin Company
(Hangzhou, China). It had an average particle size of 5–10 nm and
specific surface area of 210 ± 10 m2 g−1 .
2.2.1. Particle size and morphology of nano-TiO2
Nano-TiO2 was diluted with distilled water to a final concentra-
tion of 20 mg L−1 . After 30 min of ultrasonic treatment, nano-TiO2
was dipped by a copper screen and air-dried under sunlight for 2 h.
The particle size and crystal parameter of nano-TiO2 were detected
by transmission electron microscopy energy-dispersive X-ray spec-
troscopy (TEM-EDX). After K. brevis cultures were exposed to
nano-TiO2 (0, 10, and 100 mg L−1 ) for 24 h under similar culture
 F. Li et al. / Aquatic Toxicology 158 (2015) 1–13
parameters, nano-TiO2 morphology was observed by scanning
electron microscopy (SEM).
2.2.2. Preparation of nano-TiO2 suspension
Nano-TiO2 was suspended in F/2 medium to obtain stock solu-
tions (1000 mg L−1 (12.5 mol L−1 )) followed by 30 min of sonication,
and shaken for 2 min. Prior to the exposure experiment, the stock
solutions (1000 mg L−1 ) of nano-TiO2 were diluted to four con-
centrations, namely, 125, 250, 500, and 750 mg L−1 (1562.5, 3125,
6250, 9375 mmol L−1 ). The solutions were disinfected with UV light
before further analyses.
2.3. Effects of nano-TiO2 on algae growth
2.3.1. Shading effect
A 500 mL flask containing 250 mL of algae culture was mixed
in a 2 L flask filled with 50 mg L−1 nano-TiO2 suspension, and the
control group was filled with distilled water. The water level out-
side the 500 mL flask should be higher than that inside the flask.
All samples were prepared in triplicate. The cell number (cell ml−1 )
was counted every 24 h.
2.3.2. Growth of algae exposed to nano-TiO2 suspension
Algae cultures (pre-cultured in 5 L flask) in logarithmic phase
with cell number more than 104 cells mL−1 were diluted with
F/2 medium. Approximately 240 mL of algae was transferred into
15,500 mL flasks equally. Approximately 10 mL of these four dilu-
tions (125, 250, 500, and 750 mg L−1 ) of nano-TiO2 were added into
these flasks. The algae cultures, as well as the control without nano-
TiO2 , were treated under identical conditions. These five groups
were prepared in triplicate. The final concentrations of nano-TiO2
in the algae cultures were 0, 5, 10, 20, and 30 mg L−1 . The cell den-
sity was counted every 24 h, and the growth curve and 72 h EC50
were analyzed. The chlorophyll a (Chl-a) content was measured
after 48 h of exposure. The nano-TiO2 distributions and ultrastruc-
ture of algal cells in 0 and 20 mg L−1 nano-TiO2 suspension were
detected by TEM after 24 h of exposure.
2.4. Lipid peroxidation and site of ROS production
2.4.1. Lipid peroxidation
The MDA concentration, antioxidant enzyme (SOD and CAT)
activities, and contents of three free radicals (• OH, O2 •− , and H2 O2 )
in algal cells and suspensions were analyzed after 48 h of exposure.
2.4.2. Site of ROS production
Algae cultures were cultured as described in 2.1, and transferred
into 500 mL flasks. Firstly three inhibitors, namely, rotenone (CAS
330-54-1, analytical grade, Sigma–Aldrich, USA), diuron (CAS 83-
79-4, analytical grade, Sigma–Aldrich, USA), and dicoumarol (CAS
66-76-2, analytical grade, Sigma–Aldrich, USA), were added into
each flask. 30 min later 20 mg L−1 nano-TiO2 was added into each
flask with addition of inhibitors. Then algae cultures were exposed
to 0 and 20 mg L−1 nano-TiO2 suspensions, inhibitors, or a mixture
of the inhibitor and nano-TiO2 for 24 h under similar culture param-
eters. The final volume of algae cultures in the flasks was 250 mL,
and the groups were prepared in triplicate. The final concentrations
of rotenone, diuron, and dicoumarol were 1.25, 5, and 5 ␮mol L−1
(the concentrations at which these three inhibitors had no effects
on algae), respectively. Specific method of operation was shown in
Table 1. The ROS levels were then detected.
2.4.3. Isolated chloroplast
Algae cultures were cultured for 48 h as mentioned above.
Chlorophyll was extracted from algal cells by centrifugation,
and suspended in buffer solution (pH 8, 150 mmol L−1 NaCl,
3
100 mmol L−1 EDTA). Nano-TiO2 suspension was added, and the
final concentration of nano-TiO2 was 10 mg L−1 . The chlorophyll
suspension without nano-TiO2 was used as the control. Groups
were prepared in triplicate. Given that O2 •− is too active to detect
and • OH is the product of the secondary reaction, the • OH content
was measured in the isolated chloroplast after 24 h of exposure.
2.5. Test methods
2.5.1. Cell number and EC50
The cell number (cell mL−1 ) was counted using a hemocytome-
ter under a light microscope (Nikon, YS100, Japan) every 24 h. The
EC50 value was calculated using SPSS version 19.
2.5.2. Chl-a content
After 48 h of exposure, Chl-a of test algae was extracted in 90%
ethanol in the dark, followed by 5 min of centrifugation at 3600 rpm
for K. brevis and 6 min of centrifugation at 4200 rpm for S. costatum.
Chl-a was diluted to 10 mL with 90% ethanol. The Chl-a content
was measured at 750, 663, 645, and 630 nm with a UV–vis scanning
spectrophotometer (UV-2550, SHIMADZU, Japan), and 90% ethanol
was set as the control.
2.5.3. Preparation of cell homogenate
K. brevis cells were collected by centrifugation at 3381 rpm and
4 ◦ C for 5 min, whereas S. costatum cells were collected by centrifu-
gation at 4200 rpm and 4 ◦ C for 6 min. Cells were homogenized in
4 mL of PBS solution (pH 7.8, 4 ◦ C) by an ultrasonic cell pulverizer
(KS-500F, Kesheng, Ningbo, China) in an ice bath for 5 min. The
homogenate was used to measure the MDA content, or transferred
using 3 mL of PBS into a 10 mL plastic centrifugal tube for centrifu-
gation at 4428 rpm and 4 ◦ C for 10 min. The supernatant was used
for enzyme, ROS, and free radical assays.
2.5.4. Lipid peroxidation assays
The MDA content and SOD and CAT activities were detected
using a reagent kit (Nanjing Jiancheng Biotechnology Institute,
China) according to the manufacturer’s instructions (Deng et al.,
2009; Han et al., 2004). The MDA content was determined based
on the modified NBT method (Rocchetta et al., 2006). SOD activ-
ity was determined based on the WST-1 method. CAT activity was
determined based on the method of ammonium molybdate spec-
trophotometry.
2.5.5. ROS assays
The cell homogenate was obtained using the method described
•
in Section 2.5.3. The levels of three free radicals (i.e., • OH, O2 − ,
and H2 O2 ) and ROS were detected using a reagent kit (Nanjing
Jiancheng Biotechnology Institute, China) according to the man-
•
ufacturer’s instructions. The free radicals • OH, O2 − , and H2 O2
were detected by a UV–vis spectrophotometer (UV-2550, SHI-
•
MADZU, Japan), • OH was detected at 550 nm, O2 − was detected
at 550 nm, and H2 O2 was detected at 405 nm. ROS levels were
determined using a 2,7-dichlorofuorescin diacetate probe. ROS lev-
els were detected by a fluorescence spectrophotometer (F4600,
Hitachi, Japan) at the emission and excitation wavelengths of 525
and 488 nm, respectively.
2.5.6. TEM-EDX and SEM analysis
After 24 h of exposure, algal cells were collected by centrifu-
gation (3800 rpm, 10 min), and the samples were fixed with 2.5%
glutaraldehyde solution in 4 ◦ C for 2 h. The samples were then
washed with 0.1 M PBS (pH 7.8) by centrifugation (3800 rpm,
10 min) three times. Algal cells were fixed with 1% osmium tetrox-
ide for 1 h in 4 ◦ C, and 0.1 M PBS (pH 7.8) was added to wash the cells
by centrifugation (3800 rpm, 10 min) three times. Samples were
4 F. Li et al. / Aquatic Toxicology 158 (2015) 1–13

Table 1
A list of steps on how to find out which ETC was the site of ROS production with addition of ETC inhibitors. A: Control; B: rotenone; C: diuron; D: dicoumarol; E: nano-TiO2 ;
F: nano-TiO2 + rotenone; G: nano-TiO2 + diuron; H: nano-TiO2 + dicoumarol.

A B C D E F G H

Algae culture (mL) 250 245 245 245 245 240 240 240
62.6 ␮mol L−1 Rotenone (mL) / 5 / / / 5 / /
250 ␮mol L−1 Diuron (mL) / / 5 / / / 5 /
250 ␮mol L−1 Dicoumarol (mL) / / / 5 / / / 5
Algae culture was treated with inhibitors for 30mins
1000 mg L−1 TiO2 (mL) / / / / 5 5 5 5

dehydrated successively through a series of acetone solutions of

30%, 50%, 70%, 90%, and 100% for 10 min. The samples were embed-
ded in Epon 812 resin, and fixed in gradient temperatures of 37 ◦ C,
45 ◦ C, and 65 ◦ C for 24 h. Sample slices were prepared by a micro-
tome (Ultracut E), and stained with uranyl acetate and lead citrate.
The ultrastructures of algal cells were observed by TME-EDX (JEOL,
JEM-1200EX, Japan). The samples observed by SEM were treated
similar to the procedure mentioned above, except acetone solution
was used instead of alcohol solution. SEM (KYKY-2800B) was used
to observe samples.

2.6. Statistical analysis

The experimental data were analyzed by one-way ANOVA using

SPSS version 19. Tukey’s multiple comparison at a probability of
0.05 was also used. Statistical significance was set at p < 0.05.

3. Results

3.1. Nano-TiO2

3.1.1. Particle size of nano-TiO2 in suspension

The particle size of nano-TiO2 diluted with distilled water was
observed by TEM (Fig. 1A). The mean particle size of nano-TiO2 was
calculated to be less than 40 nm. Fig. 1B shows particle aggregation
detected by TEM-EDX. Comparison with the standard lattice index
of TiO2 showed that the aggregates were TiO2 .

3.1.2. Morphology of nano-TiO2 in algae cultures

After nano-TiO2 suspensions were added into algae cultures,
nano-TiO2 morphology in the suspensions was observed by SEM.
Fig. 2 shows nano-TiO2 aggregates on the surface of algal cells in
algae culture. The particle size of nano-TiO2 in suspensions was
bigger than that of dry powder. Algal cells were almost absolutely
covered by 100 mg L−1 nano-TiO2 . In addition, the aggregates on
the cell surface increased with increasing nano-TiO2 concentra-
tion. Nanoparticles have been used as an efficient sorbent in many
fields because of their high specific surface area. The high specific Fig. 1. Particle size of nano-TiO2 in suspension. Particle size (A) of nano-TiO2 and
surface area and strong adsorption ability of nano-TiO2 may cause its crystal parameter (B) was detected by TEM-EDX.
enwrapping of algal cells.

3.3. Effects of nano-TiO2 on algae growth

3.2. Shading effect
Aggregation of nano-TiO2 on the surface of algal cells in algae
cultures may decrease the photosynthetic abilities of algal cells and
inhibit growth. The shading effect may be a mechanism of nano-
TiO2 toxicity. Fig. 3 shows that the cell numbers of K. brevis and
S. costatum in the treatment groups changed, but were not signifi-
cantly different from those of the control. Thus, the shading effect
was not a mechanism of nano-TiO2 toxicity in algal cells. If nano-
TiO2 can enter algal cells, we then aimed to determine whether it
can inhibit algae growth.
3.3.1. Inhibition of nano-TiO2 on algae growth
The inhibition of nano-TiO2 on algae growth is shown in Fig. 4.
Fig. 4A shows the effects of nano-TiO2 on the growth of K. brevis.
The inhibitory rate increased with increasing nano-TiO2 concen-
tration, but stopped increasing with time. After 24 h of exposure,
the inhibitory rates of each group were 32%, 63%, 74%, and 84%. The
results show that K. brevis was inhibited by nano-TiO2 , and the 72 h
EC50 value was 10.69 mg L−1 .
Fig. 4B shows the effects of nano-TiO2 on the growth of S.
costatum. The inhibitory rate increased with increasing nano-TiO2
 F. Li et al. / Aquatic Toxicology 158 (2015) 1–13 5

Fig. 2. Morphology of nano-TiO2 in algae cultures. Morphology of nano-TiO2 was observed by SEM in K. brevis cultures. (A): K. brevis treated by 0 mg L−1 nano-TiO2 ; (B): K.
brevis treated by 10 mg L−1 nano-TiO2 ; (C): K. brevis treated by 100 mg L−1 nano-TiO2 .

Fig. 3. Growth of algae cells with shading effects. (A): K. brevis; (B): S. costatum.
Fig. 4. Inhibition rates of different concentrations of nano-TiO2 on the growth of
Values represent mean ± S.D. n = 3.
algae. (A): K. brevis; (B): S. costatum. Values represent mean ± S.D. n = 3.
6 F. Li et al. / Aquatic Toxicology 158 (2015) 1–13
Fig. 5. Chl-a of algae cells exposed to different concentrations of nano-TiO2 . (A):
K. brevis; (B): S. costatum. Values represent mean ± S.D. Different letters represent
significant differences between the treatment means (p < 0.05, Tukey), n = 3.
concentration, and continued to increase with time. For instance,
the inhibitory rate of the 5 mg L−1 nano-TiO2 group was 27.1%.
After 24 h of culture, it increased to 61% at the seventh day. The
other groups showed similar trends. The results show that S. costa-
tum was inhibited by nano-TiO2 , and the 72 h EC50 value was
7.37 mg L−1 .
The chlorophyll content is an indicator of the growth situation of
algal cells. The Chl-a content of algal cells exposed to different nano-
TiO2 concentrations is shown in Fig. 5. The Chl-a content of K. brevis
cells significantly decreased under treatment with 10 mg L−1 nano-
TiO2 (p < 0.05), as shown in Fig. 5A. The Chl-a contents of the control
and 30 mg L−1 groups were 448 and 209 mg m−3 , respectively. The
Chl-a content of the 30 mg L−1 group decreased by 53.3% compared
with that of the control. Fig. 5B indicates that the Chl-a content of
S. costatum cells significantly decreased with increasing nano-TiO2
concentration (p < 0.05). The Chl-a content of the 30 mg L−1 group
was 55 m−3 , and decreased by 61.5% compared with that of the
control.
3.3.2. Distribution of nano-TiO2 in algae
As shown in Fig. 6A, the cell walls of K. brevis were covered
by particles and broken down where the particles gathered. The
arrows in Fig. 6B show that the particles were found not only out-
side algal cells but also in the chloroplast inside algal cells. These
particles were analyzed by TEM-EDX (Fig. 6C). Comparison with
Table 2
The analysis of element content. A: arrows 1 in Fig. 6B; B: arrows 2 in Fig. 6B.
A
Element Weight percent (%) Atomic percent (%)
CK 28.58 67.51
Ti K 4.18 2.48
Cu K 67.23 30.02
Element Weight percent (%) Atomic percent (%)
CK 26.36 59.88
OK 6.28 10.71
Ti K 4.18 2.48
Cu K 63.93 27.45
the standard lattice index of TiO2 showed that these particles were
TiO2 . The element content of the particles marked by arrows in
Fig. 6B was also analyzed. As shown in Table 2, the weight percent
of Ti outside the cells was 4.18, which was higher than that inside
the cells. Given that the carrier of slices of algal cells was a cop-
per network, the weight percent of Cu was the highest based on
elemental analysis.
3.3.3. Ultrastructure of algal cells
The ultrastructures of algal cells were detected by TEM. S. costa-
tum is a diatom with a thick cell wall, so operation in the experiment
for algal cell immobilization failed. The ultrastructure of K. bre-
vis cells was shown in Fig. 7A. A complete cell of K. brevis with
organelles was observed, and the chloroplast exhibited a clear and
complicated architecture with numerous thylakoids (Fig. 7A and
C). Under exposure to nano-TiO2 , the stroma lamellae of chloro-
plast showed a misty appearance (Fig. 7D). Fig. 7B showed that the
treated cells had damaged cell walls.
3.4. Lipid peroxidation and ROS production
3.4.1. Lipid peroxidation assays
The membranes of algal cells were damaged based on the
ultrastructure of algal cells, as described in 3.3.4. Thus, nano-TiO2
toxicity may be related to lipid peroxidation. SOD and CAT activities
and MDA content in algal cells were detected to determine whether
nano-TiO2 causes lipid peroxidation. As shown in Fig. 8, SOD
and CAT activities and MDA content of algal cells changed under
nano-TiO2 exposure. As the nano-TiO2 concentration exceeded
10 mg L−1 , SOD activity decreased significantly (p < 0.05, Fig. 8A. By
contrast, SOD activity in S. costatum cells significantly increased
with increasing nano-TiO2 concentration (p < 0.05, Fig. 8B). CAT
activity in K. brevis cells increased significantly with increasing
nano-TiO2 concentration (p < 0.05), whereas the opposite trend was
observed in S. costatum (Fig. 8C and D). In K. brevis cells, CAT activity
was not inhibited under nano-TiO2 exposure, but promoted by the
increase in hydrogen peroxide radicals. SOD activity was inhibited
under nano-TiO2 exposure. In S. costatum cells, the changes in SOD
and CAT were consistent with the changes in hydrogen peroxide
radicals. SOD activity increased as • OH decreased, and CAT activity
decreased as • OH decreased. In K. brevis cells, the MDA contents of
the treatment groups were significantly different from those of the
control (p < 0.05, Fig. 8E), and showed an increasing trend. The MDA
content of S. costatum significantly increased with increasing nano-
TiO2 concentration (p < 0.05, Fig. 8F). The MDA content of K. brevis
and S. costatum under 30 mg L−1 nano-TiO2 exposure was 2.4- and
2.7-fold higher than that of the control, respectively.
 F. Li et al. / Aquatic Toxicology 158 (2015) 1–13 7

Fig. 6. Distribution of nano-TiO2 in algae. Arrows in Fig. 6B showed the site of nano-TiO2 entered into cells. (A, B): K. brevis treated by 20 mg L−1 nano-TiO2 ; (C): Crystal
parameter of particles detected by TEM-EDX.

Fig. 7. Ultrastructure of K. brevis cells treated by nano-TiO2 . (A, C): control cells; (B, D): cells treated by 20 mg L−1 nano-TiO2 . CW, cell wall; CHL, chloroplast.
8 F. Li et al. / Aquatic Toxicology 158 (2015) 1–13

Fig. 8. SOD, CAT activities and MDA content of algae cells. Algae were exposed to nano-TiO2 for 48 h. (A, C, E): K. brevis; (B, D, F): S. costatum. Values represent mean ± S.D.
Different letters represent significant differences between the treatment means (p < 0.05, Tukey), n = 3.

3.4.2. ROS in Fig. 9. The contents of the three free radicals demonstrated
To determine whether ROS production causes lipid peroxida- similar changes in K. brevis and S. costatum. When the nano-TiO2
tion in algal cells, the contents of three free radicals in algal cells concentration was 30 mg L−1 , the contents of • OH and H2 O2 were
under nano-TiO2 exposure were analyzed and the results are shown significantly different from those of the other groups and increased
 F. Li et al. / Aquatic Toxicology 158 (2015) 1–13 9

Fig. 9. Contents of three free radicals of algae cells. Algae were exposed to nano-TiO2 for 48 h. (A, C, E): K. brevis; (B, D, F): S. costatum. Values represent mean ± S.D. Different
letters represent significant differences between the treatment means (p < 0.05, Tukey), n = 3.

•
compared with that of the control. The O2 − contents showed no
significant differences between the treatment and control groups,
•
possibly because O2 − was too active to be detected before its
transformation. No ROS were detected in the nano-TiO2 suspen-
sion. Therefore, ROS were produced and accumulated in algal
cells.
3.5. Site of ROS production in algal cells under nano-TiO2
exposure
3.5.1. Effects of inhibitors of ETC on ROS level
To determine the site of ROS production in algal cells, the effects
of inhibitors rotenone, diuron, and dicoumarol of ETC on ROS levels
10 F. Li et al. / Aquatic Toxicology 158 (2015) 1–13

Fig. 10. Effects of inhibitors on ROS level induced by nano-TiO2 . Cells were treated with 20 mg L−1 nano-TiO2 for 12 h after pretreatment with rotenone (1.25 ␮M L−1 ),
diuron (5 ␮M L−1 ), and dicoumarol (5 ␮M L−1 ) for 30 min. A: K. brevis; B: S. costatum. (A): Control; (B): rotenone; (C): diuron; (D): dicoumarol; (E): nano-TiO2 ; (F): nano-
TiO2 + rotenone; (G): nano-TiO2 + diuron; (H): nano-TiO2 + dicoumarol. Values represent mean ± SD. Different letters represent significant differences between the treatment
groups (p < 0.05, Tukey, n = 3).

induced by nano-TiO2 are shown in Fig. 10. Cells were treated with
nano-TiO2 for 12 h (ROS level peaked in the preliminary experi-
ment) after pretreatment with rotenone, diuron, and dicoumarol
for 30 min. The fluorescence degree indicates the ROS content.
Fig. 10A shows that the ROS content in K. brevis cells significantly
increased under exposure to one of the three inhibitors or nano-
TiO2 as the control (p < 0.05). Nano-TiO2 induced more ROS than the
other three inhibitors. Groups of rotenone or dicoumarol followed
by addition with nano-TiO2 both significantly increased compared
with groups in which only one of them was added (p < 0.05). In
addition, the ROS content in the diuron with nano-TiO2 group sig-
nificantly decreased compared with that in the diuron only and
nano-TiO2 only groups (p < 0.05). Addition of diuron inhibited the
transfer of electrons in the ETC of the chloroplast, and the ROS con-
tent decreased. Thus, we considered the chloroplast to be the site
of ROS production induced by nano-TiO2 in K. brevis. In S. costa-
tum, the ROS content in the rotenone, diuron, or nano-TiO2 only
groups slightly increased compared with that of the control, except
in the dicoumarol group (Fig. 10B). This result could be due to the
high dicoumarol toxicity in S. costatum cells. Nano-TiO2 induced
Fig. 11. Effects of nano-TiO2 on hydroxyl radical production of isolated chloro-
more ROS than the other three inhibitors. Groups of one of the plast. The concentration of nano-TiO2 was 10 mg L−1 . Values represent mean ± SD
three inhibitors with the addition of nano-TiO2 slightly increased (*p < 0.05, n = 3).
compared with groups containing only one inhibitor. No significant
differences were observed among the groups. Thus, the site of ROS
production induced by nano-TiO2 in S. costatum remains unknown. nano-TiO2 on Chlamydomonas reinhardtii was 10 mg L−1 . Algal toxi-
cities of different nanoparticles were based on the types of algae and
3.5.2. Effects of nano-TiO2 on isolated chloroplast nanoparticles. The 72 h EC50 value of CuO nanoparticles on C. rein-
To prove that the chloroplast was the site of ROS production hardtii was 150.45 mg L−1 , and the NOEC ≤ 100 mg L−1 (Melegari
induced by nano-TiO2 in K. brevis cells, the • OH content of the iso- et al., 2013). Our results provide data for assessing the algal toxic-
lated chloroplast extracted from K. brevis cells was determined. ity of nano-TiO2 on marine algae. Accurate quantification of Chl-a
The • OH content induced by nano-TiO2 significantly increased by is an important step in estimating phytoplankton biomass in both
1.17-fold compared with that of the control (Fig. 11). These results marine and freshwater environments (Simon and Helliwell, 1998).
prove that the chloroplast was the site of ROS production induced The Chl-a content can be an indicator of the growth situation of
by nano-TiO2 in K. brevis cells. algal cells. In the current study, the Chl-a contents of both K. brevis
and S. costatum sharply decreased, possibly weakening their pho-
4. Discussion tosynthetic abilities. Thus, the growth of K. brevis and S. costatum
was inhibited by nano-TiO2 .
4.1. Inhibition of nano-TiO2 on algae growth
4.2. Lipid peroxidation as one of the mechanisms
To investigate the toxicity of nano-TiO2 on the growth of K.
brevis and S. costatum, several treatments were conducted. Both Some studies have reported the mechanisms by which nanopar-
K. brevis and S. costatum were inhibited, and their 72 h EC50 ticles inhibit the growth of algae. Although the shading effect is
values were 10.69 and 7.37 mg L−1 , respectively (Fig. 4). Simi- one of the mechanisms, few have been proven (Chen et al., 2012;
larly, Huang and Ismat (2005) reported that the EC50 value of Sadiq et al., 2011). Wang et al. (2008) showed that nanoparticles
 F. Li et al. / Aquatic Toxicology 158 (2015) 1–13
aggregate in seawater, thereby reducing specific surface area and
active sites of the surface. A similar phenomenon was observed
by SEM. Nano-TiO2 aggregated on the surface of algal cells after
preparation in solution, possibly because of the large specific sur-
face and high adsorption capacity of nano-TiO2 . These aggregations
may prevent chlorophyll from absorbing light and inhibit algae
growth (Schwab et al., 2011). To prove that nano-TiO2 could inhibit
algae growth by preventing light absorption of algae, the shading
effect was analyzed in our study. At 50 mg L−1 nano-TiO2 , algae
growth in the treatment groups showed no significant differences
from that of the control. Therefore, a nano-TiO2 concentration of
<50 mg L−1 could not inhibit the growth of algal cells via the shading
effect. A similar result showed that algae growth and light uti-
lization did not change in the presence of nano-TiO2 (Hund-Rinke
and Simon, 2006). Ji et al. (2011) assessed algal toxicities of nano-
ZnO and nano-TiO2 in the presence and absence of light. Their
results showed that toxicities of groups with nanoparticles in the
dark were higher than those of the groups without nanoparticles
in the dark. The shading effect was not the main factor inhibiting
algae growth. Nano-TiO2 entered the algal cells and adhered to the
chloroplast (Fig. 6). In theory, nanoparticles with particle sizes less
than cell wall apertures could go through the cell wall of algal cells
(Navarro et al., 2008). Wang et al. (2011) found that nanoparti-
cles enter algal cells by entering the apertures in the cell wall, as
well as endocytosis of the cell membrane. By contrast, our current
research indicated that nano-TiO2 might enter the cell by damag-
ing the cell wall where nanoparticles were aggregated (Fig. 7B). In
algae suspensions with nano-TiO2 , analysis of the crystal parame-
ters of particles both outside and inside algal cells proved that the
particles were TiO2 , as detected by TEM-EDX. Given that nano-TiO2
entered algal cells, algae growth was inhibited by nano-TiO2 itself.
Based on the ultrastructure of K. brevis (Fig. 7), algal cells were dam-
aged. Less organelles and more crystal particles were observed. Our
results concur with many studies that nanoparticles can enter cells.
Chen et al. (2012) claimed that nano-TiO2 was located inside algal
cells. They considered that nano-TiO2 inside algal cells might be
more toxic to the cells. Navarro et al. (2008) reported that pores
in the cell wall might be induced by nanoparticles to grow; thus,
nanoparticles could pass the cell wall through the new pores. By
contrast, our results show that nano-TiO2 might enter the cells by
damaging the cell wall. TEM images (Fig. 7) also provided direct
evidence that the cell structures were damaged by nanoparticles
inside the cells.
Lipid peroxidation induced by ROS production is one of the
main mechanisms of algal toxicity of nanoparticles (Ma et al., 2013;
Melegari et al., 2013; Rodea-Palomares et al., 2012). Antioxidant
enzymes, SOD and CAT activities, and MDA as the end product
of lipid peroxidation were detected to estimate the level of lipid
peroxidation (Han et al., 2004; Qiu et al., 2013). In this study, the
MDA contents in algal cells significantly increased with increasing
nano-TiO2 concentrations, indicating that nano-TiO2 could impose
oxidative stress and cause lipid peroxidation in algal cells (Lin et al.,
2012). Activities of SOD and CAT in K. brevis and S. costatum cells
changed, exhibiting various trends. In K. brevis cells, CAT activity
increased and SOD activity decreased with increasing nano-TiO2
concentration. The changes in SOD and CAT activities in S. costa-
tum cells were different from those in K. brevis. CAT and SOD can
both increase to remove excess ROS (Hu et al., 2014). In contrast to
the current study, increased H2 O2 inhibited SOD activity in K. bre-
vis cells and further increased H2 O2 (San Mateo et al., 1998). In S.
costatum cells, SOD activity was less by four orders of magnitudes,
and CAT activity was less by one order of magnitude than those in
K. brevis cells. As SOD and CAT activities were inhibited, the • OH
content increased by seven orders of magnitudes.
Although lipid peroxidation is considered to be caused by
intracellular ROS production, few studies have confirmed this
11
Fig. 12. Site of diuron inhibited electron transport in the chloroplast ETC. Diuron
inhibited the transfer of electrons from QA to QB .
relationship. The ROS contents in Chlorella sp. cells exposed to ROS
levels in the cells increased and were not eliminated under oxi-
dant stress caused by nanoparticles, such as nano-ZnO, thereby
increasing lipid peroxidation (Lin and Xing, 2008). In the present
study, the contents of the three free radicals increased in both K.
brevis and S. costatum cells. Moreover, no ROS were detected in
the nano-TiO2 suspensions. ROS were produced in algal cells. Thus,
the balance in the oxidation-reduction system in algal cells was
destroyed. Intracellular ROS production increased oxidative stress,
which could explain the toxic effects of nano-TiO2 on K. brevis and
S. costatum. Therefore, lipid peroxidation caused by oxidative stress
might be one of the mechanisms of nano-TiO2 in algal cells.
4.3. Chloroplast as the main site of ROS accumulation
Rotenone, diuron, and dicoumarol are inhibitors of
the mitochondrial ETC, chloroplast ETC, and membrane
oxidation–reduction ETC, respectively (Choi et al., 2002; Li
et al., 2010; Yu et al., 2007). Rotenone and diuron have been
used as ETC inhibitors in some studies. Complexes I and III of
ETC in the mitochondria are the major sites for ROS production.
Rotenone inhibits complex I and increases ROS generation in
submitochondrial particles (Li et al., 2003). The effects of diuron
on the chloroplast ETC are shown in Fig. 12. Diuron can bind with
the QB protein and inhibit PQ reduction (Laisk and Oja, 2013); thus,
the action site of diuron is between QB and QA (Choi et al., 2002).
In the present study, ROS production significantly increased under
exposure of nano-TiO2 with addition of dicoumarol/rotenone
compared with that under exposure of nano-TiO2 alone. ROS
production significantly decreased under exposure of nano-TiO2
with addition of diuron compared with that under exposure of
nano-TiO2 alone (Fig. 10A). This finding indicates that diuron could
remove excess ROS produced by nano-TiO2 , or ROS produced by
nano-TiO2 were inhibited. Given that diuron was the inhibitor
of the chloroplast ETC, ROS produced by TiO2 were found on the
chloroplast ETC. We concluded that the action site of nano-TiO2
on K. brevis cells was the chloroplast. Similar results were also
reported. Li et al. (2010) suggested that rotenone can significantly
decrease the ROS content caused by DPB-5. Similarly, a previous
study showed that H2 O2 production induced by antimycin A can
be prevented with the addition of rotenone in the mitochondria
with complex I substrates (Chen et al., 2003).
The chloroplast is the site of the affected toxicants, such as
selenite, which causes sparse substrates, dissolution of proteins,
and changes in the thylakoid system (Morlon et al., 2005). A
previous study showed that nano-TiO2 with low concentration
12 F. Li et al. / Aquatic Toxicology 158 (2015) 1–13
Fig. 13. The toxicity mechanism of nano-TiO2 in K. brevis.
under UV irradiation increases the electron transport rate, energy
conversion rate, and oxygen release of isolated chloroplast
extracted from spinach (Hong et al., 2005). Nano-TiO2 may bind
with PS II, and no effect has been found on the structure of PS II.
These findings were consistent with the results in the current study.
To prove that the chloroplast was the main site of ROS pro-
duction in K. brevis cells, we studied the effect of nano-TiO2 on
the isolated chloroplast extracted from K. brevis cells. Increased
H2 O2 contents indicated that the isolated chloroplast produced
ROS under nano-TiO2 exposure. Therefore, the chloroplast was a
site of ROS production in K. brevis cells induced by nano-TiO2 . TEM
analysis (Fig. 7) also showed that the stroma lamellae of the chloro-
plast demonstrated a misty appearance under nano-TiO2 exposure,
which explained that the chloroplast was damaged by ROS produc-
tion.
Our study is the first to report on the action site of nanoparticles
on algal cells. We proved that the site of ROS induced by nano-TiO2
in K. brevis cells was the chloroplast. The site of ROS production
induced by nano-TiO2 in S. costatum remains unknown despite the
addition of the three ETC inhibitors. However, lipid peroxidation
was detected in S. costatum cells under nano-TiO2 exposure. The
toxicity mechanism of nano-TiO2 on K. brevis is shown in Fig. 13.
First, nano-TiO2 aggregated on the surface of algal cells.
Nanoparticles then entered the cell wall by passing through the
pores or damaging the cell wall. Endocytosis of the cell mem-
brane allowed entry of the nanoparticles into the cells, and some
nanoparticles attached to the chloroplast. ROS were produced on
the ETC of the chloroplast upon nano-TiO2 exposure in cells. Excess
ROS were produced, and anti-oxidative enzymes were activated to
maintain the balance between pro-oxidation/anti-oxidation. How-
ever, this balance was destroyed by an increase in ROS production
because enzymes could not eliminate excess ROS. ROS accumulated
in algal cells, thereby causing lipid peroxidation. The ultrastructure
of cells was damaged, as evidenced by the incomplete membrane
and indistinct organelles. Finally, algae growth was inhibited. The
site of ROS production induced by nano-TiO2 in K. brevis was the
chloroplast.
The chloroplast was the site of ROS production induced by nano-
TiO2 . Results from the current study cannot determine the specific
site of the chloroplast ETC where ROS were produced.
Acknowledgements
We appreciate the financial support from the National Natu-
ral Science Foundation of China (51378480), National Key Basic
Research Program of China (2015CB453301), NSFC-Shandong Joint
Fund (U1406403), and Public Science and Technology Research
Funds Projects of Ocean (201305003).
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