Ecotoxicology and Environmental Safety 84 (2012) 155162

Contents lists available at SciVerse ScienceDirect

Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Toxicological effects of nanometer titanium dioxide (nano-TiO2) on Chlamydomonas reinhardtii

Lanzhou Chen a,b, Lina Zhou b, Yongding Liu a, Songqiang Deng b, Hao Wu b, Gaohong Wang a,n
a b

State Key Laboratory of Fresh water Ecology and Biotechnology, Institute of Hydrobiology, The Chinese Academy of Sciences, Wuhan, Hubei 430072, PR China School of Resource & Environmental Science, Wuhan University, Wuhan, 430079, PR China

a r t i c l e i n f o
Article history: Received 18 May 2012 Received in revised form 26 June 2012 Accepted 6 July 2012 Available online 9 August 2012 Keywords: Nanometer titanium dioxide Chlamydomonas reinhardtii Physiological responses Toxicological effects Electron microscopy

abstract
The toxicological effects of nanometer titanium dioxide (nano-TiO2) on a unicellular green alga Chlamydomonas reinhardtii were assessed by investigating the changes of the physiology and cytoultrastructure of this species under treatment. We found that nano-TiO2 inhibited photosynthetic efciency and cell growth, but the content of chlorophyll a content in algae did not change, while carotenoid and chlorophyll b contents increased. Malondialdehyde (MDA) content reached maximum values after 8 h exposure and then decreased to a moderately low level at 72 h. Electron microscopy images indicated that as concentrations of nano-TiO2 increased, a large number of C. reinhardtii cells were noted to be damaged: the number of chloroplasts declined, various other organelles were degraded, plasmolysis occurred, and TiO2 nanoparticles were found to be located inside cell wall and membrane. It was also noted that cell surface was surrounded by TiO2 particles, which could present an obstacle to the exchange of substances between the cell and its surrounding environment. To sum up, the effect of nano-TiO2 on C. reinhardtii included cell surface aggregation, photosynthesis inhibition, lipid peroxidation and new protein synthesis, while the response of C. reinhardtii to nano-TiO2 was a rapid process which occurs during 24 h after exposing and may relate to physiological stress system to mitigate damage. Crown Copyright & 2012 Published by Elsevier Inc. All rights reserved.

1. Introduction Nanomaterials (NMs) include natural or man-made particles, with at least one dimension of 100 nm or less, that are utilized for many purposes, particularly in industry, such as in drug delivery, medical devices, cosmetics, chemical catalysts, optoelectronics, electronics, and magnetics (Wang et al., 2008a; Aruoja et al., 2009). The increasing use of nanomaterials (NMs) has raised concerns about the effects of these substances on human health and the environment, prompting extensive investigations of NM uptake, fate and toxicity (AshaRani et al., 2009; Kahru and Dubourguier, 2010; Shvedova et al., 2010). The most distinctive feature of NMs is their size, which is an important determinant of their physicochemical properties with respect to the ease of uptake of these compounds as well as their interaction with biological tissues, particularly since NMs can result in harmful biological effects in living cells (Adams et al., 2006; Peralta-Videa et al., 2011). Ultrane particles have been shown to induce oxidative stress, and distal organ involvement (Nel et al., 2006). It is found that titanium dioxide (TiO2) and other nanoparticles can cause cell growth inhibition, lipid peroxidation

Corresponding author. Fax: 86 27 6878 0123. E-mail address: space@ihb.ac.cn (G. Wang).

and photosynthesis inhibition in algae (Wang et al., 2008a; Warheit et al., 2007; Aruoja et al., 2009), and the degree of toxicity is dependent on concentration and particle size, even in bulkier inert material (as in carbon black and TiO2) (Shvedova et al., 2010; Nel et al., 2006). But the mechanism for the function of nanoparticles on organism is still slurred. Some researches indicate that the function of nanoparticles in organisms is mediated by reactive oxygen species (ROS) production (Nel et al., 2006; Oukarroum et al., 2012; Klaine et al., 2008; Bhattacharya et al., 2010), which can cause cell damage and lipid oxidation. Wei et al. (2010) indicate that the direct contact between f-MWNT and algal cell surface (algal cell/nanoparticles aggregation) was likely responsible for reduced PSII functional cross section and oxidative stress during exponential growth. Gong et al. (2011) found that there was noticeable algal cell/NiO aggregation which may cause a reduction in the light available to those cells and thus inhibiting their growth. Recent research also showed that nanoalumina can promote the horizontal transfer of multiresistance genes mediated by plasmids across genera (Qiu et al., 2012). As one main biota in aquatic ecosystem, algae were used to asset the aquatic risk of pollution and toxin in the government environment reports (Aruoja et al., 2009; Menard et al., 2011). Table 1 showed toxicological characterization of algae under nanomaterials (NMs), which includes recent information about various nanoparticles, species, cell and physiological responds to

0147-6513/$ - see front matter Crown Copyright & 2012 Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2012.07.019

156

L. Chen et al. / Ecotoxicology and Environmental Safety 84 (2012) 155162

Table 1 Toxicological characterization of algae under nanomaterials (NMs). Nanomaterials TiO2 Species Chlamydomonas reinhardtii; Pseudokirchneriella subcapitata; Desmodesmus subspicatus; Chlorella sp Characterization for nanotoxicolgy Growth inhibition; lipid peroxidation; Stress response genes expression; Photosynthesis inhibition; Cyto-ultrastructure disorder; soluble protein synthesis; NMs aggregation on cell surface; nanoparticles penetrated cytoplasm Growth inhibition; Soluble protein synthesis; Cyto-ultrastructure disorder Growth inhibition; Photosynthesis inhibition; NMs aggregation on cell surface Growth inhibition; Lipid peroxidation; ROS production NMs aggregation on cell surface Growth inhibition; NMs aggregation on cell surface; Nanoparticles penetrated cytoplasm Growth inhibition; NMs aggregation on cell surface; Nanoparticles penetrated cytoplasm Photosynthesis inhibition; ROS production Growth inhibition; Cell membrane damage; lipid peroxidation Growth inhibition; NMs aggregation on cell surface ROS production; NMs aggregation on cell surface; photosynthesis inhibition Growth inhibition; lipid peroxidation; stress response genes expression Growth inhibition; NMs aggregation on cell surface Growth inhibition; NMs aggregation on cell surface References Wang et al. (2008a), Warheit et al. (2007), Aruoja et al. (2009), Hartmann et al. (2010), Huang et al. (2005), Griftt et al. (2008), Hund-Rinke and Simon, (2006), Ji et al. (2011), Hall et al. (2009), this study Gong et al. (2011), Wei et al. (2010), Oukarroum et al. (2012), Navarro et al. (2008) Renault et al. (2008), Perreault et al. (2012b) Aruoja et al. (2009), Wang et al.(2011), Perreault et al. (2012a), Saison et al. (2010) Rogers et al. (2010) Franklin et al. (2007), Aruoja et al. (2009), Wong et al. (2010), Ji et al. (2011) Bhattacharya et al. (2010) Wang et al. (2008a), Domingos et al. (2011) Ji et al. (2011) Ji et al. (2011)

NiO CNTs AgNP AuNP CuO

Chlorella vulgaris Dunaliella tertiolecta Chlorella vulgaris; Dunaliella tertiolect; C. reinhardtii Scenedesmus subcapitata; C. reinhardtii P. subcapitata; Microcystis aeruginosa; C. reinhardtii P. subcapitata P. subcapitata; Chlorella sp Chlorella sp.; Scenedesmus sp. C. reinhardtii Chlorella sp. Chlorella sp.

CeO2 ZnO Charged Polystyrene nanospheres Quantum dots (CdTe) Al2O3 SiO2

nanoparticles and related references cited. There are some advances about nanotoxicology of algae (Table 1). However, adaptation mechanism of cell biology and physiology in algae to nanoparticles is not clear. Since Chlamydomonas reinhardtii can offer unique advantages for genetic, biochemical, and cell biological approaches, it is one of the most used model system (Pan, 2008). Some researches indicated that nanoparticles inhibited algal growth and induced lipid peroxidation (Mroz et al., 2007; Hartmann et al., 2010; Griftt et al., 2008; Hund-Rinke and Simon, 2006). Wang et al. (2008a) reported that TiO2 caused growth inhibition and lipid peroxidation in C. reinhardtii and some stress response genes were mobilized to express highly. So we used C. reinhardtii as the experiment model to study the cell toxicity of titanium dioxide (TiO2) nanoparticles (NPs), one kind of manufactured nanosized materials, through investigating cell structure and measuring the biochemical parameters of algae in this work, and below issues were addressed: (1) to determine the effects of nano-TiO2 on algal photosynthesis and growth; (2) to study the cell ultrastructure and surface changes under TiO2; (3) to measure soluble protein and pigments content after treatment; (4) to assay lipid peroxidation.

28 7 3 mv according to Wang et al. (2008a). Inoculums were grown in 250 ml glass asks containing 100 ml of SE medium at 25 7 1 1C, 50 mE m 2 s 1 light intensity on a shaking table at 85 rpm. Five concentrations, 0.1, 1, 10, 20, 100 mg l 1 of TiO2 were added to the asks from the stock solution of nanoparticle suspensions, with an initial cell density of 104 cells ml 1 in exponential growth phase.

2.3. Measurement of growth curve The growth of C. reinhardtii was assessed by numbering algal cells in haemocytometer after different exposing times.

2.4. Determination of photosynthetic activity and pigment contents Photosynthetic activity was assessed by measuring chlorophyll uorescence (Chen et al., 2009), determined by a portable plant efciency analyzer (PEA, Hanstechs, UK). Experimental conditions were as follows: samples were kept at room temperature; dark adaptation was about 1015 min; stimulation light intensity used for experiments was half the maximum intensity (1500 mmol photon m 2 s 1). The ratio of Fv/Fm (Fv, variable uorescence; Fm, maximum uorescence; Fo, uorescence value at time 0 or 50 ms (the O transient); Fv Fm Fo) was used to indicate the photosynthetic efciency. Chlorophyll a, Chlorophyll b and carotenoids were extracted with 80% (v/v) acetone and determined according to the method of Lichtenthaler and Wellburn (1983). In brief, 2 ml algae cultures were centrifuged at 2000 rpm speed, then the superior liquid was discarded, and the samples were added 80% (v/v) acetone and placed at 4 1C during 24 h. Light absorption values of superior liquid were measured with UV-Photometer (7U-1810) at 663, 646 and 470 nm. Chlorophyll a (Ca), Chlorophyll b (Cb) and Carotenoid (Cc) contents were calculated by the formula (1). C a 12:21A663 2:81A646 C b 20:13A646 5:03A663 C c 1000A470 3:27C a 104C b =229

2. Materials and methods 2.1. Algal cultures C. reinhardtii was obtained from FACHB collection (Freshwater Algae Culture Collection of the Institute of Hydrobiology, The Chinese Academy of Sciences) and cultured in SE medium (Bristol Medium and Soilwater supernatant) (Bold, 1949) at 25 7 1 1C and 55 mE m 2 s 1 incandescent light (12:12 h light:dark) with sterilized bubbling air. 2.2. Nano-TiO2 treating method TiO2 NM powder was purchased from Sigma-Aldrich (St. Louis, MO, USA), and was a mixture of anatase and rutile and had an average BrunauerEmmettTeller (BET) surface area of 3565 m2 g 1 and an average particle size of 21 nm. The nanoparticle suspensions were prepared in 5000 mg l 1 stock solution with SE medium prior to the experiments, and the zeta potentials of the suspensions in SE medium were about

2.5. Scan electron microscopy and transmission electron microscopy The cell surface of C. reinhardtii was observed with SEM (TESCAN scan electron microscopy). After 8 h treatment with 0, 10, 20, and 100 mg/l TiO2 nanoparticles, C. reinhardtii cells were collected by centrifugation, and xed with 3% glutaraldehyde, serially dehydrated in ethanol and sputter coated with gold following standard methods. The images were scanned at high voltage of 30 kV. For each sample, at least ve elds were observed at different magnications.

L. Chen et al. / Ecotoxicology and Environmental Safety 84 (2012) 155162 The Cyto-ultrastructure of C. reinhardtii induced by TiO2 nanoparticles was observed with TEM (TECNAI transmission electron microscopy, Philips-FEI) at 200 kV. After 8 h treatment with 0, 10, 20, and 100 mg/l TiO2 nanoparticles, C. reinhardtii cells were collected by centrifugation and xed in collidine-buffered 2% glutaraldehyde containing 5 mM CaCl2 for 1 h at room temperature and postxed in 2% OsO4 and O.8% K3Fe3(CN)6 for 1 h at room temperature. Cells were then rinsed 3 times with H2O and treated for 2 h with 2% uranyl acetate. After standard dehydration, embedding (epon), and sectioning, samples were stained with lead citrate. For each sample, at least ve elds were observed at different magnications. 2.6. Cellular soluble protein and MDA content Protein concentration was determined by the method of Bradford (1976), using BAS as a standard. MDA content was assayed according to the method of He and Hader (2002). Briey, 10 ml of treated or control algae were harvested by centrifugation (3000 g for 10 min). The pellet was rinsed twice with double distilled water. Subsequently, 10 ml of 0.5 M butylated hydroxytoluene (BHT) was added to the pellet to prevent any further oxidation during storage. The pellet was placed at 80 1C in the dark until analysis. Then, 350 ml of double distilled water and 10 ml of BHT were added to each sample, which was followed by sonication for 2 min (duty cycle 10%, output control 1). The sonicated mixture was centrifuged at 10,000 g for 15 min at 4 1C and 300 ml supernatant was used for TBARS assay and 20 ml for protein determination by the Bradford method. A 60 ml sample of 70% trichloroacetic acid was added to the 300 ml supernatant and centrifuged (10,000 g for 15 min at 4 1C). An aliquot of samples of 300 ml supernatant was transferred to another microcentrifuge tube. Then, 1.5 ml acetone was added to supernatant and centrifuged at 10 000 g for 10 min at 4 1C. The pellet was dried under nitrogen in the dark. Next, 1 ml of 7% phosphoric acid, 100 ml of 6% TBA and 10 ml of 0.5 M BHT were added to the pellet and the reaction mixture was boiled for 30 min. The mixture was cooled to room temperature and then centrifuged at 10,000 g for 5 min at 4 1C. A 1 ml sample of the supernatant was used for spectrophotometric measurement. The absorbance difference between 532 and 600 nm was used to calculate the MDA formation due to lipid peroxidation (532 0.154 mM 1 cm 1 for MDA-TBA) (He and Hader, 2002). 2.7. Data analysis All data were evaluated by one-way ANOVA (Spss 6.0.1 for windows, tests: least signicant difference, Tukeys honestly signicant difference).

157

20 CK 0.1 mg/L 1 mg/L 10 mg/L 20 mg/L 100 mg/L * * 10 * * *

Cell number (105/L)

15

* * * * * * * * *

5 -10

10

20

30 40 Time (h)

50

60

70

80

0.8

Fv/Fm

0.7

CK 0.1mg/LTiO2 1mg/LTiO2 10mg/LTiO2 20mg/LTiO2 100mg/LTiO2 *

0.6

* *

* * * * *

0.5 -10

10

20

3. Results 3.1. The growth curve of Chlamydomonas reinhardtii exposed to nanoparticle TiO2 Fig. 1A indicated that there was not signicant difference between the control and the treatment group with Nano-TiO2 during 8 h exposing. When the initial concentration of TiO2 NMs was 0.1 mg l 1, there was a little increase than the control at 12 h, while there are not signicant difference between the middle dosage of groups (1, 10, 20 mg l 1) and the control. After 24 h exposing, there was signicant reduction in the treated groups and the control. 10, 20 and 100 mg l 1 Nano-TiO2 inhibited growth signicantly and the cell density of those treated groups (10, 20 and 100 mg l 1 TiO2 ) reduced gradually during experiment, which showed that cells growth was inhibited completely by high dosage TiO2. 3.2. Effect of TiO2 on photosynthetic activity of Chlamydomonas reinhardtii As shown in Fig. 1B, the photosynthetic activity (Fv/Fm, a ratio of variable to maximum uorescence, indicating activity of photosynthesis) of C. reinhardtii decreased sharply with high TiO2 concentration (of 4 1 mg/l), while no signicant change was noted at concentrations of 0.1 mg/l TiO2. After 12 h treatment, however, there was a recovery of Fv/Fm values in the C. reinhardtii samples that had received TiO2 treatment. The results also indicated that Fv/Fm values of cells with TiO2 treatment were even higher than those of the control treatments at 24, 48 and

40 30 Time (h)

50

60

70

80

Fig. 1. Evolution of growth (A) and photosynthesis activity (B) versus time in C. reinhardtii exposed to nanoparticle TiO2. Values represent mean 7 S.D.n indicates there is signicant difference between the treatment group and the control (P o 0.05, Tukey multiple comparison).

72 h (Fig. 1C). These data indicate that the effects of TiO2 on C. reinhardtii were possibly acute and that the photosynthetic system of algal cells could acclimatize to the presence of TiO2. 3.3. NPs of TiO2 aggregated on the cell wall surface of Chlamydomonas reinhardtii Comparing with the control, SEM micrographs of C. reinhardtii treated with nano-TiO2 indicated that NPs of TiO2 assembled on the cell wall surface of C. reinhardtii (Fig. 2AD). We also found that the number of aggregated NPs was dependent on the treatment concentration of nano-TiO2. NPs were noted to concentrate on the cell wall surface in the high concentration treatment group (100 mg/l). 3.4. Nano-TiO2 induction of irregular cell structures in Chlamydomonas reinhardtii Compared to the control group, algal cells from the high concentration treatment groups (100 and 20 mg/l) indicated irregular structure, including lower chloroplast volume, degraded organelles, and plasmolysis, while cell microstructures in the low concentration treatment were not affected (Fig. 2EH).

158

L. Chen et al. / Ecotoxicology and Environmental Safety 84 (2012) 155162

Fig. 2. Cell surface and ultrastructure of C. reinhardtii after exposure to nanoparticle TiO2 for 8 h. A, E: the control; B, F: 10 mg/l TiO2 treatment; C, G: 20 mg/l TiO2 treatment; D, H: 100 mg/l TiO2 treatment. Arrow showed the distribution of TiO2 NPs.

L. Chen et al. / Ecotoxicology and Environmental Safety 84 (2012) 155162

159

Nanoparticles were located on the surface of cell walls (Fig. 2FH), which conrmed the results got from SEM images. TiO2 nanoparticales also penetrated the cell wall (Fig. 2G) and plasma membrane (Fig. 2H), which may locate inside cell.
Chla (mg/L)

4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0

c

CK 0.1mg/LTiO2 1mg/LTiO2 10mg/LTiO2 20mg/LTiO2 100mg/LTiO2

f d d d b a a e d

l l k i g g j j k k i k

3.5. Effects of TiO2 on cellular soluble protein As illustrated in Fig. 3, the content of soluble protein in algae cells increased after 24 h treatment with TiO2 at 0.1, 1 and 10 mg/ l, while it decreased in cells treated with TiO2 at concentrations of 20 and 100 mg/l. After 72 h treatment, the protein content in 0.1, 1 and 10 mg/l treatment groups increased while no signicant changes were observed in groups exposed to 20 and 100 mg/l TiO2 concentration.

g gh

2 Time (d)

4.0 3.5

3.6. Photosynthetic pigments changed after NMs exposing The content of Chlorophyll a, Chlorophyll b and carotenoids in C. reinhardtii under TiO2 treatment are illustrated in Fig. 4. There were no signicant changes for Chlorophyll a in different levels of treatment groups and exposure times, while for Chlorophyll b and carotenoids content, there were obvious decreases after treating, and then there were signicant increases in treated groups after 3 and 4days exposing. The ratio of Chl a:b raised when the concentration of nano-TiO2 increased after 24 h treatment, which implied that there were more Chl a in treated algae after 24 h exposing with nano-TiO2. But this ratio reduced gradually after 48, 72 and 96 h with TiO2, which suggested that Chl a content become less in these treated cells.
Chlb (mg/L)

3.0 2.5 2.0 1.5 1.0 0.5 0.0

0mg/L 0.1mg/L 1mg/L 10mg/L 20mg/L 100mg/L

i d a bcd bcd bcd b h d f e ee bc h g ghg

bc

bcd b

bcdbc

2 Time (d)

4.0 3.5 Carotenoid (mg/L) 0mg/L 1mg/L 10mg/L 20mg/L 100mg/L 100
f e d c bc c b a de bc c de e

3.7. Effects of TiO2 on MDA content of Chlamydomonas reinhardtii The MDA content in all treatment groups (0.1, 1, 10, 20 and 100 mg/l) increased after 4 h treatment, compared to that of the control group (Fig. 5), reaching maximum values after 8 h. After 12 h treatment, however, the MDA content in every treatment group decreased, compared to values measured at 8 h. No signicant differences among these groups were noted after 72 h treatment.

3.0 2.5 2.0 1.5 1.0 0.5 0.0

j i h g g g e gh h gh f

180 160 140 Proteins (mg/L) 120 100 80 60 40 20 0

CK 0.1 1 10 20 100 d d c bb a a aa c

hg gf f e d c

2 Time (d)

Fig. 4. Soluble protein content of C. reinhardtii exposed to nanoparticle TiO2. Values represent mean 7 S.D. Those with different letter on error bar are signicantly different (P o 0.05, Tukey multiple comparison).

4. Discussion Since NPs have a number of unique characteristics, in terms of physicochemical, optical, reactive, and electrical properties, they display high interaction with organisms and potential toxicity (Peralta-Videa et al., 2011; Jin et al., 2011). Cell aggregation has been reported in cultures treated with TiO2 NMs (Wang et al., 2008a). Ji et al. (2011) showed that large aggregates of nanoparticles entrapping the algal cells were observed under the treatment of nano-ZnO and HR3 (TiO2) which may reduce the light and nutrient available to the entrapped algal cells and thus inhibit their growth. Aruoja et al. (2009) found that large aggregates were observed in the case of TiO2 nanoparticles, that entrapped almost all algal cells, while the cultures with bulk TiO2 always

c c

0h

24 h

48 h

72 h

Fig. 3. Evolution of pigment contents versus time in C. reinhardtii exposed to nanoparticle TiO2. Values represent mean 7 S.D. Those with different letter on error bar are signicantly different (P o 0.05, Tukey multiple comparison).

160

L. Chen et al. / Ecotoxicology and Environmental Safety 84 (2012) 155162

0.20 0.18 0.16 MDA (mol/mg) 0.14 0.12 0.10 0.08 0.06 0.04 -4

CK 0.1mg/LTiO2 1mg/LTiO2 10mg/LTiO2 20mg/LTiO2 100mg/LTiO2 * *

* * *

12 Time (h)

64

68

72

Fig. 5. Evolution of MDA content versus time in C. reinhardtii exposed to nanoparticle TiO2. Values represent mean 7 S.D. n indicates there is signicant difference between the treatment group and the control(P o 0.05, Tukey multiple comparison).

contained free algal cells in addition to cells entrapped in small TiO2 aggregates. They thought that TiO2 nanoparticle aggregates maybe reduce the light available to the entrapped algal cells and thus inhibite their growth. Huang et al. (2005) also indicated that P. subcapitata cells adsorbed onto their surface TiO2 nanoparticles and carried 2.3 times their own weight in TiO2 particles, and the kinetics and the extent of nano TiO2 adsorption on algae were highly dependent on pH, the maximum adsorption occurring at pH 5.5. Perreault et al. (2012) found that there were abvious aggregation of C. reinhardtii cell culture after 6 h of treatment to 0.04 g l 1 of bare CuO NPs or polymer-coated CuO NPs. SEM images of algae indicated that PS (polystyrene)nanoplastic aggregates adsorbed on the surface of algae(Bhattacharya et al.,2010), and the adsorption isotherms for Chlorella and Scenedesmus were different which could be attributed to the more rugged morphology and higher total surface area of Scenedesmus than Chlorella (Bhattacharya et al.,2010). In this study we observed cell aggregation in TiO2 treatment groups with SEM images and light microscopy, as well as evidence of TiO2 NM absorbance on some cell surfaces in the high concentration treatment groups. The TEM images of high dosage groups also supported NPs aggregation on the cell surface. This could obstruct cellular exchange with the external milieu, for example by sequestering nutrients or altering pH or redox potential. In some studies, using scanning electron microscopy (SEM) images, Metzler et al.(2011) found that a number of factors affecting the surface of algal cells including surface coverage by non-uniform multiple layers of TiO2 nanoparticles, or lipid peroxidation could cause major damage to essential processes in algae. Thus, indirect inhibition, i.e., inhibition due to various artefacts, occurs when NMs interact with the environment of the cell, which may affect cell metabolism. As a result, cell growth and activity are inhibited. In this study, we also noted a signicant decrease in growth and photosynthesis activity of algae under TiO2 treatment during 24 h exposure. Nevertheless, after 24 and 72 h exposure, photosynthetic activity in the high-dosage treatment group recovered to normal levels, while that of the low dosage groups remained at a higher level than that of the control. These recoveries may be a result of changing NM characteristics in liquid medium, or the adaptation response of algae. NM aggregation may reduce active surface sites, then impair photocatalytic activity of TiO2 NMs, and thus decrease toxicity to C. reinhardtii cells. Besides, impairment in the photocatalytic activity of TiO2 NMs over the culture

period may also be a reason for the decreased inhibitory effect on photosynthetic activity (Bhattacharya et al., 2010). It is also plausible that cellular secretion of certain macromolecules, such as polysaccharides, may affect the active sites of the NMs, which is important for detoxication of TiO2 NM by C. reinhardtii. It was found that the soluble protein content of the lowdosage groups was higher than the control, while that of the high concentration treatment groups was lower than the lowdosage groups and there was not difference between the high concentration treatment groups and the control. This suggests that more soluble protein was present in the low-dosage treated algae than the control, which may relate to de nova protein synthesis, and that can play an important role in the adaptation reaction of algal cells under NMs treatment. Gong et al. (2011) found that maximum soluble protein content occurred at 72 h and the maximum ratio of reduction of Ni presented synchronously. The reduction from nNiO to nNi may lead to weakened toxicity, so it has been proposed that some unknown enzymes of the algae play essential roles in that process, which contributes to adaptation reaction of algal cells under metal oxide NM stress. But for the high concentration treatment group, it was lower than the lowdosage groups, and the reason for this difference maybe contribute to that high concentration nano-TiO2 inhibited cell activity including protein synthesis. Besides, the soluble protein content of the low-dosage groups remained a long time from 24 to 72 h, which implied that some stress proteins were synthesized and kept high activity to mitigate Nano-TiO2 caused oxidant stress. Pigment analysis showed that chlorophyll b and carotenoid content were changed by NMs TiO2 comparing to the control. Comparing with initial culture at 0 h in the different groups, it was found that chlorophyll b and carotenoid contents were decreased at rst by TO2, then these contents increased signicantly, while the chlorophyll a content had no signicant change than the control. And the ratio of Chl a:b raised when the concentration of nano-TiO2 increased after 24 h treatment, then this ration reduced gradually after 48, 72 and 96 h treatment. Since the rst step in the degradation of chl b is to convert to chl a (Fang et al., 1998), it is possible that ROS caused by TiO2 can attack chlorophyll and some chl b is to convert to chl a, which lead to more chlorophyll a in cells. Houimli et al. (2010) also showed that NaCl-stress modies the chlorophyll b content more than that of chlorophyll a, which appears less sensitive than chlorophyll b to NaCl-stress. Because stress can increase ROS production (Wang et al., 2008b), this results in an increase in the chl a/b ratio may be involved to ROS attack. For carotenoids, it is another matter, since carotenoid is effective quenchers of tripletstate photosensitizers, singlet oxygen, and peroxy radicals (Wang et al., 2010), so algal cells use it to extinguish ROS from photochemical reaction with TiO2, and algal cells also synthesis compensatively carotenoid as an adaptation. Similar phenomena occur to various metal stresses in algae, which showed that carotenoid synthesis increase as respond to quench ROS caused by heavy metals(Bossuyt and Janssen, 2004; Nikookar et al., 2005). Another factor that indirectly inhibition of NMs effects is the production of reactive oxygen species (ROS) by nanoparticles, which can cause chemically-induced toxicity (Petit et al., 2010). The generation of ROS is a consequence of the physical structure and reactive surface chemistry of very small particles. Some reports indicate that light illumination conditions can stimulate photocatalytic activity of NPs to generate hydroxyl radicals, which are responsible for the toxicity of the material (Rogers et al., 2010; Bhattacharya et al., 2010; Oukarroum et al., 2012; Navarro et al., 2008). These NMs can also function by direct

L. Chen et al. / Ecotoxicology and Environmental Safety 84 (2012) 155162

161

inhibition, involving an interaction between NMs and the cell: these interaction can be chemical (such as inhibition of metabolism), due to membrane transport processes or mitosis, or physical (for example, mechanical damage to the cell membrane and organelles) ( Rogers et al., 2010). In our results of TEM images, we observed irregular cell structure including lower chloroplast levels and degradation of organelles as well as plasmolysis in the cells that had been treated with TiO2. These observations can be regarded as direct evidence to support the hypothesis that NMs affect cell structure. Gong et al. (2011) also indicated that C. vulgaris cells, under NiO nanoparticle stress, indicated plasmolysis, cytomembrane breakage, and thylakoid disorder. Our results added further evidence to support of these observations, based on observations using different types of metal oxide NM. Our TEM results also indicated some TiO2 NPs located inside cell wall and plasmic membrane of algae, which implied NPs inside cell may be activated to be more toxic to cells. Wang et al.(2011) found that CuO NPs located inside Microcystis aeruginosa, and dissolved organic matter (DOM) enhanced internalization of CuO NPs inside cell and increased toxicity of CuO NPs signicantly. CuO NPs can also located inside cells of C. reinhardtii (Perreault et al., 2012a) and polymer coated showed more distribution inside cell and more toxicity than bare CuO NPs. Some researches showed that gold (Au) NPs penetrated in the plasmic membrane of C. reinhardtii (Perreault et al., 2012b) and Scenedesmus subcapitata (Renault et al., 2008). Our results is the rst report about nding TiO2 NPs penetrate into cytoplasm which may be essential to asset risk of TiO2 trnasfer through the trophic chain. Nanosized TiO2 induced lipid peroxidation in the unicellular green alga C.reinhardtii and their values reached maximum peaks after 8 h treatment, after which MDA content decreased. No signicant differences were observed among these groups after 72 h treatment. Lipid peroxidation occurs as a result of attack by free radicals, such as reactive oxygen species (ROS) in biological systems (Sevanian and Ursini, 2000). Our ndings concur with results from a number of studies on this and similar topics. Wang et al. (2008b) observed that a dose-dependent increase in maximum MDA content occurred for the rst 12 h and then decreased. They thought that this level-off, or decrease, in lipid peroxidation after 12 h exposure may be a result of increasing antioxidant defense capacity or be due to aggregation of TiO2 NMs and/or the possible biomodication of TiO2 NMs by the cells (Wang et al., 2008a). Their hypothesis was supported by evidence that an increase in the antioxidant enzymes occurred after TiO2 NM treatment, as indicated by increased levels of transcriptional protein. In this study, we also found that soluble protein content (which may include the antioxidant enzymes) increased after TiO2 treatment. In conclusion, we used Chlamydomonas as an experimental model to study the effect of a particular type of manufactured nano-sized materials (nano-TiO2) on ciliated algal cells, C.reinhardtii. We found that nano-TiO2 inhibited algal photosynthesis and cell growth, and the respond of algae to nano-TiO2 was a fast change process which occurred during 24 h after exposing. In comparison with the control, the content of chlorophyll a content in treated algae did not change, while carotenoid and chlorophyll b contents increased. TiO2 induced lipid peroxidation. TEM and SEM images showed that TiO2 particles aggregated on the cell surfaces and located inside cells, which induced serious cell structure damage in C. reinhardti, including fewer chloroplasts, degradation of organelles and plasmolysis. Nano-TiO2 also induced the synthesis of soluble proteins. These effects may be result of the direct generation of ROS by photocatalytic nanoTiO2, or by the presence of nano-TiO2 in the medium which inhibited photosynthesis and induced ROS production indirectly to attack cells.

Acknowledgments The work was supported by the Chinese Natural Science Foundation (30970688, 30970446), the National Major Programs of Water Body Pollution Control and Remediation (2009ZX07103006, 2009ZX07104-005-02, 2008ZX07105-005) and the Project of Chinese Manned Spaceight.

References
Aruoja, V., Dubourguie, H., Kasemets, K., Kahru, A., 2009. Toxicity of nanoparticles of CuO, ZnO and TiO2 to microalgae Pseudokirchneriella subcapitata. Sci. Total Environ. 407, 14611468. Adams, L.K., Lyon, D.Y., Alvarez, P.J.J., 2006. Comparative eco-toxicity of nanoscale TiO2, SiO2, and ZnO water suspensions. Water Res. 40, 35273532. AshaRani, P.V., Mun, G.L.K., Hande, M.P., Valiyaveettil, S., 2009. Cytotoxicity and genotoxicity of silver nanoparticles in human cells. ACS Nano 3, 279290. Bold, H.C., 1949. The morphology of Chlamydomonas sp. nov. Bull. Torrey Bot. Club 76, 101108. Bossuyt, B.T.A., Janssen, C.R., 2004. Long-term acclimation of Pseudokirchneriella subcapitata (Korshikov) Hindak to different copper concentrations: changes in tolerance and physiology. Aquat. Toxicol. 68, 6174. Bradford, M.M., 1976. A rapid and sensitive method for the quantication of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248254. Bhattacharya, P., Lin, S.J., Turner, J.P., Ke, P.C., 2010. Physical adsorption of charged plastic nanoparticles affects algal photosynthesis. J. Phys. Chem. C 114, 1655616561. Chen, L.Z., Wang, G.H., Hong, S., Liu, A., Li, C., Liu, Y.D., 2009. UV-B-induced Oxidative Damage and Protective Role of Exopolysaccharides in Desert Cyanobacterium Microcoleus vaginatus. J. Intergr. Plant Biol. 51, 194200. Domingos, R.F., Simon, D.F., Hauser, C., Wilkinson, K.J., 2011. Bioaccumulation and Effects of CdTe/CdS Quantum Dots on Chlamydomonas reinhardtii- Nanoparticles or the Free Ions? Environ. Sci. Technol. 45, 76647669. Fang, Z., Bouwkamp, J.C., Solomos, T., 1998. Chlorophyllase activities and chlorophyll degradation during leaf senescence in non-yellowing mutant and wild type of Phaseolus vulgaris L. J. Exp. Bot. 49, 503510. Franklin, N.M., Rogers, N.J., Apte, S.C., Batley, G.E., Gadd, G.E., Casey, P.S., 2007. Comparative toxicity of nanoparticulate ZnO, bulk ZnO, and ZnCl2 to a freshwater microalga (Pseudokirchneriella subcapitata): The importance of particle solubility. Environ. Sci. Technol. 41 (24), 84848490. Gong, N., Shao, K.S., Feng, W., Lin, Z.Z., Liang, C.H., Sun, Y.Q., 2011. Biotoxicity of nickel oxide nanoparticles and bio-remediation by microalgae Chlorella vulgaris. Chemosphere 83, 510516. Griftt, R.J., Luo, J., Gao, J., Bonzongo, J.C., Barber, D.S., 2008. Effects of particle composition and species on toxicity of metallic nanomaterials in aquatic organisms. Environ. Toxicol. Chem. 27, 19721978. Hall, S., Bradley, T., Moore, J.T., Kuykindall, T., Minella, L., 2009. Acute and chronic toxicity of nano-scale TiO2 particles to freshwater sh, cladocerans, and green algae, and effects of organic and inorganic substrate on TiO2 toxicity. Nanotoxicology 3, 9197. Hartmann, N.B., Von der Kammer, F., Hofmann, T., Baalousha, M., Ottofuelling, S., Baun, A., 2010. Algal testing of titanium dioxide nanoparticles testing considerations, inhibitory effects and modication of cadmium bioavailability. Toxicology 269, 190197. He, Y.Y., Hader, D.P., 2002. UV-B-induced formation of reactive oxygen species and oxidative damage of the cyanobacterium Anabaena sp.: protective effects of ascorbic acid and N-acetyl-L-cystein. J. Photochem. Photobiol., B 66, 115124. Houimli, S.I.M., Denden, M., Mouhandes, B.M., 2010. Effects of 24-epibrassinolide on growth, chlorophyll, electrolyte leakage and proline by pepper plants under NaCl-stress. Eur. Asia J. Bio. Sci. 4, 96104. Huang, C.P., Cha, D.K., Ismat, S.S., 2005. Progress report: short-term chronic toxicity of photocatalytic nanoparticles to bacteria, algae, and zooplankton. EPA Grant Number: R831721. /http://cfpub.epa.gov/ncer_abstracts/index. cfm/fuseaction/display.abstractDetail/abstract/7384/report/0S. Hund-Rinke, K., Simon, M., 2006. Ecotoxic effect of photocatalytic active nanoparticles (TiO2) on algae and daphnids. Environ. Sci. Pollut. Res. 13, 18. Ji, J., Long, Z.F., Lin, D.H., 2011. Toxicity of oxide nanoparticles to the green algae Chlorella sp. Chem. Eng. J. 170, 525530. Jin, K.S., Joon, L.Y., Mo, K.B., Choi, Y.J., Chung, H.W., 2011. Cytotoxicity and genotoxicity of titanium dioxide nanoparticles in UVA-irradiated normal peripheral blood lymphocytes. Drug Chem. Toxicol. 34, 277284. Kahru, A., Dubourguier, H.C., 2010. From ecotoxicology to nanoecotoxicology. Toxicology 269, 105119. Klaine, S.J., Alvarez, P.J.J., Batley, G.E., Fernandes, T.F., Handy, R.D., Lyon, D.Y., Mahendra, S., McLaughlin, M.J., Lead, J.R., 2008. Nanomaterials in the environment: behavior, fate, bioavailability, and effects. Environ. Toxicol. Chem. 27, 18251851. Lichtenthaler, H.K., Wellburn, A.R., 1983. Determinations of total carotenoids and chlorophyll a and b of leaf extracts in different solvents. Biochem. Soc. Trans. (London) 63, 591592.

162

L. Chen et al. / Ecotoxicology and Environmental Safety 84 (2012) 155162

Menard, A., Drobne, D., Jemec, A., 2011. Ecotoxicity of nanosized TiO2: review of in vivo data. Environ. Pollut. 159, 677684. Metzler, D.M., Li, M.H., Erdem, A., Huang, C.P., 2011. Responses of algae to photocatalytic nano-TiO2 particles with an emphasis on the effect of particle size. Chem. Eng. J. 170, 538546. Mroz, R.M., Schins, R.P.F., Li, H., Drost, E.M., Macnee, W., Donaldson, K., 2007. Nanoparticle carbon black driven DNA damage induces growth arrest and AP-1 and NF kappa B DNA binding in lung epithelial A549 cell line. J Phys. Pharmacol. 58, 461470. Navarro, E., Piccapietra, F., Wagner, B., Marconi, F., Kaegi, R., Odzak, N., Sigg, L., Behra, R., 2008. Toxicity of silver nanoparticles to Chlamydomonas reinhardtii. Environ. Sci. Technol. 42, 89598964. Nel, A., Xia, T., Madler, L., Li, N., 2006. Toxic potential of materials at the nanolevel. Science 311, 622627. Nikookar, K., Moradshahi, A., Hosseini, L., 2005. Physiological responses of Dunaliella salina and Dunaliella tertiolecta to copper toxicity. Biomol. Eng. 22, 141146. Oukarroum, A., Bras, S., Perreault, F., Popovic, R., 2012. Inhibitory effects of silver nanoparticles in two green algae, Chlorella vulgaris and Dunaliella tertiolecta. Ecotoxicol. Environ. Saf. 78, 8085. Pan, J.M., 2008. Cilia and ciliopathies: from Chlamydomonas and beyond. Sci. China, Ser. C Life Sci. 51, 479486. Peralta-Videa, J.R., Zhao, L.J., Lopez-Moreno, M.L., Rosa, G., Hong, J., Gardea-Torresdey, J.L., 2011. Nanomaterials and the environment: a review for the biennium 20082010. J. Hazard. Mater. 186, 115. Perreault, F., Oukarroum, A., Melegari, S.P., Matias, W.G., Popovic, R., 2012a. Polymer coating of copper oxide nanoparticles increases nanoparticles uptake and toxicity in the green alga Chlamydomonas reinhardtii. Chemophere 87, 13881394. Perreault, F., Bogdan, N., Morin, M., Claverie, J., Popovic, R., 2012b. Interaction of gold nanoglycodendrimers with algal cells (Chlamydomonas reinhardtii) and their effect on physiological processes. Nanotoxicology 6, 109120. Petit, A.N., Eullaffroy, P., Debenest, T., Gagne, F., 2010. Toxicity of PAMAM dendrimers to Chlamydomonas reinhardtii. Aqua. Toxicol. 100, 187193. Qiu, Z.G., Yu, Y.M., Chen, Z.l., Jin, M., Yang, D., Zhao, Z.G., Wang, J.F., Shen, Z.Q., Wang, X.W., Qian, D., Huang, A.H., Zhang, B.C., Li, J.W., 2012. Nanoalumina promotes the horizontal transfer of multiresistance genes mediated by plasmids across genera. Proc. Nat. Acad. Sci. U.S.A. 109, 49444949.

Rogers, N.J., Franklin, N.M., Apte, S.C., Batley, G.E., Angel, B.M., Lead, J.R., Baalousha, M., 2010. Physico-chemical behaviour and algal toxicity of nanoparticulate CeO2 in freshwater. Environ. Chem. 7, 5060. Renault, S., Baudrimont, M., Mesmer-Dudon, N., Gonzalez, P., Mornet, S., Brisson, A., 2008. Impacts of gold nanoparticle exposure on two freshwater species: a phytoplanktonic alga (Scenedesmus subspicatus) and a benthic bivalve (Corbicula uminea). Gold Bull. 41, 116126. Saison, C., Perreault, F., Daigle, J.C., Fortin, K., Claverie, J., Morin, M., Popovic, R., 2010. Effect of coreshell copper oxide nanoparticles on cell culture morphology and photosynthesis (photosystem II energy distribution) in the green alga, Chlamydomonas reinhardtii. Aqua. Toxicol. 96, 109114. Sevanian, A., Ursini, F., 2000. Lipid peroxidation in membranes and low-density lipoproteins: similarities and differences. Free Radic. Biol. Med. 29, 306311. Shvedova, A.A., Kagan, V.E., Fadeel, B., 2010. Close encounters of the small kind: adverse effects of man-made materials interfacing with the nano-cosmos of biological systems. Annu. Rev. Pharmacol. Toxicol. 50, 6388. Wang, J.X., Zhang, X.Z., Chen, Y.S., Sommerfeld, M., Hu, Q., 2008a. Toxicity assessment of manufactured nanomaterials using the unicellular green alga Chlamydomonas reinhardtii. Chemosphere 73, 11211128. Wang, G.H., Chen, K., Chen, L.Z., Hu, C.X., Zhang, D.L., Liu, Y.D., 2008b. The involvement of antioxidant system in protection of desert cyanobacterium Nostoc sp. against UV-B radiation and the effects of exogenous antioxidant. Ecotoxicol. Environ. Saf. 69, 150157. Wang, G.H., Hao, Z.J., Huang, Z.B., Chen, L.Z., Li, X.Y., Hu, C.X., Liu, Y.D., 2010. Raman Spectroscopic analysis of a desert cyanobacterium Nostoc sp. in response to UV-B radiation. Astrobiology 10, 783788. Wang, Z.Y., Li, J., Zhao, J., Xing, B.S., 2011. Toxicity and internalization of CuO nanoparticles to prokaryotic alga Microcystis aeruginosa as affected by dissolved organic matter. Environ. Sci. Technol. 45, 60326040. Warheit, D.B., Hoke, R.A., Finlay, C., Donner, E.M., Reed, K.L., Sayes, C.M., 2007. Development of a base set of toxicity tests using ultrane TiO2 particles as a component of nanoparticle risk management. Toxicol. Lett. 171, 99110. Wei, L.P., Thakkar, M., Chen, Y.H., Ntim, S.A., Mitra, S., Zhang, X.Y., 2010. Cytotoxicity effects of water dispersible oxidized multiwalled carbon nanotubes on marine alga, Dunaliella tertiolecta. Aquat. Toxicol. 100, 194201. Wong, S.W.Y., Leung, P.T.Y., Djurisic, A.B., Leung, K.M.Y., 2010. Toxicities of nano zinc oxide to ve marine organisms: inuences of aggregate size and ion solubility. Anal. Bioanal. Chem. 396 (2), 609618.