Ecotoxicology and Environmental Safety 84 (2012) 155–162

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Ecotoxicology and Environmental Safety

0147-65

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n Corr

E-m

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

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

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

a State Key Laboratory of Fresh water Ecology and Biotechnology, Institute of Hydrobiology, The Chinese Academy of Sciences, Wuhan, Hubei 430072, PR Chinab 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 2012Available online 9 August 2012

Keywords:

Nanometer titanium dioxide

Chlamydomonas reinhardtii

Physiological responses

Toxicological effects

Electron microscopy

13/$ - see front matter Crown Copyright & 2

x.doi.org/10.1016/j.ecoenv.2012.07.019

esponding author. Fax: þ86 27 6878 0123.

ail address: space@ihb.ac.cn (G. Wang).

a b s t r a c t

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 cyto-

ultrastructure of this species under treatment. We found that nano-TiO2 inhibited photosynthetic

efficiency 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 formany 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 raisedconcerns about the effects of these substances on human healthand the environment, prompting extensive investigations of NMuptake, fate and toxicity (AshaRani et al., 2009; Kahru andDubourguier, 2010; Shvedova et al., 2010).

The most distinctive feature of NMs is their size, which is animportant determinant of their physicochemical properties withrespect to the ease of uptake of these compounds as well as theirinteraction with biological tissues, particularly since NMs canresult in harmful biological effects in living cells (Adams et al.,2006; Peralta-Videa et al., 2011). Ultrafine particles have beenshown to induce oxidative stress, and distal organ involvement(Nel et al., 2006). It is found that titanium dioxide (TiO2) and othernanoparticles can cause cell growth inhibition, lipid peroxidation

012 Published by Elsevier Inc. All

and photosynthesis inhibition in algae (Wang et al., 2008a;Warheit et al., 2007; Aruoja et al., 2009), and the degree oftoxicity is dependent on concentration and particle size, even inbulkier inert material (as in carbon black and TiO2) (Shvedovaet al., 2010; Nel et al., 2006). But the mechanism for the functionof nanoparticles on organism is still slurred. Some researchesindicate that the function of nanoparticles in organisms is mediatedby 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 algalcell surface (algal cell/nanoparticles aggregation) was likely respon-sible for reduced PSII functional cross section and oxidative stressduring exponential growth. Gong et al. (2011) found that there wasnoticeable algal cell/NiO aggregation which may cause a reductionin the light available to those cells and thus inhibiting their growth.Recent research also showed that nanoalumina can promote thehorizontal transfer of multiresistance genes mediated by plasmidsacross genera (Qiu et al., 2012).

As one main biota in aquatic ecosystem, algae were used toasset the aquatic risk of pollution and toxin in the governmentenvironment reports (Aruoja et al., 2009; Menard et al., 2011).Table 1 showed toxicological characterization of algae undernanomaterials (NMs), which includes recent information aboutvarious nanoparticles, species, cell and physiological responds to

rights reserved.

Table 1Toxicological characterization of algae under nanomaterials (NMs).

Nanomaterials Species Characterization for nanotoxicolgy References

TiO2 Chlamydomonas reinhardtii;

Pseudokirchneriella subcapitata;

Desmodesmus subspicatus; Chlorella sp

Growth inhibition; lipid peroxidation; Stress

response genes expression; Photosynthesis inhibition;

Cyto-ultrastructure disorder; soluble protein

synthesis; NMs aggregation on cell surface;

nanoparticles penetrated cytoplasm

Wang et al. (2008a), Warheit et al. (2007),

Aruoja et al. (2009), Hartmann et al. (2010),

Huang et al. (2005), Griffitt et al. (2008),

Hund-Rinke and Simon, (2006),

Ji et al. (2011), Hall et al. (2009), this study

NiO Chlorella vulgaris Growth inhibition; Soluble protein synthesis;

Cyto-ultrastructure disorder

Gong et al. (2011),

CNTs Dunaliella tertiolecta Growth inhibition; Photosynthesis inhibition;

NMs aggregation on cell surface

Wei et al. (2010),

AgNP Chlorella vulgaris; Dunaliella tertiolect;

C. reinhardtii

Growth inhibition; Lipid peroxidation;

ROS production NMs aggregation on cell surface

Oukarroum et al. (2012),

Navarro et al. (2008)

AuNP Scenedesmus subcapitata;

C. reinhardtii

Growth inhibition; NMs aggregation on cell

surface; Nanoparticles penetrated cytoplasm

Renault et al. (2008),

Perreault et al. (2012b)

CuO P. subcapitata;

Microcystis aeruginosa; C. reinhardtii

Growth inhibition; NMs aggregation on cell surface;

Nanoparticles penetrated cytoplasm Photosynthesis

inhibition; ROS production

Aruoja et al. (2009), Wang et al.(2011),

Perreault et al. (2012a), Saison et al. (2010)

CeO2 P. subcapitata Growth inhibition; Cell membrane damage;

lipid peroxidation

Rogers et al. (2010)

ZnO P. subcapitata;

Chlorella sp

Growth inhibition; NMs aggregation on cell surface Franklin et al. (2007), Aruoja et al. (2009),

Wong et al. (2010), Ji et al. (2011)

Charged Polystyrene

nanospheres

Chlorella sp.; Scenedesmus sp. ROS production; NMs aggregation on

cell surface; photosynthesis inhibition

Bhattacharya et al. (2010)

Quantum dots (CdTe) C. reinhardtii Growth inhibition; lipid peroxidation; stress

response genes expression

Wang et al. (2008a), Domingos et al. (2011)

Al2O3 Chlorella sp. Growth inhibition; NMs aggregation on cell surface Ji et al. (2011)

SiO2 Chlorella sp. Growth inhibition; NMs aggregation on cell surface Ji et al. (2011)

L. Chen et al. / Ecotoxicology and Environmental Safety 84 (2012) 155–162156

nanoparticles and related references cited. There are someadvances about nanotoxicology of algae (Table 1). However,adaptation mechanism of cell biology and physiology in algae tonanoparticles is not clear. Since Chlamydomonas reinhardtii canoffer unique advantages for genetic, biochemical, and cell biologi-cal approaches, it is one of the most used model system (Pan,2008). Some researches indicated that nanoparticles inhibited algalgrowth and induced lipid peroxidation (Mroz et al., 2007;Hartmann et al., 2010; Griffitt et al., 2008; Hund-Rinke andSimon, 2006). Wang et al. (2008a) reported that TiO2 causedgrowth inhibition and lipid peroxidation in C. reinhardtii and somestress response genes were mobilized to express highly. So weused C. reinhardtii as the experiment model to study the celltoxicity of titanium dioxide (TiO2) nanoparticles (NPs), one kindof manufactured nanosized materials, through investigating cellstructure and measuring the biochemical parameters of algae inthis work, and below issues were addressed: (1) to determine theeffects of nano-TiO2 on algal photosynthesis and growth; (2) tostudy the cell ultrastructure and surface changes under TiO2; (3) tomeasure soluble protein and pigments content after treatment;(4) to assay lipid peroxidation.

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 Soil–water supernatant) (Bold, 1949)

at 2571 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 Brunauer–Emmett–Teller (BET)

surface area of 35–65 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

2873 mv according to Wang et al. (2008a). Inoculums were grown in 250 ml glass

flasks containing 100 ml of SE medium at 2571 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 flasks 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 fluorescence

(Chen et al., 2009), determined by a portable plant efficiency analyzer (PEA,

Hanstechs, UK). Experimental conditions were as follows: samples were kept at

room temperature; dark adaptation was about 10–15 min; stimulation light inten-

sity used for experiments was half the maximum intensity

(1500 mmol photon m�2 s�1). The ratio of Fv/Fm (Fv, variable fluorescence; Fm,

maximum fluorescence; Fo, fluorescence value at time 0 or 50 ms (the O transient);

Fv¼Fm�Fo) was used to indicate the photosynthetic efficiency.

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).

Ca ¼ 12:21A663�2:81A646

Cb ¼ 20:13A646�5:03A663

Cc ¼ ð1000A470�3:27Ca�104CbÞ=229

ð1Þ

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 fixed with 3% glutaralde-

hyde, 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 five fields were observed at different magnifications.

0.6

0.7

0.8

*

***

**

**

Fv/Fm

CK0.1mg/LTiO21mg/LTiO210mg/LTiO220mg/LTiO2100mg/LTiO2

-105

10

15

20

*

**

*

*

*

**

***

*

**

Cel

l num

ber (

105 /

L)

Time (h)

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

0 10 20 30 40 50 60 70 80

L. Chen et al. / Ecotoxicology and Environmental Safety 84 (2012) 155–162 157

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 fixed in collidine-buffered 2%

glutaraldehyde containing 5 mM CaCl2 for 1 h at room temperature and postfixed 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 five fields were observed at different magnifications.

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). Briefly, 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 absor-

bance difference between 532 and 600 nm was used to calculate the MDA

formation due to lipid peroxidation (£532¼0.154 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 significant difference, Tukey’s honestly significant difference).

0.5

-10Time (h)

0 10 20 30 40 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 mean7S.D.n indi-

cates there is significant difference between the treatment group and the control

(Po0.05, Tukey multiple comparison).

3. Results

3.1. The growth curve of Chlamydomonas reinhardtii exposed to

nanoparticle TiO2

Fig. 1A indicated that there was not significant differencebetween the control and the treatment group with Nano-TiO2

during 8 h exposing. When the initial concentration of TiO2 NMswas 0.1 mg l�1, there was a little increase than the control at12 h, while there are not significant difference between themiddle dosage of groups (1, 10, 20 mg l�1) and the control. After24 h exposing, there was significant reduction in the treatedgroups and the control. 10, 20 and 100 mg l�1 Nano-TiO2 inhib-ited growth significantly and the cell density of those treatedgroups (10, 20 and 100 mg l�1 TiO2 ) reduced gradually duringexperiment, which showed that cells growth was inhibitedcompletely 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, aratio of variable to maximum fluorescence, indicating activity ofphotosynthesis) of C. reinhardtii decreased sharply with high TiO2

concentration (of41 mg/l), while no significant change wasnoted 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 alsoindicated that Fv/Fm values of cells with TiO2 treatment wereeven higher than those of the control treatments at 24, 48 and

72 h (Fig. 1C). These data indicate that the effects of TiO2 on C.

reinhardtii were possibly acute and that the photosyntheticsystem 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 onthe cell wall surface of C. reinhardtii (Fig. 2A–D). We also foundthat the number of aggregated NPs was dependent on thetreatment concentration of nano-TiO2. NPs were noted to con-centrate on the cell wall surface in the high concentrationtreatment 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 highconcentration treatment groups (100 and 20 mg/l) indicatedirregular structure, including lower chloroplast volume, degradedorganelles, and plasmolysis, while cell microstructures in thelow concentration treatment were not affected (Fig. 2E–H).

B

DC

A

E F

G H

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) 155–162158

3.5

4.0

llkkkk

CK0.1mg/LTiO21mg/LTiO2

L. Chen et al. / Ecotoxicology and Environmental Safety 84 (2012) 155–162 159

Nanoparticles were located on the surface of cell walls (Fig. 2F–H),which confirmed the results got from SEM images. TiO2 nano-particales also penetrated the cell wall (Fig. 2G) and plasmamembrane (Fig. 2H), which may locate inside cell.

00.0

0.5

1.0

1.5

2.0

2.5

3.0jj ii

hggghg

f edddd

baa

cChl

a (m

g/L)

Time (d)

10mg/LTiO220mg/LTiO2100mg/LTiO2

1 2 3 4

3.5. Effects of TiO2 on cellular soluble protein

As illustrated in Fig. 3, the content of soluble protein in algaecells 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 of20 and 100 mg/l. After 72 h treatment, the protein content in 0.1,1 and 10 mg/l treatment groups increased while no significantchanges were observed in groups exposed to 20 and 100 mg/lTiO2 concentration.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

d

ih

gghhgf

eeebc

dbcdbcd

bcd

bcdbcbcdbcc b

bba

Chl

b (m

g/L)

Time (d)

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

0 1 2 3 4

4.0

3.6. Photosynthetic pigments changed after NMs exposing

The content of Chlorophyll a, Chlorophyll b and carotenoids inC. reinhardtii under TiO2 treatment are illustrated in Fig. 4. Therewere no significant changes for Chlorophyll a in different levels oftreatment groups and exposure times, while for Chlorophyll b andcarotenoids content, there were obvious decreases after treating,and then there were significant increases in treated groups after3 and 4days exposing. The ratio of Chl a:b raised when theconcentration of nano-TiO2 increased after 24 h treatment, whichimplied that there were more Chl a in treated algae after 24 hexposing with nano-TiO2. But this ratio reduced gradually after48, 72 and 96 h with TiO2, which suggested that Chl a contentbecome less in these treated cells.

0.5

1.0

1.5

2.0

2.5

3.0

3.5

ji

hghghh ggg

ffee edede

d cccc bbc ba

Car

oten

oid

(mg/

L)

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

3.7. Effects of TiO2 on MDA content of Chlamydomonas reinhardtii

The MDA content in all treatment groups (0.1, 1, 10, 20 and100 mg/l) increased after 4 h treatment, compared to that of thecontrol group (Fig. 5), reaching maximum values after 8 h. After12 h treatment, however, the MDA content in every treatmentgroup decreased, compared to values measured at 8 h. Nosignificant differences among these groups were noted after72 h treatment.

0

20

40

60

80

100

120

140

160

180 hg

gf

fe

ddd

cccccc

b b

aaaa

72 h48 h24 h0 h

Pro

tein

s (m

g/L)

CK0.111020100

Fig. 3. Evolution of pigment contents versus time in C. reinhardtii exposed to

nanoparticle TiO2. Values represent mean7S.D. Those with different letter on

error bar are significantly different (Po0.05, Tukey multiple comparison).

0.0

Time (d)0 1 42 3

Fig. 4. Soluble protein content of C. reinhardtii exposed to nanoparticle TiO2.

Values represent mean7S.D. Those with different letter on error bar are

significantly different (Po0.05, Tukey multiple comparison).

4. Discussion

Since NPs have a number of unique characteristics, in terms ofphysicochemical, optical, reactive, and electrical properties, theydisplay high interaction with organisms and potential toxicity(Peralta-Videa et al., 2011; Jin et al., 2011). Cell aggregation hasbeen reported in cultures treated with TiO2 NMs (Wang et al.,2008a). Ji et al. (2011) showed that large aggregates of nanopar-ticles entrapping the algal cells were observed under the treat-ment of nano-ZnO and HR3 (TiO2) which may reduce the light andnutrient available to the entrapped algal cells and thus inhibittheir growth. Aruoja et al. (2009) found that large aggregateswere observed in the case of TiO2 nanoparticles, that entrappedalmost all algal cells, while the cultures with bulk TiO2 always

-4

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

**

*

*

**

MD

A (µ

mol

/mg)

Time (h)

CK0.1mg/LTiO21mg/LTiO210mg/LTiO220mg/LTiO2100mg/LTiO2

0 4 8 12 64 68 72

Fig. 5. Evolution of MDA content versus time in C. reinhardtii exposed to

nanoparticle TiO2. Values represent mean7S.D. n indicates there is significant

difference between the treatment group and the control(Po0.05, Tukey multiple

comparison).

L. Chen et al. / Ecotoxicology and Environmental Safety 84 (2012) 155–162160

contained free algal cells in addition to cells entrapped in smallTiO2 aggregates. They thought that TiO2 nanoparticle aggregatesmaybe reduce the light available to the entrapped algal cells andthus inhibite their growth. Huang et al. (2005) also indicated thatP. subcapitata cells adsorbed onto their surface TiO2 nanoparticlesand carried 2.3 times their own weight in TiO2 particles, and thekinetics and the extent of nano TiO2 adsorption on algae werehighly dependent on pH, the maximum adsorption occurring atpH 5.5. Perreault et al. (2012) found that there were abviousaggregation of C. reinhardtii cell culture after 6 h of treatment to0.04 g l�1 of bare CuO NPs or polymer-coated CuO NPs. SEMimages of algae indicated that PS (polystyrene)nanoplastic aggre-gates adsorbed on the surface of algae(Bhattacharya et al.,2010),and the adsorption isotherms for Chlorella and Scenedesmus weredifferent which could be attributed to the more rugged morphol-ogy and higher total surface area of Scenedesmus than Chlorella

(Bhattacharya et al.,2010). In this study we observed cell aggrega-tion in TiO2 treatment groups with SEM images and light micro-scopy, as well as evidence of TiO2 NM absorbance on some cellsurfaces in the high concentration treatment groups. The TEMimages of high dosage groups also supported NPs aggregation onthe cell surface. This could obstruct cellular exchange with theexternal milieu, for example by sequestering nutrients or alteringpH or redox potential. In some studies, using scanning electronmicroscopy (SEM) images, Metzler et al.(2011) found that anumber of factors affecting the surface of algal cells – includingsurface coverage by non-uniform multiple layers of TiO2 nano-particles, or lipid peroxidation – could cause major damage toessential processes in algae. Thus, indirect inhibition, i.e., inhibi-tion due to various artefacts, occurs when NMs interact with theenvironment of the cell, which may affect cell metabolism. As aresult, cell growth and activity are inhibited.

In this study, we also noted a significant decrease in growthand photosynthesis activity of algae under TiO2 treatment during24 h exposure. Nevertheless, after 24 and 72 h exposure, photo-synthetic activity in the high-dosage treatment group recoveredto normal levels, while that of the low dosage groups remained ata higher level than that of the control. These recoveries may be aresult of changing NM characteristics in liquid medium, or theadaptation response of algae. NM aggregation may reduce activesurface sites, then impair photocatalytic activity of TiO2 NMs, andthus decrease toxicity to C. reinhardtii cells. Besides, impairmentin the photocatalytic activity of TiO2 NMs over the culture

period may also be a reason for the decreased inhibitory effecton photosynthetic activity (Bhattacharya et al., 2010). It is alsoplausible that cellular secretion of certain macromolecules, suchas polysaccharides, may affect the active sites of the NMs, whichis important for detoxification of TiO2 NM by C. reinhardtii.

It was found that the soluble protein content of the low-dosage groups was higher than the control, while that of thehigh concentration treatment groups was lower than the low-dosage groups and there was not difference between the highconcentration treatment groups and the control. This suggeststhat more soluble protein was present in the low-dosagetreated algae than the control, which may relate to de nova

protein synthesis, and that can play an important role in theadaptation reaction of algal cells under NMs treatment. Gonget al. (2011) found that maximum soluble protein contentoccurred at 72 h and the maximum ratio of reduction ofNi presented synchronously. The reduction from nNiO to nNimay lead to weakened toxicity, so it has been proposedthat some unknown enzymes of the algae play essential rolesin that process, which contributes to adaptation reactionof algal cells under metal oxide NM stress. But for the highconcentration treatment group, it was lower than the low-dosage groups, and the reason for this difference maybe con-tribute to that high concentration nano-TiO2 inhibited cellactivity including protein synthesis. Besides, the soluble pro-tein content of the low-dosage groups remained a long timefrom 24 to 72 h, which implied that some stress proteins weresynthesized 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, itwas found that chlorophyll b and carotenoid contents weredecreased at first by TO2, then these contents increased signifi-cantly, while the chlorophyll a content had no significant changethan the control. And the ratio of Chl a:b raised when theconcentration of nano-TiO2 increased after 24 h treatment, thenthis ration reduced gradually after 48, 72 and 96 h treatment.Since the first 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 canattack chlorophyll and some chl b is to convert to chl a, whichlead to more chlorophyll a in cells. Houimli et al. (2010) alsoshowed that NaCl-stress modifies the chlorophyll b content morethan that of chlorophyll a, which appears less sensitive thanchlorophyll b to NaCl-stress. Because stress can increase ROSproduction (Wang et al., 2008b), this results in an increase in thechl a/b ratio may be involved to ROS attack. For carotenoids, it isanother matter, since carotenoid is effective quenchers of triplet-state photosensitizers, singlet oxygen, and peroxy radicals (Wanget al., 2010), so algal cells use it to extinguish ROS from photo-chemical reaction with TiO2, and algal cells also synthesis com-pensatively carotenoid as an adaptation. Similar phenomenaoccur to various metal stresses in algae, which showed thatcarotenoid synthesis increase as respond to quench ROS causedby heavy metals(Bossuyt and Janssen, 2004; Nikookar et al.,2005).

Another factor that indirectly inhibition of NMs effects is theproduction 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 structureand reactive surface chemistry of very small particles. Somereports indicate that light illumination conditions can stimulatephotocatalytic activity of NPs to generate hydroxyl radicals,which are responsible for the toxicity of the material (Rogerset 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) 155–162 161

inhibition, involving an interaction between NMs and the cell:these interaction can be chemical (such as inhibition of metabo-lism), due to membrane transport processes or mitosis, orphysical (for example, mechanical damage to the cell membraneand organelles) ( Rogers et al., 2010). In our results of TEM images,we observed irregular cell structure – including lower chloroplastlevels and degradation of organelles – as well as plasmolysis inthe cells that had been treated with TiO2. These observations canbe regarded as direct evidence to support the hypothesis thatNMs affect cell structure. Gong et al. (2011) also indicated that C.

vulgaris cells, under NiO nanoparticle stress, indicated plasmoly-sis, cytomembrane breakage, and thylakoid disorder. Our resultsadded further evidence to support of these observations, based onobservations using different types of metal oxide NM. Our TEMresults also indicated some TiO2 NPs located inside cell wall andplasmic membrane of algae, which implied NPs inside cell may beactivated to be more toxic to cells. Wang et al.(2011) found thatCuO NPs located inside Microcystis aeruginosa, and dissolvedorganic matter (DOM) enhanced internalization of CuO NPs insidecell and increased toxicity of CuO NPs significantly. CuO NPs canalso located inside cells of C. reinhardtii (Perreault et al., 2012a)and polymer coated showed more distribution inside cell andmore toxicity than bare CuO NPs. Some researches showed thatgold (Au) NPs penetrated in the plasmic membrane of C. rein-

hardtii (Perreault et al., 2012b) and Scenedesmus subcapitata

(Renault et al., 2008). Our results is the first report about findingTiO2 NPs penetrate into cytoplasm which may be essential toasset risk of TiO2 trnasfer through the trophic chain.

Nanosized TiO2 induced lipid peroxidation in the unicellulargreen alga C.reinhardtii and their values reached maximum peaksafter 8 h treatment, after which MDA content decreased. Nosignificant differences were observed among these groups after72 h treatment. Lipid peroxidation occurs as a result of attack byfree radicals, such as reactive oxygen species (ROS) in biologicalsystems (Sevanian and Ursini, 2000). Our findings concur withresults from a number of studies on this and similar topics. Wanget al. (2008b) observed that a dose-dependent increase in max-imum MDA content occurred for the first 12 h and thendecreased. They thought that this level-off, or decrease, in lipidperoxidation after 12 h exposure may be a result of increasingantioxidant defense capacity or be due to aggregation of TiO2 NMsand/or the possible biomodification of TiO2 NMs by the cells(Wang et al., 2008a). Their hypothesis was supported by evidencethat an increase in the antioxidant enzymes occurred after TiO2

NM treatment, as indicated by increased levels of transcriptionalprotein. In this study, we also found that soluble protein content(which may include the antioxidant enzymes) increased afterTiO2 treatment.

In conclusion, we used Chlamydomonas as an experimentalmodel to study the effect of a particular type of manufacturednano-sized materials (nano-TiO2) on ciliated algal cells, C.rein-

hardtii. We found that nano-TiO2 inhibited algal photosynthesisand cell growth, and the respond of algae to nano-TiO2 was a fastchange process which occurred during 24 h after exposing. Incomparison with the control, the content of chlorophyll a contentin treated algae did not change, while carotenoid and chlorophyllb contents increased. TiO2 induced lipid peroxidation. TEM andSEM images showed that TiO2 particles aggregated on the cellsurfaces and located inside cells, which induced serious cellstructure damage in C. reinhardti, including fewer chloroplasts,degradation of organelles and plasmolysis. Nano-TiO2 alsoinduced the synthesis of soluble proteins. These effects may beresult of the direct generation of ROS by photocatalytic nano-TiO2, or by the presence of nano-TiO2 in the medium whichinhibited photosynthesis and induced ROS production indirectlyto attack cells.

Acknowledgments

The work was supported by the Chinese Natural ScienceFoundation (30970688, 30970446), the National Major Programsof Water Body Pollution Control and Remediation (2009ZX07103-006, 2009ZX07104-005-02, 2008ZX07105-005) and the Project ofChinese Manned Spaceflight.

References

Aruoja, V., Dubourguie, H., Kasemets, K., Kahru, A., 2009. Toxicity of nanoparticlesof CuO, ZnO and TiO2 to microalgae Pseudokirchneriella subcapitata. Sci. TotalEnviron. 407, 1461–1468.

Adams, L.K., Lyon, D.Y., Alvarez, P.J.J., 2006. Comparative eco-toxicity of nanoscaleTiO2, SiO2, and ZnO water suspensions. Water Res. 40, 3527–3532.

AshaRani, P.V., Mun, G.L.K., Hande, M.P., Valiyaveettil, S., 2009. Cytotoxicity andgenotoxicity of silver nanoparticles in human cells. ACS Nano 3, 279–290.

Bold, H.C., 1949. The morphology of Chlamydomonas sp. nov. Bull. Torrey Bot. Club76, 101–108.

Bossuyt, B.T.A., Janssen, C.R., 2004. Long-term acclimation of Pseudokirchneriellasubcapitata (Korshikov) Hindak to different copper concentrations: changes intolerance and physiology. Aquat. Toxicol. 68, 61–74.

Bradford, M.M., 1976. A rapid and sensitive method for the quantification ofmicrogram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 72, 248–254.

Bhattacharya, P., Lin, S.J., Turner, J.P., Ke, P.C., 2010. Physical adsorption ofcharged plastic nanoparticles affects algal photosynthesis. J. Phys. Chem. C114, 16556–16561.

Chen, L.Z., Wang, G.H., Hong, S., Liu, A., Li, C., Liu, Y.D., 2009. UV-B-inducedOxidative Damage and Protective Role of Exopolysaccharides in DesertCyanobacterium Microcoleus vaginatus. J. Intergr. Plant Biol. 51, 194–200.

Domingos, R.F., Simon, D.F., Hauser, C., Wilkinson, K.J., 2011. Bioaccumulation andEffects of CdTe/CdS Quantum Dots on Chlamydomonas reinhardtii- Nanoparti-cles or the Free Ions? Environ. Sci. Technol. 45, 7664–7669.

Fang, Z., Bouwkamp, J.C., Solomos, T., 1998. Chlorophyllase activities and chlor-ophyll degradation during leaf senescence in non-yellowing mutant and wildtype of Phaseolus vulgaris L. J. Exp. Bot. 49, 503–510.

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 afreshwater microalga (Pseudokirchneriella subcapitata): The importance ofparticle solubility. Environ. Sci. Technol. 41 (24), 8484–8490.

Gong, N., Shao, K.S., Feng, W., Lin, Z.Z., Liang, C.H., Sun, Y.Q., 2011. Biotoxicity ofnickel oxide nanoparticles and bio-remediation by microalgae Chlorellavulgaris. Chemosphere 83, 510–516.

Griffitt, R.J., Luo, J., Gao, J., Bonzongo, J.C., Barber, D.S., 2008. Effects of particlecomposition and species on toxicity of metallic nanomaterials in aquaticorganisms. Environ. Toxicol. Chem. 27, 1972–1978.

Hall, S., Bradley, T., Moore, J.T., Kuykindall, T., Minella, L., 2009. Acute and chronictoxicity of nano-scale TiO2 particles to freshwater fish, cladocerans, andgreen algae, and effects of organic and inorganic substrate on TiO2 toxicity.Nanotoxicology 3, 91–97.

Hartmann, N.B., Von der Kammer, F., Hofmann, T., Baalousha, M., Ottofuelling, S.,Baun, A., 2010. Algal testing of titanium dioxide nanoparticles testingconsiderations, inhibitory effects and modification of cadmium bioavailability.Toxicology 269, 190–197.

He, Y.Y., Hader, D.P., 2002. UV-B-induced formation of reactive oxygen species andoxidative damage of the cyanobacterium Anabaena sp.: protective effects ofascorbic acid and N-acetyl-L-cystein. J. Photochem. Photobiol., B 66, 115–124.

Houimli, S.I.M., Denden, M., Mouhandes, B.M., 2010. Effects of 24-epibrassinolideon growth, chlorophyll, electrolyte leakage and proline by pepper plants underNaCl-stress. Eur. Asia J. Bio. Sci. 4, 96–104.

Huang, C.P., Cha, D.K., Ismat, S.S., 2005. Progress report: short-term chronictoxicity 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 nano-particles (TiO2) on algae and daphnids. Environ. Sci. Pollut. Res. 13, 1–8.

Ji, J., Long, Z.F., Lin, D.H., 2011. Toxicity of oxide nanoparticles to the green algaeChlorella sp. Chem. Eng. J. 170, 525–530.

Jin, K.S., Joon, L.Y., Mo, K.B., Choi, Y.J., Chung, H.W., 2011. Cytotoxicity andgenotoxicity of titanium dioxide nanoparticles in UVA-irradiated normalperipheral blood lymphocytes. Drug Chem. Toxicol. 34, 277–284.

Kahru, A., Dubourguier, H.C., 2010. From ecotoxicology to nanoecotoxicology.Toxicology 269, 105–119.

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 environ-ment: behavior, fate, bioavailability, and effects. Environ. Toxicol. Chem. 27,1825–1851.

Lichtenthaler, H.K., Wellburn, A.R., 1983. Determinations of total carotenoids andchlorophyll a and b of leaf extracts in different solvents. Biochem. Soc. Trans.(London) 63, 591–592.

L. Chen et al. / Ecotoxicology and Environmental Safety 84 (2012) 155–162162

Menard, A., Drobne, D., Jemec, A., 2011. Ecotoxicity of nanosized TiO2: review ofin vivo data. Environ. Pollut. 159, 677–684.

Metzler, D.M., Li, M.H., Erdem, A., Huang, C.P., 2011. Responses of algae tophotocatalytic nano-TiO2 particles with an emphasis on the effect of particlesize. Chem. Eng. J. 170, 538–546.

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 andAP-1 and NF kappa B DNA binding in lung epithelial A549 cell line. J Phys.Pharmacol. 58, 461–470.

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, 8959–8964.

Nel, A., Xia, T., Madler, L., Li, N., 2006. Toxic potential of materials at the nanolevel.Science 311, 622–627.

Nikookar, K., Moradshahi, A., Hosseini, L., 2005. Physiological responses ofDunaliella salina and Dunaliella tertiolecta to copper toxicity. Biomol. Eng. 22,141–146.

Oukarroum, A., Bras, S., Perreault, F., Popovic, R., 2012. Inhibitory effects of silvernanoparticles in two green algae, Chlorella vulgaris and Dunaliella tertiolecta.Ecotoxicol. Environ. Saf. 78, 80–85.

Pan, J.M., 2008. Cilia and ciliopathies: from Chlamydomonas and beyond. Sci.China, Ser. C Life Sci. 51, 479–486.

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 reviewfor the biennium 2008–2010. J. Hazard. Mater. 186, 1–15.

Perreault, F., Oukarroum, A., Melegari, S.P., Matias, W.G., Popovic, R., 2012a.Polymer coating of copper oxide nanoparticles increases nanoparticles uptakeand toxicity in the green alga Chlamydomonas reinhardtii. Chemophere 87,1388–1394.

Perreault, F., Bogdan, N., Morin, M., Claverie, J., Popovic, R., 2012b. Interaction ofgold nanoglycodendrimers with algal cells (Chlamydomonas reinhardtii) andtheir effect on physiological processes. Nanotoxicology 6, 109–120.

Petit, A.N., Eullaffroy, P., Debenest, T., Gagne, F., 2010. Toxicity of PAMAMdendrimers to Chlamydomonas reinhardtii. Aqua. Toxicol. 100, 187–193.

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. Nanoaluminapromotes the horizontal transfer of multiresistance genes mediated byplasmids across genera. Proc. Nat. Acad. Sci. U.S.A. 109, 4944–4949.

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 nanoparticulateCeO2 in freshwater. Environ. Chem. 7, 50–60.

Renault, S., Baudrimont, M., Mesmer-Dudon, N., Gonzalez, P., Mornet, S., Brisson,A., 2008. Impacts of gold nanoparticle exposure on two freshwater species: aphytoplanktonic alga (Scenedesmus subspicatus) and a benthic bivalve(Corbicula fluminea). Gold Bull. 41, 116–126.

Saison, C., Perreault, F., Daigle, J.C., Fortin, K., Claverie, J., Morin, M., Popovic, R.,2010. Effect of core–shell copper oxide nanoparticles on cell culture morphol-ogy and photosynthesis (photosystem II energy distribution) in the green alga,Chlamydomonas reinhardtii. Aqua. Toxicol. 96, 109–114.

Sevanian, A., Ursini, F., 2000. Lipid peroxidation in membranes and low-densitylipoproteins: similarities and differences. Free Radic. Biol. Med. 29, 306–311.

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 ofbiological systems. Annu. Rev. Pharmacol. Toxicol. 50, 63–88.

Wang, J.X., Zhang, X.Z., Chen, Y.S., Sommerfeld, M., Hu, Q., 2008a. Toxicityassessment of manufactured nanomaterials using the unicellular green algaChlamydomonas reinhardtii. Chemosphere 73, 1121–1128.

Wang, G.H., Chen, K., Chen, L.Z., Hu, C.X., Zhang, D.L., Liu, Y.D., 2008b. Theinvolvement of antioxidant system in protection of desert cyanobacteriumNostoc sp. against UV-B radiation and the effects of exogenous antioxidant.Ecotoxicol. Environ. Saf. 69, 150–157.

Wang, G.H., Hao, Z.J., Huang, Z.B., Chen, L.Z., Li, X.Y., Hu, C.X., Liu, Y.D., 2010. RamanSpectroscopic analysis of a desert cyanobacterium Nostoc sp. in response toUV-B radiation. Astrobiology 10, 783–788.

Wang, Z.Y., Li, J., Zhao, J., Xing, B.S., 2011. Toxicity and internalization of CuOnanoparticles to prokaryotic alga Microcystis aeruginosa as affected by dis-solved organic matter. Environ. Sci. Technol. 45, 6032–6040.

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 ultrafine TiO2 particles as acomponent of nanoparticle risk management. Toxicol. Lett. 171, 99–110.

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 nano-tubes on marine alga, Dunaliella tertiolecta. Aquat. Toxicol. 100, 194–201.

Wong, S.W.Y., Leung, P.T.Y., Djurisic, A.B., Leung, K.M.Y., 2010. Toxicities of nanozinc oxide to five marine organisms: influences of aggregate size and ionsolubility. Anal. Bioanal. Chem. 396 (2), 609–618.