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Environmental Pollution 180 (2013) 63e70

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Environmental Pollution

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Ecotoxicity of non-aged and aged CeO2 nanomaterials towardsfreshwater microalgae

Nicolas Manier a,*, Anne Bado-Nilles a,b, Patrice Delalain a, Olivier Aguerre-Chariol a,Pascal Pandard a

a INERIS (Unités EXES, ECOT et NOVA), Parc Technologique Alata, BP2, 60550 Verneuil-en-Halatte, FrancebUniversité Reims Champagne-Ardenne, EA Unité Interactions Animal-Environnement, Moulin de la Housse, B.P. 1039, 51687 Reims, France

a r t i c l e i n f o

Article history:Received 5 November 2012Received in revised form23 April 2013Accepted 25 April 2013

Keywords:Cerium dioxideAlterated nanoparticlesAgglomerationPseudokirchneriella subcapitataGrowth inhibitionFlow cytometryEnvironmental scanning electronmicroscope

* Corresponding author.E-mail addresses: nicolas.manier@ineris.fr, nmanie

0269-7491/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.envpol.2013.04.040

a b s t r a c t

The ecotoxicity of artificially alterated cerium dioxide nanoparticles (nano-CeO2) suspensions wasdetermined using the freshwater microalgae growth inhibition test. The agglomeration or aggregationstate of the alterated suspensions was followed because it represents one of the obvious modificationswhen nanoparticles reached the environment. In addition, its influence on the ecotoxicity of nano-particles is currently not well-addressed. Our results showed that the suspensions were stable within thefirst 24 h and then agglomerate up to 10 mm after 3 and 30 days. The inhibitory effect on the growth ofexposed algae was however similar whatever the tested suspension. This supports the fact that theagglomeration state of nano-CeO2, in our conditions, has few influences on the ecotoxicity toward theseorganisms. The EC50 values were 5.6; 4.1 and 6.2 mg L�1, after exposure to non aged, 3 and 30 days agedsuspensions respectively. The interaction between algal cells and nano-CeO2 was also addressed.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

As engineered nanoparticles (NPs) and nanomaterials (NMs)have diverse benefits, they have been extensively used in a broadrange of applications and cover most of the industrial sectors aswell as the medicine, the agricultural and the environmental areas(Tiede et al., 2008; Liu, 2006). Release of these emerging com-pounds is inevitable and looking at the possible routes for NPs orNMs to enter into the environment, the aquatic compartment(including both water columns and sediments) can be substantiallyexposed. The assessment of the environmental effects of nano-compounds has, consequently, become a major issue worldwide,and in the last five to ten years, an increasing number of studiesconcerning the NPs or NMs hazard assessment were published(Kahru and Dubourguier, 2009; Menard et al., 2011; Lapresta-Fernández et al., 2012). Among the research challenges to assessthe intrinsic properties of NMs and NPs, it appears necessary tostudy the effect of their whole life cycle, including both the initialforms (pristine form) and physico-chemically modified form

r@gmail.com (N. Manier).

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resulting from an alteration process (i.e aggregated or agglomer-ated forms as well as altered forms). To date, most of the studiesfocused on the hazard properties of pristine materials, trying tomaintain particles as free as possible in the studying media. Look-ing at the currently published works, few studies concerning thefate and effect of residual products resulting from an alterationprocess are available (Labille et al., 2010).

Among NPs, cerium dioxide NPs (nano-CeO2) are widely used innumber of applications such as sun cream, outdoor paint, woodcare products as well as fuel catalysts (Brar et al., 2010).Widespreaduse of nanosized CeO2 could imply a significant release of thischemical into the environment, leading to a potential for increasedenvironmental exposure. For such reason, nano-CeO2 is one of theselected NPs for priority testing within the sponsorship program ofthe Working Party of Manufactured Nanomaterials of the Organi-zation for Economic Cooperation and Development (OECD, 2008).

Authors showed that cerium dioxide nanoparticles can beharmful to the organisms living in the freshwater compartment(algae, micro-invertebrates and insect larva), unlike bulk material.Acute and chronic ecotoxicity as well as genotoxic effects werealready reported (Van Hoecke et al., 2009; Lee et al., 2009; Manieret al., 2011; Roger et al., 2010; Rodea-Palomares et al., 2011; Garciaet al., 2011; Rodea-Palomares et al., 2012). Nevertheless, most of the

N. Manier et al. / Environmental Pollution 180 (2013) 63e7064

published data concerned a cerium dioxide nanopowder freshlysuspended in water by various methods (i.e magnetic stirring, useof sonication) but no information was currently available about thealterated or agglomerated forms of such NPs.

In the present study, our aim is to contribute to understand theinfluence of an artificial alteration of cerium dioxide NPs on theirbehavior in water and their ecotoxicity toward freshwater organ-isms. Thus, the effects of commercially available cerium dioxidenanomaterial suspensions in water, subjected or not to artificialalteration process, were investigated toward the freshwatermicroalgae: Pseudokirchneriella subcapitata. The interaction be-tween algae and nano-CeO2 were subsequently investigated usingflow cytometry and Environmental Electron Scanning Microscopy(E-SEM) coupled with energy dispersive X-ray microanalyses(EDAX).

2. Materials and methods

2.1. Cerium dioxide nanomaterials

A commercially available nano-CeO2 composite (Nanobyk�, Byk company) wasobtained as 248 g L�1 stable suspension inwater. This nanomaterial is composed of aceria core, coated with triammonium citrate layer. The primary particle size isaround 10 nm (supplier data). Semi-quantitative multi-element analysis (ICP-MS7500, Agilent) was performed to obtain a detailed characterization of the elementalcomposition of the Nanobyk suspension (Table 1S). This product is designed forindustrial applications and intended to be used as architectural coating to improvescratch and UV protection for outdoor paints or for wood care products. The sus-pension was maintained in cool place (20 �C) and in the dark in order to avoidmodifications (as coating alteration or agglomeration) before the experiments.Transmission Electron Micrograph of the diluted product in high purity water isgiven in Fig. 1.

2.2. Preparation of the suspension and alteration process

For the ecotoxicity tests, an initial nano-CeO2 suspension (31.25 mg L�1) wasprepared by dilution of the commercial suspension into the algal growth medium(OECD, 2011). The nano-CeO2 suspension was artificially alterated at room tem-perature (20 �C � 1 �C) under magnetic stirring (300 rpm; using a 5 cm magneticstirring rod) and lighting (4000e5000 lux, using 36 W/840, 3350 lumens cool whitetubes) for three days and up to one month. In order to avoid differences in hydro-dynamic or in interaction with the glass vessel, all the preparations were performedin exactly the same vessels.

2.3. Physico-chemical characterization of the tested suspensions

Evolution of the secondary particle size (i.e agglomerate or aggregate size) wasfollowed using Dynamic Light Scattering (DLS; NanoZS, Malvern Instruments�). Foreach analysis, measurements were done in triplicate, directly after sampling. Non

Fig. 1. TEM micrograph of the initial nano-CeO2 suspension in milliQ water.(bar ¼ 100 nm).

invasive back scatter detection at 173� with a HeeNe laser (l ¼ 633 nm) as lightsource was used. The aggregates or agglomerates sizes were determined by the NonNegative Least Squares (NNLS) analysis method at 25 �C, after an equilibration timeof 60 s. Each measurement is an average of 11 runs of 10 s. The larger agglomerate oraggregate sizes (i.e after 3 days of alteration) were determined by laser diffraction(MasterSizer 2000, Malvern Instrument�). Zeta potential of the different suspen-sions (z) was also followed through time (NanoZS, Malvern Instruments�). The zetapotential was calculated using the Smoluchowski approximation. Each measure-ment is an average of 30 runs of 10 s. All measurements were performed in triplicate.

Transmission electronmicroscope (TEM), (CM 12, Philips�) was used to visualizethe agglomeration or aggregation state of nano-CeO2 in the non aged and agedcerium dioxide suspensions. For TEM, carbon coated grids were hydrophilised usingan Emitech K100X glow discharge apparatus. The glow discharge was performed for180 s at an air pressure of 10�1 mbar and an electric current of 40mA. This treatmentis applied to TEM grids prior to the suspension deposition. It prevents most of theartifactual agglomeration phenomenon during the drying of the suspensions on theTEM grids (Dubochet et al., 1982).

2.4. Freshwater microalgae growth inhibition test

The algal growth inhibition tests (OECD, 2011) were carried out on the greenmicroalga species: Pseudokirchneriella subcapitata (Korshikov), (formerly known asSelenastrum capricornutum, Printz). Prior to the start of growth inhibition experi-ments, the algal cells were cultured in the same growth medium used for the test.Algae are allowed to grow on an orbital shaker at 22 � 1 �C for 3 days in order toensure that they are in the exponential growth phase when used to inoculate thetest solutions. The OECD algal growth medium was used to prepare a range ofconcentrations (from 0.2 to 25 mg CeO2 L�1) of the different nano-CeO2 stock sus-pensions, which were then inoculated to a density of 8 � 103 cells mL�1. All thestudied concentrations were performed in triplicates and six controls, containingonly OECD test medium and algae, were included in each test run. All the tests wereconducted at 22 �C � 1 �C under cool white light (5000e6000 lux), under contin-uous illumination. These conditions allowed to obtain typical control growth rates ofat least 1.4e1.6 d�1 during the 72 h of incubation. All test runs performed fulfilledthe validity criteria of the OECD test guideline 201 (OECD, 2011).

Fluorescence of algal cells was recorded every 24 h and subsequent number ofcells per mL was determined for each condition. Negative control samples con-taining nano-CeO2 suspensions at the highest concentration and without alga wereused in order to take into account the potential interferences of nano-CeO2 on algalcells fluorescence. No fluorescence from the nano-CeO2 suspensions was recordedduring the experiments, whatever the suspension tested. Growth rate inhibitionswere then determined by comparison with control.

Concentration-response curves as the calculation of toxicity parameters (ECx)were determined using the logistic Hill model. The 95% confidence intervals wereestimated using a “bootstrap” simulation method. All the statistical analyses wereperformed using the REGTOX� software v.7.0.5 for Microsoft Excel (Vindimian,2005).

2.5. Flow cytometry analyses

At the end of the exposure period (i.e 72 h), the interaction betweenP. subcapitata algal cells and nano-CeO2 was evaluated for all the tested conditions toassess whether the NPs are acted in or onto the cell. This interaction was analyzedusing a flow cytometer (Cyan� ADP flow cytometer; Beckman Coulter). The loga-rithmic side scatter and FL1 intensities, indicating the cellular density of microalgae(granularity) and the green cell fluorescence respectively, were recorded.

2.6. Environmental scanning electron microscope observations

Environmental scanning electron microscope (E-SEM, FEI Quanta 400) coupledwith energy dispersive X-ray microanalyses system (EDAX) was used to directlyobserve the non-exposed and exposed algae after the 72 h exposure period, withoutany preparation (e.g. freezing, lyophilisation and/or coating as in classical mode). Avolume of 120 mL of algae suspensionwas laid down on the E-SEMmount previouslycovered by a double sided adhesive copper tape and using a Peltier heating/coolingstage in order to avoid displacement during wetting. The temperature was kept con-stant equal to 10 �C and the pressure equal to 474 Pa, in order to reach 37% of relativehumidity. The observations of algal cells were performed using Secondary Electronmode with a high voltage of 20 kV. The X-rays Microanalysis EDAX Genesis softwarewas used to analyze the cerium presence and its localization on the algal cells.

3. Results

3.1. Characterization of the nano-CeO2 suspensions

Size distribution and TEM observations of the nano-CeO2 sus-pensions are given in Fig. 2. Focusing on the non-aged suspension,

Fig. 2. Size distribution of cerium dioxide nanoparticles in OECD algae growth medium. The graphic a. presents the size evolution during the first 28 h (measurement by DLS,NanoZS, Malvern instrument�). Pictures b. illustrate the agglomeration state at T¼0 (beginning of the ageing process) (pictures b.1), after three days of alteration (picture b.2) andafter one month of alteration (picture b.3). Graphics c. and d. present the particles distribution in the three days and one month aged suspension, respectively (measurement bylaser diffraction, Mastersizer 2000, Malvern instrument�).

Fig. 3. Growth rate inhibition of Pseudokirchneriella subcapitata exposed for 72 h to thenon aged, three days and the one month aged cerium dioxide nanoparticle suspen-sions. Experimental data (mean values and replicate values) are shown together withestimated concentrationeresponse curves obtained by fitting with the logistic Hillmodel (plain curve ¼ non aged suspension; small dots curve ¼ three days aged sus-pension and large dots curve ¼ one month aged suspension).

N. Manier et al. / Environmental Pollution 180 (2013) 63e70 65

the DLS measurement (in intensity) showed a bimodal distributionof the nano-CeO2 particles in the algal growth medium. A firstpopulation of particles centered on 11 nm (predominant peak inintensity) was observed, with some agglomerates and/or aggre-gates up to 20 nm. A second population of larger agglomerates and/or aggregates, centered on 229 nm, was also recorded in this non-aged suspension. These DLS measurements were confirmed by theTEM observations (Fig. 2b.1). Moreover, it was shown that theparticles were mainly clustered in agglomerates which appeared tobe loose with weakly-bound particles, instead of strongly-bound orfused particles as observed in aggregates of NPs. This suspensionwas then subjected to ageing process and the agglomerates sizeevolution was followed up to 72 h. The suspension was quite stableup to 24 h, with a slight increase in particle size distribution from 11to 20 nm (predominant peak in intensity), when considering thepredominant particle population. After 24 h, the size distributionincreased rapidly. The agglomerate size distribution recorded after28 h of ageing showed agglomerate size centered on 580 nm(predominant peak in intensity). After 3 days of alteration, thenano-CeO2 suspension appeared slightly cloudy. Laser diffractionmeasurement reported a particle size distribution centered on7 mm,with some very large agglomerates up to 10 mm (Fig. 2c.). TEMobservations also confirmed these measurements (Fig. 2b.2). Asobserved for the non-aged suspension, the NPs in the 3 days agedsuspension were mainly clustered in agglomerates with weakly-bound particles. Similar to the 3 days ageing suspension, the 30days aged suspensions presented an agglomerate size distributioncentered on 7 mmwith some very large and loose agglomerates upto 10e20 mm (Fig. 2b.3). Zeta potential measurements showed nosignificant difference along the first 30 h of ageing process. A zetapotential of �21 mV � 1.4 mV (3 successive measurements) wasrecorded at the beginning of the alteration process, whereas a zetapotential of �25 mV � 0.4 mV were recorded after 30 h. Because ofthe very high agglomeration after 30 h of alteration, the zeta po-tential values couldn’t be estimated correctly beyond this alterationtime. Analytical measurement of the freshly prepared suspension(ICP-OES, according to ISO 11885 (ISO, 2009)) confirmed the CeO2concentration (>90% of the nominal concentration), allowing toexpress the ecotoxicological results as nominal concentrations.

3.2. Effects on the growth of P. subcapitata

The results of growth inhibition tests using P. subcapitata areillustrated by Fig. 3. Focusing on the non-aged suspension, a clearconcentrationeresponse relationship was recorded with a calcu-lated 72-h EC50 ¼ 5.6 mg L�1 (IC95% ¼ 4.8e6.4). As shown in Fig. 3,similar concentration-response curves were recorded for both agedsuspensions. The EC50 values recorded from the different runs withthe three days and one month aged suspensions were quite similarto the values recorded for the non-aged suspensions. The calculated72-h EC50 [3 days aged susp.] ¼ 4.1 mg L�1 (IC95% ¼ 3.7e4.5) and the 72-h EC50 [30 days aged susp.] ¼ 6.2 mg L�1 (IC95% ¼ 5.5e6.9). The calcu-lated EC10 and EC20 values and their respective IC95% (Table 1)suggest that the 30 days ageing suspension appears weakly lessecotoxic, at least at the lower concentrations tested. Nevertheless,in the absence of repeatability studies, it is difficult to conclude on

Table 172 h-ECx values calculated for the growth inhibition test (in mg L�1 [IC95%]).

Non aged 3 days aged 30 days aged

EC10 0.7 [0.5e1.0] 0.5 [0.4e0.7] 1.8 [1.5e2.5]EC20 1.5 [1.2e1.9] 1.1 [0.9e1.3] 2.8 [2.4e3.6]EC50 5.6 [4.8e6.4] 4.1 [3.7e4.5] 6.2 [5.5e6.9]

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the biological significance of such slight difference. The EC50 valuescalculated for the non aged and 30 days aged suspensions are notstatistically different. Also, considering the biological variationobserved on the reference substance used in our laboratory (vari-ation coefficient ¼ 20%; n ¼ 16 independent assays from 2010 to2012), the ECx values calculated for these experiments can not beregarded as different.

3.3. Interaction between nano-CeO2 and algal cells

The interaction between algal cells and nano-CeO2 in thedifferent suspensions was studied by flow cytometry and E-SEMobservations. As evident by an increase of the intensity of sidescatter (SSC; Fig. 4b, c) as compared to the control (Fig. 4a) withoutmodulation in FL1 fluorescence, a significant interaction (i.eadsorption and/or uptake) between nano-CeO2 and the microalgae

Fig. 4. Results of flow cytometer analyses. (a), (b) and (c) present the cytogram of SS (log s6.25 mg L�1 (b) and 25 mg L�1 (c). The graphic (d) present the percentage of more complex cconcentrations of non aged suspension, the three days aged suspension and the one month aof nano-CeO2 concentration in the media, whatever the suspension tested.

P. subcapitata exposed to the different concentrations of NPs (0.2e25 mg L�1) was observed, whatever the experimental conditionstested (non-aged suspension; three days and one month agedsuspensions). A pronounced concentration-dependent increase inthe SSC intensity (indicating the cell granularity) was exhibitedwhatever the age of the nano-CeO2 suspension tested (Fig. 4d). Inthe absence of NPs, no more complex cells were detected in thealgae population whereas in the presence of nano-CeO2, the per-centage of more complex cells increase at 0.4%, 0.5%, 1.5%, 5.4%,11.1%, 27.7%, 81.30% and 93.91% after exposure to 0.2, 0.39, 0.78,1.56, 3.13, 6.25, 12.5 and 25 mg mL�1 of the non-aged nano-CeO2suspensions, respectively. Similar tendencies were reported afterexposure to the three days and one month aged suspensions,despite a slight difference noticed after exposure to the highestconcentrations (i.e 6.25, 12.25 and 25 mg L�1), when focusing onthe percentage of more complex cells. The additional E-SEM ob-servations (Fig. 5) revealed that after 72 h of exposure to the nano-CeO2 suspensions, a part of the algal cells population was coveredby a large amount of cerium dioxide nanoparticles, as confirmed bythe X-ray microanalyses (Fig. 5b, c, d). Agglomerates of algal cellsand cerium dioxide were sometimes observed. No clear differencebetween the non-alterated and alterated suspension was noticed.The algal cells observed in the control group were all clear and freeand obviously no cerium was detected (Fig. 5a).

cale) and FL1 (log scale) for control (a) and exposed cell to then on aged suspension atells (mean � standard deviation; n ¼ 3) in the algae population exposed to the differentged suspension. These results show that the cell complexity increase with the increase

N. Manier et al. / Environmental Pollution 180 (2013) 63e70 67

4. Discussion

This work aimed at studying the effect of commercially availablecerium dioxide nanoparticles, subjected or not to an artificialalteration process. To the best of our knowledge, it is one of the firstreports in the literature focusing on that point. The main questionto address was: are alterated nanoparticles less or more toxic thanthe pristine form?

As presented in the result section, the ageing process led todifferent agglomeration states in the suspensions, which mainlydiffer in terms of agglomerates size. The non-aged suspension wascharacterized by small agglomerates whereas three days and onemonth aged suspensions have shown very large and loose ag-glomerates up to 10 mm. The quick and important agglomerationalready observed 24 after the start of the ageing process may beexplained by the possible alteration of the triammonium citratecoating layer. Uncoated ceria nanoparticles are known to highlyagglomerate in water, especially when the pH reaches a range 7e8(Keller et al., 2010; Manier et al., 2011), like in our test conditions.Change in the zeta potential of alterated NPs was already reportedby Labille et al. (2010) who studied the physico-chemical modifi-cations of titanium NPs coated with Al(OH)3 and poly-dimethylsiloxane layers. These authors have shown that the zetapotential modification was associated to the degradation of thecoating layer. Despite no clear modification of the zeta potentialwas recorded up to 30 h of alteration, the degradation of the coatinglayer can not be excluded thereafter. In this work, the alteration ofthe coating layer may be one of the possible mechanisms leading tothe agglomeration of CeO2 particles in the aquatic medium. Furtherspecific work on the coating modification for this nano-CeO2should however be performed to fully address this hypothesis. Itwas also shown that the time corresponding to three days of agingwas sufficient to get a steady state in term of agglomeration state ofnano-CeO2. No further agglomeration was observed after threedays.

In this work, the cerium dioxide nanoparticle suspensions wereclearly ecotoxic toward the freshwater microalgae: P. subcapitata.Moreover, given the very low solubility of the cerium dioxide inwater, as already reported by Roger et al. (2010) and Van Hoeckeet al. (2009), it can be assumed that most of the effects recordedwere related to nano-CeO2 particles and therefore, the effects of thedissolved cerium can be considered as negligible in our experi-ments. These results corroborate previously published works oncerium dioxide nanoparticles and freshwater microalgae (VanHoecke et al., 2009; Roger et al., 2010; Manier et al., 2011), even ifthe ECx values recorded by the above-mentioned authors were notconsistent from one study to another and certainly dependent tothe different form of nano-CeO2 tested. The observations madeduring the alteration process suggest an agglomeration of theparticles during the exposure period. However, during the first24 h, the non-alterated suspension is expected to be quite stableand differs from the alterated ones in terms of agglomeration state.If we postulate that the non alterated NPs would be more toxicbecause of the presence of very small agglomerates, a major effectwould be recorded toward the algal cells within the first 24 h.Therefore, a lag phase or a higher reduction of the growth inhibi-tion should be recorded within the first 24 h, regarding to the algaeexposed to the non-alterated suspension. However, the resultsobtained after 24 h of exposure to the different suspensionsshowed no differences in terms of growth or number of cells,whatever the concentration tested (Fig. 1S). Our previous work(Manier et al., 2011) on cerium dioxide nanopowder (primaryparticles size ¼ 25 nm) indicated a concentration-effect relation-ship with a mean EC50 value around 14 mg L�1 whatever themethods used to disperse the nanopowder and consequently

whatever the agglomerate size recorded. Taking into considerationthese results and those presented above, it appears obvious that thesecondary particle size (i.e aggregation and/or agglomeration state)of the nano-CeO2 have a few influence on the toxicity observedwhen considering the microalgae growth inhibition. Similar con-siderations were also reported by Kühnel et al. (2009), who showedthat the agglomeration of tungsten carbide nanoparticles does notprevent uptake and toxicity towards a rainbow trout gill cell line. Inour work, this can be explained by the fact that the particles fromthe aged suspensions appeared to be mainly weakly bound, form-ing rather loose agglomerates than aggregates with bonded parti-cles. The surface area and the subsequent potential interaction ofthe particles with the algal cells are influenced by the agglomera-tion and or aggregation state. In the case of aggregates, the activesurface area is reduced by chemical bonds between particles. Theatoms involved in these chemical bonds are not available to interactwith cells or organisms. However, in the case of agglomerates, theparticles are linked in a loose manner, which keeps the particlessurface area available for interactions. Thus, although the ceriumdioxide nanoparticles are not pristine in the alterated suspension,the available surface area and consequently the possible in-teractions with the algal cells are probably similar to the non-agedand less agglomerated particles.

On the contrary, questions remained concerning the importanceof the primary particle size. The calculated EC50 values afterexposure to nano-CeO2 of 10 nm in primary particle size weremorethan 2.5 fold lower compare to the EC50 values recorded with thenano-CeO2 particles of 25 nm in primary particle size and morethan 65 fold lower when compared to the bulk material (primaryparticle size ¼ 5 mm; Fig. 2S). This finding supports the fact that theecotoxicity toward the freshwater algae is related to the primaryparticles size and consequently to the available surface area of theparticles, as already suggested for cerium dioxide nanoparticles(Van Hoecke et al., 2009). The primary size-dependant effects werealso suggested by authors for other nanoparticles as TiO2(Hartmann et al., 2010).

The second aim of this paper was to give some answers con-cerning the interaction between cerium dioxide nanoparticle andthe freshwater microalgae. The flow cytometry analyses showedthat the percentage of more complex cells in the algal populationincreased with the increase of nano-CeO2 concentrations. Changesin cells complexity (granularity) can be explained bymorphologicalmodifications of the cells, possible interactions with nano-CeO2 onthe surface of the cells or a possible internalization of the NPs insidethe cells. In previous studies by Suzuki et al. (2007) and Zucker et al.(2010), the flow cytometry method was used to evaluate the uptakeor to detect titanium NPs in mammalian cells. Zucker et al. (2010)reported that TiO2 nanoparticles can be detected in culturedhuman-derived retinal pigment epithelial cells. They observed anincrease in the cells granularity (SSC) with the increase of TiO2concentration, which was proved to be related to nano-TiO2 inter-nalization in the cells. The use of flow cytometry to assess theinteraction between NPs and cells was also published for bacteria(Salmonella Typhimurium), (Kumar et al., 2011). These authors re-ported an increase of the cell granularity with the increase of ZnOand TiO2 NPs concentrations. These observations were directlyrelated to an uptake of the NPs by the cell, as evident by theadditional TEM studies. Moreover, these authors also showed thatthe smaller agglomerates were uniformly distributed inside thecells (including the dividing cells), while some larger agglomeratedparticles adhered outside the membrane. From our results, thedirect interaction of the NPs with algal cells can be seen as one ofthe possible explanation for the growth inhibition recorded.

The E-SEM observations and X-ray microanalyses (Fig. 5) haveclearly demonstrated that the nano-CeO2 are able to directly

Fig. 5. Representative E-SEM pictures of algae cells taken after 72 h of exposure (a ¼ control, b ¼ 6.25 mg L�1 of the non aged suspension, c ¼ 6.25 mg L�1 of the three days agedsuspension and d ¼ 6.25 mg L�1 of the one month aged suspension). Cerium oxide in association with salt (P, Ca) was indentified in close contact at the surface of the exposed algaeby X-ray microanalyses (b.1, c.1 and d.1). Arrows indicate the location of the X-ray measurement.

N. Manier et al. / Environmental Pollution 180 (2013) 63e7068

N. Manier et al. / Environmental Pollution 180 (2013) 63e70 69

interact with the algal cells surface and in some cases completelyentrap the algal cells, despite there was no evidence of cerium di-oxide nanoparticle internalization. In other hand, such kind of closeinteraction between nano-CeO2 and the algal cells was recordedwhatever the suspension tested (not aged, 3 days or one monthaged suspension), which also suggests that, the agglomerate sizehave a few influence on the interaction between cerium dioxidenanoparticles and the algal cells. Taken into account the reportfrom Kumar et al. (2011) and the presence of a cell wall inP. subcapitata, we also believe that the uptake of cerium dioxide NPsby the cell is limited, especially for the aged suspension with verylarge NPs agglomerates. To date, the internalization of nanoparticleby P. subcapitata has never been reported and the close interactionbetween algal cells and nanoparticles is not consistent in the actualpublished data. TEM observations by Van Hoecke et al. (2009)showed that cerium dioxide aggregates (suspended in OECDalgae growth medium) can cluster around the algal cells. Theseauthors found, however, no uptake and suggested that there is nostrong adsorption to the algal cells. In a previous work, same au-thors did not observe considerable adsorption of two SiO2 nano-particles (LUDOX LS and LUDOX TM40; prepared in OECD algalgrowth medium, pH stabilized with 3-(N-morpholino)propane-sulfonic acid buffer) on the outer surface of the P. subcapitata cells(Van Hoecke et al., 2008). On the contrary, Rodea-Palomares et al.(2011) reported that cerium dioxide nanoparticles were stronglyadsorbed onto the surface of the cyanobacterium Anabaena (strainCPB4337) and onto P. subcapitata cell walls (suspensions preparedin Allen and Arnon modified medium diluted 1/10 and adjusted topH 6 with 2 mM HEPES for Anabaena and in OECD growth mediumat pH 8 for P. subcapitata). These authors did not find any evidenceof cerium dioxide nanoparticles uptake and/or internalization bycells, neither in the cyanobacterium nor in the algal cells. In addi-tion, Hartmann et al. (2010) reported that nano-TiO2 (suspended inISO algae growth medium, similar to the OECD growth medium)can be highly attached to the surface of P. subcapitata cell wall andsimilar observations were published by Aruoja et al. (2009) whoobserved that nano-TiO2 (prepared in OECD growth medium) cancover the algal surfaces to a larger extent than the bulk particles do.This observation highlights the fact that systematic studies ofnanoparticles of different sizes, shapes and coating should be per-formed in order to better understand the mechanisms of action ofthese products.

Focusing on the observations related above, we believe that theclose adhesion of nano-CeO2 on the algal cells may lead to directphysical effects, such as cell membrane disruption, or indirect ef-fect such as the reduction of the available light necessary to thealgal growth (shading effect) or the limitation of the nutrientintake by the algal cells. This suggestion is strengthened by somerecently published works from Roger et al. (2010), Rodea-Palomares et al. (2012) and our additional observations using X-ray microanalyses. Firstly, concerning the cell disruption andoxidative stress induced by cerium dioxide nanoparticles, Rogeret al. (2010) showed an increase of the membrane permeabilityof P. subcapitata cells exposed to nano-CeO2, in a higher extentcompared to the bulk material. The oxidative stress, as one of themechanisms involved for cerium dioxide nanoparticles toxicity,was also pointed out for the bacteria E. coli (Thill et al., 2006) andon cultured human lung epithelial cells (BEAS-2B cells), (Park et al.,2008). In addition, Rodea-Palomares et al. (2012) recently pub-lished that cerium dioxide nanoparticles had a significant impacton the primary photochemical process of the photosystems II ofP. subcapitata, which can be related to ROS generation in the algalcells. Secondly, concerning the nutrient depletion, the X-ray mi-croanalyses revealed that the large amount of cerium dioxidenanoparticles adsorbed on the algal cell was, in each case,

associated with certain constituents of the algal growth medium,especially phosphate and calcium. The cerium dioxide and phos-phate complexation was already mentioned as one of the possibleexplanation of the growth inhibition observed in P. subcapitata.Indeed, Roger et al. (2010) showed concentration-dependantdepletion in phosphate from the algal growth media in presenceof nano-CeO2. Moreover, they indicated that the interaction be-tween nano-CeO2 and phosphate was greater compared to thebulk material, was rapid and occurred at the beginning of theexposure period. Finally, such kind of interaction between nano-particles and growth medium nutrients (especially phosphate andzinc) was also suggested for other nanoparticles such as nano-TiO2(Aruoja et al., 2009).

5. Conclusion

In conclusion, this work has shown that even alterated andhighly agglomerated, a cerium dioxide nanoparticles suspensioncan stay as ecotoxic as a non-alterated suspension, when focusingto the growth inhibition of a freshwater microalgae. The inhibitorymode of action of nano-CeO2 on the growth of these algae may bemainly mediated by a physical effect due to a close adsorption onthe cell surface, which was observed whatever the suspensiontested.We believed that this interaction between algal cells and theparticles is dependent of the primary size of the particles andconsequently of the available surface area. Such close interactioncan then induce direct and non specific physical damages likeabrasive effects, as well as indirect membrane damage due tooxidative stress. The finding of Roger et al. (2010) and the obser-vations made in our study concerning the nano-CeO2 and saltcomplexation at the surface of the cells, also suggest that the algalgrowth inhibition is partially due to the alteration in the transportof nutrients and metabolites across the cell membrane, which canlead to cell disruption. Further works involving other microalgaesuch as algae without cell wall should be conducted to addressthe important question of the interaction between algal cells andthe NPs.

Acknowledgments

This work was financially supported by the French Ministry incharge of Ecology and Sustainable Development, within theframework of the IMPECNANO program (Environmental impact ofnanomaterials e exposure through their life cycle and ecotoxicity).The authors would like to thank the anonymous reviewers, whosecomments at the reviewing stage greatly enhanced the clarity ofthe manuscript.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.envpol.2013.04.040.

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