Chemosphere 87 (2012) 1388–1394

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Chemosphere

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Polymer coating of copper oxide nanoparticles increases nanoparticles uptakeand toxicity in the green alga Chlamydomonas reinhardtii

François Perreault a, Abdallah Oukarroum a, Silvia Pedroso Melegari a,b, William Gerson Matias b,Radovan Popovic a,⇑a Department of Chemistry, University of Quebec in Montreal, Case Postal 8888, Succursale Centre-Ville, Montreal, QC, Canada H3C 3P8b Laboratório de Toxicologia Ambiental, LABTOX – Depto. de Engenharia Sanitária e Ambiental, Universidade Federal de Santa Catarina, Campus Universitário, CEP 88040-970,Florianópolis, SC, Brazil

a r t i c l e i n f o

Article history:Received 28 June 2011Received in revised form 24 January 2012Accepted 19 February 2012Available online 23 March 2012

Keywords:Copper oxide nanoparticleChlamydomonas reinhardtiiNanotoxicologyReactive oxygen speciesPhotosynthesis

0045-6535/$ - see front matter � 2012 Elsevier Ltd. Adoi:10.1016/j.chemosphere.2012.02.046

Abbreviations: CS-CuO, ‘‘Core–Shell’’ polymer-coacles; H2DCFDA, 20 ,70-dichlorodihydrofluorescein diaceICP-AES, inductively coupled plasma atomic emissionticles; P.I., performance index of photosystem II; PSII,oxygen species; TEM, transmission electron microsco⇑ Corresponding author. Tel.: +1 514 987 3000x846

E-mail address: popovic.radovan@uqam.ca (R. Pop

a b s t r a c t

Copper oxide nanoparticles (CuO NPs) are frequently used in a polymer-coated form, to be included inpaints or fabrics for antimicrobial properties. Their application in antifouling paints may lead to the con-tamination of aquatic ecosystems. However, the toxicological risk of NPs in the environment is hard toevaluate due to a lack of knowledge on the mechanisms of NP interaction with biological systems. In thisstudy, we investigated the effect of polymer coating on CuO NP toxicity in the green alga Chlamydomonasreinhardtii by comparing bare and polymer-coated CuO NPs prepared from the same CuO nanopowder.Both CuO NP suspensions were toxic to C. reinhardtii after 6 h treatment to concentrations of 0.005–0.04 g L�1. Bare and polymer-coated CuO NPs induced a decrease of Photosystem II activity and the for-mation of reactive oxygen species. Polymer-coated CuO NP was found to be more toxic than the uncoatedCuO NP. The higher toxicity of CS-CuO NP was mainly associated with the increased capacity of polymer-coated CuO NP to penetrate the cell compared to bare CuO NPs. These results indicates that the hightoxicity of polymer-coated CuO NPs in algal cells results of intracellular interactions between NPs andthe cellular system.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Nanotechnology has been a major field of innovation in the re-cent decade and nanomaterials are quickly becoming part ofnumerous products of industrial, medical and personal uses(Schmid and Riediker, 2008; Peralta-Videa et al., 2011). The wide-spread use of nanomaterials may lead to the contamination ofaquatic ecosystems by nano-sized contaminants (Klaine et al.,2008). However, the ecotoxicological risks of nanomaterials arehard to evaluate because the mechanisms of toxicity of nano-sizedmaterials are still not understood (Dhawan et al., 2009). Toxicolog-ical research has identified several key physicochemical character-istics of nanomaterials that can be related to toxicity. These includesize, shape, composition, aggregation and solubility, especially fornanomaterials composed of toxic metals (Griffit et al., 2008; John-

ll rights reserved.

ted copper oxide nanoparti-tate; HSM, high salt medium;spectroscopy; NPs, nanopar-

photosystem II; ROS, reactivepy.7; fax: +1 514 987 4054.ovic).

ston et al., 2010). For example, toxicity of nano-sized ZnO to thegreen alga Pseudokirchneriella subcapitata was found to be solelydue to particles solubilisation into toxic Zn2+ ions (Franklin et al.,2007). In addition, surface properties, such as total surface area,surface charge, surface reactivity and functionalisation were alsoidentified as major factors in the toxicity of nanomaterials (Vermaand Stellacci, 2010; Zhu et al., 2010). These properties are depen-dent to nanomaterials’ surface composition, which is often modi-fied to favour specific applications (Sperling and Parak, 2010). Thechange of surface properties of nanomaterials can be expected tohave an important role on how they interact with biologicalsystems.

Copper oxide NPs (CuO NPs) are frequently used for their anti-microbial and biocidal properties (Cioffi et al., 2005; Ren et al.,2009). In the aquatic environment, this type of NP may representan important source of contamination due to its application inantifouling paints used on boats and immersed structures. Thesepaints consist of a polymeric film made mostly of acrylic and styre-nic monomers covering CuO NPs (Almeida et al., 2007). Antifoulingpaints were found to be very efficient in preventing the attachmentof algae, shellfish and other organisms to the hull of boats (Yebraet al., 2004). However, decomposition of CuO NP-based paints

F. Perreault et al. / Chemosphere 87 (2012) 1388–1394 1389

results in the release of their components in the aquatic environ-ment (reviewed in Turner, 2010). Recently, it was shown that poly-mer-coated, core–shell (CS) CuO NPs are toxic to aquaticphotosynthetic organism (Perreault et al., 2010; Saison et al.,2010). In these studies, using microalgae and aquatic macrophytes,toxicity of CS-CuO NPs was found to be caused by the nanopartic-ulate fraction. Consequently, release of polymer-coated CuO NPsinto the aquatic environment may have severe consequences onphotosynthetic organisms, the first step of the aquatic trophicchain.

In our previous report, we showed that CS-CuO NPs were moretoxic to algal cells than bare, uncoated CuO NPs. This effect wasinterpreted as the result of aggregation and precipitation of bareCuO NPs in the media, decreasing their capacity to interact with al-gal cells (Saison et al., 2010). However, polymer coating may altertoxicity in many different ways. For the same NP size, differentpolymer coatings were found to have different effects on rootgrowth of Lolium multiflorum (Cheng et al., 2011). Polymer-coatedCdSe nanocrystals were also found to have an increased toxicitydue to higher NP precipitation on the cell surface (Kirchner et al.,2005). Consequently, to understand the mechanisms of toxicityof CS-CuO NPs in algal cells, it is necessary investigate the effectof polymer coating on the interaction between NPs and algal cells.

In this report, we investigated the effect of polymer coating ofCuO NPs on its toxicity to the alga Chlamydomonas reinhardtii. Bareand polymer-coated CuO NPs were characterised to evaluate theeffect of polymer coating on different properties relevant for NPtoxicity. The two NP suspension were then investigated to deter-mine how the presence of polymer coating alters the interactionof NPs with algal cells, their intracellular uptake and the resultingtoxicity in algae.

2. Materials and methods

2.1. Algal cultures

C. reinhardtii (CC-400) was obtained from the ChlamydomonasCenter (Duke University, USA) and cultivated in 1 L batch culturein high salt medium (HSM) as previously described (Saison et al.,2010). When the culture was in the exponential growth phase,samples having a cell density of 1 � 106 cells mL�1 were used fortreatments. Culture cell densities were determined with a Multi-sizer Z3 (Beckman Coulter Inc., USA).

2.2. NP synthesis

CuO nanopowder was obtained from MTI Corporation (USA)and used for the preparation of both bare CuO and polymer-coatedCuO NP suspensions. Raw nanopowder has a particle diameter of30–40 nm and purity of over 99%, according to the manufacturer.Bare CuO NP stock suspension was prepared in HSM and sonicatedbefore use during 3 min with a sonicator equipped with a microtip(VibraCell 400 W, Sonics & Materials Inc., USA). Polymer-coated(poly(styrene-co-butyl acrylate)) CS-CuO NP suspension was pre-pared by the protocol previously described (Daigle and Claverie,2008). Thermogravimetric analysis of CS-CuO NPs indicated aweight composition of 67% core (CuO) and 33% shell (polymer).Transmission electronic microscopy (TEM) of CS-CuO NPs showeda shell thickness of 14 nm for an average particle size of 81 nm. Fordetailed characterisation data, see Daigle and Claverie (2008).

2.3. NP treatment

Algal cells were exposed to bare and CS-CuO NPs based on thesame amount of CuO. Algae were exposed 6 h to 0.0004, 0.004,

0.01, 0.02 and 0.04 g L�1 of CuO NPs in a volume of 50 mL in HSMmedia. Illumination and temperature conditions were the same asused for algal culture but it should be noted that NP treatmentshad a higher turbidity due to NP density, which reduced the lightintensity passing through the culture by 40% at 0.4 g L�1, as mea-sured with a Li-250 light metre (Li-COR Inc., USA). Such light atten-uation had no effect on the measurement of the toxicity indicatorsused in this study (see Sections 2.5 and 2.6) due to the short expo-sure time used (6 h). Toxicity results were evaluated on the basis ofmass concentrations (g L�1) and total surface area (cm2 L�1).

For treatment of algal cells to the soluble Cu fraction, CuO andCS-CuO NP suspensions were prepared in the same way as for treat-ments and, after 6 h, the nanoparticulate fraction was removed bycentrifugation (Daigle and Claverie, 2008; Manusadzianas et al.,2012). NP suspensions were centrifuged (12,000g, 30 min) andthe supernatant was collected and used for algal exposure.

2.4. Characterisation of the NP suspensions in the culture media

Characterisation of the NP suspensions was done in the HSMmedia for a [CuO] of 0.04 g L�1 (see discussion). Particle distributionwas determined by dynamic light scattering with a ZetaPlus parti-cle sizer (Brookhaven Instruments Corporation, USA). Zeta potentialof NPs in the HSM media was determined by the electrophoreticmobility method with the ZetaPlus system. Soluble Cu fractionwas determined by Inductively Coupled Plasma – Atomic EmissionSpectroscopy (ICP–AES) analysis of the HSM media where the par-ticle fraction was removed by centrifugation as described in Section2.3. The supernatant was collected, acidified and further filtered(0.22 lm) before ICP–AES to remove any remaining particulatematter (e.g. polymer residue found in the CS-CuO NPs’ superna-tant). Photocatalytic capacity was measured by the photodegrada-tion of methylene blue. NP suspensions in HSM (0.04 g L�1) withmethylene blue (10 mg L�1) were exposed to the same conditionsdescribed for algal culture and degradation of methylene bluewas determined after 6 h by measuring the absorbance at 665 nm(Song and Zhang, 2010).

2.5. Intracellular formation of ROS

The intracellular ROS level was determined using the fluores-cent dye 20,70-dichlorodihydrofluorescein diacetate (H2DCFDA)(Invitrogen Molecular Probe, USA). H2DCFDA stock solution(10 mM) was prepared in ethanol in the dark. H2DCFDA is anon-polar compound and diffuses into algal cells where it istransformed by intracellular esterase and H2O2 into the polar, fluo-rescent 20,70-dichlorofluorescein which has a maximum emissionbetween 517 and 527 nm. After 6 h treatment, 1 mL samples of al-gal cultures were exposed 15 min to 0.2 mM H2DCFDA in the dark.Intracellular ROS level was determined by measuring the fluores-cence emission at 530 nm with a flow cytometer (FacScan; BectonDickinson Instruments, USA). Cytometry results were analysedusing the WinMDI 2.8 software. Algal cells were separated fromnoncellular particles by using a relationship between particle sizeand red fluorescence level, originating from chlorophyll (Chl)fluorescence reemission.

2.6. Chl a fluorescence measurements

Photosystem II (PSII) electron transport was evaluated by mea-suring the Chl a fluorescence rise. After 6 h treatment, total Chl con-tent (a + b) was determined spectrophotometrically, according toWellburn (1994). Before fluorescence measurements, samples weredark adapted for 30 min. Under a weak green light, an aliquot (5 mgof Chl) was placed on a 13 mm glass fibre filter (Millipore, USA)using low pressure filtration to obtain a humidified uniform layer

Fig. 1. Bare and polymer coated (CS-CuO) NPs size distribution in HSM media, for a[CuO] of 0.04 g L�1.

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of algal cells. Rapid, polyphasic rise of Chl a fluorescence was mea-sured with a Plant Efficiency Analyser fluorimeter (Handy-PEA,Hansatech Ltd., UK) using a 6 s saturating flash (3500 lmol pho-tons m�2 s�1). The fluorescence intensity at 50 ls was consideredas the O value (F50ls), fluorescence intensities for J and I transientswere determined at 2 (F2ms) and 30 ms (F30ms), respectively, andmaximum fluorescence yield P was considered as the maximal va-lue of fluorescence intensity (FM) under saturating illumination(Strasser et al., 2004). PSII performance index was calculated asP.I. = RC/ABS � ((FM–F50ls)/F50ls) � ((1 � VJ)/VJ). In this equation,RC/ABS is an indicator of the number of active PSII reaction centersper Chl unit and is calculated as RC/ABS = [(F2ms–F50ls)/4 � (F300ls–F50ls)] � (FM–F50ls)/FM. VJ is the relative value of fluorescence at Jtransient, indicating the accumulation of reduced PSII primary elec-tron acceptor, QA, and is determined as the ratio VJ = (F2ms–F50ls)/(FM–F50ls) (Strasser et al., 2004).

2.7. Light microscopy

To determine morphological changes in the algal culture, a dropof algal culture was placed on a glass microscope slide andobserved using a Nikon Eclipse TS100 microscope. Pictures weretaken with a Pixelink camera at 40� magnification. Brightnessand contrast in the pictures were modified with the Image J soft-ware (NIH Image, USA) for better clarity.

2.8. TEM analysis of intracellular CuO NPs

After 6 h treatment, 50 mL of control and NPs-treated cultures([CuO] = 0.04 g L�1) were centrifuged and the supernatant dis-carded. Without disturbing the pellet, cells were washed with500 lL of washing buffer (0.1 M cacodylate, 0.1% CaCl2, pH 7.2)for 10 s. The washing buffer was removed and 500 lL of fixationbuffer (2.5% glutaraldehyde in 0.1 M Wash buffer) was added. Cellswere fixed overnight at 4 �C. After glutaraldehyde fixation, the pel-let was washed three times 10 min with the washing buffer, andstained with osmium tetroxide (1% OsO4, 1.5% KFeCN in water)during 2 h at 4 �C. Then, the pellet was washed with nanopurewater (3 � 10 min), dehydrated with increasing concentrations ofacetone (30%, 50%, 70%, 80%, 90% and 100% acetone, 3 � 10 mineach). Dehydrated samples were infiltrated with epon at roomtemperature with increasing concentrations of epon in acetone:1:1 (overnight), 2:1 (4 h), 3:1 (overnight). Then, the pellet wasplaced in pure epon, left 4 h under vacuum and then heated 48 hat 58 �C. Hardened pellets were cut into 0.6 lm thick slices andplaced on a gold grid for TEM analysis. No secondary fixationwas used to obtain a better visualisation of NPs inside the cell.Samples were visualised with a FEI Tecnai 12 120 kV microscopeand pictures taken with a Gatan 792 Bioscan 1 � 1 k Wide AngleMultiscan CCD camera. The elemental nature of the metallic aggre-gates found inside the cell was determined by energy-dispersiveX-ray spectroscopy of the same epon-fixed samples using a PhilipsCM200 200 kV TEM equipped with a EDAX Genesis EDS system.

2.9. Intracellular copper concentration

Algae were separated from NPs using a modified density equilib-rium method (Eroglu and Melis, 2008). Sucrose solutions (20%, 40%,60%, 80%, 100% and 120%) were prepared in the HSM media used foralgal culture. Sucrose gradient was done in a Beckman centrifuga-tion tube inclined at a 30� angle. Beginning with the highest sucroseconcentration, 3 mL of each sucrose solution were slowly placed inthe tube, resulting in 6 layers of different density. After 6 h treat-ment, 50 mL of control and NP-treated cultures ([CuO] = 0.04 g L�1)was centrifuged and the pellet slowly placed on top of the sucrosegradient. The tubes were centrifuged at 1000 rpm during 20 min in

a swinging-bucket 5810R centrifuge (Eppendorf, Canada), resultingin a clear separation between algal cells and CuO NPs (whichformed a pellet at the bottom of the tube). The algal layer was recu-perated with a glass Pasteur pipette and filtered on a 0.45 lM filterpreviously dried and weighted. To remove Cu weakly bound to thecell surface or the filter, 3 � 10 mL of 10 mM ethylenediaminetetra-acetic acid in HSM medium was slowly passed through the filter.Filters were dried at 95 �C for 24 h, weighted to calculate algaldry weight and then placed in acid-washed glass tubes in which4 mL HNO3 and 500 lL H2O2 was added. The samples digested48 h at room temperature before being diluted to 20% HNO3 innanopure water for ICP-AES quantification of Cu. Cu concentrationswere normalised to dry weight.

2.10. Data analysis and statistics

All experiments were done in four replicates. Means and stan-dard deviations were estimated for each treatment. Significant dif-ferences between treatments were determined using a randompermutation test. All statistical analysis were done with the R soft-ware (v2.13.0, R Core group, 2011) and p values less than 0.05 wereconsidered as significant.

3. Results

3.1. Characterisation of the NP suspensions in the media

The effect of polymer coating on size distribution, solubility,charge and photocatalytic capacity of the NP suspensions wasinvestigated in the HSM media used for treatments. Size distribu-tion was determined by dynamic light scattering of the NP suspen-sion at the highest CuO concentration tested (0.04 g L�1). Both NPsuspensions were prepared from the same nanopowder, whichhad an average size of 30–40 nm (data not shown). However, whenbare CuO NPs were suspended in HSM media, we found a particlesize distribution centred around 148 nm, indicating the formationof aggregates (Fig. 1). When NPs were coated with polymer, parti-cle size was found to be centred around 65.4 nm, with most of theNPs below 100 nm. Therefore, CS-CuO NPs were more stable in theHSM media than bare CuO NPs, resulting in a smaller particle size.

Polymer coating of CuO NPs was also found to alter NP solubil-ity. After removal of the particulate fraction by centrifugation, sol-uble Cu concentration was found to be 70% higher in the bare CuONP media (1.55 ± 0.08 mg L�1) than in the CS-CuO NP media(0.9 ± 0.04 mg L�1), for a total CuO concentration of 0.04 g L�1

F. Perreault et al. / Chemosphere 87 (2012) 1388–1394 1391

(p < 0.05). This soluble Cu fraction constitued a small percentage(2.8% for CS-CuO NPs and 4.8% for bare CuO NPs) of the total Cucontent of the treatment.

Photocatalytic capacity, measured by the light-stimulated deg-radation of methylene blue by CuO NPs, was found to be low forboth NP. After a 6 h treatment to 0.04 g L�1, under the same condi-tions used for treatments, methylene blue photodegradation wasof 10.7 ± 4.1 and 9.4 ± 4.1% of control sample, for bare and CS-CuO NPs, respectively. Photocatalytic degradation of methyleneblue was not significantly different between bare CuO and CS-CuO NPs (p > 0.05). Similarly, surface charge, measured as the zetapotential by the electrophoretic mobility method, was not alteredby the presence of the polymer coating on the CuO NPs. In theHSM media, for a [CuO] of 0.04 g L�1, zeta potential was found tobe �51.1 ± 5.1 and �58.9 ± 3.1 mV, for the bare and CS-CuO NPsuspensions, respectively (p > 0.05).

3.2. NPs toxicity in C. reinhardtii

Toxicity of bare and CS-CuO NPs was investigated using twoindicators of toxicity: ROS formation and inhibition of PSII activity.These indicators were previously shown to be sensitive to NPs ef-fects in algae (Wang et al., 2008; Petit et al., in press). Toxicity ofbare and CS-CuO NPs was compared on the basis of the same CuOconcentration. After 6 h treatment, CS-CuO NPs were found to in-duce the formation of ROS in C. reinhardtii cells. ROS formationwas significant at a concentration of only 0.004 g L�1 CS-CuO NPs(p < 0.05) and reached as high as 392 ± 12% of control for0.04 g L�1 (Fig. 2A). Concomitantly, PSII electron transport, evalu-ated by the P.I., was decreased by CuO NP treatments. After 6 htreatment to 0.04 g L�1 of CS-CuO NPs, P.I. was decreased to13 ± 12% compared to control (p < 0.05) (Fig. 2A). Such decreaseindicates that CS-CuO NP reduced PSII capacity to convert light into

Fig. 2. Formation of ROS (-) and change in PSII performance index (P.I., - - -) in C.reinhardtii cells exposed 6 h to bare CuO NPs (d) and polymer-coated CS-CuO NPs(h). NPs effect is compared on the basis of mass concentration (A) and total surfacearea (B).

photosynthetic electron transport. However, when C. reinhardtiiwas exposed to the same mass concentrations of bare CuO NPs, tox-icity was found to be lower using both ROS formation and PSII elec-tron transport. After 6 h treatment to 0.04 g L�1 of bare CuO NPs,ROS formation was increased to 160 ± 15% compared to control,while P.I. was decreased to 78 ± 20% compared to control(Fig. 2A). Bare and polymer-coated CS-CuO NPs were also comparedon the basis of total surface area, which was previously found to bea more useful parameter for the comparison of nanomaterials’ tox-icity (Oberdörster et al., 2005). When evaluated on a total NP sur-face area per L, CS-CuO NPs still induced higher toxicity than bareCuO NPs for the same surface area (Fig. 2B).

3.3. Interaction of NPs with algal cells

The interaction of CuO NPs with algal cells was investigated byvisual changes in the cell culture morphology using light micros-copy. When algae were exposed to either bare CuO or CS-CuONPs, large aggregates composed of algal cells and CuO NPs couldbe observed (Fig. 3). Aggregation was found to be more importantin the cell culture exposed to CS-CuO NPs, which had more aggre-gates of a larger size than the cell culture exposed to bare CuO NPs(Fig. 3).

Penetration of CuO NPs inside algal cells was determined byTEM microscopy of epon-embedded cells after 6 h treatment.TEM micrographs revealed the ultrastructure of the cell, with thelarge single chloroplast occupying most the cell volume (Fig. 4).In NP-treated cells, large metallic aggregates can be observed.The aggregates were several hundreds of nm in size and localisedonly in the cytoplasm of the cell. It should be noted that the ab-sence of a secondary fixation did not allowed for the identificationof specific cytosolic organelle; however the metallic aggregates ap-pear to be enclosed in a membrane structures that can be hypothe-tized to be the vacuoles (Fig. 4, insert). To confirm that theseaggregates were CuO NPs, energy dispersive X-ray spectroscopywas used. The main elements composing the aggregates werefound to be Cu and O, confirming the CuO NP accumulation insidethe cell (Fig. 5). The presence of X-ray peaks representing C, P, Cland Ca can be attributed to biological components of the cell orthe epon resin used for sample embedding (Cl), and the Au peaksto the gold grid holding the sample. These results confirms thatboth types of CuO NPs were able to cross the biological membranesand accumulate in the cell.

Accumulation of CuO NPs inside the cell was analysed quantita-tively by the measure of total Cu content in algal cells. When C.reinhardtii was exposed to either bare or CS-CuO NPs, an importantuptake of Cu was found. Cu uptake in the presence of NPs was sig-nificantly more important than when algae were exposed to onlythe soluble Cu fraction of both NPs solution, which further con-firms that CuO penetrated the cells in a particulate form (Fig. 6).When total intracellular Cu content is compared between culturesexposed to bare and CS-CuO NPs, it appears evident that uptake ismore important for CS-CuO NPs than for bare CuO NPs. Total Cucontent in CS-CuO NP-treated cells was 6.5 times higher than inbare CuO NP-treated cells.

4. Discussion

Several mechanisms are thought to participate in the toxicinteractions of metal NPs with cellular systems. Solubilisation ofNPs into free metal ions is usually considered as one of the mostcommon mechanisms of toxicity for several types of NP (Franklinet al., 2007; Aruoja et al., 2009). However, some effects can alsobe attributed to specific properties of nano-sized particles, suchas the capacity of NPs to cross biological membrane (Miao et al.,

Fig. 3. Aggregation of C. reinhardtii cell culture for control or after 6 h of treatment to 0.04 g L�1 of bare CuO NPs or polymer-coated CuO NPs.

Fig. 4. TEM images of C. reinhardtii cells for control and after 6 h of treatment to 0.04 g L�1 of bare CuO NPs or polymer-coated CuO NPs. Cellular structures are identified asthe pyrenoid (Pyr), the chloroplast (C), starch granules (S) and CuO NPs aggregates (NP). Insert (right picture): enlarged view of a NP aggregate.

Fig. 5. Energy-dispersive X-ray spectroscopy of metallic aggregates found in thecytoplasm of CuO NPs-treated C. reinhardtii cells.

Fig. 6. Intracellular total copper concentration in C. reinhardtii cells for control andafter 6 h of treatment to 0.04 g L�1 of bare CuO NPs or polymer-coated CuO NPs.Letters (a, b, c, d) indicate results statisticaly different from each other.

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2011) or to adsorb on the cell surface (Zeyons et al., 2009). Aggre-gation of algal culture appears to be a common effect of NP-algainteractions, as it was observed for CuO (this study), NiO, CeO2

and gold-glycodendrimers NPs (Gong et al., 2011; Rodea-Palo-mares et al., 2011; Perreault et al., 2012). Some nanomaterialsare also able to generate ROS via photocatalytic processes (Hund-Rinke and Simon, 2006). Toxicological evaluation of metal NP tox-icity must consider all these factors to determine the mechanism ofNP interactions with cellular systems.

In our study, exposure of C. reinhardtii to CS-CuO NPs resulted ina stronger formation of ROS and greater inhibition of PSII activitythan when algae were exposed to bare CuO NPs. Exposure of algaeto CS-CuO NPs also resulted in a more aggregated state of cells andhigher intracellular uptake of NPs. The specific mechanism of CuO

NPs toxicity is still a matter of investigation, although previousstudies suggested a primary role of oxidative stress for the toxicityof different NPs (Xia et al., 2006; Saison et al., 2010). In addition,oxidative stress induced by quantum dots and TiO2 NPs in C. rein-hardtii was not found to be associated with PSII photoinhibitionprocesses (Wang et al., 2008). In our study, the higher inductionof ROS compared to PSII inhibition, as well as the absence of NPin the chloroplast, support these conclusions, indicating that ROSformation may be the primary mechanism for CS-CuO NPs in algae.

In our study, release of Cu ions from CuO NPs was found forboth coated and bare NPs, although CS-CuO NPs solubilisation

F. Perreault et al. / Chemosphere 87 (2012) 1388–1394 1393

was lower. Soluble Cu fraction represented between 2% and 5% oftotal Cu content of the treatments. Solubilisation of CuO NPs isdependent on the media and may range from no detectable releaseto 25% of total Cu (Aruoja et al., 2009; Buffet et al., 2011; Shi et al.,2011). In our conditions, CuO NPs solubilisation can therefore beconsidered as low. On the other hand, cellular uptake of CuO NPswas high, particularly for CS-CuO NPs. Higher uptake of CS-CuONPs by algal cells can be associated with their higher toxicity, sinceCu release and photocatalytic activity were not significantly higherfor CS-CuO NPs compared to bare CuO NPs. This may indicate that,in algae, CuO NP toxicity is due to intracellular interactions of NPs.Such effects may take place either via direct interaction of NPs withcellular components or via indirect effects due to intracellular sol-ubilisation, also called Trojan Horse effect (Studer et al., 2010). Thisconclusion is in agreement with other findings indicating thatintracellular uptake is an important part of silver NPs toxicity in al-gae (Miao et al., 2011). Similar conclusions were also found in theduckweed Landoltia punctata, where higher uptake rate of CuO NPswas the major cause for the higher toxicity of CuO NPs comparedto bulk CuO particles (Shi et al., 2011). In our study, the higher up-take of polymer-coated CS-CuO NPs may be explained by the smal-ler size of CS-CuO NPs compared to the larger bare CuO NPsaggregates. Higher cellular uptake of smaller particles or aggre-gates was also observed in vitro using the human lung epithelialcell line A549 (Andersson et al., 2011). However, it is also possiblethat some interactions between the polymer coating and the cellsurface may facilitate the penetration of CS-CuO NPs inside algalcells.

In conclusion, we found that the main effect of the polymercoating on CuO NP suspension was to decrease NPs aggregation,resulting in a lower aggregate size. We showed that CS-CuO NPswere more toxic to C. reinhardtii than bare CuO NPs at the samemassic concentration or total surface area unit. The higher toxicityof CS-CuO NPs was shown to be mainly caused by an higher uptakeof CS-CuO NPs, which accumulated as large aggregates insidemembrane structures in the cytosol. The results of this study indi-cate that, in algae, CuO NPs toxicity may be caused by intracellularinteractions between NPs and the cellular system.

Acknowledgements

This work was supported by research grants awarded to R.Popovic by the Natural Sciences and Engineering Research Councilof Canada (NSERC, Canada). F. Perreault is supported by a NSERCPh.D fellowship. S.P. Melegari was supported by a Coordenaçãode Aperfeiçoamento de Pessoal de Nível Superior (Brasil) post-doc-toral fellowship. The authors thank Pr. Jérôme Claverie (UQAM) forproviding the CS-CuO NPs.

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