Environmental impact of sunscreen nanomaterials: Ecotoxicity and genotoxicity of altered TiO2 nanocomposites on Vicia faba

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Environmental Pollution 159 (2011) 2515e2522

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Environmental Pollution

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Environmental impact of sunscreen nanomaterials: Ecotoxicityand genotoxicity of altered TiO2 nanocomposites on Vicia faba

Anne-Sophie Foltête a,c,*, Jean-François Masfaraud a,c, Emilie Bigorgne a,c, Johanne Nahmani a,c,Perrine Chaurand b,c, Céline Botta b,c, Jérôme Labille b,c, Jérôme Rose b,c, Jean-François Férard a,c,Sylvie Cotelle a,c

a Laboratoire des Interactions Ecotoxicologie, Biodiversité, Ecosystèmes (LIEBE), Université Paul Verlaine-Metz, CNRS UMR 7146, Campus Bridoux,Avenue du Général Delestraint, 57070 Metz, FrancebCentre Européen de Recherches et d’Enseignement des Géosciences de l’Environnement (CEREGE), UMR 6635 CNRS/Aix-Marseille Université,Europôle de l’Arbois, 13545 Aix-en-Provence, Francec iCEINT, International Consortium for the Environmental Implications of Nanotechnology, F-13545 Aix-en-Provence Cedex 04, France1

a r t i c l e i n f o

Article history:Received 8 March 2011Received in revised form12 June 2011Accepted 15 June 2011

Keywords:RutileNanoparticlesAlterationTitanium dioxideRisk assessment

* Corresponding author.E-mail addresses: [email protected] (A.-S. Foltê

Masfaraud), [email protected] (E. Buniv-metz.fr (J. Nahmani), [email protected] (P. Cha(C. Botta), [email protected] (J. Labille), [email protected](J.-F. Férard), [email protected] (S. Cotelle).

1 http://www.i-ceint.org.

0269-7491/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.envpol.2011.06.020

a b s t r a c t

Mineral sunscreen nanocomposites, based on a nano-TiO2 core, coated with aluminium hydroxide anddimethicone films, were submitted to an artificial ageing process. The resulting Altered TiO2 Nano-composites (ATN) were then tested in the liquid phase on the plant model Vicia faba, which was exposed48 h to three nominal concentrations: 5, 25 and 50 mg ATN/L. Plant growth, photosystem II maximumquantum yield, genotoxicity (micronucleus test) and phytochelatins levels showed no change comparedto controls. Oxidative stress biomarkers remained unchanged in shoots while in roots, glutathionereductase activity decreased at 50 mg ATN/L and ascorbate peroxidase activity decreased for 5 and25 mg ATN/L. Nevertheless, despite the weak response of biological endpoints, ICP-MS measurementsrevealed high Ti and Al concentrations in roots, and X-ray fluorescence micro-spectroscopy revealedtitanium internalization in superficial root tissues. Eventual long-term effects on plants may occur.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

With the fastly growing number of commercial products incor-porating or made of manufactured nanoparticles (MNPs), thedispersion of MNPs in the environment is not science fiction butalready occurring, causing ethical, sociological (Jacobs et al., 2010)and potentially environmental problems, even if those are difficultto assess (Aschberger et al., 2011; Baun et al., 2008; Farré et al., 2011;Navarro et al., 2008; Nowack, 2009; Nowack and Bucheli, 2007).Measurements from Kaegi et al. (2008) represent the first directevidence of the release of synthetic nanoparticles from MNPsapplications into the environment. They showed the leaching oftitanium dioxide (TiO2) nanoparticles from house facades treatedwith paint containing nano-TiO2 under the influence of atmospheric

te), [email protected] (J.-F.igorgne), Johanne.nahmani@urand), [email protected](J. Rose), [email protected]

All rights reserved.

conditions. A model of the behaviour and expected concentrationsof nano-TiO2 in the different compartments of the environment isproposed by Mueller and Nowack (2008) and by Gottschalk et al.(2010), reviewed by Menard et al. (2011).

TiO2 nanoparticles are more and more included in sunscreencreams for their good UV filter properties (AFSSET, 2010). Like othercosmetic products, sunscreen components are likely to end up inbathing waters, wastewaters and waste disposals, and finally intothe whole environment. During sewage treatment, TiO2 nano-particles seem to show a close affinity for activated sludge, andconsequently, the greater part of these nanomaterials are expectedto end up in soils through sewage sludge application (Johnson et al.,2011; Kiser et al., 2009).

The photocatalytic properties of TiO2 nanoparticles are reallyproblematic once these particles released into the environment.Indeed, they are powerful reactive oxygen species (ROS) producerswhen exposed to ultraviolet radiations (Reijnders, 2008). In plants,bare TiO2 nanoparticles can show either positive or negative effects(Brar et al., 2010; Ma et al., 2010). In sunscreens, nano-TiO2 particlesare surface modified to inhibit ROS generation (Reijnders, 2006;Wakefield et al., 2004) and prevent toxic effects, but the stabilityof the protective layers and the effects of such altered structures on

A.-S. Foltête et al. / Environmental Pollution 159 (2011) 2515e25222516

ecosystems remain largely unknown. The possibility of the regainof photocatalytic properties by sunscreen nano-TiO2 after use wasproved by Barker and Branch (2008), who noticed an acceleratedweathering (100-fold) of the coating of prepainted steel sheets bynano-TiO2-containing sunscreens used by workers during handlingof the materials.

Auffan et al. (2010) and Labille et al. (2010) have artificiallymimicked the natural expected alteration of T-Lite SF (BASF),a nanocomposite entering in sunscreen formula. T-Lite are based ona nano-TiO2 core coated with aluminium hydroxide Al(OH)3 andpolydimethylsiloxane (PDMS) films. In the continuity of thesestudies, we investigated the biological effects of their Altered TiO2Nanocomposites (ATN) on a plant model, the broad bean Vicia faba.This model was chosen for its well-known sensitivity to pollutants.Indeed it has been used by numerous authors to assess the eco-toxicity, and particularly the genotoxicity of substances, radiations,or liquid or solid polluted matrix (Cotelle et al., 1999; Manier et al.,2009; Minouflet et al., 2005; Radetski et al., 2004; Ünyayar et al.,2006).

After exposing V. faba to ATN suspensions, ecotoxicological andgenotoxicological endpointswere assessed. Titaniumandaluminiumconcentrations were determined in roots, and titanium internaliza-tion was studied by X-ray fluorescence micro-spectroscopy.

2. Material and methods

2.1. Nanocomposites artificial ageing

Mineral sunscreen nanocomposites, based on a TiO2 core (10 nm � 50 nm),coated with aluminium hydroxide and polydimethylsiloxane layers, were submittedto an alteration process detailed by Labille et al. (2010). Briefly, 100 mg nano-composite powder (provided by the manufacturer) were introduced in 250 mLultrapure water and kept agitated (690 rpm) under a white light (400 W Philips�

Master HPI-T Plus) to mimic solar spectrum during 48 h. The pH was not buffered,the volume was constant. After alteration, the resulting mixture was rested for 48 hand the supernatant of stable altered TiO2 nanocomposites (ATN) was used for thetests.

2.2. ATN dilution and size characterization

ATN suspension (120 mg/L) was diluted in a water mix (50% Evian� Frenchmineral water: 50% ultrapure water, v:v) to the following concentrations: 5, 25 and50 mg ATN/L. Evian� is a weakly mineralized water. The predominant ions are Ca2þ

and HCO�3 . The 50% dilution provided a mix with low ionic strength (4.8 mM)

minimizing particle aggregation (Petosa et al., 2010), but sufficiently mineralized toensure an acceptable plant physiological state. At the beginning of the experiment,the pH was adjusted to 6.5 with HNO3. The mediumwas not buffered because of therapid ATN aggregation in every tested buffer.

Laser diffraction was used to measure ATN size distribution in the mixture ofultrapure and mineral waters (50:50, v:v) at 25 mg/L during the first 5 h. Theapparatus used was a Malvern Mastersizer S (Malvern Instruments, Malvern, UK)displaying a measuring range from 50 nm to 900 mm. Two separate particle groupswere specifically studied: particles below 700 nm, considered as stable colloidalsuspension, and particles over 10 mm, considered as aggregates undergoing rapidsettling.

Turbidity measurements were used to determine the limit conditions forcolloidal stability of the ATN dispersion as a function of its concentration. Thesuspension was diluted from 5 to 50 mg/L in the mixture of ultrapure and mineralwaters (50:50, v:v), and left 24 h in the dark to allow settling of particles. Thesupernatant turbidity was then measured using a Hach 2100 AN turbidimeter.

2.3. Plant germination and treatment

Commercial dry seeds of V. faba (var. Aguadulce, Fabre�) were used for theexperiments. In accordance with the French standard NF T90-327 (AFNOR, 2004),seeds were soaked in deionized water for 24 h, allowed to germinate between twolayers of wet cotton at 22 �C for 3 days. After removing the primary root tips, theseedlings were suspended in aerated Hoagland’s solution at 22 �C for 4 days topromote the development of secondary roots.

Each V. faba plantlet was placed in an individual pot (Wheaton� glass) con-taining 100 mL of the diluted suspension. During exposure, plant roots and ATNsuspension were protected from light while leaves were placed under plant growthlights (125 W, Envirolite�, 6400 K). Four individuals were used per concentration,randomly disposed to balance a potentially unequal light distribution. Exposure

lasted 48 h without renewing exposure medium. The pots were briefly agitated 2times a day to resuspend the particles. Experiments were conducted in a glove box,at 20 �C � 1.

The dilution medium was used as a negative control. Maleic hydrazide 10 mM(MH; CAS 123-33-1) was used as a positive control for genotoxicity. CdCl2 0.1 mM(CAS 10108-64-2) was used as a positive control for root elongation and as a refer-ence metal for thiol levels in tissues and oxidative stress biomarkers (Béraud et al.,2007; Lin et al., 2007).

2.4. Plant harvesting

After 48 h of exposure, secondary roots were carefully rinsed twice withultrapure water and elongation of three secondary roots per plant was assessed bythe difference between their length before and after exposure.

For micronucleus test, In accordance with the French standard NF T90-327(AFNOR, 2004), root tips were excised and fixed overnight in Carnoy solution (25%glacial acetic acid: 75% ethanol, v:v) at þ4 �C and then stored in 70% ethanolat þ4 �C.

Photosystem II maximum quantum yield was assessed in leaves through Fv/Fmratio using a portable Handy PEA (Handy-Plant Efficiency Analyser, HansatechInstruments, Norfolk, UK). Fv/Fm ratio is commonly used as an indicator of overallplant physiological status.

Fresh weight of shoots was determined. Shoots and roots were divided into 2groups: the first group was frozen in liquid nitrogen and stored at �80 �C forglutathione, phytochelatins and oxidative stress biomarker measurements, thesecond group was frozen at �20 �C for Ti and Al determinations.

2.5. Metal analyses

Al and Ti concentrations in roots were determined. After drying at 105 �C andmineralization in HFeHClO4, metal concentrations in tissues were measured by ICP-MS.

2.6. Element location in plants

In order to localize the metal (Ti) in the plant tissues, X-Ray Fluorescence micro-spectroscopy (m-XRF) analysis was performed on secondary roots exposed to50 mg ATN/L, fixed in Carnoy solution and kept at þ4 �C in 70% ethanol. To distin-guish between Ti trapped in the mucilage from Ti included in the root tissues, theremainingmucilagewas removed from certain roots following the protocol of Brams(1969) after slight adaptation. Roots conserved in 70% ethanol were rinsed 10 min inultrapure water, and then immersed in 1 M NH4Cl (pH 4.15) for 10 min. After that,they were rinsed 2�10min in ultrapurewater before being put back in 70% ethanol.

Resulting “withmucilage” and “withoutmucilage” roots were then embedded inresin and cross sections were obtained with a diamond wire saw. m-XRF measure-ments were carried out at the lab-scale on a microscope (XGT7000, Horiba JobinYvon) equipped with an X-ray guide tube producing a finely focused beam witha 10 mm spot size (Rh X-ray tube, accelerating voltage of 30 kV, current of 1 mA).Major elements (P, S, and Ca) were also detected by m-XRF and images of their spatialdistribution were obtained. Aluminium mapping was not possible because of thelow m-XRF sensitivity for light element detection.

2.7. Biochemical analyses

Activities of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6),guaiacol peroxidase (GPX, EC 1.11.1.7), ascorbate peroxidase (APX, EC 1.11.1.11) andglutathione reductase (GR, EC 1.6.4.2) were measured according to Qiu-Fang et al.(2005) for SOD, to Chaoui et al. (1997) for CAT, to Roy et al. (1996) for GPX, and toGarcía-Limones et al. (2002) for APX and GR, each of them modified by Dazy et al.(2008).

Lipid peroxidation was evaluated by the thiobarbituric assay of Aravind andPrasad (2003). Briefly, 900 mL of a 0.5% TBA in 20% TCA (w/v) solution were addedto 100 mL of plant extract. The mixture was incubated at 95 �C for 30 min and thereaction was stopped by transferring tubes on ice. The non-specific absorbance at600 nm was subtracted from the 532 nm absorbance and 3 ¼ 155 mM�1 cm�1 wasused for the calculation of malondialdehyde (MDA) level.

Phytochelatins (PC) 2, 3, 4, 5 and glutathione contents in plant extracts weremeasured by HPLC according to the derivatization method of Courbot et al. (2004).PC standardswere commercially provided (Anaspec, San Jose, USA). Protein contentswere determined according to Bradford (1976) using bovine serum albumin asa standard.

2.8. Micronucleus frequency

As described in the French standard NF T90-327(AFNOR, 2004), the excised roottips were hydrolyzed in 1 N HCl at 60 �C for 6 min, squashed between a slide and itscoverslip and stained with 1% aceto-orcein solution. Micronucleus frequencies werescored from 1000 cells per root tip under a 400� microscope magnification. Twoslides were prepared for each of the 4 replicates and a total of 8000 mitotic cells

y = 226.37x + 3.9752R2 = 0.9978

y = 873.55x + 14.313R2 = 0.9657

0

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0 0.1 0.2 0.3 0.4 0.5 0.6Ti or Al in solution (mmol/L)

Ti o

r A

l in

ro

ots (m

mo

l/kg

d

.w

.)

TiAl

*

*

*

*

*

Fig. 2. Ti or Al concentrations in V. faba secondary roots after 48 h exposure withrespect to their concentrations in exposure suspensions. *Statistically significantlydifferent from corresponding controls after logarithmic transformation, Dunnett’st-test, p < 0.05.

Ti/Al ratio in mother solution: [Ti] = 8.5682 [Al]

120

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A.-S. Foltête et al. / Environmental Pollution 159 (2011) 2515e2522 2517

from 8 separate slides per experimental group were observed under blindfoldconditions.

2.9. Statistical analyses

Before comparisons were run, care was taken to check for normality of variabledistributions (ShapiroeWilk test) and homogeneity of variances (Levene test). AOne-way ANOVA was performed between ATN-exposed samples and negativecontrols, followed by Dunnett’s t-test, risk level 5%.

Student t-test was performed between negative controls and MH or CdCl2treated plants.

Root metal concentrations showed no homoscedasticity, that is why a loga-rithmic transformation was performed before testing. Data were analysed usingStatistica 7.0 software (StatSoft).

3. Results and discussion

3.1. ATN physico-chemical properties

Al and Ti total concentrations in 100 mg/L ATN mothersuspension were respectively 3.44 and 52.29 ppm (�5%), asdetermined by ICP-AES after acid digestion. These concentrationscorrespond to 1.09 � 0.055 mmol/L of Ti and 0.127 � 0.006 mmol/Lof Al. Dissolved Al in the mother suspension was below 0.2 mg/L,corresponding to a maximum of 7.4 mM of Al dissolved in thesolution. This rate was not depending to the pH (5e9).

Physico-chemical properties of ATN were detailed by Labilleet al. (2010) and Auffan et al. (2010). These authors display thatthe original nanocomposites are not completely degraded: thePDMS outer coating is significantly altered and desorbed, leavingaluminium hydroxide at the surface of the TiO2 nanoparticlesconstituting the ATN. Moreover, no dissolved aluminium isdetectable in solution. Labille et al. (2010) showed that the ATN areconstituted of aggregates characterized by a size larger than100 nm and an isoelectric point around 7.3 typical for Al(OH)3surface coating. These properties confer to the residues a weaklypositive surface charge at environmental pH and a tendency toaggregate in most common surface waters.

Our results were in accordance with their observations: duringthe first 5 h after dilution (25 mg ATN/L), ATN tended to formaggregates larger than 10 mm in size. The volume fraction ofparticles over 10 mm increased from 17 to 88%, while the volumefraction of particles below 700 nm decreased from 14 to 3% (Fig. 1).We can also see that the relative turbidity of the suspensiondramatically decreased when ATN concentration increased: 35.39,16.47 and 3.73 for 5, 25 and 50 mg ATN/L, respectively. Therefore,ATN suspensions aggregated very rapidly, and the higher ATNconcentration was, the more rapidly aggregation and sedimenta-tion occurred. This tendency of ATN to self aggregation is certainlydue to a pH close to the isoelectric point of the ATN, favouringscreening of the interparticle electrostatic repulsions. Concerningionic strength, Labille et al. (2010) find a critical coagulation

0

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40

60

80

100

0 1 2 3 4 5Time (Hours)

Vo

lum

e p

erc

en

ta

ge

Fig. 1. Time evolution of particles size in the 5 first hours after dilution of ATN mothersuspension to 25 mg ATN/L in the mixture of ultrapure and mineral waters (50:50, v:v).White triangles: particles below 700 nm; Black squares: particles over 10 mm.

concentration for ATN suspension of 20 mM NaCl or 8 mM MgCl2.Nevertheless, according to these authors, a slow coagulation isinitiated by 5 mM NaCl or 2 mM MgCl2, corresponding to ionicstrengths of 5 and 6 mM, respectively. In comparison, our exposurematrix bore an ionic strength of 4.8 mM. Hence, to our sense, ionicstrength played a minor role in ATN aggregation.

Besides, the kinetics of this aggregation mechanism is alsodetermined by the ATN concentration, which controls their colli-sion frequency, favouring faster aggregation and sedimentation athigher concentration. This effect is evidenced by the supernatantrelative turbidities as a function of the ATN concentration. Theturbidity after 24 h settling indeed decreases in a logarithmic waywhen the particle number increases. The equation describing therelative turbidity (t) as a function of the ATN concentration (c) is thefollowing: t ¼ �13.4ln(c) þ 57.566; the correlation coefficient isR2 ¼ 0.98715. However turbidity measurements gave directevidence of colloids remaining in suspension. This implies differentpotential exposure routes and/or bioaccessibilities of the ATNaccording to their concentration.

0

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0 20 40 60 80 100 120 140 160

Al (mmol/kg)

Ti (m

mo

l/kg

)

5 mg/L ATN

25 mg/L ATN

50 mg/L ATN

Fig. 3. Relationship between Ti and Al in Vicia faba secondary roots after 48 h exposureto different ATN concentrations. Dotted line: Ti/Al ratio in exposure suspension.

A.-S. Foltête et al. / Environmental Pollution 159 (2011) 2515e25222518

During exposure, root exudates could also have exerted anaction on ATN physico-chemical properties, probably acceleratingATN aggregation.

At the end of the exposure, the pH of the exposure suspensionwas comprised between 6.18 and 6.87 without any clear correlationwith ATN concentration.

3.2. Ti and Al contents in tissues

Titanium and aluminium concentrations in plant roots arerepresented in Fig. 2. Titanium concentrations were highlydifferent from controls regardless of ATN concentration.Aluminium concentrations were significantly different only for 25and 50 mg ATN/L. A strong positive linear correlation exists

Fig. 4. m-XRF analyses of roots after 48 h exposure to 50 mg ATN/L. (a) Ca, S, P and Ti distribuand Ti distribution in two cross sections and S/Ti combined image (total counting time of 15 �root (mucilage removed) and P/Ca/Ti combined image (total counting time of 20 � 600 s, 12P/Ca/Ti combined image to ensure a better readability.

between Ti (r ¼ 0.9661, p < 0.001) or Al (r ¼ 0.6707, p ¼ 0.004)concentrations in the ATN suspension and their respectiveconcentrations in plant roots. Fig. 3 shows that whatever ATNconcentration was, every individual contained a Ti/Al ratio smallerthan the one existing in the exposure suspension. This implies thatAl was internalized in a relatively larger extent than Ti into plantroots. This may be due to the higher dissolution constant ofAl(OH)3 coating compared to TiO2 core in the ATN, which favouredmore soluble and bioavailable Al species, particularly in rootmicroenvironment. Indeed, this one is acidified by the release ofHþ ions by the root (Hinsinger et al., 2003; Yan et al., 1992) andsome of the Al agglomerated to the roots can have been partiallydissolved in contact with this special environment and preferen-tially absorbed comparing to Ti.

tion in entire root (total counting time of 15 � 600 s, 256 px2, 1 px ¼ 16 mm). (b) Ca, S, P600 s, 128 px2, 1 px ¼ 8 mm). (c) P, Ca and Ti distribution in cross section of the treated

8 px2, 1 px ¼ 40 mm). S was not detected. Contrast and luminosity were modified in the

0.0

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0 5 25 50 C dATN (mg/L)

ro

ot e

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atio

n (c

m)

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0.00.2

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0 5 25 50ATN (mg/L)

fresh

w

eig

ht (g

)

0.70

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0.74

0.76

0.78

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0.84

0 5 25 50ATN (mg/L)

Fv/F

m

a

b

c

Fig. 5. (a): Secondary root elongation (mean � SD) in V. faba exposed for 48 h to ATN(3 � 4 individuals). (b): Shoot fresh weight (4 individuals, mean � SD). (c): Fv/Fm ratioin leaves (4 individuals, mean � SD). Differences are not statistically significant(ANOVA, p < 0.05) except for positive control Cd. (*Student t-test, p < 0.05).

A.-S. Foltête et al. / Environmental Pollution 159 (2011) 2515e2522 2519

Fig. 4a shows the spatial distribution of Ti along an entire rootexposed to 50 mg ATN/L. Since the X-ray beam penetrates throughthe sample, Ti retention at the root surface (probably in the muci-lage) or Ti included in the root tissues cannot be discriminated.Nevertheless, the chemical images of cross sections show high Ticoncentrations, particularly in root periphery. Moreover Ti seems to

Table 1Malondialdehyde and oxidative stress enzymes activities in shoot tissues of Vicia faba planfor the activity that inhibits nitro blue tetrazolium photoreduction by 50% compared witANOVA, p < 0.05. Cadmium treated plants not statistically significantly different from co

Control 5 mg/L

MDA � 10�8 (mol/g fw) 4.93 � 2.02 4.69 � 0.84CAT � 10�4 (mol/min/g fw) 8.40 � 3.48 2.49 � 1.37SOD (U/g fw) 27.00 � 7.87 16.17 � 2.32GR � 10�6 (mol/min/g fw) 3.60 � 1.09 2.61 � 0.38GPX � 10�6 (mol/min/g fw) 0.915 � 0.176 0.884 � 0.157APX � 10�6 (mol/min/g fw) 3.90 � 1.97 2.72 � 0.45Proteins (mol/g fw) 1.89 � 0.78 2.74 � 0.43

have been internalized in the peripheral tissues, at least in the 100outermost micrometres of the root tissues (Fig. 4b). But it is diffi-cult, from these chemical images, to precisely identify the interfacebetween the mucilage and the root tissues: an additional treatmentwas performed on the roots in order to totally remove themucilage.m-XRF mapping of this treated root (cross section) revealed theremaining presence of Ti (Fig. 4c). From these results, partial Tipenetration in root tissues can be validated.

We just showed through m-XRF analysis performed on rootsexposed to 50 mg ATN/L that high titanium concentrations existedin root peripheral tissues. Yet, ATN particles, principally under theform of aggregates over 10 mm, were not likely to penetrate into theroot cells. In fact, nanoparticles with a diameter greater than 6 or7 nm have few chances to pass through the cell wall pores (Andrewet al., 1997; Asli and Neumann, 2009; Baron-Epel et al., 1988; Car-pita et al., 1979; Proseus and Boyer, 2005). V. faba root cell wallpores have an average diameter of 10e14 nm that can reach 20 nm(Hylmö, 1955, 1958). Such coating could result from the adsorptionof the ATN onto the external tissues of the root and/or their trap-ping by mucilage. This phenomenon could reasonably be expectedconsidering the opposite surface charges of ATN (þ) (Labille et al.,2010) and cell walls (�) at pH ¼ 6.5, enabling electrostatic attrac-tions. The deposit of particles in the outer root tissues, leading tothe filling of the cell pores and interstices, bothering or blocking thenormal circulation of water and molecules of interest for the plant,is called “clogging” by Asli and Neumann (2009), who alreadysupposed such a phenomenon on maize primary roots exposed to30 nm TiO2 nanoparticles colloidal suspension. This coating couldexplain the huge titanium concentrations we observed in V. fabaroots (Fig. 2). It also could increase the smallest ATN particles intakeby endocytosis via root hair (Ove�cka et al., 2005), or ATN diffusionin the root tissues through the intercellular space without enteringthe cells. Indeed,100 nm in size TiO2 particles have shown ability tobe transferred from roots towards the leaves in Nicotiana tabacum(Ghosh et al., 2010).

3.3. Biological effects of ATN on the plants

Both rutile and anatase nano-TiO2 (4e6 nm diameter) canpromote growth in spinach (Gao et al., 2008; Ma et al., 2010; Yanget al., 2006; Zheng et al., 2005). Nevertheless, in our study usingnano-rutile, root elongation was not changed by ATN (Fig. 5a), andshoot biomass showed a slight tendency to decrease for 25 and50 mg/L ATN (Fig. 5b). Besides, nano-anatase with 4e6 nm diam-eter have been proven to increase photosystem II efficiency inspinach after spraying on the leaves (Su et al., 2007; Zheng et al.,2007a). In that case, nanoparticles were shown to have beeninternalized in the chloroplast and bound to photosystem II (Zhenget al., 2007b). Once again, in our work, Fv/Fm ratio did not changeunder ATN exposure (Fig. 5c).

Shoot MDA levels and antioxidant enzyme activities were notmodified following exposure to ATN comparatively to control group.In Cd-exposed plants similar results were obtained (Table 1). Thiol

tlets after 48 h exposure to ATN (mean� SD). SOD units are U/g fw, where 1 U standsh tubes lacking enzyme extract. No statistically significant difference from controls,ntrols, Student t-test, p < 0.05.

25 mg/L 50 mg/L CdCl2

5.56 � 2.07 4.65 � 1.29 5.69 � 1.214.74 � 5.48 4.38 � 2.82 7.48 � 4.66

20.80 � 9.33 17.39 � 4.16 29.86 � 13.722.93 � 0.57 3.09 � 0.53 2.76 � 0.71

0.928 � 0.205 0.848 � 0.120 1.024 � 0.3103.46 � 0.96 4.23 � 0.00 3.59 � 0.862.00 � 1.16 2.57 � 0.59 1.93 � 0.79

Table 2Malondialdehyde and oxidative stress enzyme activities in root tissues of Vicia faba plantlets after 48 h exposure to ATN (mean� SD). SOD units are U/g fw, where 1 U stands forthe activity that inhibits nitro blue tetrazolium photoreduction by 50% compared with tubes lacking enzyme extract. *Statistically significantly different from controls, ANOVAfollowed by Dunnett’s t-test, p < 0.05. #Cadmium treated plants statistically significantly different from controls, Student t-test, p < 0.05.

Control 5 mg/L 25 mg/L 50 mg/L CdCl2

MDA � 10�8 (mol/g fw) 2.26 � 0.27 2.41 � 1.82 3.39 � 1.32 2.85 � 0.51 5.50 � 1.55#CAT � 10�7 (mol/min/g fw) 3.56 � 0.94 3.11 � 0.53 3.44 � 0.95 3.77 � 0.49 5.81 � 1.07#SOD (U/g fw) 13.83 � 2.57 12.84 � 2.73 15.47 � 6.74 13.44 � 2.58 34.38 � 13.91#GR � 10�6 (mol/min/g fw) 1.177 � 0.131 1.157 � 0.131 0.846 � 0.476 0.243 � 0.424* 0.674 � 0.303#GPX � 10�5 (mol/min/g fw) 1.717 � 0.125 1.748 � 0.227 1.681 � 0.561 1.787 � 0.229 3.053 � 0.415#APX � 10�6 (mol/min/g fw) 5.388 � 0.868 3.711 � 0.517* 3.689 � 0.315* 4.062 � 1.294 7.741 � 3.704Proteins (mol/g fw) 0.729 � 0.166 0.606 � 0.225 0.539 � 0.265 0.547 � 0.200 0.340 � 0.120#

50 14

mcn *

A.-S. Foltête et al. / Environmental Pollution 159 (2011) 2515e25222520

concentrations in shoots were neither modified by ATN nor by Cd(data not shown). In roots, lipid peroxidation, MDA levels signifi-cantly increased in Cd-exposed individuals but not in ATN-exposedindividuals (Table 2). CAT, SOD and GPX enzymes responded simi-larly to MDA levels (Table 2). On the contrary, GR enzyme and totalproteins decreased in Cd-exposed roots. In ATN-exposed plants, GRactivity decreased significantly at 50 mg ATN/L (ANOVA p ¼ 0.0037).Nevertheless, APX activity significantly decreased (ANOVAp¼ 0.0433) only for 5 and 25mgATN/L. In other studies, anatase andrutile nano-TiO2 enhanced superoxide dismutase, catalase andperoxidase activities and decreasedmalondialdehyde in chloroplastsexposed to UV (Hong et al., 2005; Zheng et al., 2008). Malondialde-hyde could also be increased in Allium cepa roots (Ghosh et al., 2010).To the contrary, our study showed that in shoots, none of oxidativestress biomarkerwasaffectedbyATN. Evenwhenexposed toCd, anti-oxidative stress biomarkers in shoots remained unchanged, sug-gesting a limited transfer from the root tissue. In roots exposed toATN, none of these biomarkers was either increased, but rootsremained hidden from light during exposure and, to our knowledge,no element in the literature deal with a similar case. Anotherhypothesis could be that the decrease of GR and APX may be due todissolved aluminium, but in the literature, Al at lowconcentrations inhigher plants is mostly responsible for an enhanced oxidant stressenzyme activity and/or expression (Boscolo et al., 2003; Darkó et al.,2004; Dipierro et al., 2005; Sharma and Dubey, 2007; �Simonovi�cováet al., 2004; Tamás et al., 2006). Therefore, this hypothesis is noteither convincing.

Total GSH in roots was not affected in ATN-treated individuals(Fig. 6). GSH/GSSG ratio remained constant (data not shown). PC2,PC3 and PC4 synthesis was stimulated in Cd-treated roots but not inATN-treated ones.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 5 25 50 CdATN (mg/L)

Th

io

ls (µ

mo

l / g

fresh

w

eig

ht)

GSHPC2PC3PC4

*

*

*

Fig. 6. Thiol concentrations (GSH and PCs, mean � SD) in roots of V. faba exposed 48 hto ATN (4 individuals). Differences between ATN-treated and control individuals arenot statistically significant, ANOVA, p < 0.05. Cd: reference metal. *Statisticallysignificant from controls (Student t-test, p < 0.05).

Not surprisingly, positive control (MH) induced a 13-fold increaseof micronucleus frequency and a decrease of mitotic index (8.6 and5.5% in negative and positive controls respectively). Exposure ofV. faba roots to ATN did not lead to any genotoxicity (Fig. 7). Celldivision, assessed by mitotic index, was not modified too.

In the literature, photogenotoxicity of nano-TiO2 was proven(Nakagawa et al., 1997), but data are contradictory in plants. In A.cepa, no genotoxicity was found, but only the mitotic index wasmodified in root meristems (Klan�cnik et al., 2011). To the contrary,Ghosh et al. (2010) proved genotoxic effects of 100 nm in size TiO2particles in A. cepa roots and N. tabacum leaves.

Comparing effects in plants of bare TiO2 nanoparticles with theeffects of altered nanocomposites observed in this study leads us tosuggest that no or very few bare nanoparticles were released fromATN during our experiment. To summarize, no biomarker wassignificantly modified by ATN in shoots, and only few ones in roots,without any clear concentration response relationship. Moreover,neither genotoxicity nor phytochelatin synthesis was seen in roots.Despite a high quantity of aluminium and titanium oxides in roots,the quasi-absence of neither effects nor defence reaction noticed inplants would be in accordance with the hypothesis according towhich the metals internalized in the roots were still under a bio-logically inert form. Nevertheless, since Ti/Al ratio was alwayslower in tissues than in exposure suspension (Fig. 3), we can thinkthat ATN structures presented by Auffan et al. (2010) and Labilleet al. (2010) were not under unchanged form in tissues, sincealuminium seemed to have been partly solubilized.

0

5

10

15

20

25

30

35

40

45

0 5 25 50 MH

ATN (mg/L)

mcn

/ 1 000 cells

0

2

4

6

8

10

12

MI (%

)

MI

*

Fig. 7. Micronucleus frequency and mitotic index (mean � SD) in V. faba roots exposed48 h to ATN (2 � 4 individuals). Differences between ATN-treated and control indi-viduals are not statistically significant, ANOVA, p < 0.05. MH: positive control.*Statistically significantly different from controls (Student t-test, p < 0.05).

A.-S. Foltête et al. / Environmental Pollution 159 (2011) 2515e2522 2521

Ti and Al metals remained in the root peripheral tissues (Fig. 4band c). We can suppose that metals found in root tissues representa very small proportion of ATN, but our results did not indicatewhether ATN particles were effectively internalized in root cells.Perhaps they only stayed in intercellular matrix. The small part ofATN internalized in root tissues and the high quantity of ATNagglomerated on the root surface could have exerted a mechanicaction, for example by blocking cell connections or cell wall pores(Asli and Neumann, 2009; Ma et al., 2010), which could explain theslight decrease of shoot biomass for 25 and 50 mg/L ATN and thedecrease of GR and APX activities in roots.

4. Conclusion

To our knowledge, this work is the first published study relatedto the effects of altered commercial sunscreen nanocomposites inplants. High metal concentrations were detected in the rootsexposed to ATN, and titanium internalization was even observed inthe root tissues. However no biological effect was observed asopposed to results from the literature obtained on bare nano-TiO2.Therefore, for TiO2 nanoparticles risk assessment, this study showedthat apparently, no bare nanoparticlewas released fromcommercialnanocomposite, which indicate that the behaviour of the nano-particles is strongly affected by their surface modifications. Ourresults were obtained for exposure concentrations fortunately farabove the expected concentrations of suchmaterials inwastewatersor surface waters (Gottschalk et al., 2010; Mueller and Nowack,2008) and like for every substance, toxicological data have to becompared with the real concentration found in the environment(Nowack, 2009). Moreover, it is noteworthy to specify that inwaterswith pH upper than 7.3, ATN will hold a negative charge (Labilleet al., 2010), leading electronic repulsiveness with cells andpossibly reducing the clogging phenomenon. Other phenomena likeorganic-matter adsorption could also lead to the creation of nega-tively charged particles playing a role in the clogging.

Therefore, sunscreen nanocomposites seem not to exert dele-terious effects on our plant model in 48 h, but the observedimportant clogging onto the roots and above all, titanium inclusionin root tissues are worrying phenomena. Indeed, one can imaginelong-term effects in the plant. A deep improvement of knowledgeabout long-term effects of such manufactured nanocompositestowards living organisms is seriously needed.

Acknowledgements

The authors thank the French National Program NANOALTER(INSU/EC2CO/CYTRIX), the French Agency for the Environment andEnergy Management (ADEME, Paris, France) and Region Lorrainefor financial support.

Appendix. Supplementary data

Supplementary data related to this article can be found online atdoi:10.1016/j.envpol.2011.06.020.

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