Metal oxide particles catalyze photo-oxidation in environmental media

NaNomaterials aNd the eNviroNmeNt

40

Research Article • DOI: 10.2478/nanome-2013-0003 • nAnOme • 201 • 40–47

* E-mail: [email protected]

metal oxide particles catalyze photo-oxidation in environmental media

Analytical Science division, National Physical Laboratory, Teddington, TW11 0LW, UK

Caterina Minelli*, Ratna Tantra

Received 30 January 2013Accepted 21 March 2013

AbstractMetal oxide particles in the submicron and nanometer range endow a wide range of consumer products with unique properties. The widespread use of such products raises concerns on potential toxicity of these materials to man and the environment. Besides their size, the photo-catalytic properties of metal oxide particles are of particular concern. By utilizing molecular probes with tailored optical properties, we investigated the photo-catalytic properties of seven TiO2 (anatase), ZnO and CeO2 manufactured particles in environmental media. Controlled experiments confirmed that the particles catalyzed photo-oxidation and photo-production of reactive oxygen species (ROS), while no ROS generation was observed when the ionic form of the materials was used in place of the particles. While affecting their aggregation and sedimentation, the type of media was not found to strongly influence the photo-catalytic behavior of the particles. Within the size range that was investigated, ZnO particles resulted in the highest production of ROS, while anatase particles possessed the highest oxidative ability. Possible explanations of such behavior are suggested.

KeywordsManufactured nanoparticles • Nano-ecotoxicology • Reactive oxygen species • Photo-catalysis • Molecular probe • TiO2 • ZnO • CeO2

© Versita Sp. z o.o.

1. Introduction.

Due to their unique physical properties, engineered submicron-

and nano-scale materials are used to impart enhanced

functionality and improved performance to a wide range of

consumer products, from sunscreen lotions, to antibacterial

clothing, to fuel additives1. Since TiO2 exhibits enhanced

photocatalytic properties, it traditionally finds applications in

products such as self-cleaning glasses, water and air remediation

devices, and paints [1]. The photo-catalytic properties of these

materials are enhanced when their size is confined at the sub-

micrometer length scale. This finding boosted the use of metal

oxide nanomaterials in commercial products. TiO2 and other

metal-oxide manufactured submicron- and nano-particles

(mnPs) were also found suitable for enhanced sunscreen

formulations because of their ability to effectively screen from

ultraviolet (UV) radiation while at the same time being transparent,

these properties making the sunscreen products appealing

to a mass market. The widespread use of nanomaterials in

consumer products implies increased exposure for man and

their presence in the environment through a variety of routes,

including industrial waste, transportation, use, storage and

1 Woodrow Wilson International Center for Scholars and Pew Charitable Trusts. The Project on Emerging Nanotechnologies http://www.nanotechproject.org/ (29th January 2013).

disposal [2]. A qualitative risk assessment of carbon nanotubes,

TiO2 and Ag mnPs in the environment conducted in Switzerland

showed that the predicted environmental concentration of TiO2

mnPs in water was close to or higher than the relative predicted

no-effect concentration [3]. The trophic transfer of mnPs and

their biomagnifications have been demonstrated [4]. Such

studies urge the development of appropriate methodology

for the assessment of the environmental effects of mnPs and

potential associated risks. Knowledge in the field is limited and

standardization of risk assessment methodologies challenging

[5]. Currently, enormous efforts are invested worldwide in the

development of coherent approaches to assess the potentially

harmful impact of mnPs on man and the environment [6]. Among

all mnP physicochemical characteristics, the ability to generate

reactive oxygen species (ROS) and cause oxidative stress has

been indicated as an important mechanism for mnP toxicity.

For example, oxidative behavior and uncontrolled production of

ROS are potential hazards to man and the environment when

mnPs interface biological tissues. ROS are capable of damaging

cell membranes [7], alter cell functions [8] and reacting with

molecules in aquatic environments [9,10]. The ability to assess

and quantify such behavior in aqueous environment is thus

of primary importance in evaluating the impact of mnPs on

the environment. On the contrary, it has been observed that

for materials such an ZnO mnPs, photocatalytic mechanisms

3

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Photocatalytic activity of metal oxide nanoparticles

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stored at 5ºC and their temperature allowed to equilibrate at

room temperature before use. The media were bubbled with

compressed air before use to ensure homogeneous levels of

oxygen in the media. For the experiments performed in presence

of Ar, molecular oxygen was firstly removed from the media by

vacuum pumping and then Ar gas was bubbled through the

media.

2.2. MNP sample preparationThe ZnO and CeO2 materials were used as supplied under

the OeCD sponsorship programme Research into the Safety

of Manufactured Nanomaterials2. The mnP samples used in

this work are listed in Table 1. mnP powders were dispersed

2 OECD Database on Research into the Safety of Manufactured Nanomaterials. http://www.oecd.org/env/ehs/nanosafety/oecddatabaseonresea rch in tothesa fe t yo fmanu fac tu rednanomate r i a l s .htm#Database_on_Research_General_Information (30 January 2013).

of toxicity are probably the most understudied as the majority

of in-vitro and in-vivo toxicity studies were conducted under

ambient laboratory lighting which contains negligible or no UV

radiation [11]. In spite of the need for procedures to assess these

properties having been highlighted [11,12], to date there are no

standard methods to assess the photo-oxidative behavior of

nanomaterials in environmentally relevant media. The range of

nanomaterials covered by the relevant literature is limited (these

includes CeO2 [13], TiO2 [10,14-16] nanoparticulates, fullerene

[9,17] and carbon nanotubes [18]) and the lack of standardization

makes comparing the properties of different materials difficult.

Furthermore, this type of information is more urgent for those

materials that have already reached the market as products or

through their incorporation into consumer products. This work

focuses on the assessment of photo-oxidative behavior of seven

commercially available TiO2, ZnO and CeO2 mnPs in solution

and their ability to catalyze the photo-production of specific ROS

(Figure 1A). The ROS that we studied include superoxide radicals

(·O2-), hydrogen peroxide (H2O2) and hydroxyl radical (·OH). We

conducted our analysis in DI water, seawater and a medium

used for in-vivo toxicological assessments [19]. Both static and

dynamic conditions were simulated during the experiments to

reflect different aquatic scenarios such as ponds and rivers.

2. experimental procedure.

2.1. Media experiments were conducted in 3 media, namely DI water,

seawater and in vivo medium. The latter is the medium utilized in

in vivo experiments on Daphnea Magna organisms as described

in OeCD directives (see table 1 of supporting information

for additional information). media for mnP dispersion were

Figure 1. (A) Two of the possible pathways to the production of different kinds of ROS by reduction and oxidation. (B) Typical experimental set-up utilized for the assessment of ROS production by MNP photocatalysis.

Table 1. List of MNPs with measured size and ζ-potential.

MNP Name and supplier CodeCrystallite size by XRD* (nm)

Mean of the weight distribution by DCS (nm)

ζ-potential(mV)

DI water Seawater In vivo medium

DI water* In vivo medium**

TiO2

Anatase nanopowder (Sigma, mO)

T1 10.5 350±40 420±30 440±30 7±5 -16±5

ZnO

nanosun ZnO (microniser Pty,

Australia)Z1 24.1 277±7 510±40 500±200

25±4(-36±4)

5±4(-13±5)

ZnO micron (Sigma, mO)

Z2 41.5 590±30 660±20 631±520±5

(-41±6)-5±4

(-36±5)

Z-cote (BASF, Germany)

Z3 41.5 193±3 309±10 269±1624±4

(-51±5)1.3±3

(-31±5)

CeO2

nanograin Ceria (Umicore, Belgium)

C1 33.3 135±4 187.6±1.7 146±5 33±2 -1.2±3

Ceria (Sigma, mO) C2 33.3 570±80 650±80 630±40 -7±6 -15±3

Ceria Dry (Antaria, Australia)

C3 10.3 340±50 520±90 400±30 28±2 -17±3

* See Figure S1 of supporting information for XRD patterns.**Values in parenthesis were measured after 30 min solar irradiation.



C. Minelli, R. Tantra

42

on average (109 ± 1) Wm-2 using an optical power/energy meter

(newport, model 842-Pe). The samples were contained in 10

mL glass beakers over a magnetic stirrer, under the central area

of the lamp of the solar simulator (Figure 1B). Seven beakers

were fitted in one single measurement, and the irradiation was

measured to be homogeneous in this area. A probe was added

to the mnP solution during or after the irradiation. Typically, 1 mL

aliquots were extracted from the mnP solution after each period

of irradiation. every 10 min period of irradiation was followed by

a 5 min dark period to reduce sample heating.

2.6. Optical measurementsWhere appropriate, mnPs were precipitated by centrifugation in

samples for optical characterization. Absorption spectra were

acquired with a Lambda 850 UV-Vis spectrometer supported

by UV Winlab software (Perkin elmer, mA). Optical absorption

was also measured using a plate-reader Victor3 1420 multilabel

counter supported by Perkin elmer Wallac 1420 software.

Fluorescence spectra were acquired with a Perkin elmer LS 55

Luminescence Spectrometer and with the plate reader.

3. Results and discussion

3.1. Physico-chemical characterization:The mnP samples utilized for this study are listed in Table 1,

together with the measured crystallite size, average particle

size (as the mean of the particle size weight distribution) and

ζ-potential. The physico-chemical properties of the materials

utilized in this study have been extensively characterized and the

results were published elsewhere [19]. We report here only part

of the characterization that most related to the ability to photo-

produce ROS. For example surface area, and thus size of the

mnPs, is considered a key parameter in the evaluation of photo-

production of ROS in mnPs [10,25,26]. However, it is also known

that morphology and crystal defects, of which we have limited

information, influence the photo-catalytic behavior of mnPs [27]

and therefore differences within mnPs of the same chemical

family are expected. It is also expected that the medium the

mnPs are dispersed in may affect some of the physico-chemical

properties of mnPs. To test this hypothesis we characterized the

behavior of mnPs in a range of media relevant to environmental

studies, in order to produce results that are directly relevant to

real world scenarios. For example, the effect of water salinity on

mnPs was deduced by comparing their properties in DI water

and seawater. Furthermore, experiments performed in medium

traditionally used for in vivo assessment of mnP toxicity with

Daphnea magna allowed direct correlation of our results with

nano-ecotoxicologic in vivo studies. All media exhibited neutral

or moderately alkaline pH (see supporting information, Table S1).

Although all mnP samples had a crystallite size of below 50 nm

as measured by XRD, DCS measurements indicated also the

presence of agglomerates of few hundred nanometers in size,

indicating that particles are partially polycrystalline or aggregated.

Seawater and to some extent the in vivo medium induced

further particle aggregation. These results are consistent with

in accordance to OeCD directives, the protocol is published

elsewhere [19,20]. Briefly, the mnP powder was worked into a

paste before the addition of the medium, to reach a typical final

mnP concentration of 50 mg/L. An ultrasonic probe (CPX 130,

Cole-Parmer Instruments, IL) was then used to further disperse

the mnPs (190 J in 10s, repeated twice).

2.3. Sample characterizationX-ray diffraction (XRD) was performed with D5000 diffractometer

(Siemens, Germany) which consisted of a theta-theta goniometer

and a custom-made specimen stage. The X-ray source used for

these measurements was the Cu-Kα operating at 40 kV and 30

mA. Particle size was expressed as the average mean equivalent

spherical particle diameter of the particle weight distribution as

measured by differential centrifugal sedimentation (DCS) with a

DC 20000 Disc Centrifuge (CPS Instrument, FL). The reported

value was the average of three measurements and the error the

associated standard deviation. Zeta-potential was measured on

irradiated and non mnP samples with a Zetasizer nano (malvern

Instrments, UK) in DI water and in vivo medium diluted 1:10.

2.4. ROS probesUnspecific oxidation was measured utilizing KI (Sigma, mO) as

a probe in 100 mm typical final concentration in mnP solutions

[13,21]. Tri-iodide concentration was calculated by using

26000 m-1cm-1 [21] for the molar extinction coefficient at 352

nm. 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-

5-carboxanilide inner salt (XTT, Sigma, mO), 1 mm typical

concentration, was used to quantify the photo-production of

·O2- [22]. The probe was added to the mnP solutions before

irradiation. The molar extinction coefficient was assumed to be

21600 m-1cm-1 at 470 nm [22]. The concentration of H2O2 was

measured by using the probe Amplex Red that reacts with H2O2

in a 1:1 stoichiometry in presence of horseradish peroxidase

(molecular Probes, OR) to produce red-fluorescent resorufin

[23]. The probe was added to the mnP solution after irradiation,

owing to the higher stability of H2O2. For the same reason, a

H2O2 standard calibration curve was prepared by utilizing H2O2

samples with increasing concentration. Resorufin concentration

was measured both by its fluorescent emission at 590 nm and its

absorption at 560 nm. However, absorption measurements were

preferred due to quenching of the fluorescence that we observed

for the higher concentrations of H2O2. 100 μm Coumarin (Sigma,

mO) was used to assess the photo-production of ·OH [24].

Formation of ·OH was measured both by fluorescence and

absorption spectroscopy of hydroxyl-coumarin and coumarin at

277 nm and 460 nm respectively.

2.5. IrradiationmnP solutions were irradiated under a 1 kW Solar Simulator

(newport Corporation, CT), with personal wavelength correctionTm

certificate by newport. The xenon lamp of the solar simulator

was fitted with an Am1.5G filter to minimize the mismatch of

the lamp from the solar spectra. The irradiance of the solar

simulator was measured for every experiment and found to be




43

(Figure 2A). In our experiments, the tri-iodide signal was found to

increase linearly with the concentration of the mnPs (Figure 2B)

and time of irradiation (result not shown), while no tri-iodide

anions were detected in control mnP solutions incubated for the

same duration in dark conditions (Figure 2B). notably, Figure 2B

and 2C show that traces of tri-iodide ions were also detected

in solution containing no mnP but subjected to irradiation. We

suggest this is the result of photochemical formation of ROS in

water, although alteration of the KI probe due to irradiation is

also plausible (see supporting information, Figure S3). Figure 2C

compares the production of tri-iodide ions for the 7 mnP

samples in three different media. T1 mnPs exhibited the highest

oxidative activity in all media, followed by sample C1 in DI water.

For the other samples, the oxidative behavior was limited, with

values comparable to those measured in DI water. These results

are in general agreement with Liao et al. [30], who suggested

that increased recombination of photo-induced charges is at

the origin of the differences of photocatalytic properties of TiO2

and ZnO mnPs, TiO2 having higher photocatalytic ability. The

use of seawater as a dispersant mainly caused a suppression

of tri-iodide formation, with the exception of sample Z3 in which

case we measured an increase in tri-iodide concentration.

The in vivo medium generally caused a reduction in tri-iodide

production with respect to DI-water. The increased ion density in

seawater and in vivo medium with respect to DI water may imply

that electrons and holes at the mnP surface are more prone to

being neutralized, with consequent reduction of the overall mnP

the high conductivity values measured for seawater (supporting

information, Table S1), which indicate the presence of high ion

concentration in solution leading to enhanced aggregation.

ζ-potential values resulted within the range –30 mV and 30 mV,

indicating that mnPs are unstable in DI water and in vivo medium

(measurements could not be performed in seawater due to the

high conductivity), which is consistent with the fact that all mnP

dispersions incurred sedimentation, the highest rates having

been measured for seawater and the lowest for DI water (results

not shown). These results suggest that in scenarios where mnPs

are dispersed in static aquatic environments, they tend to rapidly

sediment, with consequently low probability of being irradiated by

sunlight. On the contrary, little variation in turbidity was observed

for mnP samples in simulated dynamic conditions, with minimal

variation in the temporal window from 20 to 80 minutes from

mnP dispersion (see supporting information, Figure S2). Photo-

catalytic experiments were therefore performed under simulated

dynamic conditions and within this temporal window, such that

variations in mnP concentration were minimized. Overall, the

properties of the family of ZnO mnPs shown in Table 1 did not

differ significantly from those of the CeO2 mnP family. However,

according to recent toxicity tests conducted on the same set of

ZnO and CeO2 mnPs, ZnO mnPs are more toxic than the CeO2

mnPs [19]. The reasons for such differences will be discussed

later.

3.2. General oxidative behaviorWhen metal oxide mnPs are irradiated by UV [15], electron-

hole pairs are generated upon absorption of a photon with

energy equal or greater than the mnP band-gap. TiO2, ZnO

and CeO2 have band-gaps between 3.1 eV and 3.3 eV, which

means electron-hole pairs can be generated by irradiation with

UV light form the sun. The electrons and holes that reach the

mnP’s surface have the ability to respectively reduce and oxidize

molecules adsorbed on the surface. In particular, the reduction

of oxygen molecules leads to the production of ·O2-, whose

further reduction generates other ROS such as H2O2. The holes

have the ability to directly oxidize molecules in the surroundings,

including surface hydroxyl groups and hydroxide ions formed

from water auto-ionization, which generates ·OH radicals

(Figure 1A). Two ·OH radicals can then combine to produce

H2O2. mnPs can therefore oxidize surrounding molecules via

two distinct routes, namely direct oxidation by means of the

holes and indirect oxidation via ROS action. A third oxidation

route exists for ceria mnPs owing to two possible oxidation

states of Ce atoms. Their ability to reversibly switch between

the forms Ce3+ and Ce4+ endows them with interesting redox

properties and oxidase-like behavior was found to enhance in

acidic solutions and for smaller size particles [28]. Other studies

have shown the antioxidant properties of CeO2 mnPs that act as

ROS scavengers [29].

The non-specific photo-oxidative activity of the mnPs was

studied using KI as a colorimetric probe in solution [13,21]. When

iodide anions are oxidized, tri-iodide anions form, which exhibit

a typical yellow color and an optical absorption peak at 352 nm

Figure 2. KI assay for unspecific oxidative behavior of MNPs. (A) Relative absorption of 50 mg/L T1 MNP samples in DI water after 30 min of irradiation in presence of 0.1M KI. The peak at 352 nm is indicative of tri-iodide formation. Prior the experiment, the water was saturated with air (gray solid line) or argon gas (dotted line). In one sample saturated with air, 160 SOD units/mL were added (black solid line). (B) Tri-iodide concentration measured from absorption at 352 nm of spectra such as in A for solutions of T1 MNPs with increasing concentration and subjected to dark or 20 min irradiation in presence of 0.1 M KI. (C) Comparison of tri-iodide concentrations measured for the seven MNP samples in three different media after 60 min irradiation. [MNP] = 50 mg/L; [KI] = 0.1 M.




44

spectroscopic measurements mainly performed on the reacted

forms of the probes. Some of these probes are routinely used in

cell biology to study the metabolic activity of cells. For example,

tetrazolium salts are used to assess cell proliferation [35]. Similar

approaches were also used to study the catalytic properties of

materials. For example, coumarin was used to quantify photo-

degradation occurring at TiO2 surfaces via ·OH-mediated

mechanisms [36] and to measure the generation of ·OH by

photo-excitation of TiO2 mnPs [24]. Tetrazolium salts were used

to detect ·O2- photo-produced in water by single walled carbon

nanotubes [18]. molecular probes with tailored optical properties

were also used to assess algal toxicity of CeO2 mnPs [13].

The presence of ·O2- was detected by using tetrazolium salts

as colorimetric probes. This probe was chosen for its specificity

to ·O2- and for its good solubility in (warm) water [34]. By specific

reaction with ·O2-, XTT molecules are converted into XTT formazan

molecules (Figure 3A) as indicated by the typical red color

and optical absorption peak at 470 nm (Figure 3B). The molar

concentration of XTT formazan increased linearly with the time

of irradiation for all mnPs, so that it was possible to estimate a

rate of ·O2- photo-production. As the type of media used was not

found to significantly influence the production of ·O2- (Figure 3C),

most of the experiments that will be presented were performed in

oxidative ability.

A more in depth study on the origin of the oxidative behavior

of the mnPs was attempted, the results of which are shown in

Figure 2A for TiO2 mnPs. Two DI water solutions were prepared

by saturating them with compressed air and with argon gas

respectively prior the addition of the mnPs and the KI probe.

After irradiation of the dispersions, tri-iodide production was 20%

lower for the mnPs dispersed in Ar-saturated water (Figure 2A,

dotted line). The same experiment was performed in presence

of ZnO (Z3) and CeO2 (C1) mnPs, and the production of tri-

iodide dropped by 44% and 40% respectively (see supporting

information, Figure S3). This result suggests that iodide ion

oxidation is due in part to the action of ROS that generate from

interaction of electrons with molecular oxygen and that this route

is less important in TiO2 with respect to ZnO and CeO2 mnPs.

To reinforce our hypothesis, a third experiment was performed

for TiO2 samples by using compressed-air saturated water to

which Superoxide Dismutase (SOD) was added. SOD is a class

of enzyme capable of converting ·O2- into oxygen and H2O2.

In this case, the production of tri-iodide was reduced by 10%

with respect to the first experiment (Figure 2A, black solid line),

confirming once again the role of ROS generated from oxygen

reduction in the oxidation of the KI probe. It is important to observe

that H2O2 will also contribute to iodide ion oxidation. Furthermore,

the reaction of molecular oxygen with photogenerated electrons

is known to suppress hole recombination, as well as improving

photostimulated charge separation in TiO2 mnPs [31], therefore

a major oxidative activity of the holes is expected in presence

of molecular oxygen. Hydroxyl radicals formed by hole reaction

can also contribute to iodide ion oxidation in presence of SOD,

but we will show in the next section that their contribution to

oxidation is not expected to be more significant in TiO2 mnPs

than ZnO mnPs. This is in agreement with various works which

identify the holes as the primary source of oxidation in TiO2

materials [1,32]. The results of this experiment suggest that

both direct and indirect oxidation of iodide ions occurs and that

direct oxidation of iodide ions by holes that are formed by photo-

irradiation and reach the material’s surface is more important in

TiO2 samples with respect to ZnO and CeO2 samples [1]. The use

of KI probe can therefore provide not only a comparative method

to evaluate the general oxidative behavior of mnPs, but also to

identify possible oxidative pathway mechanisms when used in

combination with Ar- or O2- saturated media.

3.3. Photoproduction of ROSSuperoxide is the first radical species produced by the reduction

of diatomic oxygen. Further reduction of ·O2- leads to a chain

formation of other ROS, namely H2O2 and ·OH, as shown in

Figure 1A. With the exception of H2O2, ROS tend to have short

lifetimes in the range of microseconds to milliseconds. This

property makes the detection and quantification of specific

ROS challenging. We detected and quantified ROS produced

by photo-catalysis in mnP solutions by using molecular probes

that change optical properties upon reaction with specific

ROS [33,34]. The presence of ROS was thus inferred from

Figure 3. (A) Chemical formula of XTT salt and XTT formazan. The latter forms upon interaction of XTT salt with superoxide radical molecules. (B) Absorption of XTT formazan measured in samples of Z3 MNPs subjected to different durations of irradiation. (C) XTT formazan concentration of Z3 MNPs dispersed in different media and subjected to irradiation (calculated from adsorption at 470 nm). (D) Rate of production of XTT formazan for 7 MNPs samples and control in DI water, measured during a period of 60 min of irradiation. (E) Comparison of XTT formazan production for a Z3 sample and one containing an equivalent Zn weight of Zn2+ ions.




45

could be detected. This effect contributes to explain the lower

levels of H2O2 concentration measured with respect to the ·O2-

concentration.

The presence of ·OH was measured by using the probe

coumarin, which reacts specifically with ·OH to form the

fluorescent molecule hydroxyl-coumarin [24]. ·OH are formed by

reduction of H2O2 via photo-generated electrons. Alternatively,

·OH are formed by reaction of a hole with hydroxide ions or

hydroxyl groups at the surface of mnPs. Figure 4C shows

the adsorption spectra of coumarin before and after 60 min

irradiation in presence of different mnPs in DI water, while in

Figure 4D the coumarin concentration is plotted for increasing

irradiation times. The control measurements indicated that the

coumarin probe was degraded to some extent also in absence

of mnPs. However, its degradation was significantly higher in

presence of mnPs, ZnO mnPs being the materials resulting

in the most degradation, followed by TiO2 and CeO2. These

results are consistent with the previous assays, suggesting

once again that ROS production is the main oxidative pathway

for ZnO mnPs. In the case of TiO2 mnPs, instead, we suggest

that the holes are mainly responsible for the photo-production

of ·OH, in agreement with the results of the KI assay which

indicated that direct reaction with holes had a major role in

the oxidative ability of these mnPs. In the case of CeO2 mnPs

oxido-reduction of the surface Ce atoms may be responsible

for the formation of ·OH, but further investigation is required to

support this hypothesis.

In general, it is not fully understood if the overall oxidative

ability (as shown by KI assay) or ROS production play the major

role in mnP toxicity [11,42]. For example, some toxicity studies

based on ROS photo-production have shown ZnO mnPs to

be more toxic than TiO2 mnPs in aqueous environments

DI water. The rate of ·O2- production was measured for all seven

mnP samples under the same experimental conditions (Figure

3D). All ZnO mnP samples exhibited elevated production of ·O2-

compared to the other types of materials, while no significant

·O2- production was detected for CeO2 mnP samples. Samples

that were irradiated in the absence of mnPs did not show

significant formation of XTT formazan, highlighting that it is the

photocatalytic action of mnPs responsible for the production of

radicals. To reinforce this finding, we repeated the experiments

substituting the ZnO mnPs with an equivalent amount by weight

of Zn2+ ions. ZnO mnPs are known to dissolve in solution [37]

and dissolved Zn2+ ions are thought to be largely responsible for

the toxicity that ZnO exhibits [11,37,38]. Figure 3e shows that no

·O2- was produced in the sample containing Zn2+ ions, supporting

the finding that Zn2+ toxicity follows toxicity pathways unrelated

to ROS production [39]. We conclude that the production of ·O2-

is catalyzed by the mnPs.

It is interesting to observe that, while samples T1 and C1

exhibited the highest oxidative behavior with the KI assay,

their production of ·O2- was relatively low compared to the ZnO

mnPs. Other works have found no direct correlation between

the oxidative ability of mnPs and their ROS production [40,41].

This observation reinforces that the electron-hole pairs photo-

generated within the different mnPs follow different routes and

that direct oxidation plays the major role.

While ZnO mnPs are known for being soluble in solution,

CeO2 and TiO2 are regarded as insoluble materials. We suggest

that surface defects resulting from loss of Zn2+ ions from the

lattice promote oxygen reduction. This hypothesis is consistent

with the significant levels of XTT formazan measured in absence

of irradiation (Figure 3C, data-point at time 0). Furthermore,

ZnO mnP ζ-potentials were measured to be more negative

when irradiation occurred (Table 1), suggesting that irradiation

enhanced mnP dissolution. We also observed that ZnO mnPs

were not able to catalyze the photo-production of ·O2- four

weeks after dispersion in solution, supporting the hypothesis

that the losses of Zn2+ ions deeply affect the surface chemistry

of the mnPs. The high electron density at the ZnO mnP surface

may also have been responsible for recombination of photo-

generated holes, explaining the low levels of direct oxidation of

the KI probe observed in the previous experiments.

Due to the high production of ·O2-, ZnO mnPs are also

expected to catalyze the formation of higher levels of H2O2

and ·OH, which form by reduction of ·O2-, compared to CeO2

and TiO2 mnPs. Figure 4A shows the production of H2O2 photo-

catalyzed by the mnPs after 60 minutes of irradiation for different

concentrations of mnPs. As expected, the production of H2O2

increased linearly with the concentration of ZnO mnPs, while

TiO2 and CeO2 mnPs did not exhibit significant production of

H2O2 (Figure 4A and B). It is worth observing that while in the

previous assays the irradiation was performed in presence of

the probes in solution, the Amplex Red probe was added after

the irradiation. This means that while all the photo-produced

·O2- could potentially react with the XTT, part of the H2O2 may

have been further reduced to ·OH and eventually H2O before it

Figure 4. (A) H2O2 production photo-catalyzed by different MNPs in DI water after 60 min of irradiation as a function of MNP concentration. (B) H2O2 photo-production for all the MNP solutions in DI water for different times of irradiation. (C) Coumarin absorption spectra before and after irradiation, in presence and non of different MNP samples. (D) Degradation of coumarin (calculated from adsorption at 277 nm) as a function of the time of irradiation for different MNP samples in DI water and control.




46

characteristics such as size, surface chemistry and propensity

to aggregate may influence their photo-catalytic properties.

The methodologies described here can be employed for

screening the photo-oxidative behavior of mnPs, in preparation

and support of expensive in vivo toxicological assessments.

Furthermore, these findings are important to achieve a deeper

understanding of the real biological damage that these materials

can cause in relation to their photo-oxidative ability and will

contribute to the formulation of effective solutions to contrast

their potential toxicity.

Acknowledgment

This work was conducted as part of PROSPecT, which is a

public-private partnership between DeFRA, ePSRC and TSB

and the nanotechnology Industries Association (nIA Ltd.)

and its members, and was administered by the DeFRA LInK

Programme. We thank Dr Tony Fry for the XRD measurements,

and Alex Shard and Andy Wain for the insight they offered in the

interpretation of the results.

[43-45], although the role of dissolved zinc needs to be

elucidated [45,46]. The development of relative simple protocols

for the assessment of the photo-catalytic properties of mnPs

in environmentally relevant media such those presented in

this work can support in vitro and in vivo investigation of mnP

toxicity and potentially offer further insights into mnP toxicity

mechanisms. Standardization of such protocols will facilitate

the comparison of the toxic effects of different mnPs in relation

to their photo-catalytic properties.

4. Conclusions

This study showed that mnPs have the ability to catalyze the

photo-oxidation of molecules in their surroundings. When

dispersed in the environment, these materials can therefore

potentially harm biomolecules and living organisms they may

interface. We have demonstrated that the mechanisms of

photo-oxidative behavior differ for TiO2, ZnO and CeO2 mnPs.

Furthermore, different behaviors were also observed within

the same family of mnPs, suggesting that specific particle

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Metal oxide particles catalyze photo-oxidation in environmental media

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