comparative phototoxicity of nanoparticulate and bulk zno to a free-living

Williams a aDepartment of Environmental Health Science, College of Public Health, The University of Georgia, Athens, GA 30602, USA bDepartment of Plant and Soil Sciences, University of K

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Comparative phototoxicity of nanoparticulate and bulk ZnO to a free-living

nematode Caenorhabditis elegans: The importance of illumination mode

and primary particle size

H Ma a,*,1 , N.J Kabengi b , P.M Bertsch b , J.M Unrine b , T.C Glenn a , P.L Williams a

aDepartment of Environmental Health Science, College of Public Health, The University of Georgia, Athens, GA 30602, USA

bDepartment of Plant and Soil Sciences, University of Kentucky, Lexington, KY 40546, USA

a r t i c l e i n f o

Article history:

Received 17 November 2010

Received in revised form

14 March 2011

Accepted 16 March 2011

Keywords:

Nanoparticulate ZnO

Bulk ZnO

Phototoxicity

Reactive oxygen species (ROS)

Caenorhabditis elegans

a b s t r a c t

The present study evaluated phototoxicity of nanoparticulate ZnO and bulk-ZnO under natural sunlight (NSL) versus ambient artiﬁcial laboratory light (AALL) illumination to a free-living nematode Caeno-rhabditis elegans Phototoxicity of nano-ZnO and bulk-ZnO was largely dependent on illumination method as 2-h exposure under NSL caused signiﬁcantly greater mortality in C elegans than under AALL This phototoxicity was closely related to photocatalytic reactive oxygen species (ROS) generation by the ZnO particles as indicated by concomitant methylene blue photodegradation Both materials caused mortality in C elegans under AALL during 24-h exposure although neither degraded methylene blue, suggesting mechanisms of toxicity other than photocatalytic ROS generation were involved Particle dissolution of ZnO did not appear to play an important role in the toxicity observed in this study Nano-ZnO showed greater phototoxicity than bulk-Nano-ZnO despite their similar size of aggregates, suggesting primary particle size is more important than aggregate size in determining phototoxicity

1 Introduction

Nanoparticulate metal oxides are being used within a great

variety of applications due to their novel optical, magnetic, and

electronic properties ( Zhou et al., 2006 ) With widespread use of

these manufactured nanoparticles, concerns about their potential

impact on the environment and human health have been raised

( Colvin, 2003 ) Although still in its infancy, toxicity studies on

manufactured metal oxide nanoparticles (such as TiO2, ZnO) are

expanding rapidly ( Hall et al., 2009; Jiang et al., 2008; Brunet et al.,

2009; Reeves et al., 2008; Franklin et al., 2007; Wang et al., 2009;

Ma et al., 2009 ).

Toxicity of manufactured nanoparticles may be attributed to

several different modes of action: chemical toxicity based on

chemical composition (e.g., release of toxic ions); surface catalyzed

reactions (e.g., formation of reactive oxygen species (ROS)); or

stress of stimuli caused by the surface, size, and shape of the

particles ( Wang et al., 2009; Nel et al., 2006 ) Dissolution of

nanoparticles resulting in the release of toxic ions has been found

to play an important role in eliciting toxicity in both metal oxides ( Franklin et al., 2007; Wang et al., 2009; Ma et al., 2009 ) and quantum dots ( Priester et al., 2009; King-Heiden et al., 2009 ) Generation of ROS by nanoparticles interacting with environmental agents (e.g., UV) represents another important mode of action for metal oxide nanoparticles with photocatalytic activities such as TiO2or ZnO, as high concentration of ROS causes oxidative stress and can eventually elicit toxicity in biological systems ( Applerot

et al., 2009 ) A wealth of studies have demonstrated phototoxicity

of TiO2nanoparticles in a broad range of biological systems, from bacteria ( Brunet et al., 2009; Sunada et al., 2003; Adams et al.,

2006 ) to mammalian cell lines ( Sayes et al., 2006; Gopalan et al.,

2009 ) The underlying mechanism of bactericidal activity of TiO2

to Escherichia coli K-12 cells has been revealed to be associated with lipid peroxidation induced by ROS generation under UV irradiation ( Maness et al., 1999 ) ZnO nanoparticles are similar to TiO2 nano-particles regarding their photodynamic and antibacterial proper-ties ( Daneshvar et al., 2007 ), and are receiving increasing application in numerous areas such as electronics, rubber additives, medicine, biosensors, personal-care products, etc However, studies on phototoxicity of manufactured ZnO nanoparticles have been very limited except for those reporting their antibacterial effects ( Jones et al., 2008 ) A recent study by Gopalan et al (2009)

* Corresponding author

E-mail addresses:mah77@uga.edu,ma.hongbo@epa.gov(H Ma)

1 Present address: Mid-Continent Ecology Division, United States Environmental

Protection Agency, Duluth, MN 55804, USA Tel.:þ1 218 529 5071

Contents lists available at ScienceDirect Environmental Pollution

j o u r n a l h o m e p a g e : w w w e l s e v i e r c o m / l o c a t e / e n v p o l

Environmental Pollution 159 (2011) 1473e1480

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found that genotoxicity of ZnO nanoparticles to human sperm and

lymphocytes was enhanced by UV irradiation Given the increasing

applications of ZnO nanoparticles and its consequent release to the

environment either by intended disposal or accidental release, it is

essential to understand their potential phototoxicity to the natural

biota.

The objective of the current study is to evaluate phototoxicity of

nanoparticulate ZnO (nano-ZnO) and its bulk counterpart

(bulk-ZnO) to a free-living nematode Caenorhabditis elegans under natural

sunlight (NSL) versus ambient arti ﬁcial laboratory light (AALL)

illumination Aqueous ZnCl2was used as control A representative

species of the nematode phylum which is of great ecological

signi ﬁcance, together with its thoroughly understood biology, short

life cycle, and ease of culture in the laboratory, C elegans has served

as a good model for both terrestrial and aquatic receptors for

eco-toxicological studies for a wide range of environmental toxicants

( Leung et al., 2008 ) More recently, it has also been used for

eco-toxicological studies on manufactured nanoparticles ( Ma et al.,

2009; Wang et al., 2009 ) To help elucidate the possible

mecha-nism of the phototoxicity (i.e., localization of ROS toxicity),

photo-catalytic activity/ROS generation of the ZnO particles and lipid

peroxidation in the nematodes were also measured The hypotheses

are that: (i) phototoxicity of nano-ZnO will be greater under NSL

than under AALL, and this phototoxicity will be positively correlated

with photocatalytic activity/ROS generation of the nanoparticles;

(ii) nano-ZnO will have greater phototoxicity than bulk-ZnO, as

smaller particles have a greater surface area per unit mass than

larger particles and thus may be more effective in generating ROS

( Applerot et al., 2009 ) Natural sunlight instead of arti ﬁcial UV light

was used to activate the nanoparticles because exposure under NSL

is a more realistic scenario under which organisms might be

exposed to nanoparticles released to the environment.

2 Materials and methods

2.1 Nano-ZnO and bulk-ZnO sample preparation

Powdered nanoparticulate ZnO (NanoGardÒzinc oxide) was purchased from Alfa

Aesar (Ward Hill, MA, USA) with a stated size of 40e100 nm Stock suspensions of

nano-ZnO and bulk-ZnO (Mallinckrodt; Phillipsburg, NJ) (100 mg/l (80% Zn)) were

prepared by sonication for 2 h in an ultrasonic bath (Branson; Danbury, CT) Speciﬁc

surface areas (SSA) of both materials were determined by BrunauereEmmeteTeller

(BET) method and were found to be 17.0 and 4.2 m2/g for nano-ZnO and bulk-ZnO,

respectively Reagent grade ZnCl2(Mallinckrodt; Phillipsburg, NJ) was used to make

ZnCl2stock solution Test solutions were freshly diluted from the stock which was

sonicated for 30 min prior to dilution to ensure proper dispersion of the materials Both

stock suspension and dilutions were made in K-medium (0.032 M KCl, 0.051 M NaCl,

pH 5.8e6.0) (Williams and Dusenbery, 1990), an aqueous medium used for C elegans

based bioassays The pH of the test solutions was measured using a pH meter

2.2 Particle characterization

The nanoparticulate and bulk ZnO were characterized using a variety of

analytical techniques Transmission electron microscopy (TEM, FEI/Philips Electron

Optics, Eindhoven, The Netherlands) was used to characterize particle morphology

and measure the primary particle size Approximately 100 particles from four

representative images of each material were measured and average sizes of the

particles were reported Dynamic light scattering (DLS) was performed using

a photo correlation spectrophotometer (Malvern Instruments, Worcestershire, UK)

to determine the dh(hydrodynamic diameter) of the particles or aggregates To

analyze aggregates> 1mm in diameter, suspensions of nanoparticles were

exam-ined by differential interference contrast microscopy (DIC) using a motorized

microscope (Nikon Instruments; Melville, NY) equipped with a 40 objective lens

and a cooled CCD monochrome camera At least 100 aggregates were recorded for

each material and average diameters were calculated

Dissolution of nano-ZnO or bulk-ZnO in suspensions was assessed byﬁltration

through regenerated cellulose membranes with a 3000 Da nominal molecular

weight cutoff (approximately 0.9 nm) using a centrifugalﬁltration device Three ml

of nano-ZnO or bulk-ZnO solutions at 100 mg/l were added to theﬁlter units and

centrifuged (Eppendorf 5810R; Westbury, NY) for 30 min at 3220 g Filtrates were

collected and analyzed for Zn concentration using an inductively coupled plasma

ﬁltrates was determined by ﬁltering ZnCl2 solutions of similar concentrations through the device

2.3 C elegans toxicity assay

C elegans (wild type N2) was obtained from Caenorhabditis Genetics Center (Minneapolis, MN) The nematode culture maintenance and generation of age-synchronized worms followed the description by Donkin and Williams (Donkin and Williams, 1995) Toxicity test was conducted for three materials: nano-ZnO, bulk-ZnO, and ZnCl2 For each material, exposure was conducted under three different illumination conditions: NSL, AALL, and in dark All tests were conducted in 24-well tissue culture plates Each test consisted of six concentrations of test substance (4, 8,

20, 40, 60, 80 mg/l Zn, corresponding to 5, 10, 25, 50, 75, 100 mg/l ZnO and 8, 17, 42,

84, 125, 167 mg/l ZnCl2) and a control, with three replicate wells for each concen-tration A 1.0 ml aliquot of test solution was added to each well which was subse-quently loaded with 10 (1) nematodes Light intensity was measured using

a digital luxmeter (Precision Mastech, Kowloon, Hong Kong, China) For exposure under NSL, the plates (without lids) were left under direct sunlight for 2 h on the outside ledge of a window facing southwest in the laboratory on bright days (25 1.5C average temperature, UV index 4e5) in October in Athens, GA (33570

1900N, 832205900W) Temperature of the exposure solution was recorded before and after exposure, and was found to increase by 2e3C; and the water loss in the exposure solution was estimated to be less than 5% after 2-h NSL exposure The average incident illuminance during the test period was 15,000 250 lux Mortality was monitored following the 2-h NSL exposure The exposure was continued for an extended 22 h under AALL by relocating the plates on a bench in the laboratory, which does not receive any sunlight Mortality was monitored again This 2-h NSL followed by 22-h AALL exposure allows for detection of possible synergistic toxicity

of the nanoparticles if different modes of action are involved under these two irradiation conditions For exposure under AALL, the plates (without lids) were placed on a bench in the laboratory with room temperature 22 0.5C The ambient laboratory lighting usesﬂuorescent lamps, and had an intensity of 525 25 lux during the test period For exposure in dark, the plates were covered by aluminum foil and placed in a cabinet at ambient room temperature Mortality was monitored

at 2 h and 24 h for exposure under AALL and in dark The plates were observed under

a dissecting microscope and the nematodes were counted and scored as live or dead following established protocole nematodes that did not move in response to

a gentle touch by a metal wire were counted dead (Williams and Dusenbery, 1990) 2.4 Photocatalytic activity measurement

In parallel to C elegans toxicity assay, photocatalytic activity of the three materials was determined under different illumination methods by measuring photodegradation of methylene blue (MB) in aqueous solution (Shen et al., 2008; Jang et al., 2006) Degradation of MB by photocatalysts such as ZnO or TiO2 involves generation of radicals such as O2andOH (Houas et al., 2001; Lachheb et al.,

2002); therefore, degradation of the dye can be used as an indicator for photo-catalytic activity/ROS generation of these materials A series of concentrations of each material were prepared in an identical manner as those for nematode bioassay; one ml of 25 mg/l MB was added to each well The plates were incubated for 30 min

to allow for equilibrium for the dye sorption to particles Prior to exposure, the plates were gently shaken by hand for a few minutes to homogenize the solution MB concentrations were measured at the beginning, after 2 h and 24 h of exposure, using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wil-mington, DE) at 665 nm A negative control using a series of MB concentrations in K-medium under AALL was also included

2.5 Lipid peroxidation measurement Lipid peroxidation in C elegans after exposure to nano-ZnO and bulk-ZnO under NSL or AALL was measured by the thiobarbituric acid assay (TBARS) for malon-dialdehyde (MDA) using an OxiSelect TBARS Assay Kit (Cell Biolabs, San Diego, CA)

C elegans exposure for lipid peroxidation measurement was similar to the toxicity assay except that larger number of nematodes (w0.5 ml worm pellet) was used and only one nano-ZnO or bulk-ZnO concentration (25 mg/l) was tested After exposure, nano-ZnO or bulk-ZnO solutions were removed and the worms were rinsed with M9 buffer for three times, then immediately frozen at80C until TBARS analysis TBARS analysis was performed following the manufacturer’s instructions Fluoro-metric measurement of MDA was obtained using a Bio-Tek Synergy 4 Hybrid Multi-Mode Microplate Reader at 540 nm excitation and 590 nm emission MDA content was standardized by total nematode protein which was measured using bicincho-ninic acid (BCA) assay (Thermo Scientiﬁc, Rockford, IL, USA)

2.6 Data analysis All data reported were based on three independent experiments Median lethal concentrations (LC50s) and corresponding 95% CIs were calculated by Probit anal-ysis using TOXSTAT software (WEST, Inc, 1994) One-way ANOVA was used to

H Ma et al / Environmental Pollution 159 (2011) 1473e1480 1474

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peroxidation between NSL and AALL exposure Correlation between C elegans

mortality and % MB degradation was assessed using Pearson’s correlation

coefﬁcients

3 Results

3.1 Particle characterization

Signi ﬁcant particle aggregation was observed for both

nano-ZnO and bulk-nano-ZnO ( Fig 1 ) A quantitative measurement of

approximately 100 particles indicated an average primary particle

size of 60 25 and 550 256 (Mean SD, n ¼ 100) nm for

nano-ZnO and bulk-nano-ZnO, respectively The average diameters of rotation

for aggregates were 2.79 1.84 and 2.43 1.59 (Mean SD,

n > 100) m m for nano-ZnO and bulk-ZnO, respectively The size

distribution was similar between the two materials ( Fig 2 ) The

working solutions of nano-ZnO and bulk-ZnO had pH values of

6.5 e7.0 Dissolution of nano-ZnO and bulk-ZnO was estimated to be

7.1% and 4.8% respectively after correction for Zn ions recovery in

the ﬁltration system.

3.2 Toxicity of nano-ZnO and bulk-ZnO

Concentration-dependent mortality in C elegans was observed

after 2-h exposure to nano-ZnO or bulk-ZnO under NSL ( Fig 3 (A)

and (B)) At an identical concentration, nano-ZnO caused greater

mortality than ZnO The 2-h LC50s for nano-ZnO and

bulk-ZnO were 38 (95% CI: 30, 45) and 65 (95% CI: 55, 78) mg/l of Zn,

respectively ( Table 1 ) No mortality occurred after 2-h exposure to ZnCl2under NSL For both materials, an extended 22-h exposure under AALL caused a signi ﬁcant increase in mortality compared to the initial 2-h exposure under NSL ( Fig 3 (A) and (B)) With the 2-h NSL and 22-h AALL exposure combined, the 24-h LC50s for nano-ZnO and bulk-nano-ZnO were 17 (95% CI: 10, 25) and 38 (95% CI: 29, 50) mg/l of Zn, respectively ( Table 1 ) Again, this extended exposure under AALL did not cause mortality in C elegans for ZnCl2.

Fig 1 TEM images of nano-ZnO and bulk-ZnO in K-medium (pH¼ 6.8): (A) 10 mg/l nano-ZnO, (B) 100 mg/l nano-ZnO, with an estimated particle size of 60 25 nm (n ¼ 100),

256 nm (n ¼ 100)

Fig 2 Aggregate size distribution of nano-ZnO and bulk-ZnO suspensions using DIC microscopy The average diameters of rotation for these aggregates were 2.79 1.84 and 2.43 1.59 (Mean SD, n > 100)mm for 100 mg/l nano-ZnO and bulk-ZnO, respectively

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Neither nano-ZnO nor bulk-ZnO caused mortality in C elegans

after 2-h exposure under AALL, but both induced mortality after 24-h

exposure ( Fig 3 (A) and (B)) For nano-ZnO, this 24-h toxicity under

AALL was lower than 2-h toxicity under NSL; however, there was no

signi ﬁcant difference between these two types of toxicity for

bulk-ZnO LC50s could not be determined for the 24-h AALL exposure

because of insuf ﬁcient mortality; however, an extrapolation from the

concentration-response curves suggested that the LC50 for

nano-ZnO was approximately 60 e80 mg/l of Zn and that for bulk-ZnO was

even higher No mortality was observed for nano-ZnO or bulk-ZnO

exposure in the dark, regardless of the duration of exposure.

Mortality in control nematodes was below 10% for all tests conducted.

3.3 Methylene blue degradation

Methylene blue degradation was used as an indicator of

pho-tocatalytic activity/ROS generation by the ZnO particles ZnCl2did

not degrade MB, regardless of the illumination method or exposure

duration Under 2-h NSL exposure, concentration-dependent MB

degradation was observed for both nano-ZnO ( Fig 4 (A)) and

bulk-ZnO ( Fig 4 (B)) The maximum MB degradation (%) by nano-ZnO

and bulk-ZnO were 67% and 45%, respectively ( Table 1 ) A strong

positive correlation was found between C elegans mortality and MB

degradation (%) for both nano-ZnO (r ¼ 0.94, p ¼ 0.002, n ¼ 7) and

bulk-ZnO (r ¼ 0.90, p ¼ 0.005, n ¼ 7) MB degradation did not occur

during the extended 22-h exposure under AALL ( Table 1 ) Under

AALL, nano-ZnO and bulk-ZnO degraded MB by 7.5% and 5.2%,

respectively during 2-h exposure, and this degradation did not

increase as the exposure extended to 24 h ( Table 1 ) No MB

degradation by nano-ZnO or bulk-ZnO was observed in the dark,

regardless of the duration of exposure.

3.4 Lipid peroxidation

Elevated lipid peroxidation in C elegans exposed to 25 mg/l

nano-ZnO or bulk-ZnO compared to control was found under all

exposure conditions ( Fig 5 ) Signi ﬁcantly greater lipid peroxidation caused by nano-ZnO than bulk-ZnO was only observed after 2-h NSL exposure Neither nano-ZnO nor bulk-ZnO caused statistically different lipid peroxidation between 2-h NSL and 2-h AALL sure Enhanced lipid peroxidation was also observed when expo-sure was extended from 2 h to 24 h, under both NSL and AALL.

4 Discussion Although several studies have investigated toxicity of ZnO nanoparticles to a variety of environmentally relevant species ( Franklin et al., 2007; Wang et al., 2009; Ma et al., 2009; Reddy

et al., 2007; Brayner et al., 2010 ), few have considered their phototoxicity ( Adams et al., 2006 ) The current study found that phototoxicity of nano-ZnO was substantially enhanced under NSL illumination compared to AALL or in the dark using C elegans as test organism Concurrent photodegradation of MB under NSL sug-gested this phototoxicity is associated with photoactivation/ROS generation of the nanoparticles A strong positive correlation (r ¼ 0.94, p ¼ 0.002, n ¼ 7) between C elegans mortality and MB degradation during 2-h NSL exposure suggested that photocatalytic activity of the nanoparticles may be a predictor for phototoxicity Several studies have documented increased antibacterial activity of ZnO nanoparticles under ambient laboratory light ( Applerot et al., 2009; Jones et al., 2008 ) or natural sunlight ( Adams et al., 2006 )

as compared to dark conditions, presumably due to photoactivation

of the nanoparticles The present study, however, demonstrates that photocatalytic ROS generation by ZnO nanoparticles under NSL can cause mortality to a terrestrial/aquatic organism e the nema-tode C elegans.

Phototoxicity of the ZnO nanoparticles is strictly dependent on illumination mode Nano-ZnO has band gap energy of 3.37 ev, equivalent to a wavelength of 368 nm ( Wang, 2004 ) Upon excita-tion by light with a wavelength less than 368 nm, electron ehole pairs are generated on the ZnO surface The holes in the valence band can react with H2O or hydroxide ions adsorbed on the surface

to produce hydroxyl radicals, and the electrons in the conduction band can reduce O2 to produce superoxide ions ( Maness et al.,

1999 ) Both of these radicals are extremely reactive with contact-ing biological molecules Photoactivation and the consequent phototoxicity of nano-ZnO occurred under NSL but not AALL The spectrum of natural sunlight used in this study can be roughly represented by a typical solar radiation spectrum ( supplemental Fig 1(A) ), although the dosimetry and spectrum of solar radiation

in a particular geographical location can be affected by a number of factors, such as, geographical conditions, atmospheric variability, weather conditions, etc ( Diamond et al., 2002 ) It is estimated that approximately 8 e9% of the total solar energy emitted from the sun

Nano-ZnO (mg/l)

0 20 40 60 80 100 120

Bulk-ZnO (mg/l)

2h NSL 2h AALL 2h NSL+22h AALL 24h AALL

Fig 3 Toxicity of nano-ZnO (A) and bulk-ZnO (B) to C elegans under different illumination method (NSL-natural sunlight, AALL-ambient artiﬁcial laboratory light)

Table 1

LC50s for nano-ZnO and bulk-ZnO and the corresponding maximum degradation of

methylene blue (MB) by the two materials under different illumination method and

exposure time

2-h NSL 2-h AALL 2-h NSL+ 22-h AALL 24-h AALL

LC50 (%95 CI, mg/l Zn)

Nano-ZnO 38 (30,45) NA 17 (10, 25) 60e80

Bulk-ZnO 65 (55,78) NA 38 (29,50) >60e80

Maximum % MB degradation (MeanSEM, n¼3)

Nano-ZnO 66.94.5* 7.53.0 70.35.3* 9.84.2

Bulk-ZnO 44.66.1* 5.21.1 44.9+4.7* 6.43.3

NSL-natural sunlight, AALL-ambient artiﬁcial laboratory light.*p< 0.01

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falls in the UV region of the electromagnetic spectrum (6.3% UVA

(320 e400 nm), 1.5% UVB (290e320 nm)) ( Frederick, 1995 )

There-fore, the natural sunlight could ef ﬁciently activate ZnO

nano-particles and produce ROS, as indicated by signi ﬁcant MB

degradation (70 4%), and consequently cause toxicity to the

nematodes In contrast, ZnO nanoparticles were marginally

acti-vated under AALL, as suggested by a subtle MB degradation of

7 3%, and caused no mortality to the nematodes The AALL used

typical “white cool” ﬂuorescent lamps, and a typical light spectrum

of these lamps ( supplemental Fig 1(B) ) contains negligible amount

of UV ( <400 nm) light It is suggested that UV exposure from sitting

under ﬂuorescent lights for eight hours is equivalent to only one

minute of sun exposure ( Lytle et al., 1993 ) Thus, the AALL cannot

activate the nanoparticles to cause toxicity These ﬁndings have

important implication in risk assessment for photoactive

nano-particles As most toxicity assays are conducted under standard

laboratory conditions, which do not perceive all risk factors;

exposure under natural sunlight conducted in this study represents

at least some range of potential exposure that may occur in the

environment.

It should be noted that the ZnO concentrations tested in

the present study are relatively high compared to those found in

the environment Current estimates of ZnO concentrations in the

environment range from the low m g/l to a few hundred m g/l ( Boxall

et al., 2007 ), and a more recent study by Gottschalk et al (2009)

reported modeled nano-ZnO concentrations in the environment

to be approximately 10 ng/l in natural surface water and 1 m g/l in

treated wastewater However, as ZnO nanoparticles are being widely used in a broad range of applications including personal-care products such as cosmetics and sunscreens, it is expected that their concentrations in the environment will increase continually ( Daughton and Ternes, 1999 ) Furthermore, results from these relatively high concentrations serve as a foundation for further investigation on phototoxicity of these nanoparticles at environ-mental relevant concentrations using more sensitive endpoints at cellular/subcellular and molecular levels.

Elevated lipid peroxidation after 2-h NSL exposure to both nano-ZnO and bulk-nano-ZnO con ﬁrmed that the phototoxicity is related to ROS generation by the ZnO particles and oxidative stress in the nematodes Although the current study did not directly identify the location of the phototoxicity in C elegans, two different modes of action may be proposed First, the ROS generation and resulting toxicity may occur at the surface of the nematodes Surface acting toxicity is not a new concept in ecotoxicology as certain toxicants can be adsorbed to the exterior surface of the organism and elicit toxicity ( Handy et al., 2008a ) This process has been implicated in the toxicity of TiO2nanoparticles to trout ( Federici et al., 2007 ) The

C elegans cuticle has an evenly distributed net negative charge at neutral pH ( Himmelhoch and Zuckerman, 1983 ), which may enhance particle aggregation on the surface of the animals via electrostatic interactions ( Wang et al., 2009 ) Particle aggregates attached onto the nematode cuticle was observed during exposure The C elegans cuticle is an extracellular matrix with a major component of collagen ROS such as superoxide radical anion or hydroxyl radical has been reported to cause damage to calf skin collagen by degrading the protein ( Monboisse and Borel, 1992 ) It is possible that intensive ROS generation by nano-ZnO under NSL caused lethal toxicity to C elegans through damaging the cuticle Another possibility is that nanoparticles were ingested by C elegans and the toxicity occurred internally C elegans unselectively ingest bacteria and ﬁne particles that are less than 5 m m in size ( Donkin and Dusenbery, 1993 ), and its transparency and small body diam-eter allow for adequate UV penetration ( Coohill et al., 1988; Mills and Hartman, 1998 ) A measure of oxidative stress (e.g., lipid per-oxidation) within the nematodes may help to differentiate these two modes of action If ROS generation and phototoxicity occurred internally, oxidative stress would be a good indicator of the observed toxicity Greater lipid peroxidation in the nematodes after 2-h NSL exposure to nano-ZnO was consistent with the greater mortality caused by the nano-ZnO compared to bulk-ZnO, sug-gesting that the internal-acting mechanism might have been involved However, lipid peroxidation alone is not suf ﬁcient to differentiate the two possible modes of action as oxidative stress within a multicellular organism may be mediated by a variety of factors in addition to the photocatalytic ROS generation Studies

0.000 0.050 0.100 0.150 0.200 0.250

Nano-ZnO (mg/l)

0.000 0.050 0.100 0.150 0.200 0.250

Bulk-ZnO (mg/l)

2h NSL 2h AALL 2h NSL+22h AALL 24h AALL

Fig 4 Methylene blue degradation by nano-ZnO (A) and bulk-ZnO (B) under different illumination methods (NSL-natural sunlight, AALL-ambient artiﬁcial laboratory light)

0

0.3

0.6

0.9

1.2

1.5

1.8

2.1

2.4

NSL+22h AALL 24h AALL

Nano

Bulk

∗

Fig 5 Lipid peroxidation in C elegans after exposure to 50 mg/l nano-ZnO and

bulk-ZnO under NSL or AALL (NSL-natural sunlight, AALL-ambient artiﬁcial laboratory light)

(* p < 0.001, compared to control)

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using more direct and speci ﬁc in-situ and/or in vivo techniques for

ROS identi fication including dichlorofluorescein fluorescence assay

(H2DCFDA) ( Xie et al., 2006; Kim et al., 2009 ) and Aminophenyl

ﬂuorescein assay (APF) ( Setsukinai et al., 2003 ) are currently

undergoing to further understand the underlying mechanism of

the phototoxicity.

As phototoxicity of nano-ZnO associated with ROS generation

occurred in as quickly as 2 h under NSL, toxicity of nano-ZnO under

AALL was only observed after 24-h exposure This 24-h toxicity

under AALL was lower than 2-h phototoxicity under NSL ( Table 1 ),

and seemed to be mediated by mechanisms different from

photo-activation as no signi ﬁcant MB degradation occurred concurrently

( Table 1 ) Dissolution and release of ionic zinc from nanoparticulate

ZnO has been recognized as a major contributor to its toxicity to

freshwater alga ( Franklin et al., 2007 ) and ﬁsh embryos ( Zhu et al.,

2008 ) Dissolution of nano-ZnO in the current study was estimated

to be 7.1% at the highest concentration tested, equivalent to 5.6 mg/l

of Zn This amount of zinc does not seem to be able to cause

mortality in C elegans, as aqueous ZnCl2at higher concentrations

did not cause mortality during 24-h exposure Elevated lipid

per-oxidation in C elegans exposed to nano-ZnO under AALL when the

exposure time was extended from 2 h to 24 h was consistent with

the dramatically increased toxicity ( Fig 3 ), suggesting that

oxida-tive stress is involved in this toxicity This non-photocatalytic

toxicity of the nanosized ZnO was also observed during the

extended 22-h exposure under AALL following 2-h NSL exposure,

causing a synergistic effect in C elegans mortality under the test

conditions when both NSL and AALL illumination were involved.

Zhu et al (2009) reported that toxicity of nanosized ZnO on

developing zebra ﬁsh embryos and larvae under ambient laboratory

conditions was not solely a result of particle dissolution; and the

authors proposed that the nano-ZnO aggregates might have elicited

toxicity by increasing ROS formation and/or compromising the

cellular oxidative stress response upon interaction with the

bio-logical system This particle-dependent, non-photocatalytic toxicity

of ZnO nanoparticles to aquatic organisms certainly warrants

further investigation.

Many studies on metal oxide particles such as TiO2 and ZnO

have demonstrated that nanoparticles induce greater toxicity than

their larger counterparts at equivalent mass concentrations, using

test species from rats/mice to bacteria ( Oberdörster et al., 2000;

Oberdöerster et al., 2005; Applerot et al., 2009; Jones et al.,

2008 ) Findings from the present study were in good agreement

with these studies, that the nanosized ZnO had greater MB degra-dation capability and phototoxicity compared to bulk-ZnO at the same mass dose The primary particle size of bulk-ZnO (550 256 nm) was approximately 10 times that of nano-ZnO (60 25 nm), and the latter had a speciﬁc surface area about four times of the former Several authors have suggested that total surface area may be a more appropriate dose metric to describe dose eresponse relationships than mass dose, especially when evaluating poorly soluble particles ( Oberdörster et al., 2000; Jiang

et al., 2008 ) Therefore, the methylene blue degradation and

C elegans mortality after 2-h NSL exposure were also plotted toward total surface area of the two materials ( Fig 6 ) Surprisingly,

MB degradation did not show signi ficant difference between nano-ZnO and bulk-nano-ZnO when normalized to unit surface area, and the bulk-ZnO seemed to be even more toxic than nano-ZnO This suggests that the greater photocatalytic ROS generation and phototoxicity observed in nano-ZnO is a pure effect of surface area rather than “nano-specific” effects (e.g., enhanced surface reac-tivity) This is not surprising given that the size of nano-ZnO particles (60 25 nm) used in this study is still rather large It is mostly at 10 nm and smaller that nano-speci fic effects to start to appear for ZnO particles ( Oberdöerster et al., 2005 ) Similarly,

a comprehensive study of the effect of particle size on ROS gener-ation in anatase TiO2found that a sharp increase in ROS generation per unit surface area occurs only for particles with a size range of

10 e30 nm, and relatively constant ROS generation per unit surface area are observed for particles below 10 nm and above 30 nm ( Jiang

et al., 2008 ).

In addition to primary particle size, aggregation of nanoparticles

in aqueous medium may strongly impact their reactivity ( Handy

et al., 2008b ), nanoparticle ebiological system interactions, and toxicity ( Grassian et al., 2007 ) Aggregate size has been found to be

a determining factor in the uptake and response of immortalized brain microglia to nano-TiO2 ( Long et al., 2006 ) and in the bioavailability of nanoparticles to plant roots, algae, and fungi ( Navarro et al., 2008 ) Nano-ZnO and bulk-ZnO formed similar-sized aggregates (approximately 2 microns) in test solution in the current study, yet the former exhibited greater phototoxicity than the later at an identical mass concentration Therefore, primary particle size appears to be more important than aggregate size in determining phototoxicity of ZnO particles This may be related to the greater accessible surface area of nanosized ZnO than its bulk counterparts in terms of ROS generation.

0

20

40

60

80

100

120

Nano ZnO Bulk ZnO

0.000 0.030 0.060 0.090 0.120

0.150

0.180 0.210

Nano ZnO bulk ZnO

Fig 6 Toxicity of nano-ZnO and bulk-ZnO to C elegans (A) and methylene blue degradation (B) by the two materials after 2-h NSL exposure when dose is expressed as total surface area (NSL-natural sunlight, AALL-ambient artiﬁcial laboratory light)

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5 Conclusions

This paper demonstrates that phototoxicity of nano-ZnO and

bulk-ZnO was dramatically enhanced under natural sunlight

illu-mination as compared to arti ﬁcial laboratory light illumination.

This phototoxicity was well-correlated with photocatalytic ROS

generation of the ZnO particles, suggesting that photocatalytic

activity of such metal oxide nanoparticles may be a predictor of

their phototoxicity This phototoxicity under natural sunlight has

great implications for the environmental risk assessment of such

metal oxide nanoparticles as most toxicity assays are conducted

under standard laboratory conditions which do not perceive all risk

factors, whereas nanoparticles spilled or disposed into the

envi-ronment will inevitably be exposed to sunlight.

This study also found toxicity of both nano-ZnO and bulk-ZnO

under ambient laboratory conditions, which only occurred in

a longer timeframe and was less in magnitude as compared to the

toxicity under natural sunlight This toxicity was not related to

photocatalytic ROS generation Dissolution of the ZnO particles to

ionic zinc did not seem to be a major contributor to the observed

toxicity either This non-photocatalytic, particle-dependent toxicity

warrants further investigation These ﬁndings suggest that toxicity

of nanoparticles may be mediated by multiple mechanisms or

modes of action, depending on the physicochemical properties of

the nanoparticles as well as exposure conditions Toxicity resulting

from either mode of action should not be neglected during the risk

assessment of these nanoparticles.

Lastly, through comparing the phototoxicity of nano-ZnO and

bulk-ZnO by taking into account their primary particle size and

agglomerate size in the test medium, this study suggests that

primary particle size seems to be more important than aggregate

size in determining phototoxicity.

Acknowledgments

This work was supported by the United States Environmental

Protection Agency through Science to Achieve Results Grant

number 832530 The authors acknowledge Dr Stephen Diamond

for his constructive suggestions for improving the manuscript The

authors also acknowledge the Department of Physiology and

Pharmacology at The University of Georgia for use of the Bio-Tek

Synergy 4 microplate reader.

Appendix Supplementary data

Supplementary data related to this article can be found online at

doi:10.1016/j.envpol.2011.03.013

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Tiêu đề	Comparative phototoxicity of nanoparticulate and bulk ZnO to a free-living nematode Caenorhabditis elegans: The importance of illumination mode and primary particle size
Tác giả	H. Ma, N.J. Kabengi, P.M. Bertsch, J.M. Unrine, T.C. Glenn, P.L. Williams
Trường học	The University of Georgia
Chuyên ngành	Environmental Health Science
Thể loại	bài báo
Năm xuất bản	2011
Thành phố	Athens

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