research article toxic effects of nickel oxide bulk and nanoparticles on the aquatic...

Research ArticleToxic Effects of Nickel Oxide Bulk and Nanoparticles onthe Aquatic Plant Lemna gibba L

Abdallah Oukarroum1 Lotfi Barhoumi2 Mahshid Samadani1 and David Dewez1

1Departement de Chimie Universite du Quebec a Montreal CP 8888 Succursale Centre-Ville Montreal QC Canada H3C 3P82Faculte des Sciences de Bizerte Universite de Carthage Jarzouna 7021 Bizerte Tunisia

Correspondence should be addressed to David Dewez dewezdaviduqamca

Received 25 June 2014 Revised 14 September 2014 Accepted 29 September 2014

Academic Editor Rishi Shanker

Copyright copy 2015 Abdallah Oukarroum et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The aquatic plant Lemna gibba L was used to investigate and compare the toxicity induced by 30 nm nickel oxide nanoparticles(NiO-NPs) and nickel(II) oxide as bulk (NiO-Bulk) Plants were exposed during 24 h to 0ndash1000mgL of NiO-NPs or NiO-BulkAnalysis of physicochemical characteristics of nanoparticles in solution indicated agglomerations of NiO-NPs in culture mediumand a wide size distribution was observed Both NiO-NPs and NiO-Bulk caused a strong increase in reactive oxygen species(ROS) formation especially at high concentration (1000mgL)These results showed a strong evidence of a cellular oxidative stressinduction caused by the exposure to NiO Under this condition NiO-NPs and NiO-Bulk induced a strong inhibitory effect onthe PSII quantum yield indicating an alteration of the photosynthetic electron transport performance Under the experimentalconditions used it is clear that the observed toxicity impact was mainly due to NiO particles effect Therefore results of this studypermitted determining the use of ROS production as an early biomarker of NiO exposure on the aquatic plant model L gibba usedin toxicity testing

1 Introduction

The specific properties of engineered metallic nanoparti-cles (NPs) are directly related to their nanosize chemicalcomposition shape and surface charge which permitted anextensive range of application in nanotechnology industriesHowever these NPs received recently considerable attentionin order to manage their safe use and several studies con-cerning the characterization of NPs demonstrated potentialcytotoxicity for many aquatic organisms by causing a cellularoxidative stress effect [1ndash3] Indeed it was suggested for thetoxicity mechanism and intensity of NPs suspension to behighly dependent on their physicochemical proprieties inaqueous solution Several studies showed that the toxicity ofNPs was directly related to their intrinsic properties of mate-rial itself such as their relative hydrodynamic size concen-tration surface chemistry shape and the chemical character-istics of the exposure medium (eg pH and ionic strength)[4ndash7] Several studies reported that the toxicity mecha-nism was related to the release of ions from nanoparticles

andor their direct interaction to membranes causinginhibitory effects on cellular functions [8ndash10] Howeverothers studies suggested that their toxic effects were directlyrelated to NPs penetration and oxidation into the cellularsystem [11ndash13] These NPs will inevitably be released intoaquatic ecosystems throughwastewater output alteringwaterquality and representing a risk of toxicity for freshwaterorganisms Particularly the effects of NPs on aquatic plantshave been widely discussed in recent years since they are eco-logically at the food base of trophic chains in the ecosystem[1 14]Therefore the exposition of NPs in aquatic ecosystemsrepresents a biological risk of toxicity which needs to beevaluated at cellular level for aquatic plants by considering thebioavailability and the uptake of NPs in aqueous solution

Nickel oxide nanoparticles (NiO-NPs) possess uniqueproperties compared to its bulk Nickel(II) oxide (NiO-Bulk)which can be used in industrial products promoting inno-vative applications [15] Recently it has been reportedthatNiO-NPs were able to be easily transported into biologicalsystems inducing both cytotoxic and genotoxic effects [16]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 501326 7 pageshttpdxdoiorg1011552015501326

2 BioMed Research International

However the bioaccumulation effect of NiO-NPs on aquaticplant system has not been extensively examined since onlyvery few data are available concerning their toxicity

In this toxicological study we used the duckweed Lemnagibba L as a model organism because of its rapid growthsmall size and floating leaves having a high capacity to uptakecontaminants Recently duckweeds were widely used for thetesting of substances based on growth inhibition [17] sincethey were known for a long time to be widespread aquaticmacrophytes as a source of food for many aquatic organismsof higher trophic level [18] Therefore the aim of this workwas to evaluate and compare the toxic effects of NiO-NPs andNiO-bulk by using Lemna gibba as an aquatic bioindicatorParticularly we investigated the induction of ROS and thechange of the photosynthetic activity when plants wereexposed during 24 h to a range of concentrations between0 and 1000mgL Therefore we were able to determine theuse of the production of ROS as an early biomarker of NiOexposure before the deterioration of photosynthesis and plantgrowth Finally this study permitted defining the toxicity riskof NiO-NPs suspensions and NiO-bulk on the viability ofduckweeds

2 Material and Methods21 BiologicalMaterial Theaquatic plant Lemna gibba L wasobtained from the Canadian Phycological Culture Centre(formerly UTCC 310) Plants were grown in an inorganicculture medium according to the Organisation for EconomicCooperation and Development (OECD) guidelines (AAPgrowth medium) This nutritive medium consisted ofthe following salts NaNO

3 510mgL MgCl

2sdot6H2O

240mgL CaCl2sdot2H2O 90mgL MgSO

4sdot7H2O 290mgL

K2HPO4sdot3H2O 30mgL H

3BO3 37mgL MnCl

2sdot4H2O

83mgL FeCl3sdot6H2O 32mgL Na

2EDTAsdot2H

2O 60mgL

ZnCl2 66 120583gL CoCl

2sdot6H2O 29 120583gL Na

2MoO4sdot2H2O

145 120583gL CuCl2sdot2H2O 024120583gL NaHCO

3 300mgL

Before the medium was autoclaved for sterilization its pHwas adjusted to 65plusmn01 using 01MHCl Ionic strength for Lgibba culture medium was 425 10minus3 and was calculated withchemical equilibrium model software Visual MINTEQ 30

Experiments were done in a growing chamber CONV-IRON (Controlled Environments Limited Winnipeg MBCanada) having a 168 h lightdark photoperiod A light irra-diance of 100 120583mol of photons mminus2 sminus1 was provided by coolwhite fluorescent lamps (Sylvania GRO-LUX F40GSWS)

22 Nickel Oxide Nanoparticles and Nickel(II) OxideNickel(II) oxide was purchased from BDH LaboratorySupplies (UK) and it was used as NiO-Bulk Nearly sphericalnickel nanopowder (NiO-NPs) was purchased from MTICorporation (Richmond CA USA) According to themanufacturer the diameter of NiO-NPs was 30 nm puritywas 999 and the specific surface area was 50ndash80m2gIn this study NiO-NPs size distribution in L gibba culturemedium was determined by dynamic light scattering (DLS)with a ZetaPlus Particle Sizer (Brookhaven InstrumentsCorporation USA) using 90Plus Particles Sizing SoftwareVer 420 A stock suspension of 1000mgL was prepared and

sonicated before use during 3min with an ultrasonicatorin order to homogenize the suspension of nanoparticlesZeta potential in the culture media was determined bythe electrophoretic mobility method with the ZetaPlussystem Measurements were done at 24 h of exposure inL gibba medium under growth conditions NiO-NPs weresuspended in L gibba culture medium to a concentration of1mgmL and absorbance spectra were measured by usinga UV-VIS spectrophotometer (Lambda 40 Perkin Elmer)To determine the solubility of NiO-NPs suspensions of0ndash1000mgL were prepared and incubated for 24 h in thecondition as described above for algal culture NiO-NPssuspensions were then centrifuged at 12000 g for 30minThe supernatant was removed with care and filtered throughcellulose filters (022120583m) in order to remove before analysisany possible agglomerated nanoparticulate matter Thequantification of soluble Ni was done by atomic absorptionspectrometry using a Varian SpectrAA 220 FS system

23 Treatment Conditions Triple-fronded L gibba plants inthe exponential growth phase were used for experimentsFive triple-fronded L gibba plants in three replicates wereexposed during 24 h in Petri dishes containing growthmedium having initial NiO-NPs or NiO-Bulk concentrationsof 0 1 10 100 and 1000mgL under the same condition asdescribed above

24 Reactive Oxygen Species (ROS) Formation The ROS for-mation was measured by using the cell permeable indicator2101584071015840dichlorodihydrofluorescein diacetate (H

2DCFDA) [19]

Cellular esterases hydrolyze the probe to the nonfluorescent2101584071015840dichlorodihydrofluorescein (H

2DCF) which is better

retained in the cell In the presence of ROS and cellularperoxidases H

2DCF is transformed to the highly fluorescent

2101584071015840dichlorofluorescein (DCF) Plants were washed threetimes with half-strength Hutnerrsquos medium and treated with5 120583M H

2DCFDA for 30min at 25∘C [20] Each treatment

and control were treated with 5 120583M of H2DCFDA in 1mL of

solutionTheDCFfluorescence signal wasmeasured using anexcitation wavelength at 485 nm and an emission wavelengthat 530 nm The DCF fluorescence was measured directly onintact plants and the fluorescence signal was normalizedby fresh weight All fluorescence data were collected usinga fluorescence plate reader (SpectraMax M2e Multi-ModeMicroplate Reader)

25 Chl 119886 Fluorescence Transients Chl 119886 fluorescence mea-surements were conducted at room temperature with aportable fluorimeter ldquoPlant Efficiency Analyzerrdquo (Handy-PEA Hansatech Instruments Ltd Kingrsquos Lynn Norfolk UK)All L gibba plants were dark adapted for at least 15min beforethe measurements were started to allow all PSII reaction cen-ters to be in open state (reoxidized) and the electron transportchain to be fully oxidized The measurement consisted of asingle strong 1 s light pulse of 3000120583mol of photons mminus2 sminus1and excitation intensity sufficient to ensure closure of all PSIIreaction centers which was provided by an array of threeultrabright red light-emitting diodes (peak at 650 nm) in theinstrument

BioMed Research International 3

Diameter (nm)

Part

icle

s fra

ctio

n

0

20

40

60

80

100

0 200 400 600 800 1000 1200 1400

24h 596

(a)

0

05

1

15

2

25

3

400 500 600 700 800

Nanopure water

L gibba culture medium

Abso

rban

ce (r

el u

nits)

Wavelength (nm)

(b)

Figure 1 (a) Size distribution of NiO-NPs suspensions prepared in culture medium of L gibba and measured at 24 h of incubation (b)Absorbance spectra of NiO-NPs suspensions in L gibba culture medium (1mgmL)

26 Fluorescence Parameters Based on the fluorescence tran-sients measured during the first second of illuminationseveral biophysical expressions leading to a description ofa photosynthetic sample in a given physiological state wereevaluated One of the parameters calculated is the maximumyield of primary photochemistry of PSII (119865

119881119865

119872) It corre-

sponds to the efficiency by which an absorbed photon willbe trapped by PSII reaction centers [21] The absorption ofphotons (ABS) per active reaction center (RC) showing theantenna size was estimated by the ratio ABSRC The per-formance index of PSII activity (PI) which consider all PSIIphotochemical reactions was one of the Chl 119886 fluorescenceparameters providing useful and quantitative informationabout the complete state of photosynthesis and the organismvitality The formula expression of the performance index ofPSII activity was well explained in reference [22]

27 Data Analysis and Statistics For all treatments meanswere determined for each treatment condition Significantdifferences between control and treated samples were deter-mined by using one-way analysis of variance (ANOVA) andmultiple comparison of Tukeyrsquos test where 119875 values less than005 (119875 lt 005) were considered to be significantly different

3 Results and Discussion

Since metallic nanoparticles represent hazardous aquaticcontaminants the toxic potential of NiO-NPs needs to bedetermined for the development of bioassays used in toxicityrisk assessment In this regard the aquatic plant L gibbarepresents a sensitive organism in toxicity testing In thistoxicological investigation we clearly showed the potentialsource of toxicity of NiO-NPs suspensions in comparison to

NiO-Bulk Indeed nickel(II) oxide is known to be practicallyinsoluble in water limiting considerably the release of freeionic Ni and its bioavailability to aquatic organisms On theother hand the physicochemical conditions of the aqueoussolution may alter the properties of NiO-NPs modifyingtheir bioavailability by changing their surface of contactcharge and solubility Particle size measurements showedthat NiO-NPs formed rapidly a wide size distribution ofagglomerations when suspended in L gibba culture mediumwhich was found to be stable in the culture medium duringthe entire experimental exposure (Figure 1) The mediandiameter of particle size distribution for nanoparticles sus-pension in culturemediumwas 596 nm Such agglomerationswere formed due to physicochemical properties of the Lgibba culture medium such as the pH of 65 and thecomposition of nutrients causing an ionic strength of 425 times10minus3 Such conditions were affecting the interaction of NiO-NPs with inorganic ions by changing the surface charge ofnanoparticlesHere we noted that the charge at NiO-NPssurface was negative in L gibba culture medium equallingminus2681 (plusmn05) Indeed it has been previously reported thatnanoparticles were forming agglomerates in testing mediaby changing the surface charge of nanoparticles due toionic strength [7 14 23 24] Moreover the formation ofagglomerates was affecting the solubility of NiO-NPs in Lgibba culture medium since our results indicated very lowconcentrations of ionic form of nickel in the soluble fractionin comparison to total mass of nanoparticles (Table 1) Suchlow solubility of NiO-NPs was also observed when theywere suspended in other culture media [25 26] Indeed theformation of agglomerates caused their sedimentation at thebottom of the experimental flask decreasing their surfacecontact with the medium and their solubilization Therefore


Table 1 Change in dissolved free ionic nickel (in mgL) releasedfrom different concentrations of NiO-NPs suspensions in L gibbaculture medium at 24 h of treatment

[NiO-NPs] mgL [Free ionic Ni] mgL1 na10 00510 plusmn 005100 110 plusmn 061000 593 plusmn 02

0

200

400

600

800

1000

1200

1400

0 1 10 100 1000

ROS

indu

ctio

n (

to co

ntro

l)

Concentration (mgL)

NiO-NPsNiO-Bulk

lowast

lowastlowastlowast

Figure 2 Change in the production of reactive oxygen species(ROS) for L gibba plants exposed during 24 h to different concen-trations of NiO-Bulk or NiO-NPs suspensions Asterisks indicatestatistical significance between control and treatment (119875 lt 005)

toxic effects of NiO-NPs need to be directly related to theirphysicochemical characteristics such as their solubility in theL gibba culture medium

For environmental protection agencies the increaserelease of nanoparticles into the aquatic environment repre-sents a risk for the integrity of the food web through theirinteractions with various microorganisms invertebrates andfish With such concern many studies showed the evidenceof the bioaccumulation effects of metallic nanoparticles inaquatic organisms inwhich the cytotoxicity impact was espe-cially indicated by the induction of oxidative stress and theinhibition of growth [27] In the present study our bioassay oftoxicity focused on the production of intracellular ROS usingthe plant model L gibba Indeed the production of ROS wasused as an early and rapid biomarker of the presence of NiO-NPs cytotoxicity already at 24 h of treatment It is importantto notice that the visible spectrum of NiO-NPs (in Figure 1)showed no specific band at 530 nm suggesting that the ROSformation was mainly due to the highly fluorescent com-pound 2101584071015840-dichlorofluorescein (DCF)Our results indicateda significant increase in intracellular ROS concentrationswhen L gibba plants were exposed to NiO-NPs suspensions(Figure 2) Particularly the ROS formation increased by 90

compared to control (119875 lt 005) at the lowest concentrationof NiO-NPs Moreover the strongest fluorescence signal ofthe ROS sensor (H

2DCFDA) was found for L gibba plant

exposed to 1000mgL indicating an increased production ofROS by 5 times compared to control (119875 lt 005) Concerningthe effect of NiO-Bulk the production of ROS was similarto NiO-NPs under the testing exposure concentration rangeof 1ndash100mgL However when L gibba plant was exposed to1000mgL of NiO-Bulk or NiO-NPs the production of ROSwas 2 times higher for NiO-Bulk in comparison to NiO-NPsThese results clearly indicated that the induction of oxidativestress toxicity was not directly related to NiO-NPs solubilityTherefore NiO-NPs were uptaken into L gibba plant systemcausing the production of intracellular ROS directly throughphotocatalysis or indirectly through molecular cell damagesIn an earlier study researchers explored the bioaccumula-tion effects of NiO-NPs on algal cells of Chlorella vulgaris[26] They observed that NiO-NPs formed deposits in thecytoplasm which provoked growth inhibition and cellularultrastructure alterations after 72 h of exposure to 10ndash50mgLof NiO-NPs However our results indicated that NiO-NPspossess a different toxicitymechanism in comparison to othermetallic nanoparticles for which it was suggested that theirsolubilization into free metal ions was the main mechanismof toxicity [10 28]

Moreover the toxic effect of NiO-NPs was investigatedon the photosynthetic functions and compared to NiO-Bulkeffects The chlorophyll fluorescence rise measured from allleaves displayed the typical OJIP transients when plottedon a logarithmic time scale (Figure 3) The fluorescencetransients showed two steps between O and P the J transientat about 2ms and the I transient at 30ms It was previouslydemonstrated that theO-J phasewas strongly light dependenton the reduction of quinoneQA [22] and the J-P rise reflectedthe reduction of the rest of the electron transport chain afterQA [29] When L gibba plants were exposed to 1000mgL ofNiO-NPs or NiO-Bulk effects it was noticed that the induc-tion of the OJIP fluorescence transients differed significantlyin comparison to control plants (Figure 3) Under this experi-mental condition results demonstrated an inhibitory effect ofNiO-NPs and NiO-Bulk on the photochemical activity of thephotosystem II (PSII) which was indicated by the reductionin the quantum yield of PSII electron transport (Figure 4)This alteration suggested damages to the structural andfunctional properties of PSII Furthermore the increase ofABSRC ratio indicated an increase in the inactivation ofPSII reaction centers which was related to a downregulationmechanism to dissipate the excess of excitation energy fromchlorophyll molecules (Figure 4) Similarly the reductionof the overall process of PSII photochemistry and electrontransport activity shown by the decrease in PI parametervalue indicated that both NiO-NPs and NiO-Bulk were ableto alter directly the photosynthetic performance of L gibbaplant

Previously an in vitro study on PSII submembrane frac-tions isolated from spinach showed that NiCl

2at millimolar

range of concentration had a strong inhibitory effect onPSII reaction center functions and oxygen evolution activity[30] In another study using two duckweed species Spirodela


0

1

2

3

4

5

6 NiO-NPsFl

uore

scen

ce in

tens

ity (r

el u

nits)

Time (ms)001 01 1 10 100 1000

O

J

I

P

(a)

Control1

10

100

1000

Fluo

resc

ence

inte

nsity

(rel

uni

ts)

Time (ms)

0

1

2

3

4

5

6

001 01 1 10 100 1000

NiO-Bulk

(b)

Figure 3 Fast Chl 119886 fluorescence OJIP transients exhibited when L gibba plants were exposed to 0 1 10 100 and 1000mgL of NiO-Bulk orNiO-NPs suspensions (119899 = 5) The symbols used are O J I and P representing respectively the fluorescence intensities at 50 120583s 2ms 30msand 200ms

0

20

40

60

80

100

120 FVFM

Concentration (mgL)1 10 100 1000

lowast

Chl a

fluo

resc

ence

par

amet

ers v

alue

( o

f con

trol)

(a)

0

50

100

150

200

250ABSRC


lowast

Chl a

fluo

resc

ence

par

amet

ers v

alue

( o

f con

trol)

(b)

Concentration (mgL)

NiO-NPsNiO-Bulk

0

20

40

60

80

100

120

140

1 10 100 1000

PI

lowastChl a

fluo

resc

ence

par

amet

ers v

alue

( o

f con

trol)

(c)

Figure 4 Change in the Chl 119886 fluorescence parameters such as the maximum quantum yield of primary photochemistry (119865119881119865

119872) and the

ratio between light-harvesting chlorophyll antenna size and the number of active PSII reaction centers (ABSRC) and the performance indexof PSII activity (PI) for L gibba plants exposed during 24 h to different concentrations of NiO-Bulk or NiO-NPs suspensions (119899 = 5)


polyrhiza clone SJ and Lemna minor clone St it was shownthat NiCl

2(the most soluble form of Ni) induced a strong

decrease in the contents of both chlorophylls 119886 and b and alsoan inhibition of fresh weight over a 7-day test period [31]However our toxicological investigation indicated that overa 24 h toxicity testing period the toxicity impact of NiO wasnot induced by the free ionic form of Ni Nevertheless undera longer time of treatment such as 7 days it is possible that thesoluble fraction containing free ionic Ni may also contributeto the cellular toxic effects

In this study we clearly showed the potential sourceof toxicity of NiO-NPs suspensions in comparison to NiO-Bulk The aquatic plant Lemna gibba demonstrated to bea valuable bioindicator of NiO cellular toxicity which wasindicated by the deterioration of photochemical activities ofphotosynthesis and the induction of oxidative stress Underthe experimental conditions used it is clear that the observedtoxicity impact was mainly due to NiO particles effectTherefore results of this study permitted determining theuse of ROS production as an early toxicity biomarker of NiOexposure to L gibba plant

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors acknowledge the financial support providedby NSERC National Research Council of Canada as anindividual Discovery Grant to Professor David Dewez

References

[1] S J Klaine P J J Alvarez G E Batley et al ldquoNanomaterialsin the environment behavior fate bioavailability and effectsrdquoEnvironmental Toxicology andChemistry vol 27 no 9 pp 1825ndash1851 2008

[2] I Bhatt and B N Tripathi ldquoInteraction of engineered nanopar-ticles with various components of the environment and possiblestrategies for their risk assessmentrdquo Chemosphere vol 82 no 3pp 308ndash317 2011

[3] J R Peralta-Videa L Zhao M L Lopez-Moreno G de la RosaJ Hong and J L Gardea-Torresdey ldquoNanomaterials and theenvironment a review for the biennium 2008ndash2010rdquo Journal ofHazardous Materials vol 186 no 1 pp 1ndash15 2011

[4] R J Griffitt J Luo J Gao J-C Bonzongo and D S BarberldquoEffects of particle composition and species on toxicity ofmetallic nanomaterials in aquatic organismsrdquo EnvironmentalToxicology and Chemistry vol 27 no 9 pp 1972ndash1978 2008

[5] Y Ju-Nam and J R Lead ldquoManufactured nanoparticles anoverview of their chemistry interactions and potential environ-mental implicationsrdquo Science of the Total Environment vol 400no 1ndash3 pp 396ndash414 2008

[6] T XiaMKovochichM Liong et al ldquoComparison of themech-anism of toxicity of zinc oxide and cerium oxide nanoparticlesbased on dissolution and oxidative stress propertiesrdquoACSNanovol 2 no 10 pp 2121ndash2134 2008

[7] J Jiang G Oberdorster and P Biswas ldquoCharacterization ofsize surface charge and agglomeration state of nanoparticledispersions for toxicological studiesrdquo Journal of NanoparticleResearch vol 11 no 1 pp 77ndash89 2009

[8] C Levard E M Hotze G V Lowry and G E BrownldquoEnvironmental transformations of silver nanoparticles impacton stability and toxicityrdquo Environmental Science and Technologyvol 46 no 13 pp 6900ndash6914 2012

[9] A Oukarroum L Barhoumi L Pirastru and D Dewez ldquoSilvernanoparticle toxicity effect on growth and cellular viability ofthe aquatic plant Lemna gibbardquo Environmental Toxicology andChemistry vol 32 no 4 pp 902ndash907 2013

[10] F Perreault M Samadani and D Dewez ldquoEffect of solublecopper released from copper oxide nanoparticles solubilisationon growth and photosynthetic processes of Lemna gibba LrdquoNanotoxicology vol 8 no 4 pp 374ndash382 2014

[11] J Fabrega S R Fawcett J C Renshaw and J R LeadldquoSilver nanoparticle impact on bacterial growth effect of pHconcentration and organic matterrdquo Environmental Science andTechnology vol 43 no 19 pp 7285ndash7290 2009

[12] L Y Yin Y W Cheng B Espinasse et al ldquoMore than theions the effects of silver nanoparticles on Lolium multiflorumrdquoEnvironmental Science and Technology vol 45 no 6 pp 2360ndash2367 2011

[13] F Perreault R Popovic and D Dewez ldquoDifferent toxicitymechanisms between bare and polymer-coated copper oxidenanoparticles in Lemna gibbardquo Environmental Pollution vol185 pp 219ndash227 2014

[14] I Rodea-Palomares K Boltes F Fernandez-Pinas et alldquoPhysicochemical characterization and ecotoxicological assess-ment of CeO

2nanoparticles using two aquatic microorgan-

ismsrdquo Toxicological Sciences vol 119 no 1 pp 135ndash145 2011[15] P Song D Wen Z X Guo and T Korakianitis ldquoOxidation

investigation of nickel nanoparticlesrdquoPhysical ChemistryChem-ical Physics vol 10 no 33 pp 5057ndash5065 2008

[16] R Magaye and J Zhao ldquoRecent progress in studies of metallicnickel and nickel-based nanoparticlesrsquo genotoxicity and car-cinogenicityrdquo Environmental Toxicology and Pharmacology vol34 no 3 pp 644ndash650 2012

[17] OECD ldquoLemna sp Growth Inhibition Testrdquo Guidelines for thetesting of Chemicals OECD 2006

[18] M A Lewis ldquoUse of freshwater plants for phytotoxicity testinga reviewrdquo Environmental Pollution vol 87 no 3 pp 319ndash3361995

[19] I B Gerber and I A Dubery ldquoFluorescence microplate assayfor the detection of oxidative burst products in tobacco cellsuspensions using 2101584071015840-dichlorofluoresceinrdquo Methods in CellScience vol 25 no 3-4 pp 115ndash122 2004

[20] T S Babu T A Akhtar M A Lampi S TripuranthakamD G Dixon and B M Greenberg ldquoSimilar stress responsesare elicited by copper and ultraviolet radiation in the aquaticplant Lemna gibba Implication of reactive oxygen species ascommon signalsrdquo Plant and Cell Physiology vol 44 no 12 pp1320ndash1329 2003

[21] G H Krause and E Weis ldquoChlorophyll fluorescence andphotosynthesis the basicsrdquo Annual Review of Plant Physiologyand Plant Molecular Biology vol 42 no 1 pp 313ndash349 1991

[22] R J Strasser A Srivastava and M Tsimilli-Michael ldquoAnalysisof the chlorophyll a fluorescence transientrdquo inChlorophyll Fluo-rescence A Signature of Photosynthesis G G Papageorgiou Ed


vol 19 of Advances in Photosynthesis and Respiration pp 321ndash362 Kluwer Academic Publishers Dodrecht The Netherlands2004

[23] E J Gubbins L C Batty and J R Lead ldquoPhytotoxicity of silvernanoparticles to Lemna minor Lrdquo Environmental Pollution vol159 no 6 pp 1551ndash1559 2011

[24] A Oukarroum S Bras F Perreault and R Popovic ldquoInhibitoryeffects of silver nanoparticles in two green algae Chlorellavulgaris and Dunaliella tertiolectardquo Ecotoxicology and Environ-mental Safety vol 78 pp 80ndash85 2012

[25] C Ispas D Andreescu A Patel D V Goia S Andreescuand K N Wallace ldquoToxicity and developmental defects ofdifferent sizes and shape nickel nanoparticles in zebrafishrdquoEnvironmental Science and Technology vol 43 no 16 pp 6349ndash6356 2009

[26] N Gong K Shao W Feng Z Lin C Liang and Y SunldquoBiotoxicity of nickel oxide nanoparticles and bio-remediationby microalgae Chlorella vulgarisrdquo Chemosphere vol 83 no 4pp 510ndash516 2011

[27] S Ma and D Lin ldquoThe biophysicochemical interactions atthe interfaces between nanoparticles and aquatic organismsadsorption and internalizationrdquo Environmental Sciences Pro-cesses amp Impacts vol 15 no 1 pp 145ndash160 2013

[28] S K Misra A Dybowska D Berhanu S N Luoma and EValsami-Jones ldquoThe complexity of nanoparticle dissolution andits importance in nanotoxicological studiesrdquo Science of the TotalEnvironment vol 438 pp 225ndash232 2012

[29] G Schansker S Z Toth and R J Strasser ldquoMethylviologen anddibromothymoquinone treatments of pea leaves reveal the roleof photosystem I in the Chl 119886 fluorescence riseOJIPrdquoBiochimicaet Biophysica Acta (BBA)mdashBioenergetics vol 1706 no 3 pp250ndash261 2005

[30] S Boisvert D Joly S Leclerc S Govindachary J Harnoisand R Carpentier ldquoInhibition of the oxygen-evolving complexof photosystem II and depletion of extrinsic polypeptides bynickelrdquo BioMetals vol 20 no 6 pp 879ndash889 2007

[31] K-J Appenroth K Krech A Keresztes W Fischer and HKoloczek ldquoEffects of nickel on the chloroplasts of the duck-weeds Spirodela polyrhiza and Lemna minor and their possibleuse in biomonitoring and phytoremediationrdquoChemosphere vol78 no 3 pp 216ndash223 2010

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Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


However the bioaccumulation effect of NiO-NPs on aquaticplant system has not been extensively examined since onlyvery few data are available concerning their toxicity

In this toxicological study we used the duckweed Lemnagibba L as a model organism because of its rapid growthsmall size and floating leaves having a high capacity to uptakecontaminants Recently duckweeds were widely used for thetesting of substances based on growth inhibition [17] sincethey were known for a long time to be widespread aquaticmacrophytes as a source of food for many aquatic organismsof higher trophic level [18] Therefore the aim of this workwas to evaluate and compare the toxic effects of NiO-NPs andNiO-bulk by using Lemna gibba as an aquatic bioindicatorParticularly we investigated the induction of ROS and thechange of the photosynthetic activity when plants wereexposed during 24 h to a range of concentrations between0 and 1000mgL Therefore we were able to determine theuse of the production of ROS as an early biomarker of NiOexposure before the deterioration of photosynthesis and plantgrowth Finally this study permitted defining the toxicity riskof NiO-NPs suspensions and NiO-bulk on the viability ofduckweeds

2 Material and Methods21 BiologicalMaterial Theaquatic plant Lemna gibba L wasobtained from the Canadian Phycological Culture Centre(formerly UTCC 310) Plants were grown in an inorganicculture medium according to the Organisation for EconomicCooperation and Development (OECD) guidelines (AAPgrowth medium) This nutritive medium consisted ofthe following salts NaNO

3 510mgL MgCl

2sdot6H2O

240mgL CaCl2sdot2H2O 90mgL MgSO

4sdot7H2O 290mgL

K2HPO4sdot3H2O 30mgL H

3BO3 37mgL MnCl

2sdot4H2O

83mgL FeCl3sdot6H2O 32mgL Na

2EDTAsdot2H

2O 60mgL

ZnCl2 66 120583gL CoCl

2sdot6H2O 29 120583gL Na

2MoO4sdot2H2O

145 120583gL CuCl2sdot2H2O 024120583gL NaHCO

3 300mgL

Before the medium was autoclaved for sterilization its pHwas adjusted to 65plusmn01 using 01MHCl Ionic strength for Lgibba culture medium was 425 10minus3 and was calculated withchemical equilibrium model software Visual MINTEQ 30

Experiments were done in a growing chamber CONV-IRON (Controlled Environments Limited Winnipeg MBCanada) having a 168 h lightdark photoperiod A light irra-diance of 100 120583mol of photons mminus2 sminus1 was provided by coolwhite fluorescent lamps (Sylvania GRO-LUX F40GSWS)

22 Nickel Oxide Nanoparticles and Nickel(II) OxideNickel(II) oxide was purchased from BDH LaboratorySupplies (UK) and it was used as NiO-Bulk Nearly sphericalnickel nanopowder (NiO-NPs) was purchased from MTICorporation (Richmond CA USA) According to themanufacturer the diameter of NiO-NPs was 30 nm puritywas 999 and the specific surface area was 50ndash80m2gIn this study NiO-NPs size distribution in L gibba culturemedium was determined by dynamic light scattering (DLS)with a ZetaPlus Particle Sizer (Brookhaven InstrumentsCorporation USA) using 90Plus Particles Sizing SoftwareVer 420 A stock suspension of 1000mgL was prepared and

sonicated before use during 3min with an ultrasonicatorin order to homogenize the suspension of nanoparticlesZeta potential in the culture media was determined bythe electrophoretic mobility method with the ZetaPlussystem Measurements were done at 24 h of exposure inL gibba medium under growth conditions NiO-NPs weresuspended in L gibba culture medium to a concentration of1mgmL and absorbance spectra were measured by usinga UV-VIS spectrophotometer (Lambda 40 Perkin Elmer)To determine the solubility of NiO-NPs suspensions of0ndash1000mgL were prepared and incubated for 24 h in thecondition as described above for algal culture NiO-NPssuspensions were then centrifuged at 12000 g for 30minThe supernatant was removed with care and filtered throughcellulose filters (022120583m) in order to remove before analysisany possible agglomerated nanoparticulate matter Thequantification of soluble Ni was done by atomic absorptionspectrometry using a Varian SpectrAA 220 FS system

23 Treatment Conditions Triple-fronded L gibba plants inthe exponential growth phase were used for experimentsFive triple-fronded L gibba plants in three replicates wereexposed during 24 h in Petri dishes containing growthmedium having initial NiO-NPs or NiO-Bulk concentrationsof 0 1 10 100 and 1000mgL under the same condition asdescribed above

24 Reactive Oxygen Species (ROS) Formation The ROS for-mation was measured by using the cell permeable indicator2101584071015840dichlorodihydrofluorescein diacetate (H

2DCFDA) [19]

Cellular esterases hydrolyze the probe to the nonfluorescent2101584071015840dichlorodihydrofluorescein (H

2DCF) which is better

retained in the cell In the presence of ROS and cellularperoxidases H

2DCF is transformed to the highly fluorescent

2101584071015840dichlorofluorescein (DCF) Plants were washed threetimes with half-strength Hutnerrsquos medium and treated with5 120583M H

2DCFDA for 30min at 25∘C [20] Each treatment

and control were treated with 5 120583M of H2DCFDA in 1mL of

solutionTheDCFfluorescence signal wasmeasured using anexcitation wavelength at 485 nm and an emission wavelengthat 530 nm The DCF fluorescence was measured directly onintact plants and the fluorescence signal was normalizedby fresh weight All fluorescence data were collected usinga fluorescence plate reader (SpectraMax M2e Multi-ModeMicroplate Reader)

25 Chl 119886 Fluorescence Transients Chl 119886 fluorescence mea-surements were conducted at room temperature with aportable fluorimeter ldquoPlant Efficiency Analyzerrdquo (Handy-PEA Hansatech Instruments Ltd Kingrsquos Lynn Norfolk UK)All L gibba plants were dark adapted for at least 15min beforethe measurements were started to allow all PSII reaction cen-ters to be in open state (reoxidized) and the electron transportchain to be fully oxidized The measurement consisted of asingle strong 1 s light pulse of 3000120583mol of photons mminus2 sminus1and excitation intensity sufficient to ensure closure of all PSIIreaction centers which was provided by an array of threeultrabright red light-emitting diodes (peak at 650 nm) in theinstrument


Diameter (nm)

Part

icle

s fra

ctio

n

0

20

40

60

80

100

0 200 400 600 800 1000 1200 1400

24h 596

(a)

0

05

1

15

2

25

3

400 500 600 700 800

Nanopure water


Abso

rban

ce (r

el u

nits)

Wavelength (nm)

(b)



119881119865

119872) It corre-









0

200

400

600

800

1000

1200

1400

0 1 10 100 1000

ROS

indu

ctio

n (

to co

ntro

l)

Concentration (mgL)

NiO-NPsNiO-Bulk

lowast

lowastlowastlowast









2at millimolar



0

1

2

3

4

5

6 NiO-NPsFl

uore

scen

ce in

tens

ity (r

el u

nits)

Time (ms)001 01 1 10 100 1000

O

J

I

P

(a)

Control1

10

100

1000

Fluo

resc

ence

inte

nsity

(rel

uni

ts)

Time (ms)

0

1

2

3

4

5

6

001 01 1 10 100 1000

NiO-Bulk

(b)


0

20

40

60

80

100

120 FVFM


lowast

Chl a

fluo

resc

ence

par

amet

ers v

alue

( o

f con

trol)

(a)

0

50

100

150

200

250ABSRC


lowast

Chl a

fluo

resc

ence

par

amet

ers v

alue

( o

f con

trol)

(b)

Concentration (mgL)

NiO-NPsNiO-Bulk

0

20

40

60

80

100

120

140

1 10 100 1000

PI

lowastChl a

fluo

resc

ence

par

amet

ers v

alue

( o

f con

trol)

(c)


119872) and the









Acknowledgment


References








































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Diameter (nm)

Part

icle

s fra

ctio

n

0

20

40

60

80

100

0 200 400 600 800 1000 1200 1400

24h 596

(a)

0

05

1

15

2

25

3

400 500 600 700 800

Nanopure water


Abso

rban

ce (r

el u

nits)

Wavelength (nm)

(b)



119881119865

119872) It corre-









0

200

400

600

800

1000

1200

1400

0 1 10 100 1000

ROS

indu

ctio

n (

to co

ntro

l)

Concentration (mgL)

NiO-NPsNiO-Bulk

lowast

lowastlowastlowast









2at millimolar



0

1

2

3

4

5

6 NiO-NPsFl

uore

scen

ce in

tens

ity (r

el u

nits)

Time (ms)001 01 1 10 100 1000

O

J

I

P

(a)

Control1

10

100

1000

Fluo

resc

ence

inte

nsity

(rel

uni

ts)

Time (ms)

0

1

2

3

4

5

6

001 01 1 10 100 1000

NiO-Bulk

(b)


0

20

40

60

80

100

120 FVFM


lowast

Chl a

fluo

resc

ence

par

amet

ers v

alue

( o

f con

trol)

(a)

0

50

100

150

200

250ABSRC


lowast

Chl a

fluo

resc

ence

par

amet

ers v

alue

( o

f con

trol)

(b)

Concentration (mgL)

NiO-NPsNiO-Bulk

0

20

40

60

80

100

120

140

1 10 100 1000

PI

lowastChl a

fluo

resc

ence

par

amet

ers v

alue

( o

f con

trol)

(c)


119872) and the









Acknowledgment


References








































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




0

200

400

600

800

1000

1200

1400

0 1 10 100 1000

ROS

indu

ctio

n (

to co

ntro

l)

Concentration (mgL)

NiO-NPsNiO-Bulk

lowast

lowastlowastlowast









2at millimolar



0

1

2

3

4

5

6 NiO-NPsFl

uore

scen

ce in

tens

ity (r

el u

nits)

Time (ms)001 01 1 10 100 1000

O

J

I

P

(a)

Control1

10

100

1000

Fluo

resc

ence

inte

nsity

(rel

uni

ts)

Time (ms)

0

1

2

3

4

5

6

001 01 1 10 100 1000

NiO-Bulk

(b)


0

20

40

60

80

100

120 FVFM


lowast

Chl a

fluo

resc

ence

par

amet

ers v

alue

( o

f con

trol)

(a)

0

50

100

150

200

250ABSRC


lowast

Chl a

fluo

resc

ence

par

amet

ers v

alue

( o

f con

trol)

(b)

Concentration (mgL)

NiO-NPsNiO-Bulk

0

20

40

60

80

100

120

140

1 10 100 1000

PI

lowastChl a

fluo

resc

ence

par

amet

ers v

alue

( o

f con

trol)

(c)


119872) and the









Acknowledgment


References








































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

1

2

3

4

5

6 NiO-NPsFl

uore

scen

ce in

tens

ity (r

el u

nits)

Time (ms)001 01 1 10 100 1000

O

J

I

P

(a)

Control1

10

100

1000

Fluo

resc

ence

inte

nsity

(rel

uni

ts)

Time (ms)

0

1

2

3

4

5

6

001 01 1 10 100 1000

NiO-Bulk

(b)


0

20

40

60

80

100

120 FVFM


lowast

Chl a

fluo

resc

ence

par

amet

ers v

alue

( o

f con

trol)

(a)

0

50

100

150

200

250ABSRC


lowast

Chl a

fluo

resc

ence

par

amet

ers v

alue

( o

f con

trol)

(b)

Concentration (mgL)

NiO-NPsNiO-Bulk

0

20

40

60

80

100

120

140

1 10 100 1000

PI

lowastChl a

fluo

resc

ence

par

amet

ers v

alue

( o

f con

trol)

(c)


119872) and the









Acknowledgment


References








































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of








Acknowledgment


References








































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of
















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

research article toxic effects of nickel oxide bulk and nanoparticles on the aquatic...

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