effects of copper nanoparticles exposure in the mussel mytilus galloprovincialis

Published: September 27, 2011

r 2011 American Chemical Society 9356 dx.doi.org/10.1021/es200955s | Environ. Sci. Technol. 2011, 45, 9356–9362

ARTICLE

pubs.acs.org/est

Effects of Copper Nanoparticles Exposure in the MusselMytilus galloprovincialisTania Gomes,† Jos�e P. Pinheiro,‡ Ibon Cancio,§ Catarina G. Pereira,† C�atia Cardoso,† andMaria Jo~ao Bebianno†,*†CIMA, Faculty of Science and Technology, University of Algarve, Campus de Gambelas, 8005-139 Faro, Portugal‡CBME, Faculty of Science and Technology, University of Algarve, Campus de Gambelas, 8005-139 Faro, Portugal§Dept. Zoology & Animal Cell Biology, Scholl of Science and Technology, University of the Basque Country, E-48080 Bilbao, Spain

’ INTRODUCTION

Nanotechnology is a rapid growing field that comprises theresearch and development of particles <100 nm. As nanotech-nology start to come on line with larger scale production andincreasing applications, it is inevitable that nanomaterials andtheir byproducts end up in the aquatic environment where theycan induce short and long-term effects in aquatic organisms.1,2

Copper is an essential metal, with a role as a cofactor innumerous enzymes (cytochrome oxidase, superoxide dismutase,among others) that is toxic when present in higher concentra-tions than those necessary for organisms.3�6 Soluble forms ofCu have been extensively investigated on its bioavailability andeffects in aquatic organisms (e.g., refs 4�6). In the nanoform,copper is increasingly used in various applications such as air andliquid filtration, wood preservation, bioactive coatings, and coat-ings on integrated circuits and batteries and thermal and elec-trical conductivity. Additionally, these Cu nanoparticles are appliedin several products as inks, skin products, and textiles mainly dueto their bactericide properties.7�11 The toxicity of copper oxidenanoparticles (CuONPs) was assessed in several test organisms,namely, bacteria (Vibrio fischeri, Escherichia coli, Staphylococcusaureus, Bacillus subtilis), protozoa (Tetrahymena thermophila),crustaceans (Daphnia magna, Thamnocephalus platyurus, Daph-nia pulex, and Ceriodaphnia dubia), algae (Pseudokirchneriella

subcapitata), and zebrafish (Danio rerio), showing a cytotoxiceffect in all these species.7�10,12�16 However, there is a severelack of information on the potential effects of CuO NPs in bi-valve species, as well as its behavior in aqueous environments.

Despite the rapid emerging literature on the production ofreactive oxygen species (ROS) and oxidative stress as main ef-fects of NPs exposure, its mechanisms of toxicity need furtherclarification in invertebrate species.1,2,17,18 Recent studies havesuggested that oxidative stress may be the cause of CuO NPscytotoxicity in bacteria, daphnids, zebrafish, as well as in humanlung cells.7,9,11,14,15 Biomarkers that have been used as earlywarning signals of the presence of contaminants in aquaticenvironments are important tools to assess the toxic effects ofnanomaterials in aquatic organisms.1,4,5,9 Filter-feeding molluscssuch as Mytilus sp. are a target group for the uptake of nano-particles present in the aquatic environment. They have beenwidely used in the assessment of water quality due to their abilityto accumulate conventional contaminants in the dissolved or thesuspended form.1,2,7,19 Due to their filter feeding habits, bivalves

Received: March 22, 2011Accepted: September 27, 2011Revised: September 26, 2011

ABSTRACT: CuO NPs are widely used in various industrialand commercial applications. However, little is known abouttheir potential toxicity or fate in the environment. In this studythe effects of copper nanoparticles were investigated in the gillsof mussels Mytilus galloprovincialis, comparative to Cu2+. Mus-sels were exposed to 10 μgCu 3 L

�1 of CuONPs and Cu2+ for 15days, and biomarkers of oxidative stress, metal exposure andneurotoxicity evaluated. Results show that mussels accumulatedcopper in gills and responded differently to CuONPs and Cu2+,suggesting distinct modes of action. CuONPs induced oxidativestress in mussels by overwhelming gills antioxidant defensesystem, while for Cu2+ enzymatic activities remained unchangedor increased. CuONPs andCu2+ originated lipid peroxidation inmussels despite different antioxidant efficiency. Moreover, an induction of MT was detected throughout the exposure in musselsexposed to nano and ionic Cu, more evident in CuO NPs exposure. Neurotoxic effects reflected as AChE inhibition were onlydetected at the end of the exposure period for both forms of copper. In overall, these findings show that filter-feeding organisms aresignificant targets for nanoparticle exposure and need to be included when evaluating the overall toxicological impact ofnanoparticles in the aquatic environment.

9357 dx.doi.org/10.1021/es200955s |Environ. Sci. Technol. 2011, 45, 9356–9362

Environmental Science & Technology ARTICLE

gill epithelium is the main interface between the organism andthe surrounding environment, being the primary pathway ofexposure to environmental contaminants. In bivalves exposed tonanoparticles, gills seem to be the first targeted organ, either bydirect passage or particle uptake (e.g., refs 1,2,7,14). Therefore, inthis work, the toxic effects of CuONPs in gills of musselsMytilusgalloprovincialis were evaluated using as end points biomarkersof oxidative stress (antioxidant enzymes SOD, CAT, and GPX,and lipid peroxidation), metal exposure (MT), and neurotoxicity(AChE). These effects were compared with mussels exposed toionic Cu since the mode of action of Cu accumulation in thisspecies is well understood.

’EXPERIMENTAL SECTION

Nanoparticles Characterization. Copper oxide nanoparti-cles (<50 nm) stock solution was prepared in ultrapure water,sonicated for 30 min and kept in constant shaking to reach a con-centration of 10 μgCu 3 L

�1. Ionic copper stock solution (Cu2+)was prepared identically but not sonicated. The particles size wascharacterized using transmission electron microscopy (TEM)and dynamic light scattering (DLS). For TEM analysis, CuONPs were diluted in ultrapure water and sonicated to keep theparticles in solution and avoid aggregation. A drop of the dilutionat 32 ppm was allowed to dry on a Ni grid cover and examined at80 KV. The range of particles sizes was determined throughanalysis of 250 NPs randomly selected. Images were recordedusing a JEOL JEM-1230 TEM equipped with a digital cameramodel 785 ES1000W Erlangshen CCD. Additionally, particlesize and agglomerates, as well as behavior in natural seawaterduring 12 h were followed using DLS. The hydrodynamic radii ofthe nanoparticles were determined using an ALV apparatus withAr ion lased (514.5 nm). Diluted particle dispersions (100 μg 3L

�1

CuO NPs) were measured at 90� and intensity fluctuations ana-lyzed automatically and in a single run by an ALV-7000 digitalcorrelator. The temperature was controlled (20( 0.1 �C) using aHaake Phoenix-II heater/circulator with a C30P cooling bath,withHaake Sil 180mineral oil. The temperature was read directlyfrom the decalin bath using a Platinum Pt100 temperature sensor.Laboratory Exposure.MusselsMytilus galloprovincialis (61.7(

8.4 mm) were collected in South of Portugal and acclimated dur-ing 7 days in natural seawater at constant temperature and aera-tion. Afterward, fifty mussels were placed in tanks filled withseawater in a triplicate design (around 2.5 mussels/L) and exposedto 10 μgCu 3 L

�1 of CuO NPs and Cu2+ along with a controlgroup kept in clean seawater, for a period of 15 days. The copperconcentration selected was environmentally relevant.5,20 Waterwas changed every 12 h (to avoid nanoparticles aggregation)with redosing after each change. Temperature (17.8 ( 1.1 �C),salinity (36.3 ( 0.2), oxygen saturation (97.8 ( 4.9%), and pH(7.8 ( 0.1) were measured daily. Mussels were collected fromcontrols, CuO NPs and Cu2+ in the beginning of the experimentand after 3, 7, and 15 days of exposure. Nomortality was detectedduring the exposure period. After sampling, gills were dissectedand immediately frozen in liquid nitrogen and stored at�80 �Cuntil further use.Metal Analysis. Copper concentrations were determined in

water samples from CuO NPs and Cu2+ exposures after a periodof 12 h before water renewal and redosing. Total copper concen-trations from both exposures were determined after acid digestionwith 2% nitric acid (HNO3), while dissolved copper from CuONPs exposure was determined after water filtration (0.02 μm

filter, Anotop 25, Whatman) and acid digestion.14 Cu in all watersamples and on dried (80 �C) gills of mussels after wet digestedwith HNO3were analyzed by graphite furnace atomic absorptionspectrometry (AAS AAnalyst 800, Perkin-Elmer). Quality assur-ance was checked using a standard reference material (LobsterHepatopancreas) provided by the National Research Council,Canada—TORT II. The mean ( standard deviation (106.8 (2.5 μg 3 g

�1) was similar to the certificated value (106.0 (10.0 μg 3 g

�1). Quality assurance was checked using a standard ref-erencematerial (Lobster Hepatopancreas) provided by theNationalResearch Council, Canada—TORT II. The mean ( standarddeviation (106.8 ( 2.5 μg 3 g

�1) was similar to the certificatedvalue (106.0 ( 10.0 μg 3 g

�1).Enzymatic Activities. Superoxide dismutase, catalase and

glutathione peroxidase activities were measured in the gills cyto-solic fraction. Superoxide dismutase activity (SOD) was deter-mined by the absorption of the reduction of cytochrome c by thexanthine oxidase/hypoxanthine system at a wavelength of550 nm.21 Catalase activity (CAT) was a result of the decreaseof the absorbance at 240 nm due to hydrogen peroxide con-sumption, using a molar extinction coefficient of 40M�1 cm�1.22

Total glutathione peroxidase (GPX) was measured followingNADPH oxidation at 340 nm in the presence of excess glu-tathione reductase, reduced glutathione and cumene hydroper-oxide as substrate.23

Metallothioneins. Gills were homogenized in three volumesof Tris-HCl buffer (0.02 M, pH 8.6) and centrifuged at 30 000gfor 45 min (4 �C). The supernatant was separated from thepellet, and two aliquots were used for lipid peroxidation and totalprotein determination. The remaining supernatant was heat-treated at 80 �C and recentrifuged at 30 000g for 45 min (4 �C).An aliquot of the heat-treated cytosol was used for the quantifica-tion of MT concentration by differential pulse polarography.24

Acetylcholinesterase. Gills were homogenized on ice in fivevolumes of a Tris-HCl buffer (100 mM, pH 8.0) containing 10%Triton and centrifuged at 12 000g for 30 min (4 �C). Thiscolorimetric method is based on the coupled enzyme reaction ofacetylthiocholine as the specific substrate for AChE and 5,50-dithio-bis-2-nitrobenzoate as an indicator for the enzyme reac-tion at 450 nm.25

Lipid Peroxidation. Lipid peroxidation (LPO) was assessedby determining malondialdehyde (MDA) and 4-hydroxyalkenals(4-HNE) concentrations upon the decomposition by polyunsa-turated fatty acid peroxides using malondialdehyde bis-(tetra-metoxypropan) as a standard.26

Total Protein Concentration. Total protein content of gillswas measured by the Lowry method27 using Folin’s Reagent andBovine Serum Albumin (BSA) as a standard.Statistical Analysis. The data obtained was tested using one-

way analysis of variance (ANOVA) or the Kruskal�Wallis OneWay Analysis of Variance on Ranks. If significant, pairwise multiple-comparison procedures were conducted, using the Tukey test orthe Dunn’s method. Linear regression was also applied, to verifyexisting relationships between variables. Statistical significancewas set at p < 0.05 and analyses were performed using SigmaPlot10.Principal Component Analysis (PCA) was applied to evaluate

the relationship between copper concentrations, antioxidant enzy-mes activities, MT concentrations, AChE activity and LPO levelsin the gills of control and exposed mussels along the period ofexposure. Computations were performed using XLStat2009.



’RESULTS AND DISCUSSION

To our knowledge this is the first study that focused on theeffects of CuO NPs in the gills ofM. galloprovincialis. The nano-particles used are spherical in shape and not strongly aggregated,with a mean diameter of 31 ( 10 nm (Figure 1A and B). Theparticle size distribution of CuO NPs obtained by DLS showedpolydisperse aggregates (polydispersity index between 0.26 and0.48) characterized by single particles with sizes from30 to 40 nm toaggregates ranging from 238 to 338 nm. The higher size of CuONPs suspended in seawater obtained by DLS compared to TEMis due to the propensity of these particles to aggregate in aqueousstate. This finding is supported by other studies that used CuONPs, some of which from the same manufacturer.7,13�15,28

As the number of CuO NPs applications increase, it is likelythat they will end up in the environment, and in significantquantities. However, emissions of NPs to the aquatic environ-ment are difficult to detect and quantify, and no available dataexists on CuO NPs. In our study, more than 50% of the nominalconcentration of 10 μgCu 3 L

�1 added in the nano or ionic formwas removed from the water column after the 12 h exposure(53% for CuO NPs and 66% for Cu2+). The lost of this amountof copper may be due either to the presence of the mussels,copper dissolution or nanoparticles aggregation and sedimenta-tion.13,14 Of the total Cu concentration (4.8 ( 01 μgCu 3 L

�1)obtained from the CuO NPs exposure, less than 1% of the initial

added dose is present in the dissolved form, indicating that mostof the Cu present in solution is in the nanoparticulate form.Other authors using CuO NPs also showed lower dissolutionfrom nanocopper, suggesting that Cu toxicity is mainly due toCuO NPs.7,13�15

The bioaccumulation of NPs in invertebrates provide valuableknowledge on NPs bioavailability and allow more realistictoxicity information.1,2,29 Data about internal exposure concen-trations and accumulation of NPs in various tissues on chronicexposure of aquatic organisms is practically inexistent.2,29 In thisstudy, the exposure to CuO NPs resulted in a significant accumula-tion of copper in mussel gills with time (9.8 ( 1.9 to 12.5( 1.4μg 3 g

�1 dw, Figure 1C). In mussels exposed to Cu2+, accumula-tion only occurred in the first week (16.9 ( 2.4 and 15.5 ( 2.4μg 3 g

�1 dw, p < 0.05), followed by a decrease at the end of theexperiment, to levels similar to control (p > 0.05, Figure 1C).This decrease is indicative of the elimination rate of Cu in bi-valves through detoxification processes,3,19 whereas in thoseexposed to CuO NPs the elimination rate is slower than its accu-mulation. Mussels accumulated more copper from Cu2+ thanCuO NPs in the first week of exposure, suggesting a highercopper bioavailability from Cu2+. Mussel gills are a target organfor nanoparticles exposure, being more sensitive to metal disso-ciation from NPs than its internalization,1,2,7,14,30 nevertheless,no distinction was made between dissolved and incorporated

Figure 1. � (A) Transmission electron microscopic image of CuO nanoparticles at 32 ppm inMilli-Q water. (B) Particle size distribution histogram ofCuO NPs obtained from TEM images. (C) Copper concentrations in gills of musselsM. galloprovincialis from controls and exposed to CuO NPs andCu2+ for 15 days in a dry weight tissue basis (average ( Std). Capital and lower letters represent statistical differences between treatments in eachexposure day and for each treatment during the exposure duration, respectively (p < 0.05).



copper particles. Several studies have shown copper accumula-tion in bivalves tissues (e.g., refs 4,5,31), however, no data existson the accumulation of copper from NPs exposure.

Chemical reactivity, as well as specific surface characteristicsconfers nanomaterials the capacity to generate ROS by mereinteraction with subcellular structures and by directing its reacti-vity to subcellular compartments. In the case of metal nanopar-ticles, the physical contact between cells and particles may causechanges in the vicinity of the contact area and increase the dis-solution of metals or generate extracellular ROS.7,9,17 Copper,being a redox active metal, has the capacity to produce ROSthrough Fenton-type reactions leading to the production ofoxyradicals that activate/inhibit several antioxidant enzymes.3�9

The activities of SOD, CAT and GPX were used along with lipidperoxidation to assess the oxidative status of mussel gills exposedto CuO NPs and Cu2+ (Figures 2 and 3).

SOD, CAT and GPX activities changed after exposure toCuO NPs, showing that these NPs have also potent redoxproperties with the capacity to generate ROS (Figure 2). InCuO NPs exposed mussels, SOD activity increased linearly(7.5 U 3mg�1prot 3 d

�1, r = 0.99, p < 0.05) in the first 7 days,indicative of the formation of superoxide anions. CAT wasonly induced after 3 days of NPs exposure (43%) while GPXactivity remained unchanged and similar to unexposed mussels(p > 0.05). The induction of GPX after a week of NPs exposure

(15.8 ( 3.1 to 21.3 ( 1.7 nmol 3min 3mg�1prot) suggeststhe detoxification of hydroperoxides possibly associated withincreased levels of hydroxyl radicals originated by CuO NPs,whereas at the beginning SOD and CAT levels may have beensufficient to counteract the overproduction of ROS. The SODand CAT similar antioxidant efficiencies were supported by thePCA analysis (Figure 4A) that shows a significant correlationin the first week of exposure. After two weeks, both SOD andCAT activities decreased (38 and 33% of inhibition, p < 0.05)in mussels exposed to CuO NPs, whereas GPX continued toincrease. These inhibitory effects suggest an overproduction ofROS that could have led to the degeneration of the enzymes.These ROS can be available to react with Cu2+ from CuO NPsdissolution, leading to the formation of hydroxyl radicals gen-erated from H2O2 under Cu

+ exposure through the Fenton andHaber Weiss reactions, possibly leading to SOD and CAT inactiva-tion.5,6 These data are in line with recent observations that showthat CuO NPs cytotoxicity is mediated by oxidative stress, alter-ing the antioxidant capacity of cells against ROS. In human lungepithelial cells, CuO NPs (80 μg 3 cm

�2, 4 h, 30 nm) blocked the

Figure 2. Superoxide dismutase (A), catalase (B), and glutathioneperoxidase (C) activities in gills of mussels M. galloprovincialis fromcontrol and exposed to CuONPs and Cu2+ for 15 days (average( Std).Capital and lower letters represent statistical differences betweentreatments in each day of exposure and for each treatment during theexposure duration, respectively (p < 0.05).

Figure 3. Metallothionein concentrations (A), inhibition of acetycho-linesterase activity (B) and lipid peroxidation (C) in gills of musselsM. galloprovincialis from controls and exposed to CuONPs and Cu2+ for15 days (average ( Std). Capital and lower letters represent statisticaldifferences between treatments in each exposure day and for eachtreatment during the exposure duration, respectively (p < 0.05).Asterisks represent statistical differences between control and exposedmussels (p < 0.05).



antioxidant defenses by inhibiting CAT and GR activities and in-creasing GPX or SOD and CAT activities after exposure to 10, 25,and 50 μg 3mL

�1 for 24 h (52.5( 10.2 nm).11,28,32 In bivalves, theonly existing data on antioxidant efficiency are of Cu2+ exposure.

Mussels exposed to Cu2+ showed different antioxidant re-sponses (Figure 2) with the enzymatic activities unchanged orincreased (Figure 2). SOD activity was activated during thewhole experiment (171% increase by day 15) resulting in theformation of superoxide radicals. CAT activity only increasedafter 3 days of exposure (36%) and remained unchanged fromday 7 until the end of the experiment, at levels similar to controls(p > 0.05). As mentioned above, this result can be associated withthe involvement of Cu in Fenton and Haber Weiss reactions,leaving no substrate available for CAT activation, or to the induc-tion of other components of the antioxidant defense system.5,6 Likefor CAT, GPX activity was induced in the first 3 days of exposure(25.0 ( 1.7 nmol 3min 3mg�1prot, p < 0.05) remaining un-changed until the end of the experiment, always higher than thatin control. This increase in GPX activity suggests a further detoxi-fication of ROS combined with the action of MT; either by ROSscavenging (day 7) or Cu detoxification (day 15), justifying CATunaltered activities. The PCA analysis shows a clear associationbetween GPX activity and Cu2+-exposed mussels, validating the en-hancement of this enzyme activity to neutralize ROS (Figure 4A).Similar results were detected in mussels exposed to 60 μgCu 3L

�17

for 3 weeks and in the clam R. decussatus exposed to 0.5 and2.5 μgCu 3L

�1 Cu for 3 days.5

Metallothioneins are low-molecular weight cysteine-rich pro-teins induced by metals that can also act as an oxygen speciesscavengers, participating in antioxidant processes protectingcells from oxidative stress.19,20,31 Although information on MTbehavior upon exposure to CuO NPs is nonexistent, the role ofMT in ionic/soluble Cu detoxificationmechanisms is well under-stood in bivalves, either by controlling its intracellular availabilityor by detoxifying excessive metal concentrations.4,5,20,31,33 Inmussels exposed to CuONPs,MT increased linearly with time ofexposure, with an induction rate of 0.3 mg 3 g

�1prot 3 d�1 (r =

0.99, p < 0.05), reflecting not only the role of this protein in Cuhomeostasis and detoxification (Figure 3A), but also a possibleinvolvement in gills antioxidant defense system that can explainthe absence of SOD and CAT responses (day 15). Only twostudies addressed the role of MT in bivalve species: inC. virginicaexposed to silver nanoparticles (16 μg 3 L

�1-1.6 ng 3 L�1, 15 (

6 nm) an increase in MT expression was associated with silvermetabolism or to the increase of oxyradicals and in C. flumineaexposed to gold nanoparticles (1.6� 103�1.6� 105 AuNP/cell,10 nm) to protect cells against gold-induced oxidative stress.34,35

In mussels exposed to Cu2+, MT levels also increased in thefirst week of exposure with a lower induction rate (0.2 mg 3 g

�1-prot 3 d

�1, r = 0.99, p < 0.05) when compared to CuO NPs(Figure 3A), denoting its importance in Cu metabolism, as alsoseen by the close association betweenCu concentrations andMTin the PCA (Figure 4). Contrarily to the response for CuO NPs,MT decreased in the gills of mussels exposed to Cu2+ at the endof the experiment (6.7 mg 3 g

�1prot), suggesting a role of MT incopper detoxification, which is in agreement with the copperaccumulation results in mussel gills (Figure 1C). Cu can bind toMT to form insoluble Cu�MT complexes that precipitate intolysosomes and are eliminated by exocytosis.3,20,31 Similar resultswere detected in R. decussatus31 and Crassostrea gigas20 exposedto 50 μgCu 3 L

�1 and 0.5�5 μgCu 3 L�1, respectively.

Acetylcholinesterase is a biomarker of exposure to organo-phosphorus pesticides that can also be inhibited by a diverserange of metals, including copper.4,5,33 A dose-dependent de-crease of this enzyme after Cu2+exposure is well established inbivalve species, as in R. decussatus (75 μgCu 3 L

�1, 5 days)5 andmussels (40 μg 3 L

�1 and 60 μg 3 L�1, 1 and 3 weeks).33,4 In this

study, inhibition of AChE was observed in CuO NPs and Cu2+

exposed mussels (Figure 3B) only at the end of the experiment,with a 34% and 53% inhibition, respectively (p < 0.05), alsoconfirmed by the PCA (Figure 4). The high affinity of Cu tosulfur donor groups can cause AChE inhibition by binding to itsthiol residues, as in MT.5 These results confirm the specificity ofAChE response to Cu exposure, either in the nano or ionic form.The neurotoxic effects of nanoparticles in M. edulis exposed to1 mg 3 L

�1 Fe NPs (5�90 nm, 12 h) showed no significantdifferences in AChE activity.36 Nevertheless, one study showedthat AChE has the potential to be used as a biomarker for CuONPs (25 nm), because of its strong AChE inhibition (76%) andlow median inhibitory concentration (4 mg 3 L

�1).37

Significant variations of enzymatic activities exist betweencontrol and Cu-exposed mussels throughout the experimentalperiod suggesting that gills responded differently to both formsof copper (Figure 2). The overall PCA analysis (Figure 4) in-dicates a clear separation between control and Cu-exposedmussels. Unexposed mussels, as well as those exposed to Cu2+

are closely associated at different times of exposure (day 3, 7,and 15) showing similar biomarker tendency. As for CuO NPsexposed mussels, a clear separation of the sampling periods

Figure 4. � Principal component analysis (PCA) of copper accumula-tion and the battery of biomarkers in gills of musselsM. galloprovincialisfrom controls and exposed to CuONPs and Cu2+ for 15 days. A, PC1 vsPC2; B, PC1 vs PC3.



occurred, suggesting a marked different behavior betweenmusselgills response with time of exposure. Failure of antioxidant defensesto counteract ROS produced by both forms of Cu either by beinginhibited or overwhelmed can interrupt the balance between theantioxidant/prooxidant system in mussels leading to oxidativedamage of biomolecules.4�6 One of the best known effects of ex-cess Cu is the peroxidative damage to membrane lipids, triggeredby the reaction of lipid radicals and oxygen to form peroxylradicals that can alter membrane fluidity and permeability or attackother intracellular molecules.4�6 Despite different antioxidant effi-ciency, LPO increased linearly with time inmussels exposed toCuONPs and Cu2+ (Figure 3C), with induction rates of 36.8 nmol 3 g

�1

prot 3d�1 (r = 0.99; p < 0.05) and 49.7 nmol 3 g

�1prot 3 d�1 (r =

0.97, p < 0.05), respectively. In the first three days of CuO NPsexposure, SODandCAT activities proved to be antioxidant efficientand prevent deleterious effects in lipids of cellular membranes,confirmed by the relative proximity of these mussels to the controlgroup in the PCA analysis (Figure 4A). In the remaining period,CuO NPs seems to continuously increase ROS production activat-ing the combined action of antioxidant defenses (SOD, CAT, GPX,and MT) until a point where the antioxidant capacity was over-whelmed causing SOD andCAT inactivation and a continuousMTand GPX increase. Although GPX andMT can remove most of theROS by increasing its activities, they cannot compete with hydroxylradicals’ generation via the Fenton reaction thereby causing anincrease in LPO levels. In mussels exposed to Cu2+, antioxidantenzymes were activated during the whole exposure period (exceptCAT) alongwith an increase inMT levels leading to a detoxificationprocess by the end of the exposure, nevertheless, not enough toprevent LPO. These results are in agreement with the PCA thatshows a clear association between copper concentrations in gills andLPO levels, as well as withMT andGPX (Figure 4). In human cellsand E. coli exposed to CuO NPs (30�50 nm) their toxicity wasrelated to oxidative stress, mediated by lipid peroxidation, oxida-tive lesions and increase of intracellular ROS11,13,15,28,32 Evidencethat LPO occurs after Cu exposure was also observed in severalbivalve species, as clams and mussels.4�6

Altogether, our results support the conclusion that oxidativestress is a significant mechanism of toxicity for CuONPs7,9,11,15,32,38

and that its mode of action appears distinct from Cu2+. In otheraquatic organisms (V. fisheri, D. magna, T. platyurus, P. subcapitata,T. thermophila) CuO NPs (∼30 nm) showed a higher toxicitywhen compared to its ionic/soluble form, associated with Cuions dissolution.9,10,12 Nevertheless, the dissolution of Cu ionsdo not fully explain the toxicity of CuO NPs in zebrafish,7,14

human cell cultures,11,29,32 or daphnids15 exposed to particleswith similar size (30�50 nm), where other mechanisms derivedfrom the particle effect had to be considered (e.g., oxidative stressdue to ROS formation). In our study, a combination of theparticle effect and ions dissolution can account for the differencesin the toxic effects exerted by CuO NPs along the exposureperiod. Mussel gills can be taking up dissolved Cu released fromthe particles combined with a cellular uptake of nanoparticlesaggregates. CuO NPs can pass the cellular membrane, enter insidethe cell, dissolve rapidly and release high concentrations of ionssufficient to disrupt Cu homeostasis and generate radicals.1,14,28,38

This NPs mechanism of toxicity named “Trojan horse-typemechanism” was identified in cell cultures.38,39 The increas-ing copper concentrations in mussel gills can be indicative ofan increasing rate of exposure that leads to a continuous releaseof Cu from the NPs. The reaction time of gills cells is slower thanthe particle dissolution and uptake leading to enzymatic

breakdown and to a continuous increase in MT levels, whereasin mussels exposed to Cu2+, this metal is eliminated more rapidlyvia MT detoxification pathway. Another fraction of the CuONPscan be taken up by endocytosis and their toxicological responsecontrolled by surface processes (ROS, adsorption).1,2,38 Thepresence of CuONPs aggregates in suspension (as seen by DLS)facilitate a continuous source of NPs that can either be dissolvedor incorporated, leading to a continuous ROS generation (intraand/or extracellular), that increases with time of exposure. Acorrelation between formation of larger aggregates and biomar-ker responses with increasing time of exposure was suggested inM. galloprovincialis exposed to nano carbon black, C60 fullerenes,nano-TiO2 and nano-SiO2.

40 A more efficient and rapid captureand ingestion of NPs in aggregated form was also observed inmussels and oysters exposed to polystyrene NPs when comparedto those in suspension.29 As for M. edulis, NPs from glass wooland Fe are taken up by gills epithelial cells as pathways of uptakeby diffusion or by endocytosis, independently of the size of theaggregates.37,41 Aggregation has a crucial role in nanoparticlestoxicity, and the cumulative effects of the dissociation ofmetal ions, size and surface-area properties of these particlescannot be discarded and need further clarification in CuONPs mechanisms.1,2,29,35,40

Despite the information given by acute experiments, they donot provide complete information about the interactions ofnanomaterials with classical test species and there is a need todirect research toward invertebrate tests using long-term expo-sure to better understand NPs toxicity mechanisms.2,9,13,15 As forCuO NPs, most of the data available concerns acute toxicityacross a wide spectrum of aquatic species,7�10,12�16 and thisstudy is one of the first to address long-term effects of these NPsin this species. Overall our results show that mussels represent atarget for environmental exposure to nanoparticles where ex-posure duration may be a contributing factor in NPs mediatedtoxicity. In summary, long-term exposure to CuO NPs causeoxidative stress in gills of mussels as evidenced by the breakdownof the antioxidant defense system and lipid peroxidation, as well asacetylcholinesterase inhibition andmetallothionein induction.Never-theless the underlying mechanisms associated with biomarkers res-ponses are still uncertain, and the observed oxidative stress may dueto an association between the nanoparticle effect and the dissociationof copper ions from the nanoparticles. Future research is required tounderstand the mechanisms of CuO NPs toxicity in aquatic organ-isms, where the uptake and accumulation of CuO NPs in othermussel tissues should be considered, as well as the importance ofbioavailability and particle aggregation for long periods of time.

’AUTHOR INFORMATION

Corresponding Author*Phone: (+351) 289800100; fax: (+351) 289800069; e-mail:[email protected].

’ACKNOWLEDGMENT

This research was supported by a Foundation of Science andTechnology PhD Grant (SFRH/BD/41605/2007).

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