Differential Oxidative Stress of Octahedral and Cubic Cu2O Micro

Aug 15, 2012 - We demonstrated the differential influences of two crystallographic Cu2O nanocrystals on the antioxidant process. Specifically, octahed...
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Differential Oxidative Stress of Octahedral and Cubic Cu2O Micro/ Nanocrystals to Daphnia magna Wenhong Fan,† Xiaolong Wang,† Minming Cui,† Dongfeng Zhang,† Yuan Zhang,‡ Tao Yu,‡ and Lin Guo*,† †

Key Laboratory of Bio-Inspired Smart Interfacial Science and Technology of Ministry of Education, School of Chemistry and Environment, Beihang University, Beijing 100191, PR China ‡ Chinese Research Academy of Environmental Sciences, Beijing 100012, PR China S Supporting Information *

ABSTRACT: This study attempts to understand the impact of different shapes of an individual micro/nanomaterial on their biotoxicities to aquatic organisms. Two differently shaped Cu2O micro/nanocrystals (cubes and octahedrons with side lengths of 900 nm) were exposed to Daphnia magna for 72 h, afterward several antioxidant biomarkers such as reactive oxygen species (ROS), catalase (CAT), total antioxidant capacity (T-AOC), and malondialdehyde (MDA) in D. magna were measured. We demonstrated the differential influences of two crystallographic Cu2O nanocrystals on the antioxidant process. Specifically, octahedral Cu2O nanocrystals showed a higher level of oxidative stress, possibly because of its larger surface area and higher reaction activity of the octahedron. The biomarker results further showed that the oxidative stress and antioxidant mechanism process involved three stagesantioxidant response, oxidation inhibition, and antioxidant inactivation. Furthermore, the accumulation of MDA was mainly responsible for the ROS-induced toxicity.

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INTRODUCTION Nanoparticles (NPs) have numerous applications in consumer and health care products because of their unique physical and chemical properties, such as small size, rapid diffusion, high specific areas, and reactivity in the liquid or gas phase.1,2 Engineered NPs tend to end up in aquatic environments through many pathways, causing an increased concern over the aquatic biota after their uptake of NPs.3−6 Nano-Cu2O has long been studied because its unique properties make it a candidate material for photovoltaic and photocatalysis, or a negative electrode material for lithium-ion batteries.7 Recently, we have succeeded in delicately controlling the crystallographic structure of Cu2O nanocrystals. For instance, a simple Cu2O cube is converted into octahedron via selective absorption of crystal polyvinylpyrrolidone (PVP) on the surface. These Cu2O nanocrystals differ in many properties and can be used to explore the differences in biotoxicity effects among nanocrystals of various shapes and surface facets.8 The toxicity of NPs is theoretically expected to be different from that of traditional materials because of their extremely small size and high surface-volume ratio.9,10 For instance, traditional TiO2 has been utilized for many years as a safe substance, but nano-TiO2 is hazardous to aquatic organisms.11 Theories have been proposed to explain such phenomena including generation of reactive oxygen species (ROS) leading to inflammation, DNA damage, and cell death;12 binding with macromolecules making them dysfunctional;13 and functioning © 2012 American Chemical Society

as a source of soluble metal ions, enhancing their bioavailability.14 The toxicity of NPs to aquatic organisms might be attributed to three aspects, such as the presence of NPs, the released soluble ions, and the free radicals generated by NP suspension.15 The underlying physicochemical mechanisms leading to the toxicity of NPs to aquatic organisms are still under debate. Some studies showed that the toxicity of NPs was mainly caused by the release of soluble ions,16−18 while other studies showed that NPs have the ability to penetrate into cells or migrate to various organs and tissues, and may cause damage to the biological systems through other mechanisms; therefore the toxicity cannot be simply attributed to the dissolved ions.19,20 Thus, a study on the roles of soluble ions, NPs, and ROS within the organisms is important for a better understanding of the mechanisms of NP toxicity. The physical structure of micro/nanomaterials contributes to their properties. To achieve particular functions or applications, it is necessary to precisely manipulate the physicochemical characteristics of NPs. Moreover, previous studies showed that the physicochemical characteristics of NPs, such as size, surface area, surface modification, and radical formation can significantly influence their toxicity.21−23 Lovern and Klaper10 have studied dispersed nanosized TiO2 and indicated that the Received: Revised: Accepted: Published: 10255

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Exposure of D. magna to Cubic and Octahedral Cu2O Nanocrystals. The suspensions of the octahedral and cubic Cu2O nanocrystals were prepared as described before. According to the results of dissolution (Supporting Information Table S1), the exposure concentration was set to 15 μg L−1 (octahedron) and 43 μg L−1 (cube) to give dissolved Cu concentrations for the octahedral and cubic shapes of 1.7 μg and 1.8 μg L−1 after equilibrium, respectively. The water sample without Cu2O nanocrystals was used as the control. Daphnids aged 14 d were added to each nanomaterial suspension at a density of 1 individual/10 mL in each beaker and exposed to the Cu2O nanocrystals for 3 d. The daphnids were not fed during the exposure to minimize the impacts of any produced feces on the behavior of the nanocrystals. At 6, 12, 24, 36, 48, and 72 h, 60 daphnids were collected from each suspension and depurated in ultrapure water. The same tests were conducted with the control (unexposed) daphnids as the initial condition. Immobilized daphnids were considered to be inhibited. The number of them was counted after 72 h and the immobilization ratio was calculated. Three replicates were performed for each treatment. Bioaccumulation of Cu in D. magna. Ten daphnids were dried at 80 °C to constant weight and then digested in 68% nitric acid (HNO3, Aristar grade) at 110 °C until the digestion tube was dry and the dissolution was complete. The digestion tube was washed with 2% HNO3, and all the washing solutions were transferred to a volumetric flask and diluted for Cu analysis via ICP-MS. The metal bioaccumulation was calculated based on the dry weight (dry wt) of the daphnids (μg g−1 dry wt).27 Scanning Electron Microscopy and Energy X-ray Dispersive Spectrometry Analysis. The detail for this part is provided in the Supporting Information (SI). Metallothionein Concentration. Twenty exposed daphnids were weighed after removal of residual water from the surface of their bodies. The daphnid tissues were homogenized through ultrasonication in a 0.5 mL sucrose buffer (0.25 mol L−1 sucrose, 0.1 mol L−1 Tris-HCl, pH 8.6) and centrifuged at 16 000 × g in a refrigerated centrifuge for 20 min. The supernate was collected and diluted to 1.5 mL with a homogenate and used for MT measurement, which was performed using the modified silver saturation method.28 The supernatant fluid was mixed with 0.5 mL of 20 μg mL−1 Ag+ solution. After 10−20 min of incubation at room temperature, 0.1 mL of red blood cell hemolysate was pipetted into the mixture. The newly homogenized solution was heated in a boiling water bath for 5 min and subsequently centrifuged at 1200 × g for 5 min. The process steps from hemolysate addition to centrifugation were repeated twice before the collection of the final supernate via a 5 min centrifugation at 16 000 × g. The final supernate was digested with 68% HNO3 as described above and was then diluted. The Ag concentration was determined using ICP-MS. Two replicates from each treatment and one blank (without D. magna) were analyzed using the same process described above. The MT content in the samples was calculated using the equation

toxicity may be directly related to size. Pan et al.24 have reported that the morphology of nanotrititanate statistically influences its cytotoxicity and genotoxicity. Indeed, an investigation on the effects of nanomaterials with different physicochemical characteristics on aquatic organisms is of great interest. Here we conducted a comprehensive toxicity assessment of octahedral and cubic Cu2O nanocrystals to further assess the ecological impact and toxicity mechanism of Cu2O nanocrystals with different shapes. This assessment aimed to determine whether the dissolved Cu ions or NPs are more important in evaluating Cu2O nanocrystal toxicity to aquatic organisms. The model freshwater zooplankton Daphnia magna was used as the test organism at the same concentration of dissolved Cu ions. This assessment included a 72 h exposure., with measurements of bioaccumulations at 3, 6, 12, 24, 36, and 72 h. Five biomarkers were evaluated, including metallothionein (MT), ROS content, catalase (CAT) activity, total antioxidant capacity (T-AOC) and malondialdehyde (MDA). MT was chosen since it can indicate a defense mechanism of many toxic metal ions. Other biomarkers were chosen because they can reflect the responses and outcomes of oxidative stress. The results will provide important insights into the broad impacts of NPs on aquatic environments.

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EXPERIMENTAL SECTION

Preparation and characterization of Cu2O nanocrystals. Cu2O nanocrystal samples were prepared according to the previous study. 8 Crystal microstructural analyses were performed using FESEM (Hitachi-S4300) with an accelerating voltage of 20 kV. Water. The environmental water sample used in the current study was obtained from Huo Qi Ying Bridge, China (latitude 39°58′5.58″, longitude 116°16′53.3″). The pH of the water was 8.5, and its total organic carbon was 3.4 ± 0.1 mg L−1. The concentrations of Cu, Cd and Zn in the water sample was 18.1 ± 0.24 ng L−1, 0.13 ± 0.06 ng L−1, and 38.3 ± 5.6 ng L−1, respectively, as determined by inductively coupled plasma mass spectroscopy (ICP-MS; VG PQ2 TURBO). All water samples were filtered through a 1.2 μm membrane prior to use. Model Organism. D. magna used in the current study were maintained for 2 years and were cultured at 23 °C with a light: dark cycle of 16:8 h in the environmental chamber. The green algae Scenedesmus obliquus were fed to the daphnids at a concentration of 1−2 × 105 cells mL−1 per day. The algae were grown in an artificial WC medium25 and collected through centrifugation at the exponential growth stage. The sensitivity of D. magna was tested using potassium dichromate prior to the experiments. Dissolution of Octahedral and Cubic Cu2O Nanocrystals in the Water. The suspensions of the nanocrystals (100 μg L−1) were prepared based on a procedure in which 0.2 mg of Cu2O nanocrystals was added to 500 mL water. The pH of the medium was adjusted to 6.8 by adding dilute nitric acid solution since the gut pH of D. magna was neutral. Three parallel samples were set up for each nanocrystal shape (cubic and octahedral). After rapid mixing, the flasks were placed on a shaker at 150 rpm. A 10 mL aliquot was withdrawn at 10, 30, 60, 90, 120, 180, and 360 min. These samples were centrifuged at 12 000 rpm for 10 min.26 The Cu concentration in the supernate was determined by ICP-MS. The data are shown in Supporting Information Table S1.

μg of MT per g of tissue =

c Ag + × V × 7000 ww × 18 × 108

where cAg+ is the Ag+ concentration in the final mixture, V is the total volume of the final mixture, and ww is the wet weight of D. magna. 10256

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Figure 1. FESEM images and corresponding three-dimensional geometry models of Cu2O nanoparticles (a, cubes; b, octahedrons). Panels c1−c3 are the TEM images and the corresponding selected area electron diffraction (SAED) patterns with the electron beams parallel to [100], [110], and [111] of cubic-shaped Cu2O crystals, while panels d1-d3 are the counterpart with octahedral-shaped Cu2O crystals. Panels e and f are the atomic arrangement in (100) and (111) planes of the Cu2O structure, respectively.

Determination of ROS Using DCF Fluorescence. The amount of ROS generated in D. magna was determined using H2DCFDA.29 A 25 mM H2DCFDA stock solution (in methanol) was diluted with environmental water/ultrapure water (1:1) to a final concentration of 10 mM. Only live D. magna were used for the determination of ROS. One environmental water/ultrapure water (1:1) control was performed in addition to the treatment series. At each time point, 10 daphnids were transferred to approximately 1 mL of exposure solution to a 48-well cluster plate. The exposure solution was carefully aspirated off, leaving the animals in each well. One milliliter of 10 mM H2DCFDA was immediately added to each well in the cluster plate. Fluorescence was monitored using a CytoFluor fluorometer (Millipore Inc., Bedford, MA) with an excitation wavelength of 485 nm and an emission wavelength of 530 nm, both with a bandwidth of 20 nm. Fluorescence was measured after 4 h of incubation with the dye. After the fluorescence analysis, the daphnids were transferred to a Whatman 3 M blotting paper (Fisher Scientific) to dry for 15 min and then weighed. Fluorescence was divided by the fresh weight. Determination of CAT capacity, T-AOC, and MDA in D. magna. Twenty exposed daphnids were weighed after

removing the water on their body surfaces. Tissues of D. magna were homogenized through ultrasonication in 0.5 mL of sucrose buffer (0.25 M sucrose, 0.1 M Tris-HCl, pH 8.6) and centrifuged at 16 000 × g for 20 min. The supernatant fluid was diluted to 1.5 mL with a homogenate, and 1 mL supernatant fluid was used to determine CAT activities, T-AOC, and MDA. CAT activities, T-AOC, and MDA content in D. magna were assayed using commercially available kits according to the manufacturer’s protocol (Nanjing Jiancheng Bioengineering Institute, China). CAT activity was calculated and expressed as U/mgprot (nmol H2O2 consumed/s/mg protein).30 T-AOC was measured on the basis of the deoxidation ability of Fe3+ to Fe2+. The values were expressed as U/mg prot (nmol Fe3+ deoxidated/min/mg protein).31 MDA assays were determined using the thiobarbituric acid colorimetric method.32 The MDA concentrations were expressed as nmol MDA/mg protein.

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RESULTS AND DISCUSSION Characterization of Cu2O Nanocrystals. The fieldemission scanning electron microscopy (FESEM) images of Cu2O nanocrystals are shown in Figure 1. The synthesis and characterization of these NPs have already been reported.8 The side lengths of both the cube and the octahedron were 10257

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eliminate the impact of soluble ions. Under such conditions, a significant difference in the Cu accumulation of D. magna exposed to cubic and octahedral Cu2O nanocrystals was found. On the basis of our results, ingestion via the digestive tract was the main pathway of entry of the nanomaterials into D. magna.34 Cu accumulation in the two exposure systems immediately increased within the first 24 h and then decreased slightly. In earlier studies of accumulation and elimination of dietary trace metal, rapid assimilation and trophic transfer in the first 24 h and release within 24 h−72 h was found, which was consistent with our results.35 The shape and size of the Cu2O nanocrystal agglomerates in the midguts of D. magna are shown in Figure 3a, and the presence of Cu was confirmed through the

approximately 900 nm. The shape of Cu2O nanocrystals can be precisely controlled by taking advantage of the selective surface stabilization of PVP on {111} surfaces of cuprite Cu2O. The surface area ratio of {111} to {100} was delicately tuned by simply adjusting the amount of the PVP added, which resulted in a systematic shape evolution and formation of a variety of Cu2O architectures. As illustrated in the three-dimensional model in Figure 1, the cubic Cu2O consists of six {100} facets, and the octahedral Cu2O has eight {111} facets. TEM and SAED analysis are also used to characterize the morphologies and structures of the crystals furthermore (Figure 1c and Figure 1d). As shown in Figure 1e and f, along the (100) direction, the periodicity was defined as two layers. Layer 1 was composed of O, whereas Cu atoms dominated layer 2. With respect to the (111) direction, three atom layers consisted of one period, where the Cu layer was sandwiched between two layers of O atoms. Thus, the three layers were considered to be in the same surface. Every two Cu atoms had a dangling bond perpendicular to the {111} planes. For the crystallographic surface structure of Cu2O, only {100} sets had 100% oxygen-terminated surfaces, which implies a minimum energy state. Furthermore, {111} surfaces may possess a high-energy status because of the exposure of Cu atoms with dangling bonds. X-ray powder diffraction (XRD) (Rigaku, Dmax2200, CuKα) was used for this structural determination.8 Bioaccumulation and Immobilization of D. magna Exposed to Cubic and Octahedral Cu2O Nanocrystals. The Cu accumulation in D. magna caused by exposure to the two types of Cu2O nanocrystals for 72 h is shown in Figure 2.

Figure 3. SEM image of the internal intestines of D. magna after a 3 d exposure and microanalysis of accumulated Cu2O nanocrystals in the intestines of D. magna using an energy dispersive spectrometer. The arrows are pointing to the Cu2O agglomerates verified by EDS. More details are available in Supporting Information (SI).

microanalysis report of the energy dispersive spectrometer (Figure 3b). Details are provided in the Supporting Information (Figure S1). After 72 h, the immobilization ratios were calculated to evaluate the toxicity, and were 40% ± 3.0% for octahedral Cu2O nanocrystals and 25% ± 1.0% for cubes. These results demonstrated the difference in the toxicity of these two nanocrystals. Octahedrons showed a higher toxicity than the cubes statistically. Induction of MT. MT concentrations, as a promising biomarker for metal pollution, were investigated. Figure 4 illustrates that the MT concentrations of D. magna in the two exposure systems were significantly higher than those of the controls and increased with exposure time. The MT concentration in the cubic Cu2O nanocrystal system increased from 22 μg g−1 wet wt to 37.6 μg g−1 wet wt. This result showed no significant difference (p < 0.01) to that in the octahedral Cu2O nanocrystal system (22 μg g−1 wet wt to 38.7 μg g−1). These findings indicated that only the dissolved metal ions induced an immediate increase in MT level. Induction of MT during metal exposure is usually thought to be a protection mechanism in aquatic organisms by binding to the excess of metal ions.36 The presence of NPs had little effect on MT induction, presumably because Cu2O nanocrystals in the intestines dissolved quickly and the Cu ions can enter the

Figure 2. Bioaccumulation of Cu in D. magna during a 3 d exposure to two different crystallographic characters of Cu2O nanocrystals. Each point is expressed as mean ± standard deviation (n = 3).

The accumulated Cu in D. magna exposed to the two different shapes of Cu2O nanocrystal suspensions was statistically higher than those in the control group significantly. The accumulated Cu in the cubic and octahedral Cu2O nanocrystal suspensions increased from (204 and 203) μg g−1 dry wt to (292 and 500) μg g−1 dry wt, respectively, after 36 h. Cu accumulates in D. magna via two potential pathways. First, D. magna may feed on the particles 0.4 μm−40 μm in size,33 which included the Cu2O nanocrystals used in the present study. Second, the metal ions dissolved by NPs can be absorbed by the organisms from the aqueous phase. Dissolved Cu2+ was maintained at the same concentration in the two different nanocrystal suspensions to 10258

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Figure 4. MT activities in D. magna during a 3 d exposure to two different crystallographic characters of Cu2O nanocrystals. Each point is expressed as mean ± standard deviation (n = 3).

cells easily. By contrast, the Cu2O nanocrystals of 900 nm size cannot enter the cells directly via endocytosis. The formation of MT has been considered as the main detoxification mechanism for organisms via the interaction of thiol groups with the offending metal. In the two exposure systems, the same MT level was inducted, but the immobilization ratios were not similar at the same condition. Thus, toxicity cannot simply be interpreted as the effect of dissolved ions. Potential toxicity of NPs should be further considered. Influence of Different Crystallographic Cu2O Nanocrystals on the Antioxidant Process. The main potential mechanism of NP toxicity is thought to be via oxidative stress with ROS, which damages lipids, carbohydrates, proteins, and DNA.37,38 To interpret the difference in the toxicity between these two shapes of Cu2O, it is worthwhile to detect ROS, CAT activities, T-AOC, and MDA in D. magna during the exposure (Figure 5). The intensity of the dichlorofluorescein (DCF) fluorescence was used to indicate the production of ROS. A time-dependent increase in ROS generation in D. magna was observed from 0 to 48 h in the two exposure systems. At 36 h, the ROS level in the cubic Cu2O nanocrystal system was 275% of the initial level, whereas it was 758% of the initial condition in the octahedral Cu2O nanocrystal system. After 36 h, the ROS generation increased by 10 times in the two exposure systems. A similar variation in the trend of two antioxidative biomarkers (CAT activities and T-AOC) was found (Figure 5) for both octahedrons and cubes. CAT activities increased to 40.32 U/ mg prot (cubic) and 33.16 U/mg prot (octahedral) within the first 12 h, and then decreased to the initial level after 36 h for both nanocrystals. At 12 h, T-AOC increased to 14.70 U/ mgprot, which was 450% of the initial level, then decreased to 40%−70% of the initial level for the octahedral Cu2O nanocrystals. T-AOC increased to 280% of the initial level at 24 h, and then decreased to the same level for the cubic Cu2O nanocrystals. The induction of CAT usually indicated that excess H2O2 needed to be decomposed in order to maintain the balance of free radicals in D. magna. Once the balance was destructed, as manifested by the reduction of CAT activity, the residual H2O2 would cause damages in D. magna. The role of T-AOC was similar, but it also included the function of various

Figure 5. ROS, CAT, T-AOC, and MDA in D. magna during a 3 d exposure to two different crystallographic characters of Cu2O nanocrystals: octahedrons (a) and cubes (b). Each point is expressed as mean ± standard deviation (n = 3).

nonenzymes and small molecule antioxidants, for example, a variety of vitamins, amino acids.39 MDA was a good general indicator of lipid peroxidation and its accumulation was considered a marker for oxidative stress. Figure 5 shows that the MDA concentrations in D. magna exposed to the Cu2O nanocrystals increased with exposure time. Furthermore, the MDA concentration in the cubic Cu2O nanocrystal system increased to 254% of the initial level, which was significantly lower (p < 0.01, one-way ANOVA) than that in the octahedral Cu2O nanocrystal system (337% of the initial level). To further compare the effects of two different shapes of Cu2O nanocrystals on oxidative stress, it was necessary to first remove the possible influence of different Cu accumulations. Thus, ROS and MDA concentrations in D. magna were first divided by the amounts of Cu accumulation. At most time points, ROS and MDA concentrations per unit Cu accumulation for the octahedron were higher than those for the cube (Figure 6), suggesting that the octahedrons caused a markedly higher level of oxidative stress. The difference of the inactivation activities existed between the Cu2O cubic crystals and the octahedral ones was related to the specific surface area and the atom arrangements of exposed crystallographic facets. The octahedrons had more specific surface area and its atom arrangements made the surface energy higher than cubes.40 As reported previously (Figure 1c−f), Cu has a dangling bond perpendicular to the {111} facets, and the {100} facets have O-terminated surfaces. To minimize the total surface energy, biochemical reactions will tend to occur on the higher energy surfaces {111}. Therefore, the interaction activity in the octahedrons is statistically stronger than that in the 10259

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standing hypothesis is that Cu reacts with endogenous H2O2 to generate hydroxyl radicals in a process analogous to the Fenton reaction, and it can catalyze the transfer of electrons from a donor biomolecule to an acceptor, such as O2 to generate O2−• or hydroxy radicals (·OH) in the digestive system (Figure 7).45

Figure 7. Proposed roles of Cu in the pathways of ROS formation.

These free radicals from a variety of sources had led to the development of a series of defense mechanisms in organisms. CAT is a typical enzymatic antioxidant that can dismutate to convert H2O2 to water and oxygen. T-AOC can represent nonenzymatic antioxidants including ascorbic acid (vitamin C), α-tocopherol (vitamin E), glutathione, and other antioxidants. The protection of these antioxidants against oxidative stress is reported in numerous studies. But with the passage of time, the excessive ROS micromolecules entered cell membranes easily and even acted on them directly. These ROS were destructive to the cell membrane, cytoplasm, and DNAs, which then led to the immobilization of D. magna.46,47 The increasing accumulation of MDA could confirm the above discussions. On the basis of the analysis above, the process of oxidative stress can be described as a hierarchical hypothesis that may be divided into three phases: antioxidant response, oxidation inhibition, and antioxidant inactivation.2 Phase I, antioxidant response, refers to 0 h−12 h of the exposure. It was the lowest level of oxidative stress which can be associated with the induction of antioxidant and detoxification enzymes. ROS can be eliminated through enzymatic and nonenzymatic action. The few accumulated MDA in this phase shows that the organism was not damaged yet. This balance between ROS and antioxidants is essential for the survival of organisms and their health. Phase II refers to 12 h−36 h of the exposure. During this phase, ROS concentration continued to increase, while enzymes started to be reduced. The balance was broken, and continuous ROS generation was not inhibited through the antioxidant action of cells. Meanwhile, MDA accumulated quickly. We can conjecture that other forms of injury, such as protein denaturation and DNA damage, may have also occurred. Phase III refers to 36 h−72 h of the exposure. This phase was the highest level of oxidative stress. ROS generation was uncontrollable because of the breakdown of antioxidant action. Protective response was inactivated and overtaken by inflammation and cytotoxicity. Defects or aberrancy of this protective response pathway may determine disease susceptibility during the exposure. High ROS concentration can be a fatal mediator of damage to cell structures, nucleic acids, lipids, and proteins.48,49 Studies of different types of NPs and various testing organisms shared a common characteristics of hierarchical hypothesis oxidative stress model. Bioresponse aggravated gradually over time or with increasing dose, which indicated oxidative stress developed from low level to high

Figure 6. ROS and MDA content is divided by the amount of Cu accumulation in D. magna during a 3 d exposure to two different crystallographic characters of Cu2O nanocrystals. ROS/Cu accumulation (a) and MDA/Cu accumulation (b). Each point is expressed as mean ± standard deviation (n = 3).

cubes; copper terminated would be rather unstable due to the active interaction with the hydroxyl group, thus generating more ROS. Our results were in good accordance with the above analysis. Ren et al. tested the antibacterial activities of different shaped Cu2O crystals and showed that Cu2O octahedral crystals exhibited higher activity in inactivating E. coli than the Cu2O cubic ones.41 In this study, the immobilization ratio was 40% for octahedral Cu2O nanocrystals and 25% for cubic Cu2O nanocrystals at a similar exposure concentration. In this case, the concentration of dissolved Cu was considered to have no toxic effects,42 which confirmed that the toxicity of nanocrystals was mainly caused by the agglomerates. Antioxidant Process by Cu2O Nanocrystals in D. magna. We analyzed the variation tendency of the biomarkers to elucidate the antioxidant process by Cu2O nanocrystals. As shown in Figure 5, the exposure to different types of Cu2O nanocrystal suspensions induced significant changes in ROS and enzymatic biomarkers of D. magna. ROS and MDA accumulation increased gradually with the CAT activities, and T-AOC first increased and then decreased with exposure time. These results indicated that Cu2O nanocrystals in digestive tissues could generate ROS through various biochemical processes. During these processes, Cu, a redox-active transition metal that could cycle between two redox states, oxidized cupric and reduced cuprous states, can be used.43,44 A long10260

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level. The duration of each phase may be highly dependent on doses, nanoparticle types and aquatic species.50,51 To conclude, nanocrystal Cu2O entered into D. magna mainly via the digestive tract. The production of MT showed that the toxicity of NPs can be divided into two types, dissolved ions and particles. The octahedral Cu2O nanocrystals caused higher immobilization ratio and generated more ROS per unit Cu accumulation than the cubic Cu2O. These results indicated that the two shaped nanocrystals caused different toxicity effects to cladocerans probably due to their differences in specific surface area and surface activity. The variation tendency of ROS, CAT, T-AOC, and MDA in D. magna strongly suggested the hierarchical antioxidant process. Our study provided strong evidence of the antioxidation mechanism and suggested that the different types of introduced nanomaterials can significantly affect the toxicity of nanoparticles on aquatic organisms. Future studies should address the impacts of specific factors such as the shape or specific surface area on the biotoxicity of nanoparticles, with a better control of other confounding factors.

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ASSOCIATED CONTENT

S Supporting Information *

Dissolution of octahedral and cubic Cu2O nanocrystals in the water sample (Table SI) and additional details of the scanning electron microscopy and energy X-ray dispersive spectrometry analysis (Figure S1). This information is available free of charge via the Internet at http://pubs.acs.org/.

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AUTHOR INFORMATION

Corresponding Author

*Pone: (86)-10-82338162.Fax: (86)-10-82338162. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China “Water environmental quality evolution and water quality criteria in lakes” (No. 2008CB418201), the Natural Science Foundation of China (No. 40871215), and the Natural Science Foundation of Beijing (No. 8092019).

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