Effects of CuO nanoparticles on Lemna minor
© Song et al. 2016
Received: 3 November 2015
Accepted: 7 January 2016
Published: 27 January 2016
Copper dioxide nanoparticles (NPs), which is a kind of important and widely used metal oxide NP, eventually reaches a water body through wastewater and urban runoff. Ecotoxicological studies of this kind of NPs effects on hydrophyte are very limited at present. Lemna minor was exposed to media with different concentrations of CuO NPs, bulk CuO, and two times concentration of Cu2+ released from CuO NPs in culture media. The changes in plant growth, chlorophyll content, antioxidant defense enzyme activities [i.e., peroxidase (POD), catalase (CAT), superoxide dismutase (SOD) activities], and malondialdehyde (MDA) content were measured in the present study. The particle size of CuO NPs and the zeta potential of CuO NPs and bulk CuO in the culture media were also analyzed to complementally evaluate their toxicity on duckweed.
Results showed that CuO NPs inhibited the plant growth at lower concentration than bulk CuO. L. minor roots were easily broken in CuO NPs media under the experimental condition, and the inhibition occurred only partly because CuO NPs released Cu2+ in the culture media. The POD, SOD, and CAT activities of L. minor increased when the plants were exposed to CuO NPs, bulk CuO NPs and two times the concentration of Cu2+ released from CuO NPs in culture media, but the increase of these enzymes were the highest in CuO NPs media among the three kinds of materials. The MDA content was significantly increased compared with that of the control from 50 mg L−1 CuO NP concentration in culture media.
CuO NPs has more toxicity on L. minor compared with that of bulk CuO, and the inhibition occurred only partly because released Cu2+ in the culture media. The plant accumulated more reactive oxygen species in the CuO NP media than in the same concentration of bulk CuO. The plant cell encountered serious damage when the CuO NP concentration reached 50 mg L−1 in culture media. The toxicology of CuO NP on hydrophytes must be considered because that hydrophytes are the basic of aquatic ecosystem.
Nano-technology has a strong claim to be regarded as the first important advance in technology of the third millennium (Robert 2012). Given the rapid development of nanotechnology, an increasing risk of human and environmental exposure to nanotechnology-based materials is apparent. However, data on the potential environmental effects of nanoparticles (NPs) are scarce (Clément et al. 2013; Zhang et al. 2013), particularly on the effects and mechanisms of these NPs on higher plants (Nair et al. 2010; Song et al. 2012; Miralles et al. 2012).
Metal oxide NPs are manufactured at a large scale for both industrial and household use (Aitken et al. 2006; Xia et al. 2013). CuO NPs, an important kind of metal oxide NPs, are used in catalysis, gas sensors, solar energy conversion, high-temperature superconductors, and field-emission emitters (Chowdhuri et al. 2004; Yin et al. 2005; Dar et al. 2008; Jammi et al. 2009). With such large-scale applications, CuO NPs will inevitably reach bodies of water through waste water and urban runoff. Therefore, understanding the risks of this kind of NPs to aquatic ecosystems is essential. The toxicity study of CuO NPs on aquatic organisms has drawn considerable attention in recent years. Aquatic creatures, such as fish, algae, bacteria, and crustaceans, are adversely affected by CuO NPs (Kahru and Dubourguier 2010; Gunawan et al. 2011; Zhao et al. 2011; Li et al. 2012). However, the toxic effects of CuO NPs on hydrophytes are scare at present. Aquatic macrophytes are important for oxygen production, nutrient cycling, water quality control, sediment stabilization, and shelter for aquatic organisms and wildlife (Mohan and Hosetti 1999); these plants are vital in maintaining the stability of aquatic ecosystems. Thus, the toxic effect of CuO NPs on aquatic plants should be studied on time.
Lemna minor, a duckweed species, is a widespread, free-floating aquatic macrophyte. L. minor is a food source for waterfowl and a shelter for small aquatic invertebrates. L. minor grows fast and reproduces more rapidly than other vascular plants. Because of these characteristics, duckweed is often used in water body restoration and ecotoxicological studies (Song et al. 2012; Žaltauskaitė and Norvilaitė 2013). To study the toxicity effect of CuO NPs on L. minor, the macro growth and microphysical response of L. minor exposed to CuO NPs in several concentrations were investigated compared with those of L. minor exposed to bulk CuO and soluble Cu2+. These physical indexes include the peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) activities of this floating plant, as well as its malondialdehyde (MDA) and chlorophyll contents, under different treatments.
Plant materials, growth conditions, and treatment procedures
Composition of the modified Steinberg medium
Concentration (mg L−1)
Characterization of CuO NPs and bulk CuO
CuO NPs and bulk CuO were purchased from Shanghai Jingchun Reagent Limited Company, China. CuO NPs, with the purity is greater than 99.5 %, particle diameter is 40 nm and a surface area is 25–40 m2 g−1.The purity of bulk CuO is more than 99.9 %, particle diameter is 10 μm. The morphology of the CuO NPs was examined by transmission electron microscopy (TEM, JEOL 100CX, Japan). The particle diameter of CuO NPs in solution and the zeta potential of CuO NPs and bulk CuO in solution were measured using a 90 plus particle size analyzer (DR-525, Brookhave Instruments Corporation, USA) at 12 h after media preparation. The Cu2+ concentration that the nano-CuO released to the culture media was measured by ICP-MS 7500ce, Agilent, USA at 24 h after media preparation.
The experiment media were divided into three treatments. The media for treatment 1 consisted of 1/10 modified Steinberg medium with 0, 10, 50, 100, 150, and 200 mg L−1 CuO NPs, which were ultrasonicated for 30 min. The media for treatment two consisted of 1/10 modified Steinberg medium with 0, 10, 50, 100, 150, and 200 mg L−1 bulk CuO. The media for treatment three were 1/10 modified Steinberg medium with an amount of CuCl2 that supplied twice the Cu2+ concentration released from CuO NPs in treatment 1 media. The tests were performed in 500 mL beakers containing 200 mL media. The pH of all of the culture media was adjusted to 6.5.
Enzyme extraction and chlorophyll determination
To obtain the enzyme extract, 500 mg of whole plant was homogenized in 5 mL cold potassium phosphate buffer (0.1 M, pH 7.8). The homogenate was centrifuged at 15,000g (4 °C) for 15 min in a refrigerant centrifuge. The supernatant was used as the enzyme extract. The enzyme extraction was conducted at 4 °C.
Chlorophyll content was measured using the duckweed fronds. The fronds were whetted and distilled in ethanol (96 %), and then the extracts were measured spectrophotometrically at 665, 649, and 470 nm (Zhao 2000a).
For POD, the mixture consisted of 50 mM potassium phosphate buffer (pH 7.0), 1 mL; 0.2 % H2O2, 2 mL; 0.2 % guaiacol, 0.95 mL; and enzyme extract, 50 µL. The enzyme activity was measured by monitoring the increase in absorbance at 470 nm during polymerization of guaiacol into tetraguaiacol. CAT activity was measured spectrophotometrically by following the consumption of H2O2 at 240 nm (Liu 2006). SOD was measured using SOD detection kit that was produced in Nanjing Jiancheng Bioengineering Institute. Assay was carried out according to the specification of the detection kit. MDA activity was measured according to Zhao (2000b). The enzyme extracts of 1.5 and 2.5 mL thiobarbituric acid (TBA, 0.5 %) were boiled for 20 min, and then centrifuged. Afterwards, the supernatant was measured spectrophotometrically at 532, 600, and 450 nm.
Results were expressed as mean ± standard deviation (SD). The obtained data were evaluated by Student’s t test compared with their corresponding control (0 mg L−1 CuO) using Prism 5.0 statistical package. The statistical significance was considered at p < 0.05.
Results and discussions
Characterization of CuO NPs and the particles in media
The CuO NPs aggregated to form larger sizes in the culture media. The aggregations are likely driven by the divalent ions and low zeta potential (Griffitt et al. 2007). The divalent ion effect can be due to NP bridging via ionic bonds to form NP–M +–NP (where M + is the salt cation), with Na+ in the culture media, thereby promoting NP aggregation (Wang et al. 2011a). In addition, divalent cations such as Mg2+ and Ca2+ (also present in the culture media) have been shown to induce NP aggregation (Akaighe et al. 2012). Agglomerates forming a neck between two or more particles create an area of negative surface curvature, and nucleation occurs at this interface under equilibrium conditions. This action can result in the fusion of the agglomerates and a reduction in total particle surface area (Chang et al. 2012).
Effects of CuO NPs, bulk CuO, and Cu2+ on the growth of L. minor
The micro-growth of L. minor indicated that CuO NPs exhibited greater effect on the growth of L. minor than the bulk CuO in the same concentration. the effect of CuO NPs on L. minor growth partly because of the Cu2+ releasing in culture media.
Effects of CuO NPs, bulk CuO, and Cu2+ on the chlorophyll content of L. minor fronds
Effects of CuO NPs, bulk CuO, and Cu2+ on antioxidant defense enzymes and MDA of L. minor
The effects of CuO NPs, bulk CuO, and twice concentration released from CuO NPs in culture media on the protective enzymes (i.e., POD, CAT, and SOD) of L. minor was also examined, as well as the MDA content. The production of active oxygen species is a biochemical change that possibly occurs when plants are subjected to harmful stress conditions. The chloroplasts and mitochondria of plant cells are important intracellular generators of reactive oxygen species (ROS). Internal O2 concentration is high during photosynthesis, and chloroplasts are particularly prone to generate ROS; therefore, these cytotoxic ROS can remarkably disrupt normal metabolism through oxidative damage of lipids, nucleic acids, and proteins. Deleterious effects of ROS and lipid peroxidation products are counteracted by an antioxidant defense system (Pejic´ et al. 2009). These damages can be examined by analyzing the changes of certain antioxidant enzymes, such as SOD, CAT, and POD.
Copper dioxide NPs aggregated in culture media. The stability of media with bulk CuO was higher than that of media with the same concentration of CuO NPs. CuO NPs released Cu2+ in culture media.
CuO NPs, bulk CuO, and Cu2+ decreased the growth of L. minor, and the effects of these three treatments on L. minor roots were more significant than the influence of the treatments on L. minor fronds. The effect of bulk CuO was not as remarkable as that of CuO NPs, and the effect of CuO NPs was partly due to the Cu2+ released from CuO NPs in the culture media.
L. minor cells exposed to CuO NPs accumulated more ROS compared with the plant cells exposed to the same concentration of bulk CuO. The plant cells accumulated ROS in CuO NP media partly because CuO NPs released Cu2+ in the culture media. The plant cell encountered serious damage when the CuO NP concentration was 50 mg L−1.
The present study received financial support from the National Natural Science Foundation of China (NSFC) (81560536), National Natural Science Foundation of China (NSFC) (81560614), Science and Technology Research Program of Shihezi University (RCZX201331), Science and Technology Research Program of Shihezi University(RCZX201445), Open Program of Shanghai Key Laboratory of Atmospheric Particle Pollution Prevention(LAP3) (FDLAP14003), Open Fund from Xinjiang Key Laboratory of Biological Resources and Genetic Engineering and Major Science (XJDX0201_2014_10) and Technology Program for Water Pollution Control and Treatment (2012ZX07201002).
GS, WH and YG designed carried out the experiments, YW designed the experiments, analyzed and interpreted data. LL, ZZ, QN and RM drafted the manuscript. LM and HW analyzed and interpreted data, and helped to draft the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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