Ecotoxicology and Environmental Safety 104 (2014) 302–309
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Effect of silver nanoparticles on rice (Oryza sativa L. cv. KDML 105) seed germination and seedling growth Pakvirun Thuesombat a, Supot Hannongbua b, Sanong Akasit b, Supachitra Chadchawan c,n a
Nanoscience and Technology Program, Graduate School, Chulalongkorn University, Bangkok 10330, Thailand Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand c Center of Excellence in Environment and Plant Physiology, Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand b
art ic l e i nf o
a b s t r a c t
Article history: Received 8 September 2013 Received in revised form 14 March 2014 Accepted 19 March 2014 Available online 15 April 2014
With the advances in nanotechnology, silver nanoparticles (AgNPs) have been applied in many industries, increasing their potential exposure level in the environment, yet their environmental safety remains poorly evaluated. The possible effects of different sized AgNPs (20, 30–60, 70–120 and 150 nm diameter) on jasmine rice, Oryza sativa L. cv. KDML 105, were investigated at different concentrations (0.1, 1, 10, 100 and 1000 mg/L) upon seed germination and seedling growth. The results revealed that the level of seed germination and subsequent growth of those seedlings that germinated were both decreased with increasing sizes and concentrations of AgNPs. Based on the analysis of AgNPs accumulation in plant tissues, it implied that the higher uptake was found when the seeds were treated with the smaller AgNPs, 20 nm diameter AgNPs, but it was trapped in the roots rather than transported to the leaves. These resulted in the less negative effects on seedling growth, when compared to the seed soaking with the larger AgNPs with 150 nm diameter. The negative effects of AgNPs were supported by leaf cell deformation when rice seeds were treated with 150-nm-diameter AgNP at the concentration of 10 or 100 mg/L during seed germination. These results further strengthen our understanding of environmental safety information with respect to nanomaterials. & 2014 Elsevier Inc. All rights reserved.
Keywords: Silver nanoparticle Rice Nanotoxicology
1. Introduction With the advancement in nanotechnology, silver NPs (AgNPs) have been applied in many industries, including daily products and medical products. Worldwide, the present production of AgNPs is estimated to be around 500 tons per year and this is predicted to increase over the next few years (Mueller and Nowack, 2008). AgNPs may be released into the environment by several routes, including during their synthesis, incorporation of the AgNPs into other goods, and recycling or disposal of these goods and AgNPs. NPs have been shown to have higher and unique toxicity than their corresponding bulk materials (Shi et al., 2011). Thus, a better knowledge of nanomaterials, including their mode of interaction, uptake, accumulation and impact on the biosystems, and on their control measures to avoid nanopollution in ecological systems, is of concern and increasing importance (Navarro et al., 2008a). AgNPs have a dimension of 1–100 nm. The most important influences in determining the degree of bioaccumulation of AgNPs are likely to be similar to those that influence metal contaminants
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Corresponding author. Fax: þ662 2528979. E-mail addresses: [email protected], [email protected] (S. Chadchawan). http://dx.doi.org/10.1016/j.ecoenv.2014.03.022 0147-6513/& 2014 Elsevier Inc. All rights reserved.
(Garcia-Alonso et al., 2011). In plants, NPs are adsorbed to plant surfaces and taken up through natural nano or micrometer scale plant openings. Several pathways exist or are predicted for NP association and uptake in plants was explained (Dietz and Herth, 2011). AgNPs released into an aquatic environment can be a source of dissolved/suspended silver ions or metals, respectively, and so potentially exert toxic effects on aquatic organisms (Handy et al., 2008; Nowack, 2009). The impact of various types of NPs on higher plants has also been examined (Hong and Otaki, 2006; Navarro et al., 2008b; Zhu et al., 2008; Lin et al., 2009; Seeger et al., 2009). When growing, plants absorb relatively large amounts of essential and nonessential elements, which at certain concentrations may be toxic. Once stored within the plants, beneficial or toxic elements can be transferred along the food chain to consumers. Rice (Oryza sativa L.) is an important human food crop worldwide and is considered to be a model for monocot species for molecular biology research (Chhun et al., 2003), and so it is a reasonable and suitable species for research on nanomaterial safety. Studies on the toxicity of NPs are still emerging and have shown negative effects on the growth and development of plants. Lin and Xing (2007) analyzed the phytotoxicity of five types of multiwalled NPs (MWCNT, Al, Al2O3, Zn and ZnO) on the seed germination and seedling root growth in six higher plant species (Raphanus sativus, Lolium perenne, Lactuca sativa, Brassica napus,
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Zea mays and Cucumis sativus). Seed germination was not affected, except for the inhibition of Z. mays by ZnONPs and L. perenne by ZnNPs at high levels (2000 g/mL). However, the inhibition of root growth was found to vary greatly among the different types of NPs and plants, but was correlated to the NP concentrations. With respect to L. perenne, where ZnONPs caused a significant decrease in the seedling biomass, root tip shrinkage and collapse of the root epidermis, the cell internalization and translocation of was found to not be directly correlated with their dissolution in the bulk nutrient solution or rhizosphere (Lin and Xing, 2007). Wu et al. (2012) evaluated the effects of the metal oxide nanoparticles, CuO, NiO, TiO2, Fe2O3, and Co3O4 on germination index of lettuce, radish and cucumber and it was found that CuO and NiO showed the strongest inhibitory effects on these seed germination. The cytotoxicity of MWCNTs on rice suspension cells was found to correlate with an increase in the level of reactive oxygen species and could be reversed by the addition of ascorbic acid, a primary antioxidant (Tan et al., 2009). Nano-CuO (Shaw and Hossain, 2013) and nanoZnO toxicity (Dimkpa et al., 2012) were also determined in rice and wheat, respectively. These metal nanoparticles increased the reactive oxygen species in plant tissues, as well. Some reports have shown positive or no effects of NPs on plants. The effect of TiO2NPs on the photochemical reaction of chloroplasts of spinach, Spinacia oleracea, was to induce an increased Hill reaction and chloroplast activity that accelerated the oxidation-reduction process in chloroplasts (Zheng et al., 2005). Moreover, the non-cyclic photophosphorylation activity was higher. The explanation proposed for this is that the TiO2NPs might enter the chloroplasts where the catalyzed oxidationreduction reactions might then accelerate the electron transport and oxygen evolution (Hong et al., 2005). In addition, an increased germination rate and germination index for S. oleracea was noted after exposure to TiO2NPs at 0.25–4 percent (w/v) but not with larger TiO2 particles at the same concentrations. During the growth of the plants the dry weight and chlorophyll formation were both increased by exposure to the TiO2NPs (Zheng et al., 2005). The C70 NPs might have entered the plant roots via osmotic pressure and capillary forces and then entered through the cell wall pores and translocated through intercellular plasmadesmata (Lin et al., 2009). The phytotoxicity of silver nanoparticle (AgNPs) was evaluated in Arabidopsis thaliana, and it was found that AgNPs were apoplastically transported and aggregated at plasmodesmata (Geisler-Lee et al., 2013). From the concern about the potential toxicity of NPs, researchers have tried to test for cytotoxicity on living cells, including those from plants, animals, and microorganisms (Tan et al., 2009). Rice plants are becoming an interesting testing model for evaluating NPs because rice is the staple food crop of over half the world's population. If rice plants exposed to NPs revealed adverse effects it may suggest a potential impact on plant development and the food chain, which could in turn impact on humans, animals and the environment. Therefore, in this research the effect of different sized AgNPs at different concentrations on rice seedlings was evaluated in terms of the level of seed germination and the subsequent growth and leaf morphology of the seedlings. The obtained results should aid in leading to a further development of the nanomaterial safety information and the mechanism of AgNPs response in plants. 2. Materials and methods
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volume was adjusted to 100 mL to obtain a stock solution of AgNO3 at the concentration of 0.1 M. Food grade sucrose was bought from a local supermarket. Sodium hydroxide and nitric acid were purchased from Merck KGaA 64271 (Darmstadt, Germany). All chemicals were used as-received without further purification. The water used in all experiments was de-ionized water. 2.2. Synthesis of AgNPs Ten milliliter of 0.1 M AgNO3 in nitric acid, derived as outlined in Section 2.1, was mixed with 5 mL of 50 mM sucrose in a 100-mL beaker and left for 45 min at room temperature for partial hydrolysis of the sucrose to glucose and fructose for the subsequent aldehyde-mediated reduction of Ag þ to Ag0. Then, 5 mL of NaOH at a concentration of 0.3, 0.6, 0.9, 1.2, 1.5, 1.8, 2.1, 2.4, 2.7 or 3.0 M was added and mixed. The obtained silver powder is silver nanoparticles of various sizes. To ensure the complete conversion of silver ion into silver nanoparticle was performed according to Wongravee et al. (2013) by addition of reducing agent, NaBH4 into Ag colloids. If Ag þ ion remains in the colloids, the intensity in extinction spectra (at 400 nm) will increase. The spectra obtained suggested no Ag þ remained in the system after the synthesis of AgNPs. With the complete conversion of Ag þ to Ag nanoparticle, the concentration of Ag nanoparticle stock solution was 5350 mg/L (approximately 5000 mg/L), and this stock solution was used to prepare the AgNP at the indicated concentration. 2.3. Characterization of the AgNPs The size of the hydrated AgNPs obtained from each NaOH concentration (Section 2.2) was characterized by dynamic light scattering (DLS) using a particle size analyzer (Microtrac Zetztrac model NPA 152-31a-0000-000-20M). Each AgNP powder was washed with pure water five times by repeated centrifugation/ resuspension and then dispersed into pure water at 5 mg/500 mL by sonication for 1 min prior to immediately being analyzed by DLS using DLS;ELS 8000 Otsuka Electronics Osaka Japan. In addition the anhydrous morphology and particle size of the AgNPs was evaluated with JEOL JSM-6510 a scanning electron microscope and Hitachi H-7650, transmission electron microscope. The suspension of AgNP powder was washed and re-dispersed into pure water by sonication as outlined above, and then the suspension was dropped on the cleaned brass stub and air dried prior to examination. 2.4. Plant materials and growing condition Jasmine rice (O. sativa) cultivar Khao Dawk Mali 105 (KDML 105) was used as the test plant to investigate the effect of AgNPs on rice seed germination and seedling growth. AgNPs with a diameter of 20, 30–60, 70–120 and 150 nm were used, each at five aqueous suspension concentrations of 0.1, 1, 10, 100 and 1,000 mg/L, plus the control (0 mg/mL). Seed germination and seedling growth was performed in a greenhouse under natural light and temperature. Rice seeds (150 per treatment) were soaked in the aqueous AgNP suspension of the specific AgNP size and concentration for 24 h, and then sand germinated in a 12.5-cm diameter pot. After 7 days of germination, WP No. 2 solution was added to the pot as the nutrient source for seedling growth and grown on sand for another 7 days before transferred to a fresh hydroponic WP No. 2 solution. Then, they were grown in hydroponics system for 7 days in order to get the healthy root system and make them appropriate for root harvest for root growth determination. The WP No. 2 solution consisted of 580 mg/l KNO3, 500 mg/l CaSO4, 450 mg/l MgSO4 7H2O, 250 mg/l Triple super phosphate, 100 mg/l (NH4)2SO4, 160 mg/l Na2EDTA, 120 mg/l FeSO4 7H2O, 15 mg/l MnSO4 H2O, 5 mg/l H3BO3, 1.5 mg/l ZnSO4 7H2O, 1 mg/l KI, 0.1 mg/l Na2MoO4 2H2O, 0.05 mg/l CuSO4 5H2O, and 0.05 mg/l CoCl2 6H2O. 2.5. Silver nanoparticles accumulation After germination for 21 days, the treated rice seedlings were dried at 301C for 4 days and collected for the AgNP accumulation analysis by inductively coupled plasma optical emission spectrometry (ICP-OES; PerkinElmer Optima 4300 DV, German). A representative sample of up to 0.5 g is digested in 9 mL of concentrated nitric acid and 3 mL of hydrofluoric acid for 15 min using the microwave heating system. The temperature profile is specified to permit specific reactions and incorporates reaching 180 7 5 1C in approximately less than 5.5 min and remaining at 1807 5 1C for 9.5 min for the completion to specific reaction (Dugo et al., 2012; Kailasa and Wu, 2012; Ojeda and Rojas, 2013). After cooling the vessel contents were filtered, centrifuged and then decanted. Then the sample volume was adjusted and analyzed by ICP-OES.
2.1. Materials for AgNP synthesis 2.6. Effect of AgNPs on rice anatomy Silver nitrate (AgNO3) stock solution was prepared by dissolving 1.07 g of silver metal in 50 mL nitric acid. The solution was gently heated and stirred until all the silver metal was completely dissolved, then allowed to cool to 20 1C whereupon the
Transverse sections were made of selected leaves from 21-day-old seedlings grown without exposure to AgNPs (control) or after exposure as seeds for 24 h to
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Fig. 1. TEM images of AgNPs. (A) 20 nm, (B) 30–60 nm, (C) 70–120 nm and (D) 150 nm. 20-nm- and 150-nm-diameter AgNPs at 10 mg/L and 100 mg/L (Section 2.4). Transverse cross-sections of 80 mm thick were made using a plant microtome MT-3, and specimens were examined and photographed under white light using an Olympus DP70 photomicroscope.
2.7. Experimental design, data collection and statistical analysis A completely randomized design (CRD), with five replicates and four plants per replicate, was used to determine the effect of AgNPs on rice seedling growth. The growth parameters, in terms of the shoot and root fresh weights, shoot and root dry weights, plant height and root length, were measured at 21 days after germination. The results are expressed as the mean7 one standard error (SE) of five replicates and data were analyzed using one-way analysis of variance (ANOVA) with Dunnett's post hoc test to determine the significance relative to the control. In all cases, p o0.05 was considered significant.
3. Results and discussion 3.1. Characterization of the AgNPs The AgNPs derived from the aldehyde reduction of AgNO3 using sucrose in the presence of HNO3 followed by different NaOH concentrations (Section 2.2) were analyzed by DLS and SEM. In the absence of the NaOH the solutions remained clear, but showed a
gray precipitate of NPs upon addition of the different final NaOH concentrations (Supplementary Fig. S1). Sizes of AgNPs depending on amount of NaOH were added. The AgNP used in the experiments was shown in Fig. 1. 3.2. Effect of AgNP concentrations on rice seed germination and seedling growth Various sizes (20, 30–60, 70–120 and 150 nm) and concentrations, ranging from 0.1, 1.0, 10, 100, and 1000 mg/L, of AgNPs were studied for the effects on rice seedling growth. The similar patterns for dosage response were found in all AgNP type tested (Supplementary Table S1). AgNPs, with a 20 nm diameter were selected as a representative size to show the effects of varying the AgNP concentration on rice seed germination and seedling growth as it showed the least negative effects of seedling growth, when compared to other types of AgNPs effects. Exposure of the rice seeds to the 20-nm-diameter AgNPs showed a clear and dose-dependent inhibitory effect on their subsequent germination success and on the growth of those seedlings that did germinate (Fig. 2). After being continuously grown in a hydroponic system for 1 week (21-day-old post germination), the seedling growth was determined in terms of
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Fig. 2. The effects of 24-h seed soaking in AgNPs at different concentrations (0 (control), 0.1, 1.0, 10.0, 100.0, and 1000.0 mg/L) upon the (A, B) seedling growth at 14 day after germination. Images shown are representative of those seen from five independent trials. Effect of the AgNP concentration on 4-week-old seedlings, derived after seedsoaking in 20-nm-diameter AgNPs at the indicated concentration, were examined for (C) shoot fresh weight, (D) shoot dry weight, (E) root fresh weight, (F) root dry weight, (G) shoot height, and (H) root length. Data are shown as the mean þ 1 SE and are derived from five independent trials. Means with a different lowercase letter above them are significantly different (P o0.05).
the shoot and root fresh and dry weights and root length/height. AgNPs at all concentrations affected the seedling growth, although this was not statistically significant at 10 mg/L for all parameters measured. The absence of an inhibitory effect of AgNPs at 10 mg/L may result from some AgNP effects of the unknown mechanism, which was enough to compensate the growth inhibitory effects due to the action of AgNP. The higher concentrations of AgNPs (100 and 1000 mg/L) strongly inhibited both the shoot and root growth (especially as dry weight), with a more marked inhibition of the shoot growth
than the root growth (Fig. 2C–H). The shoot dry weight was reduced by 35–78 percent with 100–1000 mg/L AgNPs (Fig. 2D), while the root dry weight was reduced by 23–26 percent (Fig. 2F) compared to the no AgNP control. Similar trends were also found in the shoot height (Fig. 2G) and root length (Fig. 2H). Plant roots have been considered as the main route of plant's exposure to NPs may lead to physical or chemical toxicity in plants (Anjum et al., 2013). Growth inhibitory effects were also found when nano-CuO was treated to rice seedlings. Nano-CuO was shown to cause the severe oxidative burst and root membrane damage (Shaw and
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Fig. 3. The effects of 24-h seed soaking in water (c, control) or in AgNPs at 10 mg/L of a diameter of 20 (1), 30–60(2), 70–120 (3) and 150 (4) nm. The effects of (A, B) seedling growth at 14 day after germination. Images shown are representative of those seen from five independent trials. The effect of AgNPs size on 4-week-old seedlings, derived after -soaking in 10 mg/L AgNPs of various sizes (20, 30–60, 70–120, and 150 nm diameter) or in distilled water alone (control (c)), were examined for (C) shoot fresh weight, (D) shoot dry weight, (E) root fresh weight, (F) root dry weight, (G) shoot height, and (H) root length. Data are shown as the meanþ 1 SE and are derived from five independent trials. Means with a different lowercase letter above them are significantly different (Po 0.05).
Hossain, 2013). The dose dependent response of AgNPs on growth was also found in Arabidopsis thaliana and it was shown that AgNPs could be transported into root tissues and caused the brown root tip phenotype, indicating toxicity in root meristem (Geisler-Lee et al., 2013). The rice roots were also turned brown, when treated with AgNPs (data not shown). The inhibitory effects of AgNPs on rice seed germination was contrast to the evidence found in the previous study, which showed that MWCNT, Al2O3, Al at 2000 mg/L had no effects on seed germination of radish, rape, ryegrass, lettuce corn, and cucumber (Lin and Xing, 2007). This suggested that rice may be
susceptible to metal nanoparticle than other plants or the AgNPs are more toxic to plants than MWCNT, Al2O3 and Al nanoparticles. 3.3. Effect of the AgNP size on the germination and growth of rice seedlings With respect to the effect of the size of the AgNPs on rice seedling growth, the 2-week-old rice seedlings exposed to the different sized AgNPs (20, 30–60, 70–120, and 150 nm diameter) at 10 mg/L are presented in (Fig. 3). This concentration (10 mg/L) was selected because showed the lowest effect (Section 3.2). It was clearly seen
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that the larger AgNPs (70–120 and 150 nm diameter) showed a greater level of inhibition of seed germination and seedling growth (for those that did germinate) than the smaller AgNPs (Fig. 3). The AgNPs of all sizes at 10 mg/L did not show any significant effects on both the shoot and root fresh weights of 21-day-old seedlings (Fig. 3C and E), although the numerical trend was to reduce the fresh weight with increasing AgNP sizes. Moreover, the addition of AgNPs of any size decreased the dry weight, plant height and root length (Fig. 3D, F–H), and so revealed the negative effects of AgNP sizes on rice seedling growth. The size dependent effects of AgNPs were also found in Arabidopsis root growth (Geisler-Lee et al., 2013). The larger AgNP had the stronger growth inhibition effects than the smaller ones, which was consistent with our report. 3.4. Bioaccumulation of AgNPs in plant tissues After seed soaking, the rice seeds were germinated on sand and then transferred to the nutrient solution. The carried over amount
Fig. 4. The concentration of AgNPs carried over into the nutrient solution. Data are shown as the mean7 1 SE and are derived from three independent trials. Means with a different lowercase letter above them are significantly different (Po 0.05).
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of AgNPs in the nutrient solution was determined as shown in Fig. 4. The nutrient solution with seedlings germinated from treated seeds with 0.1 or 1.0 mg/L AgNPs at any sizes did not contain AgNPs at the detectable level. At higher concentration of AgNPs, the higher concentration of contaminated AgNPs in nutrient solution was found (Fig. 4). However, the carried over concentration was less than 3000 fold dilution of AgNP concentration for the seed-soaking. Various concentrations of AgNPs were found in leaf and root tissues depending on sizes and concentration of AgNP treatment. As low as 1 mg/L of 20 nm diameter AgNP could be uptaked by rice seedlings as it could be detected in root tissues. However, no detectable level of AgNPs was found in leaves. This indicated that AgNPs were trapped in the roots and AgNP transport may be inhibited by some mechanisms. When seeds were exposed to the higher concentration of 20 nm diameter AgNPs, the higher accumulation was found. The AgNPs accumulated in leaves was found when seeds were treated with 100 or 1000 mg/L (Fig. 5A). The increase in AgNP accumulation was found when the seeds were treated with higher concentration of AgNPs. This is similar to the evidence found in other crop plants (Lee et al., 2012). When seeds were treated with the larger sizes of AgNPs at 1 mg/L, no detectable level of AgNPs was found in both roots and shoots. At higher concentration, 10 mg/L AgNPs with 70–150 nm in diameter, AgNP treated seeds developed with the accumulation of AgNPs in both shoot and root tissues (Fig. 5C and D). However, seeds treated with 10 mg/L of 30–60 nm AgNPs resulted in AgNP accumulation in roots only (Fig. 5B). AgNP treatment in Arabidopsis showed the accumulation of Ag0 at root tip at low concentration of the small AgNP (diameter of 20 nm and 40 nm), but when it was treated with the low concentration of AgNP with 80 nm diameter, no Ag0 was detected at root tip. Ag0 was detected both on surface and in internal tissues (Geisler-Lee et al., 2013). This supports the hypothesis that roots tend to trap the smaller AgNP than the larger ones. Moreover, the study in ryegrass with 20 nm ZnO-NP was found that the NP was associated with root surface (Lin and Xing, 2008). This is
Fig. 5. Bioaccumulation of AgNPs in root and leaf tissues when seeds were treated with 20 (A), 30–60 (B), 70–120 (C), and 150 (D) nm AgNPs at various concentrations. Data are shown as the mean 7 1 SE and are derived from three independent trials. Means with a different lowercase letter above them are significantly different (P o 0.05).
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Fig. 6. The effects of seed exposure to (A, B) 20-nm- and (C, D) 150-nm-diameter AgNPs at a concentration of (A, C) 10 mg/L or (B, D) 100 mg/L on the leaf anatomy of 21-dold rice seedlings, compared with the (E) control plants. Arrows show abnormal of parenchyma cell, and scale bars represent 200 μm. Transverse section images shown (20 X magnification) are representative of those seen from at least 50 such sections per sample and five independent samples.
consistent with our result that when rice seeds were treated with low concentrations (0.1–10 mg/L) of the small AgNPs (with diameter of 20 and 30–60 nm), Ag could be detected in root tissue only (Fig. 5A and B), while they were treated with the larger AgNPs (with diameter of 70–12, and 150 nm) (Fig. 5C and D) at10 mg/L Ag was detected in both root and leaf tissues, suggesting that the transport from root to shoot occurred in these treatments. The long distance transport of NP has been detected in Arabidopsis and Phalaenopsis sp. (Hischemö ller et al., 2009). Based on these data, it suggested that the smaller sizes of AgNPs had the higher ability for penetrating into plant roots, as the smaller sizes could be found at higher concentration in root tissues. AgNPs can enter into plant cells either through endocytosis or nonendocytosis penetration (Navarro et al., 2008a). However, the larger size AgNPs could be transported more efficiently to the shoots,
resulting in the more negative effects on plant growth due to the larger AgNP exposure (Fig. 3). 3.5. Effect of AgNPs on the rice seedling leaf anatomy Since exposure of rice seeds to the 20-nm-diameter AgNPs at 10 mg/L had almost no significant effect on the rice seed germination level or subsequent seedling growth, while 100 mg/L of 150nm-diameter AgNP had a significant inhibitory effect on both, then the anatomical changes in 21-day-old seedling leaves after exposure of the seeds to 20- or 150-nm-diameter AgNPs at 10 and 100 mg/L were evaluated. The 20-nm-diameter AgNPs at 10 mg/L showed only a slight effect on the rice leaf anatomy (Fig. 6A), whereas at 100 mg/L (Fig. 6B), and the larger 150-nm-diameter AgNPs at 10 mg/L
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(Fig. 6C) and 100 mg/L (Fig. 6D) all showed clear alterations in the seedling leaf anatomy in the mid-rib area, especially in the parenchyma cells connecting the upper and lower vascular bundles. In the control plants, about five to seven layers of parenchyma cells were found (Fig. 6E), while more layers of smaller cells were found in the AgNP-treated seedlings (Fig. 6A–D). In addition, cell disruption was also evident when seeds were exposed to the larger (150-nm-diameter) sized AgNPs at the higher concentration of 100 mg/L (Fig. 6D). These results are consistent with that reported for ZnONPs and TiO2NPs, where the larger sized NPs caused more cell damage than the smaller ones (Kumar et al., 2011). The mechanism of penetration of AgNPs is not fully understood, they could either traverse the cell membrane or diffuse through the lipid bilayer. The cell membrane is permeable to different substances that can diffuse through it. A recent report demonstrated that MWCNTs showed similar possible cytotoxic effects on rice suspension cells (Tan et al., 2009). 4. Conclusion This study demonstrated that exposure of rice seeds to AgNPs had a clear phytotoxic effect on the rice seedlings. The effect of AgNPs on rice (O. sativa cv. KDML 105) was dependent upon the size and concentration of the AgNPs. Increasing the AgNP concentration over the range of 0.1 to 1000 mg/L increased the inhibition level of seed germination and subsequent seedling growth, especially at the higher level of 100 and 1000 mg/L, for each size of AgNPs. Likewise, increasing the size of the AgNPs over the 20–150 nm diameter range increased the inhibition effect upon seed germination and seedling growth. The inhibition effects were due to both the penetration and transport of AgNPs through plant tissues. For the application of AgNPs, sizes and concentrations should be under consideration for environmental safety. Acknowledgments The authors are grateful to the Center of Innovation of Chulalongkorn University, Thailand for financial support. Finally, we would like to thank all members of CE in Environment and Plant Physiology for their contributions to this project. CE was supported by Ratchadaphiseksomphot Endowment Fund, Chulalongkorn University. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2014.03.022. References Anjum, N.A., Gill, S.S., Duarte, A.C., Pereira, E., Ahmad, I., 2013. Silver nanoparticles in soil-plant systems. J. Nanoparticle Res. 15. Chhun, T., Taketa, S., Tsurumi, S., Ichii, M., 2003. Interaction between two auxinresistant mutants and their effects on lateral root formation in rice (Oryza sativa L.). J. Exp. Botany 54, 2701–2708. Dietz, K.-J., Herth, S., 2011. Plant nanotoxicology. Trends Plant Sci. 16, 582–589.
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Effect of silver nanoparticles on rice (Oryza sativa L. cv. KDML 105) seed germination and seedling growth Pakvirun Thuesombat a, Supot Hannongbua b, Sanong Akasit b, Supachitra Chadchawan c,n a
Nanoscience and Technology Program, Graduate School, Chulalongkorn University, Bangkok 10330, Thailand Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand c Center of Excellence in Environment and Plant Physiology, Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand b
art ic l e i nf o
a b s t r a c t
Article history: Received 8 September 2013 Received in revised form 14 March 2014 Accepted 19 March 2014 Available online 15 April 2014
With the advances in nanotechnology, silver nanoparticles (AgNPs) have been applied in many industries, increasing their potential exposure level in the environment, yet their environmental safety remains poorly evaluated. The possible effects of different sized AgNPs (20, 30–60, 70–120 and 150 nm diameter) on jasmine rice, Oryza sativa L. cv. KDML 105, were investigated at different concentrations (0.1, 1, 10, 100 and 1000 mg/L) upon seed germination and seedling growth. The results revealed that the level of seed germination and subsequent growth of those seedlings that germinated were both decreased with increasing sizes and concentrations of AgNPs. Based on the analysis of AgNPs accumulation in plant tissues, it implied that the higher uptake was found when the seeds were treated with the smaller AgNPs, 20 nm diameter AgNPs, but it was trapped in the roots rather than transported to the leaves. These resulted in the less negative effects on seedling growth, when compared to the seed soaking with the larger AgNPs with 150 nm diameter. The negative effects of AgNPs were supported by leaf cell deformation when rice seeds were treated with 150-nm-diameter AgNP at the concentration of 10 or 100 mg/L during seed germination. These results further strengthen our understanding of environmental safety information with respect to nanomaterials. & 2014 Elsevier Inc. All rights reserved.
Keywords: Silver nanoparticle Rice Nanotoxicology
1. Introduction With the advancement in nanotechnology, silver NPs (AgNPs) have been applied in many industries, including daily products and medical products. Worldwide, the present production of AgNPs is estimated to be around 500 tons per year and this is predicted to increase over the next few years (Mueller and Nowack, 2008). AgNPs may be released into the environment by several routes, including during their synthesis, incorporation of the AgNPs into other goods, and recycling or disposal of these goods and AgNPs. NPs have been shown to have higher and unique toxicity than their corresponding bulk materials (Shi et al., 2011). Thus, a better knowledge of nanomaterials, including their mode of interaction, uptake, accumulation and impact on the biosystems, and on their control measures to avoid nanopollution in ecological systems, is of concern and increasing importance (Navarro et al., 2008a). AgNPs have a dimension of 1–100 nm. The most important influences in determining the degree of bioaccumulation of AgNPs are likely to be similar to those that influence metal contaminants
n
Corresponding author. Fax: þ662 2528979. E-mail addresses: [email protected], [email protected] (S. Chadchawan). http://dx.doi.org/10.1016/j.ecoenv.2014.03.022 0147-6513/& 2014 Elsevier Inc. All rights reserved.
(Garcia-Alonso et al., 2011). In plants, NPs are adsorbed to plant surfaces and taken up through natural nano or micrometer scale plant openings. Several pathways exist or are predicted for NP association and uptake in plants was explained (Dietz and Herth, 2011). AgNPs released into an aquatic environment can be a source of dissolved/suspended silver ions or metals, respectively, and so potentially exert toxic effects on aquatic organisms (Handy et al., 2008; Nowack, 2009). The impact of various types of NPs on higher plants has also been examined (Hong and Otaki, 2006; Navarro et al., 2008b; Zhu et al., 2008; Lin et al., 2009; Seeger et al., 2009). When growing, plants absorb relatively large amounts of essential and nonessential elements, which at certain concentrations may be toxic. Once stored within the plants, beneficial or toxic elements can be transferred along the food chain to consumers. Rice (Oryza sativa L.) is an important human food crop worldwide and is considered to be a model for monocot species for molecular biology research (Chhun et al., 2003), and so it is a reasonable and suitable species for research on nanomaterial safety. Studies on the toxicity of NPs are still emerging and have shown negative effects on the growth and development of plants. Lin and Xing (2007) analyzed the phytotoxicity of five types of multiwalled NPs (MWCNT, Al, Al2O3, Zn and ZnO) on the seed germination and seedling root growth in six higher plant species (Raphanus sativus, Lolium perenne, Lactuca sativa, Brassica napus,
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Zea mays and Cucumis sativus). Seed germination was not affected, except for the inhibition of Z. mays by ZnONPs and L. perenne by ZnNPs at high levels (2000 g/mL). However, the inhibition of root growth was found to vary greatly among the different types of NPs and plants, but was correlated to the NP concentrations. With respect to L. perenne, where ZnONPs caused a significant decrease in the seedling biomass, root tip shrinkage and collapse of the root epidermis, the cell internalization and translocation of was found to not be directly correlated with their dissolution in the bulk nutrient solution or rhizosphere (Lin and Xing, 2007). Wu et al. (2012) evaluated the effects of the metal oxide nanoparticles, CuO, NiO, TiO2, Fe2O3, and Co3O4 on germination index of lettuce, radish and cucumber and it was found that CuO and NiO showed the strongest inhibitory effects on these seed germination. The cytotoxicity of MWCNTs on rice suspension cells was found to correlate with an increase in the level of reactive oxygen species and could be reversed by the addition of ascorbic acid, a primary antioxidant (Tan et al., 2009). Nano-CuO (Shaw and Hossain, 2013) and nanoZnO toxicity (Dimkpa et al., 2012) were also determined in rice and wheat, respectively. These metal nanoparticles increased the reactive oxygen species in plant tissues, as well. Some reports have shown positive or no effects of NPs on plants. The effect of TiO2NPs on the photochemical reaction of chloroplasts of spinach, Spinacia oleracea, was to induce an increased Hill reaction and chloroplast activity that accelerated the oxidation-reduction process in chloroplasts (Zheng et al., 2005). Moreover, the non-cyclic photophosphorylation activity was higher. The explanation proposed for this is that the TiO2NPs might enter the chloroplasts where the catalyzed oxidationreduction reactions might then accelerate the electron transport and oxygen evolution (Hong et al., 2005). In addition, an increased germination rate and germination index for S. oleracea was noted after exposure to TiO2NPs at 0.25–4 percent (w/v) but not with larger TiO2 particles at the same concentrations. During the growth of the plants the dry weight and chlorophyll formation were both increased by exposure to the TiO2NPs (Zheng et al., 2005). The C70 NPs might have entered the plant roots via osmotic pressure and capillary forces and then entered through the cell wall pores and translocated through intercellular plasmadesmata (Lin et al., 2009). The phytotoxicity of silver nanoparticle (AgNPs) was evaluated in Arabidopsis thaliana, and it was found that AgNPs were apoplastically transported and aggregated at plasmodesmata (Geisler-Lee et al., 2013). From the concern about the potential toxicity of NPs, researchers have tried to test for cytotoxicity on living cells, including those from plants, animals, and microorganisms (Tan et al., 2009). Rice plants are becoming an interesting testing model for evaluating NPs because rice is the staple food crop of over half the world's population. If rice plants exposed to NPs revealed adverse effects it may suggest a potential impact on plant development and the food chain, which could in turn impact on humans, animals and the environment. Therefore, in this research the effect of different sized AgNPs at different concentrations on rice seedlings was evaluated in terms of the level of seed germination and the subsequent growth and leaf morphology of the seedlings. The obtained results should aid in leading to a further development of the nanomaterial safety information and the mechanism of AgNPs response in plants. 2. Materials and methods
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volume was adjusted to 100 mL to obtain a stock solution of AgNO3 at the concentration of 0.1 M. Food grade sucrose was bought from a local supermarket. Sodium hydroxide and nitric acid were purchased from Merck KGaA 64271 (Darmstadt, Germany). All chemicals were used as-received without further purification. The water used in all experiments was de-ionized water. 2.2. Synthesis of AgNPs Ten milliliter of 0.1 M AgNO3 in nitric acid, derived as outlined in Section 2.1, was mixed with 5 mL of 50 mM sucrose in a 100-mL beaker and left for 45 min at room temperature for partial hydrolysis of the sucrose to glucose and fructose for the subsequent aldehyde-mediated reduction of Ag þ to Ag0. Then, 5 mL of NaOH at a concentration of 0.3, 0.6, 0.9, 1.2, 1.5, 1.8, 2.1, 2.4, 2.7 or 3.0 M was added and mixed. The obtained silver powder is silver nanoparticles of various sizes. To ensure the complete conversion of silver ion into silver nanoparticle was performed according to Wongravee et al. (2013) by addition of reducing agent, NaBH4 into Ag colloids. If Ag þ ion remains in the colloids, the intensity in extinction spectra (at 400 nm) will increase. The spectra obtained suggested no Ag þ remained in the system after the synthesis of AgNPs. With the complete conversion of Ag þ to Ag nanoparticle, the concentration of Ag nanoparticle stock solution was 5350 mg/L (approximately 5000 mg/L), and this stock solution was used to prepare the AgNP at the indicated concentration. 2.3. Characterization of the AgNPs The size of the hydrated AgNPs obtained from each NaOH concentration (Section 2.2) was characterized by dynamic light scattering (DLS) using a particle size analyzer (Microtrac Zetztrac model NPA 152-31a-0000-000-20M). Each AgNP powder was washed with pure water five times by repeated centrifugation/ resuspension and then dispersed into pure water at 5 mg/500 mL by sonication for 1 min prior to immediately being analyzed by DLS using DLS;ELS 8000 Otsuka Electronics Osaka Japan. In addition the anhydrous morphology and particle size of the AgNPs was evaluated with JEOL JSM-6510 a scanning electron microscope and Hitachi H-7650, transmission electron microscope. The suspension of AgNP powder was washed and re-dispersed into pure water by sonication as outlined above, and then the suspension was dropped on the cleaned brass stub and air dried prior to examination. 2.4. Plant materials and growing condition Jasmine rice (O. sativa) cultivar Khao Dawk Mali 105 (KDML 105) was used as the test plant to investigate the effect of AgNPs on rice seed germination and seedling growth. AgNPs with a diameter of 20, 30–60, 70–120 and 150 nm were used, each at five aqueous suspension concentrations of 0.1, 1, 10, 100 and 1,000 mg/L, plus the control (0 mg/mL). Seed germination and seedling growth was performed in a greenhouse under natural light and temperature. Rice seeds (150 per treatment) were soaked in the aqueous AgNP suspension of the specific AgNP size and concentration for 24 h, and then sand germinated in a 12.5-cm diameter pot. After 7 days of germination, WP No. 2 solution was added to the pot as the nutrient source for seedling growth and grown on sand for another 7 days before transferred to a fresh hydroponic WP No. 2 solution. Then, they were grown in hydroponics system for 7 days in order to get the healthy root system and make them appropriate for root harvest for root growth determination. The WP No. 2 solution consisted of 580 mg/l KNO3, 500 mg/l CaSO4, 450 mg/l MgSO4 7H2O, 250 mg/l Triple super phosphate, 100 mg/l (NH4)2SO4, 160 mg/l Na2EDTA, 120 mg/l FeSO4 7H2O, 15 mg/l MnSO4 H2O, 5 mg/l H3BO3, 1.5 mg/l ZnSO4 7H2O, 1 mg/l KI, 0.1 mg/l Na2MoO4 2H2O, 0.05 mg/l CuSO4 5H2O, and 0.05 mg/l CoCl2 6H2O. 2.5. Silver nanoparticles accumulation After germination for 21 days, the treated rice seedlings were dried at 301C for 4 days and collected for the AgNP accumulation analysis by inductively coupled plasma optical emission spectrometry (ICP-OES; PerkinElmer Optima 4300 DV, German). A representative sample of up to 0.5 g is digested in 9 mL of concentrated nitric acid and 3 mL of hydrofluoric acid for 15 min using the microwave heating system. The temperature profile is specified to permit specific reactions and incorporates reaching 180 7 5 1C in approximately less than 5.5 min and remaining at 1807 5 1C for 9.5 min for the completion to specific reaction (Dugo et al., 2012; Kailasa and Wu, 2012; Ojeda and Rojas, 2013). After cooling the vessel contents were filtered, centrifuged and then decanted. Then the sample volume was adjusted and analyzed by ICP-OES.
2.1. Materials for AgNP synthesis 2.6. Effect of AgNPs on rice anatomy Silver nitrate (AgNO3) stock solution was prepared by dissolving 1.07 g of silver metal in 50 mL nitric acid. The solution was gently heated and stirred until all the silver metal was completely dissolved, then allowed to cool to 20 1C whereupon the
Transverse sections were made of selected leaves from 21-day-old seedlings grown without exposure to AgNPs (control) or after exposure as seeds for 24 h to
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Fig. 1. TEM images of AgNPs. (A) 20 nm, (B) 30–60 nm, (C) 70–120 nm and (D) 150 nm. 20-nm- and 150-nm-diameter AgNPs at 10 mg/L and 100 mg/L (Section 2.4). Transverse cross-sections of 80 mm thick were made using a plant microtome MT-3, and specimens were examined and photographed under white light using an Olympus DP70 photomicroscope.
2.7. Experimental design, data collection and statistical analysis A completely randomized design (CRD), with five replicates and four plants per replicate, was used to determine the effect of AgNPs on rice seedling growth. The growth parameters, in terms of the shoot and root fresh weights, shoot and root dry weights, plant height and root length, were measured at 21 days after germination. The results are expressed as the mean7 one standard error (SE) of five replicates and data were analyzed using one-way analysis of variance (ANOVA) with Dunnett's post hoc test to determine the significance relative to the control. In all cases, p o0.05 was considered significant.
3. Results and discussion 3.1. Characterization of the AgNPs The AgNPs derived from the aldehyde reduction of AgNO3 using sucrose in the presence of HNO3 followed by different NaOH concentrations (Section 2.2) were analyzed by DLS and SEM. In the absence of the NaOH the solutions remained clear, but showed a
gray precipitate of NPs upon addition of the different final NaOH concentrations (Supplementary Fig. S1). Sizes of AgNPs depending on amount of NaOH were added. The AgNP used in the experiments was shown in Fig. 1. 3.2. Effect of AgNP concentrations on rice seed germination and seedling growth Various sizes (20, 30–60, 70–120 and 150 nm) and concentrations, ranging from 0.1, 1.0, 10, 100, and 1000 mg/L, of AgNPs were studied for the effects on rice seedling growth. The similar patterns for dosage response were found in all AgNP type tested (Supplementary Table S1). AgNPs, with a 20 nm diameter were selected as a representative size to show the effects of varying the AgNP concentration on rice seed germination and seedling growth as it showed the least negative effects of seedling growth, when compared to other types of AgNPs effects. Exposure of the rice seeds to the 20-nm-diameter AgNPs showed a clear and dose-dependent inhibitory effect on their subsequent germination success and on the growth of those seedlings that did germinate (Fig. 2). After being continuously grown in a hydroponic system for 1 week (21-day-old post germination), the seedling growth was determined in terms of
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Fig. 2. The effects of 24-h seed soaking in AgNPs at different concentrations (0 (control), 0.1, 1.0, 10.0, 100.0, and 1000.0 mg/L) upon the (A, B) seedling growth at 14 day after germination. Images shown are representative of those seen from five independent trials. Effect of the AgNP concentration on 4-week-old seedlings, derived after seedsoaking in 20-nm-diameter AgNPs at the indicated concentration, were examined for (C) shoot fresh weight, (D) shoot dry weight, (E) root fresh weight, (F) root dry weight, (G) shoot height, and (H) root length. Data are shown as the mean þ 1 SE and are derived from five independent trials. Means with a different lowercase letter above them are significantly different (P o0.05).
the shoot and root fresh and dry weights and root length/height. AgNPs at all concentrations affected the seedling growth, although this was not statistically significant at 10 mg/L for all parameters measured. The absence of an inhibitory effect of AgNPs at 10 mg/L may result from some AgNP effects of the unknown mechanism, which was enough to compensate the growth inhibitory effects due to the action of AgNP. The higher concentrations of AgNPs (100 and 1000 mg/L) strongly inhibited both the shoot and root growth (especially as dry weight), with a more marked inhibition of the shoot growth
than the root growth (Fig. 2C–H). The shoot dry weight was reduced by 35–78 percent with 100–1000 mg/L AgNPs (Fig. 2D), while the root dry weight was reduced by 23–26 percent (Fig. 2F) compared to the no AgNP control. Similar trends were also found in the shoot height (Fig. 2G) and root length (Fig. 2H). Plant roots have been considered as the main route of plant's exposure to NPs may lead to physical or chemical toxicity in plants (Anjum et al., 2013). Growth inhibitory effects were also found when nano-CuO was treated to rice seedlings. Nano-CuO was shown to cause the severe oxidative burst and root membrane damage (Shaw and
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Fig. 3. The effects of 24-h seed soaking in water (c, control) or in AgNPs at 10 mg/L of a diameter of 20 (1), 30–60(2), 70–120 (3) and 150 (4) nm. The effects of (A, B) seedling growth at 14 day after germination. Images shown are representative of those seen from five independent trials. The effect of AgNPs size on 4-week-old seedlings, derived after -soaking in 10 mg/L AgNPs of various sizes (20, 30–60, 70–120, and 150 nm diameter) or in distilled water alone (control (c)), were examined for (C) shoot fresh weight, (D) shoot dry weight, (E) root fresh weight, (F) root dry weight, (G) shoot height, and (H) root length. Data are shown as the meanþ 1 SE and are derived from five independent trials. Means with a different lowercase letter above them are significantly different (Po 0.05).
Hossain, 2013). The dose dependent response of AgNPs on growth was also found in Arabidopsis thaliana and it was shown that AgNPs could be transported into root tissues and caused the brown root tip phenotype, indicating toxicity in root meristem (Geisler-Lee et al., 2013). The rice roots were also turned brown, when treated with AgNPs (data not shown). The inhibitory effects of AgNPs on rice seed germination was contrast to the evidence found in the previous study, which showed that MWCNT, Al2O3, Al at 2000 mg/L had no effects on seed germination of radish, rape, ryegrass, lettuce corn, and cucumber (Lin and Xing, 2007). This suggested that rice may be
susceptible to metal nanoparticle than other plants or the AgNPs are more toxic to plants than MWCNT, Al2O3 and Al nanoparticles. 3.3. Effect of the AgNP size on the germination and growth of rice seedlings With respect to the effect of the size of the AgNPs on rice seedling growth, the 2-week-old rice seedlings exposed to the different sized AgNPs (20, 30–60, 70–120, and 150 nm diameter) at 10 mg/L are presented in (Fig. 3). This concentration (10 mg/L) was selected because showed the lowest effect (Section 3.2). It was clearly seen
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that the larger AgNPs (70–120 and 150 nm diameter) showed a greater level of inhibition of seed germination and seedling growth (for those that did germinate) than the smaller AgNPs (Fig. 3). The AgNPs of all sizes at 10 mg/L did not show any significant effects on both the shoot and root fresh weights of 21-day-old seedlings (Fig. 3C and E), although the numerical trend was to reduce the fresh weight with increasing AgNP sizes. Moreover, the addition of AgNPs of any size decreased the dry weight, plant height and root length (Fig. 3D, F–H), and so revealed the negative effects of AgNP sizes on rice seedling growth. The size dependent effects of AgNPs were also found in Arabidopsis root growth (Geisler-Lee et al., 2013). The larger AgNP had the stronger growth inhibition effects than the smaller ones, which was consistent with our report. 3.4. Bioaccumulation of AgNPs in plant tissues After seed soaking, the rice seeds were germinated on sand and then transferred to the nutrient solution. The carried over amount
Fig. 4. The concentration of AgNPs carried over into the nutrient solution. Data are shown as the mean7 1 SE and are derived from three independent trials. Means with a different lowercase letter above them are significantly different (Po 0.05).
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of AgNPs in the nutrient solution was determined as shown in Fig. 4. The nutrient solution with seedlings germinated from treated seeds with 0.1 or 1.0 mg/L AgNPs at any sizes did not contain AgNPs at the detectable level. At higher concentration of AgNPs, the higher concentration of contaminated AgNPs in nutrient solution was found (Fig. 4). However, the carried over concentration was less than 3000 fold dilution of AgNP concentration for the seed-soaking. Various concentrations of AgNPs were found in leaf and root tissues depending on sizes and concentration of AgNP treatment. As low as 1 mg/L of 20 nm diameter AgNP could be uptaked by rice seedlings as it could be detected in root tissues. However, no detectable level of AgNPs was found in leaves. This indicated that AgNPs were trapped in the roots and AgNP transport may be inhibited by some mechanisms. When seeds were exposed to the higher concentration of 20 nm diameter AgNPs, the higher accumulation was found. The AgNPs accumulated in leaves was found when seeds were treated with 100 or 1000 mg/L (Fig. 5A). The increase in AgNP accumulation was found when the seeds were treated with higher concentration of AgNPs. This is similar to the evidence found in other crop plants (Lee et al., 2012). When seeds were treated with the larger sizes of AgNPs at 1 mg/L, no detectable level of AgNPs was found in both roots and shoots. At higher concentration, 10 mg/L AgNPs with 70–150 nm in diameter, AgNP treated seeds developed with the accumulation of AgNPs in both shoot and root tissues (Fig. 5C and D). However, seeds treated with 10 mg/L of 30–60 nm AgNPs resulted in AgNP accumulation in roots only (Fig. 5B). AgNP treatment in Arabidopsis showed the accumulation of Ag0 at root tip at low concentration of the small AgNP (diameter of 20 nm and 40 nm), but when it was treated with the low concentration of AgNP with 80 nm diameter, no Ag0 was detected at root tip. Ag0 was detected both on surface and in internal tissues (Geisler-Lee et al., 2013). This supports the hypothesis that roots tend to trap the smaller AgNP than the larger ones. Moreover, the study in ryegrass with 20 nm ZnO-NP was found that the NP was associated with root surface (Lin and Xing, 2008). This is
Fig. 5. Bioaccumulation of AgNPs in root and leaf tissues when seeds were treated with 20 (A), 30–60 (B), 70–120 (C), and 150 (D) nm AgNPs at various concentrations. Data are shown as the mean 7 1 SE and are derived from three independent trials. Means with a different lowercase letter above them are significantly different (P o 0.05).
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Fig. 6. The effects of seed exposure to (A, B) 20-nm- and (C, D) 150-nm-diameter AgNPs at a concentration of (A, C) 10 mg/L or (B, D) 100 mg/L on the leaf anatomy of 21-dold rice seedlings, compared with the (E) control plants. Arrows show abnormal of parenchyma cell, and scale bars represent 200 μm. Transverse section images shown (20 X magnification) are representative of those seen from at least 50 such sections per sample and five independent samples.
consistent with our result that when rice seeds were treated with low concentrations (0.1–10 mg/L) of the small AgNPs (with diameter of 20 and 30–60 nm), Ag could be detected in root tissue only (Fig. 5A and B), while they were treated with the larger AgNPs (with diameter of 70–12, and 150 nm) (Fig. 5C and D) at10 mg/L Ag was detected in both root and leaf tissues, suggesting that the transport from root to shoot occurred in these treatments. The long distance transport of NP has been detected in Arabidopsis and Phalaenopsis sp. (Hischemö ller et al., 2009). Based on these data, it suggested that the smaller sizes of AgNPs had the higher ability for penetrating into plant roots, as the smaller sizes could be found at higher concentration in root tissues. AgNPs can enter into plant cells either through endocytosis or nonendocytosis penetration (Navarro et al., 2008a). However, the larger size AgNPs could be transported more efficiently to the shoots,
resulting in the more negative effects on plant growth due to the larger AgNP exposure (Fig. 3). 3.5. Effect of AgNPs on the rice seedling leaf anatomy Since exposure of rice seeds to the 20-nm-diameter AgNPs at 10 mg/L had almost no significant effect on the rice seed germination level or subsequent seedling growth, while 100 mg/L of 150nm-diameter AgNP had a significant inhibitory effect on both, then the anatomical changes in 21-day-old seedling leaves after exposure of the seeds to 20- or 150-nm-diameter AgNPs at 10 and 100 mg/L were evaluated. The 20-nm-diameter AgNPs at 10 mg/L showed only a slight effect on the rice leaf anatomy (Fig. 6A), whereas at 100 mg/L (Fig. 6B), and the larger 150-nm-diameter AgNPs at 10 mg/L
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(Fig. 6C) and 100 mg/L (Fig. 6D) all showed clear alterations in the seedling leaf anatomy in the mid-rib area, especially in the parenchyma cells connecting the upper and lower vascular bundles. In the control plants, about five to seven layers of parenchyma cells were found (Fig. 6E), while more layers of smaller cells were found in the AgNP-treated seedlings (Fig. 6A–D). In addition, cell disruption was also evident when seeds were exposed to the larger (150-nm-diameter) sized AgNPs at the higher concentration of 100 mg/L (Fig. 6D). These results are consistent with that reported for ZnONPs and TiO2NPs, where the larger sized NPs caused more cell damage than the smaller ones (Kumar et al., 2011). The mechanism of penetration of AgNPs is not fully understood, they could either traverse the cell membrane or diffuse through the lipid bilayer. The cell membrane is permeable to different substances that can diffuse through it. A recent report demonstrated that MWCNTs showed similar possible cytotoxic effects on rice suspension cells (Tan et al., 2009). 4. Conclusion This study demonstrated that exposure of rice seeds to AgNPs had a clear phytotoxic effect on the rice seedlings. The effect of AgNPs on rice (O. sativa cv. KDML 105) was dependent upon the size and concentration of the AgNPs. Increasing the AgNP concentration over the range of 0.1 to 1000 mg/L increased the inhibition level of seed germination and subsequent seedling growth, especially at the higher level of 100 and 1000 mg/L, for each size of AgNPs. Likewise, increasing the size of the AgNPs over the 20–150 nm diameter range increased the inhibition effect upon seed germination and seedling growth. The inhibition effects were due to both the penetration and transport of AgNPs through plant tissues. For the application of AgNPs, sizes and concentrations should be under consideration for environmental safety. Acknowledgments The authors are grateful to the Center of Innovation of Chulalongkorn University, Thailand for financial support. Finally, we would like to thank all members of CE in Environment and Plant Physiology for their contributions to this project. CE was supported by Ratchadaphiseksomphot Endowment Fund, Chulalongkorn University. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2014.03.022. References Anjum, N.A., Gill, S.S., Duarte, A.C., Pereira, E., Ahmad, I., 2013. Silver nanoparticles in soil-plant systems. J. Nanoparticle Res. 15. Chhun, T., Taketa, S., Tsurumi, S., Ichii, M., 2003. Interaction between two auxinresistant mutants and their effects on lateral root formation in rice (Oryza sativa L.). J. Exp. Botany 54, 2701–2708. Dietz, K.-J., Herth, S., 2011. Plant nanotoxicology. Trends Plant Sci. 16, 582–589.
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