Ecotoxicology and Environmental Safety 100 (2014) 131–137
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Zinc oxide nanoparticles delay soybean development: A standard soil microcosm study Sung-Ji Yoon a,1, Jin Il Kwak a,1, Woo-Mi Lee a, Patricia A. Holden b, Youn-Joo An a,n a b
Department of Environmental Science, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea Bren School of Environmental Science and Management, University of California, Santa Barbara, CA, USA
art ic l e i nf o
a b s t r a c t
Article history: Received 17 July 2013 Received in revised form 11 October 2013 Accepted 15 October 2013 Available online 2 December 2013
Soybean is an important crop and a source of food for humans and livestock. In this study, for the first time, the long-term effects of zinc oxide (ZnO) nanoparticles on the growth, development, and reproduction of soybean [Glycine max (L.) Merrill] were evaluated in a standard soil microcosm study. The soil was treated with 0, 50, or 500 mg/kg (dry weight) of ZnO nanoparticles. The growth and development of soybean plants were tracked during a cultivation period of 8–9 weeks under greenhouse conditions. Soybean development was damaged in both treatment groups, particularly in the group that received 500 mg/kg ZnO nanoparticles. In comparison with the control group, the roots and shoots of soybeans in treatment groups were shorter and had smaller surface area and volume. Furthermore, the plants in the 500 mg/kg treatment group did not form seeds. ZnO nanoparticles negatively affected the developmental stages and reproduction of soybean plants in a soil microcosm. & 2013 Elsevier Inc. All rights reserved.
Keywords: Glycine max Microcosm Nanoparticles Soil Soybean Zinc oxide
1. Introduction Nanotechnology has enabled great advances in electronic, environmental, cosmetic, pharmaceutical, and material applications (Nel et al., 2006; Nowack and Bucheli, 2007). However, its adoption may also cause problems. The expansion of the nanotechnology industry means that nanoparticles can be directly and indirectly released into water and soil ecosystems during production, consumption, and disposal (Nel et al., 2006; Navarro et al., 2008; Lee and An, 2010). The use of zinc oxide nanoparticles (ZnO NPs) is increasing in personal care products and consumer goods, such as cosmetics, clothing, sunscreens, and bottle coatings (Tsuji et al., 2009). Ecotoxicity studies using ZnO NPs are needed to understand the potential impacts of increasing ZnO levels in water and soil ecosystems. Previous studies examined the effects of ZnO NPs in terrestrial plants such as radishes, rape, ryegrass, lettuce, corn (Lin and Xing, 2007), cucumber (Lin and Xing, 2007; Kim et al., 2011), zucchini (Stampoulis et al., 2009), mouse-ear cress (Lee et al., 2010), mung and gram (Mahajan et al., 2011), garden cress and faba bean (Manzo et al., 2011), garlic (Shaymurat et al., 2012), and onion (Kumari et al., 2011). At this time, only one study has observed biotransformation and
n
Corresponding author. Tel.: þ 82 2 2049 6090; fax: þ 82 2 2201 6295. E-mail address: [email protected] (Y.-J. An). 1 Equal contribution.
0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2013.10.014
́ ez-Moreno et al., 2010) genotoxicity of ZnO NPs in soybean (Lop during hydroponic germination. One study has assessed the bioaccumulation and translocation of ZnO NPs from organic farm soil into leaves and beans during plant growth (Priester et al., 2012). Soybean is an important crop and is widely cultivated. Exposure of plants to NPs may cause uptake, translocation, bioaccumulation, and biotransformation of NPs in the food chain. The mechanisms of absorption, transportation, and accumulation of NPs in crop plants are not well known, and have been reported only by Priester et al. (2012) and Du et al. (2011). Most studies have only evaluated crop plants to the germination stage, and have not examined the complete developmental cycle (Rico et al., 2011). Few ecosystem-level microcosm studies of NPs have been carried out. Soil microcosm studies of NPs have mostly reported the effects of NPs on microbial communities and plants. Ge et al. (2011) reported that ZnO and TiO2 NPs negatively affected microbial biomass, diversity, and community composition in unplanted soil microcosms. Kim et al. (2009) reported that ZnO NPs decreased microbial community diversity and the biomass of Zea mays in a microcosm. Other studies have evaluated the effects of Ag NPs, SiO2 NPs (Kumar et al., 2011, 2012), iron oxide magnetic NPs (Fe3O4 and γ-Fe2O3) (He et al., 2011), and fullerene (C60) (Nyberg et al., 2008) on the microbial community. Shah and Belozerova (2009) reported the effects of Si NPs, Pd NPs, Au NPs, and Cu NPs on the soil microbial community and on lettuce. López-Moreno et al. (2010) reported that root growth of soybean was decreased at a ZnO NP concentration of 500 mg/L
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in Millipore water. In addition, Lin and Xing (2007) reported that the root lengths for six crops exposed to ZnO NPs at a concentration of 2000 mg/L in suspensions were significantly inhibited. Aquatic microcosm studies of NPs have evaluated copper oxide (CuO) (Pradhan et al., 2011), Ag (Bradford et al., 2009; Mühling et al., 2009; Pradhan et al., 2011), zero-valent iron (Barnes et al., 2010), and TiO2 NPs (Battin et al., 2009) in microbial communities. One mesocosm study evaluated the effect of Au NPs in an estuarine food web (Ferry et al., 2009). To the best of our knowledge, this is the first study to evaluate the long-term effect of ZnO NPs on the growth, development, and reproduction of soybean (Glycine max (L.) Merrill) in a standard soil microcosm. We focused on the effects of ZnO NPs on the developmental stages of soybean, and evaluated whole soybean growth, including total length, surface area, average diameter, stem volume, and root volume. In addition, we measured the bioaccumulation of Zn in the roots, stems, and leaves of soybeans.
for 24 h (for sand) or air-dried at room temperature for 3 days (for peat moss). Soil was prepared with 0, 50, or 500 mg/kg (dry weight) ZnO NPs from stock soils corresponding to 100 times each test concentration, in five replicates. Stock soils of
2. Materials and methods 2.1. NP preparation of soil ZnO NPs (Sigma-Aldrich, Inc.), with a purity4 97 percent and a particle size o50 nm, were mixed with the test soil at specified doses. The test soil used was the OECD standard soil (OECD, 1984) and was composed of 69.5 percent sand, 20 percent kaolin, 10 percent peat moss, and 0.5 percent calcium carbonate. Sand and peat moss were sieved through a 2-mm mesh sieve and oven-dried at 105 1C
Fig. 2. Vegetative and reproductive developmental stages of soybean for control, low, and high ZnO NP treatments. Five replicates were prepared for each concentration. Vegetative stages: BG¼ before germination, VE¼ emergence, VC¼ cotyledon, V1¼first trifoliate, V2¼second trifoliate, V3¼ third trifoliate, V4¼ fourth trifoliate, V5¼fifth trifoliate, and V6¼ flowering initiation. Reproductive stages: R1¼beginning to bloom and first flower, R2¼ full bloom and flower in top two nodes, R3¼ emergence of pod and 3/16" pod in top four nodes, R4¼ full pod and 3/4" pod in top four nodes, R5¼1/8" seed in top four nodes, and R6¼full-size seed in top four nodes.
Fig. 1. Soybean stem growth for control (A, closed circles), low (B, diamonds), and high (C, squares) ZnO NP treatments before harvest. Soybeans were harvested on day 57 for the high treatment and day 65 for the control and low treatment groups. Error bars represent the standard deviation of the mean (n ¼5 plants).
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ZnO NPs were made by alternating layers of OECD standard soil with ZnO NP powder, then mixing them evenly using a hand mixer for 10 min and a roller mixer for 4 h. A hand mixer and several mixing tools were used to homogeneously dilute the stock soil into test soil. Soil pH was measured in the control soil and in soils amended with ZnO NPs using a pH probe in a 1:2 w/v soil:water paste. The pH of all soils ranged from 4.8 to 5.2.
2.2. Test plants Soybean seeds [G. max (L.) Merrill] were purchased in 2010 (Paju Agricultural Products Corporation, Kyunggi-do, Korea). The seeds were surface-sterilized in 5 percent sodium hypochlorite solution for 3 min and then rinsed five times in sterile distilled water.
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2.3. Microcosm design Microcosms consisted of plastic cylinders (internal diameter, 18 cm; height, 21 cm), packed with ZnO NP-amended soils (described above) and kept in a greenhouse. Five replicates of each concentration were prepared. Before the microcosms were filled with soil, each cylinder was lined with a plastic bag containing a drain hole, and leachate containers were placed on the bottom. Each microcosm was filled with 2 kg (dry weight) of OECD standard soil amended with ZnO NPs. All treatments were sprayed with 1111 mL of N-free Hoagland’s solution (Hoagland and Arnon, 1950) and aged in a greenhouse in darkness for 9 days. After aging, three holes were made at a depth of 1 cm in the surface soil, and three seeds were planted in each hole. After 11 days of germination, excess seedlings were removed from each microcosm, leaving one seedling. Every 37 1 days, either N-free Hoagland’s solution (pH 6.0) or tap water was added to maintain 35 percent
Fig. 3. Soybean growth after control, low, and high ZnO NP treatments, at harvest in terms of total plant length, surface area, average diameter, and volume for (A) root and (B) stem. Root scanning images for (C) control, (D) 50, and (E) 500 mg/kg (dry weight base) ZnO NPs. Error bars represent one standard deviation from the mean (n¼ 5 replicates). Significant differences from controls (p o0.05) are marked with an asterisk.
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water content. Microcosms were rotated every day and kept at 25–30 1C. Stem growth and developmental stages of soybeans were recorded throughout the experimental period. The experimental periods were 57 days for the 500 mg/kg group, and 65 days for the control and 50 mg/kg groups. Soybeans in the control group and the 50 mg/kg group were harvested when the developmental stage of plants reached reproductive stage 6. The plants that received 500 mg/kg ZnO NPs began to wilt before they reached that stage, so they were harvested earlier.
growth were unaffected), but the developmental stage was inhibited at the time of formation of the first trifoliate (V1, Fig. 2). Some individuals from the 500 mg/kg ZnO NP treatment stopped developing and were unable to reach the reproductive stage, and other individuals reached the bloom stage but did not show normal development (Fig. 2).
2.4. Plant analyses
3.2. Effect of ZnO NPs on growth of whole plant
During growth, stem length and developmental stage were recorded, and developmental stages were separated into vegetative stages (1–6) and reproductive stages (1–6). After harvest, the total length, surface area, average diameter, stem volume, and root volume were measured. A root scanner was used to measure the root volume (WinRHIZOReg 2008, Regent Instruments Inc., Canada). Total biomass of roots, stems, leaves, and seeds were measured.
The growth of soybean at the different concentrations of ZnO NPs was assessed according to the length, surface area, diameter, and biovolume of the roots and the main stem at harvest (Fig. 3). The root length was decreased by 10 and 89 percent in the 50 and 500 mg/kg ZnO NPs treatments, respectively (Fig. 3A). The EC50 value of root length was 160 (141–182) mg/kg. In comparison with the control, the final root surface area was decreased by 13 and 88 percent, and the root volume was decreased by 16 and 87 percent in the 50 mg/kg and 500 mg/kg groups, respectively. The EC50 values were 156 (135– 180) and 151 (128–177) mg/kg for the surface area and volume of the roots, respectively. In addition, compared with the control, the treatment groups had fewer root hairs (data not shown). In comparison with the control, the final root surface area was decreased by 13 and 88 percent, and the root volume was decreased by 16 and 87 percent in the 50 and 500 mg/kg groups, respectively. The average EC50 values were 156 (135–180) and 151 (128–177) mg/kg for the surface area and volume of the roots, respectively. Stem length of soybean was not significantly affected in the 50 mg/kg group, but the stem growth in the 500 mg/kg group was inhibited by 76 percent, and the EC50 value was 227 mg/kg (Fig. 3B). In comparison with the control group, the surface area of the stem was decreased by 5 and 82 percent, the volume of the stem was inhibited by 9 and 88 percent, and the average diameter was reduced by 7 and 25 percent, in the 50 and 500 mg/kg groups, respectively. The EC50 values were 192 (166–222) and 165 (145–188) mg/kg for the surface area and volume of the stem, respectively. Thus, the stem growth of soybean was significantly inhibited by the 500 mg/kg ZnO NP treatment. Stem growth at high concentrations of ZnO NPs may be reduced by either ZnO NPs or Zn ions; the mechanism of toxicity remains unknown. After harvest, the soybean biomass was evaluated by measuring the dry weights of roots, stems, leaves, and seeds. The soybean biomass generally decreased with increasing ZnO NPs (see the supplementary data; Table S1). The biomasses of seeds were
2.5. Sample preparation for transmission electron microscopy Plant samples for transmission electron microscopy (TEM) analysis were prepared using an adaptation of the method used by Lee et al. (2012). Root samples were obtained from the root tip. Stem samples were obtained from the aboveground part of the plant. The developmental stages of the stem samples were R6 and V1 for the control and the 500 mg/kg group, respectively. The samples were observed using field-emission TEM (JEM 2100F, JEOL, Tokyo, Japan) with an energydispersive spectrometry resolution of 142 eV. 2.6. Metal analysis The analysis of dissolved Zn in the soil used 5 g of wet soil and 15 mL of distilled water added to the soil and mixed using a vortex mixer. The samples were centrifuged at 3000 rpm for 15 min, and supernatants were filtered with Whatmans No. 2 filter paper, a 0.2-μm nylon filter, and a stirred filter (molecular weight cut-off: 30,000 Da). The filtrates were analyzed with ICP-AES (JY 138; Ultrace, Jobin Yvon, France). The harvested plants were analyzed for accumulation of Zn in tissues such as root, stem, and leaf, and to assess the transfer of Zn from the soil to the above-ground plant. The plants were washed with distilled water and separated into a maximum of six parts (root and nodes 1–5). Each separated sample was dried at 65 1C for 24 h, added to 3 mL of HNO3, and heated at 120 1C. After heating, distilled water was added to the sample and it was heated three times to evaporate the acid. Then 10 mL of distilled water was added to the sample and it was filtered with Whatmans No. 2 filter paper. The filtrates were analyzed for total Zn using ICP-AES. 2.7. Statistical analyses The median effective concentrations (EC50) for growth of soybean were estimated using the trimmed Spearman–Karber method with the software SPEARMAN (USEPA, 1999), provided by the U.S. EPA Center for Exposure Assessment Modeling. A 95 percent significance level (p o 0.05) was used for all comparisons.
3.1. Effects of ZnO NPs on stem growth and developmental stages At the 3-week mark and throughout the remaining the exposure period, stem growth of soybean was significantly inhibited in the group that received 500 mg/kg ZnO NPs, whereas the growth in the group that received 50 mg/kg ZnO NPs was similar to that of the control (Fig. 1). The plants grown with 500 mg/kg ZnO NPs showed early wilting, etiolation, and spots (Fig. 1). In addition, their developmental stage was significantly affected (Fig. 2). However, the difference in growth between the control and exposure groups was not observed initially; one potential reason is that zinc may be associated with activity of auxin, a plant growth hormone (Skoog, 1940). In order to evaluate their developmental stage, plants were classified in the vegetative stage or reproductive stage. All of the developmental stages, including the development of trifoliate leaves, blooms, and pods and seeds, were reached at almost the same time in the control group and the 50 mg/kg ZnO NP group. Development of plants in the 500 mg/kg ZnO NP treatment was similar to that of plants in the other groups until day 14 (i.e., germination and seedling
Bioaccumulated Zn in plant (mg/kg dry weight)
7000
3. Results and discussion
*
6000 5000
*
control 50 mg/kg 500 mg/kg
4000
*
*
1000
500
0
Root
Stem
Leaf
Parts of plant Fig. 4. Bioaccumulation of Zn in the roots, stems, and leaves of soybeans. Error bars represent one standard deviation from the mean (n ¼5 replicates). Significant differences from controls (po 0.05) are marked with an asterisk. The large standard errors in some plant samples may be attributed to the difference in the developmental stages of samples.
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Fig. 5. Transmission electron micrographs (TEMs) of the stem (A–D) and root (E–H) of soybean: (A) control group stem cell, (B, C) stem cell exposed to 500 mg/kg (dry soil basis) ZnO NPs, (D) energy-dispersive spectroscopy (EDS) spectrum of an electron-dense spot in the TEM scan in (C), (E) control group root cell, (F, G) root cell exposed to 500 mg/kg (dry soil basis) ZnO NPs, and (H) EDS spectrum of an electron-dense spot in the TEM scan in (G).
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44 721 and 377 24 mg/kg for the control and 50 mg/kg groups, respectively. Seeds were not produced by any of the 500 mg/kg plants. Zn plays an important role in the growth and development of plants and is an essential element for enzymatic activities (Vallee, 1976). Leaves commonly require a concentration of 15–20 mg/kg Zn for growth (Broadley et al., 2007). However, excess Zn can be toxic to plants, and plant stress is closely related to uptake of Zn or exposure concentration (Takkar and Mann, 1978; Fang and Kao, 2000; Vaillant et al., 2005; Broadley et al., 2007). Takkar and Mann (1978) reported that Zn concentrations greater than 60 ppm in wheat and over 81 ppm in maize markedly reduced yields. Fang and Kao (2000) found that excess Zn enhanced peroxidase activity, which was related to defense functions and stress-induced lignification in rice leaves. Vaillant et al. (2005) observed growth inhibition and reduction in photosynthetic activity in Datura species exposed to Zn. 3.3. Bioaccumulation of Zn Bioaccumulation of Zn in soybean varied with the concentrations of ZnO NP treatments (Fig. 4). In the control, the concentrations of accumulated Zn in soybean were 105 770, 79 735, and 17 714 mg/kg dry weight for leaves, roots, and stems, respectively. These amounts reflected a high background level of total Zn in the control soil (10 77 mg/kg as soil dry weight). In the 50 mg/kg group, the concentrations of accumulated Zn were 710 7529, 6117 263, and 247 7233 mg/kg for leaves, roots, and stems, respectively. Bioaccumulation of Zn in the control group and the 50 mg/kg group increased in the following order: stems, roots, and leaves. The accumulated Zn in the 500 mg/kg group was 894 7376, 3797 7809, and 4772 7 1541 mg/kg for leaves, roots, and stems, respectively. The Zn content of the plants in the two treatment groups was higher than that of the control. These findings agree with those of Du et al. (2011). The transfer of Zn from the root to the upper stem (nodes 1–5) was not significantly correlated in the replicates of all concentrations (see the supplementary data; Fig. S1). Consequently, bioaccumulation of Zn increased with the high concentration of ZnO NPs. In previous studies, associations between uptake or exposure concentration of Zn and toxicity were proven (Takkar and Mann, 1978; Fang and Kao, 2000; Souza and Rauser, 2003; Vaillant et al., 2005). TEM images showed Zn deposits in stem and root (Fig. 5) cells from soybean seedlings that were exposed to 500 mg/kg ZnO NPs. The box in Fig. 5C shows electron-dense deposits in the stem cells exposed to 500 mg/kg ZnO NP. The Zn deposits were found primarily in vacuoles, which play a role in detoxification and transport of heavy metals (Martinoia et al., 2007). In one stem cell, an electron-dense deposit was verified to include Zn using energydispersive spectrometry (Fig. 5C and D). TEM images of root cells from the control and 500 mg/kg groups showed features that were similar to one another (Fig. 5E–H). The electron-dense deposits were observed in cells that were exposed to ZnO NPs (Fig. 5F). The storage of Zn in these deposits could be related to a detoxification process (Brooks and White, 1995). As expected, Zn was not detected in the control group by TEM or energy-dispersive spectrometry (see the supplementary data; Fig. S2). 3.4. Ionization of Zn in the harvest soil The concentrations of ionized (filterable, through a stirred filter) Zn in the harvest soil were 0.0970.01, 0.2870.17, and 0.2270.03 mg Zn2 þ /kg (dry weight basis) in the control, 50 mg/ kg, and 500 mg/kg groups, respectively. These results indicated that the dissolved Zn2 þ from ZnO NPs was very low. The Zn2 þ ions act as nutrients to plants (Broadley et al., 2007). Therefore, the adverse
effects on plant growth were mainly caused by ZnO NPs. However, further studies of bulk and ion treatments are needed because our measurements of ionized Zn were taken post-harvest and may not represent what the plants experienced during two months of growth.
4. Conclusions To investigate the effects on growth, reproduction, and development of ZnO NPs on soybean [G. max (L.) Merrill], plants were grown with ZnO NPs for 65 days (in the control and 50 mg/kg groups) or for 57 days (in the 500 mg/kg group) in a standard soil microcosm. High accumulations of Zn were associated with the inhibition of elongation and volumetric growth and noticeable effects on development of soybean. Microscopic analysis revealed electron-dense deposits in stem and root cells exposed to ZnO NPs. However, ZnO NPs were not discernible in the soybean roots and stem cells exposed to 500 mg/kg ZnO NPs in this study. It is possible that ZnO NPs were dissolved in plant cells because of the long duration of this experiment (57 days), but this explanation was not verified by any specific experiments. In conclusion, ZnO NPs negatively influenced the developmental stages and reproduction of soybean in a soil microcosm.
Acknowledgments This work was supported by the Konkuk University in 2013. The authors thank the Korean Basic Science Institute for electron microscopic analyses.
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Zinc oxide nanoparticles delay soybean development: A standard soil microcosm study Sung-Ji Yoon a,1, Jin Il Kwak a,1, Woo-Mi Lee a, Patricia A. Holden b, Youn-Joo An a,n a b
Department of Environmental Science, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea Bren School of Environmental Science and Management, University of California, Santa Barbara, CA, USA
art ic l e i nf o
a b s t r a c t
Article history: Received 17 July 2013 Received in revised form 11 October 2013 Accepted 15 October 2013 Available online 2 December 2013
Soybean is an important crop and a source of food for humans and livestock. In this study, for the first time, the long-term effects of zinc oxide (ZnO) nanoparticles on the growth, development, and reproduction of soybean [Glycine max (L.) Merrill] were evaluated in a standard soil microcosm study. The soil was treated with 0, 50, or 500 mg/kg (dry weight) of ZnO nanoparticles. The growth and development of soybean plants were tracked during a cultivation period of 8–9 weeks under greenhouse conditions. Soybean development was damaged in both treatment groups, particularly in the group that received 500 mg/kg ZnO nanoparticles. In comparison with the control group, the roots and shoots of soybeans in treatment groups were shorter and had smaller surface area and volume. Furthermore, the plants in the 500 mg/kg treatment group did not form seeds. ZnO nanoparticles negatively affected the developmental stages and reproduction of soybean plants in a soil microcosm. & 2013 Elsevier Inc. All rights reserved.
Keywords: Glycine max Microcosm Nanoparticles Soil Soybean Zinc oxide
1. Introduction Nanotechnology has enabled great advances in electronic, environmental, cosmetic, pharmaceutical, and material applications (Nel et al., 2006; Nowack and Bucheli, 2007). However, its adoption may also cause problems. The expansion of the nanotechnology industry means that nanoparticles can be directly and indirectly released into water and soil ecosystems during production, consumption, and disposal (Nel et al., 2006; Navarro et al., 2008; Lee and An, 2010). The use of zinc oxide nanoparticles (ZnO NPs) is increasing in personal care products and consumer goods, such as cosmetics, clothing, sunscreens, and bottle coatings (Tsuji et al., 2009). Ecotoxicity studies using ZnO NPs are needed to understand the potential impacts of increasing ZnO levels in water and soil ecosystems. Previous studies examined the effects of ZnO NPs in terrestrial plants such as radishes, rape, ryegrass, lettuce, corn (Lin and Xing, 2007), cucumber (Lin and Xing, 2007; Kim et al., 2011), zucchini (Stampoulis et al., 2009), mouse-ear cress (Lee et al., 2010), mung and gram (Mahajan et al., 2011), garden cress and faba bean (Manzo et al., 2011), garlic (Shaymurat et al., 2012), and onion (Kumari et al., 2011). At this time, only one study has observed biotransformation and
n
Corresponding author. Tel.: þ 82 2 2049 6090; fax: þ 82 2 2201 6295. E-mail address: [email protected] (Y.-J. An). 1 Equal contribution.
0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2013.10.014
́ ez-Moreno et al., 2010) genotoxicity of ZnO NPs in soybean (Lop during hydroponic germination. One study has assessed the bioaccumulation and translocation of ZnO NPs from organic farm soil into leaves and beans during plant growth (Priester et al., 2012). Soybean is an important crop and is widely cultivated. Exposure of plants to NPs may cause uptake, translocation, bioaccumulation, and biotransformation of NPs in the food chain. The mechanisms of absorption, transportation, and accumulation of NPs in crop plants are not well known, and have been reported only by Priester et al. (2012) and Du et al. (2011). Most studies have only evaluated crop plants to the germination stage, and have not examined the complete developmental cycle (Rico et al., 2011). Few ecosystem-level microcosm studies of NPs have been carried out. Soil microcosm studies of NPs have mostly reported the effects of NPs on microbial communities and plants. Ge et al. (2011) reported that ZnO and TiO2 NPs negatively affected microbial biomass, diversity, and community composition in unplanted soil microcosms. Kim et al. (2009) reported that ZnO NPs decreased microbial community diversity and the biomass of Zea mays in a microcosm. Other studies have evaluated the effects of Ag NPs, SiO2 NPs (Kumar et al., 2011, 2012), iron oxide magnetic NPs (Fe3O4 and γ-Fe2O3) (He et al., 2011), and fullerene (C60) (Nyberg et al., 2008) on the microbial community. Shah and Belozerova (2009) reported the effects of Si NPs, Pd NPs, Au NPs, and Cu NPs on the soil microbial community and on lettuce. López-Moreno et al. (2010) reported that root growth of soybean was decreased at a ZnO NP concentration of 500 mg/L
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in Millipore water. In addition, Lin and Xing (2007) reported that the root lengths for six crops exposed to ZnO NPs at a concentration of 2000 mg/L in suspensions were significantly inhibited. Aquatic microcosm studies of NPs have evaluated copper oxide (CuO) (Pradhan et al., 2011), Ag (Bradford et al., 2009; Mühling et al., 2009; Pradhan et al., 2011), zero-valent iron (Barnes et al., 2010), and TiO2 NPs (Battin et al., 2009) in microbial communities. One mesocosm study evaluated the effect of Au NPs in an estuarine food web (Ferry et al., 2009). To the best of our knowledge, this is the first study to evaluate the long-term effect of ZnO NPs on the growth, development, and reproduction of soybean (Glycine max (L.) Merrill) in a standard soil microcosm. We focused on the effects of ZnO NPs on the developmental stages of soybean, and evaluated whole soybean growth, including total length, surface area, average diameter, stem volume, and root volume. In addition, we measured the bioaccumulation of Zn in the roots, stems, and leaves of soybeans.
for 24 h (for sand) or air-dried at room temperature for 3 days (for peat moss). Soil was prepared with 0, 50, or 500 mg/kg (dry weight) ZnO NPs from stock soils corresponding to 100 times each test concentration, in five replicates. Stock soils of
2. Materials and methods 2.1. NP preparation of soil ZnO NPs (Sigma-Aldrich, Inc.), with a purity4 97 percent and a particle size o50 nm, were mixed with the test soil at specified doses. The test soil used was the OECD standard soil (OECD, 1984) and was composed of 69.5 percent sand, 20 percent kaolin, 10 percent peat moss, and 0.5 percent calcium carbonate. Sand and peat moss were sieved through a 2-mm mesh sieve and oven-dried at 105 1C
Fig. 2. Vegetative and reproductive developmental stages of soybean for control, low, and high ZnO NP treatments. Five replicates were prepared for each concentration. Vegetative stages: BG¼ before germination, VE¼ emergence, VC¼ cotyledon, V1¼first trifoliate, V2¼second trifoliate, V3¼ third trifoliate, V4¼ fourth trifoliate, V5¼fifth trifoliate, and V6¼ flowering initiation. Reproductive stages: R1¼beginning to bloom and first flower, R2¼ full bloom and flower in top two nodes, R3¼ emergence of pod and 3/16" pod in top four nodes, R4¼ full pod and 3/4" pod in top four nodes, R5¼1/8" seed in top four nodes, and R6¼full-size seed in top four nodes.
Fig. 1. Soybean stem growth for control (A, closed circles), low (B, diamonds), and high (C, squares) ZnO NP treatments before harvest. Soybeans were harvested on day 57 for the high treatment and day 65 for the control and low treatment groups. Error bars represent the standard deviation of the mean (n ¼5 plants).
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ZnO NPs were made by alternating layers of OECD standard soil with ZnO NP powder, then mixing them evenly using a hand mixer for 10 min and a roller mixer for 4 h. A hand mixer and several mixing tools were used to homogeneously dilute the stock soil into test soil. Soil pH was measured in the control soil and in soils amended with ZnO NPs using a pH probe in a 1:2 w/v soil:water paste. The pH of all soils ranged from 4.8 to 5.2.
2.2. Test plants Soybean seeds [G. max (L.) Merrill] were purchased in 2010 (Paju Agricultural Products Corporation, Kyunggi-do, Korea). The seeds were surface-sterilized in 5 percent sodium hypochlorite solution for 3 min and then rinsed five times in sterile distilled water.
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2.3. Microcosm design Microcosms consisted of plastic cylinders (internal diameter, 18 cm; height, 21 cm), packed with ZnO NP-amended soils (described above) and kept in a greenhouse. Five replicates of each concentration were prepared. Before the microcosms were filled with soil, each cylinder was lined with a plastic bag containing a drain hole, and leachate containers were placed on the bottom. Each microcosm was filled with 2 kg (dry weight) of OECD standard soil amended with ZnO NPs. All treatments were sprayed with 1111 mL of N-free Hoagland’s solution (Hoagland and Arnon, 1950) and aged in a greenhouse in darkness for 9 days. After aging, three holes were made at a depth of 1 cm in the surface soil, and three seeds were planted in each hole. After 11 days of germination, excess seedlings were removed from each microcosm, leaving one seedling. Every 37 1 days, either N-free Hoagland’s solution (pH 6.0) or tap water was added to maintain 35 percent
Fig. 3. Soybean growth after control, low, and high ZnO NP treatments, at harvest in terms of total plant length, surface area, average diameter, and volume for (A) root and (B) stem. Root scanning images for (C) control, (D) 50, and (E) 500 mg/kg (dry weight base) ZnO NPs. Error bars represent one standard deviation from the mean (n¼ 5 replicates). Significant differences from controls (p o0.05) are marked with an asterisk.
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water content. Microcosms were rotated every day and kept at 25–30 1C. Stem growth and developmental stages of soybeans were recorded throughout the experimental period. The experimental periods were 57 days for the 500 mg/kg group, and 65 days for the control and 50 mg/kg groups. Soybeans in the control group and the 50 mg/kg group were harvested when the developmental stage of plants reached reproductive stage 6. The plants that received 500 mg/kg ZnO NPs began to wilt before they reached that stage, so they were harvested earlier.
growth were unaffected), but the developmental stage was inhibited at the time of formation of the first trifoliate (V1, Fig. 2). Some individuals from the 500 mg/kg ZnO NP treatment stopped developing and were unable to reach the reproductive stage, and other individuals reached the bloom stage but did not show normal development (Fig. 2).
2.4. Plant analyses
3.2. Effect of ZnO NPs on growth of whole plant
During growth, stem length and developmental stage were recorded, and developmental stages were separated into vegetative stages (1–6) and reproductive stages (1–6). After harvest, the total length, surface area, average diameter, stem volume, and root volume were measured. A root scanner was used to measure the root volume (WinRHIZOReg 2008, Regent Instruments Inc., Canada). Total biomass of roots, stems, leaves, and seeds were measured.
The growth of soybean at the different concentrations of ZnO NPs was assessed according to the length, surface area, diameter, and biovolume of the roots and the main stem at harvest (Fig. 3). The root length was decreased by 10 and 89 percent in the 50 and 500 mg/kg ZnO NPs treatments, respectively (Fig. 3A). The EC50 value of root length was 160 (141–182) mg/kg. In comparison with the control, the final root surface area was decreased by 13 and 88 percent, and the root volume was decreased by 16 and 87 percent in the 50 mg/kg and 500 mg/kg groups, respectively. The EC50 values were 156 (135– 180) and 151 (128–177) mg/kg for the surface area and volume of the roots, respectively. In addition, compared with the control, the treatment groups had fewer root hairs (data not shown). In comparison with the control, the final root surface area was decreased by 13 and 88 percent, and the root volume was decreased by 16 and 87 percent in the 50 and 500 mg/kg groups, respectively. The average EC50 values were 156 (135–180) and 151 (128–177) mg/kg for the surface area and volume of the roots, respectively. Stem length of soybean was not significantly affected in the 50 mg/kg group, but the stem growth in the 500 mg/kg group was inhibited by 76 percent, and the EC50 value was 227 mg/kg (Fig. 3B). In comparison with the control group, the surface area of the stem was decreased by 5 and 82 percent, the volume of the stem was inhibited by 9 and 88 percent, and the average diameter was reduced by 7 and 25 percent, in the 50 and 500 mg/kg groups, respectively. The EC50 values were 192 (166–222) and 165 (145–188) mg/kg for the surface area and volume of the stem, respectively. Thus, the stem growth of soybean was significantly inhibited by the 500 mg/kg ZnO NP treatment. Stem growth at high concentrations of ZnO NPs may be reduced by either ZnO NPs or Zn ions; the mechanism of toxicity remains unknown. After harvest, the soybean biomass was evaluated by measuring the dry weights of roots, stems, leaves, and seeds. The soybean biomass generally decreased with increasing ZnO NPs (see the supplementary data; Table S1). The biomasses of seeds were
2.5. Sample preparation for transmission electron microscopy Plant samples for transmission electron microscopy (TEM) analysis were prepared using an adaptation of the method used by Lee et al. (2012). Root samples were obtained from the root tip. Stem samples were obtained from the aboveground part of the plant. The developmental stages of the stem samples were R6 and V1 for the control and the 500 mg/kg group, respectively. The samples were observed using field-emission TEM (JEM 2100F, JEOL, Tokyo, Japan) with an energydispersive spectrometry resolution of 142 eV. 2.6. Metal analysis The analysis of dissolved Zn in the soil used 5 g of wet soil and 15 mL of distilled water added to the soil and mixed using a vortex mixer. The samples were centrifuged at 3000 rpm for 15 min, and supernatants were filtered with Whatmans No. 2 filter paper, a 0.2-μm nylon filter, and a stirred filter (molecular weight cut-off: 30,000 Da). The filtrates were analyzed with ICP-AES (JY 138; Ultrace, Jobin Yvon, France). The harvested plants were analyzed for accumulation of Zn in tissues such as root, stem, and leaf, and to assess the transfer of Zn from the soil to the above-ground plant. The plants were washed with distilled water and separated into a maximum of six parts (root and nodes 1–5). Each separated sample was dried at 65 1C for 24 h, added to 3 mL of HNO3, and heated at 120 1C. After heating, distilled water was added to the sample and it was heated three times to evaporate the acid. Then 10 mL of distilled water was added to the sample and it was filtered with Whatmans No. 2 filter paper. The filtrates were analyzed for total Zn using ICP-AES. 2.7. Statistical analyses The median effective concentrations (EC50) for growth of soybean were estimated using the trimmed Spearman–Karber method with the software SPEARMAN (USEPA, 1999), provided by the U.S. EPA Center for Exposure Assessment Modeling. A 95 percent significance level (p o 0.05) was used for all comparisons.
3.1. Effects of ZnO NPs on stem growth and developmental stages At the 3-week mark and throughout the remaining the exposure period, stem growth of soybean was significantly inhibited in the group that received 500 mg/kg ZnO NPs, whereas the growth in the group that received 50 mg/kg ZnO NPs was similar to that of the control (Fig. 1). The plants grown with 500 mg/kg ZnO NPs showed early wilting, etiolation, and spots (Fig. 1). In addition, their developmental stage was significantly affected (Fig. 2). However, the difference in growth between the control and exposure groups was not observed initially; one potential reason is that zinc may be associated with activity of auxin, a plant growth hormone (Skoog, 1940). In order to evaluate their developmental stage, plants were classified in the vegetative stage or reproductive stage. All of the developmental stages, including the development of trifoliate leaves, blooms, and pods and seeds, were reached at almost the same time in the control group and the 50 mg/kg ZnO NP group. Development of plants in the 500 mg/kg ZnO NP treatment was similar to that of plants in the other groups until day 14 (i.e., germination and seedling
Bioaccumulated Zn in plant (mg/kg dry weight)
7000
3. Results and discussion
*
6000 5000
*
control 50 mg/kg 500 mg/kg
4000
*
*
1000
500
0
Root
Stem
Leaf
Parts of plant Fig. 4. Bioaccumulation of Zn in the roots, stems, and leaves of soybeans. Error bars represent one standard deviation from the mean (n ¼5 replicates). Significant differences from controls (po 0.05) are marked with an asterisk. The large standard errors in some plant samples may be attributed to the difference in the developmental stages of samples.
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Fig. 5. Transmission electron micrographs (TEMs) of the stem (A–D) and root (E–H) of soybean: (A) control group stem cell, (B, C) stem cell exposed to 500 mg/kg (dry soil basis) ZnO NPs, (D) energy-dispersive spectroscopy (EDS) spectrum of an electron-dense spot in the TEM scan in (C), (E) control group root cell, (F, G) root cell exposed to 500 mg/kg (dry soil basis) ZnO NPs, and (H) EDS spectrum of an electron-dense spot in the TEM scan in (G).
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44 721 and 377 24 mg/kg for the control and 50 mg/kg groups, respectively. Seeds were not produced by any of the 500 mg/kg plants. Zn plays an important role in the growth and development of plants and is an essential element for enzymatic activities (Vallee, 1976). Leaves commonly require a concentration of 15–20 mg/kg Zn for growth (Broadley et al., 2007). However, excess Zn can be toxic to plants, and plant stress is closely related to uptake of Zn or exposure concentration (Takkar and Mann, 1978; Fang and Kao, 2000; Vaillant et al., 2005; Broadley et al., 2007). Takkar and Mann (1978) reported that Zn concentrations greater than 60 ppm in wheat and over 81 ppm in maize markedly reduced yields. Fang and Kao (2000) found that excess Zn enhanced peroxidase activity, which was related to defense functions and stress-induced lignification in rice leaves. Vaillant et al. (2005) observed growth inhibition and reduction in photosynthetic activity in Datura species exposed to Zn. 3.3. Bioaccumulation of Zn Bioaccumulation of Zn in soybean varied with the concentrations of ZnO NP treatments (Fig. 4). In the control, the concentrations of accumulated Zn in soybean were 105 770, 79 735, and 17 714 mg/kg dry weight for leaves, roots, and stems, respectively. These amounts reflected a high background level of total Zn in the control soil (10 77 mg/kg as soil dry weight). In the 50 mg/kg group, the concentrations of accumulated Zn were 710 7529, 6117 263, and 247 7233 mg/kg for leaves, roots, and stems, respectively. Bioaccumulation of Zn in the control group and the 50 mg/kg group increased in the following order: stems, roots, and leaves. The accumulated Zn in the 500 mg/kg group was 894 7376, 3797 7809, and 4772 7 1541 mg/kg for leaves, roots, and stems, respectively. The Zn content of the plants in the two treatment groups was higher than that of the control. These findings agree with those of Du et al. (2011). The transfer of Zn from the root to the upper stem (nodes 1–5) was not significantly correlated in the replicates of all concentrations (see the supplementary data; Fig. S1). Consequently, bioaccumulation of Zn increased with the high concentration of ZnO NPs. In previous studies, associations between uptake or exposure concentration of Zn and toxicity were proven (Takkar and Mann, 1978; Fang and Kao, 2000; Souza and Rauser, 2003; Vaillant et al., 2005). TEM images showed Zn deposits in stem and root (Fig. 5) cells from soybean seedlings that were exposed to 500 mg/kg ZnO NPs. The box in Fig. 5C shows electron-dense deposits in the stem cells exposed to 500 mg/kg ZnO NP. The Zn deposits were found primarily in vacuoles, which play a role in detoxification and transport of heavy metals (Martinoia et al., 2007). In one stem cell, an electron-dense deposit was verified to include Zn using energydispersive spectrometry (Fig. 5C and D). TEM images of root cells from the control and 500 mg/kg groups showed features that were similar to one another (Fig. 5E–H). The electron-dense deposits were observed in cells that were exposed to ZnO NPs (Fig. 5F). The storage of Zn in these deposits could be related to a detoxification process (Brooks and White, 1995). As expected, Zn was not detected in the control group by TEM or energy-dispersive spectrometry (see the supplementary data; Fig. S2). 3.4. Ionization of Zn in the harvest soil The concentrations of ionized (filterable, through a stirred filter) Zn in the harvest soil were 0.0970.01, 0.2870.17, and 0.2270.03 mg Zn2 þ /kg (dry weight basis) in the control, 50 mg/ kg, and 500 mg/kg groups, respectively. These results indicated that the dissolved Zn2 þ from ZnO NPs was very low. The Zn2 þ ions act as nutrients to plants (Broadley et al., 2007). Therefore, the adverse
effects on plant growth were mainly caused by ZnO NPs. However, further studies of bulk and ion treatments are needed because our measurements of ionized Zn were taken post-harvest and may not represent what the plants experienced during two months of growth.
4. Conclusions To investigate the effects on growth, reproduction, and development of ZnO NPs on soybean [G. max (L.) Merrill], plants were grown with ZnO NPs for 65 days (in the control and 50 mg/kg groups) or for 57 days (in the 500 mg/kg group) in a standard soil microcosm. High accumulations of Zn were associated with the inhibition of elongation and volumetric growth and noticeable effects on development of soybean. Microscopic analysis revealed electron-dense deposits in stem and root cells exposed to ZnO NPs. However, ZnO NPs were not discernible in the soybean roots and stem cells exposed to 500 mg/kg ZnO NPs in this study. It is possible that ZnO NPs were dissolved in plant cells because of the long duration of this experiment (57 days), but this explanation was not verified by any specific experiments. In conclusion, ZnO NPs negatively influenced the developmental stages and reproduction of soybean in a soil microcosm.
Acknowledgments This work was supported by the Konkuk University in 2013. The authors thank the Korean Basic Science Institute for electron microscopic analyses.
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