Biol Trace Elem Res (2012) 149:419–424 DOI 10.1007/s12011-012-9434-5
Effects Silver Nanoparticles and Magnetic Field on Growth of Fodder Maize (Zea mays L.) Ali Asghar Berahmand & Ali Ghafariyan Panahi & Hossein Sahabi & Hassan Feizi & Parviz Rezvani Moghaddam & Nasser Shahtahmassebi & Amir Fotovat & Hossein Karimpour & Omran Gallehgir
Received: 20 October 2011 / Accepted: 17 April 2012 / Published online: 4 May 2012 # Springer Science+Business Media, LLC 2012
Abstract Two experiments were done in 2008 and 2009 to study the effects of magnetic field and silver nanoparticles on fodder maize (Zea mays L.). These experiments were done with seven treatments based on a randomized complete block design in four replications. The treatments were as follows: magnetic field and silver nanoparticles+Kemira fertilizer (T1), magnetic field and silver nanoparticles+ Humax fertilizer (T2), magnetic field and silver nanoparticles (T3), Kemira fertilizer (T4), Librel fertilizer (T5), Humax fertilizer (T6), and a control (T7). Results showed that fresh yield was higher in treatments T3 and T4. Treatments T3 and T4 had increased maize fresh yields of 35 and 17.5 % in comparison to the control, respectively. The dry A. A. Berahmand : A. Ghafariyan Panahi Khorasan Razavi Education Administration, Mashhad, Iran A. A. Berahmand e-mail:
[email protected] A. Ghafariyan Panahi e-mail:
[email protected] H. Sahabi Torbat Heydarieh Technical and Engineering Faculty, Torbat Heydarieh, Iran e-mail:
[email protected] H. Feizi (*) : P. Rezvani Moghaddam : H. Karimpour Department of Agronomy, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran e-mail:
[email protected] P. Rezvani Moghaddam e-mail:
[email protected]
matter yield of those plants exposed to magnetic field and silver nanoparticles was significantly higher than that from any of the other treatments. Magnetic field and silver nanoparticle treatments (T3 and T1) showed higher percentages for ears, and the lowest percentages were found in treatments T7 and T5. In general, the soil conditions for crop growth were more favorable in 2009 than in 2008, which caused the maize to respond better to treatments tested in the study; therefore, treatments had more significant effects on studied traits in 2008 than in 2009. Keywords Fodder maize . Magnetic field . Silver nanoparticles . Yield
H. Karimpour e-mail:
[email protected] N. Shahtahmassebi Physics Department, Faculty of Sciences and Nano Research Center, Ferdowsi University of Mashhad, Mashhad, Iran e-mail:
[email protected] A. Fotovat Soil Science Department, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran e-mail:
[email protected]
O. Gallehgir Department of Agriculture, Payame Noor University, Genaveh, Iran e-mail:
[email protected]
420
Berahmand et al.
Introduction Nanoparticles are materials small enough to fall within the nanometric scale, with at least one of their dimensions being less than a few hundred nanometers. This minute scale of nanoparticles represents significant changes in their physical properties compared with those in bulk materials. Most of these changes are related to the appearance of quantum effects as size decreases and are the origin of phenomena such as super paramagnetism [1]. Nanoparticles have enhanced reactivity due to a greater proportion of surface atoms relative to the interior of a structure [2, 3]. As such, insoluble substances can exhibit drastically enhanced solubility when particle size is less than 100 nm. In addition, materials with dimensions less than 5 nm exhibit unique magnetic/optical properties, electronic states, and catalytic reactivity that differ from equivalent bulk materials [4]. Zhu et al. [5] noted that pumpkin plants watered with Fe3O4 nanoparticles in a 500-mg L−1 solution showed no visible phytotoxicity, but root-to-shoot nanoparticle translocation was detected magnetometrically. Lin and Xing [6] indicated that natural organic matter and humic acid (100 mg L−1) increased the stability of multi-wall carbon nanotubes and Al2O3 nanoparticle solutions. Musante and White [7] stated that Ag nanoparticles reduced biomass and transpiration of Cucurbita pepo by 66–84 % compared with bulk Ag. The Ag ion concentration was 4.4–10 times greater in nanoparticles than in bulk particle solutions. Lei et al. [8] stated that nanoanatase (TiO2) reduced antioxidant stress in spinach chloroplasts by reducing H2O2, superoxide radicals, and malonyldialdehyde content, while increasing superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase, and catalase activities. Silver nanoparticles release Ag+, which has been reported to interact with cytoplasmic components and nucleic acids to inhibit respiratory enzymes and to interfere with membrane permeability [9]. Physiological activity of Ag+ from nanoparticles is also a possibility. Ag+, generally applied as silver thiosulfate, effectively inhibits ethylene-mediated processes, such as flower senescence and abscission [10, 11]. As with other cations (e.g., K+, Ca2+), positive effects on plant stem hydraulic conductivity of Ag+ are possible [12]. Ohkawa et al. [13] reported that silver-containing compounds extended the vase life of cut roses. Lu et al. [9] reported that a pulse treatment of 250 mg L−1 nanoparticles had a phytotoxic effect. However, pulse treatments for 1 h with 50- and 100-mg L−1
nanoparticle solutions extended vase life and suppressed reduction in fresh weight during the vase period. Amounts of water uptake and water loss by the cut flowers were reduced under treatment with silver nanoparticles. The average Ag content of soil is 100 ng g−1, ranging from 10 to 5,000 ng g−1. Since Ag ions have a high affinity for sulphydryl, amino, and imino groups, it is believed that uptakes of these ions result in their binding to and/or mixing with membrane constituents and possibly active sites on some enzymes thereby altering membrane permeability [14]. In bean (Phaseolus vulgaris), corn (Zea mays), and tomato (Lycopersicon esculentum) plants, a low Ag+ concentration (50 nM) inhibited shoot uptake of the ions. In the roots, Ca uptake increased while P and S uptakes decreased [14]. Some physical treatments often only change the course of some physiological processes in seeds and plants, which increase their vigor and contribute to enhanced development of a plant [15, 16]. It has been stated that the positive effect of magnetic treatment may be due to paramagnetic properties of some atoms in plant cells and pigments such as chloroplasts [17]. Magnetic properties of molecules determine their ability to attract and then change the energy of a magnetic field in other types of energy and to transfer this energy to other structures in plant cells, thus activating them [17]. Cakmak et al. [18] reported higher seed germination and growth rates of bean and wheat plants from exposure of seeds to a permanent 7-mT magnetic field. It has been reported that maize seeds exposed to a 150-mT magnetic field encouraged shoot development and led to increases in germination, fresh weight, and shoot length [19]. The magnetic field may play an important role in cation uptake capacity and has a positive effect on immobile plant nutrient uptake, such as Ca and Mg, but negative electrical charges on plants inhibited the uptakes of anions such as P and S [20]. Based on these findings, this experiment was done to study the responses of fodder maize to silver nanoparticles and magnetic field combinations in comparison with commercial fertilizers in field conditions during experiments done over two years.
Materials and Methods Site Description This study was carried out at the Razavi Research and Technology Institute in 2008 and the Agricultural Faculty
Table 1 Physicochemical properties of soil samples in 2008 and 2009 Soil sample CaCO3 (%) OC (%) Zn Fe Mn mg kg−1 2008 2009
10 10
0.75 0.51
Cu
K
P
1.1 6.1 13.3 0.75 200 5 1.5 5.9 11 1.1 133 20.5
N (total) N (available) SP (%) pH
882 921
10 12
31.5 28.4
7.7 8.71
EC (dS m−1) Texture
1.10 1.09
Sandy clay loam Loam
Effects Silver Nanoparticles and Magnetic Field on Fodder Maize
of Ferdowsi University of Mashhad, Iran, in 2009 (latitude, 36° 15′ N; longitude, 56° 28′ E; and altitude, 985 m). The average annual rainfall was 252 mm, and the long-term average minimum and maximum air temperatures were 6 and 22°C, respectively. A soil sample was taken from a depth of 0–30 cm before planting. Soil properties were determined using the following procedures: available nitrogen was analyzed using the KCl extraction procedure; total nitrogen by the Kjeldal method; phosphorus by the Olsen procedure; potassium by ammonium acetate extraction; soil texture by the hydrometric method [21]; Fe, Mn, Zn, and Cu by the DTPA-TEA procedure [22]; organic carbon using the method suggested by Walky and Black [21]; and CaCo3 by neutralization with acid [23]. Physical and chemical properties of the soil were determined in the Soil and Plant Analysis Laboratory, Ferdowsi University of Mashhad, Iran (Table 1). Treatment Description and Data Collection These experiments tested seven treatments based on a randomized complete block design in four replications. The treatments were as follows: magnetic field and silver nanoparticles+Kemira commercial fertilizer (T1), magnetic field and silver nanoparticles+Humax commercial fertilizer (T2), magnetic field and silver nanoparticles (T3), Kemira fertilizer (T4), Librel commercial fertilizer (T5), Humax fertilizer (T6), and a control (T7). In each plot, a distance of 75 cm was set between the rows, and the final plant density was 11.1 plants per square meters. The maize variety was SC 704. A plot size of 3.5×6 m was used. For all treatments, nitrogen fertilizer (as urea) on the basis of 250 and 250 kg ha−1; phosphorus fertilizer (as phosphate ammonium), 250 and 150 kg ha−1; and potassium fertilizer (as potassium sulfate), 120 and 50 kg ha−1, were applied in 2008 and 2009, respectively. Pesticides, herbicides, and fungicides were not used for controlling pests, diseases, and weeds during the growing seasons. Weeds were managed by hand weeding throughout the growing season. Ingredients of Humax fertilizer consisted of 12 % humic acid, 3 % folic acid, and 3 % K2O. Components of Kemira and Librel fertilizers were 20 % K2O, 20 %N, and 20 % P2O5 and micronutrients (Fe, Zn, Mn, Cu, Mo, B, and Mg). Humax and Kemira fertilizers were applied as fertigation and Librel fertilizer by foliar application according to factory recommendations. After seed emergence, magnetic field treatment was done by employing magnet pieces with dimensions of 3× 1 cm and strength of 10 mT, located adjacent to or near each plant on the soil’s surface. At the same time, 40 gha−1 of colloidal nanosilver was used in the irrigation water for the silver nanoparticle treatment. The average size of silver nanoparticles was around 20 nm, determined by transition electron microscope (TEM) in the Central Laboratory of Ferdowsi University of Mashhad,
421
Fig. 1 Image of silver nanoparticle size by TEM
Iran (Fig. 1). Agronomic traits of maize such as fodder fresh yield, fodder dry yield, plant dry matter, plant height, and plant components (leaf, stem, and ear) were measured at harvest time. Statistical Analysis Analysis of variance was performed on a randomized complete block design. Data were analyzed using MSTAT-C software. Significant difference levels were calculated for all measured traits, and the means were compared with the multiple-range Duncan test.
Results and Discussion Effects of Silver Nanoparticles and Magnetic Field on Fodder Yield The treatments affected most studied traits of maize in 2008, but not in 2009 (Table 2). Results indicated that treatments of silver nanoparticles with magnetic field (T3) had the highest fodder fresh yield (74.5 tons ha−1) followed by the Kemira fertilizer treatment (T4) (64.9 tons ha−1) in 2008. Silver nanoparticles with magnetic field treatment (T3) showed about 35 % more fresh yield in comparison to the control. Although the greatest fodder fresh yield was in T3 in 2008, it did not show significant difference compared to the other treatments in 2009 (Table 3). It has been reported that germination and early growth of maize seedlings improved when seeds were exposed to a continuously stationary magnetic field [24]. It was shown that a combination of nanosized SiO2 and TiO2 could increase nitrate reductase enzyme in soybean (Glycine max), improve its absorption ability of water and fertilizer, promote its antioxidant system, and in fact accelerate its germination and growth [25]. The treatments in this experiment were not affected significantly for dry matter percentage of maize in either year
422
Berahmand et al.
Table 2 Analysis of variance (mean of squares) of fresh yield, dry matter, and dry matter yield of fodder maize Source of variation 2008
2009
Degree of freedom Fresh yield Dry matter Dry matter yield Degree of freedom Fresh yield Dry matter Dry matter yield Replication Treatment Error
3 6 18
116.9 205.9a 45.5
3.6 4.5ns 1.8
13.6 25.1a 3.7
3 6 18
129.3 209.8ns 52.0
4.1 5.8ns 3.9
16.6 27.5ns 5.8
ns Not significantly different a
Significantly different at the 5 % probability level
Table 3 Effects of silver nanoparticles and magnetic field on yield and dry matter of maize Treatment 2008
2009
Fresh yield Dry matter (%) Dry matter yield Plant height (cm) Fresh yield Dry matter (%) Dry matter yield Plant height (cm) (tons ha−1) (tons ha−1) (tons ha−1) (tons ha−1) T1 T2 T3 T4 T5 T6 T7 Mean LSD
61.7 61.6 74.5 64.9 55.9 53.9 55.2 61.1 10.02
29.38 29.19 29.65 27.32 28.33 27.26 27.36 28.35 1.98
18.1 18.1 22.1 17.8 16.0 14.7 15.1 17.4 2.87
259.5 260.9 261.3 259.3 258.8 259.3 257.5 259.5 2.62
65.9 58.3 69.2 55.4 57.2 69.0 58.3 61.9 19.34
37.8 36.8 36.0 34.3 36.3 35.5 34.0 35.8 4.7
25.0 21.2 24.3 18.9 20.6 24.6 19.8 22.05 7.03
206.9 203.3 210.2 171.1 187.6 194.7 185.7 194.2 44.94
T1 silver nanoparticles and magnetic field+Kemira commercial fertilizer, T2 silver nanoparticles and magnetic field+Humax commercial fertilizer, T3 silver nanoparticles and magnetic field, T4 Kemira commercial fertilizer, T5 Librel commercial fertilizer, T6 Humax commercial fertilizer, and T7 control
Table 4 Effects of silver nanoparticles and magnetic field on proportion of maize plant components based on dry matter percentage
T1 silver nanoparticles and magnetic field+Kemira commercial fertilizer, T2 silver nanoparticles and magnetic field +Humax commercial fertilizer, T3 silver nanoparticles and magnetic field, T4 Kemira commercial fertilizer, T5 Librel commercial fertilizer, T6 Humax commercial fertilizer, and T7 control
Treatment
2008
2009
Leaf proportion (%)
Stem proportion (%)
Ear proportion (%)
Leaf proportion (%)
Stem proportion (%)
Ear proportion (%)
T1 T2 T3
29.1 30.2 27.6
38.9 42.3 35.9
32.0 37.5 36.6
29.5 31.0 29.3
34.4 33.2 29.3
36.0 35.8 41.3
T4 T5 T6 T7 Mean LSD
30.4 34.2 31.9 33.3 31.0 3.03
41.0 46.7 42.3 43.3 41.5 4.81
28.7 19.1 25.8 33.4 30.4 7.31
31.8 31.7 29.9 33.1 30.9 6.99
34.4 34.6 32.6 34.4 33.3 7.32
33.7 33.6 37.5 32.4 35.8 12.76
Effects Silver Nanoparticles and Magnetic Field on Fodder Maize
(Table 3). The highest fodder dry matter yield was achieved with magnetic field treatment in combination with silver nanoparticles (T3). Lower yields were in the controls and in 2008 in T6, whereas these treatments did not significantly affect fodder dry matter yield in 2009 (Table 3). Plant height was not significantly affected by treatments in either year (Table 3). Magnetic field may play an important role in cation uptake capacity and had a positive effect on immobile nutrient uptake by a plant, for example with Ca and Mg, but negative electrical charges on plants inhibited the uptake of anions such as P and S [20]. It is most probable that texture, fertility level, and cation exchangeable capacity of the soil affected plant response to the treatments tested in this study. It was also revealed that suspended TiO2 contents in soil suspensions were positively correlated with the dissolved organic carbon and clay contents of the soil, but were negatively correlated with ionic strength, pH, and zeta potential [26]. Therefore, it seems that because of the higher fertility of the soil in 2009 compared to 2008, the effect of treatments on maize was more significant in 2008 due to differences between the soil characteristics in the two locations such as texture and fertilizer level. Vashisth and Nagarajan [27] stated that magnetic field increased seedling dry weight of 1-month-old chickpea plants. De Souza et al. [28] reported that electromagnetic treatments led to a significant increase in leaf area, leaf dry weight, mean fruit weight, fruit yield per plant, and fruit yield of tomato per area. Effects of Silver Nanoparticles and Magnetic Field on Maize Plant Components The ear percentage of maize mainly determines the nutritional value of animal feed. Results of this study revealed that the greatest leaf proportions in plants were 33.1, 31.8, and 31.7 % in T7, T4, and T5, respectively, and the lowest was in T3 treatment (29.3 %). Also, the lowest stem proportion in plants was in the T3 treatment (29.3 %). In contrast, the T3 treatment produced the highest ear proportion (41.3 %), while the lowest ear proportion (32.4 %) was found in the control group in 2008 (Table 4). It seems that use of silver nanoparticles and magnetic field (T3) led to better availability and uptake of soil nutrients for plants, improving the growth and development of fodder maize and enrichment of that fodder. Therefore, it can be concluded that produced fodder maize had better quality in terms of its suitability for animal feed from the T3 treatment in 2008. Similar trends were also found in 2009. The lowest leaf and stem proportion and the greatest ear proportion were observed in T3 treatment in 2009 (Table 4). It has been reported that maize seeds exposed to a 150-mT magnetic field encouraged shoot development and led to increased germination, fresh weight, and shoot length [17]. In an
423
experiment by Racuciu et al. [29], a stimulatory effect on plants was demonstrated on maize seeds exposed to a low static magnetic field (50 mT) at their early growth stages; effects were enhancement of fresh weight, level of assimilatory pigments as well as the chlorophyll ratio, average level of nucleic acids, and an increased seedling length. Also, Koontz and Berle [14] reported that a low Ag+ concentration (50 nM) inhibited shoot uptake of the ions on bean, corn, and tomato plants. In the roots, Ca uptake increased while P and S uptakes decreased. Other studies reported that nanosized TiO2 can promote plant photosynthesis and nitrogen metabolism and greatly improve the growth of spinach at an appropriate concentration [30–33]. Conclusions In general, applications of a combination of silver nanoparticles and magnetic field led to improved quantitative yields of fodder maize, especially in 2008. Similar effects were demonstrated on the studied traits in 2009. But results were more significant in 2008 than 2009. It is probable that there were more suitable soil conditions for crop growth in 2009 than in 2008 and that this caused the lower response from plants to the treatments tested in this experiment. The treatment combining silver nanoparticles and magnetic field (T3) most effectively improved the quality of fodder maize for animal feed compared to other treatments. It is recommended that further studies are done on the influence of magnetic field and silver nanoparticles alone on maize. Additional research is recommended on reasons for stimulated plant growth from this mode of action by silver nanoparticles in combination with a magnetic field. References 1. Gonzalez-melendi P, Fernandez-pacheco R, Coronado MJ, Coredor E, Testillano PS, Risueno MC, Marquina C, Ibarra MR, Rubiales D, Perez-Deluque A (2008) Nanoparticles as smart treatment-delivery systems in plants: assessment of different techniques of microscopy for their visualization in plant tissues. Ann Bot 101:187–195 2. Handy RD, Kamme F, Lead JR, Hasselov M, Owen R, Crane M (2008) The ecotoxicology and chemistry of manufactured nanoparticles. Ecotoxicology 17:287–314 3. Handy RD, Owen R, Valsami-Jones E (2008) The ecotoxicology of nanoparticles and nanomaterials: current status, knowledge gaps, challenges, and future needs. Ecotoxicology 17:315– 325 4. Auffan M, Rose J, Wiesner MR, Bottero J (2009) Chemical stability of metallic nanoparticles: a parameter controlling their potential cellular toxicity in vitro. Environ Pollut 157:1127–1133 5. Zhu H, Han J, Xiao QJ, Jin Y (2008) Uptake, translocation, and accumulation of manufactured iron oxide nanoparticles by pumpkin plants. J Environ Monit 10:685–784 6. Lin D, Xing B (2007) Phytotoxicity of nanoparticles: inhibition of seed germination and root growth. Environ Pollut 150:243–250
424 7. Musante C, White JC (2010) Toxicity of silver and copper to Cucurbita pepo: differential effects of nano and bulk-size particles. Environ Toxicol. doi:10.1002/tox. 1-8 8. Lei Z, Mingyu S, Xiao W, Chao L, Chunxiang Q, Liang C, Hao H, Xiao-qing L, Fashui H (2008) Antioxidant stress is promoted by nano-anatase in spinach chloroplasts under UV-B radiation. Biol Trace Elem Res 121:69–79 9. Lu J, Cao P, He S, Liu J, Li H, Cheng G, Ding Y, Joyce DC (2010) Nano-silver pulse treatments improve water relations of cut rose cv. movie star flowers. Postharvest Biol Technol 57:196–202 10. Altman SA, Solomos T (1995) Differential respiratory and morphological responses of carnations pulsed or continuously treated with silver thiosulfate. Postharvest Biol Technol 5:331–343 11. Ichimura K, Yoshioka S, Yumoto-Shimizu H (2008) Effects of silver thiosulfate complex (STS), sucrose and combined pulse treatments on the vase life of cut snapdragon flowers. Environ Control Biol 46:155–162 12. Van Ieperen W (2007) Ion-mediated changes of xylem hydraulic resistance in plant: fact or fiction? Trends Plant Sci 12:137–142 13. Ohkawa K, Kasahara Y, Suh J (1999) Mobility and effects on vase life of silver containing compounds in cut rose flowers. Hort Sci 34:112–113 14. Koontz HV, Berle KL (1980) Silver uptake, distribution and effect on calcium, phosphorus and sulfur uptake. Plant Physiol 65:336– 339 15. Podlesny J, Misiak L, Koper R (2001) Concentration of free radicals in faba bean seeds after the pre-sowing treatment of the seeds with laser light. Int Agrophysics 15:185–189 16. Podlesny J, Pietruszewski S, Podleoen A (2004) Efficiency of the magnetic treatment of broad bean seeds cultivated under experimental plot conditions. Int Agrophysics 18:65–71 17. Aladjadjiyan A (2010) Influence of stationary magnetic field on lentil seeds. Int Agrophysics 24:321–324 18. Cakmak T, Dumlupinar R, Erdal S (2009) Acceleration of germination and early growth of wheat and bean seedlings grown under various magnetic field and osmotic conditions. Bioelectromagnetics 30:1–10 19. Aladjadjiyan A (2002) Study of the influence of magnetic field on some biological characteristics of Zea mays. J Central Eur Agric 3:89–94
Berahmand et al. 20. Esitken A, Turan M (2004) Alternating magnetic field effects on yield and plant nutrient element composition of strawberry (Fragaria ananassa cv. Camarosa). Soil Plant Sci 54:135–139 21. Page AL, Miller RH, Keeney DR (1982) Methods of soil analysis. Part2: Chemical and microbiological properties. Am Soc Agronomy, Soil Sci Am Publisher, Madison 22. Lindsay WL, Norvell WA (1978) Development of a DTPA soil test for zinc, iron, manganese, and copper. Soil Sci Soc Am J 42:421–428 23. Richards LA (1954) Diagnosis and improvement of saline and alkali soil. USDA. Agriculture Handbook No. 60. Washington, USA 24. Florez M, Carbonell MV, Martinez E (2007) Exposure of maize seeds to stationary magnetic fields: effects on germination and early growth. Environ Exp Bot 59:68–75 25. Lu CM, Zhang CYWuJQ, Tao MX (2002) Research of the effect of nanometer on germination and growth enhancement of Glycine max and its mechanism. Soybean Sci 21:168–172 26. Peralta-Videa JR, Zhao L, Lopez-Moreno ML, dela Rosa G, Hong J, Gardea-Torresdey JL (2011) Nanomaterials and the environment: a review for the biennium 2008–2010. J Hazard Mater 186:1–15 27. Vashisth A, Nagarajan S (2008) Exposure of seeds to static magnetic field enhances germination and early growth characteristics in chickpea (Cicer arietinum L.). Bioelectromagnetics 29:571–578 28. De Souza A, Garcí D, Sueiro L, Gilart F, Porras E, Licea L (2006) Pre-sowing magnetic treatments of tomato seeds increase the growth and yield of plants. Bioelectromagnetics 27:247–257 29. Racuciu M, Creanga D, Horga I (2006) Plant growth under static magnetic field influence. Romanian J Phys 53:353–359 30. Hong F, Zhou J, Liu C, Yang F, Wu C, Zheng L, Yang P (2005) Effects of nano-TiO2 on photochemical reaction of chloroplasts of spinach. Biol Trace Elem Res 105:269–279 31. Hong F, Yang F, Liu C, Gao Q, Wan Z, Gu F, Wu C, Ma Z, Zhou J, Yang P (2005) Influence of nano-TiO2 on the chloroplast aging of spinach under light. Biol Trace Elem Res 104:249–260 32. Yang F, Liu C, Gao F, Su M, Wu X, Zheng L, Hong F, Yang P (2007) The improvement of spinach growth by nano-anatase TiO2 treatment is related to nitrogen photoreduction. Biol Trace Elem Res 119:77–88 33. Zheng L, Hong F, Lu S, Liu C (2005) Effect of nano-TiO2 on strength of naturally aged seeds and growth of spinach. Biol Trace Elem Res 105:83–91
