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Synthetic apatite nanoparticles as a phosphorus fertilizer for soybean (Glycine
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max)
Ruiqiang Liu* and Rattan Lal
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Carbon Management &Sequestration Center, School of Environment & Natural Resources, the
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Ohio State University, 210 Kottman Hall, 2021 Coffey Road, Columbus, Ohio 43210, USA.
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*Corresponding author. Tel.: +1 614 292 5678; fax: +1 614 556 8942
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E-mail address:liu.603@osu.edu (R. Liu)
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Supplemental Online Material-1 (SOM-1)
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Supplementary Material and Method
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Lettuce Seed germination test
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The phytotoxicity of nHA was estimated using the 5-d seed germination test (Bowers et al.,
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1997). Tests were conducted using buttercrunch lettuce (Lactuca sativa) seeds purchased from a
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local nursery (Columbus, Ohio, USA). Test seeds were selected for uniform size and color after
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screening through a #20 sieve to remove the small seeds. Seed germination tests were conducted
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by placing 10 test seeds in 10 ml of nHA solution (as prepared or diluted) contained in clear,
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polystyrene Petri dishes (9 × 50 mm) with leak-proof covers. Five replicates of 10 seeds each
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were prepared for each solution. Control treatments (5 replicates) were conducted in a similar
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fashion but using distilled water. Test containers were incubated at 25 ◦C under a light-dark
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cycle of 18 h and 6 h, respectively, at ~ 38 mE m-2 s-1 for a period of 5 d (incubator
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manufactured by Percival Scientific Inc.). After 5 d, the total number of germinated seeds was
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recorded and the percent germination determined for each elutriate. The germination was
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considered successful if the primary root length was ≥ 3 mm. The final length of each
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germinated seedling was measured using a caliper (Bet-Art Products).
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Supplementary results and discussion
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Assessment of HA nanoparticles toxicity by lettuce seed germination test
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Although neither calcium or phosphate, nor apatite is believed to be toxic to plants or other
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organisms at normal concentrations, the toxicity of nHA should be specifically tested to address
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the public concerns on introducing synthesized nanoparticles to the environment. It has been
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widely accepted that the toxicity of a type of nanoparticles might be very different from the bulk
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counterpart due to the higher specific surface areas and the higher reactivity of the former
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(Taylor and Walton, 1993; Lee et al., 2010; Ma et al., 2010). Moreover, nanoparticles could
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reduce or totally block the porous structures or intercellular spaces in plant organs and thus affect
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associated physiological activities (e.g., nutrients uptake and transport) (Ma et al., 2010).
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However, beneficial effects of some engineered nanoparticles (e.g., TiO2, SiO2, and multiple
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wall carbon nanotubes) on plant growth have also been reported (Lin et al., 2004;
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Khodakovskaya et al., 2009; Su et al., 2009).
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Supplementary Fig. 1 shows responses of lettuce seeds and seedlings to DI water, nHA as
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prepared, and the DI-water diluted nHA solutions. The data indicate that lettuce seeds in DI
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water had an average total germination of 98% while those in nHA solution had an average
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germination of 86%. Total germination in diluted nHA solutions ranged from 84 – 98%.
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Statistical analysis suggests that lettuce seeds germination in those solutions were not
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significantly different (S. Fig. 1a), showing that the nHA, as mild as DI water, did not exhibit
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any acute toxic or inhibitory effect on plants and should be an environmentally friendly product.
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Elongation of the germinated lettuce seedlings also supports this conclusion (S. Fig. 1b). The
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seedlings were 4.3 cm long on average in DI water, which was very close to the values of 4.6 cm
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grown in the nHA solution and 3.4-5.0 cm found in the diluted nHA solutions. The seedlings
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even grew better in some of the nHA solutions than in DI water. For example, 5.0 cm-elongation
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was observed in 80% nHA solution, which was 18% longer than that in DI water, suggesting that
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the nHA could provide P nutrient to lettuce seedlings and promote the growth. However, nutrient
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P might not needed in high amount on the early stage of lettuce growth so that the results of
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germination test only are not enough to support the benefit roles of nHA solution as a P fertilizer.
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But the germination tests at least indicate that the nHA is not toxic to plants and as benign as DI
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water. Applications of engineered nHA in the field should be safe to the environment and the
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ecosystem.
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Supplementary Figure Caption
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Supplementary Figure 1. Effects of nano-sized hydroxyapatite (nHA) on germination of
lettuce seeds (a, top) and elongation of the germinated seedlings (b, bottom). DIW: Deionized
water; 100% nHA: nHA as pared; 80% nHA: diluted nHA solution with DIW and so on.
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a
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DIW
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nHA
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nHA
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Supplementary Figure 1. Effects of nano-sized hydroxyapatite (nHA) on germination of lettuce seeds (a, top) and elongation of the germinated
seedlings (b, bottom). DIW: Deionized water; 100% nHA: nHA as pared; 80% nHA: diluted nHA solution with DIW and so on.
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Supplementary References
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Bowers, N., Pratt, J.R., Beeson, D., & Lewis, M. Comparative evaluation of soil toxicity using
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lettuce seeds and soil ciliates. Environ. Toxic. Chem. 16, 207-213 (1997).
Khodakovskaya, M. et al. Carbon nanotubes are able to penetrate plant seed coat and
dramatically affect seed germination and plant growth. ACS Nano 3, 3221-3227 (2009).
Lee, C.W. et al. Developmental phytotoxicity of metal oxide nanoparticles to Arabidopsis
thaliana. Environ. Toxicol. Chem. 29, 669-675 (2010).
Lin, B. et al. Effects of TMS (nanostructured silicon dioxide) on growth of Changbai Larch
seedlings. J. For. Res. CHN 15,138-140 (2004).
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Ma, X, Geiser-Lee, J., Deng, Y. & Kolmakov, A. Interactions between engineered nanoparticles
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(ENPs) and plants: phytotoxicity, update and accumulation. Sci. Total Environ. 408, 3053-
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3061 (2010).
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Su, M. et al. Promotion of nano-anatase TiO2 on the spectral responses and photochemical
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activities of D1/D2/Cyt b559 complex of spinach. Spectrochim. Acta. Part A Mol. Biomol.
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Spectrosc. 72, 1112-1116 (2009).
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Taylor, R. & Walton, D.R.M. The chemistry of fullerenes. Nature 363, 685-693 (1993).
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