The influence of coated nanoCeO2 on the phenol content in wheat and pea

Abstract

Introduction The use of nanomaterials in various commercial products and industrial processes has increased. Although the application of nanoparticles has great importance, some of them can be risky to human health and the environment. The unregulated usage of nanoparticles can result in excessive accumulation in sediments, soils, air and aquatic environments, endangering terrestrial plants1. Cerium oxide nanoparticles (nanoCeO2) have been extensively investigated due to the excellent oxygen storage capacities on the basis of the redox transition between Ce3+ and Ce4+ and formation of oxygen vacancies on their surface2. The effect of nanoCeO2 on individual organisms and the ecosystem in general are not sufficiently explored and the literature on the toxicity of nanoCeO2 in edible plants is contradictory. NanoCeO2 is very stable in soil and different environmental media and has been found to transfer within plant tissues unaltered. It is very likely that it interacts with plants in nanoparticulate forms3. In this research we used CeO2, naked and coated with three different carbohydrates (glucose, pullulan or levan), to study their effect on phenol content, as an important indicator of plant stress4.5, in aboveground plant organs in two different plants (wheat and pea). Materials and methods NanoCeO2 was synthetized using self-propagating room temperature (SPRT) method by the procedure of Matović et al6. Briefly, Ce(NO3)3∙6H20 and NaOH, as starting materials, were hand mixed in a mortar with a pestle for about 5-10 min. The obtained product was rinsed three times for 10 min with deionized water and twice with ethanol in a centrifuge at 4200 rpm. The powders were dried overnight at 70 ˚C. NanoCeO2, synthesized by SPRT method, was subsequently coated with three different carbohydrates to obtain glucose-, levan- and pullulan-coated nanoCeO2 (G-CeO2, L-CeO2 and P-CeO2). Suspensions of these nanoparticles prepared in DI water at concentration of 200 mg/L were used for all experiments. The suspensions of nanoparticles were characterized by X-ray diffraction (XRD) and transmission electron microscopy (TEM). XRD (Figure 1) confirmed the nanoCeO2 cristalinity and TEM (Figure 2) provided data on particle size and shape. Wheat (Triticum aestivum) and pea (Pisum sativum) plants were treated with all types of nanoCeO2 in two different ways – 1) during germination (after which the plants were grown in hydroponics without nanoparticles) and 2) during growth in hydroponics (without the treatment during germination). For each treatment (control, nanoCeO2 and G-CeO2, L-CeO2 and P-CeO2) during germination four replicates of 20 seeds were placed in Petri dishes. Seeds of all plant species were placed in 10 mL treatment suspension prepared in distilled water. Germinated seeds were transferred to hydroponics (MS/2 medium) and grown for the next 3 weeks under 24 ± 2°C temperature regime and 16/8h photoperiod. After that the aboveground parts of plants were cut off and phenols were isolated by the procedure7. Statistical analysis was done using nonparametric Kruskal-Wallis test to explore the differences between levels of phenols in the aboveground part of wheat and pea. Post-hoc intergroup comparisons of phenols’ level (between control and treatments) were performed by the Mann-Whitney test using SPSS 13.0 software. Results The physiochemical properties of the synthesized nanoCeO2 were analyzed using XRD and HRTEM methods. The XRD patterns of obtained nanoCeO2 are shown in Figure 1. Samples exhibited typical peaks corresponding to planes which are the typical of face centered. All samples shows broadness peaks, explained due their synthesis procedure at low temperature, but not shift were found following this synthesis procedure even further coating process.

Figure 1. XRD pattern of uncoated nanoCeO2 and coated G-, L- and P-CeO2 For uncoated nanoCeO2 HRTEM images (Figure 2) demonstrated an average size between 8-13 nm, which is slightly increased for the coated systems. This can be explained by the obviously coating effect which apparently aggregates them, especially in L-CeO2. In all nanoCeO2 was found a crystalline structure with a spacing of 0.333 nm (Figure 2E), which indicate face-centered cubic crystallographic structure8.

Figure 2. HRTEM images of the obtained nanoCeO2 (A) Uncoated nanoCeO2, (B) L-CeO2, (C) G-CeO2 and (D) P-CeO2. Histogram of nanoparticles size distribution is shoven as inset in (A). Treatment of wheat with four different nanoCeO2, uncoated and coated, did not show statistically significant differences in total phenol content of the aboveground plant parts, regardless of the timing of its application (during germination or growth in hidroponics). On the other hand, it can be observed (Figure 3) that total phenol content increased in pea plants treated with L-CeO2 and P-CeO2 during germination. Between the other treatments in pea plants there were no statistically significant differences, as well as in comparison with control. These results show that the treatment with different nanoCeO2 has stronger effect on plant metabolism when applied during germination compared to its application during growth in hydroponics. Although different coating of selected nanoparticles improves their biocompatibility, the results show that it can influence the difference in plant response.

Figure 3. Effect of nanoCeO2, G-CeO2, L-CeO2 and P-CeO2 treatment during germination/growth on phenol content in the aboveground parts of wheat and pea plants (Mean±SE; * indicates statistically significant differencies in comparison with control, p<0.05) Conclusions We investigated the ecotoxicity of different nanoparticles using changes in phenol content as the main indicator of plant stress. The results have shown stimulating effect of the L-CeO2 and P-CeO2 nanoparticles on pea phenol content, while there was no effect on wheat phenols, in the aboveground plant parts. This indicates that the effect of nanoparticles depends on plant species and of particle coating, meaning that their toxicity may vary for different plant species. The impact of nanoparticles on morphological parameters, total antioxidative activity and changes in phenolic profile, remains to be investigated.