Effects of Titanium Dioxide (TiO2) and EDTA on Growth and Biochemical Properties of Spinach (Spinacia oleracea)

An emerging field of nanotechnology in recent years is the use of nanoparticles and nanomaterials in agricultural systems which is due to their excellent mechanical, electrical, optical, surface properties, crop protection and nano-fertilizers. Titanium dioxide (TiO2) is a class of nanoparticles which widely used in the food industry, cosmetics, papers, pharmaceuticals, plastics and industrial raw materials. The widespread industrial application of TiO2 is due to its white pigment, ultraviolet blocking property, and chemical features commonly used to alleviate pollutants concentration in water, soil and air. Owing to its increasing use in the industry, a large part of TiO2 residues are released into the environment, and currently, TiO2 nanoparticles are being considered an emerging environmental contaminant. However, there have been a number of studies reporting beneficial effects of TiO2 on growth and physiological traits of crops. It has been postulated that the TiO2-induced improvement of crop growth is not merely related to the promotion of photosynthesis; other biochemical processes especially nitrogen metabolism are also involved in this event. Ethylene diamine tetraacetic acid (EDTA) is a widely used as a chelating agent, i.e., the chemical is able to sequester metal ions such as Ca2+ and Fe3+. EDTA is used as nitrogen source for doping of TiO2 nanoparticles which improves TiO2 photocatalytic features. The present study was conducted to investigate the effects of TiO2 nanoparticles and EDTA on growth indices and biochemical parameters in spinach (Spinacia oleracea). For detailed evaluation of treatment effects, different concentrations of TiO2 nanoparticles were sprayed on spinach leaves and the samples were collected in a time course.

A factorial experiment was carried out in the form of completely randomized design (CRD) with three replications. Soil samples were taken before cultivation of spinach (S. oleracea) seeds (Var VIROFLAY) and analyzed for nutrients’ concentration. Treatments include different levels of TiO2 (T1=0, T2=0.05mg/l and T3=0.1mg/l) and two concentrations of EDTA (E1=0 and E2=130mg/l) sprayed on spinach plants in research greenhouse of agriculture faculty, Ferdowsi University of Mashhad. Aqueous solutions of nanoparticles were treated by ultrasound for 10 min to enhance homogeneity. The solutions were sprayed on the plant at six- leaves stage. The plant samples were taken before reproductive phase for measurement of biochemical parameters. Nitrogen content of plant samples was measured by PDV 500 Macro- Kjeldahl device; Potassium content was determined by 310c flame photometer; phosphorus concentration in plant samples was measured by spectrophotometer model 2100. Chlorophyll and carotenoid contents were measured by the method proposed by Lichtenthaler (1978). For analysis of growth parameters, plant samples were taken a week after TiO2 treatments and leaf area, shoot fresh/dry weight, stem length, internode length, root area, root fresh/dry weight and total root diameter were measured.

Application of 0.05mg/l of TiO2 nanoparticles without EDTA resulted in 13.5% and 9.48% increase in nitrogen and protein; respectively, however by increasing nanoparticles to 0.1mg/l, nitrogen and protein content in the treated plants were respectively reduced to 21% and 19.57% of those of control group (p<0.01). Phosphorus content of the treated plants was decreased in both concentrations of TiO2 with the higher reduction in 0.1mg/l of TiO2. Potassium content showed a 6.63% increase by applying 0.05mg/l of TiO2; however, by increasing TiO2 to 0.1mg/l, potassium content was decreased to 12%. EDTA increased nitrogen and protein content by 5.95% and 1.9%, respectively; however phosphorus and potassium contents were reduced by 8.7% and 5.65%, respectively. Interaction of 0.05mg/l TiO2 and 130mg/l EDTA resulted in increasing nitrogen, protein and potassium content (14%, 6.8% and 15%; respectively) and reduction of phosphorus (19.9%). Application of 0.1mg/l TiO2 and 130mg/l EDTA decreased significantly nitrogen, protein, potassium and phosphorus to 25.44%, 26.75%, 39.22% and 27.8%; respectively. Interaction of TiO2 and EDTA increased dry/fresh weight, diameter and total area of spinach plants. Our results are in agreement with those reported by others. Spinach shoot dry and fresh weights were enhanced by application of 0.05mg/l TiO2; while stem length was increased at both 0.05mg/l and 0.1mg/l TiO2 significantly. Interaction of TiO2 and EDTA was also followed by increasing in growth parameters. This finding is according with the results reported by other authors. These contradictory results suggest that effects of TiO2 on plant growth and physiology don’t follow a clear-cut trend and other factors may play important roles in this story.

In this study, we investigated the influence of TiO2 nanoparticles on growth and biochemical properties in spinach. In general, the results indicated that application of 0.05mg/l of TiO2 has a significant promoting effect on the studied traits. Increasing TiO2 concentration to 0.1mg/l was followed by negative effects that may be attributed to poisonous effect of extra-concentration of this nanoparticle on DNA replication, enzymatic activity and cell proliferation. Coincidence of NR activity and growth changes supports the crucial role of nitrogen metabolism in mediating TiO2 effects on spinach growth. More notably, application of EDTA enhanced positive impacts of the nanoparticles. This synergy may be due to the fact that EDTA acts as an N-source and improves photocatalytic properties of TiO2 nanoparticles.