1. A method of enhancing fluoride hyper accumulation efficiency of a plant for improving the soil quality, said method comprising the steps of treating the seedlings of said plant with different Fluoride concentrations followed by Iron nanoparticles and chelating agent wherein said Iron nanoparticle is present in the range of 170 to 220 mgkg-1 while said chelating agent is present in the range of 15 to 25 mgkg-1.

2. The method as claimed in claim 1, wherein said method comprises steps: a) Surface sterilization of said plant seedlings by 10% H2SO4 for about 10 minutes followed by rinsing with pure water; b) Sowing of said sterilized seeds under soil approximately at the depth of 1cm followed by treatment with different fluoride concentrations after approximately 10 days of said sowing; c) Ultrasonicating said sterilized seeds for about 30 minutes with the suspension of Iron Oxide nano-particles suspended in distilled water; d) Treating the soil of said plant seedlings with Iron nanoparticles present in the range of 170 to 220 mgkg-1 followed by irrigating said plant seedlings with water; e) Treating said plant seedlings with Ethylene Diamine Tetra acetic Acid (EDTA) present in the range of 15 to 25 mgkg-1 followed by irrigating said plant seedlings with water

3. The method as claimed in claim 1, wherein said plant comprises Prosopis juliflora.

4. The method as claimed in claim 1, wherein said Iron nanoparticle comprises Fe3O4

5. The method as claimed in claim 1, wherein said chelating agent comprises Ethylene Diamine Tetra Acetic Acid.

6. The method as claimed in claim 1, wherein said Iron nanoparticle is present in the concentration of 200 mgkg-1

7. The method as claimed in claim 1, wherein said Ethylene Diamine Tetra acetic Acid is present in the concentration of 20 mgkg-1

8. The method as claimed in claim 1, wherein said Fluoride concentrations comprises 25, 50, 75, 100 mgkg-1 soil. , Description:Field of the Invention: The present invention is in the field of phyto-remediation. The Invention in particular provides a method of enhancing the hyperaccumulation tendency of a plant, along with enhancing its biomass and improving the soil property. Background of the Invention: The following background discussion includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art. Fluoride contamination is a worldwide problem. Many parts of the world are severely affected by this contamination and still there is no cure of fluorosis. F pollution in water, soil and vegetation is the main cause of environmental toxicity. In water, F react with some of the elements, and then turns into salts, finally deposit on soil as sediments (Molla et al., 2007). F at high concentration in soils can cause various forms of toxicity which has negative effects on plants, animals and human health through the food chain (Stevens et al., 1997). F is known to cause dental and skeletal fluorosis when its concentration is higher than 1.5 mg L-1 in drinking water (Teotia and Teotia, 1994). The available methods for F removal are:

1. Reverse Osmosis (RO)

2. Forward Osmosis (FO)

3. Evaporation

4. Ion-exchange

5. Adsorption

6. Precipitation The conventional remediation technologies are expensive and some of the techniques do not remove F effectively. Therefore, it is important to develop economically viable and more effective method to decontaminate soils from F contamination. In recent years, use of plants in the reclamation of F contamination has gained considerable importance. Phytoremediation can be defined as the use of plants for the extraction, immobilization, containment or degradation of contaminants (Macek et al., 2000). The most significant advantage of phytoremediation is its low cost and effectiveness (Abdul and Schroder, 2009). It can be up to 1000-fold cheaper compared to conventional methods. It has been estimated that phytoextraction to clean up one acre of sandy loam soil to a depth of 55 cm will cost 60,000-100,000$ compared to 400,000$ for conventional using traditional soil removal methods (Salt et al., 1995). The cost and effectiveness of the filtration techniques are still not satisfactory and further improvements are required. The limitations of existing defluoridation technologies are: a) Precipitation: This method involves the precipitation of sparingly soluble F salt as insoluble fluorapatite by the addition of chemicals (coagulants and coagulant aids). Most common materials used in the technique are Aluminium salts (e.g. Alum), lime and Poly Aluminium Hydroxy sulphate. The best example of precipitation technique is Nalgonda Technique developed by National Environmental Engineering Research Institute (NEERI), Nagpur in 1961. It involves addition of Aluminium salts, lime and bleaching powder followed by rapid mixing, flocculation, sedimentation, filtration and disinfection. Limitations: Required dosages of chemical are high, requirement of trained labor, sludge produced after treatment is high, high concentration of aluminium is released in water during process which may cause Alzheimer’s syndrome. b) Adsorption: This technique is mainly based on the adsorption of F ions on the surface of an active agent. The commonly used adsorbent materials for this process are activated alumina, rice husk, bone charcoal, activated carbon and serpentine. Limitations: Toxic waste produced, highly pH dependant, adsorbents develop fouling smell, effectiveness decreases with time. c) Membrane process: In this process, on the basis of molecular shape and size, particles are isolated using extraordinarily composed semi-permeable membrane. The common membrane processes for F removal are reverse osmosis (RO), nano-filtration, electrodialysis. Limitations: Expensive, large amount of water is rejected as brine, high electricity consumption and removes all ions including essential minerals, water membrane cost is high, fouling of membrane takes place, membrane has to be changed periodically, F ion is not removed due to its small size. d) Ion-exchange: Synthetic chemicals, namely, cation and anion exchange resins have been used for F removal from water. A strongly fundamental anion-exchange resin can be used for F removal containing quaternary ammonium functional groups. Limitations: Contamination prone, expensive, water wastage is high, large volume regenerate required for cation and anion exchange resins regeneration. The cost and effectiveness of the F removal techniques are still not satisfactory. The chemical process suffers with the drawback of being expensive, producing harmful byproducts, requires high amount of energy, reject large amount of water during the process, does not remove Fluoride (F) at permissible limit, technically complex and is also not recyclable. Hence, there is a need of an efficient, greener and cost effective process for the removal of fluoride contamination from the soil. Object(s) of the Invention: A primary object of the present invention is to overcome the drawback/s associated with the prior art. Yet another object of the present invention is to provide an easy and efficient method of remediation of Fluoride contamination from soil and water as well. Yet another object of the present invention is to provide easy and efficient method of remediation of Fluoride contamination from soil and water using hyper-accumulator plant. Yet another object of the present invention is to provide a method of enhancing the hyperaccumulation efficiency of Fluoride hyperaccumulator plant Prosopis juliflora. Yet another object of the present invention is to provide a method for balancing the pH level of the soil by changing it from alkaline to acidic (7.30-6.37), which is required for nutrient availability and metal solubility in soil. Yet another object of the present invention is provide a method for improving the micro and macro nutrients such as EC, N, P, K, Mn, Zn and Cu etc. showing an overall increase in soil quality suitable for agriculture purpose and recycling. Yet another object of the present invention is to provide cost-effective and renewable method to reduce fluoride contamination from soil and water as well. Yet another object of the present invention is to provide a method for improving the surrounding environment without hampering the ecology in natural way. Yet another object of the present invention is to provide a method for improving the soil property and enhancing and recycling of the biomass Summary of the Invention: In an aspect of the Invention, there is provided a method of enhancing fluoride hyper accumulation efficiency of a plant for improving the soil quality, said method comprising the steps of treating the seedlings of said plant with different Fluoride concentrations followed by Iron nanoparticles and chelating agent wherein said Iron nanoparticle is present in the range of 170 to 220 mgkg-1 while said chelating agent is present in the range of 15 to 25 mgkg-1. The method comprises steps: a) Surface sterilization of said plant seedlings by 10% H2SO4 for about 10 minutes followed by rinsing with pure water; b) Sowing of said sterilized seeds under soil approximately at the depth of 1cm followed by treatment with different fluoride concentrations after approximately 10 days of said sowing; c) Ultrasonicating said sterilized seeds for about 30 minutes with the suspension of Iron Oxide nano-particles suspended in distilled water; d) Treating the soil of said plant seedlings with Iron nanoparticles present in the range of 170 to 220 mgkg-1 followed by irrigating said plant seedlings with water; e) Treating said plant seedlings with Ethylene Diamine Tetra acetic Acid (EDTA) present in the range of 15 to 25 mgkg-1 followed by irrigating said plant seedlings with water Brief Description of the Drawings To further clarify advantages and features of the present invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof, which is illustrated in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope. The invention will be described and explained with additional specificity and detail with the accompanying drawings in which: Figure 1 illustrates Layout of constructed wetlands for Nano-phytoremediation technology using Prosopis juliflora (P1, P2 and P3= Prosopis juliflora plants grown) Figure 2 illustrates schematic diagram of nano-phytoremediation technology Figure 3 illustrates F accumulation efficiency of P. juliflora after NPs treatment (A) root and (B) shoot Figure 4 illustrates Effect of NPs on F treated P. juliflora plant (A) translocation and (B) bioaccumulation factor. Figure 5 illustrates Light microscope analysis for (A) root, (B) shoot and (C) leaf of P. juliflora plant Figure 6 illustrates (A) and (B) show the FESEM image and EDS spectrum of root sample. While (C) and (D) show the FESEM image and EDS spectrum of shoot sample and (E) EDX spectrum of leaf of P. juliflora plant Figure 7 illustrates translocation of F with NPs in P. juliflora plant Figure 8 illustrates F accumulation efficiency of P. juliflora after NPs treatment with EDTA (A) root and (B) shoot Figure 9 illustrates Effect of NPs with EDTA on F treated P. juliflora plant (A) translocation and (B) bioaccumulation factor Detailed Description of the Invention For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates. It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof. Throughout the patent specification, a convention employed is that in the appended drawings, like numerals denote like components. The present Invention provides a method of enhancing fluoride hyper accumulation efficiency of a plant for improving the soil quality. In an embodiment, the method comprises the steps of treating the seedlings of said plant with different Fluoride concentrations followed by Iron nanoparticles and chelating agent wherein said Iron nanoparticle is present in the range of 170 to 220 mgkg-1 while said chelating agent is present in the range of 15 to 25 mgkg-1. In an embodiment, the method comprises steps of: a) Surface sterilization of said plant seedlings by 10% H2SO4 for about 10 minutes followed by rinsing with pure water; b) Sowing of said sterilized seeds under soil approximately at the depth of 1cm followed by treatment with different fluoride concentrations after approximately 10 days of said sowing; c) Ultrasonicating said sterilized seeds for about 30 minutes with the suspension of Iron Oxide nano-particles suspended in distilled water; d) Treating the soil of said plant seedlings with Iron nanoparticles present in the range of 170 to 220 mgkg-1 followed by irrigating said plant seedlings with water; e) Treating said plant seedlings with Ethylene Diamine Tetra acetic Acid (EDTA) present in the range of 15 to 25 mgkg-1 followed by irrigating said plant seedlings with water The method enhances the phytoremediation efficiency of Fluoride contaminated soil and productivity of biomass. The method not just removes Fluoride contaminants from the soil but also improves soil property for further use in agricultural purpose and recycling the biomass for various applications. In an embodiment, the plant comprising but not limited to Prosopis juliflora. In another embodiment, the Iron nanoparticle comprises but not limited to Fe3O4. In another embodiment, the chelating agent comprises but not limited to Ethylene Diamine Tetra Acetic Acid. In another embodiment, the Iron nanoparticle is present in the concentration of 200 mgkg-1 In another embodiment, the Ethylene Diamine Tetra acetic Acid is present in the concentration of 20 mgkg-1 In another embodiment, the fluoride concentrations comprises 25, 50, 75, 100 mgkg-1 soil. The method is technically advantageous in terms of removing fluoride content from the soil. P. juliflora plant under normal condition removes F upto 34.13%, however, the method of the present Invention improves this procedure and is able to remove F from soil by 82.97%. Application of Fe3O4 nanoparticle improves the removal efficiency by P.juliflora plant by 68.04% and further enhanced to 82.97% by addition of EDTA chelating agent. The treatment with nanoparticles and EDTA increased the root length of the plant and thus improved the deep root system that helps in phytoremediation. The method can be utilized for soil F removal for agricultural purposes, water defluoridation for drinking purposes. The advantages of the present invention are following:

1. The P.juliflora plant removes F upto 34.13%, but our invention improves this procedure and is able to remove F from soil by 82.97%. Application of Fe3O4 NP improves the removal efficiency by P.juliflora plant by 68.04%, which is further enhanced to 82.97% by addition of EDTA chelating agent.

2. Productivity of plant biomass is enhanced.

3. The benefit of biological systems instead of chemical process, with no residual output that are harmful to the environment.

4. Minimum time consuming process and energy consumption.

5. Cost benefit wetland constructed as compared to conventional technology.

6. Extremely low amount required for maintenance.

7. Constructed wetland system is not emitted pollution so it is free from any contaminates.

8. Soil and water quality are improved as previous time comparison in all aspects (mineral content, low amount of F and other factor).

9. The method based on the treatment of NAF, Fe3O4 nanoparticle and EDTA is highly simple, efficient and cost effective.

10. The method also increases productivity of plant biomass

11. The method also improves soil quality in terms of mineral content, low amount of Fluoride and other factor The Invention is further described with the help of non-limiting examples: The below mentioned experiments shows that the method demonstrates dramatic improvement in P. juliflora in terms of its ability to accumulate F. It was reported that P. juliflora plant removes F upto 34.13% (Saini et al., 2012), but present invention improved this procedure and removed F from soil by 82.97%. Application of Fe3O4 NP improved the removal efficiency by P.juliflora plant by 68.04%, further enhanced to 82.97% by addition of EDTA chelating agent. The present invention not just removes Fluoride contaminants from the soil but also improves the soil property for further use in agricultural purpose and recycling the biomass for various applications. EXAMPLE 1 Soil characteristics of F contaminated wetlands analysis Soil samples were collected from constructed wetland and stored in cold temperature at 4 °C. The pH of soil was determined as described by Allen (1989). Available N in soil was estimated by alkaline permanganate method (Subbiah and Bajaj, 1962). Available P was extracted by using Olsen’s reagent and estimated through spectrophotometer after developing the blue color by ascorbic method (Olsen, 1982). Available K was extracted with neutral ammonium acetate and estimated by flame photometer (Schollenberger and Simon, 1945). The DTPA extractable Fe, Mn, Zn and Cu metals in soil were estimated by using atomic absorption spectrophotometer (AAS) (Lindsay, 1978). The results are tabulated below: TABLE. 1 Physiochemical characteristics of constructed wetlands before invention Mineral content (0-5cm) S.No pH E.C N P K Fe Mn Zn Cu F T0 7.52±0.02 1.38±0.05 112±0.02 10.20±0.02 123±0.03 1.00±0.02 0.35±0.03 1.34±0.01 1.22±0.55 41.23±0.05 T1 7.51±0.03 1.39±0.01 116±0.04 10.62±0.03 108±0.02 1.11±0.02 1.73±0.05 1.73±1.22 1.09±0.34 42.19±0.08 T2 7.27±0.05 1.43±0.04 114±0.00 11.51±0.02 125±0.03 1.10±0.01 2.12±0.01 1.71±0.43 3.12±0.44 45.89±0.07 T3 7.90±0.06 1.87±0.02 115±0.00 10.61±0.02 114±0.04 1.73±0.02 1.76±0.00 1.72±1.00 2.18±0.23 35.78±0.05 T4 7.42±0.02 1.43±0.01 115±0.01 11.59±0.01 133±0.05 1.73±0.02 2.33±0.01 3.73±0.00 3.14±1.22 42.97±0.07 T5 7.63±0.02 1.58±0.02 114±0.02 10.22±0.02 128±0.02 1.86±0.01 1.77±0.02 1.77±0.23 2.67±0.12 41.59±0.04 T6 7.20±0.00 1.76±0.03 94±0.02 09.31±0.02 141±0.01 1.85±0.02 1.94±0.03 1.67±0.34 3.06±1.55 44.86±0.06 T7 8.00±0.03 1.26±0.03 113±0.04 10.61±0.03 139±0.02 2.27±0.01 2.23±0.01 3.33±0.54 2.75±0.55 38.66±0.02 T8 7.12±0.02 1.74±0.03 93±0.04 10.51±0.01 156±0.04 2.43±0.02 2.54±0.02 1.39±0.66 3.95±0.06 38.67±0.07 Note: Values are mean of three replicates and ± SD (Electrical conductivity-E.C dSm-1), (Sodium-N, Phosphorus-P, Potassium-K kgha-1), (Iron-Fe, Manganese-Mn, Zinc-Zn, Copper-Cu mgkg-1), T0-Control, T1-25 mgkg-1 F, T2-25 mgkg-1 F+ 200 mgkg-1Fe3O4, T3-50 mgkg-1 F, T4-50 mgkg-1 F+200 mgkg-1 Fe3O4, T5-75 mgkg-1 F, T6-75 mgkg-1 F+200 mgkg-1 Fe3O4, T7-100 mgkg-1 F, T8-100 mgkg-1 F+200 mgkg-1 Fe3O4. The present invention showed the alkaline nature of the utilized soil. All the micro and macro nutrients were reported in lower quantity due the alkalinity of the present soil. The average amount of F content in soil was 41.31 mgkg-1. EXAMPLE 2 Wetland construction and soil treatment Soil wetland was constructed with 2m2-length*width and 0.15 m depth. Prosopis juliflora plant seeds were collected from Central Arid Zone Research Institute (CAZRI), Jodhpur (Rajasthan) India. Seeds were surface sterilized with 10% H2SO4 for 10 min and rinsed with millipore water. The soil on the test area was excavated, the soil was automatically homogenized and filled in twenty seven plots (length*breath=2m*1m=2m2), which are bordered at the sides to prevent lateral flow between the plots. After washing, the seeds were immediately placed at 1 cm depth in soil (six seeds at one line in a bad) (FIG. 1). Treatment After 10 days the seedlings were treated with different F concentrations, 25, 50, 75, 100 mgkg-1 soil. The iron oxide nanoparticles (Fe3O4) were suspended in distilled water and dispersed with the aid of ultrasonication (PCI Analytics, Probe ultrasonicator) for 30 min for treatment. After 3 days, 200 mgkg-1 NPs treatment was given to soil of different F concentrations. NPs were not provided to the control plant. After the NPs application, plants are irrigated with water only. Roots and shoots of treated and control plants were harvested 120 days after the NPs treatment. F content of roots and shoots of plant was determined by McQuaker and Gurney (1977) protocol using fluoride ion-selective electrode (Czarnowski et al., 1996). The translocation (TF) and bioaccumulation factor (BF) were measured as described by Zhao and McGrath (2003). Total F content in plant and remaining soil samples was calculated by alkali fusion-ion technique (Niu et al. 2007). [BF = {F concentration in shoot}/ {F concentration in soil}] [TF = {F concentration in shoot}/ {F concentration in root}] The remaining F in soil was calculated by subtracting F accumulated in plant root and shoot from total F treatment. FIG. 2 demonstrates the layout of nano-phytoremediation technology. Microscopic observations Root and shoot samples were prepared for scanning electron microscopy (SEM). Collected samples were fixed in 2.5% glutaraldehyde in 0.05 M potassium phosphate buffer (pH 7.1) for 8 h, and dehydrated in an ethanol series (Johansen, 1940). Plant samples were first observed under light microscope (OLYMPUS, CH20i) for preliminary observation. The samples coating was done with platinum for 60 s by a Sputter Coater and observed by field emission scanning electron microscopy (FESEM MIRA3 TESCAN) coupled to an energy-dispersive X-ray (EDX) spectroscope. The elemental compositions of root, shoot and leaf were analyzed by EDX. The highest F accumulated in P. juliflora plant was 48.00 and 48.15 mgkg-1 soil in root and shoot at 141.31 mgkg-1 NaF (100+ 41.31)respectively (FIG. 3). The remaining F in soil is shown in FIG. 3 (C). Therefore, the accumulation of F in both root and shoot of P. juliflora is detected higher than control plant (only F treatment). The calculated T.F and B.F. were 1.00 and 1.06 for NPs treated P. juliflora plant at different F concentrations (FIG. 4). The obtained T.F and B.F proved the accumulation of F in P. juliflora plant. The light microscope studies of root, shoot and leaf samples showed the presence of NPs in xylem of plant. The presence of NPs in leaf sample confirms the uptake and accumulation of NPs with F (FIG. 5). For the confirmation of translocation and bioaccumulation of NPs with F, FESEM analysis with EDX spectrum was done on root and shoot samples of P. juliflora plant. The FESEM images showed the presence of NPs in xylem of root and shoot samples. The presence of F with NPs was detected by EDX spectrum. Peaks of F and iron oxide NPs were reported in all root, shoot and leaf samples of P. juliflora plant (FIG. 12.6). This confirms the translocation of F along with NPs in P. juliflora plant. The experiment indicated that application of Fe3O4 NPs improves the F removal efficiency by P.juliflora plant by 68.04 %. To our knowledge, the above experiments are the first conclusive demonstration of the ability of any plant to accumulate F with the help of iron oxide NPs. FIG.7 demonstrates the translocation system of F with NPs in P. juliflora plant. EXAMPLE 3 Treatment of NPs with Ethylene Diamine Tetra Acetic Acid (EDTA) The wetland was constructed and treated with different concentration of F and NPs (see Example 2). After 3 days, P. juliflora plant was treated with 20 mmol kg-1 EDTA. After the EDTA application, plants are irrigated with water only. Roots and shoots of treated and control plants were harvested 120 days after the EDTA treatment. F content of roots and shoots of plant was determined (see Example 2). T.F and B.F was calculated and remaining F in soil was determined (see Example 2). The F accumulated in P. juliflora plant was 58.15 and 59.10 mgkg-1 soil in root and shoot, 141.31 mgkg-1 NaF (100+ 41.31) respectively (FIG. 8 (A) and (B)). The remaining F in soil was demonstrated in FIG.8 (C). The T.F and B.F were increased upto 1.01 and 2.45 with the treatment of EDTA along with NPs (FIG.9). Application of Fe3O4 NPs improves the F removal efficiency by P.juliflora plant by 68.04% (see Example 2), further enhanced to 82.97% by addition of EDTA chelating agent. The EDTA plays an important role in enhancing the solubility of metal ions in soil, therefore increases the uptake of F along with NPs. EXAMPLE 4 Soil characteristics analysis after Nano-phytoremediation All the soil characteristics were analysed (see Example 1). The results are tabulated below: TABLE. 2 Physiochemical characteristics of soil after invention S.No pH E.C N P K Fe Mn Zn Cu T0 7.41±0.00 2.44±0.05 145±5.00 14.70±2.55 136±0.05 2.75±0.05 2.69±0.50 4.77±0.01 3.08±0.00 T1 6.28±0.00 2.43±0.00 112±4.00 12.70±3.55 128±0.06 2.56±0.02 2.23±0.00 2.03±0.03 1.08±0.03 T2 6.27±0.00 2.46±0.00 94±2.00 15.50±4.00 141±1.55 2.45±0.03 2.74±0.00 3.55±0.01 3.16±0.05 T3 6.28±0.00 2.54±0.00 113±5.00 12.60±3.00 139±1.00 3.05±0.05 2.43±0.50 2.03±0.50 1.15±0.04 T4 6.78±0.00 2.54±0.00 193±4.55 16.20±3.00 146±3.44 2.53±0.04 2.78±0.60 2.55±0.55 3.55±0.02 T5 6.41±0.00 2.44±0.50 195±3.55 16.70±6.00 146±4.55 3.75±0.06 2.69±0.70 2.77±2.55 1.38±0.00 T6 6.25±0.00 4.54±0.60 193±5.22 17.60±2.00 170±6.00 4.27±0.05 3.63±0.50 4.33±0.50 3.75±0.03 T7 6.43±0.00 4.74±0.06 143±4.22 16.20±4.50 146±2.55 3.83±0.06 2.74±0.40 3.05±2.55 1.45±0.04 T8 6.03±0.00 5.64±0.00 195±5.00 17.70±1.55 173±2.00 4.12±0.40 4.35±2.00 4.66±1.00 4.36±0.50 T9 6.12±0.00 7.48±0.00 110±2.00 12.90±4.00 151±1.55 3.14±0.03 3.51±0.00 3.12±0.01 2.96±0.05 T10 6.19±0.00 8.61±0.00 173±4.55 14.20±3.00 133±3.44 4.51±0.04 3.13±0.60 2.68±0.55 3.27±0.02 T11 6.25±0.00 7.58±0.60 188±5.22 17.60±2.00 164±6.00 3.62±0.05 2.15±0.50 4.92±0.50 4.39±0.03 T12 6.21±0.00 5.34±0.00 191±5.00 17.80±1.55 169±2.00 4.92±0.40 4.42±2.00 5.16±1.00 5.29±0.50 Note: Values are mean of three replicates and ± SD (Electrical conductivity-E.C dSm-1), (Sodium-N, Phosphorus-P, Potassium-K kgha-1), (Iron-Fe, Manganese-Mn, Zinc-Zn, Copper-Cu mgkg-1, T0-Control, T1-25 mgkg-1 F, T2-25 mgkg-1 F+ 200 mgkg-1 Fe3O4, T3-50 mgkg-1 F, T4-50 mgkg-1 F+200 mgkg-1 Fe3O4, T5-75 mgkg-1 F, T6-75 mgkg-1 F+200 mgkg-1 Fe3O4, T7-100 mgkg-1 F, T8-100 mgkg-1 F+200 mgkg-1 Fe3O4, T9-25 mgkg-1F+200 mgkg-1 Fe3O4+20 mmol kg-1 EDTA, T10-50 mgkg-1F+200 mgkg-1Fe3O4+20 mmol kg-1 EDTA, T11-75 mgkg-1F+200 mgkg-1 Fe3O4+20 mmol kg-1 EDTA, T12-100 mgkg-1F+200 mgkg-1 Fe3O4+20 mmol kg-1 EDTA. The present invention demonstrated the improvement in soil quality after nano-phytoremediation. The soil pH has changed from alkaline to acidic (7.30-6.37), which is required for nutrient availability and metal solubility in soil. The EC, N, P, K, Fe, Mn, Zn and Cu were increased from 1.53-4.52, 98-157.30, 9.57-15.61, 129.66-149.38, 1.67-3.50, 1.86-3.03, 2.04-3.50 and 2.57-2.99. These micro and macro nutrients were enhanced which are beneficial for the agricultural purposes. The initial average F concentration obtained was 41.31 mgkg-1 in wetlands and after nano-phytoremediation the F concentration obtained was 24.06 mgkg-1 F in the soil.

The present invention is in the field of phyto-remediation. The Invention in particular provides a method of enhancing the hyperaccumulation tendency of a plant, along with enhancing its biomass and improving the soil property.
Background of the Invention;
The following background discussion includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
Fluoride contamination is a worldwide problem. Many parts of the world are severely affected by this contamination and still there is no cure of fluorosis. F pollution in water, soil and vegetation is the main cause of environmental toxicity. In water, F react with some of the elements, and then turns into salts, finally deposit on soil as sediments (Molla et al, 2007). F at high concentration in soils can cause various forms of toxicity which has negative effects on plants, animals and human health through the food chain (Stevens et al, 1997). F is known to cause dental and skeletal fluorosis when its concentration is higher than 1.5 mg L"1 in drinking water (Teotia and Teotia, 1994). The available methods for F removal are:
1. Reverse Osmosis (RO)
2. Forward Osmosis (FO)
3. Evaporation
4. Ion-exchange
5. Adsorption
6. Precipitation
The conventional remediation technologies are expensive and some of the techniques do not remove F effectively. Therefore, it is important to develop economically viable and more effective method to decontaminate soils from F contamination. In recent years, use of plants in the reclamation of F contamination has gained considerable importance. Phyto remediation can be defined as the use of plants for the extraction, immobilization, containment or degradation of contaminants (Macek et al, 2000). The most significant advantage of phytoremediation is its low cost and effectiveness (Abdul and Schroder, 2009). It can be up to 1000-fold cheaper compared to conventional methods. It has been estimated that

phytoextraction to clean up one acre of sandy loam soil to a depth of 55 cm will cost 60,000-100,000$ compared to 400,000$ for conventional using traditional soil removal methods (Salt et al, 1995). The cost and effectiveness of the filtration techniques are still not satisfactory and further improvements are required. The limitations of existing defluoridation technologies are:
a) Precipitation: This method involves the precipitation of sparingly soluble F salt as
insoluble fluorapatite by the addition of chemicals (coagulants and coagulant aids). Most
common materials used in the technique are Aluminium salts (e.g. Alum), lime and Poly
Aluminium Hydroxy sulphate. The best example of precipitation technique is Nalgonda
Technique developed by National Environmental Engineering Research Institute (NEERI),
Nagpur in 1961. It involves addition of Aluminium salts, lime and bleaching powder
followed by rapid mixing, flocculation, sedimentation, filtration and disinfection.
Limitations: Required dosages of chemical are high, requirement of trained labor, sludge produced after treatment is high, high concentration of aluminium is released in water during process which may cause Alzheimer's syndrome.
b) Adsorption: This technique is mainly based on the adsorption of F ions on the surface of
an active agent. The commonly used adsorbent materials for this process are activated
alumina, rice husk, bone charcoal, activated carbon and serpentine.
Limitations: Toxic waste produced, highly pH dependant, adsorbents develop fouling smell, effectiveness decreases with time.
c) Membrane process: In this process, on the basis of molecular shape and size, particles are isolated using extraordinarily composed semi-permeable membrane. The common membrane processes for F removal are reverse osmosis (RO), nano-filtration, electrodialysis. Limitations: Expensive, large amount of water is rejected as brine, high electricity consumption and removes all ions including essential minerals, water membrane cost is high, fouling of membrane takes place, membrane has to be changed periodically, F ion is not removed due to its small size.
d) Ion-exchange: Synthetic chemicals, namely, cation and anion exchange resins have been used for F removal from water. A strongly fundamental anion-exchange resin can be used for F removal containing quaternary ammonium functional groups.

Limitations: Contamination prone, expensive, water wastage is high, large volume regenerate required for cation and anion exchange resins regeneration. The cost and effectiveness of the F removal techniques are still not satisfactory.
The chemical process suffers with the drawback of being expensive, producing harmful byproducts, requires high amount of energy, reject large amount of water during the process, does not remove Fluoride (F) at permissible limit, technically complex and is also not recyclable.
Hence, there is a need of an efficient, greener and cost effective process for the removal of fluoride contamination from the soil.
Object(s) of the Invention:
A primary object of the present invention is to overcome the drawback/s associated with the
prior art.
Yet another object of the present invention is to provide an easy and efficient method of
remediation of Fluoride contamination from soil and water as well.
Yet another object of the present invention is to provide easy and efficient method of
remediation of Fluoride contamination from soil and water using hyper-accumulator plant.
Yet another object of the present invention is to provide a method of enhancing the
hyperaccumulation efficiency of Fluoride hyperaccumulator plant Prosopis juliflora.
Yet another object of the present invention is to provide a method for balancing the pH level
of the soil by changing it from alkaline to acidic (7.30-6.37), which is required for nutrient
availability and metal solubility in soil.
Yet another object of the present invention is provide a method for improving the micro and
macro nutrients such as EC, N, P, K, Mn, Zn and Cu etc. showing an overall increase in soil
quality suitable for agriculture purpose and recycling.
Yet another object of the present invention is to provide cost-effective and renewable method
to reduce fluoride contamination from soil and water as well.
Yet another object of the present invention is to provide a method for improving the
surrounding environment without hampering the ecology in natural way.
Yet another object of the present invention is to provide a method for improving the soil
property and enhancing and recycling of the biomass

Summary of the Invention;
In an aspect of the Invention, there is provided a method of enhancing fluoride hyper accumulation efficiency of a plant for improving the soil quality, said method comprising the steps of treating the seedlings of said plant with different Fluoride concentrations followed by Iron nanoparticles and chelating agent wherein said Iron nanoparticle is present in the range of 170 to 220 mgkg"1 while said chelating agent is present in the range of 15 to 25 mgkg"1'
The method comprises steps:
Surface sterilization of said plant seedlings by 10% H2SO4 for about 10
minutes followed by rinsing with pure water;
Sowing of said sterilized seeds under soil approximately at the depth of
1 cm followed by treatment with different fluoride concentrations after
approximately 10 days of said sowing;
Ultrasonicating said sterilized seeds for about 30 minutes with the
suspension of Iron Oxide nano-particles suspended in distilled water;
Treating the soil of said plant seedlings with Iron nanoparticles present
in the range of 170 to 220 mgkg"1 followed by irrigating said plant
seedlings with water;
Treating said plant seedlings with Ethylene Diamine Tetra acetic Acid
(EDTA) present in the range of 15 to 25 mgkg"1 followed by irrigating
said plant seedlings with water
Brief Description of the Drawings
To further clarify advantages and features of the present invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof, which is illustrated in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope. The invention will be described and explained with additional specificity and detail with the accompanying drawings in which:
Figure 1 illustrates Layout of constructed wetlands for Nano-phytoremediation technology using Prosopis juliflora (Pi, P2 and P3= Prosopis juliflora plants grown) Figure 2 illustrates schematic diagram of nano-phytoremediation technology

Figure 3 illustrates F accumulation efficiency of P. juliflora after NPs treatment (A) root and (B) shoot
Figure 4 illustrates Effect of NPs on F treated P. juliflora plant (A) translocation and (B)
bioaccumulation factor.
Figure 5 illustrates Light microscope analysis for (A) root, (B) shoot and (C) leaf of P.
juliflora plant
Figure 6 illustrates (A) and (B) show the FESEM image and EDS spectrum of root sample.
While (C) and (D) show the FESEM image and EDS spectrum of shoot sample and (E) EDX
spectrum of leaf of P. juliflora plant
Figure 7 illustrates translocation of F with NPs in P. juliflora plant
Figure 8 illustrates F accumulation efficiency of P. juliflora after NPs treatment with EDTA
(A) root and (B) shoot
Figure 9 illustrates Effect of NPs with EDTA on F treated P. juliflora plant (A) translocation
and (B) bioaccumulation factor
Detailed Description of the Invention
For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.
It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof. Throughout the patent specification, a convention employed is that in the appended drawings, like numerals denote like components.
The present Invention provides a method of enhancing fluoride hyper accumulation efficiency of a plant for improving the soil quality.
In an embodiment, the method comprises the steps of treating the seedlings of said plant with different Fluoride concentrations followed by Iron nanoparticles and chelating agent wherein
-6-

said Iron nanoparticle is present in the range of 170 to 220 mgkg-1 while said chelating agent is present in the range of 15 to 25 mgkg-1.
In an embodiment, the method comprises steps of:
a) Surface sterilization of said plant seedlings by 10% H2SO4 for about 10 minutes followed by rinsing with pure water;
b) Sowing of said sterilized seeds under soil approximately at the depth of 1cm followed by treatment with different fluoride concentrations after approximately 10 days of said sowing;
c) Ultrasonicating said sterilized seeds for about 30 minutes with the suspension of Iron Oxide nano-particles suspended in distilled water;
d) Treating the soil of said plant seedlings with Iron nanoparticles present in the range of 170 to 220 mgkg-1 followed by irrigating said plant seedlings with water;
e) Treating said plant seedlings with Ethylene Diamine Tetra acetic Acid (EDTA) present in the range of 15 to 25 mgkg-1 followed by irrigating said plant seedlings with water
The method enhances the phytoremediation efficiency of Fluoride contaminated soil and
productivity of biomass. The method not just removes Fluoride contaminants from the soil
but also improves soil property for further use in agricultural purpose and recycling the
biomass for various applications.
In an embodiment, the plant comprising but not limited to Prosopis juliflora.
In another embodiment, the Iron nanoparticle comprises but not limited to Fe3O4.
In another embodiment, the chelating agent comprises but not limited to Ethylene Diamine
Tetra Acetic Acid.
In another embodiment, the Iron nanoparticle is present in the concentration of 200 mgkg-1
In another embodiment, the Ethylene Diamine Tetra acetic Acid is present in the
concentration of 20 mgkg-1
In another embodiment, the fluoride concentrations comprises 25, 50, 75, 100 mgkg-1 soil.
The method is technically advantageous in terms of removing fluoride content from the soil. P. juliflora plant under normal condition removes F upto 34.13%, however, the method of the present Invention improves this procedure and is able to remove F from soil by 82.97%.
-7-

Application of Fe3O4 nanoparticle improves the removal efficiency by P.juliflora plant by 68.04% and further enhanced to 82.97% by addition of EDTA chelating agent. The treatment with nanoparticles and EDTA increased the root length of the plant and thus improved the deep root system that helps in phytoremediation. The method can be utilized for soil F removal for agricultural purposes, water defluoridation for drinking purposes.
The advantages of the present invention are following:
1. The P.juliflora plant removes F upto 34.13%, but our invention improves this procedure and is able to remove F from soil by 82.97%. Application of Fe3O4 NP improves the removal efficiency by P.juliflora plant by 68.04%, which is further enhanced to 82.97% by addition of EDTA chelating agent.
2. Productivity of plant biomass is enhanced.
3. The benefit of biological systems instead of chemical process, with no residual output that are harmful to the environment.
4. Minimum time consuming process and energy consumption.
5. Cost benefit wetland constructed as compared to conventional technology.
6. Extremely low amount required for maintenance.
7. Constructed wetland system is not emitted pollution so it is free from any contaminates.
8. Soil and water quality are improved as previous time comparison in all aspects (mineral content, low amount of F and other factor).
9. The method based on the treatment of NAF, Fe3O4 nanoparticle and EDTA is highly simple, efficient and cost effective.
10. The method also increases productivity of plant biomass
11. The method also improves soil quality in terms of mineral content, low amount of Fluoride and other factor
The Invention is further described with the help of non-limiting examples: The below mentioned experiments shows that the method demonstrates dramatic improvement in P. juliflora in terms of its ability to accumulate F. It was reported that P. juliflora plant removes F upto 34.13% (Saini et al., 2012), but present invention improved this procedure and removed F from soil by 82.97%. Application of Fe3O4 NP improved the removal efficiency by P.juliflora plant by 68.04%, further enhanced to 82.97% by addition of EDTA chelating agent. The present invention not just removes Fluoride contaminants from
-8-

the soil but also improves the soil property for further use in agricultural purpose and recycling the biomass for various applications.
EXAMPLE 1
Soil characteristics of F contaminated wetlands analysis
Soil samples were collected from constructed wetland and stored in cold temperature at 4 °C. The pH of soil was determined as described by Allen (1989). Available N in soil was estimated by alkaline permanganate method (Subbiah and Bajaj, 1962). Available P was extracted by using Olsen’s reagent and estimated through spectrophotometer after developing the blue color by ascorbic method (Olsen, 1982). Available K was extracted with neutral ammonium acetate and estimated by flame photometer (Schollenberger and Simon, 1945). The DTPA extractable Fe, Mn, Zn and Cu metals in soil were estimated by using atomic absorption spectrophotometer (AAS) (Lindsay, 1978). The results are tabulated below:
TABLE. 1 Physiochemical characteristics of constructed wetlands before invention
Mineral content (0-5 cm)

S.
No
T0
T1
T2
T3
T4
T5
T6

pH
7.52±0
.02
7.51±0
.03
7.27±0
.05
7.90±0
.06
7.42±0
.02
7.63±0
.02
7.20±0 E.C N
112±0.
02
116±0.
04
114±0.
00
115±0.
00
115±0.
01
114±0.
02
94±0.0 P K
123±0.
03
108±0.
02
125±0.
03
114±0.
04
133±0.
05
128±0.
02
141±0. Fe
1.00±0 .02 1.11±0
.02
1.10±0
.01
1.73±0
.02
1.73±0
.02
1.86±0
.01
1.85±0 Mn
0.35±0
.03
1.73±0
.05
2.12±0
.01
1.76±0
.00
2.33±0
.01
1.77±0
.02
1.94±0 Zn Cu F

1.38±0. 05
10.20± 0.02


1.34±0. 01 1.22±0.
55 41.23±0.0
5

1.39±0. 01
10.62± 0.03


1.73±1.
22 1.09±0. 34 42.19±0.0 8

1.43±0. 04
11.51±
0.02


1.71±0. 43 3.12±0. 44 45.89±0.0 7

1.87±0. 02
10.61± 0.02


1.72±1. 00 2.18±0.
23 35.78±0.0
5

1.43±0. 01
11.59± 0.01


3.73±0. 00 3.14±1.
22 42.97±0.0 7

1.58±0. 02
10.22± 0.02


1.77±0.
23 2.67±0. 12 41.59±0.0 4

1.76±0.
09.31±


1.67±0. 3.06±1. 44.86±0.0

-9-

T7 T8 .00
8.00±0
.03
7.12±0
.02 03 2
113±0.
04
93±0.0
4 0.02 01 .02 .03
139±0. 2.27±0 2.23±0
02 .01 .01
156±0. 2.43±0 2.54±0
04 .02 .02 34 55

1.26±0. 03
10.61± 0.03
3.33±0. 54 2.75±0. 55

1.74±0. 03
10.51± 0.01
1.39±0. 66 3.95±0. 06

6
38.66±0.0
2
38.67±0.0
7

Note: Values are mean of three replicates and ± SD (Electrical conductivity-E.C dSm-1), (Sodium-N, Phosphorus-P, Potassium-K kgha-1), (Iron-Fe, Manganese-Mn, Zinc-Zn, Copper-Cu mgkg-1), T0-Control, T1-25 mgkg-1 F, T2-25 mgkg-1 F+ 200 mgkg-1Fe3O4, T3-50 mgkg-1 F, T4-50 mgkg-1 F+200 mgkg-1 Fe3O4, T5-75 mgkg-1 F, T6-75 mgkg-1 F+200 mgkg-1 Fe3O4, T7-100 mgkg-1 F, T8-100 mgkg-1 F+200 mgkg-1 Fe3O4.
The present invention showed the alkaline nature of the utilized soil. All the micro and macro nutrients were reported in lower quantity due the alkalinity of the present soil. The average amount of F content in soil was 41.31 mgkg-1.
EXAMPLE 2
Wetland construction and soil treatment
Soil wetland was constructed with 2m2-length*width and 0.15 m depth. Prosopis juliflora plant
seeds were collected from Central Arid Zone Research Institute (CAZRI), Jodhpur (Rajasthan) India. Seeds were
surface sterilized with 10% H2SO4 for 10 min and rinsed with millipore water. The soil on the test area was
excavated, the soil was automatically homogenized and filled in twenty seven plots
(length*breath=2m*1m=2m2), which are bordered at the sides to prevent lateral flow between
the plots. After washing, the seeds were immediately placed at 1 cm depth in soil (six seeds at one
line in a bad) (FIG. 1). Treatment
After 10 days the seedlings were treated with different F concentrations, 25, 50, 75, 100 mgkg-1 soil. The iron oxide nanoparticles (Fe3O4) were suspended in distilled water and dispersed with the aid of ultrasonication (PCI Analytics, Probe ultrasonicator) for 30 min for treatment. After 3 days, 200 mgkg-1 NPs treatment was given to soil of different F concentrations. NPs were not provided to the control plant. After the NPs application, plants are irrigated with water only. Roots and shoots of treated and control plants were harvested 120 days after the NPs treatment. F content of roots and shoots of plant was determined by
-10-

McQuaker and Gurney (1977) protocol using fluoride ion-selective electrode (Czarnowski et
al., 1996).
The translocation (TF) and bioaccumulation factor (BF) were measured as described by Zhao
and McGrath (2003). Total F content in plant and remaining soil samples was calculated by
alkali fusion-ion technique (Niu et al. 2007).
[BF = {F concentration in shoot}/ {F concentration in soil}]
[TF = {F concentration in shoot}/ {F concentration in root}]
The remaining F in soil was calculated by subtracting F accumulated in plant root and shoot
from total F treatment. FIG. 2 demonstrates the layout of nano-phytoremediation technology.
Microscopic observations
Root and shoot samples were prepared for scanning electron microscopy (SEM). Collected samples were fixed in 2.5% glutaraldehyde in 0.05 M potassium phosphate buffer (pH 7.1) for 8 h, and dehydrated in an ethanol series (Johansen, 1940). Plant samples were first observed under light microscope (OLYMPUS, CH20i) for preliminary observation. The samples coating was done with platinum for 60 s by a Sputter Coater and observed by field emission scanning electron microscopy (FESEM MIRA3 TESCAN) coupled to an energy-dispersive X-ray (EDX) spectroscope. The elemental compositions of root, shoot and leaf were analyzed by EDX.
The highest F accumulated in P. juliflora plant was 48.00 and 48.15 mgkg-1 soil in root and shoot at 141.31 mgkg-1 NaF (100+ 41.31)respectively (FIG. 3). The remaining F in soil is shown in FIG. 3 (C). Therefore, the accumulation of F in both root and shoot of P. juliflora is detected higher than control plant (only F treatment). The calculated T.F and B.F. were 1.00 and 1.06 for NPs treated P. juliflora plant at different F concentrations (FIG. 4). The obtained T.F and B.F proved the accumulation of F in P. juliflora plant.
The light microscope studies of root, shoot and leaf samples showed the presence of NPs in xylem of plant. The presence of NPs in leaf sample confirms the uptake and accumulation of NPs with F (FIG. 5). For the confirmation of translocation and bioaccumulation of NPs with F, FESEM analysis with EDX spectrum was done on root and shoot samples of P. juliflora plant. The FESEM images showed the presence of NPs in xylem of root and shoot samples. The presence of F with NPs was detected by EDX spectrum. Peaks of F and iron oxide NPs
-11-

were reported in all root, shoot and leaf samples of P. juliflora plant (FIG. 12.6). This confirms the translocation of F along with NPs in P. juliflora plant.
The experiment indicated that application of Fe3O4 NPs improves the F removal efficiency by P.juliflora plant by 68.04 %. To our knowledge, the above experiments are the first conclusive demonstration of the ability of any plant to accumulate F with the help of iron oxide NPs. FIG.7 demonstrates the translocation system of F with NPs in P. juliflora plant.
EXAMPLE 3
Treatment of NPs with Ethylene Diamine Tetra Acetic Acid (EDTA)
The wetland was constructed and treated with different concentration of F and NPs (see Example 2). After 3 days, P. juliflora plant was treated with 20 mmol kg-1 EDTA. After the EDTA application, plants are irrigated with water only. Roots and shoots of treated and control plants were harvested 120 days after the EDTA treatment. F content of roots and shoots of plant was determined (see Example 2). T.F and B.F was calculated and remaining F in soil was determined (see Example 2).
The F accumulated in P. juliflora plant was 58.15 and 59.10 mgkg-1 soil in root and shoot, 141.31 mgkg-1 NaF (100+ 41.31) respectively (FIG. 8 (A) and (B)). The remaining F in soil was demonstrated in FIG.8 (C). The T.F and B.F were increased upto 1.01 and 2.45 with the treatment of EDTA along with NPs (FIG.9).
Application of Fe3O4 NPs improves the F removal efficiency by P.juliflora plant by 68.04% (see Example 2), further enhanced to 82.97% by addition of EDTA chelating agent. The EDTA plays an important role in enhancing the solubility of metal ions in soil, therefore increases the uptake of F along with NPs.
EXAMPLE 4
Soil characteristics analysis after Nano-phytoremediation
All the soil characteristics were analysed (see Example 1). The results are tabulated below:
TABLE. 2 Physiochemical characteristics of soil after invention
-12-

S.No
T0
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12 pH E.C N P K Fe Mn Zn Cu

7.41±0.00 2.44±0.05 145±5.00 14.70±2.55 136±0.05 2.75±0.05 2.69±0.50 4.77±0.01 3.08±0.00

6.28±0.00 2.43±0.00 112±4.00 12.70±3.55 128±0.06 2.56±0.02 2.23±0.00 2.03±0.03 1.08±0.03

6.27±0.00 2.46±0.00 94±2.00 15.50±4.00 141±1.55 2.45±0.03 2.74±0.00 3.55±0.01 3.16±0.05

6.28±0.00 2.54±0.00 113±5.00 12.60±3.00 139±1.00 3.05±0.05 2.43±0.50 2.03±0.50 1.15±0.04

6.78±0.00 2.54±0.00 193±4.55 16.20±3.00 146±3.44 2.53±0.04 2.78±0.60 2.55±0.55 3.55±0.02

6.41±0.00 2.44±0.50 195±3.55 16.70±6.00 146±4.55 3.75±0.06 2.69±0.70 2.77±2.55 1.38±0.00

6.25±0.00 4.54±0.60 193±5.22 17.60±2.00 170±6.00 4.27±0.05 3.63±0.50 4.33±0.50 3.75±0.03

6.43±0.00 4.74±0.06 143±4.22 16.20±4.50 146±2.55 3.83±0.06 2.74±0.40 3.05±2.55 1.45±0.04

6.03±0.00 5.64±0.00 195±5.00 17.70±1.55 173±2.00 4.12±0.40 4.35±2.00 4.66±1.00 4.36±0.50

6.12±0.00 7.48±0.00 110±2.00 12.90±4.00 151±1.55 3.14±0.03 3.51±0.00 3.12±0.01 2.96±0.05

6.19±0.00 8.61±0.00 173±4.55 14.20±3.00 133±3.44 4.51±0.04 3.13±0.60 2.68±0.55 3.27±0.02

6.25±0.00 7.58±0.60 188±5.22 17.60±2.00 164±6.00 3.62±0.05 2.15±0.50 4.92±0.50 4.39±0.03

6.21±0.00 5.34±0.00 191±5.00 17.80±1.55 169±2.00 4.92±0.40 4.42±2.00 5.16±1.00 5.29±0.50

Note: Values are mean of thr ee replicates and ± SD (Electrical conductivity-E.C dSm ),
(Sodium-N, Phosphorus-P, Potassium-K kgha ), (Iron-Fe, Manganese-Mn, Zinc-Zn, Copper-Cu mgkg , T0-Control, T1-25 mgkg F, T2-25 mgkg F+ 200 mgkg Fe3O4, T3-50 mgkg F, T4-50 mgkg-1 F+200 mgkg- Fe3O4, T5-75 mgkg-1 F, T6-75 mgkg-1 F+200 mgkg-1 Fe3O4, T7-100 mgkg-1 F, T8-100 mgkg-1 F+200 mgkg-1 Fe3O4, T9-25 mgkg-1 F+200 mgkg-Fe3O4+20 mmol kg EDTA, T10-50 mgkg F+200 mgkg Fe3O4+20 mmol kg EDTA, T11-75 mgkg F+200 mgkg Fe3O4+20 mmol kg EDTA, T12-100 mgkg F+200 mgkg
-1
F e3O4+20 mmol kg EDT A.
The present invention demonstrated the improvement in soil quality after nano-phytoremediation. The soil pH has changed from alkaline to acidic (7.30-6.37), which is required for nutrient availability and metal solubility in soil. The EC, N, P, K, Fe, Mn, Zn and Cu were increased from 1.53-4.52, 98-157.30, 9.57-15.61, 129.66-149.38, 1.67-3.50, 1.86-3.03, 2.04-3.50 and 2.57-2.99. These micro and macro nutrients were enhanced which are beneficial for the agricultural purposes.
The initial average F concentration obtained was 41.31 mgkg-1 in wetlands and after nano-phytoremediation the F concentration obtained was 24.06 mgkg- F in the soil.

Claims:We Claim

1. A method of enhancing fluoride hyper accumulation efficiency of a plant for improving the soil quality, said method comprising the steps of treating the seedlings of said plant with different Fluoride concentrations followed by Iron nanoparticles and chelating agent wherein said Iron nanoparticle is present in the range of 170 to 220 mgkg-1 while said chelating agent is present in the range of 15 to 25 mgkg-1.

2. The method as claimed in claim 1, wherein said method comprises steps:
a) Surface sterilization of said plant seedlings by 10% H2SO4 for about 10 minutes followed by rinsing with pure water;
b) Sowing of said sterilized seeds under soil approximately at the depth of 1cm followed by treatment with different fluoride concentrations after approximately 10 days of said sowing;
c) Ultrasonicating said sterilized seeds for about 30 minutes with the suspension of Iron Oxide nano-particles suspended in distilled water;
d) Treating the soil of said plant seedlings with Iron nanoparticles present in the range of 170 to 220 mgkg-1 followed by irrigating said plant seedlings with water;
e) Treating said plant seedlings with Ethylene Diamine Tetra acetic Acid (EDTA) present in the range of 15 to 25 mgkg-1 followed by irrigating said plant seedlings with water
3. The method as claimed in claim 1, wherein said plant comprises Prosopis juliflora.
4. The method as claimed in claim 1, wherein said Iron nanoparticle comprises Fe3O4
5. The method as claimed in claim 1, wherein said chelating agent comprises Ethylene Diamine Tetra Acetic Acid.
6. The method as claimed in claim 1, wherein said Iron nanoparticle is present in the concentration of 200 mgkg-1
7. The method as claimed in claim 1, wherein said Ethylene Diamine Tetra acetic Acid is present in the concentration of 20 mgkg-1
8. The method as claimed in claim 1, wherein said Fluoride concentrations comprises 25, 50, 75, 100 mgkg-1 soil.
, Description:Field of the Invention:
The present invention is in the field of phyto-remediation. The Invention in particular provides a method of enhancing the hyperaccumulation tendency of a plant, along with enhancing its biomass and improving the soil property.
Background of the Invention:
The following background discussion includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
Fluoride contamination is a worldwide problem. Many parts of the world are severely affected by this contamination and still there is no cure of fluorosis. F pollution in water, soil and vegetation is the main cause of environmental toxicity. In water, F react with some of the elements, and then turns into salts, finally deposit on soil as sediments (Molla et al., 2007). F at high concentration in soils can cause various forms of toxicity which has negative effects on plants, animals and human health through the food chain (Stevens et al., 1997). F is known to cause dental and skeletal fluorosis when its concentration is higher than 1.5 mg L-1 in drinking water (Teotia and Teotia, 1994).
The available methods for F removal are:
1. Reverse Osmosis (RO)
2. Forward Osmosis (FO)
3. Evaporation
4. Ion-exchange
5. Adsorption
6. Precipitation
The conventional remediation technologies are expensive and some of the techniques do not remove F effectively. Therefore, it is important to develop economically viable and more effective method to decontaminate soils from F contamination. In recent years, use of plants in the reclamation of F contamination has gained considerable importance. Phytoremediation can be defined as the use of plants for the extraction, immobilization, containment or degradation of contaminants (Macek et al., 2000). The most significant advantage of phytoremediation is its low cost and effectiveness (Abdul and Schroder, 2009). It can be up to 1000-fold cheaper compared to conventional methods. It has been estimated that phytoextraction to clean up one acre of sandy loam soil to a depth of 55 cm will cost 60,000-100,000$ compared to 400,000$ for conventional using traditional soil removal methods (Salt et al., 1995). The cost and effectiveness of the filtration techniques are still not satisfactory and further improvements are required.
The limitations of existing defluoridation technologies are:
a) Precipitation: This method involves the precipitation of sparingly soluble F salt as insoluble fluorapatite by the addition of chemicals (coagulants and coagulant aids). Most common materials used in the technique are Aluminium salts (e.g. Alum), lime and Poly Aluminium Hydroxy sulphate. The best example of precipitation technique is Nalgonda Technique developed by National Environmental Engineering Research Institute (NEERI), Nagpur in 1961. It involves addition of Aluminium salts, lime and bleaching powder followed by rapid mixing, flocculation, sedimentation, filtration and disinfection.
Limitations: Required dosages of chemical are high, requirement of trained labor, sludge produced after treatment is high, high concentration of aluminium is released in water during process which may cause Alzheimer’s syndrome.

b) Adsorption: This technique is mainly based on the adsorption of F ions on the surface of an active agent. The commonly used adsorbent materials for this process are activated alumina, rice husk, bone charcoal, activated carbon and serpentine.
Limitations: Toxic waste produced, highly pH dependant, adsorbents develop fouling smell, effectiveness decreases with time.

c) Membrane process: In this process, on the basis of molecular shape and size, particles are isolated using extraordinarily composed semi-permeable membrane. The common membrane processes for F removal are reverse osmosis (RO), nano-filtration, electrodialysis.
Limitations: Expensive, large amount of water is rejected as brine, high electricity consumption and removes all ions including essential minerals, water membrane cost is high, fouling of membrane takes place, membrane has to be changed periodically, F ion is not removed due to its small size.

d) Ion-exchange: Synthetic chemicals, namely, cation and anion exchange resins have been used for F removal from water. A strongly fundamental anion-exchange resin can be used for F removal containing quaternary ammonium functional groups.
Limitations: Contamination prone, expensive, water wastage is high, large volume regenerate required for cation and anion exchange resins regeneration. The cost and effectiveness of the F removal techniques are still not satisfactory.

The chemical process suffers with the drawback of being expensive, producing harmful byproducts, requires high amount of energy, reject large amount of water during the process, does not remove Fluoride (F) at permissible limit, technically complex and is also not recyclable.

Hence, there is a need of an efficient, greener and cost effective process for the removal of fluoride contamination from the soil.

Object(s) of the Invention:
A primary object of the present invention is to overcome the drawback/s associated with the prior art.
Yet another object of the present invention is to provide an easy and efficient method of remediation of Fluoride contamination from soil and water as well.
Yet another object of the present invention is to provide easy and efficient method of remediation of Fluoride contamination from soil and water using hyper-accumulator plant.
Yet another object of the present invention is to provide a method of enhancing the hyperaccumulation efficiency of Fluoride hyperaccumulator plant Prosopis juliflora.
Yet another object of the present invention is to provide a method for balancing the pH level of the soil by changing it from alkaline to acidic (7.30-6.37), which is required for nutrient availability and metal solubility in soil.
Yet another object of the present invention is provide a method for improving the micro and macro nutrients such as EC, N, P, K, Mn, Zn and Cu etc. showing an overall increase in soil quality suitable for agriculture purpose and recycling.
Yet another object of the present invention is to provide cost-effective and renewable method to reduce fluoride contamination from soil and water as well.
Yet another object of the present invention is to provide a method for improving the surrounding environment without hampering the ecology in natural way.
Yet another object of the present invention is to provide a method for improving the soil property and enhancing and recycling of the biomass
Summary of the Invention:
In an aspect of the Invention, there is provided a method of enhancing fluoride hyper accumulation efficiency of a plant for improving the soil quality, said method comprising the steps of treating the seedlings of said plant with different Fluoride concentrations followed by Iron nanoparticles and chelating agent wherein said Iron nanoparticle is present in the range of 170 to 220 mgkg-1 while said chelating agent is present in the range of 15 to 25 mgkg-1.

The method comprises steps:
a) Surface sterilization of said plant seedlings by 10% H2SO4 for about 10 minutes followed by rinsing with pure water;
b) Sowing of said sterilized seeds under soil approximately at the depth of 1cm followed by treatment with different fluoride concentrations after approximately 10 days of said sowing;
c) Ultrasonicating said sterilized seeds for about 30 minutes with the suspension of Iron Oxide nano-particles suspended in distilled water;
d) Treating the soil of said plant seedlings with Iron nanoparticles present in the range of 170 to 220 mgkg-1 followed by irrigating said plant seedlings with water;
e) Treating said plant seedlings with Ethylene Diamine Tetra acetic Acid (EDTA) present in the range of 15 to 25 mgkg-1 followed by irrigating said plant seedlings with water
Brief Description of the Drawings
To further clarify advantages and features of the present invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof, which is illustrated in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope. The invention will be described and explained with additional specificity and detail with the accompanying drawings in which:
Figure 1 illustrates Layout of constructed wetlands for Nano-phytoremediation technology using Prosopis juliflora (P1, P2 and P3= Prosopis juliflora plants grown)
Figure 2 illustrates schematic diagram of nano-phytoremediation technology
Figure 3 illustrates F accumulation efficiency of P. juliflora after NPs treatment (A) root and (B) shoot
Figure 4 illustrates Effect of NPs on F treated P. juliflora plant (A) translocation and (B) bioaccumulation factor.
Figure 5 illustrates Light microscope analysis for (A) root, (B) shoot and (C) leaf of P. juliflora plant
Figure 6 illustrates (A) and (B) show the FESEM image and EDS spectrum of root sample. While (C) and (D) show the FESEM image and EDS spectrum of shoot sample and (E) EDX spectrum of leaf of P. juliflora plant
Figure 7 illustrates translocation of F with NPs in P. juliflora plant
Figure 8 illustrates F accumulation efficiency of P. juliflora after NPs treatment with EDTA (A) root and (B) shoot
Figure 9 illustrates Effect of NPs with EDTA on F treated P. juliflora plant (A) translocation and (B) bioaccumulation factor

Detailed Description of the Invention
For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.
It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof. Throughout the patent specification, a convention employed is that in the appended drawings, like numerals denote like components.
The present Invention provides a method of enhancing fluoride hyper accumulation efficiency of a plant for improving the soil quality.

In an embodiment, the method comprises the steps of treating the seedlings of said plant with different Fluoride concentrations followed by Iron nanoparticles and chelating agent wherein said Iron nanoparticle is present in the range of 170 to 220 mgkg-1 while said chelating agent is present in the range of 15 to 25 mgkg-1.

In an embodiment, the method comprises steps of:
a) Surface sterilization of said plant seedlings by 10% H2SO4 for about 10 minutes followed by rinsing with pure water;
b) Sowing of said sterilized seeds under soil approximately at the depth of 1cm followed by treatment with different fluoride concentrations after approximately 10 days of said sowing;
c) Ultrasonicating said sterilized seeds for about 30 minutes with the suspension of Iron Oxide nano-particles suspended in distilled water;
d) Treating the soil of said plant seedlings with Iron nanoparticles present in the range of 170 to 220 mgkg-1 followed by irrigating said plant seedlings with water;
e) Treating said plant seedlings with Ethylene Diamine Tetra acetic Acid (EDTA) present in the range of 15 to 25 mgkg-1 followed by irrigating said plant seedlings with water
The method enhances the phytoremediation efficiency of Fluoride contaminated soil and productivity of biomass. The method not just removes Fluoride contaminants from the soil but also improves soil property for further use in agricultural purpose and recycling the biomass for various applications.
In an embodiment, the plant comprising but not limited to Prosopis juliflora.
In another embodiment, the Iron nanoparticle comprises but not limited to Fe3O4.
In another embodiment, the chelating agent comprises but not limited to Ethylene Diamine Tetra Acetic Acid.
In another embodiment, the Iron nanoparticle is present in the concentration of 200 mgkg-1
In another embodiment, the Ethylene Diamine Tetra acetic Acid is present in the concentration of 20 mgkg-1
In another embodiment, the fluoride concentrations comprises 25, 50, 75, 100 mgkg-1 soil.

The method is technically advantageous in terms of removing fluoride content from the soil. P. juliflora plant under normal condition removes F upto 34.13%, however, the method of the present Invention improves this procedure and is able to remove F from soil by 82.97%. Application of Fe3O4 nanoparticle improves the removal efficiency by P.juliflora plant by 68.04% and further enhanced to 82.97% by addition of EDTA chelating agent. The treatment with nanoparticles and EDTA increased the root length of the plant and thus improved the deep root system that helps in phytoremediation. The method can be utilized for soil F removal for agricultural purposes, water defluoridation for drinking purposes.

The advantages of the present invention are following:
1. The P.juliflora plant removes F upto 34.13%, but our invention improves this procedure and is able to remove F from soil by 82.97%. Application of Fe3O4 NP improves the removal efficiency by P.juliflora plant by 68.04%, which is further enhanced to 82.97% by addition of EDTA chelating agent.
2. Productivity of plant biomass is enhanced.
3. The benefit of biological systems instead of chemical process, with no residual output that are harmful to the environment.
4. Minimum time consuming process and energy consumption.
5. Cost benefit wetland constructed as compared to conventional technology.
6. Extremely low amount required for maintenance.
7. Constructed wetland system is not emitted pollution so it is free from any contaminates.
8. Soil and water quality are improved as previous time comparison in all aspects (mineral content, low amount of F and other factor).
9. The method based on the treatment of NAF, Fe3O4 nanoparticle and EDTA is highly simple, efficient and cost effective.
10. The method also increases productivity of plant biomass
11. The method also improves soil quality in terms of mineral content, low amount of Fluoride and other factor

The Invention is further described with the help of non-limiting examples:
The below mentioned experiments shows that the method demonstrates dramatic improvement in P. juliflora in terms of its ability to accumulate F. It was reported that P. juliflora plant removes F upto 34.13% (Saini et al., 2012), but present invention improved this procedure and removed F from soil by 82.97%. Application of Fe3O4 NP improved the removal efficiency by P.juliflora plant by 68.04%, further enhanced to 82.97% by addition of EDTA chelating agent. The present invention not just removes Fluoride contaminants from the soil but also improves the soil property for further use in agricultural purpose and recycling the biomass for various applications.

EXAMPLE 1
Soil characteristics of F contaminated wetlands analysis
Soil samples were collected from constructed wetland and stored in cold temperature at 4 °C. The pH of soil was determined as described by Allen (1989). Available N in soil was estimated by alkaline permanganate method (Subbiah and Bajaj, 1962). Available P was extracted by using Olsen’s reagent and estimated through spectrophotometer after developing the blue color by ascorbic method (Olsen, 1982). Available K was extracted with neutral ammonium acetate and estimated by flame photometer (Schollenberger and Simon, 1945). The DTPA extractable Fe, Mn, Zn and Cu metals in soil were estimated by using atomic absorption spectrophotometer (AAS) (Lindsay, 1978).
The results are tabulated below:

TABLE. 1 Physiochemical characteristics of constructed wetlands before invention

Mineral content (0-5cm)

S.No pH E.C N P K Fe Mn Zn Cu F
T0 7.52±0.02 1.38±0.05 112±0.02 10.20±0.02 123±0.03 1.00±0.02 0.35±0.03 1.34±0.01 1.22±0.55 41.23±0.05
T1 7.51±0.03 1.39±0.01 116±0.04 10.62±0.03 108±0.02 1.11±0.02 1.73±0.05 1.73±1.22 1.09±0.34 42.19±0.08
T2 7.27±0.05 1.43±0.04 114±0.00 11.51±0.02 125±0.03 1.10±0.01 2.12±0.01 1.71±0.43 3.12±0.44 45.89±0.07
T3 7.90±0.06 1.87±0.02 115±0.00 10.61±0.02 114±0.04 1.73±0.02 1.76±0.00 1.72±1.00 2.18±0.23 35.78±0.05
T4 7.42±0.02 1.43±0.01 115±0.01 11.59±0.01 133±0.05 1.73±0.02 2.33±0.01 3.73±0.00 3.14±1.22 42.97±0.07
T5 7.63±0.02 1.58±0.02 114±0.02 10.22±0.02 128±0.02 1.86±0.01 1.77±0.02 1.77±0.23 2.67±0.12 41.59±0.04
T6 7.20±0.00 1.76±0.03 94±0.02 09.31±0.02 141±0.01 1.85±0.02 1.94±0.03 1.67±0.34 3.06±1.55 44.86±0.06
T7 8.00±0.03 1.26±0.03 113±0.04 10.61±0.03 139±0.02 2.27±0.01 2.23±0.01 3.33±0.54 2.75±0.55 38.66±0.02
T8 7.12±0.02 1.74±0.03 93±0.04 10.51±0.01 156±0.04 2.43±0.02 2.54±0.02 1.39±0.66 3.95±0.06 38.67±0.07

Note: Values are mean of three replicates and ± SD (Electrical conductivity-E.C dSm-1), (Sodium-N, Phosphorus-P, Potassium-K kgha-1), (Iron-Fe, Manganese-Mn, Zinc-Zn, Copper-Cu mgkg-1), T0-Control, T1-25 mgkg-1 F, T2-25 mgkg-1 F+ 200 mgkg-1Fe3O4, T3-50 mgkg-1 F, T4-50 mgkg-1 F+200 mgkg-1 Fe3O4, T5-75 mgkg-1 F, T6-75 mgkg-1 F+200 mgkg-1 Fe3O4, T7-100 mgkg-1 F, T8-100 mgkg-1 F+200 mgkg-1 Fe3O4.

The present invention showed the alkaline nature of the utilized soil. All the micro and macro nutrients were reported in lower quantity due the alkalinity of the present soil. The average amount of F content in soil was 41.31 mgkg-1.

EXAMPLE 2
Wetland construction and soil treatment
Soil wetland was constructed with 2m2-length*width and 0.15 m depth. Prosopis juliflora plant seeds were collected from Central Arid Zone Research Institute (CAZRI), Jodhpur (Rajasthan) India. Seeds were surface sterilized with 10% H2SO4 for 10 min and rinsed with millipore water. The soil on the test area was excavated, the soil was automatically homogenized and filled in twenty seven plots (length*breath=2m*1m=2m2), which are bordered at the sides to prevent lateral flow between the plots. After washing, the seeds were immediately placed at 1 cm depth in soil (six seeds at one line in a bad) (FIG. 1).
Treatment
After 10 days the seedlings were treated with different F concentrations, 25, 50, 75, 100 mgkg-1 soil. The iron oxide nanoparticles (Fe3O4) were suspended in distilled water and dispersed with the aid of ultrasonication (PCI Analytics, Probe ultrasonicator) for 30 min for treatment. After 3 days, 200 mgkg-1 NPs treatment was given to soil of different F concentrations. NPs were not provided to the control plant. After the NPs application, plants are irrigated with water only. Roots and shoots of treated and control plants were harvested 120 days after the NPs treatment. F content of roots and shoots of plant was determined by McQuaker and Gurney (1977) protocol using fluoride ion-selective electrode (Czarnowski et al., 1996).
The translocation (TF) and bioaccumulation factor (BF) were measured as described by Zhao and McGrath (2003). Total F content in plant and remaining soil samples was calculated by alkali fusion-ion technique (Niu et al. 2007).
[BF = {F concentration in shoot}/ {F concentration in soil}]
[TF = {F concentration in shoot}/ {F concentration in root}]
The remaining F in soil was calculated by subtracting F accumulated in plant root and shoot from total F treatment. FIG. 2 demonstrates the layout of nano-phytoremediation technology.

Microscopic observations
Root and shoot samples were prepared for scanning electron microscopy (SEM). Collected samples were fixed in 2.5% glutaraldehyde in 0.05 M potassium phosphate buffer (pH 7.1) for 8 h, and dehydrated in an ethanol series (Johansen, 1940). Plant samples were first observed under light microscope (OLYMPUS, CH20i) for preliminary observation. The samples coating was done with platinum for 60 s by a Sputter Coater and observed by field emission scanning electron microscopy (FESEM MIRA3 TESCAN) coupled to an energy-dispersive X-ray (EDX) spectroscope. The elemental compositions of root, shoot and leaf were analyzed by EDX.

The highest F accumulated in P. juliflora plant was 48.00 and 48.15 mgkg-1 soil in root and shoot at 141.31 mgkg-1 NaF (100+ 41.31)respectively (FIG. 3). The remaining F in soil is shown in FIG. 3 (C). Therefore, the accumulation of F in both root and shoot of P. juliflora is detected higher than control plant (only F treatment). The calculated T.F and B.F. were 1.00 and 1.06 for NPs treated P. juliflora plant at different F concentrations (FIG. 4). The obtained T.F and B.F proved the accumulation of F in P. juliflora plant.

The light microscope studies of root, shoot and leaf samples showed the presence of NPs in xylem of plant. The presence of NPs in leaf sample confirms the uptake and accumulation of NPs with F (FIG. 5). For the confirmation of translocation and bioaccumulation of NPs with F, FESEM analysis with EDX spectrum was done on root and shoot samples of P. juliflora plant. The FESEM images showed the presence of NPs in xylem of root and shoot samples. The presence of F with NPs was detected by EDX spectrum. Peaks of F and iron oxide NPs were reported in all root, shoot and leaf samples of P. juliflora plant (FIG. 12.6). This confirms the translocation of F along with NPs in P. juliflora plant.

The experiment indicated that application of Fe3O4 NPs improves the F removal efficiency by P.juliflora plant by 68.04 %. To our knowledge, the above experiments are the first conclusive demonstration of the ability of any plant to accumulate F with the help of iron oxide NPs. FIG.7 demonstrates the translocation system of F with NPs in P. juliflora plant.

EXAMPLE 3

Treatment of NPs with Ethylene Diamine Tetra Acetic Acid (EDTA)
The wetland was constructed and treated with different concentration of F and NPs (see Example 2). After 3 days, P. juliflora plant was treated with 20 mmol kg-1 EDTA. After the EDTA application, plants are irrigated with water only. Roots and shoots of treated and control plants were harvested 120 days after the EDTA treatment. F content of roots and shoots of plant was determined (see Example 2). T.F and B.F was calculated and remaining F in soil was determined (see Example 2).
The F accumulated in P. juliflora plant was 58.15 and 59.10 mgkg-1 soil in root and shoot, 141.31 mgkg-1 NaF (100+ 41.31) respectively (FIG. 8 (A) and (B)). The remaining F in soil was demonstrated in FIG.8 (C). The T.F and B.F were increased upto 1.01 and 2.45 with the treatment of EDTA along with NPs (FIG.9).

Application of Fe3O4 NPs improves the F removal efficiency by P.juliflora plant by 68.04% (see Example 2), further enhanced to 82.97% by addition of EDTA chelating agent. The EDTA plays an important role in enhancing the solubility of metal ions in soil, therefore increases the uptake of F along with NPs.

EXAMPLE 4

Soil characteristics analysis after Nano-phytoremediation
All the soil characteristics were analysed (see Example 1). The results are tabulated below:

TABLE. 2 Physiochemical characteristics of soil after invention
S.No pH E.C N P K Fe Mn Zn Cu

T0 7.41±0.00 2.44±0.05 145±5.00 14.70±2.55 136±0.05 2.75±0.05 2.69±0.50 4.77±0.01 3.08±0.00
T1 6.28±0.00 2.43±0.00 112±4.00 12.70±3.55 128±0.06 2.56±0.02 2.23±0.00 2.03±0.03 1.08±0.03
T2 6.27±0.00 2.46±0.00 94±2.00 15.50±4.00 141±1.55 2.45±0.03 2.74±0.00 3.55±0.01 3.16±0.05
T3 6.28±0.00 2.54±0.00 113±5.00 12.60±3.00 139±1.00 3.05±0.05 2.43±0.50 2.03±0.50 1.15±0.04
T4 6.78±0.00 2.54±0.00 193±4.55 16.20±3.00 146±3.44 2.53±0.04 2.78±0.60 2.55±0.55 3.55±0.02
T5 6.41±0.00 2.44±0.50 195±3.55 16.70±6.00 146±4.55 3.75±0.06 2.69±0.70 2.77±2.55 1.38±0.00
T6 6.25±0.00 4.54±0.60 193±5.22 17.60±2.00 170±6.00 4.27±0.05 3.63±0.50 4.33±0.50 3.75±0.03
T7 6.43±0.00 4.74±0.06 143±4.22 16.20±4.50 146±2.55 3.83±0.06 2.74±0.40 3.05±2.55 1.45±0.04
T8 6.03±0.00 5.64±0.00 195±5.00 17.70±1.55 173±2.00 4.12±0.40 4.35±2.00 4.66±1.00 4.36±0.50
T9 6.12±0.00 7.48±0.00 110±2.00 12.90±4.00 151±1.55 3.14±0.03 3.51±0.00 3.12±0.01 2.96±0.05
T10 6.19±0.00 8.61±0.00 173±4.55 14.20±3.00 133±3.44 4.51±0.04 3.13±0.60 2.68±0.55 3.27±0.02
T11 6.25±0.00 7.58±0.60 188±5.22 17.60±2.00 164±6.00 3.62±0.05 2.15±0.50 4.92±0.50 4.39±0.03
T12 6.21±0.00 5.34±0.00 191±5.00 17.80±1.55 169±2.00 4.92±0.40 4.42±2.00 5.16±1.00 5.29±0.50

Note: Values are mean of three replicates and ± SD (Electrical conductivity-E.C dSm-1), (Sodium-N, Phosphorus-P, Potassium-K kgha-1), (Iron-Fe, Manganese-Mn, Zinc-Zn, Copper-Cu mgkg-1, T0-Control, T1-25 mgkg-1 F, T2-25 mgkg-1 F+ 200 mgkg-1 Fe3O4, T3-50 mgkg-1 F, T4-50 mgkg-1 F+200 mgkg-1 Fe3O4, T5-75 mgkg-1 F, T6-75 mgkg-1 F+200 mgkg-1 Fe3O4, T7-100 mgkg-1 F, T8-100 mgkg-1 F+200 mgkg-1 Fe3O4, T9-25 mgkg-1F+200 mgkg-1 Fe3O4+20 mmol kg-1 EDTA, T10-50 mgkg-1F+200 mgkg-1Fe3O4+20 mmol kg-1 EDTA, T11-75 mgkg-1F+200 mgkg-1 Fe3O4+20 mmol kg-1 EDTA, T12-100 mgkg-1F+200 mgkg-1 Fe3O4+20 mmol kg-1 EDTA.

The present invention demonstrated the improvement in soil quality after nano-phytoremediation. The soil pH has changed from alkaline to acidic (7.30-6.37), which is required for nutrient availability and metal solubility in soil. The EC, N, P, K, Fe, Mn, Zn and Cu were increased from 1.53-4.52, 98-157.30, 9.57-15.61, 129.66-149.38, 1.67-3.50, 1.86-3.03, 2.04-3.50 and 2.57-2.99. These micro and macro nutrients were enhanced which are beneficial for the agricultural purposes.
The initial average F concentration obtained was 41.31 mgkg-1 in wetlands and after nano-phytoremediation the F concentration obtained was 24.06 mgkg-1 F in the soil.