INTRODUCTION:

Phytoremediation is a newly evolving field of science and technology that uses plants to clean-up polluted soil, water, or air ^1-3. Phytoremediation is a technology for real clean-up of contaminated soils and the contaminants which are potentially toxic. It reduces the risks presented by a contaminated soil by decreasing contaminants’ bioavailability using plants as a source. The two greatest advantages of phytoremediation compared with traditional abatement methods are cost effectiveness and less ecosystem disruption. Plants may also help to stabilize contaminants by accumulating and precipitating toxic trace elements in the roots. Organic pollutants can potentially be chemically degraded and ultimately mineralized into harmless biological compounds. 2,4,6-trinitrotoluene (TNT) are widely used in military munitions.^4,5. They and their breakdown products are major human-produced contaminants in the environment; manufacturing, deployment, and improper disposal contribute to contamination. where they constitute a source of toxic, mutagenic, and carcinogenic effects on humans and other biota. In humans, high and prolonged exposures to TNT cause hyperplasia of the bone marrow leading to aplastic anemia and a drastic loss of blood platelets⁶.

The introduction of harmful substances into the environment has been shown to have many adverse effects on human health, agricultural productivity and natural ecosystems⁷. Because the costs of growing a crop are minimal compared to those of soil removal and replacement, the use of plants to remediate hazardous soils is seen as having great promise.^8-14

MATERIALS AND METHODS:

Collection of polluted soil sample:

Soil samples were obtained from polluted soil area vellore district, Tamilnadu. The 0-10 cm surface layer was collected and used for the investigation. Soil sample was placed in clean polyethylene bags and brought to the laboratory where it was air-dried for a week until totally dry and ground to pass through a 2-mm mesh stainless steel sieve. The soil was then homogenized and stored at room temperature and processed for the analysis.

Total metal concentration in soil:

For determining the total metal concentration in the soil samples, EPA method 3050B was used¹⁵. This method is not a total digestion technique; instead it will give environmentally available metals. For the digestion of the samples a representative sample of 1 gram, dry weight, was mixed with 10 ml 1:1 nitric acid, heated on a hotplate located in a fume hood and refluxed for 15 minutes at a temperature of 95^oC ± 5^oC. This was followed by digestion of samples with repeated addition of 5 ml of concentrated nitric acid, which was added until no further reaction occurred with the nitric acid. Absence of brown fumes from the solution indicates the completion of nitric acid digestion. Then the sample was digested with 30% hydrogen peroxide. Hydrogen peroxide was repeatedly added (1 ml each) to the sample until the sample appearance was unchanged. Finally, the sample was digested with 10 ml concentrated hydrochloric acid for 15 minutes. The digested sample was then filtered through Whatman No. 40 filter 28 paper and collected in a 100 ml volumetric flask and made up to 100 ml with distilled water. Proper dilutions of filtered sample were prepared and analyzed by Atomic Absorption (AA) Spectrometry. Digestion of samples were done in triplicate and performed under a hood to ensure safety.

Authentication of the plant:

Hibiscus arnottianus was authenticated by National Institute of Herbal Science, Chennai.

Phytoremediation of TNT using Plants:

Seeds of Hibiscus arnottianus was obtained from vellore district, Tamilnadu, seed province. Inorder to quantitate the amount of phytoremediation the process was carried out in the soil spiked with TNT and without TNT served as control. The concentrations of TNT selected for this experiment were 25, 50, 75 to 100 mg/kg. Initially, the seeds was soaked in water in a container for 24 hr and germinated in commercial soil for 2 weeks. The seedlings were watered daily. After 4 weeks, healthy plants with similar height were selected. Plants were later transplanted and grown in the TNT spiked 100 days (4 months). Each set was subdivided into 4 soil treatments as follows: control soil, 25 mg/kg TNT soil, 50 mg/kg TNT+soil, 75 mg/kg TNT+soil, 100 mg/kg TNT+soil. The TNT residue concentrations in spiked soil were determined and the percentage removal efficiency was calculated. The determination of TNT residue concentration was carried out during a period of 100 days (D0, D10, D20, D30, D40, D50, D60, D70, D80, D90, and D100).

Analytical Method of TNT:

For analyzing TNT in the soil treated by phytoremediation process the supernatant was separated by transferring 1 ml of the mixed liquid to a 2 ml polypropylene microcentrifuge tube containing 1 ml of 35% methanol. After centrifugation at 13000 rpm (≈ 12470 g-force) for 5 min, the supernatant was filtered through a 0.45 μm PTFE filter prior to analysis of TNT concentrations using gas chromatography.

Gas Chromatography- Mass Spectrum Analysis:

The phytoremediation of samples from the experiment at day interval at the various concentrations were subjected to GC-MS. The analysis was performed using GC SHIMADZU QP2010 system equipped with Elite-1 fused silica capillary column (Diameter: 0.25 mm, Length: 30.0 m, Film thickness: 0.25 μm. Helium gas (99.999%) was used as the carrier gas at a constant column flow rate of 1.51ml/min and an injection volume of 2μl, Ion-source temperature at 200°C and the ionization energy of 70eV with Injector temperature at 200°C. The oven temperature was at 70°C with an increase of 10 min at 300°C. Total GC running time was 35 min. Mass spectra were taken at 70eV; a scan interval of 0.5 seconds with scan range of 40 – 1000 m/z. NIST08 and WILEY8 database was used for the identification of the separated peaks. The relative percentage of each component was calculated by comparing its average peak area to the total areas.

RESULTS AND DISCUSSION:

Plant metabolism of xenobiotics involves three phases: activation (transformation), conjugation, and compartmentation^16,
17. TNT belongs to the nitroaromatics group and consists of an aromatic ring with three nitro groups. TNT in plants is probably detoxified¹⁸. The soils chemical and physical properties prior to planting in the contaminated soil were shown were analysed. The soil parameter including textures were determined for both contaminated and phytoremediated soil compared to the nutrient ratings for soil fertility. (Table .1.) Due to the differences in initial concentrations of TNT in spiked soil (100 mg/kg and 500 mg/kg), the percentages of removal efficiency was performed to compare the efficiency among the different initial concentrations of TNT in both untreated and treated spiked soil by Hibiscus arnottianus. The resultshas demonstrated the overall removal efficiency (%) of all treatments in treated soil by Hibiscus arnottianus was higher than that of untreated soil. (Fig 1) Profiles of removal efficiency in each treatment of both untreated and treated soil were not relatively similar. The highest removal efficiency was found in the treatment of 25 mg/kg TNT at all time points, and followed by 50 mg/kg. Notably at 75mg/kg and 100 mg/kg TNT were found to have less percentages of removal efficiency. The overall results showed higher removal efficiency of 25 mg/kg initial TNT concentration in soil than that of other concentration used showed and found a dramatic increase of the removal efficiency during at three month intervals and a moderate increase during after the post period of third month. It was slow down after day fourth month. In treated soil with the same experiment groups of other concentration it was found a dramatically continuous decrease in in the removal efficiency. Growth and yield parameters of the Hibiscus arnottianus reduced with increased in TNT concentrations. At 100mg/kg and the ability to absorb TNT varied with different concentration (Fig 2). The Hydrolysed products of TNT degraded by phytoremediation process was noticed at 80^th day at the concentration of 25mg/kg concentration (Fig 3). This phytoremediation method for TNT removal in contaminated soil was found to be a very promising method in the future which can be applied for the contaminated sites. In terms of half life, it showed to be more effective than other methods such as biodegradation that was previously reported by some researchers. It was reported that the thermophilic and mesophilic half-lives were 11.9 and 21.9 days for TNT remediation by biodegradation method ^{19, 20}.

Table 1 : Soil analysis

S.No	Analysis	Control soil	Phytoremediation Soil
S.No	Analysis	without treatment	Phytoremediation Soil
1	p H	7.42	7.35
2	EC	0.33	0.3
3	Organic carbon(OC)	0.98	0.33
4	Nitrogen(N)	107	108.19
5	Phosphorous	14.69	11.31
6	Potassium	74.73	87.36
7	Calcium	544.53	616.01
8	Magnesium	163.3	185.64
9	Sodium	113.58	134
10	Iron	10.88	7.51
11	Manganese	7.11	9.34
12	Copper	1.57	2.38
13	Zinc	0.74	1.16
14	Boron	0.43	0
15	Molybedenum	0	0
16	Sulphate	12.54	0
17	Humus (HA)	67.42	78.06

Fig: 1 TNT removal efficeiency at different concentrations.

(a) (b)

(c)

(d) (e)

Fig:2 Observations of Trinitrotoluene removal efficiency in treated soil with Hibiscus arnottianus (a) control soil, (b)25 mg/kg TNT soil, (c) 50 mg/kg TNT+soil, (d) 75 mg/kg TNT+soil, (e) 100 mg/kg TNT+soil.

Fig:3 Hydrolysed products of TNT degraded by phytoremediation process at 80^th day at the concentration of 25mg/kg concentration

CONCLUSION:

According to the experimental results obtained from this study, it indicated that phytoremediation for degradation and removal of TNT spiked soil has obviously more effective than either chemical methods. Regarding the time points of the complete TNT degradation the highest removal efficiency of phytoremediation was found in soil with the TNT (25 mg/kg initial TNT concentration) in treated soil Hibiscus arnottianus. Hence high explosives such as 2,4,6-trinitrotoluene (TNT) which are known as the important contaminants in the environment could be removed by phytoremediation as a cost-effective abatement.

ACKNOWLEDGEMENTS:

We are greatly indebted to Vellore Institute of Technology for the constant encouragement and help and my sincere thanks to Thiruvalluvar University for extending necessary facilities.

REFERENCES:

1. Salt DE, Smith RD, Raskin I: Phytoremediation. Annual Reviews of Plant Physiology and Plant Molecular Biology. 49; 1998: 643-668.

2. Rugh CL, Bizily SP, Meagher RB: Phytoremediation of environmental mercury pollution. In Phytoremediation of Toxic Metals: Using Plants to Clean-up the Environment. Edited by Ensley B, Raskin I. New York: John Wiley and Sons; 1999:151-169.

3. Meagher RB, Rugh CL, Kandasamy MK, Gragson G, Wang NJ: Engineered phytoremediation of mercury pollution in soil and water using bacterial genes. In Phytoremediation of Contaminated Soil and Water. Edited by Terry W, Bañuelos G. Berkeley, California: Ann Arbor Press, Inc.; 2000:201-219.

4. Best EPH, Miller JL, Larson SL. Tolerance towards explosives, and explosives removal from groundwater in treatment wetland mesocosms. Water Science and Technology. 44; 2001: 515–521

5. Hannink NK, Rosser SJ, Bruce NC. Phytoremediation of explosives. Critical Reviews in Plant Scienc.21: 2002; 511–538

6. Rosenblatt DH. Toxicology of explosives and propellants. In: Kaye SM (ed) Encyclopedia of explosives and related items. US Army Armament Research Development Committee, Dover, NJ. 1980.

7. Garbarino, J.R., Hayes, H., Roth, D., Antweider, R., Brinton, T.I. and Taylor, H. (Contaminants in the Mississippi River, U. S. Geological Survey Circular 1133, Virginia, U.S.A. www.pubs.usgs.gov/circ/circ1133/)1995.

8. Raskin I, Kumar PBAN, Dushenkov S, Salt DE. Bioconcentration of heavy metals by plants. Current Opinion in Biotechnology. 5; 1994: 285–290.

9. Salt DE, Blaylock M, Kumar PBAN, Dushenkov S, Ensley BD, Chet I, Raskin I: Phytoremediation: a novel strategy for the removal of toxic metals from the environment using plants. Bio-Technology. 13; 1996:468–474.

10. Cunningham SD, Berti WR, Huang JW: Phytoremediation of contaminated soils. Trends in Biotechnology. 13; 1995:393–397.

11. Cunningham SD, Berti WR, Huang JWW. Phytoremediation of contaminated soils. Trends in Biotechnology. 13; 1995:393–397

12. Raskin I: Plant genetic engineering may help with environmental cleanup [commentary]. Proceedings of the National Academy of Sciences. 93; 1996:3164–3166.

13. Moffat A: Plants proving their worth in toxic metal cleanup. Science. 269; 1995: 302–303.

14. Chaney RL, Brown SL, Li YM, Angle JS, Homer FA, Green CE: Potential use of metal hyperaccumulators. Mining Environmental Management. 3; 1995:9–11.

15. U.S.EPA. SW-846 EPA Method 8095: Explosives by gas chromatography. Test methods for evaluating solid waste. Office of Solid Waste, Washington, DC, 2000.

16. Sandermann H Jr. Plant metabolism of xenobiotics. Trends in Biochemical Sciences.17; 1992: 82–84

17. Schaffner A, Messner B, Langebartels C, Sandermann H. Genes and enzymes for in-planta phytoremediation of air, water and soil. Acta Biotechnology. 22; 2002:141–151.

18. Hannink NK, Rosser SJ, Bruce NC. Phytoremediation of explosives. Critical Reviews in Plant Science. 21; 2002 :511–538

19. R.T. Williams, P.S. Zeigenfuss and W.E. Sisk. Composting of explosives and propellant contaminated soils under thermophilic and mesophilic conditions. Journal of Industrial Microbiology. 9; 1992:137-144.

20. R.L. Crawford. Biodegradation of nitrated munitions compounds and herbicides by obligately anaerobic bacteria; Biodegradation of nitroaromatic compounds. In J.C. Spain. Plenum Press, New York. 49; 1995: 87-98.

Received on 05.05.2014 Modified on 06.06.2014

Research J. Pharm. and Tech. 7(8): August 2014 Page 902-905