Influence of nickel and lead applied in combination with vermicompost on growth and accumulation of heavy metals by Mentha arvensis Linn. cv.

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1 Indian Journal of Natural Products and Resources Vol. 3 (2), June 2012, pp Influence of nickel and lead applied in combination with vermicompost on growth and accumulation of heavy metals by Mentha arvensis Linn. cv. Kosi Sukhmal Chand*, Ankit Pandey and D D Patra Division of Agronomy and Soil Science Central Institute of Medicinal and Aromatic Plants, Lucknow , Uttar Pradesh, India Received 5 July 2011; Accepted 11April 2012 A pot experiment was conducted under glass house conditions during and at the Central Institute of Medicinal and Aromatic Plant, Lucknow, India. The study was conducted to evaluate the influence of different combinations of nickel (Ni) and lead (Pb) applied with and without vermicompost, on growth and yield, as well as on concentration and accumulation of Ni, Cr and Pb by Mentha arvensis Linn. cv. Kosi. The results revealed that Ni and Pb applied at 25: 25 and 25:50 ratio with vermicompost produced 40 and 52 % higher fresh herbage and root yield, respectively over control. Equal proportions of Ni and Pb ratio (50:50 ppm) exhibited negative effect on the herb and fresh root yield especially without vermicompost. Concentration and uptake of heavy metals (Pb, Ni, Cr) and micronutrients (Zn, Cu, Mn) were higher when nickel and lead were applied in combination with vermicompost over sole supply of heavy metals (Ni, Pb). Accumulation of heavy metals (Pb, Ni, Cr) were higher (24 times) in root tissues than in shoot tissues of plants whereas micronutrients (Zn, Cu, Mn) accumulation follow the reverse trend. Keywords: Mentha arvensis, Vermicompost, Medicinal, Aromatic, Nickel, Lead. IPC code; Int. cl. ( ) A61K 36/00 *Correspondent author: E mail: sukhmal1234@gmail.com Introduction Phytoremediation refers to a diverse collection of plantbased technologies that use either naturally occurring or genetically engineered plants for cleaning contaminated environments 1,2. Heavy metal (Ni, Cd, Cr Pb, etc.) contamination in agricultural land is a consequence of various anthropogenic activities including urbanization, industrialization and use of sewage water and sludge for irrigation. Large amount of heavy metals such as Ni, Cd, Cr and Pb, etc. are entering into soil due to agricultural and industrial activities. Excessive concentration of these elements in soil induces stress and responses in plant adverse effect on crops, livestock and human being. The presence of excessive amounts of heavy metals in soil is phytotoxic and could result in adverse effect on crop such as reduction in root growth, disturbances in mineral nutrition and carbohydrate metabolism 3. Some plant species tolerate and thrive in heavy metal rich soils as they can sequester excessive amounts of heavy metals in their biomass without damaging basic metabolism 1. These plants are known as hyper accumulators. Such plants can be used for phytoremediation which is an ecofriendly, less expensive technique than excavation and chemical leaching of heavy metals. Since these toxic metals are distributed in all parts of the plants, employing edible species may not be a good proposition for phytoremediation. Mentha arvensis Linn. var. piperascens Malinv. ex Holmes (menthol mint) is an industrial crop widely cultivated for its valued essential oil from which menthol is isolated. Essential oil of mint, menthol and terpenes of the dementholated oil are extensively used in food, perfumery and pharmaceutical industries. Being an essential oil bearing crop, mint can be cultivated in heavy metal polluted soils. The essential oil of mint is extracted by hydrodistillation of herb and there is limited possibility of the oil being contaminated with metals. The present experiment was conducted to study the growth performance of menthol mint on heavy metal enriched soil and influence of vermicompost as the source of organic matter on heavy metal toxicity visàvis possibility of hyperaccumulation of heavy metals. Materials and Methods A pot experiment was conducted during and at the Central Institute of Medicinal and Aromatic Plant, Lucknow, India. Ten kg of air

2 CHAND et al: INFLUENCE OF NICKEL AND LEAD WITH VERMICOMPOST ON MENTHA ARVENSIS GROWTH 257 dried and sieved (<2 mm) soil was filled in 20 cm diam. earthen pots. Soil was properly enriched with required quantity of heavy metal solution (nickel sulphate and lead acetate) before filling in pot and left for two weeks for equilibration. Treatment consisted of two levels (25, 50 ppm) of each heavy metal (Ni and Pb) with and without vermicompost and a control (no heavy metal, no vermicompost) (Table 1). Initial status of soil and vermicompost analysis are presented in Table 2. Treatments were laid in a completely randomized design (CRD) with three replications. Three healthy and uniform size suckers of Mentha arvensis cv. Kosi were transplanted in each pot. Nitrogen, phosphorus and potassium were 150, 60 and 60 mg/kg of soil through urea, single super phosphate and muriate of potash, respectively. Fifty per cent of N (urea) along with full dose of P and K were applied as basal dose. Remaining N was applied 45 days after transplanting of the suckers. Harvesting of crop The crop was harvested after three months and different growth parameters such as herb, root and oil yield were recorded. A 100 g composite fresh herb sample under each treatment was collected to estimate the essential oil by hydro distillation method in Table 1 Details of treatments Treatment Control VC 7.5 g/kg soil Ni : Pb : : 25:25 ppm Ni : Pb : : 25:25 ppm + VC 7.5 g/kg soil Ni : Pb : : 25:50 ppm Ni : Pb : : 25:50 ppm + VC 7.5 g/kg soil Ni : Pb : : 50:25 ppm Ni : Pb : : 50:25 ppm + VC.5 g/kg soil Ni : Pb : : 50:50 ppm Ni : Pb: : 50:50 ppm + VC 7.5 g/kg soil VC: Vermicompost Table 2 Initial characteristic of soil and metal concentration of vermicompost used in experiment Characteristics Initial soil status Vermicompost status Soil texture Organic carbon (%) Ec (dsm 1 ) ph Cu (ppm) Ni (ppm) Cr (ppb) Pb (ppm) Mn(ppm) Zn (ppm) Sandy loam Clevenger s apparatus 4. Essential oil yield was computed by multiplying oil content with its corresponding herb yield. Essential oil analysis For chemical profiling, essential oil samples were analyzed by Perkin Elmer GC model Auto XL fitted with FID and capillary column PE (50m x 0.32 x µm film thickness ) using a temperature program from 100 o C to 280 o 3% ramp rate and H 2 carrier at 10 psi inlet pressure. The injector and detector temperatures maintained at 220 o C and 290 o C, respectively were applied in the analyses. Turbochrom software was used for peak percentage calculation. Identification of components was based on GCMS analysis on Perkin Elmer Turbo Mass system using identical column and condition with He as a carrier gas at 10 psi inlet pressure and comparison of mass spectra with NIST and Wiley libraries. Soil and plant analysis After harvesting the crop, soil samples were collected from each pot, shade dried and analysed for micronutrients and heavy metals analysis. The plant samples were washed with distilled water, oven dried (65 C), ground and digested in diacid (HNO 3 :HClO 4 :: 10:4) mixture. Iron, manganese, zinc, copper, cadmium, nickel, lead, and chromium in acid digested plant samples and in soil extract (with DTPA) were estimated by Inductively Coupled Plasma (model Optima 5300V OES) by standard procedures as described by Page et al 5 and Lindsay and Norvell 6. Statistical analysis Pooled data of two years ( and ) and was subjected to analysis of variance (ANOVA) and least significant differences (LSD) of the treatment mean values were calculated using the F method 7. Results and Discussion Shoot, root yield and plant height Data presented in Table 3 indicated that all treatments significantly affected the fresh shoot, root and its dry weight and plant height. Highest fresh shoot and root weight were recorded in and, respectively. The sole application of vermicompost ( ) produced higher shoot yield over integrated supply of heavy metal and vermicompost except treatment. Improved shoot yield under sole application of vermicompost may be attributed to instant availability of macro and micro nutrients for

3 258 INDIAN J NAT PROD RESOUR, JUNE 2012 longer period of plant growth and retention of heavy metals (Ni and Pb) in soil by increasing cation exchange capacity. Similar results were obtained by Pande et al 8 and Prasad et al 9. Combined application of heavy metals (Ni, Pb) and vermicompost produced higher shoot and root yield as compared to sole supply of heavy metals. In treatment,, and heavy metals in combination with vermicompost fresh shoot and root yield were increased by 24,16, 3,15 and 17,72,28,7%, respectively, over the respective heavy metals treatments without vermicompost. Minimum shoot and root yield were recorded in and, respectively. Minimum fresh shoot and root yield were recorded in and, respectively. Application of Ni and 25 and 50 ppm ( ) with vermicompost recorded higher fresh root yield as compared to other Pb containing treatments (, ). Table 3 Influence of heavy metals and vermicompost on fresh and dry matter yield of shoot, root and plant height of Mentha arvensis Linn. cv. Kosi Treatment LSD (p=0.05) Fresh shoot Dry shoot Fresh root Dry root Plant height (cm) Lead has inhibitory effect on root growth hence shoot and root yield reduced. This finding is in agreement with Godbold and Kettner 10. Reduction in root growth is mainly by restricting cell division and cell elongation 11, 12. Shoot and root dry matter were affected by treatment variation and observed the highest shoot and root dry matter were recorded when plant was supplied with sole vermicompost at 7.5 g/kg soil without heavy metal. Dry matter of plant recorded in and were at par. Dry matters of root also followed the same trend. Maximum plant height was reported in. The extent of increase in height was 21 % over to Treatment. It may be probably due to lower rate of Ni and Pb application with additional supply of vermicompost. Vermicompost restrict the intake of Pb and Ni as a high source of organic matter through production of phytochelation 8,13. Thus there is increased chances of fair growth of plant without adverse effect of metals stress. Almost similar plant height was observed in, and having vermicompost. Concentration and accumulation of heavy metals (Pb, Ni, Cr) in shoot and root Concentrations and uptake of heavy metals in shoot and root were (Table 4 & 5) significantly affected by different treatments. Lead and nickel concentrations were minimum under sole vermicompost as it contained minimal quantity of these two metals. In general, Pb and Ni concentrations were higher in root as compared to shoot of M. arvensis Linn. Lead and nickel concentrations in shoot and root were higher in the treatments where vermicompost was applied with Ni and Pb as compared to that without vermicompost. Nickel in shoot portion was higher in, and Table 4 Influence of heavy metals and vermicompost on concentration and uptake of Pb, Ni and Cr in shoot of Mentha arvensis Linn. cv. Kosi Uptake in shoot (µg) Treatment Pb Ni Cr Pb Ni Cr LSD(p=0.05)

4 CHAND et al: INFLUENCE OF NICKEL AND LEAD WITH VERMICOMPOST ON MENTHA ARVENSIS GROWTH 259 which significantly decreased with and inspite of application of vermicompost. Chromium concentration in shoot and root did not follow any definite trend. Higher concentrations of Pb and Cr with vermicompost might be due to lower ph as a result of production of organic acid. Low ph promotes the availability of micronutrients and heavy metals as reported by several workers 14, 15. Results (Table 5) indicated that magnitude of accumulation of all the three metals (Pb, Ni, Cr) were higher in root tissue as compared to shoot portion. Our results corroborate with the findings of Patra et al 16 who reported that heavy metals absorbed by the plants accumulate in root then being translocated to shoot in mint crop. Addition of vermicompost was presumed to be mobilized the Pb in the rhizosphere by producing the low molecular weight organic acid, responsible for formation of organic ligands 17. Accumulation of Pb in root might be attributed to binding of this metal to exchange sites and extracellular precipitation, mainly in the form of lead carbonates, with both these mechanisms occurring in the cell walls 18. Higher accumulation of nickel in the root may be due to its sequestration in the cation exchange sites of the wall of xylem parenchyma cells and immobilization in the vacuoles of root 19. Concentration and accumulation of micronutrients (Zn, Cu, Mn) in shoot and root Zinc, copper and manganese concentration (Table 6 & 7) were lower in all the treatments over sole application of vermicompost ( ). Zinc concentration in and was at par. It has been further observed that combined supply of vermicompost with graded levels of Ni and Pb increased the concentrations of Cu, Zn and Mn compared to treatments without vermicompost. Higher availability of Cu, Mn, and Zn with vermicompost supply may be attributed to conducive Table 5 Influence of heavy metals and vermicompost on concentration and uptake of Pb, Ni and Cr in root of Mentha arvensis Linn. cv. Kosi Uptake in root (µg) Treatment Pb Ni Cr Pb Ni Cr LSD (p=0.05) Table 6 Influence of heavy metals and vermicompost on concentration and uptake of micronutrients in shoot of Mentha arvensis Linn. cv. Kosi Uptake in shoot (µg) Treatment Zn Cu Mn Zn Cu Mn LSD (p=0.05)

5 260 INDIAN J NAT PROD RESOUR, JUNE 2012 Table 7 Influence of heavy metals and vermicompost on concentration and uptake of micronutrients by Mentha arvensis Linn. cv. Kosi root Uptake in root(µg) Treatment Zn Cu Mn Zn Cu Mn LSD (p=0.05) Table 8 Concentration of micronutrients and heavy metals in post harvest soil Treatment Cr (ppb) Ni (ppm) Cu (ppm) Mn (ppm) Zn (ppm) Pb (ppm) s LSD(p=0.05) environment like lower ph of the soil due to production of organic acid and higher availability of these elements in vermicompost itself. Concentration of Zn, Cu and Mn in shoot varied in the range , and ppm, respectively and concentration was in order of Mn>Cu>Zn. Vermicompost ( ) application recorded maximum accumulation of micronutrients in the shoot biomass. Accumulation of Zn, Cu and Mn was less in Ni and Pb treatments applied without vermicompost possibly due to competition with Ni. However, application of vermicompost both under low and high levels of Ni and Pb improved the concentration and accumulation in shoot and root. Concentration and accumulation of Zn, Cu and Mn were higher in shoots as compared to roots. Concentration of micronutrients and heavy metals in post harvest soil Concentration of Cr varied from ppb (Table 8) which marginally increased over initial status in the soil. Similarly nickel and lead concentration increased due to their direct input to the soil through treatments. The lead concentration was higher as compared to nickel and chromium probably due to lower solubility and mobility of this metal in the soil. Manganese concentration declined in spite of its supply through vermicompost. Zinc did not follow any definite trend however the concentration was less as compared to its initial status in the soil. Conclusion The results indicate that menthol mint withstand heavy metal enrichment of 25 ppm (Ni and Pb) in presence of vermicompost as the source of organic matter. Growth parameters and herb yield marginally enhanced with the metals in presence of 7.5 g/kg vermicompost. In general, concentration and accumulation of heavy metals (Pb, Ni, Cr) and micronutrients (Zn, Cu, Mn) were higher with application of nickel and lead in combination of vermicompost irrespective of their levels. Accumulation of heavy metals (Pb, Ni, Cr) were higher in root tissue as compared to

6 CHAND et al: INFLUENCE OF NICKEL AND LEAD WITH VERMICOMPOST ON MENTHA ARVENSIS GROWTH 261 shoot. However, micronutrient (Zn, Cu, Mn) accumulation followed the reverse trend. Vermicompost as a source of organic chelate helped in binding Ni and Pb to certain extent, and thus increased the DTPA extractable Ni, Cr and Pb which is about 24 times higher than that without vermicompost treatments. It would therefore, appear that M. arvensis lacks hyper accumulation capacity to accumulate Ni, Cr and Pb in higher amount especially in shoot portion. This suggest that it could not be used as scavenger to cleanse the soil polluted with Ni and Pb metals but can be grown with application of vermicompost or organic matter under low heavy metal toxicity where it can be successfully cultivated without any adverse influence on the produce. References 1 Cunningham SD, JR. Shann, DE Crowley and TA Anderson, Phytoremediation of contaminated water and soil, In: E.L. Kruger, T.A. Anderson, and J.R. Coats (ed.) Phytoremediation of soil and water contaminant,. ACS symposium series 664, American Chemical Society, Washington, DC.1997, p Flathman PE and Lanza, GR, Phytoremediation: current views on an emerging green technology, J Soil Contam, 1998, 7(4), Moya JL, S Ros and I Picazo, Influence of cadmium and nickel on growth, net photosynthesis and carbohydrate distribution in rice plants, Photosynth Res, 1993, 36, Clevenger JE, Apparatus for determination of volatile oil, J Amer Pharmac Assoc, 1928, 17, Page AL Miller, RH Keeny, DR Baker, DE, Ellis, R Jr and Rhoades JD, Methods of Soil Analysis, Part 2; American Society of Agronomy Inc., Soil Science Society of America Inc., Madison, Wisconsin Lindsay WL and Norvell WA, Development of a DTPA soil test for zinc, manganese and copper, Soil Sci Soc Am J, 1978, 42, Sokal RR and Rolfs IJ, Biometry: The Principle and practices of statistics in biological research and edu, Freeman, New York Pande P, Chand S, Yadav VK, Anwar M and Patra DD, Influence of Chromium with Vermicompost on growth and accumulation by Brahmi, Comm Soil Sci Plant Anal, 2007, 38, Prasad A, Singh AK, Chand S, Chanotiya CS and Patra, DD, Effect of chromium and lead on yield, chemical composition of essential oil and accumulation of heavy metals of mint species, Comm Soil Sci Plant Anal, 2010, 41, Godbold DL and Kettner C, Lead influences root growth and mineral nutrition of Picea abies seedlings, J Plant Physiol, 1991, 139, Eun SO, Youn HS Lee Y, Lead disturb microtubule organization in the root meristem of Zea mays, Physiol Plant, 2000, 110, Malkowski E, Kita A. Galas W, Karez, W and Michael K, Lead distribution in corn seedlings (Zea mays Linn.) and its effects on growth and the concentration of potassium and calcium, Plant Growth Regul, 2002, 37, Chen C, Huang D and Liu J, Functions and toxicity of nickel in plant: Recent advances and future prospectus, Clean, 2009, 37(45), Antoniadis V, Robinson JS and Alloway BJ, Effect of short term ph fluctuations on cadmium, nickel, lead and zinc availability to rye grass in a sewage sludgeamended field, Chemosphere 2008, 71, Saygideger S, Dogan M and Kesar G, Effect of lead and ph on Lead uptake, Chlorophyll and nitrogen content of Typha latifolia L. and Ceratophyllum demersum L., Inter J Agric Biol, 2004, 1, Patra M, Bhomik, N, Bandopadhyay B and Sharma A, Comparison of mercury, lead and arsenic with respect to genotoxic effects on plant system and the development of genetic tolerance, Environ Exp Bot, 2004, 52, Jones DL and Darrah PR, Role of root derived organic acids in the mobilization of nutrients from the rhizosphere, Plant and Soil, 1994, 166, Jarvis MD and Leung DWM, Chelated lead transport in Pinus radiata: an ultastructural study, Environ Exp Bot, 2002, 48, Seregin IV and Kozhevnikova, Physiological role of nickel and its toxic effects on higher plants, Russ J Plant Physiol, 2006, 53,