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Communications in Soil Science and Plant Analysis

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Comparative Phytoremediation of ChromiumContaminated Soils by Fenugreek, Spinach, and Raya

G. S. Dheri , M. S. Brar & S. S. Malhi
a a a b

Department of Soils , Punjab Agricultural University , Ludhiana, Punjab, India

b

Agriculture and AgriFood Canada , Research Farm, Highway 6 South, P. O. Box 1240, Melfort, Saskatchewan, Canada , S0E 1A0 Published online: 06 Jun 2007.

To cite this article: G. S. Dheri , M. S. Brar & S. S. Malhi (2007) Comparative Phytoremediation of ChromiumContaminated Soils by Fenugreek, Spinach, and Raya, Communications in Soil Science and Plant Analysis, 38:11-12, 1655-1672, DOI: 10.1080/00103620701380488 To link to this article: http://dx.doi.org/10.1080/00103620701380488

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Communications in Soil Science and Plant Analysis, 38: 16551672, 2007 Copyright # Taylor & Francis Group, LLC ISSN 0010-3624 print/1532-2416 online DOI: 10.1080/00103620701380488

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Comparative Phytoremediation of Chromium-Contaminated Soils by Fenugreek, Spinach, and Raya

G. S. Dheri and M. S. Brar
Department of Soils, Punjab Agricultural University, Ludhiana, Punjab, India

S. S. Malhi
Agriculture and Agri-Food Canada, Melfort, Saskatchewan, Canada

Abstract: A glasshouse investigation was undertaken to evaluate the natural potential of fenugreek (Trigonella foenumgraecum L.), spinach (Spinacia oleracea L.), and raya (Brassica campestris L.) for cleanup of chromium (Cr) contaminated silty loam and sandy soils. Four kilograms of soil per treatment in earthen pots was treated with ve levels of chromium [0, 1.25, 2.5, 5.0, and 10.0 mg Cr kg21 soil through dipotassium chromate (K2Cr2O7], equilibrated for 21 days at eld-capacity moisture content, and then fenugreek, spinach, and raya were grown for 60 days after seeding. The concentration of diethylene triamine pentaacetic acid (DTPA)-extractable Cr increased signicantly with increasing rate of Cr application in both soils, but the increase was higher in sandy soil than in silty loam soil. The DTPA-extractable Cr in both soils decreased after harvesting of crops compared to its concentration in soil before sowing of the crops. The decrease in DTPA-extractable Cr concentration was highest in soil growing raya and least in the fenugreek-growing soil. The percent reduction in dry-matter yield (DMY) with increasing levels of added Cr in comparison to the zero-Cr control was highest for fenugreek (49 and 52%) followed by spinach (36 and 42%) and lowest for raya (29 and 34%) in silty loam soil and sandy soil, respectively. Also, the percent reduction in mean shoot yield of all crops was higher in sandy soil (41%) compared to silty loam soil (36%), when the rate of applied Cr was increased from 0 to 10 mg Cr kg21 soil. The DMY of both shoot and root was highest for raya and lowest for fenugreek. The Cr concentration in fenugreek, spinach, and raya Received 11 July 2006, Accepted 15 August 2006 Address correspondence to S. S. Malhi, Agriculture and Agri-Food Canada, Research Farm, Highway 6 South, P. O. Box 1240, Melfort, Saskatchewan, Canada S0E 1A0. E-mail: malhis@agr.gc.ca 1655

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increased with increasing level of added Cr in both soils. The concentration of Cr in both shoot and root was highest in raya, followed by spinach and fenugreek. The overall mean uptake of Cr in shoot was almost four times and in root was about two times higher in raya compared to fenugreek. The ndings indicated that family Cruciferae (raya) was most tolerant to Cr toxicity, followed by chenopodiacea (spinach) and Leguminosae (fenugreek). Because raya removed the highest amount of Cr from soil, it could be used for pytoremediation of mildly Cr-contaminated soils. Keywords: Chromium, fenugreek, phytoremediation, raya, soil texture, spinach

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INTRODUCTION Chromium (Cr) is an environmental pollutant element and ranks seventh in abundance in the earth crust (Katz and Salem 1994). The major contributors of Cr contamination are the leather tanning, electroplating, and stainless steel industries (Riley and Zachara 1991; Turick, Apel, and Carmiol 1996). India is one of the largest producers of leather, and nearly 80% of the tanneries are engaged in the chrome-tanning process (Rajamani et al. 1995). Tanning industries in India release 2000 to 3200 Mg of elemental Cr into the environment annually, with Cr concentration varying from 2000 to 5000 mg L21 of efuents (Chandra, Sinha, and Rai 1997). Efuents from such industries are often disposed into the sewage system, which is used as a source of irrigation for agricultural lands. Consumption of crop plants grown on these soils may create a serious health hazard. Long-term exposure of Cr (VI) is toxic to plants and animals (Ajmal, Nomani, and Ahmad 1984; Bishnoi et al. 1993) and causes many health hazards (Burrows 1983; Bagdon and Hazen 1991). The relative mobility of Cr in soil depends upon the pH and Eh of soil and is controlled largely by its oxidation state (Nieboer and Jusys 1988). The remediation of Cr-contaminated soils with organic amendments is based on the chemical and biological reduction of Cr (VI) to Cr (III) (Losi, Amrhein, and Fronkenberger 1994; Bolan et al., 2003). Hexavalent chromium (VI) is a strong oxidizing agent and is readily reduced in the presence of appropriate electron donors such as organic carbon and reduced iron. Recently, there has been an increasing interest in the use of phytoremediation for amelioration of heavy-metal-contaminated soils, sediments, and waters (Kumar et al. 1995; Ghosh and Singh 2005). Phytoremediation is an emerging technology and is considered for remediation of inorganic- and organic-contaminated sites because of its cost-effectiveness, aesthetic advantages, and long-term applicability, and it is a safe alternate to conventional soil cleanup. This technique involves the use of the ability of some plants to absorb and accumulate high concentrations of heavy-metal ions. Phytoremediation is best suited for a country such as India, because it requires low investment and is based on plants natural capability to take up metal ions from soil. The plants that are capable of accumulating a higher amount of heavy metals

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are used to cleanup heavy-metal-contaminated sites. These hyperaccumulator plants have the genetic potential to concentrate and remove toxic metals. The objective of this study was to evaluate the natural potential of plants belonging to three different families such as fenugreek (Trigonella foenumgraecum L.), spinach (Spinacia oleracea L.), and raya (Brassica campestris L. cv. Italica) for their effectiveness to tolerate and absorb Cr from Cr-contaminated silty loam and sandy soils.

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MATERIALS AND METHODS Collection and Processing of Bulk Soil Samples A glasshouse pot experiment was conducted at the Research Farm of Department of Soils, Punjab Agricultural University (PAU), Ludhiana, Punjab, India. Bulk surface soil samples (0 15 cm) of a silty loam soil (soil I) and a sandy soil (soil II) were collected, air dried in shade at about 308C and ground to pass through a 2-mm sieve. The processed soil samples were thoroughly mixed and analyzed for pH, electrical conductivity (EC) (Elico Solu Bridge Type CM 84 conductivity meter), organic carbon (c) (Walkley and Black 1934, calcium carbonate (Puri 1930), particle-size distribution (Kilmer and Alexander 1949). And cation exchange capacity (CEC) (Richards 1954). The concentration of diethylene triamine pentaacetic acid (DTPA) extractable Cr was measured by using the DTPA method as described by Lindsay and Norvell (1978). The texture of soil I was silty loam with 33.6% sand, 49.4% silt, and 17.0% clay. The pH, EC, CEC, OC, and CaCO3 of soil I were 7.96, 0.35 dSm21, 16.34 cmol kg21, 0.89%, and 0.73%, respectively. Soil II was sandy in texture (87.4% sand, 5.1% silt, and 7.4% clay), noncalcareous, and comparatively low in CEC (7.4 cmol kg21), EC (0.24 dSm21), organic C (0.54%), and pH (7.49). The native DTPA-extractable Cr concentrations in soil I and soil II were 0.15 and 0.08 mg kg21, respectively.

Crop Growing, Harvesting, Collection, Processing, and Analysis of Samples The experiment was conducted in a 5 3 factorial randomized complete block design with three replications. Four kilograms of soil treated with ve levels of chromium [0, 1.25, 2.5, 5.0, and 10.0 mg Cr kg21 soil through dipotassium chromate (K2CR2O7)] was lled in earthen pots lined with polyethylene sheet and allowed to equilibrate for 21 days at eld capacity soil moisture content. Deionized water was used to maintain soil moisture at eld capacity in each pot. Three crops selected for this study were fenugreek (Trigonella foenumgraecum L.), spinach (Spinacia oleracea L.), and raya

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(Brassica campestris L. cv. Italica). Prior to sowing of crops, representative soil samples were collected from each pot using a stainless steel tube auger. The soil samples were air dried in shade at about 308C, ground to pass through a 2-mm sieve, and stored in cloth bags for DTPA-extractable Cr concentration using an atomic absorption spectrophotometer (Isaac and Kerber 1971). Fifteen seeds of different crops were sown per pot after applying recommended doses of nitrogen (N), phosphorus (P), and potassium (K), respectively, at 100, 13, and 25 mg kg21 soil to raya; 35, 5, and 0 mg kg21 soil to spinach; and 65, 13, and 0 mg kg21 soil to fenugreek. After emergence, seedlings were thinned to ve plants per pot. Pots were irrigated with deionized water as required. Crop plants were harvested at 60 days after seeding (DAS). Both shoot and root samples were collected at harvesting. Shoot samples were collected by cutting with a stainless steel cutter, and roots were extracted from soil by gently washing with tap water. Before extracting the root samples, soil samples were again collected from each pot using a stainless steel tube auger. Shoot and root samples were washed sequentially with tap water, acidulated distilled water, distilled water, and deionized water to make sure that no adsorbed material remained on the root surface. The shoot and root samples were then air dried in shade and oven dried at 658C to a constant weight. After recording dry-matter yield (DMY), the samples were ground in a stainless steel Wiley mill to pass through a 0.3-mm sieve and stored in paper bags for chemical analysis. The nely ground plant samples (0.50 g shoot and 0.25 g root material) were mixed with 5 mL of 16 M nitric acid (HNO3) in 125-mL digestion tubes and allowed to react overnight. The next day, samples were digested at 1508C until the volume was reduced to 2 3 mL. After cooling, 2 mL of 30% hydrogen peroxide (H2O2) was added, and the samples were again digested until 1 2 mL of material was left. Final volume was made to 10 mL with deionized water, and concentration of Cr was determined with an atomic absorption spectrophotometer. The data were subjected to analysis of variance (ANOVA) using factorial randomized complete block design (Panse and Sukhatme 1967), and least signicant difference (LSD0.05 was used to determine signicant differences between treatment means.

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RESULTS AND DISCUSSION Effect of Applied Chromium on DTPA-Extractable Chromium in Soil before Sowing and after Harvest of Crops The concentration of DTPA-extractable Cr in soil increased signicantly with increasing rare of Cr application (Figure 1). The native concentration of Cr was lower in sandy soil (0.008 mg Cr kg21 soil) than in silty loam soil (0.15 mg Cr kg21 soil), but with the addition of different levels of Cr, the

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Figure 1. DTPA-extractable Cr in two Cr-contaminated soils before sowing of crops in a glasshouse experiment. LSD0.05: Soil 0.14, Cr 0.23, Soil Cr 0.32, Crop NS, Soil Crop NS, Crop Cr NS, Soil Crop Cr NS. NS refers to not signicant.

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sandy soil showed higher DTPA-Cr concentration in comparison to silty loam soil. The mean Cr concentration was increased by 1.32 mg Cr kg21 in silty loam soil, whereas it was increased by 2.14 mg Cr kg21 in sandy soil when the rate of applied Cr increased from 0 to 10 mg Cr kg21 soil. The overall increase in Cr concentration with Cr application over the zero-Cr control was 1.73 mg Cr kg21 soil. Also, the magnitude of increase in Cr concentration with Cr application was higher in sandy soil (28-fold) than silty loam soil (10fold). The higher concentration of native Cr in silty loam soil was probably due to its higher retention capacity, which can be further related to high concentration of clay, organic C, and CEC in silty loam soil (Sharma, Brar, and Malhi 2005). At a given level of applied Cr, the mean concentrations of DTPA-Cr in silty loam soil and sandy soil before sowing of fenugreek, spinach, and raya were almost similar (Figure 1). The DTPA-extractable Cr in both soils markedly decreased after harvesting of crops (Figure 2). The mean concentration of DTPA-extractable Cr in soil was decreased from 0.58 to 0.50 (14%) after fenugreek, from 0.58 to 0.44 (24%) after spinach, and from 0.57 to 0.35 mg Cr kg21 soil (39%) after raya in silty loam soil. In sandy soil, the corresponding decreases were from 0.83 to 0.72 mg Cr kg21 soil (13%) after fenugreek, from 0.84 to 0.64 Cr kg21 soil (24%) after spinach, and from 0.84 to 0.49 mg Cr kg21 soil (42%) after raya. The overall mean concentration of DTPA-Cr in soils with different crop species grown was decreased from 0.70 to 0.61 mg Cr kg21 soil (13%) after fenugreek, from 0.71 to 0.54 mg Cr kg21 soil (24%) after spinach, and from 0.70 to 0.42 mg CR kg21 soil (40%) after raya. This indicated that the decrease in DTPA-Cr concentration was highest in soil growing raya and lowest in soil growing fenugreek. The residual concentration of DTPA-extractable Cr in soil after harvest varied with the crop species grown. The overall mean of residual DTPA-Cr concentration after harvest was highest (0.61 mg Cr kg21 soil) in fenugreek soil and lowest (0.42 mg Cr kg21 soil) in raya soil. This indicated that raya absorbed a greater amount of Cr from soil than other crops. This might be due to the genetic differences in the efciency of the crops to absorb Cr metal. The removal of Cr by crops and its transformation into relatively unavailable forms might be the reasons for the decrease of DTPA-extractable Cr concentration in both soils after harvest of the crops. This suggested that extent and magnitude of reduction in DTPA-extractable Cr concentration in soil after harvest varied with different crop species. Similar results have been reported by Sidhu (2005).

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Effect of Applied Chromium on Dry-Matter Yield of Shoot and Root of different Crops Different crops showed considerable variation in dry-matter yield (DMY) of shoots even in the absence of Cr application in both soils (Figure 3). Shoot DMY was highest in raya, followed by spinach and fenugreek in both soils.

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Figure 2. DTPA-extractable Cr in two Cr-contaminated soils after harvest of crops in a glasshouse experiment. LSD0.05: Soil 0.06, Cr 0.09, Soil Cr 0.13, Crop 0.07, Soil Crop NS, Crop Cr 0.16, Soil Crop Cr NS. NS refers to not signicant. 1661

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1662 G. S. Dheri, M. S. Brar, and S. S. Malhi Figure 3. Dry-matter yield (DMY) of shoots of different crops grown in two CR-contaminated soils in a glasshouse experiment. LSD0.05: Soil 0.06, Cr 0.1, Soil Cr 0.13, Crop 0.08, Soil Crop 0.11, Crop Cr 0.17, Soil Crop Cr 0.24.

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The results showed that DMY of all three crops marginally increased from zero-Cr control to the initial level of applied Cr (i.e., 1.25 mg Cr kg21 soil) in both soils. Although Cr is not recognized as an essential plant nutrient, the favorable effect of applied Cr at initial level might be due to its effect on rhizosphere microorganisms as reported by Hervey (1971). As the rate of added Cr was further increased from 1.25 to 10 mg Cr kg21 soil, shoot DMY was signicantly decreased irrespective of the soil type and crop species grown. The decrease was from 3.91 to 1.73 g pot21 in fenugreek, from 4.72 to 2.84 g pot21 in spinach, and from 6.34 to 4.47 g pot21 in raya when grown in silty loam soil and from 2.96 to 1.47 g pot21 in fenugreek, from 3.96 to 2.07 g pot21 in spinach, and from 5.37 to 3.37 g pot21 in raya grown in sandy soil. The percent DMY reduction due to Cr application from the control was highest in fenugreek (49 and 52%), followed by spinach (36 and 42%), and lowest in raya (29 and 34%) in silty loam soil and sandy soil, respectively. This indicated that extent and magnitude of decrease in shoot DMY varied with crop species as well as soil type at different Cr levels. The raya was the most tolerant crop and fenugreek was the most sensitive to Cr toxicity. Therefore, raya could be used with better success for phytoremediation of Cr-contaminated soils. The crop plants grown in silty loam soil gave higher DMY in comparison to their counterparts in sandy soil, irrespective of the added Cr level. Also, the percent reduction in mean shoot DMY of all crops was higher in sandy soil (41%) compared to silty loam soil (36%) when the Cr level was increased from 0 to 10 mg Cr kg21 soil. This indicates that the extent of Cr toxicity was also inuenced by variation in the soil texture. The plants grown in sandy soil were more prone to Cr toxicity because of its higher availability in soil solution as compared to the plants grown in silty loam soil. Therefore, crop DMYs were more adversely affected in sandy soil. Sharma, Brar, and Malhi (2005) also reported a similar trend in spinach DMY with application of Cr. The toxic effect of Cr on yield and quality of different crops were reported by Nishimura and Takyashi (1982) in ryegrass and red clover, and by Yiyong, Tongchou, and Boer (1989) in raddish. Bartlett and James (1979) suggested that toxicity of Cr is due to its high oxidizing power and penetrability to biological membranes. The DMY of root varied widely among the crops even in the absence of applied Cr (Figure 4). The root DMY of crops followed the same trend as of shoot DMY when the concentration of applied Cr increased from 0 to 10 mg Cr kg21 soil. The highest DMY of root was noticed in raya, lowest in fenugreek crop, and spinach showed intermediate yield. After initial jump, the root DMY of all crops decreased in both soils with the increase in Cr level from 1.25 to 10 mg kg21 soil. However, the extent of decrease varied with crop species and soil type. The percent reduction in root DMY was 45 and 48% in fenugreek, 37 and 42% in spinach, and 22 and 31% in raya grown in silty loam soil and sandy soil, respectively. The percent decrease in mean root DMY was 33% in silty loam soil and 39% in sandy

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1664 G. S. Dheri, M. S. Brar, and S. S. Malhi Figure 4. Dry-matter yield (DMY) of roots of different crops grown in two Cr-contaminated soils in a glasshouse experiment. LSD0.05: Soil 0.04, Cr 0.06, Soil Cr NS, Crop 0.05, Soil Crop 0.07. Crop CR NS, Soil Crop Cr NS. NS refers to not signicant.

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soil. Thus, decrease in DMY was higher in sandy soil compared to silty loam soil. The decrease in root DMY was highest in fenugreek and lowest in raya. Roots of raya had the highest tendency to tolerate Cr toxicity followed by spinach and fenugreek.

Effect of Applied Chromium on Chromium Concentration in Shoot and Root of Different Crops

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The mean concentration of Cr in the shoot of all crops increased with increasing rate of Cr application (Figure 5); however, the extent of increase was different in crop species (Figure 3). The Cr concentration in shoot increased from 0.90 to 2.53 mg g21 DM (2.8 times), from 1.24 to 4.13 mg g21 DM (3.3 times), and 1.38 to 6.02 mg g21 DM (4.4 times) in fenugreek, spinach, and raya, respectively, when the rate of Cr application was raised from 0 to 10 mg Cr kg21 soil in silty loam soil. In the same order, the Cr concentration in shoot increased from 0.72 to 3.52 mg g21 DM (4.8 times), from 1.03 to 6.09 mg g21 DM (5.4 times), and from 1.26 to 8.43 mg g21 DM (6.7 times), respectively, in sandy soil. The increase in Cr concentration of shoot at higher rates of applied Cr was probably due to marked and signicant increase in available Cr in soil, which resulted in increased amount of metal being absorbed by the plants. In all crops, at a given level of applied Cr, the concentration of Cr in shoots was higher in sandy soil. The mean Cr concentration in shoots increased from 1.17 to 4.23 mg g21 DM in silty loam soil and from 1.0 to 6.01 mg g21 DM in sandy soil. The higher availability of Cr in sandy soil was due to low pH, CEC, clay concentration, and organic C. Sharma, Brar, and Malhi (2005) reported higher Cr concentration in spinach plants grown in sandy soil compared to silty clay loam soil. Narwal et al. (1993) and Khurana (2000) reported higher Cd concentration in plants due to Cd application. Henry (2000) also reported that Brassica juncea has a good ability to transport Pb from roots to shoots, and even a Pb concentration of 500 mg I21 was not phytotoxic to Brassica species. The concentration of Cr in roots varied widely among crops at different levels of applied Cr in both soils (Figure 6). The Cr concentration increased signicantly in roots of all crops in both soils with increasing levels of applied Cr. The highest Cr concentration was in roots of raya and lowest in fenugreek. The again indicated the high potential of raya to accumulate Cr. The increase in mean Cr concentration in roots was from 2.52 to 15.62 mg g21 in sandy soil and from 2.74 to 11.90 mg g21 in silty loam soil with the application of 10 mg Cr kg21 soil. This indicated that the toxic effect of Cr to plants was more prominent in sandy soil. Higher Cr concentration in roots indicated its low translocation to aboveground parts. Wallace (1989) also reported lesser translocation of Cr from root to shoot of spinach compared to absorption. The restriction of translocation of Cr to upper plant parts may be attributed to the ability of

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1666 G. S. Dheri, M. S. Brar, and S. S. Malhi Figure 5. Concentration of Cr in Shoots of different crops grown in two Cr-contaminated soils in a glasshouse experiment. LSD0.05: Soil 0.14, Cr 0.22, Soil Cr 0.31, Crop 0.17, Soil Crop 0.24, Crop Cr 0.38, Soil Crop NS. NS referes to not signicant.

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Figure 6. Concentration of Cr in roots of different crops grown in two Cr-contaminated soils in a glasshouse experiment. LSD0.05: Soil 0.12, Cr 0.19, Soil Cr 0.26, Crop 0.14, Soil Crop 0.20, Crop Cr 0.32, soil Crop Cr NS. NS refers to not signicant. 1667

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1668 G. S. Dheri, M. S. Brar, and S. S. Malhi Figure 7. Uptake of Cr by shoots of different crops grown in two Cr-contaminated soils in a glasshouse experiment. LSD0.05: Soil 0.13, Cr 0.20, Soil Cr 0.29, Crop 0.16, Soil Crop 0.22, Crop Cr 0.35, Soil Crop Cr 0.50.

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Figure 8. Uptake of Cr by roots of different crops grown in two Cr-contaminated soils in a glasshouse experiment. LSD0.05: Soil 0.23, Cr 0.37, Soil Cr 0.52, Crop 0.29, Soil Crop 0.41, Crop Cr 0.64, Soil Crop Cr 0.91. 1669

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plant roots to function as detoxicating agents by immobilizing and holding back elements that will be toxic to aboveground parts of the plant (Macnical and Beckett 1985).

Effect of Applied Chromium on Chromium Uptake in Shoot and Root of Different Crops

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The uptake of Cr in shoots varied in different crop species (Figure 7). In fenugreek, the Cr uptake increased with application of Cr up to only 5 mg kg21 in both soils. This was due to very drastic reduction in DMY of fenugreek with higher rate of Cr application. The Cr uptake in shoot was highest in raya. The mean Cr uptake followed the same trend as was observed for DMY and Cr concentration in plants. The mean Cr uptake in shoots was increased 2.5- and 3.8-fold in silty loam soil and sandy soil, respectively. The uptake of Cr in roots of all crops was signicantly affected by different levels of Cr application (Figure 8). In spite of lower DMY of roots, the mean Cr uptake by roots of all crops was higher compared to shoots. The uptake of Cr by roots of all crops increased in both soils with increasing levels of Cr application. The maximum uptake of Cr in both soils was observed in raya roots and minimum in fenugreek roots. This indicates that raya roots had the highest ability to tolerate Cr toxicity. Kumar et al. (1995) and Ebbs and Kochian (1997, 1998) also observed that Brassica species belonging to the family Cruciferae such as Brassica juncea, Brassica napus, and Brassica rapa have potential to accumulate moderate levels of heavy metals and may be used to cleanup toxic-metalcontaminated sites.

CONCLUSIONS Three plant species used in this study varied widely in their ability to absorb, accumulate, and tolerate Cr toxicity. Because the three crops belong to different botanical families such as Leguminosae (fenugreek), Chenopodiaceae (Spinach), and Cruciferae (raya), there might be some specic uptake mechanism in different plant species for heavy-metal tolerance. Family Cruciferae (raya) was most tolerant to Cr toxicity, followed by Chenopodiacea (spinach) and Leguminosae (fenugreek). Hence, raya may be used for phytoremediation of mildly Cr-contaminated soils.

ACKNOWLEDGEMENTS The rst author is grateful to Punjab Agricultural University, Ludhiana, Punjab, for awarding a Senior Merit fellowship during the course of

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investigation. Thanks are also due to Kuldeep Singh, Assistant Soil Chemist, Department of soils, PAU, Ludhiana, Punjab, for his analysis on the atomic absorption spectrophotometer.

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