Distribution and bioavailability of selenium fractions in some seleniferous soils of Punjab, India

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1 Archives of Agronomy and Soil Science December 2005; 51(6): Distribution and bioavailability of selenium fractions in some seleniferous soils of Punjab, India (Verteilung und Bioverfügbarkeit von Selenfraktionen in einigen Selen haltigen Böden der Region Punjab, Indien) K. S. DHILLON, S. K. DHILLON, & N. PAREEK Department of Soils, Punjab Agricultural University Ludhiana, Punjab, India (Received 18 November 2004; accepted 1 June 2005) Abstract Soil-solid phase associations of Se in seleniferous soils of Punjab were investigated by following sequential extraction procedures involving multiple extractions with 0.2 M K 2 SO 4 (2 times), 0.1 M Na 2 SeO 3 (4 times), 0.05 M NH 4 OH (4 times), 6 M HCl (2 times) and 9 M HNO 3 (2 times) vis-a-vis single extractions with 0.25 M KCl, 0.1 M KH 2 PO 4, 4 M HCl and concentrated HCl. Soil samples were equilibrated with 75 Se (as Na 2 SSeO kbq g 7 1 soil by incubating at field capacity moisture regime and subjecting to alternate wetting and drying cycles. Following multiple extraction procedure, out of total 75 Se added, % was present in readily available form (0.2 M K 2 SO 4 extractable); % as isotopically exchangeable (0.1M Na 2 SeO 3 extractable) and % as organic Se (0.05 M NH 4 OH extractable). Selenium extractable in K 2 SO 4 was significantly correlated with free iron (r = , p ) and CaCO 3 (r = 0.670, p ) content of the soils. Negative relationship was observed between Se uptake by maize (Zea mays L.) and ammonium hydroxide extractable (r = , p ) as well as residual Se (r = , p ) in soils. Highly positive coefficients of correlation between isotopically exchangeable Se and Se content (r = 0.851, p ) as well as its uptake by maize (r = 0.841, p ) indicated that the isotopically exchangeable form of Se may be considered as an index of bioavailable Se in seleniferous soils of Punjab. None of the fractions defined by following single extraction procedure was correlated with either the soil characteristics or Se uptake by maize plants. Multiple extraction procedure could, thus, better explain the distribution of Se in different fractions and uptake by plants. Keywords: Selenium fractions, distribution, bioavailability, seleniferous soils. Introduction Selenium is not an essential element for plant growth, but its level in forages and grains is critical for animal and human health. Selenium concentrations between and Correspondence: Dr K. S. Dhillon, Department of Soils, Punjab Agricultural University Ludhiana , Punjab, India. Tel: , ext 317. Fax: karajdhillon@yahoo.co.in or dhillon_karaj@yahoo.com ISSN print/issn online ª 2005 Taylor & Francis DOI: /

2 634 K. S. Dhillon et al. 4 5 mg kg 7 1 in the animal diet are considered as critical levels of adequacy and toxicity, respectively (Underwood 1977). Some pockets of seleniferous soils producing vegetation containing 4 5 mg Se/kg dry matter, have been identified in the Hoshiarpur and Nawanshehar districts of Punjab state in north-western India (Dhillon & Dhillon 1991). These soils have developed due to deposition of Se-rich material transported through flood water from the nearby hills of the Shiwalik range. Parent material of the soils is derived from upper Shiwalik rocks composed mainly of polymictic conglomerates of variable composition containing many unstable materials such as granite, basalt and limestone. (Dhillon & Dhillon 2003). Total Se concentration in these soils ranges from mg/kg. Animal and human health has been adversely affected by consuming plants grown on the seleniferous soils (Dhillon & Dhillon 1997). Efficient management of seleniferous agricultural land requires a good understanding of the nature and behaviour of Se in these soils. Selenium exists in different oxidation states: selenide (Se 2- ), elemental or colloidal Se (Se 0 ), selenite (Se 4+ ), selenate (Se 6+ ) and several organic combinations (McNeal & Balistrieri 1989). These chemical forms of Se differ widely in their solubility and availability to plants. Radioactive 75 Se was used as a tracer to fractionate Se into various inorganic and organic forms of Se in soils producing low-se forages (Cary et al. 1967; Hamdy & Gissel-Nielsen 1976). A sequential partial dissolution scheme was developed for partitioning Se at elevated levels in various soil samples (Chao & Sanzolone 1989). Recently sequential extraction procedure involving 0.1 M NaOH, 0.1 M Na 2 SO 3 and 30% H 2 O 2 was employed to extract dissolved volatile Se species and other organic Se associated with humic substances (Gao et al. 2000). Fate of dimethylediselenide (DMDSe) in soil was investigated by monitoring the changes in DMDSe the most important organic volatile species in the environment (Zhang & Frankenberger 2002). The present investigation was undertaken to understand the soil-solid phase association of Se in the seleniferous soils of Indian Punjab by sequential extraction of Se following the schemes proposed by Cary et al. (1967) involving exhaustive extraction with the same reagent and that of Chao & Sanzolone (1989) constituting single extraction with each of the extractants. Bioavailability of different Se fractions was evaluated by working out correlation coefficients between different fractions of Se, soil characteristics and Se uptake by maize (Zea mays L.) plants. Materials and methods Surface ( m) soil samples were collected from eight different sites representing typical seleniferous soils in the villages Simbly (District Hoshiarpur) and Barwa and Mehindpur (District Nawanshehar) in north-eastern parts of Punjab (Dhillon & Dhillon 1991). Soil samples were air-dried, ground to pass through 2 mm sieve and analysed for total Se and some important physico-chemical characteristics (see Table I). Brief description of the methods employed for determining calcium carbonate (Puri 1930) and free iron (Assami & Kumada 1959) content of soils are given below: Calcium carbonate To 10 g soil taken in a conical flask, 100 ml of distilled water, 0.5 g CaSO 4.2H 2 O and 10 ml of M AlCl 3.6H 2 O were added. After shaking the contents of the flask, 10 drops each of 1% bromothymol blue and 1% bromocresol green indicators were added. Soil suspensions were boiled on a hot plate. Appearance of a green colour shows the presence of CaCO 3, which

3 Table I. Physico-chemical characteristics of experimental soils. Bioavailable Se in seleniferous soils 635 Soils ph 1 Electrical 1 conductivity (ds m 7 1 ) Organic 2 Carbon (%) Calcium 3 carbonate (%) Free 4 Fe (%) Clay 5 (%) Total Se 6 (mg kg 7 1 ) Simbly Simbly Simbly Mehindpur Mehindpur Mehindpur Barwa Barwa ph and electrical conductivity 1:2 soil:water ratio; 2 Walkley & Black 1934; 3 Puri 1930; 4 Assami & Kumada 1959; 5 Day 1965; 6 Cummins et al was titrated against 0.25 M H 2 SO 4 until it turned golden yellow. Contents of the flask were boiled again. If the green colour appeared again, it was titrated until the permanent golden yellow colour was attained. Free iron To 1 g soil taken in an erlenmeyer flask, 3 g of sodium hydrosulphite and 100 ml of 0.02 M EDTA were added. After heating the flask at 708C on a water bath for 15 m, the soil solution was filtered and Fe was determined on a Varian Techtron atomic absorption spectrophotometer. Fractionation of soil Se The soil samples (250 g) were tagged with radioactive 75 Se as sodium selenosulphate (Na 2 SSeO kbq g 7 1 soil. Samples were brought to 50% of saturation moisture content, mixed thoroughly and then kept for air-drying in the laboratory. Six such cycles of alternate wetting and drying followed during the next eight months. Soil was mixed thoroughly twice a week during this period. It was assumed that by following repeated wetting and drying cycles and thorough mixing, isotopic equilibrium was nearly achieved. Soil samples were ground to pass through a 0.2 mm sieve before subjecting to fractionation of Se. Different fractions of Se were obtained by following the multiple extraction procedure proposed by Cary et al. (1967) and single extraction procedure of Chao and Sanzolone (1989). The only modification made in the procedure of Chao and Sanzolone (1989) was that instead of KClO 3 + conc. HCl, conc. HCl alone was used for extracting the soil. A brief description of the extraction procedures is given below. Multiple extraction procedure of Cary et al. (1967). Soil samples were subjected to sequential extraction with 0.2 M K 2 SO 4 (2 times) followed by 0.1 M Na 2 SeO 3 (4 times), 0.05 M NH 4 OH (4 times), 6 M HCl (2 times) and 9 M HNO 3 (3 times). One g portion of the soil was suspended in 25 ml of extractant in a polypropylene centrifuge tube and equilibrated the soil on end-to-end shaker at 258C. Extraction time was generally 16 h, but it varied from 1 h for HNO 3 to as much as 64 h for one extraction with Na 2 SeO 3. The samples were then centrifuged, filtered and the filtrate was counted for 75 Se.

4 636 K. S. Dhillon et al. Single sequential extraction scheme of Chao and Sanzolone (1989). One g portion of the soil was shaken with 25 ml of 0.25 M KCl for 30 m. The suspension was centrifuged, filtered and the residual soil was then extracted with 25 ml of 0.1 M KH 2 PO 4 for 30 m followed by 4 M HCl and concentrated HCl. In both the procedures, the next extractant was added to the residual soil in the centrifuge tube without intervening drying or washings. All the extractions were carried out in duplicate and the results were expressed as mean of the two values. Radioactivity of the soil extracts was measured by using multi-channel gamma ray spectrometer (Berthold make, model LB 2104). The 75 Se extracted in each fraction was calculated as per cent of total 75 Se added. Plant available Se A greenhouse experiment was conducted by growing maize (Zea mays L.) as a test crop in all the seleniferous soils collected from different locations. Four kg of air-dry soil samples were placed in polythene-lined earthen pots replicated four times. Basal dose of N (50 mg kg 7 1 ), P (25 mg P 2 O 5 kg 7 1 ), K (20 mg K 2 Okg 7 1 ) and Zn (10 mg kg 7 1 ) were supplied through urea, potassium dihydrogen orthophosphate, potassium chloride and zinc sulphate, respectively. Ten seeds of maize (cv Parbhat) were sown on 25 May in the soil maintained at field capacity moisture regime. After germination, when the seedlings were well established, five plants were retained in each pot. Irrigation was applied to maintain the soil moisture level near field capacity. The crop was harvested after 50 days of growth. Plant samples were washed free of any surface contamination. After air-drying, the samples were dried in an oven at C to a constant weight and dry matter yield was recorded. Selenium analysis Plant samples were ground to pass through a 1 mm sieve. Soil and plant samples were digested in a mixture of HNO 3 and HClO 4 in 5:2 ratio and the digests were analysed for Se by following the colourimetric method as described by Cummins et al. (1964). Statistical analysis Data pertaining to dry matter yield and Se content of plants were analysed following Completely Randomized Design (Snedecor & Cochran 1994). Results and discussion Fractions of soil Se Multiple sequential extraction procedure as proposed by Cary et al. (1967). Potassium sulphate (0.2 M K 2 SO 4 ) extractable selenium (Se) consists of selenate, selenite and organic Se compounds of low molecular weight and is termed as readily available Se (Cary et al. 1967). In seleniferous soils incubated at field capacity, the amount of 75 Se extracted in the first extraction with 0.2 M K 2 SO 4 varied from % of added 75 Se. In comparison to the first extraction, the percentage of 75 Se released from different soils in the second extraction with 0.2 M K 2 SO 4 decreased by 16 39%. The sum of the two extractions with 0.2 M K 2 SO 4 (i.e., readily available Se) varied from %; the smallest amount was released from Simbly-1 soil, and the greatest from Mehindpur-2 soil (see Table II). The average amount of

5 Bioavailable Se in seleniferous soils 637 Table II. Different fractions of Se (% of total 75 Se added) as per sequential extraction procedure proposed by Cary et al. (1967). Easily available Se Potentially available Se Difficultly available Se 0.2 M K 2 SO 4 Isotopically Exchangeable 0.1 M Na 2 SeO M NH 4 OH 6 M HCl 9 M HNO 3 Soils (2x) (4x) (4x) (2x) (3x) Residual Simbly Simbly Simbly Mehindpur Mehindpur Mehindpur Barwa Barwa total 75 Se released in 0.2 M K 2 SO 4 was the greatest for Mehindpur soils (17.9%), followed by Barwa (14.3%) and Simbly (12.5%) soils. As Fe 2 O 3 is known to have higher Se- sorption capacity than even clay minerals (Hamdy & Gissel-Nielsen 1977), the amount of 75 Se released in 0.2 M K 2 SO 4 was negatively correlated with free iron (r = , p ) content of the soils (see Figure 1a). A significant coefficient of correlation was observed between the K 2 SO 4 extractable fraction of Se and CaCO 3 content (r = 0.670, p ) of the soils. In some muck, clay loam and sandy soils from Denmark, the K 2 SO 4 soluble fraction of Se ranged from % of added 75 Se (Hamdy & Gissel-Nielsen 1976). It was further observed that soils treated with CaCO 3 had a greater percentage of readily-soluble Se than soils incubated with organic matter. Dhillon et al. (1992) reported that the total as well as hot water soluble (available) Se content of the seleniferous soils were significantly correlated with CaCO 3 (r =0.42,p and 0.43, p , respectively). In this study, the positive relationship between CaCO 3 and the available Se content of the soil as well as with that of the plants (r = 0.37, p ) suggests that the Se adsorbed on CaCO 3 particles remains easily accessible to plants for absorption and accumulation in toxic levels. Adriano (1986) has reported that Se tends to concentrate in carbonaceous debris in sand stone. The isotopically exchangeable fraction of 75 Se extracted by Na 2 SeO 3 (after removing the K 2 SO 4 -soluble fraction of Se) represents selenite, possibly adsorbed on hydrous iron oxides. The amount of 75 Se desorbed in Na 2 SeO 3 decreased during each successive extraction and the total amount of Se released during four successive extractions varied from % (see Table II). The average amount of 75 Se released in Na 2 SeO 3 was the lowest for Barwa (28.0%) followed by Mehindpur (41.6%) and Simbly (43.8%) soils. Among the different fractions of Se extracted, isotopically exchangeable Se was the greatest and it may be attributed to the fine texture of the soils. Hamdy and Gissel-Nielsen (1976) observed that in comparison to muck and sandy soils, clay loam soil contained higher amounts of isotopically exchangeable fraction. Ammonium hydroxide (0.05 M NH 4 OH) soluble fraction of Se is considered to consist of unknown organic compounds, probably protein-bound Se amino acids and adsorbed selenite or selenate on organic compounds. As the soils in the present investigation had low organic

6 638 K. S. Dhillon et al. Figure 1. Relationship between (a) K 2 SO 4 extractable Se and free iron content; (b) isotopically exchangeable and residual Se; (c) isotopically exchangeable Se and its uptake by maize; (d) NH 4 OH extractable Se and its uptake by maize in seleniferous soils of the Punjab. matter content (see Table I), the amount of NH 4 OH soluble fraction of 75 Se was appreciably lower than both the K 2 SO 4 extractable and isotopically exchangeable forms of 75 Se. The percentage of 75 Se desorbed through each successive extraction with NH 4 OH decreased as the number of extractions increased. The total amount of 75 Se desorbed through four extractions with NH 4 OH varied from % (see Table II). Barwa soils, which released the smallest average amount of isotopically exchangeable 75 Se, released the greatest average amount of NH 4 OH soluble fraction of Se (9.8%). Hamdy and Gissel-Nielsen (1976) observed that sandy soils contained a greater amount of NH 4 OH extractable Se than the muck and clay loam soils. They also proposed that the sum of isotopically exchangeable and NH 4 OH extractable fractions of Se may be considered as potentially available Se. As such Simbly soil had the greatest average amount of potentially available Se (52.1%) followed by Mehindpur (49.6%) and Barwa (37.8%) soils. The major proportion of the potentially available Se in all the soils was present as isotopically exchangeable Se. It is consistent with the observations of Hamdy and Gissel-Nielsen (1977). El-Nennah and Soliman (1982) found that % of Se in some soils of Egypt was present in potentially available form.

7 Bioavailable Se in seleniferous soils 639 The Se fraction dissolved by 6 M HCl is considered to originate from selenite occluded in sesquioxides. The major proportion of 6 M HCl extractable Se was desorbed in the first extraction and it ranged from % of added 75 Se. The total amount of 75 Se desorbed in two extractions with 6 M HCl varied from % (see Table II) and was positively correlated with clay content (r = 0.742, p ) of the soils. The 9 M HNO 3 soluble and residual Se consist of elemental Se and heavy metal selenides. A greater amount of 75 Se was released from all the soils during the first rather than the second extraction with HNO 3 ; varying from and % of added 75 Se, respectively. The total amount of HNO 3 extractable 75 Se in soils from different locations ranged from % with greater amounts being desorbed from Simbly soils. All the soil samples (except that of Barwa-1) had lower amounts of 9 M HNO 3 soluble Se than 6 M HCl extractable Se. The differences in residual 75 Se in different soils were conspicuous and it varied from % of added 75 Se (see Table II). The average amount of residual 75 Se was the greatest for Barwa (34.5%); Simbly (20.2%) and Mehindpur (20.3) soils followed. Residual 75 Se was negatively correlated with isotopically exchangeable Se (r = , p ) (see Figure 1b) and positively correlated with free iron content (r = 0.651, p ) of the soils. Hamdy and Gissel-Nielsen (1976) termed the sum of 6 M HCl extractable, 9 M HNO 3 extractable and residual Se as difficultly available form of Se. In the present investigation, this fraction of Se ranged from % of added 75 Se. Although Barwa soils had a greater amount of total native Se (av mg g 7 1, Table I), a major proportion of added 75 Se (av. 47.9%) was found in difficultly available state compared to that in Simbly (av. 35.3%) and Mehindpur (32.4%) soils. On the other hand, Simbly and Mehindpur soils though having a less amount of total native Se (see Table I), possessed a greater percentage of added 75 Se in potentially available form. Single sequential extraction procedure as proposed by Chao and Sanzolone (1989). Potassium chloride (0.25 M KCl) is known to extract water-soluble and non-specifically sorbed selenate fraction of Se. The amount of 75 Se desorbed with KCl from different soils varied from % of added 75 Se. However, no significant differences were observed in the average amount of 75 Se desorbed from the soils from different locations (see Table III). In general, a greater amount of 75 Se was released with 0.2 M K 2 SO 4 in two extractions (see Table II) compared to that desorbed through single extraction with 0.25 M KCl (see Table III). Dhillon and Dhillon (2004) found that 0.25 M KCl extractable Se in some seleniferous and non-seleniferous soils of India varied from % of added 75 Se. It was further observed that when the soils were subjected to seven repeated extractions with 0.25 M KCl, nearly 60 65% of 75 Se added to the seleniferous soils could be mobilized. The cumulative 75 Se desorbed through seven repeated extractions with 0.25 M KCl was significantly correlated with ph (r = 0.927, p ) of the soils suggesting greater availability of Se in alkaline soils. Potassium dihydrogen orthophosphate (0.1 M KH 2 PO 4 ) is an effective extractant to desorb selenite (SeO 3 2- ) specifically sorbed on oxide minerals (Hingston et al. 1971; Rajan 1979; Balistrieri & Chao 1987) and clay minerals (Bar-Yosef & Meek 1987). Selenite, though not as labile as selenate (Ahlrichs & Hossner 1987), is nevertheless available for plant uptake and also subject to leaching losses through the ligand exchange mechanism and anion competition (Parfitt 1978). The amount of 75 Se desorbed through KH 2 PO 4 varied from % (see Table III) and invariably all the soils released greater amounts of 75 Se in KH 2 PO 4 than in KCl. It may be attributed to incomplete extraction of 75 Se through single extraction with KCl. When some Indian soils were subjected to seven repeated extractions first with 0.25 M KCl and then with 0.1M KH 2 PO 4, % of added 75 Se could be extracted in KH 2 PO 4 (Dhillon & Dhillon 2004).

8 640 K. S. Dhillon et al. Table III. Different fractions of Se (% of total 75 Se added) as per sequential extraction procedure described by Chao and Sanzolone (1989). Available Se Unavailable Se Resistant Se Soils 0.25 M KCl 0.1 M KH 2 PO 4 4 M HCl Conc HCl Residual Simbly Simbly Simbly Mehindpur Mehindpur Mehindpur Barwa Barwa Chao and Sanzolone (1989) designated the sum of the fractions of Se extracted by 0.25 M KCl and by 0.1 M KH 2 PO 4 as available Se. As such, % of added 75 Se was present in the available form in these seleniferous soils. On the basis of average values, soils from Simbly contained the greatest amount of Se in the available form (26.5%) and those from Barwa the smallest (21.5%). The most important adsorbents of Se among the constituents of soils are the oxide minerals. Hydrochloric acid (4 M) is capable of dissolving to varying degrees the Fe, Mn and Al oxides, amorphous materials, carbonates and certain sulphide minerals. Selenium extracted with 4 M HCl is not readily available for plant uptake, but it has the potential to become available through chemical and microbial mobilization (Chao & Sanzolone 1989). Lowering the redox potential and ph with addition of HCl may render Fe and Mn oxides soluble, thereby releasing the associated Se into soluble form. When soil residue (after extraction with 0.25 M KCl and 0.1 M KH 2 PO 4 ) was subjected to extraction with 4 M HCl, the amount of 75 Se released from different seleniferous soils ranged from % (see Table III). The average amount of 75 Se released from three groups of soils varied from % and it was the greatest for Mehindpur soils and the lowest for Simbly soils. Simbly-3 soil, which contained the greatest amount of available 75 Se (30.7%) had the smallest amount of 4 M HCl extractable 75 Se (2.0%). Chao and Sanzolone (1989) reported that KClO 3 + concentrated HCl mixture extracts the Se present in various sulphide minerals. In the present investigation, soil residue after extraction with 4 M HCl was extracted with concentrated HCl alone; the amount of Se extracted from different soils varied from % (see Table III). The average amount of 75 Se extracted with concentrated HCl was the greatest for Mehindpur (4.1%) and the smallest for Barwa (1.7%) soils. Although Barwa soils released appreciable amounts of 75 Se in 4 M HCl (5.2%), these soils released a very less amount of Se in the concentrated HCl (1.7%). According to Chao and Sanzolone (1989), the sum of the two fractions extracted with 4 M HCl and concentrated HCl is termed as unavailable Se. In the present investigation, it varied from % in different soils. Mehindpur soils had the greatest average amount of unavailable Se (10.0%) and Barwa and Simbly had similar amounts of unavailable 75 Se (7.0%). The sum of all the fractions of extracted 75 Se when subtracted from the added 75 Se provided an estimate of the residual 75 Se. According to Chao and Sanzolone (1989) the residual form is actually the resistant fraction of Se. As expected, the amount of residual Se in different soils was conspicuously large when subjected to single extraction procedure and it

9 Bioavailable Se in seleniferous soils 641 varied from % (Table III). Barwa soils containing the greatest amount of native Se (see Table I) and possessed the major proportion of added 75 Se as residual 75 Se (71.5%). Similar observations have also been recorded by following the multiple extraction procedure. It confirms that Barwa soils contained the major proportion of native Se as residual Se. The amounts of 75 Se extracted through different extractants like 0.25 M KCl, 0.1 M KH 2 PO 4 and 4 M HCl and residual Se left thereafter were not significantly correlated with any of the soil properties. Possibly single extraction of the seleniferous soils with any of these reagents is not enough to extract Se present in different pools. While studying the pools of Se using multiple extraction procedure (seven repeated extractions with each of the 0.25 M KCl and 0.1 M KH 2 PO 4 and two with 4 M HCl), Dhillon and Dhillon (2004) observed that extraction with KCl alone can mobilize 60 65% Se present in seleniferous soils. Even KH 2 PO 4 could desorb greater amounts of Se than single extraction alone. Moreover, in the sequential extraction procedure proposed by Chao and Sanzolone (1989), after single extraction of the soil with KCl, the remainder of KCl extractable Se is extracted through the subsequent KH 2 PO 4 extraction, thus overestimating the KH 2 PO 4 extractable Se pool. The scheme proposed by Cary et al. (1967) involved repeated extractions two with 0.2 M K 2 SO 4, four with 0.1 M Na 2 SeO 3, four with 0.05 M NH 4 OH, two with 6 M HCl and three with 9 M HNO 3 and the Se fractions extracted through these reagents were significantly correlated with some of the physico-chemical characteristics of the soils. It shows that the scheme proposed by Cary et al. (1967) gives better estimates of different Se fractions than the single-extraction scheme proposed by Chao and Sanzolone (1989). Even the repeated extractions procedure used by Dhillon and Dhillon (2004) provided more meaningful information about different Se pools than the Chao and Sanzolone (1989) procedure. Dry matter yield and Se uptake by maize from seleniferous soils Accumulation of dry matter by maize plants grown in these soils varied from g pot 7 1 (see Table IV) with the smallest yield obtained in the case of Barwa-2 soil and the greatest in Mehindpur-2 soil. No significant differences were observed in the dry matter yield of maize in the three soils from Simbly, two from Barwa and Mehindpur-3. Dry matter yield obtained in Simbly-2 was significantly greater than in Barwa-2 soil. The yields of maize in Mehindpur-1 and Mehindpur-2 soils were significantly greater than those in all other soils under study. Selenium concentration of maize plants varied from 4.3 in Barwa-2 to 7.4 mg g 7 1 in Simbly-3 (Table IV). In general, Se concentration in maize was found to be the greatest in Simbly as compared to that in maize grown in the Mehindpur and Barwa soils. The selenium concentration in maize grown in Mehindpur and Barwa soils did not differ significantly. Selenium concentration of maize was positively correlated with isotopically exchangeable 75 Se (r = 0.851, p ) and potentially available 75 Se (r = 0.660, p ) and negatively correlated with residual 75 Se (r = , p ) fractions obtained through extraction procedure of Cary et al. (1967). On the other hand, no significant correlation coefficient was observed between the Se concentration of maize and the amount of 75 Se extracted through KCl or KH 2 PO 4 by following the Chao and Sanzolone (1989) fractionation procedure. Cary and Allaway (1969) observed that Se content in alfalfa was positively correlated with 0.01 M CaCl 2 (r =0.91, p ) and 0.2 M K 2 SO 4 (r = 0.90, p ) soluble Se. Gissel-Nielsen et al. (1984) observed that the Se uptake by ryegrass was not significantly correlated with any of the soil-se fractions ranging from water-soluble to 9 M HNO 3 soluble fractions. The isotopically exchangeable fraction of soil-se in active root zone approximated the fraction from which the plants absorbed Se.

10 642 K. S. Dhillon et al. Table IV. Dry matter yield, Se content and uptake by maize (Zea mays L.) grown in seleniferous soils. Dry matter yield Se content Se uptake Soils (g pot 7 1 ) (mg g 7 1 dry matter) (mg pot 7 1 ) Simbly Simbly Simbly Mehindpur Mehindpur Mehindpur Barwa Barwa LSD (p ) Selenium uptake is the product of yield and Se concentration in maize plants. Thus it was the highest in maize grown in Mehindpur-2 soil due to its highest yield. On the other hand, Se uptake by maize was significantly higher in two out of three Simbly soils as compared to that of maize grown in Barwa soils. In the later soils, both yield and Se concentration were the lowest. Selenium uptake was positively correlated with isotopically exchangeable Se (r = 0.841, p ) (see Figure 1c) and potentially available Se (r = 0.871, p ), but negatively correlated with NH 4 OH extractable Se (r = , p ) (see Figure 1d), residual Se (r = , p ) and difficultly available Se (r = , p ). Thus, the isotopically exchangeable fraction of Se should prove a better index of bioavailable Se in seleniferous soils of Punjab. Conclusion Available pools of different nutrients are generally determined by single-extraction procedures, but present investigation reveals that repeated extractions of soil with the same reagent can give better estimates of plant available pools of Se. Isotopically exchangeable Se as determined through four consecutive extractions can be a reliable index of available Se and can explain large variations in the uptake of Se by crop plants. References Adriano DC Trace elements in terrestrial environments. New York: Springer-Verlag. Ahlrichs JS, Hossner LR Selenate and selenite mobility in overburden by saturated flow. J Environ Qual 16: Assami T, Kumada K A new method of determining free iron in paddy soils. Soil Pl Fd 5: Balistrieri LS, Chao TT Selenium adsorption by goethite. Soil Sci Soc Am J 51: Bar-Yosef B, Meek D Selenium sorption by kaolinite and montmorillonite. Soil Sci 144: Cary EE, Allaway WH The stability of different forms of selenium applied to low selenium soils. Soil Sci Soc Am Proc 36: Cary EE, Wieczorek GA, Allaway, WH Reactions of selenite-selenium added to soils that produce lowselenium forages. Soil Sci Soc Am Proc 31: Chao TT, Sanzolone RF Fractionation of soil selenium by sequential partial dissolution. Soil Sci Soc Am J 53: Cummins LM, Martin JL, Maag GW, Maag DD A rapid method for the determination of selenium in biological material. Analyt Chem 36: Day PR Particle fractionation and particle size analysis. In: Black et al., editors. Methods of soil analysis, Part 1. Madison, WI: American Society of Agronomy. pp

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