Chemosphere 85 (2011) 677–682

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Chemosphere

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Technical Note

Effects of rapeseed residue on lead and cadmium availability and uptakeby rice plants in heavy metal contaminated paddy soil

Yong Sik Ok a,⇑, Adel R.A. Usman a,b, Sang Soo Lee a, Samy A.M. Abd El-Azeem a, Bongsu Choi a,c,Yohey Hashimoto d, Jae E. Yang a

a Department of Biological Environment, Kangwon National University, Chuncheon 200-701, Republic of Koreab Department of Soils and Water, University of Assiut, Assiut 71526, Egyptc Crop Environment Research Division, National Institute of Crop Science, Suwon 441-857, Republic of Koread Department of Bioresource Science, Mie University, 1577 Kurima-machiya, Mie 514-8507, Japan

a r t i c l e i n f o a b s t r a c t

Article history:Received 5 October 2010Received in revised form 16 June 2011Accepted 17 June 2011Available online 20 July 2011

Keywords:Heavy metalBioavailabilityRice paddyCrop residueGreen manureBioenergy

0045-6535/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.chemosphere.2011.06.073

⇑ Corresponding author. Tel.: +82 33 250 6443; faxE-mail address: soilok@kangwon.ac.kr (Y.S. Ok).

Rapeseed (Brassica napus L.) has been cultivated for biodiesel production worldwide. Winter rapeseed iscommonly grown in the southern part of Korea under a rice-rapeseed double cropping system. In thisstudy, a greenhouse pot experiment was conducted to assess the effects of rapeseed residue applied asa green manure alone or in combinations with mineral N fertilizer on Cd and Pb speciation in the contam-inated paddy soil and their availability to rice plant (Oryza sativa L.). The changes in soil chemical andbiological properties in response to the addition of rapeseed residue were also evaluated. Specifically,the following four treatments were evaluated: 100% mineral N fertilizer (N100) as a control, 70% mineralN fertilizer + rapeseed residue (N70 + R), 30% mineral N fertilizer + rapeseed residue (N30 + R) and rape-seed residue alone (R). The electrical conductivity and exchangeable cations of the rice paddy soil sub-jected to the R treatment or in combinations with mineral N fertilizer treatment, N70 + R and N30 + R,were higher than those in soils subjected to the N100 treatment. However, the soil pH value with theR treatment (pH 6.3) was lower than that with N100 treatment (pH 6.9). Use of rapeseed residue as agreen manure led to an increase in soil organic matter (SOM) and enhanced the microbial populationsin the soil. Sequential extraction also revealed that the addition of rapeseed residue decreased the easilyaccessible fraction of Cd by 5–14% and Pb by 30–39% through the transformation into less accessible frac-tions, thereby reducing metal availability to the rice plant. Overall, the incorporation of rapeseed residueinto the metal contaminated rice paddy soils may sustain SOM, improve the soil chemical and biologicalproperties, and decrease the heavy metal phytoavailability.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction can be the main cause of chronic and acute diseases (Yang et al.,

Heavy metal contamination in paddy soils is one of the mostserious issues confronting rice production and soil managementin Asian countries. Especially in Korea, the large paddy areas havebeen severely contaminated by Cd and Pb via effluent from minetailings and other wastes generated by closed or abandoned mines(Yang et al., 2006; Ok et al., 2007). As a result, accumulation of hea-vy metals into rice plants has produced a major environmental riskto human health. Previous studies have shown that heavy metalcontents in rice plants grown near abandoned mines are higherthan those grown in the uncontaminated areas (Ok et al., 2011a).Additionally, rice paddy management has been a public concernsince the daily ingestion of high Cd and Pb from rice productions

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: +82 33 241 6640.

2007).The investigations are currently underway to effectively reme-

diate the paddy soils contaminated with heavy metals. The immo-bilization, which transforms heavy metals into less bioavailableforms, was considered as one of the most effective ways to reme-diate the heavy metal contaminated soils (Gadepalle et al., 2007;Hashimoto et al., 2009a,b; Ok et al., 2010a,b, 2011b). Amongimmobilizing agents, organic amendments have been shown toeffectively alleviate heavy metal toxicity to plants by transformingthe metals into less available fractions (Shuman, 1999). The incor-poration of organic amendments into the heavy metal contami-nated soils could maintain soil organic matter (SOM), improvesoil physicochemical and biological properties, and increase plantproduction (Yoon et al., 2004; Kim et al., 2010). Moreover, manyprevious studies have demonstrated the benefits of utilizing organ-ic amendments for immobilization of heavy metals in the soils(Bolan et al., 2003; Walker et al., 2004; O’Dell et al., 2007; Tandyet al., 2009).

678 Y.S. Ok et al. / Chemosphere 85 (2011) 677–682

In Korea, the rapeseed plant is being cultivated as a biodiesel re-source in rice paddies under double cropping system. Typically, therapeseed plant can be cultivated in rice paddies as a winter cropafter harvesting the rice plant. Previous studies evaluated the ef-fects of adding rapeseed residue as a green manure in rice-rape-seed double cropping system to reduce N fertilizer (Choi et al.,2009). However, the effects of incorporation of rapeseed residueinto contaminated soils on heavy metal immobilization have notbeen investigated to date.

Therefore, the aim of the present study was to assess the ef-fects of rapeseed residue alone or in combinations with mineralN fertilizers on the geochemical forms of Cd and Pb in the con-taminated paddy soil, as well as on their availability to riceplants. The effects of rapeseed residue on the soil chemical andbiological properties of the contaminated paddy soil were alsoinvestigated.

2. Materials and methods

2.1. Characterization of heavy metal contaminated paddy soil

Heavy metal contaminated soil was collected from a depth of 0to 30 cm in a paddy field adjacent to Seosung mine in Seosan,Chungnam Province, Korea. Based on the previous results fromstudies (MRC, 2007; Ok et al., 2010a), many paddy fields in this re-gion have been contaminated with Cd and Pb at exceed levels ofthe Korean Standard Test due to contamination from mine tailingsand other wastes generated from Seosung mine. The soil sampleswere air dried and ground to pass through a 2-mm sieve. The se-lected chemical properties of the paddy soil used were determinedby the Korean Standard Methods (NIAST, 2000). The soil had a loa-my texture with a CEC of 15 cmolc kg�1. The soil pH was 6.3 andelectrical conductivity (EC) was 0.21 dS m�1. In addition, the aquaregia extraction revealed that the total Cd and Pb concentrations inthe paddy soil were 17 and 1246 mg kg�1, respectively, indicatingthat the selected paddy soil was highly contaminated with Cd andPb according to the Soil Environment Conservation Act from Kor-ean Ministry of Environment (<4 mg kg�1 for Cd and <200 mg kg�1

for Pb) (MOE, 2010).

2.2. Experimental design and treatments

In this study, the use of rapeseed residue as a green manure wasevaluated for reducing the availability of Cd and Pb in the heavymetal contaminated paddy soil. Rapeseed (Brassica napus cv. Sun-mang) was cultivated from October 2007 to June 2008 in a paddyfield located in Yeonggwang-gun, Chunnam Province, which is ina southwestern part of Korea. This region is primarily producingrice and has designed for the rapeseed cultivation during winterseason for biodiesel manufacturing (Nam et al., 2008). During har-vesting on June 13, 2008, the rapeseed residue consisting of shootsand roots only without seeds was collected. The rapeseed residueof shoot and root was oven dried at 70 �C for 48 h and was choppedfor the use as a green manure. The total C and N in prepared rape-seed residue were 34.02% and 0.54%, respectively, and its ratio was63:1.

Four treatments were applied to pots (75 � 45 � 20 cm) in agreenhouse, including 100% recommended conventional N inputof N100, N70 + R, N30 + R and R. The experiment was designedusing a randomized complete block design with three replications.The P and K fertilizers were also applied at the recommended lev-els of common Korean soils. The N, P and K fertilizers were appliedas urea, fused superphosphate and potassium chloride, respec-tively. The rapeseed residue was applied at a dosage of143 kg N ha�1 based on the plant N content. Rice (Oriza sativa L.

cv. Ilmibyeo) seedlings were transplanted to pots filled with trea-ted soils as succeeding crops (40 d after sowing) at a density of8 unit hill�1. The transplanted rice seedlings were grown in a plas-tic greenhouse. Rice plants were sampled during the harvestingstage or 80 d after transplantation.

2.3. Soil chemical analyses

At the end of the pot experiment, soil samples were collected,air dried and sieved through a 2-mm sieve for analysis. The chem-ical properties of the soil were determined according to the KoreanStandard Methods (NIAST, 2000). Soil pH was measured in a 1:5suspension of soil to water using a digital pH meter. The total sol-uble salt in the extract was determined using a digital electricalconductivity meter (Orion 3-Star, Thermo Scientific, USA) and soilorganic C was determined using the Walkley–Black method. Inaddition, the exchangeable form of cations (Ca2+, Mg2+, Na+ andK+) was extracted using 1 N ammonium acetate at pH 7 and thenthe concentrations of extracted cations were determined by anatomic absorption spectrophotometer (AAnalystTM 700 AAS, Per-kin Elmer, USA).

2.4. Soil biological analyses

To evaluate the effects of the additions of rapeseed residue andmineral N fertilizer on the microbial activity in the contaminatedpaddy soil, the bacteria and fungi were evaluated. Specifically, soilsamples (1 g) were diluted in 99 mL of sterilized water and thenagitated for 5 min. Next, 100 lL aliquots of the sequential dilutedsamples were spread directly onto the surface of nutrient agarmedium for bacteria or plate count agar medium for fungi. Eachtest plate was incubated at 30 �C for 48 h for bacteria and 7 d forfungi. At the end of the culture period, the colony-forming units(CFUs) were enumerated (Pepper and Gerba, 2009).

2.5. Sequential extraction of Cd and Pb in contaminated paddy soil

The geochemical forms of Cd and Pb in soils were assessed usingthe method of Tessier et al. (1979). The aliquots of 1 g soil per eachreplicate were placed in 50 mL polypropylene centrifuge tubes andweighed, then the sequential extractions were conducted for solu-ble plus exchangeable (1 M of MgCl2, pH 7), carbonate-bound (1 Mof NaOAc, pH 5), Fe–Mn oxide-bound (0.04 M of NH2OH�HCl in 25%(v/v) HOAc, pH 2 and heated at 96 �C), organic matter-bound(0.02 M HNO3 in 30% H2O2, pH 2) and the residual (digested with3:1 concentrated HCl/HNO3). Finally, the concentrations of Pband Cd were determined by an inductively coupled plasma opticalemission spectrometer (ICP-OES).

2.6. Cadmium and Pb concentrations in rice plant tissues

To measure the Cd and Pb concentrations in rice plants, thesamples were separated into stem, leave and hull. Plant sampleswere ground in a stainless steel mill and then digested with 5 mLof concentrated H2SO4 and 2 mL of 30% H2O2 (Campbell and Plank,1998; Yang et al., 2009). The Cd and Pb concentrations were mea-sured using ICP-OES. In addition, transfer factors (TF) of Cd and Pbwere calculated to determine the transfer of metals from the paddysoil to rice plant (Usman and Mohamed, 2009). The value of TF wascalculated as the ratio of the metal concentration in the parts ofrice plant (leaves + stems + hulls) to the metal concentration inthe paddy soil.

Table 1Effects of the addition of rapeseed residue alone or in combinations with mineral N fertilizer on the chemical properties of paddy soil.

Treatments pHa ECb (dS m�1) OMc (g kg�1) Exchangeable cations (cmol(+) kg�1)

Ca2+ Mg2+ Na+ K+

N100 6.9a 0.05c 22.3b 4.5b 1.8b 0.1c 0.1dN70 + R 6.4bc 0.19b 27.0a 4.9a 2.0a 0.3b 0.4cN30 + R 6.5b 0.25b 29.0a 4.9a 2.1a 0.3ab 0.5bR 6.3c 0.33a 27.6a 4.8a 2.1a 0.4a 0.5a

The same letters in the table indicate no significant difference at a 0.05 significance level, determined by Tukey’s HSD test (n = 3).a 1:5 soil to water ratio.b Electrical conductivity.c Organic matter.

Table 2Colony forming units (CFU) of fungi and bacteria and fungal/bacterial (F/B) ratio inpaddy soils as affected by the addition of rapeseed residue alone or in combinationswith mineral N fertilizer.

Treatments Fungi(CFU � 104 g�1 soil)

Bacteria(CFU � 104 g�1 soil)

F/Bratio

N100 44 ± 5 40 ± 3 1.1N70 + R 70 ± 6 40 ± 2 1.7N30 + R 71 ± 6 55 ± 4 1.4R 97 ± 10 45 ± 4 2.2

Fig. 1. Effect of the rapeseed residue alone or in combinations with mineral Nfertilizer (N100: 100% mineral N fertilizer, N70 + R: 70% mineral N fertil-izer + rapeseed residue, N30 + R: 30% mineral N fertilizer + rapeseed residue, R:rapeseed residue alone) on transfer factors (TF) of: (a) Cd and (b) Pb. (TF = mg metalcontent in rice tissues (leaves + stems + hulls)/mg metal content in contaminatedpaddy soil). The same letters above each bar indicate no significant difference at a0.05 significance level, determined by Tukey’s HSD test (n = 3).

Table 3Concentrations of cadmium (Cd) and lead (Pb) in rice plants subjected to the additionof rapeseed residue alone or in combinations with mineral N fertilizer.

Metal Treatments Concentration (mg kg�1)

Leaves Stems Hulls

Cd N100 10.6a 21.4a 10.4aN70 + R 9.3a 21.8a 10.7aN30 + R 10.0a 18.8a 8.3aR 8.5a 17.6a 6.9aN100 30.7a 18.4a 4.7a

Pb N70 + R 25.4ab 13.8ab 3.0aN30 + R 22.8ab 11.9b 2.9aR 18.6b 6.6c 3.1a

The same letters in the table indicate no significant difference at a 0.05 significancelevel, determined by Tukey’s HSD test (n = 3).

Y.S. Ok et al. / Chemosphere 85 (2011) 677–682 679

2.7. Statistical analyses

The differences among treatment groups were evaluated byone-way ANOVA with Tukey’s honest significant difference (HSD)

test using the SAS statistics program (SAS, 2003). Statistics weretested at a 0.05 significance level.

3. Results and discussion

3.1. Changes in soil chemical properties

Table 1 shows the changes in soil characteristics after eachtreatment. The addition of the R treatment or in combinations withmineral N fertilizer (i.e. N70 + R and N30 + R) lowered the soil pHwhen compared to the N100 treatment. The pH values decreasedsignificantly by 0.50, 0.40 and 0.60 units in soils treated withN70 + R, N30 + R and R, respectively, when compared to the N100treatment. These decreases in soil pH can be attributed to theacidic effect of decomposable products of organic residue of rape-seed. Specifically, the production of organic acids and the release ofH+ during the decomposition of organic amendments in the soil de-crease the soil pH (Bolan et al., 1991; Usman et al., 2004; Ok et al.,2007; Kiiya et al., 2010). In addition, the decrease in soil pH may bedue to the oxidation of mineralized NH4 to NO3 by soil microorgan-isms (Ortiz Escobar and Hue, 2008). On the contrary, the soil pHmay be possibly increased with the application of organic amend-ments due to the release of NHþ4 from organic N mineralization orthe formation of organic aluminum complexes in soil solution (Or-tiz Escobar and Hue, 2008; Kiiya et al., 2010). The values of soil ECtended to increase when the rapeseed residue was applied. Theseincreases of soil EC could be resulted from the solubilization of cat-ions and anions from rapeseed residue formed from organic mattermineralization (Madejon et al., 2001). In addition, the addition ofrapeseed residue as an organic amendment led to significantly in-crease the SOM content and exchangeable cations such as Ca2+,Mg2+, Na+ and K+ in soils. Our results agree with many studies thatthe application of plant residue into soil maintains SOM, improvesthe soil physicochemical properties, and increases nutrient avail-ability (Clark et al., 1998; Goyal et al., 1999; Tirol-Padre et al.,2007; Tejada et al., 2008).

Fig. 2. Effect of the rapeseed residue alone or in combinations with mineral Nfertilizer (N100: 100% mineral N fertilizer, N70 + R: 70% mineral N fertil-izer + rapeseed residue, N30 + R: 30% mineral N fertilizer + rapeseed residue, R:rapeseed residue alone) on geochemical forms of: (a) Cd and (b) Pb in contaminatedpaddy soil.

680 Y.S. Ok et al. / Chemosphere 85 (2011) 677–682

3.2. Changes in soil biological properties

Biological soil environment is to be sensitive to contaminantsand its characteristics are considered as indicators for determiningsoil quality (Kennedy and Papendick, 1995; Yao et al., 2003; Kirket al., 2005; Usman et al., 2005). In this study, the CFU of bacteriaand fungi and the fungal/bacteria (F/B) ratio in the metal contam-inated paddy soil were determined with the applications of rape-seed residue, mineral N fertilizer or their combinations.

Our results indicated that rapeseed residue applied as a greenmanure played a role in increasing microbial, including bacteriaand fungi, populations as shown in Table 2. The increases in soilbacteria and fungi populations were observed in soil subjected tothe R, N70 + R and N30 + R treatments when compared to soilsubjected to the N100 treatment. The highest population of fungi(97 � 104 CFU g�1) was observed in response to the addition of Rtreatment. Similarly, many studies have reported an increase infungi population when various types of green manure were ap-plied (Vinten et al., 2002; Tangjang et al., 2009). Jia et al.(2010a) found that the microbial population has a positive corre-lation with SOM. This likely occurred because an increase ofSOM in response to the application of rapeseed residue providesthe favorite environment to microbe and increases their growthand activity. Additionally, the rapeseed residue in soils can bereadily available organic C forms compared to inorganic chemi-cal fertilizers. The F/B ratio was often used to evaluate the sus-tainability of agriculture systems (de Vries et al., 2006). The F/B ratio decreased with increasing the mineral N application rate,primarily due to suppression of fungal growth. This result sup-ports previous studies of Bardgett et al. (1999), Vinten et al.(2002) and de Vries et al. (2006) showing that the applicationof mineral N fertilization reduces the F/B ratio whereas the addi-tion of organic amendments into soils increases the F/B ratio be-cause of increasing fungal growth. The organic amendmentshaving a high C/N ratio such as a rapeseed residue (C/N ratioof 63) stimulate fungal growth than bacterial growth (Vintenet al., 2002). Generally, the application of plant residue intothe contaminated paddy soil may sustain SOM, thereby improv-ing soil biological properties. Consequently, the green manureaddition and its effects on soil environments make the signifi-cant contribution to plant survival in the soils contaminatedwith heavy metals.

3.3. Bioavailability of Cd and Pb with rapeseed residue

The concentrations of Cd and Pb, and their values of TF in riceplants grown in the soils subjected to the R, N70 + R and N30 + Rtreatments were presented in Fig. 1 and Table 3, respectively.The value of TF is one of the key parameters as the measure ofan exposure degree of metal to human via plant intake. In thisstudy, the higher TF values were observed for Cd (1.94–2.49) rela-tive to those of Pb (0.02–0.04). These results show that the accu-mulation of Cd is greater than that of Pb in plants. One ofpossible reasons is that Cd has a relatively higher mobility in nat-ure and a lower retention capacity than other toxic metals (Usman,2008). However, the concentration of Pb in leaves was relativelyhigh related to its high concentration in the soil. This result canbe explained by previous investigations reporting that the higherconcentration of heavy metals in the soil is reflected by the higherconcentration of metals in the plants (Piotrowska and Chlopecka,1994; Nan et al., 2000; Jia et al., 2010b).

For treatment effects, the bioaccumulation of Cd and especiallyPb in rice tissues was less for the contaminated paddy soil with theR, N70 + R and N30 + R treatments than with the N100 treatment.Especially, the highest decrease of metal bioavailability was ob-served in the soils with the R treatment, as indicated by a lowerconcentration of metal and a lower TF value. For instance, the addi-tion of the R treatment decreased the Cd concentrations by 20%,18% and 34% in the leaves, stems and hulls of the rice plant, respec-tively, when compared to the N100 treatment. With the R treat-ment, the Pb concentrations in the contaminated paddy soil werealso decreased significantly by 39% and 64% in leaves and stemsof rice plant, respectively, when compared to those with theN100 treatment.

The addition of rapeseed residue reduced the concentrations ofCd and especially Pb by transferring a readily available form, solu-ble plus exchangeable fraction (Fig. 2). The addition of rapeseedresidue led to the redistribution of Cd and Pb forms in the contam-inated paddy soil, resulting in the transformation of heavy metalstowards more stable fractions. The readily available fractions ofmetal were decreased by 5–14% for Cd and 30–39% for Pb depend-ing on the addition of rapeseed residue when compared to theaddition of N100 treatment. In addition, the carbonate fraction ofPb had a small decrease after the addition of rapeseed residue.The highest decrease in readily available fractions of Cd and Pbwas found in soil subjected to the R treatment. In the meantime,their concentrations tended to slightly increase in the carbonatesor Fe/Mn oxides and residual fractions (62.5%). These findingsindicate that the addition of rapeseed residue generally decreasesthe easily accessible fractions of Cd and especially Pb by transform-ing them to less accessible fractions, resulting in lower metal avail-ability to rice plants.

Several studies have reported that organic matter may reducemetal availability and mobility via the redistribution of heavy met-als from the soluble or exchangeable form to fractions associatedwith organic matter, carbonates, Fe/Mn oxides or the residual frac-tions (Shuman, 1999; Walker et al., 2004). In this study, the addi-tion of rapeseed residue did not increase the concentrations of Cdand Pb in the solid-phase of organic matter. Meanwhile, the addi-tion of such organic residue favors heavy metals to react with othersolid-phase fractions, resulting in heavy metals immobilizationand thus they become less available for plant uptake. The heavymetals may be adsorbed by clay minerals, carbonates or hydrousoxides or may be precipitated as metal carbonate, hydroxide andphosphate (McLean and Bledsoe, 1992). Additionally, the redistri-bution of heavy metals among solid-phases depends on the typesof heavy metal and soil, soil pH and organic matter characteristicsrelated to the degree of humification (Shuman, 1999; Stacey et al.,2001; Hashimoto et al., 2011).

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4. Conclusions

This study showed the effects of rapeseed residue applied as agreen manure alone or in combinations with mineral N fertilizeron Cd and Pb speciation in the contaminated paddy soil and theiravailability to rice plant. A significant decrease in the bioavailabil-ity of Pb was observed in response to the addition of rapeseed res-idue. Moreover, the addition of organic materials such as arapeseed residue could alleviate heavy metals toxicity towardplants by redistributing them to less available fractions. Use of ra-peseed residue may help sustain SOM, thereby improving the soilquality and fertility in a highly contaminated paddy area. For fu-ture study, the heavy metals availability in soils with varioussources of crop residue should be investigated.

Acknowledgements

This research was supported by Basic Science Research Programthrough the National Research Foundation of Korea (NRF) fundedby the Ministry of Education, Science and Technology (2009-0071439) and by Cooperative Research Program for AgriculturalSciences & Technology Development (PJ9069612011) of the RuralDevelopment Administration in Korea. Instrumental analysis waspartly supported by Institute of Environmental Research and Cen-tral Laboratory of Kangwon National University in Korea.

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