Relative Value of Phosphate Compounds in Reducingthe Bioavailability and Toxicity of Lead in ContaminatedSoils

Jin Hee Park & Nanthi Bolan &

Mallavarapu Megharaj & Ravi Naidu

Received: 21 April 2011 /Accepted: 30 June 2011 /Published online: 15 July 2011# Springer Science+Business Media B.V. 2011

Abstract Lead forms stable compounds with phos-phate and the immobilized Pb becomes lessavailable to soil biota. In this study, we tested thebioavailabilty of Pb using earthworms (Eiseniafetida) and plants after immobilization of Pb by asoluble P compound and an insoluble rock phosphatecompound in the presence of phosphate-solubilizingbacteria (Enterobacter sp.). Rock phosphate in thepresence of phosphate-solubilizing bacteria and asoluble P compound enhanced Pb immobilization asmeasured by NH4NO3-extractable Pb concentration,thereby reduced its bioavailability as evaluated byearthworm Pb loading and sunflower (Helianthusannuus) Pb uptake under greenhouse conditions.However, soluble P treatment increased the concen-

tration of Pb in soil solution thereby inhibited theroot elongation of mustard (Brassica hirta) seed-lings. Sunflower plants in the Pb-spiked soil withoutP amendments showed symptoms of necrosis andstunting because of Pb toxicity. Both soluble andinsoluble P treatments significantly increased shootand root weight and decreased Pb concentration inshoot by more than 50% compared to the control.However, high Pb concentration in soil solution wasfound in soluble P treatment, which can be attributedto dissolved organic carbon–Pb complex formation,thereby increasing Pb mobility. The inoculation ofphosphate-solubilizing bacteria can facilitate phytos-tabilization of Pb-contaminated site.

Keywords Bioavailability . Pb immobilization .

Phosphate-solubilizing bacteria . Rock phosphate .

Earthworm

1 Introduction

Lead is considered to be a toxic element for bothplants and animals. Lead causes acute and chronictoxicity in human but acute toxicity is actually a rarecondition. The effects of Pb are related to four organsystems: haemopoietic, nervous, gastro-intestinal, andrenal (Conor 1980). The major symptoms of chronicPb toxicity are related to a neuromuscular syndromeincluding paralysis of the peripheral motor nerves andanemia (Johnson 1998). In addition, Pb poisoning can

Water Air Soil Pollut (2012) 223:599–608DOI 10.1007/s11270-011-0885-7

J. H. Park :N. Bolan :M. Megharaj :R. NaiduCentre for Environmental Risk Assessmentand Remediation, University of South Australia,Mawson Lakes, SA, Australia

J. H. Park :N. Bolan :M. Megharaj :R. NaiduCooperative Research Centre for ContaminationAssessment and Remediation of the Environment,Adelaide, SA, Australia

J. H. Park (*)Centre for Mined Land Rehabilitation,University of Queensland,Sir James Foots Building,St. Lucia, QLD 4072, Australiae-mail: uqjpark7@uq.edu.au

cause learning disabilities, attention deficit disorders,lowered IQ, and antisocial behavior in children (He etal. 2009; Lidsky and Schneider 2003).

Organisms dwelling in soil are sensitive to Pb. Forexample, Pb affects cocoon production and mortality ofearthworms. Davies et al. (2003) tested the toxicity ofvarious Pb compounds on earthworms and noticed thatPb toxicity decreased in the order nitrate>carbonate>sulfide, which is attributed to the difference in thesolubility of Pb compounds. The mean LC50 value(lethal concentration when 50% of mortality occurs)for earthworms was 5,321±275 mg Pb/kg.

The responses of plants to heavy metal toxicity varyaccording to the bioavailable concentration in soil,duration of the exposure period, growth stage of plant,and activity of plant roots and their associated micro-organisms (Ernst and Nelissen 2000; MacFarlane andBurchett 2002). Main toxic effects of Pb to plantinclude depression on seed germination, disturbance inmitosis, induction of leaf chlorosis, inhibition of rootand shoot growth, reduction in photosynthesis andtranspiration, and inhibition of DNA synthesis andenzymatic activities (Islam et al. 2007).

There are three main levels that control the bioavail-ability when metals are released to soil ecosystem,including the substrate level, uptake level, and targetlevel (Weltje 1998). The first, substrate level, deals withcompetitive sorption interactions, resulting in a specificdistribution of the interacting metals over the solid andliquid phases of the soil. This process changes thebioavailable fraction of metals. The second and thirdlevels describe interactions of metals during uptakeand at target sites within an organism, respectively(Weltje 1998). Due to these interactions, bioavailabilityand toxicity of metals to organisms are closely related.Therefore, the distribution of metals over the soilphases is critical to organisms in soil because only themetal concentration in the liquid phase is consideredavailable to them.

Since in situ Pb stabilization techniques do notdecrease total Pb content in soil, it is important toreduce Pb bioavailability as well as to achieve a long-term stabilization of Pb (Cao et al. 2002). Lead formsstable compounds with phosphate and the effective-ness of P compounds in immobilizing Pb fromcontaminated soils primarily depends on their solu-bility (Ma and Rao 1999; Park et al. 2011b). Althoughthe soluble-P treatment has often been shown toimmobilize Pb effectively in soils, the use of soluble P

as a soil amendment needs attention due to thepossibility of P leaching leading to eutrophication(Basta and McGowen 2004; Cao et al. 2002; Park etal. 2011a). Hence, soluble P sources may not be suitablefor the remediation of Pb, especially in low P-retainingsandy soils. Instead of soluble P, insoluble P sources canbe used to immobilize Pb with the aid of solubilizingagents such as phosphate-solubilizing bacteria (PSB).Phosphate-solubilizing bacteria have been shown toincrease Pb immobilization in soil by enhancing thesolubilization of insoluble P compounds (Park et al.2011b). Most research efforts evaluating the immo-bilization of heavy metals have focused on reducingtheir solubility and extractability in soils (Basta andMcGowen 2004) and only a limited work has testedthe effect of immobilization on subsequent bioavail-ability to soil biota including earthworms and plants.

In this study, Pb-contaminated soils treated with Psources in the presence or absence of PSB was testedfor the bioavailabiliy of immobilized Pb to earth-worms (Eisenia fetida) by monitoring Pb accumula-tion in earthworms. Phytoavailability and toxicity ofimmobilized Pb were tested using germination andmetal uptake experiments using sunflower as a testplant. The objective of this study was to examine thebioavailability of immobilized Pb by phosphatecompounds to earthworms and plants.

2 Methods

2.1 Soils Used

Soil used for this research was collected from anuncontaminated site and spiked with Pb. Also, a Pb-contaminated soil (600 mg Pb/kg) from a shooting rangewas used for germination experiment. The soil sampleswere air-dried and sieved (<2 mm). The spiked soilsample was prepared by mixing the soil with Pb(NO3)2solution at a concentration of 2,000 mg Pb/kg soil. Thespiked soil was incubated at 37°C with three cycles ofwetting and drying over a period of 20 days (Si et al.2006). The soil samples were analyzed for pH, Olsen P,total and NH4NO3-extractable Pb concentrations fol-lowing the methods described by Park et al. (2011b).Soil texture was analyzed using a micropipette method(Miller and Miller 1987). Soil organic matter (%) wasanalyzed with the Walkley–Black method (Nelson andSommers 1996). The maximum water-holding capacity

600 Water Air Soil Pollut (2012) 223:599–608

(WHC) of the soil was measured using the Grantmethod (Grant and Tingle 2002). The properties ofsoils are presented in Table 1.

2.2 Preparation of Bacterial Suspension

Phosphate-solubilizing bacteria, Enterobacter sp.(accession number; GQ414735) isolated from Pb-contaminated soil of a Zn and Pb smelter were usedfor this research (Park et al. 2011b). Bacteria grownin 100 mL of trypticase soy yeast medium containing(per liter) 30 g trypticase soy broth and 3 g yeastextract for 48 h were harvested by centrifugation at4,000×g/min for 20 min. The cells were resuspendedin a sterilized saline solution (8.5 g NaCl/L Milli-Qwater). Bacterial cells were washed twice with asaline solution and twice with sterile Milli-Q waterand resuspended in 100 mL of sterile Milli-Q water(ca. 1×109 colony forming unit per milliliter).

2.3 Incubation of Soils with P Amendments

The Pb spiked soils were pre-amended with soluble(KH2PO4) or insoluble P (rock phosphate (RP);800 mg P/kg soil) sources. Bacterial suspension (1%v/w) was added to the RP-amended soil and sterileMilli-Q water was added to the control instead ofbacterial suspension. Moisture content was kept at60% WHC of the soil. After incubation of soils with Psources, the soil samples were air-dried and analyzedfor pH, EC, and 1 M NH4NO3-extractable Pb asdescribed in Section 2.1. Water-extractable Pb con-centration was analyzed by inductively coupledplasma mass spectroscopy (ICP-MS, Agilent) afterextracting soil solution (soil/water=1:5). Dissolvedorganic carbon was analyzed using an automatic totalorganic carbon analyzer (Model 1010, O.I. Analytical,USA) after extracting soil solution. Phosphate-solubilizing bacterial population was measured bythe plate-counting method and the total microbial

activity was measured using dehydrogenase enzymeactivity (Casida et al. 1964; Singh and Singh 2005).

2.4 Germination Test

The germination test was conducted using Pb-contaminated shooting range soil to examine theeffect of P amendments on Pb toxicity to plants (DiSalvatore et al. 2008). Germination rate and rootelongation have been used to represent the rapidbioavailability (i.e., phytotoxicity) test because oftheir simplicity, sensitivity, and low cost (Wang et al.2001). After incubation of soil (30 g) with P amend-ments and PSB, surface sterilized yellow mustard(Brassica hirta) seeds (15 per plate) were placed onthe surface of soils in Petri dish and incubated in agrowth chamber at 25°C in dark condition. Seedgermination rate and root elongation were checkedafter 4 days of incubation of seeds. The percentage ofrelative seed germination (RSG, Eq. 1), relative rootgrowth (RRG, Eq. 2), and germination index (GI,Eq. 3) were calculated (Ko et al. 2008).

RSG %ð Þ ¼ number of seed germination in treatment

number of seed germination in the control� 100

ð1Þ

RRG %ð Þ ¼ mean root length per seedling in treatment

mean root length per seedling in the control� 100

ð2Þ

GI %ð Þ ¼ %seed germination�%root germination

100%ð3Þ

2.5 Earthworm Ecotoxicity Test

Adult earthworms were washed and depurated for24 h before exposure to soils. Ten mature E. fetida

Table 1 Properties of soils used

Soil Texture WHC (%) pH NH4NO3 extractablePb (mg/kg)

Total Pb(mg/kg)

Olsen P(mg/kg)

Organicmatter (%)

Shooting range soil Sandy loam 38.5 5.88 28.7 346 3.78 0.70

Pb spiked soil Silt loam 73.0 5.23 42.7 2000 3.74 10.9

Water Air Soil Pollut (2012) 223:599–608 601

were weighed and added to each of three replicatesand cultured in 100 g of pretreated soils with Pamendments and PSB at 18°C with continuous light.At the end of 2 weeks, earthworms were depurated for24 h, weighed and put into 75-mL glass tubes. Fivemilliliters of HNO3 was added into the glass tube andkept overnight. On the next day, the tubes were heatedfirst at 75°C for 30 min and then at 100°C for 30 min.After digestion, the samples were diluted with Milli-Qwater and Pb concentration was analyzed by ICP-optical emission spectroscopy (OES, Agilent).

2.6 Phytoavailability Test

Pre-incubated silt loam soils (400 g) with P amend-ments and PSB placed into 600-mL opaque plasticpots. Rhizon sampler (one per pot) was horizontallyplaced in 2 cm from the bottom of the pot to extractpore water. Surface-sterilized sunflower (Helianthusannuus) seeds (five seeds per pot) were sown directlyinto the soils on 09 Feb 2009 and the pots werecovered with transparent plastic bags for 3 days tomaintain the moisture content. Sunflower was chosento assess the bioavailability of Pb in this studybecause of its high biomass, rapid growth (Usmanand Mohamed 2009), and ability to take up Pb (Lin etal. 2009). The number of seedlings was thinned totwo per pot 1 week after germination. The sunflowerswere grown in greenhouse with natural light at 28°C.The pots were watered every day and plants wereharvested at 6 weeks after germination (02 April2009). Three milliliters of soil solution was takenusing the rhizon sampler at 4, 7, 14, 21, 28, 42, and56 days after the establishment of the pot experimentand analyzed for metals using ICP-MS and fordissolved organic carbon (DOC) using an automatictotal organic carbon analyzer.

The plant parameters monitored in this experimentwere selected based on the ecological effects testguidelines of the US Environmental Protection Agency(1996). Shoot height and visual phytotoxicity wererecorded on the day of harvest. Roots were manuallyextracted from soil. After harvesting the plants, theshoots and roots were separated, washed with Milli-Qwater, oven-dried, and ground. Dry weights of shootsand roots were measured after harvesting plants. Theground plant material (0.500 g) was weighed directlyinto a 75-mL digestion tube with 5 mL of concentratednitric acid and left to cold digest in a fume cupboard

overnight. After keeping the samples overnight, thetubes were heated using a temperature-controlleddigestion block (AI Scientific Block DigestionSystem AIM 500, Australia) programmed to slowlyincrease the temperature to 140°C until ca. 1 mL ofdigest remained in the tube. The tubes were broughtto room temperature prior to dilution with Milli-Qwater. The samples were mixed thoroughly andfiltered with a syringe filter and analyzed for metalsby ICP-OES.

2.7 Statistical Analysis

All experiments were conducted with three replicates.The data collected were analyzed statistically usingSPSS 17 software (IBM, Australia). Duncan’s multi-ple range test was used to compare the means of thetreatments, variability in the data were expressed asthe standard deviation, and a P<0.05 was consideredto be statistically significant. Relationships betweendifferent data were evaluated by means of simplelinear regression analysis. The acceptance limit wasset to 95% significance level.

3 Results and Discussion

3.1 Lead Immobilization in Soil

Ammonium nitrate-extractable Pb concentration sig-nificantly decreased when soluble P and insoluble Pwere added to soil (Fig. 1), indicating the immobili-zation of Pb. Ammonium nitrate (1 M) extraction hasbeen used to assess the influence of the amendmentson the labile metal pools (Friesl et al. 2003).Inoculation of PSB was found to have an enhancedability for immobilizing Pb with RP in soil. However,slightly acidic pH (5.7) of the soil might have alsocontributed to RP dissolution because low soil pHenhances solubility of RP (Table 2). Water-extractablePb concentration did not coincide with NH4NO3-extractable Pb concentration (Fig. 1). Water-extractable Pb concentration was significantly higherin soluble P-treated soils than other treatments. Water-extractable Pb concentration was highly related withDOC concentration of soil (R2=0.72, P<0.05).Turpeinen et al. (2000) showed a significant positivecorrelation between the solubility of Pb and theconcentration of DOC in soil solution. Soluble P

602 Water Air Soil Pollut (2012) 223:599–608

amendment increased DOC concentration of soilsolution. It was reported that phosphate sorption tothe soil increased DOC in solution because phosphateions compete for surface sites occupied by organiccompounds (Giesler et al. 2005; Park et al. 2011a).

Some PSB were found in uninoculated soils becausethe soils were not sterilized prior to the inoculation ofPSB, but the number of PSB was significantly higher inPSB inoculated soil than the control (Table 2). Soildehydrogenase activity increased with bacteria inocu-

lation. Although several PSB species occur in soils,their numbers are usually not high enough espe-cially in Pb-contaminated soils to solubilize P inorder to achieve the immobilization of Pb. There-fore, inoculation of PSB at a much higher concen-tration than that normally found in soil is necessaryto achieve Pb immobilization and P uptake byplants (Rodríguez and Fraga 1999).

3.2 Seed Germination

There was no significant difference in seed germina-tion rate (Table 3). Root elongation was significantlylower in soluble P-treated soil due to high concentra-tion of water-soluble Pb. It is known that rootelongation is more sensitive to soluble metal concen-tration than seed germination (Valerio et al. 2007).Cell division at the root tip and cell elongation in theextension zone is affected by the presence of metals(Arduini et al. 1994). Di Salvatore et al. (2008)reported that 0–1024 μM of Pb solution showed noeffect on seed germination of lettuce (var. trocadero),but root elongation was inhibited by the increasing Pbconcentration. The RSG, RRG, and GI were higher inthe RP-treated soils than the control, indicatingreduced Pb phytotoxicity of RP-treated soil (Table 3).However, GI index of soluble P-treated soil was lowerthan untreated control. Although soluble P treatmentimmobilized Pb by reducing NH4NO3-extractable Pb,the high water-soluble Pb concentration in thistreatment inhibited germination parameters whereasRP with PSB treatment enhanced root elongation dueto low water-soluble Pb concentration.

3.3 Earthworm Pb Loading

Both soluble and insoluble P treatments significantlyreduced earthworm Pb loading in soil compared to thecontrol (Fig. 2). Reduced Pb concentration by RP, RPwith PSB, and soluble P was 5.9%, 10.2%, and14.4%, respectively, compared to the control. Ownbyet al. (2005) investigated the bioavailability of Pb tothe earthworm, E. fetida, after amending a Pb- andZn-contaminated soil with four different forms of Pcompounds. Triple superphosphate at 5,000 mg P/kgdecreased Pb bioavailability as measured by Pbaccumulation in soil, but 5,000 mg P/kg of RP didnot decrease Pb bioavailability to earthworms becauseZn probably competed with Pb for immobilization

Table 2 pH, DHA and PSB population of soils after incubationwith P amendments and PSB

Treatments pH DHA (mg TPF/kgdry soil/24 h)

PSB (CFU/gdry soil)

S(Control) 5.71±0.023 a 7.43±1.42 a 2.8×102

SRP 5.70±0.060 a 32.4±1.12 b 4.8×103

SRPB 5.73±0.015 a 37.9±5.50 b 9.3×106

SP 5.76±0.015 a 21.0±0.32 c 6.8×104

Data are the means±standard deviation of three replicates.Means with the same letter within a column are not signif-icantly different at p<0.05 by Duncan’s multiple range testusing SPSS 17 (S soil, SRP soil+rock phosphate, SRPB soil+rock phosphate+PSB, SP soil+KH2PO4)

DHA dehydrogenase activity, TPF triphenyl formazan, PSBphosphate-solubilizing bacteria, CFU colony forming unit

TreatmentsS SRP SRPB SP

Pb c

once

ntra

tion

(mg/

kg)

0

1

2

3

4

5

615

20

25

30

35

NH4NO3Water

a

b

c

d

b b b

a

Fig. 1 Ammonium nitrate- and water-extractable Pb concentra-tion of soil after earthworm experiment. Means with the sameletter within a phase are not significantly different at p<0.05 byDuncan’s multiple range test using SPSS 17. Each valuerepresents the mean of three replicates with standard deviationshown by error bars (S soil, SRP soil+rock phosphate, SRPBsoil+rock phosphate+PSB, SP soil+KH2PO4)

Water Air Soil Pollut (2012) 223:599–608 603

due to the low level of P released from RP. Therefore,the inoculation of PSB is likely to enhance thesolubilization of RP, thereby improving Pb immobiliza-tion and reducing Pb toxicity to soil organisms even inthe presence of other metal contaminants. Although weamended a much reduced amount of P (800 mg P/kg)into soil compared to the study of Ownby et al. (2005;5,000 mg P/kg soil), PSB inoculation solubilized RPand reduced Pb bioaccumulation by earthworm. Therewas no mortality of earthworms in the soil becausesignificant mortality has been reported only whenearthworm Pb load exceeded ca. 100 mg/kg ofearthworm (Davies et al. 2003). However, there wasweight reduction in earthworms after the incubation ofearthworms in Pb-contaminated soil (Fig. 2). Weight

reduction was also regarded as an indicative factor ofPb toxicity to earthworms.

Positive relationships between earthworm tissuemetal concentration and total soil or soil solutionmetal concentration have been reported (Nahmani etal. 2007). Earthworm Pb accumulation was comparedwith NH4NO3-extractable Pb concentration of soil.Ammonium nitrate-extractable Pb concentration insoil was positively correlated with earthworm Pbloading (Fig. 3, R2=0.63, p<0.01). Davies et al.(2003) showed that both water- and DTPA-extractablePb of spiked soil positively correlated with Pbaccumulation in earthworms. Similarly, Suthar et al.(2008) collected metal-contaminated soils and earth-worms dwelling in these soils, and analyzed DTPA-extractable metals of soils and metals in earthwormbodies. They obtained a significant positive correla-tion between Pb concentration in worm tissues andsurrounding soils (R2=0.63, p<0.01).

Table 3 The percentage of relative seed germination (RSG), relative root growth (RRG) and germination index (GI) with yellowmustard in soil after Pb immobilization

Samples Germination rate (%) Root length (cm) RSG (%) RRG (%) GI (%)

S 97.8 a 0.80±0.083 ab – – –

SRP 100 a 1.0±0.034 a 102 126 128

SRPB 100 a 0.87±0.204 a 102 110 112

SP 97.8 a 0.59±0.059 b 100 74.0 74.0

Data are the means of three replicates. Means with the same letter within a column are not significantly different at p<0.05 byDuncan’s multiple range test using SPSS 17 (S soil, SRP soil+rock phosphate, SRPB soil+rock phosphate+PSB, SP soil+KH2PO4)

Treatments

Pb c

once

ntra

tion

of e

arth

wor

m b

ody

(mg/

kg)

0

20

40

60

80

100

Red

uctio

n in

ear

thw

orm

wei

ght (

%)

0

10

20

30

40

50

a

ab

S SRP SRPB SP

b b

Fig. 2 Pb concentration (bars) and weight reduction (line) ofearthworms after 2 weeks of exposure in soils. Means with thesame letter within a phase are not significantly different at p<0.05by Duncan’s multiple range test using SPSS 17. Each valuerepresents the mean of three replicates with standard deviationshown by error bars (S soil, SRP soil+rock phosphate, SRPBsoil+rock phosphate+PSB, SP soil+KH2PO4)

NH4 NO3 extractable Pb concentration of treated soils (mg/kg)

16 18 20 22 24 26 28 30 32 34

Pb c

once

ntra

tion

of e

arth

wor

m b

ody

(mg/

kg)

55

60

65

70

75

80

Y=0.74X+47.03

R2 =0.63

Fig. 3 Correlation between NH4NO3-extractable Pb concen-tration of treated soils and earthworm Pb accumulation

604 Water Air Soil Pollut (2012) 223:599–608

3.4 Plant Growth and Lead Uptake

Sunflower plants in Pb-spiked soil without Pamendments showed symptoms of necrosis andstunting. Although Pb is not lethally toxic to plantsat some level, chlorosis, necrosis, and stuntedgrowth are the common visual symptoms of Pbtoxicity (Chen et al. 2009; Tarradellas et al. 1997).The effect of Pb on plant growth depends on Pbavailability and plant species. Lead mainly affectsseed germination, seedling growth, photosynthesis,plant water status, mineral nutrition, and enzymaticactivities (Patra et al. 2004).

Both soluble and insoluble P treatments signifi-cantly increased shoot and root weight. Shoot dryweight increased by 315%, 329%, and 541% for RP(SRP), RP with PSB (SRPB), and KH2PO4 (SP)treatments, respectively, compared to the control (S)(Fig. 4). Chen et al. (2009) amended Pb-contaminatedsoil with a number of soil amendments including Pcompounds at various levels and tested Pb uptake bycauliflower (Brassica oleracea L.). The addition of Pamendments increased the shoot and root biomass,which was attributed to both decrease in Pb toxicityand increase in P nutrition (Chen et al. 2009). Cao etal. (2009) also reported increase in soybean (Glycinemax L.) shoot biomass and decrease in metalaccumulation when phosphoric acid and RP were

amended together into a Cu-, Zn-, and Pb-contaminated soil. However, there was no increasein biomass for RP-only treatment, which might becaused by low availability of P from RP. Biomassincrease in P-treated soils in this study might beattributed to both reduction in Pb toxicity as Plowered NH4NO3-extractable Pb, and increased Psupply as indicated by high P concentration of plantsin P treated soils (Fig. 5).

Phosphorus amendments significantly reducedshoot and root Pb concentration (Fig. 6). SRP, SRPB,and SP decreased Pb concentration of sunflower shootby 57%, 64%, and 76%, respectively, compared to S.There was a sixfold higher accumulation of Pb in rootthan in shoot. It has often been reported that Pbaccumulation occurs more in root than in shoot(Caille et al. 2005; Chen et al. 2009). Similar toshoot Pb concentration, the root Pb concentration wasalso significantly reduced in RP and soluble Ptreatments. Ammonium nitrate-extractable Pb concen-tration for various treatments in soil was correlatedwith Pb concentration in plant shoot (R2=0.75 and0.66 for shoot and root, respectively, p<0.05).

Metal uptake by plants depend mainly on theconcentration and speciation of the metal in soil solution,diffusion of metals from bulk soil to the root surface,entry into the root, and finally, transfer from root to shoot(Patra et al. 2004). To assess the effect of P treatment on

Pot treatmentS SRP SRPB SP

Wei

ght (

g)

0

1

2

3

4Dry shoot weightDry root weight

c

c

b

b

b

b

a

a

Fig. 4 Average shoot and root biomass per plant. Means withthe same letter within a category are not significantly differentat p<0.05 by Duncan’s multiple range test using SPSS 17. Eachvalue represents the mean of three replicates with standarddeviation shown by error bars (S soil, SRP soil+rockphosphate, SRPB soil+ rock phosphate+PSB, SP soil+KH2PO4)

Pot treatment

S SRP SRPB SP

P co

ncen

trat

ion

of p

lant

(m

g/kg

)

0

500

1000

1500

2000

2500

3000

Shoot Root

bcc

c bc

b b

a

a

Fig. 5 Phosphorus concentration in shoot and root ofsunflower. Means with the same letter within a categoryare not significantly different at p<0.05 by Duncan’s multiplerange test. Each value represents the mean of three replicateswith standard deviation shown by error bars (S soil, SRP soil+rock phosphate, SRPB soil+rock phosphate+PSB, SP soil+KH2PO4)

Water Air Soil Pollut (2012) 223:599–608 605

translocation of Pb from root to shoot, root to shoot Pbconcentration ratio was calculated for various treatments(Chen et al. 2009). The calculated root to shoot ratiowas 6.00, 8.68, 10.5, and 12.1 for S, SRP, SRPB, andSP, respectively. Low (less than one) root to shoot ratioindicates a ready transfer of Pb from root to shoot (Chenet al. 2009). Phosphorus treatment and PSB reduced thetransport of Pb from root to shoot as indicated by higherroot to shoot ratio compared to the control. Wong andLau (1985) also reported a reduction in the transport ofPb from root to shoot in Brassica chinensis andRaphanus sativus with increasing P concentration,which may be attributed to the immobilization of metalsin the vascular system (Padmavathiamma and Li 2010).Metals form carbonate or phosphate precipitates inapoplastic (extracellular) and symplastic (intracellular)compartments in the root (Raskin et al. 1997).

Although many studies have shown that soluble Ptreatments such as phosphoric acid, single superphos-phate and potassium dihydrogen phosphate, andinsoluble P treatments such as RP and hydroxyapatitesignificantly reduce Pb uptake of plants, no study hasexamined the effect of PSB in Pb bioavailability byinsoluble P compounds. Hettiarachchi and Pierzynski(2002) investigated the effect of P treatment on Pbphytoavailability in metal-contaminated soil. Theaddition of P as triple superphosphate significantlyreduced Pb concentration in shoot of sudax (Sorghumvulgare L.) and Swiss chard (Beta vulgaris L.).

Brown et al. (2004) observed a reduction in Pbconcentration of tall fescue with the treatments oftriple superphosphate, phosphoric acid, and RP.Although P addition is the most effective treatmentin Pb immobilization, it is likely to exacerbate theproblem of eutrophication of surface water byexcessive P (Brown et al. 2004). Hence, soluble Psources may not be suitable for the remediation of Pb-contaminated soils. Instead of soluble P, insoluble Psources can be used to immobilize Pb with the aid ofsolubilizing agents such as PSB.

Lead concentration in pore water decreased withsunflower growth, which may be attributed to theplant uptake of Pb from pore water (Fig. 7). Leadconcentration of pore water in soluble P-amended soilwas higher than other treatments. This trend wasconsistent with the water-extractable Pb resultsobtained in Section 3.1. The higher Pb concentrationin pore water in soluble P treatment was attributed toDOC concentration in pore water. There was asignificant correlation (R2=0.80, p<0.05) betweenDOC and Pb concentrations in pore water, indicatingthat DOC formed a complex with Pb, thus increasingPb mobility as described in Section 3.1. Phosphate-solubilizing bacteria inoculation with RP reduced Pbconcentration in soil solution up to 40 days of sunflowergrowth (Fig. 7). This effect can be explained by Psolubilization by PSB from RP and subsequentimmobilization of Pb through lead–phosphate precip-itation (Park et al. 2011b, c).

Pot treatmentS SRP SRPB SP

Pb c

once

ntra

tion

(mg/

kg b

iom

ass)

0

50

100

150

200

250

300

900

1200

1500

1800

2100ShootRoot

a

b bc

a

bc

d

Fig. 6 Lead concentration in shoot and root of sunflower.Means with the same letter within a category are notsignificantly different at p<0.05 by Duncan’s multiple rangetest using SPSS 17. Each value represents the mean of threereplicates with standard deviation shown by error bars (S soil,SRP soil+rock phosphate, SRPB soil+rock phosphate+PSB,SP soil+KH2PO4)

Time (days)

0 10 20 30 40 50 60

Pb c

once

ntra

tion

in p

ore

wat

er (

mg/

L)

0

1

2

3

4

5

S SRPSRPB SP

Fig. 7 Changes in Pb concentration of pore water with plantgrowth. Each value represents the mean of three replicates withstandard deviation shown by error bars (S soil, SRP soil+rockphosphate, SRPB soil+ rock phosphate+PSB, SP soil+KH2PO4)

606 Water Air Soil Pollut (2012) 223:599–608

4 Conclusions

Phosphate-solubilizing bacteria with RP significantlyenhanced Pb immobilization and subsequently reducedPb toxicity to soil organisms. Insoluble P compoundswith PSB can achieve the immobilization of Pb byslowly releasing P, and also provide P and Ca nutrientsto plants. Phosphorus application is likely to enhancethe remediation of Pb contaminated sites by bothimmobilizing Pb and initiating vegetation. While Pbimmobilization reduces phytotoxicity, vegetation isessential to keep the soil containing the immobilizedPb intact (Hettiarachchi and Pierzynski 2002). Vegeta-tion cover initiates soil development on the contami-nated site and helps to achieve extensive and long-termphytostabilization (Mertens et al. 2004). Since naturalestablishment of vegetation in metal-contaminatedsoils is difficult due to phytotoxicity, immobilizationof toxic metals is needed prior to vegetation. There-fore, immobilization of Pb using P compounds andPSB can promote vegetation of metal-contaminatedbarren sites.

Acknowledgment This study was sponsored by Coopera-tive Research Centre for Contamination Assessment andRemediation of the Environment (CRC CARE), Australia incollaboration with the University of South Australia. Theauthors thank Dr. Mohammad Rahman for technical assis-tance with the ICP analysis.

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