effects of soil amendments on growth and metal uptake by ocimum gratissimum grown in...

Effects of Soil Amendments on Growth and Metal Uptakeby Ocimum gratissimum Grown in Cd/Zn-Contaminated Soil

Rattanawat Chaiyarat & Rujira Suebsima &

Narupot Putwattana & Maleeya Kruatrachue &

Prayad Pokethitiyook

Received: 6 August 2009 /Accepted: 6 April 2010 /Published online: 19 May 2010# Springer Science+Business Media B.V. 2010

Abstract Hydroponic and pot experiments were con-ducted to assess the uptake of heavy metals (Cd and Zn)by a common crop plant, African basil, Ocimumgratissimum. In addition, the effects of soil amend-ments, hydroxyapatite (HA) and cow manure on plantgrowth and metal accumulations were compared. In thehydroponic study, plants were exposed to variousconcentrations of Cd (2.5 and 5 mg L−1) and Zn (10and 20 mg L−1) for 15 days. O. gratissimum wasshown to be a Cd accumulator more than a Znaccumulator. Cadmium concentration in its shootsexceeded 100 mg kg−1. In the pot experiments, soilsfrom a heavily Cd-contaminated site (Cd 67.9 mg kg−1

and Zn 2,886.8 mg kg−1) were treated with cowmanure and HA at the rates of 10% and 20% (w/w),and 0.75 and 1.5% (w/w), respectively. Plants were

grown in the greenhouse for 3 months. The addition ofcow manure resulted in the highest biomass productionand the lowest accumulations of Cd in plant parts,while HA was more efficient than cow manure inreducing Zn uptake. Leaves of African basil showed adecreased Cd concentration from 1.5 to 0.3 mg kg−1

(cow manure) and decreased Zn concentration from69.3 to 34 mg kg−1 (HA). This clearly demonstratesthe efficiency of HA and cow manure in reducingmetal content in leaves of plants grown on high metal-contaminated soil to acceptable or close to acceptablevalues (0.2 mg kg−1 for Cd, 99.4 mg kg−1 for Zn).

Keywords Ocimum gratissimum . Cadmium . Zinc .

Cowmanure . Hydroxyapatite

1 Introduction

Heavy metal accumulation in soils is of concern inagricultural production due to its adverse effects on foodquality, crop growth, and environmental health (Islam etal. 2007). In Mae Sot District, Tak Province, westernThailand, areas of known nonferrous mineralizationadjacent to rice-based agricultural systems and high Cdcontamination of paddy has been reported (Simmons etal. 2005). This Cd contamination is associated withsuspended sediment transported to fields via irrigation(Simmons et al. 2005).

Crop plants growing on heavy metal-contaminatedsoils can accumulate high concentrations of trace

Water Air Soil Pollut (2011) 214:383–392DOI 10.1007/s11270-010-0430-0

R. Chaiyarat :R. Suebsima :N. Putwattana :P. PokethitiyookDepartment of Biology, Faculty of Science,Mahidol University,Bangkok, Thailand

M. KruatrachueDepartment of Biology, Faculty of Science,Mahidol University International College,Bangkok, Thailand

R. Chaiyarat (*)Faculty of Environment and Resource Studies,999 Phuthamonthon 4 Road, Salaya Phutthamonthon,Nakhon Pathom 73170, Thailande-mail: [email protected]

elements that cause serious health risks to consumers(Islam et al. 2007). High concentrations of Cd, Zn,Cu, and Pb have been reported in several crop species(Yang et al. 2002; Ni et al. 2002; Long et al. 2003;Xiong and Wang 2005; Zheljazkov et al. 2006). InThailand, excess Cd was discovered in rice, soybeans,and garlic, grown in the vicinity of a Zn mining areawhere Cd occurs as an accessory mineral (Simmons etal. 2005).

Various soil amendments, both inorganic andorganic, have been used to immobilize heavy metalsin contaminated soils. Inorganic materials that havebeen tested with success to reduce Cd availability toplants include rock phosphate, apatite, hydroxyapatite(HA), iron and manganese oxides and oxyhydroxides,and liming agents (Keller et al. 2005). Organicamendments are low-cost adsorbents that will addplant nutrients and improve soil water-holding capacityand may also help reduce phytotoxicity resulting inthe increases in plant growth and survival (Levy etal. 1999; Li et al. 2001). Farmyard manure, cow orpig manure, and compost decrease bioavailability ofheavy metals in soil and crop plants (Ram andVeerloo 1985; Narwal and Singh 1998; Walker et al.2003; Pichtel and Bradway 2008).

Members of the Ocimum species have beencultivated as cash crops for herbal tea and essentialoils in Europe. In Thailand and elsewhere inSoutheast Asia, the leaves of Ocimum basilicum, O.citriodurum, O. tenuiflorum, and O. gratissimum areused in cooking or eaten fresh. Earlier studies havesuggested that some essential aromatic and medicinalcrops might be capable of accumulating heavy metalsfrom contaminated soil (Zheljazkov et al. 2006). Thepresent investigation therefore aimed to (1) determinethe uptake and tolerance of O. gratissimum to Cd andZn in hydroponic and pot experiments and (2)evaluate the efficiency of soil amendments (HA andcow manure) in reducing Cd and Zn concentrationsin O. gratissimum leaves of plants grown in Cd- andZn-contaminated soils.

2 Materials and Methods

2.1 Hydroponic Culture and Growth Condition

Twelve-week-old seedlings (20-cm height) of O.gratissimum (African basil) were obtained from a

farm in Bangkok, Thailand. Plants of uniform sizeand appearance were transferred to plastic containerscontaining 1 L of modified Hoagland’s solution withlow phosphate (0.2 mM KH2PO4) and no EDTA atpH 5.5 and acclimated for 1 week. Subsequently, theywere transferred to modified Hoagland’s solution withvery low phosphate (0.01 mM KH2PO4) at pH 5.5,which contained either 2.5 mg Cd L−1, 5.0 mg CdL−1, 10 mg Zn L−1, and 20 mg Zn L−1, supplied as Cd(NO3)2 and ZnSO4 (MERCK, Demstadt, Germany).For each treatment, three replicates of O. gratissimumwere cultivated in each plastic container. Plants grownin nutrient solution without metals served as controls.All plastic containers were put on shelves equipped withsupplementary lighting (4,600 lx, 12-h photoperiod, 27–28°C) in a randomized complete block design. Thesolutions (without aeration) were changed every 5 daysto prevent depletion of metals and nutrients. Plantswere harvested after 15 days. The concentration ofheavy metals in solution was measured before andafter the solution was changed. Plant samples wereharvested, washed with tap water twice, and rinsedwith deionized water. They were divided into leaves,stems, and roots and oven-dried (70°C) for 2 days toa constant weight. The dry weight of each plant partwas measured.

2.2 Pot Culture and Soil Amendments

Soil samples were collected from a Cd-contaminatedsite at Phadae Village, Mae Sot District, Tak Province,at 0- to 20-cm depth. Soil was air-dried and thenground to pass through a 2-mm nylon mesh sieve.Organic matter was determined by Walkley-Blacktitration (Walkley and Black 1934), total N by theKjeldhal method (Black 1965), available P by theBray II method (Bray and Kurtz 1945), available Kby an atomic absorption spectrophotometer afterextraction with NH4OAc (ICARDA 2001), Ca andMg by an atomic absorption spectrophotometer with1 N ammonium acetate pH 7.0 (Pratt 1965), cationexchange capacity (CEC) by sodium saturation(Chapman 1965), DTPA-extraction followed Lindsayand Norvell (1978), and pH by a pH meter (HI 221,Hanna Instruments)

The soil was treated with two levels of hydroxy-apatite (HA) and cow manure and then incubated for4 weeks. Synthetic HA, Ca5(PO4)3OH (Fluka; pH 7.1;SO4

−2≤0.1%; Cl−, Ni, Co≤0.05%; Na≤0.04%; K≤

384 Water Air Soil Pollut (2011) 214:383–392

0.01%; Zn, Cd, Pb, Fe, and Cu≤0.005%) waspurchased from Sigma-Aldrich Chemicals (UK).Cow manure was air-dried for 1 week and groundbefore analysis and use. The pH of the amendmentswas measured in 1:10 water extracts. After mixingsoil with either cow manure or HA, it was furtherincubated for another 4 weeks, undergoing threecycles of saturation with deionized water and air-drying. Five treatments were set up: Cd/Zn soilwithout amendments; Cd/Zn soil+0.75% HA; Cd/Znsoil + 1.5% HA; Cd/Zn soil + 10% cow manure; andCd/Zn soil + 20% cow manure. HA was applied on a0.75% and 1.5% w/w basis (7.5 and 15 g kg−1 soil,respectively), while cow manure was applied at a rateof 10% and 20% w/w basis (100 and 200 g kg−1 soil,respectively).

The soil was transferred into free-draining 2-kgplastic pots (8 inches in diameter), which had twopieces of 0.1-mm plastic screen at the bottom to retainthe fine particles. Each pot was placed in a separatedish to prevent leaching of metals. Seedlings of O.gratissimum were transplanted into the pots. Oneplant per pot represented one replicate, and therewere three replicates for each treatment. The potswere arranged in a randomized complete blockdesign in a greenhouse (25–28°C, 68% relativehumidity, natural sunlight, 12-h/12-h photoperiod).Plants were watered daily and fertilized (50%strength Hoagland’s solution) once per week. Plantsamples were harvested after 30, 60, and 90 days ofgrowth. At harvest, plants were carefully removedfrom the pots and washed thoroughly with tapwater to remove any attached soil particles. Thenthey were rinsed again with deionized water,divided into leaves, stems, and roots, and oven-dried (70°C) for 2 days to a constant weight. Thedry weight of each plant part was determined.

2.3 Heavy Metals Analyses

To determine Cd and Zn accumulation, dry plantsamples were ground with a mortar and pestle to afine powder and sieved through a 2-mm nylon meshsieve. One-gram dry weight of plant samples wasdigested with 10 mL of a mixture of nitric/perchloricacid (2:1 v/v) (Johnson and Ulrich 1959), andimpurities were removed by filtration through aWhatman No. 42 filter paper. Cadmium and Znconcentration in plant samples were measured by a

flame atomic absorption spectrophotometer (VarianceSpectra AA 55 B).

Soil samples were ground and passed through a2-mm nylon mesh sieve. Half a gram of soil sampleswas digested with 5 mL of aqua regia (Ure 1995), andimpurities were removed by filtration. After digestion,Cd and Zn concentrations in plant and soil sampleswere measured by flame atomic absorption spectro-photometer. Bioavailable Cd and Zn were determinedby diethylene-triamine-penta-acetic acid (DTPA)extraction following Lindsay and Norvell (1978).The standard references of plant and soil materials(soil, MS IHS 3; plant, MS IHS Rp) from theInternational Water Management Institute (IWMI),Bangkok, Thailand, were used to verify the accuracyof metal determination.

2.4 Data Analysis

Growth performance of O. gratissimum was indi-cated by the biomass production and survival rate(Rotkittikhun et al. 2007).

The translocation factor (TF) indicates the plant’sability to translocate heavy metals from the roots tothe harvestable aerial parts (Mattina et al. 2003). Itwas calculated on a dry weight basis.

TF ¼ metal concentration in shoot mg kg�1ð Þmetal concentration in root mg kg�1ð Þ

2.5 Statistical Analysis

Data were expressed as means with standard devia-tion (SD). One-way analysis of variance (ANOVA;SPSS 11.5 computer software [SPSS, Chicago, IL])was used to test the effect of heavy metals and soilamendments on growth and metal contents in plantsand soils. If the F-value showed significant differ-ences (p≤0.05), means were compared using the leastsignificant difference (LSD) method.

3 Results

3.1 Hydroponic Study

After 15 days, across all Cd and Zn treatments, O.gratissimum exhibited 100% survival. There were no

Water Air Soil Pollut (2011) 214:383–392 385

significant differences (p>0.05) in dry biomassproduction between Cd- or Zn-treated plants and thatof controls.

O. gratissimum accumulated metals (Cd, Zn) inroots more than shoots, and the metal concentrationsignificantly increased (p≤0.05) with the increase ofmetal concentration in the solution. The highest Cdaccumulation was found in plants exposed to5 mg L−1 Cd solution (362.2 mg kg−1 in shoots,2,948.8 mg kg−1 in roots) and 20 mg L−1 Zn solution(749.4 mg kg−1 in shoots, 3,765.3 mg kg−1 in roots;Table 1). None of the plants grown in Cd- and Zn-amended nutrient solutions exhibited TF valuesgreater than 1.0 (Table 1).

3.2 Pot Study

3.2.1 Soil Characteristics

The main physicochemical characteristics of Cd/Znsoil and cow manure are shown in Table 2. The pH ofCd/Zn soil was near neutral (7.3) with an EC value of0.5 dS m−1. The soil texture was sandy loam withmoderate organic matter content (2.5%), moderatelevel of P (17 mg kg−1), low level of K (70 mg kg−1),and high levels of Ca (3,200 mg kg−1) and Mg(200 mg kg−1). The total N was 0.1%, and the CECwas 13.8%. The soil contained high levels of Cd(67.9 mg kg−1) and Zn (2,886.8 mg kg−1). Incomparison, cow manure had a higher pH (7.5),higher EC (4.1 dS m−1), much higher CEC (51%),

and organic matter (29.2%). Cow manure containedhigher total N (1.6%), P, K, Ca, and Mg (6,600,10,500, 19,900, and 6,100 mg kg−1, respectively) andlow Cd concentration (0.5 mg kg−1), which did notexceed that of agricultural soils (Cd 0.1–0.6 mg kg−1;Kabata-Pendias 2001). Slightly elevated Zn concen-tration (262 mg kg−1; agricultural soils 20–110 mg kg; Kabata-Pendias 2001) was observed incow manure.

Table 3 shows a comparison of some physico-chemical properties and concentrations of Cd and Znin Cd/Zn soil and HA and cow manure-amendedsoils. The addition of 10% and 20% cow manureresulted in lower pH values (from 7.3 to 7.0–7.1) andmuch higher values of EC (3.2 and 4.1 dS m−1,respectively) and CEC (21.8% and 20.2%, respec-tively) when compared with Cd/Zn soil (EC 0.5 dS m−1,CEC 13.8%) and Cd/Zn soil with HA (EC 0.6-0.9dS m−1, CEC 14.4%–14.8%)

When the total and DTPA-extractable concentrationsof Cd and Zn among all treatments were comparedbefore planting, the addition of cowmanure resulted in asignificant reduction (p≤0.05) of total Cd (10.9%–20.2%) and total Zn (10.2%–22.1%) as compared withthe un-amended control. However, decreases in DTPA-extractable Cd (11.7%–17.4%) and increases in DTPA-extractable Zn (5.5%–7.5%) were observed (Table 3).The addition of 0.75% and 1.5% HA did notsignificantly (p>0.05) change the Cd concentrations(both total and DTPA-extractable), but resulted in18.4%–18.6% increases in DTPA-extractable Zn.

Table 1 Growth performance and heavy metals (Cd, Zn) accumulation in O. gratissimum exposed to various concentrations of heavymetals for 15 days

Metal concentration(mg L−1)

Survivalrate (%)

Dry biomassproduction (g plant−1)

Metal accumulation (mg kg−1) TF

Start Finish Leaf Stem Shoot Root

Cd

0 100 1.4±0.7a 2.5±0.5a 0.0±0.0a 0.0±0.0a 0.0±0.0a 0.0±0.0a 0.0

2.5 100 1.4±0.2a 2.2±0.3a 18.9±7.1b 117.6±59.7b 136.5±59.3b 1,646.4±275.3b 0.08

5 100 1.4±0.5a 2.3±0.1 a 24.7±11.3b 337.5±10.5c 362.2±93.5c 2,948.8±280.5c 0.12

Zn

0 100 1.4±1.2a 2.5±0.5a 0.0±0.0a 0.0±0.0a 0.0±0.0a 0.0±0.0a 0.0

10 100 1.3±0.7a 2.0±0.6a 229.8±39.5b 261.8±61.6b 491.6±66.8b 3,998.9±66.8d 0.12

20 100 1.4±0.3a 2.3±0.4a 263.6±82.3b 485.8±10.2c 749.4±106.3c 3,765.3±506.9d 0.19

Data with different letter in the same column indicate a significant different at 5% level according to Duncan’s test


Total and DTPA-extractable Cd and Zn concen-trations in soil at each monthly harvest are shown inTable 4. The addition of cow manure significantlydecreased the total and DTPA-extractable Cd concen-tration throughout the 3-month experiment (p≤0.05).There was no change in total Zn concentration, but asignificant decreases in DTPA-extractable Zn wereobserved at each harvest (p≤0.05). HA, on the otherhand, did not have the effect on total Cd and Znconcentrations, but a significant decrease of DTPA-extractable Cd and Zn was observed at each harvest(p≤0.05; Table 4).

3.2.2 Growth Performance

There was no significant difference in plant biomassproduction (DW) of O. gratissimum cultivated in

un-amended soil over the entire 3-month period (p>0.05; Table 5). The low survival rate and nominalchange in dry biomass production indicated severephytotoxicity of Cd/Zn soil.

The results indicated that at 1- and 2-monthsampling, no significant difference (p>0.05) in drybiomass production was observed among the amendedtreatments (Table 5). At 2 months, the application ofcow manure (20%) resulted in the highest biomassproduction (23 g plant−1) compared with only 5.6 gplant−1 from the Cd/Zn treatment. At 3 months, plantsgrown in both cow manure and HA-amended soilsexhibited much higher biomass production than thosegrown on Cd/Zn soil (p≤0.05). The highest biomassproduction was found in plants grown on cow manure-amended soils (48.5–59.1 g plant−1; Table 5). Theresults also indicated that O. gratissimum cultivated inamended soils increased biomass over the 3-monthperiod.

3.2.3 Metals Accumulation

O. gratissimum accumulated metals (Cd, Zn) in rootsmore than shoots (Table 6). The addition of cowmanure or HA resulted in decreased Zn (months 1–3)and Cd (months 2–3) accumulation compared withplants grown on Cd/Zn soil (p≤0.05). Cow manureresulted in the lowest accumulation of Cd in theshoots (1.1–1.3 mg kg−1 in stems, 0.3 mg kg−1 inleaves), while HA resulted in the lowest Zn accumu-lation (23.6–24.7 mg kg−1 in stems, 34.0 mg kg−1 inleaves) after 3 months of treatment (Table 6). In thecow manure treatment, 82% decrease in shoot Cdconcentration and 61%–63% decrease in root Cdconcentration when compared with control groupwere observed. In addition, it also resulted in 56%–

Table 2 Physical and chemical properties of Cd/Zn soil andcow manure used in the pot experiment

Parameter Cd/Zn soil Cow manure

pH 7.3 7.5

EC (dS m−1) 0.5 4.1

CEC (%) 13.8 51

Organic matter (%) 2.5 29.2

Soil texture Sandy loam –

Element concentrations

n (%) 0.1 1.6

P (mg kg−1) 17 6,600

Ca (mg kg−1) 3,200 19,900

Mg (mg kg−1) 200 6,100

K (mg kg−1) 70 10,500

Zn (mg kg−1) 2,886.8 262

Cd (mg kg−1) 67.9 0.5

Table 3 pH, EC, CEC, and total and extractabe of Cd and Zn in different treatments before plant growth experiment

Treatment pH EC (dS m−1) CEC (%) Cd concentration (mg kg−1) Zn concentration (mg kg−1)

Total Extractable Total Extractable

Cd/Zn 7.3 0.5 13.8 67.9a 23.6a 2,886.8a 240.7c

Cd/Zn + 0.75% HA 7.4 0.6 14.8 66.0a 24.6a 2,817.8a 285.0a

Cd/Zn + 1.5% HA 7.1 0.9 14.4 66.4a 23.4a 2,891.8a 285.4a

Cd/Zn + 10% cow manure 7.0 3.2 21.8 60.5b 20.8b 2,593.4b 254.6b

Cd/Zn + 20% cow manure 7.1 4.1 20.2 54.2c 19.5b 2,250.2c 258.8b

Data with different letter in the same column indicate a significant different at 5% level according to Duncan’s test


68% decrease in Zn root concentration when com-pared with control. The addition of HA yielded thehighest percentage of decrease (52%–53%) in shootZn concentration when compared with control.

4 Discussion

Hydroponic studies are a quick way to estimate themetal tolerance and accumulation in plants. The resultsof the present study showed that O. gratissimum grownhydroponically was tolerant to Cd and Zn andaccumulated relatively high concentrations of thesemetals in its roots. The translocation factor (TF) valuesfor both Cd and Zn were less than 1 indicating a poorability of this plant to translocate Cd and Zn fromroots to shoots. This is also reflected in the soil potexperiments.

Phytoextraction efficiency for Cd and Zn has beenreported for various aromatic and medicinal plants(Zheljazkov and Nielsen 1996b; Zheljazkov andWarman 2003) such as coriander, dill, chamomile,peppermint basil, hyssop, lemon balm, and sage(Zheljazkov et al. 2008). Earlier studies suggestedthat some aromatic and medicinal crops were capable

of accumulating heavy metals from contaminated soil(Zheljazkov and Nielsen 1996a, b; Scora and Chang1997; Zheljazkov and Warman 2003). In comparison,O. gratissimum accumulated higher Cd concentrationin shoots (362.2 at 5 mg L−1 Cd) compared with otherspecies of aromatic plants grown in 6 mg L−1 Cdsolution (CdCl2) such as O. basilicum or sweet basil(149.5 mg kg−1), Mentha x piperita or peppermint(19.4 mg kg−1) and Salvia offinalis or sage(41.3 mg kg−1) (Zheljazkov et al. 2008). Even thoughAfrican basil accumulated more than 100 mg kg−1 Cdin their shoots, the TF value was still less than 1.Cadmium in shoots of this plant did not reach thehigh-concentration characteristic of hyperaccumula-tors (Brown et al. 1995), but was similar to concen-trations found in known metal accumulators such asBrassica juncea, B. rapa, B. napus, Ipomoea aqua-tica, and Festuca rubra (Ebbs and Kochian 1997;Wang et al. 2008).

The Zn accumulation in shoots of O. gratissimum(749.4 at 20 mg L−1 Zn) was relatively similar to thatin Chinese cabbage B. pekinensis (494 at 25 mg L−1

Zn) and pakchoi B. chinensis (925 at 25 mg L−1 Zn)(Islam et al. 2007). This is much lower than in Znhyperaccumulators (≥10,000 mg kg−1, Baker and

Soil treatment Cd concentration (mg kg−1) Zn concentration (mg kg−1)

Total Extractable Total Extractable

Month 1

1. Cd/Zn 61.2±1.0a 21.7±1.4a 2,514.8±17.5b 203.1±21.3ab

2. Cd/Zn + 0.75% HA 62.4±0.5a 18.7±0.4b 2,512.3±55.2b 188.4±4.8ab

3. Cd/Zn + 1.5% HA 61.9±2.3a 19.1±1.2b 2,755.6±211.3a 193.3±22.2a

4. Cd/Zn + 10% cow manure 58.2±2.5b 14.7±0.4c 2,515.4±125.0b 156.0±4.4c

5. Cd/Zn + 20% cow manure 57.4±0.2b 15.0±0.4c 2,490.5±103.9b 178.4±20.0bc

Month 2

1. Cd/Zn 63.4±0.2a 20.8±1.3a 2,580.3±75.3ab 206.9±13.9a

2. Cd/Zn + 0.75% HA 62.1±0.5ab 18.9±1.0ab 2,591.2±11.1ab 182.6±16.3b

3. Cd/Zn + 1.5% HA 62.6±1.0ab 17.8±1.5bc 2,599.6±70.2a 189.4±16.2b

4. Cd/Zn + 10% cow manure 57.8±5.9bc 16.1±1.0c 2,432.9±140.1bc 165.3±22.2c

5. Cd/Zn + 20% cow manure 54.3±2.5c 15.7±2.0c 2,395.6±91.3c 166.7±27.1c

Month 3

1. Cd/Zn 62.8±0.4a 22.2±3.0a 2,420.8±337.9ab 216.9±52.4a

2. Cd/Zn + 0.75% HA 64.6±1.8a 18.7±0.7a 2,570.7±79.6ab 184.5±25.8b

3. Cd/Zn + 1.5% HA 63.9±1.3a 17.0±0.6a 2,652.1±53.0a 189.6±14.9b

4. Cd/Zn + 10% cow manure 58.0±2.9b 17.6±0.7b 2,381.3±60.9ab 160.0±17.0c

5. Cd/Zn + 20% cow manure 55.2±3.6b 16.7±0.9b 2,279.3±159.5b 168.8±3.5bc

Table 4 Total and DTPA-extractable Cd and Znconcentrations in soils withvarious amendments for3 months

Data with different letter inthe same column indicate asignificant different at 5%level according to Duncan’stest


Brooks 1989). However, the results of the experimentindicated that if O. gratissimum was grown in ahighly heavy metal-contaminated medium, both Cdand Zn may accumulate in shoots above the maxi-mum permissible concentrations for human consump-tion (Zheljazkov et al. 2008).

Results from soil-grown specimens reflect real-world conditions much better than those fromhydroponics (McGrath et al. 2001). The concentra-tions of Cd (67.9 mg kg−1) and Zn (2,886.8 mg kg−1)in soils at Tak were high compared with those in otheragricultural soils (0.1–0.6 mg kg−1 for Cd and 20–110 mg kg−1 for Zn; Kabata-Pendias 2001). Thepresent study shows that cow manure was moreeffective than HA in reducing Cd and Zn concen-

trations in soil. The addition of cow manure resultedin the decreases in total and DTPA-extractable Cd andZn concentrations in soil when compared with un-amended Cd/Zn soil. This is partly due to the dilutioneffect when cow manure was mixed with Cd/Zn soil(Friedland 1989). In addition, the reduction of metalsmay be due to chelation, complexation, and absorp-tion between metals and organic matter in cowmanure (Friedland 1989; Ross 1994). HA amendmentresulted in the reduction of only DTPA-extractable Cdand Zn. This may be due to the small rates ofapplication (0.75% and 1.5%) and the extremely highconcentrations of Cd and Zn in soil. Boisson et al.(1999) demonstrated that the most effective HAconcentration in reducing DTPA-extractable Cd andZn concentrations was 5% HA. Keller et al. (2005)also showed similar results with Cd. They stated thatthe HA amendment was more efficient when Cdconcentrations in soil were low. At higher soil Cdconcentrations, the amount of amendment added wasnot sufficient to immobilize all available Cd.

In the present study, both cow manure and HAtreatments significantly improved the survival rate ofAfrican basil as compared with the un-amendedcontrol. Cow manure application increased the drybiomass production by 8- to 10-fold, while HA onlyincreased the dry biomass 4-fold. Similar findingswere reported by Walker et al. (2003) and Clemente etal. (2003). The major benefits of manure addition tosoil are related to the increased organic matter contentand biological activity. Organic matter from manureacts as a nutrient pool, improves nutrient cycling,increases CEC and buffer capacity, reduces compac-tion, improves soil physical properties, and reducesmetal phytotoxicity (Stewart et al. 2000). HA, on theother hand, had no effect on soil properties, butresulted in reduction of phytotoxicity owing to adecrease of exchangeable amount of Cd and Zn(Mench et al. 1998).

Both cow manure and HA significantly loweredCd and Zn accumulations especially in the shoots(leaves and stems). Cow manure applications (10%and 20%) reduced Cd accumulation 5-fold in leaves,6-fold in stems and 4-fold in roots. Cadmiumconcentration in leaves decreased from 1.5 to0.3 mg kg−1. Cd/Zn soil with cow manure showeddecreases in Cd and Zn concentrations (both total andextractable). On the other hand, HA was moreefficient than cow manure in reducing Zn uptake (2-

Table 5 Growth performance of O. gratissimum grown in Cd/Zn soil with various amendments for 3 months

Soil treatment Survivalrate (%)

Dry biomassproduction(g plant−1)

Month 0

1. Cd/Zn (control) 100 1.8±0.2a

2. Cd/Zn + 0.75% HA 100 1.7±0.1a

3. Cd/Zn + 1.5% HA 100 1.6±0.9a

4. Cd/Zn + 10% cow manure 100 1.8±0.5a

5. Cd/Zn + 20% cow manure 100 1.7±0.2a

Month 1

1. Cd/Zn (control) 100 5.4±0.2a

2. Cd/Zn + 0.75% HA 100 4.2±0.9a

3. Cd/Zn + 1.5% HA 100 7.5±0.3a

4. Cd/Zn + 10% cow manure 100 6.9±0.3a

5. Cd/Zn + 20% cow manure 100 11.8±1.1a

Month 2

1. Cd/Zn (control) 100 5.6±0.2a

2. Cd/Zn + 0.75% HA 100 12.4±4.9b

3. Cd/Zn + 1.5% HA 100 17.0±2.4b

4. Cd/Zn + 10% cow manure 100 17.4±1.1b

5. Cd/Zn + 20% cow manure 100 23.0±1.2c

Month 3

1. Cd/Zn (control) 66.7 6.2±0.5a

2. Cd/Zn+0.75% HA 88.9 23.9±4.9b

3. Cd/Zn + 1.5% HA 88.9 24.0±1.9b

4. Cd/Zn + 10% cow manure 88.9 59.1±4.0c

5. Cd/Zn + 20% cow manure 88.9 48.5±5.9c

Data with different letter in the same column indicate asignificant different at 5% level according to Duncan’s test


fold). Zinc concentration in leaves was decreasedfrom 69.3 to 34 mg kg−1.

Several studies have demonstrated the efficiency ofcow manure and HA in reducing heavy metalaccumulation in plants (Ram and Veerloo 1985;Narwal and Singh 1998; Boisson et al. 1999;Clemente et al. 2003, 2005; Walker et al. 2003;Keller et al. 2005; Pichtel and Bradway 2008).Organic amendments can decrease heavy metalbioavailability, shifting them from plant availableforms to fractions associated with organic matter,carbonates, or metal oxides (Walker et al. 2003).Additionally, organic matter amendments to metalcontaminated soils can have an ameliorative effect dueto increased surface area and an increase in the numberof specific adsorption sites (Shuman 1999) and also thedilution effect when they are mixed with metalcontaminated soil (Friedland 1989; Lozano-cerezo etal. 1999; Chiu et al. 2006). Reduction of Cd and Zn

availability to plants by the application of HA has beenreported in several studies (Chlopecka and Adriano1997; Boisson et al. 1999; Knox et al. 2001, 2003;Keller et al. 2005). Zinc and Cd sorption to HA isexplained by surface complexation and coprecipitation,presumably combined with ion exchange and soliddiffusion resulting in a decrease of the exchangeableamount of these metals (Mench et al. 1998). However,excess HA (5%) can cause new growth inhibitions inplants and decreased uptake of nutrients leading todeficiency problems (Boisson et al. 1999). Hence, HAapplication can be an effective remediation techniquebut needs to be approached with care (Boisson et al.1999). In addition, HA may be too expensive to beused on a large scale.

The lowest Cd concentration in leaves of O.gratissimum grown on 20% cow manure-amendedsoil was 0.3 mg kg−1, which was slightly above themaximum level for Cd set by the Commission of the

Table 6 Cadmium and Zn accumulations in O. gratissimum grown in Cd/Zn soil with various amendments for 3 months

Soil treatment Cd accumulation (mg kg−1) TF Zn accumulation (mg kg−1) TF

Leaf Stem Shoot Root Leaf Stem Shoot Root

Month 0

1. Cd/Zn (control) 0 0 0 0 – 50.2±17.9a 30.3±2.8a 80.5±20.7a 107.6±6.4a –

2. Cd/Zn + 0.75% HA 0 0 0 0 – 41.6±11.4a 33.3±2.5a 74.9±23.9a 99.7±17.3a –

3. Cd/Zn + 1.5% HA 0 0 0 0 – 28.1±4.2a 28.9±1.5a 57.0±5.7a 95.6±24.6a –

4. Cd/Zn + 10% cow manure 0 0 0 0 – 34.1±9.5a 21.8±5.5a 55.9±15.0a 66.3±15.2a –

5. Cd/Zn + 20% cow manure 0 0 0 0 – 53.7±13.9a 31.9±4.2a 85.6±18.1a 95.6±12.1a –

Month 1

1. Cd/Zn (control) 1.9±0.2a 2.1±0.8a 4.0±0.8a 21.8±4.0a 0.18 93.4±9.3a 89.9±11.6a 183.3±13.4a 401.8±37.9a 0.45

2. Cd/Zn + 0.75% HA 1.8±1.0a 1.6±0.9a 3.4±0.1a 17.1±3.8a 0.20 89.0±27.9a 65.6±27.5b 154.6±30.0ab 364.3±48.0a 0.42

3. Cd/Zn + 1.5% HA 1.5±0.3a 2.2±0.9a 3.7±0.6a 25.4±3.7a 0.15 56.1±0.4b 62.7±13.8b 118.8±13.8b 450.6±42.6a 0.26

4. Cd/Zn + 10% cow manure 2.6±1.1a 2.3±1.7a 4.9±1.7a 29.1±1.6a 0.17 105.7±47.1a 65.9±18.6b 171.6±45.2a 404.1±18.2a 0.42

5. Cd/Zn + 20% cow manure 2.5±0.3a 1.5±1.2a 4.0±1.5a 20.2±8.3a 0.20 81.6±33.6a 52.0±6.0b 134.0±27.6ab 460.6±33.2a 0.29

Month 2


2. Cd/Zn + 0.75% HA 1.0±0.6bc 3.8±0.4a 4.8±0.9ab 20.0±3.0bc 0.24 41.2±7.5bc 61.5±20.0a 102.7±27.4b 465.9±106.1c 0.22

3. Cd/Zn + 1.5% HA 1.4±0.4ab 3.1±0.3a 4.5±0.3bc 25.0±4.8b 0.18 67.1±18.9ab 37.8±5.4b 104.9±14.8b 625.1±27.1b 0.17

4. Cd/Zn + 10% cow manure 1.2±0.5bc 2.8±1.4a 4.0±0.9bc 17.6±4.0bc 0.23 62.9±18.5ab 52.4±15.3a 115.2±18.6ab 390.4±7.6c 0.30

5. Cd/Zn + 20% cow manure 0.4±0.1c 2.6±0.4a 3.0±0.4c 13.8±2.5c 0.22 35.7±12.4c 30.3±6.0b 66.0±15.7c 22.3±66.9d 0.30

Month 3


2. Cd/Zn + 0.75% HA 0.4±0.1b 3.4±0.9b 3.8±1.0b 25.1±3.8b 0.15 34.0±6.4b 24.7±1.6bc 58.6±7.9c 499.1±49.4b 0.12

3. Cd/Zn + 1.5% HA 0.5±0.0b 3.1±0.3b 3.6±0.3b 19.4±1.0bc 0.19 34.0±9.1b 23.6±2.9bc 57.6±11.8c 311.5±61.1c 0.17

4. Cd/Zn + 10% cow manure 0.3±0.2b 1.3±0.4c 1.6±0.5c 12.9±3.2c 0.12 47.6±6.7b 32.7±10.0b 80.4±16.0b 260.5±29.0d 0.31

5. Cd/Zn + 20% cow manure 0.3±0.1b 1.1±0.2c 1.4±0.2c 13.5±1.8c 0.10 39.7±9.3b 37.5±13.7b 77.2±17.9b 195.4±10.8d 0.40

Data with different letter in the same column indicate a significant different at 5% level according to Duncan’s test.


European Communities (2001) and the Codex Ali-mentarius Commission (2004) (0.2 mg kg−1 for leafyvegetables and fresh herbs). With the application ofHA, the lowest Zn concentration in leaves was34 mg kg−1 (much below the recommended maxi-mum limits for vegetables 99.4 mg kg−1; CodexAlimentarius Commission 2001). Even though thereduction of Cd and Zn bymanure and HA amendmentsclearly resulted from the decrease in bioavailable metalsfor the plants, the attribution to the dilution effect causedby the significant increase of shoot biomass cannot beruled out. The results of the present study clearlyindicated the great efficiency of both soil additives inlowering metal concentrations in plant parts grown onhighly metal contaminated soil to acceptable or close toacceptable values. Further studies involving field trialsgrowing African basil, O. gratissimum, on Cd/Zn-contaminated sites with the application of cow manureor HA should be implemented. Since African basil haslong been a traditional crop for the commercialproduction of essential oil or as a culinary herb, theanalysis of Cd and Zn in essential oil and seeds shouldbe performed.

Acknowledgements This research work was supported bythe Center on Environmental Health, Toxicology and Manage-ment of Toxic Chemicals under Science & TechnologyPostgraduate Education and Research Development Office(PERDO) of the Ministry of Education and the Office of theNational Research Council of Thailand (NRCT). We aregrateful to Asst. Prof. Philip Round for assistance with theproof reading of the manuscript.

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