Download - Research Article Cadmium Phytoremediation by …downloads.hindawi.com/journals/bmri/2013/324830.pdfDepartment of Chemistry, COMSATS Institute of Information Technology, Abbottabad,

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 324830 9 pageshttpdxdoiorg1011552013324830

Research ArticleCadmium Phytoremediation by Arundo donax L fromContaminated Soil and Water

Maria Sabeen1 Qaisar Mahmood1 Muhammad Irshad1 Iftikhar Fareed2 Afsar Khan3

Farid Ullah1 Jamshaid Hussain1 Yousaf Hayat4 and Sobia Tabassum5

1 Department of Environmental Sciences COMSATS Institute of Information Technology Abbottabad 22060 Pakistan2Department of Natural Resource Engineering and Management University of Kurdistan Hewler Kurdistan Iraq3 Department of Chemistry COMSATS Institute of Information Technology Abbottabad Pakistan4Department of Mathematics Statistics and Computer Sciences KPK Agricultural University Peshawar Pakistan5 Interdisciplinary Research Centre in Biomedical Materials COMSATS Institute of Information Technology Defence RoadOff Raiwind Road Lahore Pakistan

Correspondence should be addressed to Qaisar Mahmood mahmoodzjugmailcom

Received 14 November 2013 Revised 8 December 2013 Accepted 8 December 2013

Academic Editor Fernando Barbosa Jr

Copyright copy 2013 Maria Sabeen et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The potential of Arundo donax L for phytoextraction of cadmium (Cd) from contaminated soil and water was probed The plantswere grown under greenhouse conditions in pots containing a nutrient solution or soil with increasing doses of Cd (0 50 100250 500 750 and 1000120583g Lminus1) for 21 days The growth and physiology of plants were evaluated at the end of the experiment ThemaximumCd content in root was 300 120583g gminus1 during hydroponics experiments over 230120583g gminus1 in soil experiment Cd concentrationin stem was 262 120583g gminus1 at 750 120583g Lminus1 supplied Cd in hydroponics over 1912 120583g gminus1 at 1000 in soil experiment The maximum Cdconcentration in leaves from hydroponics was 187 120583g gminus1 Relatively low Cd uptake occurred during soil experiment with lowtranslocation factor (TF) values Both Bioaccumulation Factor (BF) and TF values for hydroponics were greater than 1 The IC

50

values of ABTS and DPPH showed that both time and increasing Cd concentrations affected the production of antioxidants withlower half maximal inhibitory concentration (IC

50

) value on the 21st days A donax showed better potential for Cd remediation ofaquatic environments

1 Introduction

Consequent to global industrialization heavymetal pollutionis a widespread problem which has become a major environ-mental concern due to hazardous effects on human and envi-ronmental health In industrialized societies heavy metalsare the world over environmental contaminants Air or waterpollution by metals varies from soil pollution because heavymetals persevere in soil for a longer time period as comparedwith the other compartment of the biosphere [1] In the latestdecades the yearly global release of heavy metals attained22000 t (metric ton) for cadmium 939000 t for copper783000 t for lead and 1350000 t for zinc [2] Cadmium(Cd) a highly toxic metal pollutant of soils inhibits root andshoot growth and yield production affects nutrient uptake

and homeostasis is frequently accumulated by agriculturallyimportant crops and then enters the food chain with asignificant potential to impair animal and human health [3]The mutagenic aptitude of toxic heavy metals causes DNAdamage and probably causes carcinogenic effects in animalsand humans [4]The prime health perils coupled with Cd aredamage to the neurological system having indications likeuncontrollable shaking muscle wasting partial blindnessand deformities in children exposed in the womb [5]

Many phytoremediation technologies have been used forthe remediation of polluted soils and water throughout theWorld [6] Phytoremediation costs almost one-fourth of theother physical and chemical methods of pollutant treatmentThe major advantages of the process include improvementof the soil quality as it is driven by solar energy thus suitable

2 BioMed Research International

to most regions and climates cost effective and technicallyfeasible process plants serve as sufficient biomass for rapidremediation promote high rhizosphere activity and finallyrestoration in a reasonable time frame of 2 to 3 years

The use of low cost fast growing indigenous plants withefficient biomass producing plant species such as Arundodonax L is highly desirable for phytoremediation of metalcontaminated sites and waters It is cultivable throughoutAsia Southern Europe North Africa and theMiddle East forthousands of years with local names of ldquoGiant Reedrdquo ldquoNurrrdquoldquoNurrurdquo or ldquoNurrordquo [7ndash9] It is considered as one of the mostbiologically productive of all communities [10] Previousexperiments on giant reed suggested that the stem height anddiameter number of nodes fresh and dry weight of leavesand net photosynthesis were not affected indicating thatplants tolerated the high concentrations of Cd and Ni [11]As giant reed plants are very promising energy plants theycan be cultivated in contaminated soils to provide biomass forenergy production purposes [11] Our research group isexploring the potential of this plant in environmental reme-diation of various heavy metals at high concentrationsThe plant showed some potential against arsenic [7ndash9]and chromium [10] remediation The specific objective ofthe current study was to investigate the phytoremediationability of A donax for cadmium remediation and to comparethe Cd removal from contaminated soil and water

2 Materials and Methods

21 Plant Material The plant material used for the presentstudy was A donax L with the aim to evaluate its responsesto cadmium added to liquid mediumThe plant material wascollected near PMA road Abbottabad Khyber PakhtunkhwaPakistan Plants were collected for the heavy metal analysisThe soil-grown plants used for hydroponics tests were takenfrom growth of young meristematic buds grown in sterileaqueous medium [8] The young plants were transplanted inplastic trays containing 12 strength basal nutrient solutions

22 Experimental Design The phytoremediation ability of Adonax was compared in Cd contaminated soil and aqueoussolution The Cd containing aqueous solution was preparedby dissolving cadmium chloride salt in the double distilledwater Various Cd treatments were given in triplicates toexperimental plants (both in hydroponics and soil) Cdtreatments included 0 (control) 50 100 250 500 750 and1000120583g Lminus1 Each pot contained 4 A donax L plants withan average 250 g biomass on fresh weight basis Two kinds ofexperiments were performed in randomized complete blockdesign (RCBD) with three replications for plants grown inHoagland solution [12] and in soil Plants with healthy anduniform shoots of almost equalmorphological characteristicswere immersed in nutrient solution for three weeks Plantswere grown under greenhouse conditions in the laboratorywith Hoagland solution continuously aerated and renewedafter every 2-3 days with an addition of 100ndash200mL ofnutrient solution Table 1 shows the average values of variousplant characteristics

Table 1 Average values of variousmorphological parameters beforecadmium treatment

Parameters ValuesPlant height (cm) 55 plusmn 12

No of leaves per plant 87 plusmn 18

No of nodes per plant 33 plusmn 22

Average root length per plant (cm) 8 plusmn 2

Toxicity symptoms Leaf burning

23 Growth Parameters Various growth parameters of Adonax were studied under cadmium stress including plantheight (cm) number of leaves per plant number of nodesper plant dry weight average root length (cm) and toxicitysymptoms prior to and after the cadmium treatments

24 Physiological Parameters The physiological parametersof the plants included photosynthetic pigments and thedetermination of antioxidants

241 Photosynthetic Pigments At the time of harvest freshleaves of both of various treatments fromboth kinds of exper-iments were collected for the determination of chlorophyll achlorophyll b and carotene contents Leaves were cut intosmall pieces and 05 g of the sample was put into the glasstest tubesThen 10mL of 80 acetone was added to the tubesand kept overnight for complete extraction Photosyntheticpigments were determined spectrophotometrically using thevisible wavelengths of 663 645 and 480 nm for chlorophyll achlorophyll b and carotene respectively [12]

242 Antioxidant Determination To determine antioxidantactivity another parallel set of experiment was conductedIn this experiment four replicates each of 250 gm againsteach treatment was planted in a pot Each pot was givenHoaglandrsquos solution and the respective treatment of cadmiumabove mentioned After each week one set was taken out andnew leaves were countedThese plants were placed outside forshade dry

All the plants were ground until uniform size Now thissample was dipped in methanol for 4 to 5 days The plantextract was filtered and methanol was recovered by passingit through rotary The thick left over extract was taken outin Petri dishes and placed in fume hood The thick jelly likeextract was stored in sample bottles for further analysis

DPPH Free Radical Scavenging Assay 22-diphenyl-1-picrylhydrazyl free radical (DPPH) scavenging activity ofcrude extract and various fractions were estimated by stan-dard DPPH assay protocol with certain modifications [13]The reaction mixture contains 05mL of test samples and25mL of DPPH in methanol Concentration of DPPH was100 120583M in the reaction mixture These reaction mixtureswere incubated for 30min at 37∘C The absorbance wasmeasured at 517 nm using spectrophotometer (5000 IrmecoGmbH D-21496 Geesthacht Germany) Percent radicalscavenging activity or percent antioxidant index (AI)

BioMed Research International 3

by sample treatment was determined by comparison withmethanol treated control group An IC

50

value denotes theconcentration of sample which is required to scavenge 50DPPH free radicals Propyl gallate was used as positivecontrols

ABTS+ Assay Total antioxidant activity was evaluated apply-ing an improved 221015840-azinobis-3-ethyl-benzothiazoline-6-sulfonic acid cation (ABTS) decolorization assay by Re et al[14] ABTS+ radical cation (ABTS+) was produced by reactingABTS stock solution (7mM) with 245mM potassium per-sulphate and allowing the solution to stand in the dark atroom temperature for 12ndash14 hours before use For the studyof total antioxidant activity the solution was diluted withethanol to an absorbance of 070 (plusmn002) at 374 nm Percent-age inhibitionwas calculated by using the following equation

Percentage inhibition = [1 minusabsorbancesample

absorbancecontrol] times 100

(1)

IC50

values were calculated based on various determinationsof these antioxidants by supposing that reduced IC

50

valueswould indicate a greater oxidative stress in the plant causedby absorbed metal content

25 Bioaccumulation Factors The phytoextraction ability ofA donax L plants was assessed using both the translocationfactor (TF) and the bioaccumulation factor (BF) as follows

(1) Translocation factor

TF = [Cd]shoot[Cd]root

(2)

(2) Bioaccumulation factor

BF = [Cd]shoot[Cd]solution

(3)

26 Analytical Procedure The harvested plants were sepa-rated into stems leaves and branches and the fresh weightwas recorded For dry weight determination plant materialwas oven-dried at 70∘C for 24 h weighed ground with pestleand mortar and sieved at 01mm nylon sieve Plant sampleswere digested throughwet digestion formetals determinationusing the HNO

3

HClO4

A 05 g sample was taken in 100mLconical flask and 10mL of Perchloric and Nitric acid mixture(1 2 ratios) was added to each conical flask and left overnightNext day glass funnels were placed at the mouth of eachflask in such a way that funnel stem stayed at least one inchabove the surface of liquidThe flasks were then placed on hotplate and the temperature was gradually increased to allowfor effective digestion It took about 15 to 20 minutes whenHNO

3

volatilized as nitrous oxide fumes and then whitefumes of Perchloric acid came out from the flaskThe solutionin flask was white in color at that stage After digestionthe flasks were removed from the hot plate allowed to cooland few milliliters of distilled water was added The digested

Table 2 Physicochemical properties of the soil used in experiment

Characteristic ValuepH 68Available nitrogen (mg kgminus1) 754Available potassium (mg kgminus1) 621Available phosphorus (mg kgminus1) 78Total nitrogen (g kgminus1) 086Total phosphorus (g kgminus1) 07Total potassium (g kgminus1) 173Organic matter (g kgminus1) 214

material was then transferred to 50mL volumetric flask andthe volume was made up to 50mL with deionized water Thereadings weremeasured on Perkin Elmer Atomic AbsorptionSpectrometer-700 [15]

27 Soil Analysis The soil used in the experiment wascollected from an experimental field in COMSATS Instituteof Information Technology Abbottabad The soil was sievedto remove roots pebbles and other unwanted materials Thesoil was analyzed for various physic-chemical parametersaccording to [8] and presented in Table 2

At the end of growing periods soil samples from tworeplicates were oven-dried at 70∘C Soil sample (1 g) wasdigested on a hot plate with 15mL nitric acid and 10mLhydrogen peroxide The digests were brought to 50mL withdeionized distilled water and the impurities were removed byfiltration A total Cd content was analyzed by Perkin ElmerAtomic Absorption Spectrometer-700

28 Statistical Analysis All determinations were performedin triplicate and mean values are presented in the resultsOne-way analysis of variance (ANOVA) was carried out forboth the experiments separately using SAS 83 software (SASIns Inc Cary USA) Treatment means were portioned usingLeast Significant Difference (LSD) at appropriates 120572 value(005)

3 Results

31 Cadmium Content of Plant The results of the cadmiumconcentration in the different plant parts grown in contam-inated water were presented in Figure 1(a) In root the cad-mium uptake had linear relation to the increasing suppliedcadmium concentration Root Cd contents at various treat-ments significantly (119875 lt 005) differed The maximum cad-mium uptake in stem was noted at 750120583g Lminus1 (2628120583g gminus1)However the cadmium concentration at 1000 120583g Lminus1 wassignificantly (119875 lt 005) different from all other treatmentsexcept 750 120583g Lminus1 Cd accumulation in leaves was the maxi-mum at 1000 120583g Lminus1 (1875120583g Lminus1) and was significantly (119875 lt005) different from the rest of the treatments For thetreatments 50 to 500120583g Lminus1 the leaf cadmium content was inthe range of 48 to 12983120583g gminus1 Overall theCd accumulationpattern in various plant organs was as follows root gt stem gt


0 200 400 600 800 1000 1200

0

50

100

150

200

250

300

350

Root Stem

Leaves Culture medium

Cd concentration (120583g Lminus1)

Cd

conc

entr

atio

n in

pla

nt p

arts

(120583g g

minus1)

(a)

0 200 400 600 800 1000 1200

0

100

200

300

400

Root Stem

Leaves Soil medium

Cd

conc

entr

atio

n in

pla

nt p

arts

(120583g g

minus1)

Cd concentration (120583g Lminus1) in soil

(b)

Figure 1 (a) The accumulation of cadmium in various plant parts in hydroponics experiment (b) The accumulation of cadmium in variousplant parts during soil experiment

leaf (Figure 1(a)) The left over Cd content in the aqueousmedium was in the range of 12 to 1864120583g Lminus1 for varioustreatments The maximum left over cadmium concentration(1864 120583g Lminus1) was noted for 1000 120583g Lminus1 supplied cadmium(Figure 1(a))

The results of the cadmium concentration in plant partsgrown in contaminated soil were presented in Figure 1(b)ThemaximumCd concentration in roots of soil grown plants was230 120583g gminus1 at 1000120583g gminus1 supplied Cd content The plant rootCd content was significantly (119875 lt 005) different from sup-plied Cd contents of 500 120583g gminus1 and below For stem themax-imum cadmium uptake occurred at 1000 120583g gminus1 which was191 120583g gminus1 which was significantly (119875 lt 005) The stem cad-mium concentration at 750120583g Lminus1 was 99 120583g gminus1 significantly(119875 lt 005) different from the values at 250120583g gminus1 and lowersupplied Cd treatments (Figure 1(b)) The maximum uptakein leaves of soil grownplants occurred at 500120583g gminus1 treatmentwhich was 138 120583g gminus1 and was significantly (119875 lt 005)different from the rest of treatments At higher treatments(750 to 1000120583g Lminus1) the leaf cadmium contents were 81 and53120583g gminus1 respectively and were nonsignificantly different(119875 gt 005) Like hydroponics the accumulation pattern ofcadmium in various plant organswas as follows rootgt stemgtleaf (Figure 1(b)) The left over cadmium concentration inthe soil medium was in the range of 27 to 343 120583g gminus1 soil forvarious supplied cadmium concentrations

32 Effect of Cadmium on Photosynthetic Pigments Theeffects of cadmium on the photosynthetic pigments that ischlorophyll a chlorophyll b and carotenes of plants grownin hydroponics were presented in Figure 2(a) The maximumchlorophyll a content was observed at Cd treatment of250120583g Lminus1 (Figure 2(a)) The amount of chlorophyll b hadsimilar trend like chlorophyll a with the maximum value

(096mg gminus1) at 250120583g Lminus1 supplied Cd (Figure 2(a)) As faras carotene content was concerned its amount was themaximum (155mg gminus1) at 100 120583g Lminus1 Further increase in thesupplied Cd did not cause any significant (119875 lt 005) increasein carotene content However the amount of carotene wassignificantly (119875 lt 005) different than control and 50120583g Lminus1(Figure 2(a))

The effects of cadmium on the photosynthetic pigmentsin soil grown plants were presented in Figure 2(b) Thechlorophyll a content was the maximum at Cd treatment of1000 120583g gminus1 The amount of chlorophyll b increased up to250120583g gminus1 where its value was 021mg gminus1 and then it showeda decline towards the maximum Cd content in soil As far ascarotene content was concerned amount was the maximum(074mg gminus1) at 100 120583g gminus1 supplied Cd in soil Furtherincrease in the supplied cadmium did not cause any increasein carotene content (Figure 2(b))

33 Effect of Cadmium on Bioconcentration Factors Theresults of bioconcentration factors for plants grown in hydro-ponics were presented in Figure 3(a) The translocation andbioaccumulation factors increased up to 750 120583g Lminus1 Cd in thehydroponics culture (Figure 3(a)) Overall BF and TF valueswere in the range of 03 to 30 and 1 to 228 respectivelyfor various Cd treatments The highest values were TF = 16and BF = 3000 for 750120583g Lminus1 Cd treatment Howeverboth TF and BF decreased at 1000 120583g Lminus1 and some toxicitysymptoms appeared in the plants receiving that Cd treatment(Figure 3(a)) Both bioconcentration factor values for soilgrown plants were presented in Figure 3(b)The translocationand bioaccumulation factors increased as a function of Cdconcentration up to 500120583g gminus1 supplied Cd however itdecreased a little at higher Cd content in soil (Figure 3(b))Overall the values of BF and TF were in the range of 07 to067 and 12 to 13 respectively for various Cd treatments


0 200 400 600 800 1000 120000

05

10

15

20

25

Chlorophyll a Chlorophyll b

Carotene


Chlo

roph

yll c

once

ntra

tion

(mg g

minus1)

(a)


Carotene

0 200 400 600 800 1000 1200

00

05

10

15

20

Chlo

roph

yll c

once

ntra

tion

(mg g

minus1)

minus15

minus10

minus05

Cd concentration (120583g gminus1)

(b)

Figure 2 (a) The effect of cadmium concentrations on chlorophyll content in hydroponics experiment (b) The effect of cadmiumconcentrations on chlorophyll content during soil experiment

0 200 400 600 800 1000 1200

Fact

or v

alue

s

0

5

10

15

20

25

30

35

Translocation factor Bioaccumulation factor


(a)

0 200 400 600 800 1000 1200

Fact

or v

alue

s

00

02

04

06

08

10

12

14

16



(b)

Figure 3 (a) Relative bioconcentration factors at various cadmium treatments in hydroponics (b) Relative bioconcentration factors at variouscadmium treatments in soil

The highest values were TF = 13 and BF = 071 for the Cdtreatment of 500120583g gminus1 Translocation factors were abovethe reference value (10) for hyperaccumulation however BFvalues were below 1 (Figure 3(b))

34 Effects of Cadmium on Growth Characteristics Growthperformance of the plant in reference to plant height nodesinternodes tillers fresh weight and number of leaves ofplants from hydroponics is depicted in Figure 4(a) Theresults showed that there was no significant (119875 lt 005)increase in plant height root length nodes and internodes oftreated plants at all levels of Cd treatments However some

increase in the fresh weight leaves and tillers of the treatedplants was observed For leaves the number significantlyincreased up to 100 120583g Lminus1 supplied Cd The number wassignificantly different at all other treatments except 250 and500120583g Lminus1 supplied Cd level It implied that Cd initiallyenhanced the growth of plants up to the concentration of300 120583g Lminus1 in hydroponics culture however at higher con-centrations the growth of the plantwas reduced (Figure 4(a))Growth performance of the plant in reference to plantheight tillers fresh weight and number of leaves duringthe soil experiment is depicted in Figure 4(b) A trend ofgrowth similar to hydroponics experiment was observed


0 200 400 600 800 1000 1200

Num

ber o

f lea

ves a

nd fr

esh

wei

ght (

g)

0

50

100

150

200

250

300

350

Num

ber o

f till

ers

0

1

2

3

4

5

Leaves Fresh weight

Tillers


(a)

Leaves Fresh weight

Tillers

0 200 400 600 800 1000 1200

Num

ber o

f lea

ves a

nd fr

esh

wei

ght (

g)

0

100

200

300

400

Num

ber o

f till

ers

0

1

2

3

4

5

6

7

120583g gminus1)Cd concentration in soil (

(b)

Figure 4 (a) Effect of cadmium on fresh weight number of leaves and tillers of treated plants growth in hydroponics culture (b) Effect ofcadmium on fresh weight number of leaves and tillers of treated plants growth in in soil

The observation that increase in number of tillers withoutincrease in plant height (stunted growth) indicated Cd stress

35 Antioxidant Assays Antioxidants are chemical com-pounds that can bind to free oxygen radicals preventingthese radicals from damaging healthy cellsThe present studyinvolved the determination of one of the most commonlyused organic radicals for the evaluation of antioxidant effi-ciency of pure compounds and complex mixtures is theradical cations derived from ABTS and DPPH These radicalcations could be generated by enzymatic chemical and elec-trochemical means The IC

50

measured at 7th 14th and 21stdays of the experiment were presented in Figures 5 and 6TheIC50

values of ABTS showed that both time and increasingCd concentrations strongly inhibited the production of ABTS(Figure 5) which was indicated by lower IC

50

value on 21stday A similar trend was obvious for DPPH up to 500120583g Lminus1supplied Cd (Figure 6) However a relative increase in IC

50

was observed at higher supplied Cd contents (gt500120583g Lminus1)showing that the concentration of these antioxidants mightbe greater with increasingly higher Cd exposure

4 Discussion

The present study employed the use of A donax to treat Cdmetal in hydroponics and soil contaminated environmentsOverall the results indicated that the plant is useful for thetreatment of Cd contaminated wastewaters However betteruptake was observed in hydroponics cultures as comparedwith soil environment The maximum plant root Cd contentwas 300 120583g gminus1 in hydroponics experiment as compared with230 120583g gminus1 in soil experiment Likewise Cd concentration instem for hydroponics culture was 262120583g gminus1 at 750120583g Lminus1supplied Cd over 1912 120583g gminus1 at 1000 in soil experiment In

0 200 400 600 800 1000 12000

10

20

30

40

50

After 7 days After 14 days

After 21 days

Cd concentrations (120583g Lminus1)

IC50

valu

es

Figure 5 The effect of cadmium concentrations on the IC50

valuesagainst antioxidant activity of ABTS

case of leaves the maximum Cd concentration for hydro-ponics was 187 120583g gminus1 at 1000120583g Lminus1 supplied Cd In soilexperiment the Cd concentration in leaves was 137120583g gminus1at 500120583g Lminus1 Relatively low Cd uptake occurred during soilexperiment which consequently resulted in low TF values Itwas opposite in case of hydroponics where BF and TF valueswere always greater than 1 Both factors values were above thereference value (10) for hyperaccumulation

A donax L (giant reed Poaceae) is a potentially high-yielding nonfood crop which can be used for the productionof energy paper pulp and wooden building materials [11] Itis a robust invasive perennial grass wild growing in southernEuropean regions and other Mediterranean countries [16] Itis also very common in Pakistan Giant reed can easily adapt


0 200 400 600 800 1000 12000

20

40

60

80

100

120

140


After 21 days

IC50

valu

es



valuesagainst antioxidant activity of DPPH

to different ecological conditions and grow in all types of soils[17] The plant has been evaluated for the phytoremediationability towards arsenic contamination [7] It was suggestedthat A donax plants may be employed to treat water contain-ing arsenic concentrations up to 600120583g Lminus1 [7]

Increasing metal concentrations in the wastewaters andsoils adjacent to industrial regions of the world are a seriousthreat to the natural environmental sustainability Amongthe heavy metals cadmium is of special concern due to itspotential toxicity to biota at low concentrations [18] The useof indigenous plants like A donax is very promising to com-bat metal toxicity in soils and wastewaters Different plantspecies have different capacities for uptaking and toleratingthe heavy metals like cadmium and others [19 20]Themetalhyperaccumulators show an extra aptitude for accumulatingthe large quantity of metals in their aerial parts [21] Thisspecial characteristic of the metal hyperaccumulators makethem extremely appropriate for phytoremediation that is touse plants for cleaning up the polluted soils In the precedingdecade many studies have been accomplished to explore themechanisms liable for the better metal uptake and tolerancevia natural hyperaccumulators as model plant species [22] Ingeneral the metal hyperaccumulation in plants is acknowl-edged as a mishmash of high metal uptake coupled with animproved tissue tolerance against the detrimental effects ofhigher metal concentrations through a better antioxidativeresponse and sequestration at the cellular level [23] Reme-diation of heavy metals contaminated that soil may possiblybe carried out using physicochemicals processes such asion-exchange precipitation reverse osmosis evaporationand chemical reduction however the procedures requisiteexternal man-made resources and expensive [1]

Plants absorb toxic metals translocate and accumulatethem in roots and shoots and finally resist to metal con-tamination thus remediate contaminated environments [24]Phytoremediation is a growing field of research in environ-mental studies because of the advantages of its environmentalfriendliness safe cost effectiveness and the possibility of

harvesting the plants for the extraction of absorbed con-taminants such as metals that cannot be easily biodegradedfor recycling among others [7] Moreover it is based on theecological engineering principlesThemost effective phytore-mediation plants are those classified as hyperaccumulators[25] and accumulators Hyperaccumulators are character-ized based on four features First the concentration in theshoots (stems or leaves) of a hyperaccumulator should be10000 120583g gminus1 for Zn and Mn above 1000 120583g gminus1 dry mass forAs Pb Cu Ni and Co 100 120583g gminus1 for 1 Cd and 1 120583g gminus1 forAu [7 8 26] Second is translocation factor (concentration inshootsrootsgt1) metal concentrations in the shoots of a plantshould be higher than those in the roots [7] Third is bioac-cumulation factor (concentration in planthabitat gt1) [7]and lastly it is tolerance ability a hyperaccumulator shouldhave high tolerance to toxic contaminants A donax L cantolerate arsenic concentrations up to 600120583g Lminus1 without toxi-city symptoms appeared on the plantThe appearance of sometoxicity symptoms in the leaves roots and slow growth at1000 120583g Lminus1 revealed that althoughA donax L cannot toleratebut still accumulate and volatilize as concentration above600120583g Lminus1 [7ndash9]

The results suggested that Cd contamination affected thephotosynthetic pigments to some extent Detailed studiesindicate that heavy metals have effects on photosyntheticpigments in plants Heavy metals are known to interferewith chlorophyll synthesis either through direct inhibition ofan enzymatic step or by inducing deficiency of an essentialnutrient [27] An important indicator which determines pho-tosynthesis intensity is chlorophyll content in plant leavesCadmium markedly suppresses chlorophyll accumulation inleaves [28] Carotenoid actively participates in photosynthesisas well and it was shown that content and ratio of carotenoidsare strictly changed under impact of different stresses [29]However it has been determined that carotenoids are lesssensitive to the impact of cadmium as compared to chloro-phylls [30] The success of phytoextraction is inherentlydependent on several plant characteristics the two mostimportant being the ability to accumulate large quantities ofbiomass rapidly and the capacity to accumulate large quanti-ties of environmentally important metals in the shoot tissue[31] Effective phytoextraction requires both plant geneticability and the development of optimal agronomic practicesincluding (1) soil management practices to improve the effi-ciency of phytoextraction and (2) cropmanagement practicesto develop a commercial cropping system The present studyshowed that A donax has potential to remediate the Cdcontaminated environments as indicated by its high tissue Cdconcentrations and BF and TF values higher than 1 especiallyfor hydroponics experiment The reason behind less Cduptake in soil experiment may be that it may have beenadsorbed in soil particles or it is leaching from rhizosphereas the experiment was conducted in sandy soil

The IC50

value was defined as the concentration ofthe sample necessary to cause 50 inhibition which wasobtained by interpolation from linear regression analysis [32]A lower IC

50

value is associatedwith a higher radical scaveng-ing activity The IC

50

values of ABTS showed that both time


and increasing Cd concentrations affected the productionof ABTS which was indicated by lower IC

50

value on 21stdays A similar trend was obvious for DPPH upto 500120583g Lminus1supplied Cd However a relative increase in IC

50

wasobserved at higher supplied Cd contents (gt500 120583g Lminus1) show-ing that the concentration of these antioxidants might begreater with increasingly higher Cd exposure One of themost commonly used organic radicals for the evaluationof antioxidant efficiency of pure compounds and complexmixtures is the radical cation derived from 221015840-azinobis-3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) [33] Thedifferences among all studiedmorphological parameterswerestatistically nonsignificant likewise the fresh and dryweightsof control and experimental plants were also nonsignificantReduced growth was noted in control plants which maypossibly be due to the fact that the plants were grown in potsandnot in the fieldA donax is a grasswithC3 photosyntheticpathway unlike other grasses (eg switchgrass and miscant-hus) with C4 pathway [34] Physiological processes such asphotosynthesis and water status are sensitive to heavymetals[35] in several plant species Heavy metals have been foundto inhibit electron transport in photosynthetic systems [36]Photosynthetic rates of A donax were unaffected by thetreatments indicating that the photosynthetic systemwas notharmed and showed a strong tolerance of this plant to theincreased heavy metal concentrations in the soil The meanvalues of giant reed Pn rates found in this study were higherthan those usual forC3 plants (18ndash20120583molCO

2

mminus2 sminus1) [37]Rossa et al [38] in a comparative study on photosynthesis offive C3 and three C4 grasses found thatA donax had high Pnrates higher than the other grasses (370120583molCO

2

mminus2 sminus1)under similar environmental conditions

5 Conclusions

The phytoremediation ability of Arundo donax to treatcadmium contamination was compared in hydroponics andsoil environmentsThe plant is useful for the treatment of Cdcontaminated wastewaters both in hydroponics and in soilenvironments However better uptake was observed inhydroponics cultures as compared with soil environmentBoth BF and TF values for hydroponics were greater than1 which confirmed its suitability for aquatic contaminatedenvironments At higher Cd exposure plant showed someantioxidative stress as the concentration of antioxidants wasgreater with increasing Cd exposure

Conflict of Interests

The authors have neither any conflict of interests nor anyfinancial gain from the present work

References

[1] M M Lasat ldquoPhytoextraction of toxic metals a review ofbiological mechanismsrdquo Journal of Environmental Quality vol31 no 1 pp 109ndash120 2002

[2] O V Singh S Labana G Pandey R Budhiraja and R K JainldquoPhytoremediation an overview of metallic ion decontamina-tion from soilrdquo Applied Microbiology and Biotechnology vol 61no 5-6 pp 405ndash412 2003

[3] L S Di Toppi and R Gabbrielli ldquoResponse to cadmium inhigher plantsrdquo Environmental and Experimental Botany vol 41no 2 pp 105ndash130 1999

[4] C Baudouin M Charveron R Tarroux and Y Gall ldquoEnviron-mental pollutants and skin cancerrdquo Cell Biology and Toxicologyvol 18 no 5 pp 341ndash348 2002

[5] WHO ldquoHealth and environment in sustainable developmentrdquoReport WHO Geneva Switzerland 1997

[6] M A Kamran Amna R Mufti et al ldquoThe potential of theflora from different regions of Pakistan in phytoremediation areviewrdquo Environmental Science and Pollution Research 2013

[7] N Mirza Q Mahmood A Pervez et al ldquoPhytoremediationpotential of Arundo donax in arsenic-contaminated syntheticwastewaterrdquo Bioresource Technology vol 101 no 15 pp 5815ndash5819 2010

[8] N Mirza A Pervez Q Mahmood and S S Ahmad ldquoPhytore-mediation of arsenic (As) andmercury (Hg) contaminated soilrdquoWorld Applied Sciences Journal vol 8 pp 113ndash118 2010

[9] N Mirza A Pervez Q Mahmood M M Shah and M NShafqat ldquoEcological restoration of arsenic contaminated soilby Arundo donax Lrdquo Ecological Engineering vol 37 no 12 pp1949ndash1956 2011

[10] S Kausar Q Mahmood I A Raja et al ldquoPotential of Arundodonax to treat chromium contaminationrdquo Ecological Engineer-ing vol 42 pp 256ndash259 2012

[11] E G Papazoglou G A Karantounias S N Vemmos and D LBouranis ldquoPhotosynthesis and growth responses of giant reed(Arundo donax L) to the heavymetals Cd andNirdquo EnvironmentInternational vol 31 no 2 pp 243ndash249 2005

[12] D Liu T Q Li X F Jin X E Yang E Islam and Q Mah-mood ldquoLead induced changes in the growth and antioxidantmetabolism of the lead accumulating and non-accumulatingecotypes of Sedum alfrediirdquo Journal of Integrative Plant Biologyvol 50 no 2 pp 129ndash140 2008

[13] M S Blois ldquoAntioxidant determinations by the use of a stablefree radicalrdquo Nature vol 181 no 4617 pp 1199ndash1200 1958

[14] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology andMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[15] R John P Ahmad K Gadgil and S Sharma ldquoHeavy metaltoxicity effect on plant growth biochemical parameters andmetal accumulation by Brassica juncea Lrdquo International Journalof Plant Production vol 3 no 3 pp 65ndash75 2009

[16] N El Bassam Energy Plant Species Their Use and Impact onEnvironment and Development Earthscan LLC WashingtonDC USA 1998

[17] R E Perdue ldquoArundo donaxmdashsource of musical reeds andindustrial celluloserdquo Economic Botany vol 12 no 4 pp 368ndash404 1958

[18] P Das S Samantaray and G R Rout ldquoStudies on cadmiumtoxicity in plants a reviewrdquo Environmental Pollution vol 98 no1 pp 29ndash36 1997

[19] V Bert I Bonnin P Saumitou-Laprade P de Laguerie and DPetit ldquoDo Arabidopsis halleri from nonmetallicolous popula-tions accumulate zinc and cadmiummore effectively than thosefrom metallicolous populationsrdquo New Phytologist vol 155 no1 pp 47ndash57 2002


[20] N Roosens N Verbruggen P Meerts P Ximenez-Embun andJ A C Smith ldquoNatural variation in cadmium tolerance and itsrelationship to metal hyperaccumulation for seven populationsof Thlaspi caerulescens from western Europerdquo Plant Cell andEnvironment vol 26 no 10 pp 1657ndash1672 2003

[21] A J M Baker and P L Walker ldquoEcophysiology of metaluptake by tolerant plantsrdquo in Heavy Metal Tolerance in PlantsEvolutionary Aspects A J Shaw Ed pp 155ndash177 CRC PressBoca Raton Fla USA 1999

[22] E Lombi F J Zhao S P McGrath S D Young and G ASacchi ldquoPhysiological evidence for a high-affinity cadmiumtransporter highly expressed in aThlaspi caerulescens ecotyperdquoNew Phytologist vol 149 no 1 pp 53ndash60 2001

[23] B P Shaw S K Sahu and R K Mishra ldquoHeavy metal inducedoxidative damage in terrestrial plantsrdquo in Heavy Metal Stress inPlantsmdashfrom Biomolecules to Ecosystems M N V Prasad Edpp 84ndash126 Springer New York NY USA 2nd edition 2004

[24] V Mudhal N Madaan and A Mudhal ldquoHeavy metals inplants phytoremediation plants used to remediate heavy metalpollutionrdquoAgricultural and Biological Journal of North Americavol 1 pp 40ndash46 2010

[25] C Y Wei and T B Chen ldquoArsenic accumulation by twobrake ferns growing on an arsenic mine and their potential inphytoremediationrdquo Chemosphere vol 63 no 6 pp 1048ndash10532006

[26] M Srivastava L Q Ma and J A G Santos ldquoThree new arsenichyperaccumulating fernsrdquo Science of the Total Environment vol364 no 1ndash3 pp 24ndash31 2006

[27] F van Assche and H Clijsters ldquoEffect of metals on enzymeactivity in plantsrdquo Plant Cell and Environment vol 13 no 3pp 195ndash206 1990

[28] J Moreno-Caselles R Moral A Perez-Espinosa and M DPerez-Murcia ldquoCadmium accumulation and distribution incucumber plantrdquo Journal of Plant Nutrition vol 23 no 2 pp243ndash250 2000

[29] B Demmig-Adams and W W Adams III ldquoThe role of xan-thophyll cycle carotenoids in the protection of photosynthesisrdquoTrends in Plant Science vol 1 no 1 pp 21ndash26 1996

[30] N NeeluM KumarM Tomar and A K Bhatnagar ldquoInfluenceof cadmium on growth and development of Vicia faba LinnrdquoIndian Journal of Experimental Biology vol 38 no 8 pp 819ndash823 2000

[31] E A Pilon-Smits ldquoPhytoremediationrdquo Annual Review of PlantBiology vol 56 pp 15ndash39 2005

[32] YQingming P Xianhui KWeibao et al ldquoAntioxidant activitiesofmalt extract frombarley (HordeumvulgareL) toward variousoxidative stress in vitro and in vivordquoFoodChemistry vol 118 no1 pp 84ndash89 2010

[33] C A Rice-Evans and N J Miller ldquoTotal antioxidant status inplasma and body fluidsrdquo Methods in Enzymology vol 234 pp279ndash293 1994

[34] I Lewandowski J M O Scurlock E Lindvall andM ChristouldquoThe development and current status of perennial rhizomatousgrasses as energy crops in the US and Europerdquo Biomass andBioenergy vol 25 no 4 pp 335ndash361 2003

[35] S Monni C Uhlig O Junttila E Hansen and J Hyny-nen ldquoChemical composition and ecophysiological responsesof Empetrum nigrum to aboveground element applicationrdquoEnvironmental Pollution vol 112 no 3 pp 417ndash426 2001

[36] J M Becerril A Munoz-Rueda P Aparicio-Tejo and CGonzalez-Murua ldquoThe effects of cadmium and lead on pho-tosynthetic electron transport in clover and lucernerdquo PlantPhysiology and Biochemistry vol 26 pp 913ndash918 1989

[37] H Mohr and P Schopfer Plant Physiology C4 and CAM PlantsSpringer Berlin Germany 1995

[38] B Rossa A V Tuffers G Naidoo and D J von WillertldquoArundo donax L (Poaceae)mdasha C3 species with unusually highphotosynthetic capacityrdquo Botanica Acta vol 111 no 3 pp 216ndash221 1998

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


to most regions and climates cost effective and technicallyfeasible process plants serve as sufficient biomass for rapidremediation promote high rhizosphere activity and finallyrestoration in a reasonable time frame of 2 to 3 years

The use of low cost fast growing indigenous plants withefficient biomass producing plant species such as Arundodonax L is highly desirable for phytoremediation of metalcontaminated sites and waters It is cultivable throughoutAsia Southern Europe North Africa and theMiddle East forthousands of years with local names of ldquoGiant Reedrdquo ldquoNurrrdquoldquoNurrurdquo or ldquoNurrordquo [7ndash9] It is considered as one of the mostbiologically productive of all communities [10] Previousexperiments on giant reed suggested that the stem height anddiameter number of nodes fresh and dry weight of leavesand net photosynthesis were not affected indicating thatplants tolerated the high concentrations of Cd and Ni [11]As giant reed plants are very promising energy plants theycan be cultivated in contaminated soils to provide biomass forenergy production purposes [11] Our research group isexploring the potential of this plant in environmental reme-diation of various heavy metals at high concentrationsThe plant showed some potential against arsenic [7ndash9]and chromium [10] remediation The specific objective ofthe current study was to investigate the phytoremediationability of A donax for cadmium remediation and to comparethe Cd removal from contaminated soil and water

2 Materials and Methods

21 Plant Material The plant material used for the presentstudy was A donax L with the aim to evaluate its responsesto cadmium added to liquid mediumThe plant material wascollected near PMA road Abbottabad Khyber PakhtunkhwaPakistan Plants were collected for the heavy metal analysisThe soil-grown plants used for hydroponics tests were takenfrom growth of young meristematic buds grown in sterileaqueous medium [8] The young plants were transplanted inplastic trays containing 12 strength basal nutrient solutions

22 Experimental Design The phytoremediation ability of Adonax was compared in Cd contaminated soil and aqueoussolution The Cd containing aqueous solution was preparedby dissolving cadmium chloride salt in the double distilledwater Various Cd treatments were given in triplicates toexperimental plants (both in hydroponics and soil) Cdtreatments included 0 (control) 50 100 250 500 750 and1000120583g Lminus1 Each pot contained 4 A donax L plants withan average 250 g biomass on fresh weight basis Two kinds ofexperiments were performed in randomized complete blockdesign (RCBD) with three replications for plants grown inHoagland solution [12] and in soil Plants with healthy anduniform shoots of almost equalmorphological characteristicswere immersed in nutrient solution for three weeks Plantswere grown under greenhouse conditions in the laboratorywith Hoagland solution continuously aerated and renewedafter every 2-3 days with an addition of 100ndash200mL ofnutrient solution Table 1 shows the average values of variousplant characteristics

Table 1 Average values of variousmorphological parameters beforecadmium treatment

Parameters ValuesPlant height (cm) 55 plusmn 12

No of leaves per plant 87 plusmn 18

No of nodes per plant 33 plusmn 22

Average root length per plant (cm) 8 plusmn 2

Toxicity symptoms Leaf burning

23 Growth Parameters Various growth parameters of Adonax were studied under cadmium stress including plantheight (cm) number of leaves per plant number of nodesper plant dry weight average root length (cm) and toxicitysymptoms prior to and after the cadmium treatments

24 Physiological Parameters The physiological parametersof the plants included photosynthetic pigments and thedetermination of antioxidants

241 Photosynthetic Pigments At the time of harvest freshleaves of both of various treatments fromboth kinds of exper-iments were collected for the determination of chlorophyll achlorophyll b and carotene contents Leaves were cut intosmall pieces and 05 g of the sample was put into the glasstest tubesThen 10mL of 80 acetone was added to the tubesand kept overnight for complete extraction Photosyntheticpigments were determined spectrophotometrically using thevisible wavelengths of 663 645 and 480 nm for chlorophyll achlorophyll b and carotene respectively [12]

242 Antioxidant Determination To determine antioxidantactivity another parallel set of experiment was conductedIn this experiment four replicates each of 250 gm againsteach treatment was planted in a pot Each pot was givenHoaglandrsquos solution and the respective treatment of cadmiumabove mentioned After each week one set was taken out andnew leaves were countedThese plants were placed outside forshade dry

All the plants were ground until uniform size Now thissample was dipped in methanol for 4 to 5 days The plantextract was filtered and methanol was recovered by passingit through rotary The thick left over extract was taken outin Petri dishes and placed in fume hood The thick jelly likeextract was stored in sample bottles for further analysis

DPPH Free Radical Scavenging Assay 22-diphenyl-1-picrylhydrazyl free radical (DPPH) scavenging activity ofcrude extract and various fractions were estimated by stan-dard DPPH assay protocol with certain modifications [13]The reaction mixture contains 05mL of test samples and25mL of DPPH in methanol Concentration of DPPH was100 120583M in the reaction mixture These reaction mixtureswere incubated for 30min at 37∘C The absorbance wasmeasured at 517 nm using spectrophotometer (5000 IrmecoGmbH D-21496 Geesthacht Germany) Percent radicalscavenging activity or percent antioxidant index (AI)



50





(1)

IC50


50





(2)



(3)


3

HClO4


3








3 Results



0 200 400 600 800 1000 1200

0

50

100

150

200

250

300

350

Root Stem



Cd

conc

entr

atio

n in

pla

nt p

arts

(120583g g

minus1)

(a)

0 200 400 600 800 1000 1200

0

100

200

300

400

Root Stem

Leaves Soil medium

Cd

conc

entr

atio

n in

pla

nt p

arts

(120583g g

minus1)


(b)









0 200 400 600 800 1000 120000

05

10

15

20

25


Carotene


Chlo

roph

yll c

once

ntra

tion

(mg g

minus1)

(a)


Carotene

0 200 400 600 800 1000 1200

00

05

10

15

20

Chlo

roph

yll c

once

ntra

tion

(mg g

minus1)

minus15

minus10

minus05


(b)


0 200 400 600 800 1000 1200

Fact

or v

alue

s

0

5

10

15

20

25

30

35



(a)

0 200 400 600 800 1000 1200

Fact

or v

alue

s

00

02

04

06

08

10

12

14

16



(b)






0 200 400 600 800 1000 1200

Num

ber o

f lea

ves a

nd fr

esh

wei

ght (

g)

0

50

100

150

200

250

300

350

Num

ber o

f till

ers

0

1

2

3

4

5

Leaves Fresh weight

Tillers


(a)

Leaves Fresh weight

Tillers

0 200 400 600 800 1000 1200

Num

ber o

f lea

ves a

nd fr

esh

wei

ght (

g)

0

100

200

300

400

Num

ber o

f till

ers

0

1

2

3

4

5

6

7


(b)




50



50


50


4 Discussion


0 200 400 600 800 1000 12000

10

20

30

40

50


After 21 days


IC50

valu

es






0 200 400 600 800 1000 12000

20

40

60

80

100

120

140


After 21 days

IC50

valu

es









The IC50


50


50




50


50


2


2


5 Conclusions




References












































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



50





(1)

IC50


50





(2)



(3)


3

HClO4


3








3 Results



0 200 400 600 800 1000 1200

0

50

100

150

200

250

300

350

Root Stem



Cd

conc

entr

atio

n in

pla

nt p

arts

(120583g g

minus1)

(a)

0 200 400 600 800 1000 1200

0

100

200

300

400

Root Stem

Leaves Soil medium

Cd

conc

entr

atio

n in

pla

nt p

arts

(120583g g

minus1)


(b)









0 200 400 600 800 1000 120000

05

10

15

20

25


Carotene


Chlo

roph

yll c

once

ntra

tion

(mg g

minus1)

(a)


Carotene

0 200 400 600 800 1000 1200

00

05

10

15

20

Chlo

roph

yll c

once

ntra

tion

(mg g

minus1)

minus15

minus10

minus05


(b)


0 200 400 600 800 1000 1200

Fact

or v

alue

s

0

5

10

15

20

25

30

35



(a)

0 200 400 600 800 1000 1200

Fact

or v

alue

s

00

02

04

06

08

10

12

14

16



(b)






0 200 400 600 800 1000 1200

Num

ber o

f lea

ves a

nd fr

esh

wei

ght (

g)

0

50

100

150

200

250

300

350

Num

ber o

f till

ers

0

1

2

3

4

5

Leaves Fresh weight

Tillers


(a)

Leaves Fresh weight

Tillers

0 200 400 600 800 1000 1200

Num

ber o

f lea

ves a

nd fr

esh

wei

ght (

g)

0

100

200

300

400

Num

ber o

f till

ers

0

1

2

3

4

5

6

7


(b)




50



50


50


4 Discussion


0 200 400 600 800 1000 12000

10

20

30

40

50


After 21 days


IC50

valu

es






0 200 400 600 800 1000 12000

20

40

60

80

100

120

140


After 21 days

IC50

valu

es









The IC50


50


50




50


50


2


2


5 Conclusions




References












































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0 200 400 600 800 1000 1200

0

50

100

150

200

250

300

350

Root Stem



Cd

conc

entr

atio

n in

pla

nt p

arts

(120583g g

minus1)

(a)

0 200 400 600 800 1000 1200

0

100

200

300

400

Root Stem

Leaves Soil medium

Cd

conc

entr

atio

n in

pla

nt p

arts

(120583g g

minus1)


(b)









0 200 400 600 800 1000 120000

05

10

15

20

25


Carotene


Chlo

roph

yll c

once

ntra

tion

(mg g

minus1)

(a)


Carotene

0 200 400 600 800 1000 1200

00

05

10

15

20

Chlo

roph

yll c

once

ntra

tion

(mg g

minus1)

minus15

minus10

minus05


(b)


0 200 400 600 800 1000 1200

Fact

or v

alue

s

0

5

10

15

20

25

30

35



(a)

0 200 400 600 800 1000 1200

Fact

or v

alue

s

00

02

04

06

08

10

12

14

16



(b)






0 200 400 600 800 1000 1200

Num

ber o

f lea

ves a

nd fr

esh

wei

ght (

g)

0

50

100

150

200

250

300

350

Num

ber o

f till

ers

0

1

2

3

4

5

Leaves Fresh weight

Tillers


(a)

Leaves Fresh weight

Tillers

0 200 400 600 800 1000 1200

Num

ber o

f lea

ves a

nd fr

esh

wei

ght (

g)

0

100

200

300

400

Num

ber o

f till

ers

0

1

2

3

4

5

6

7


(b)




50



50


50


4 Discussion


0 200 400 600 800 1000 12000

10

20

30

40

50


After 21 days


IC50

valu

es






0 200 400 600 800 1000 12000

20

40

60

80

100

120

140


After 21 days

IC50

valu

es









The IC50


50


50




50


50


2


2


5 Conclusions




References












































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0 200 400 600 800 1000 120000

05

10

15

20

25


Carotene


Chlo

roph

yll c

once

ntra

tion

(mg g

minus1)

(a)


Carotene

0 200 400 600 800 1000 1200

00

05

10

15

20

Chlo

roph

yll c

once

ntra

tion

(mg g

minus1)

minus15

minus10

minus05


(b)


0 200 400 600 800 1000 1200

Fact

or v

alue

s

0

5

10

15

20

25

30

35



(a)

0 200 400 600 800 1000 1200

Fact

or v

alue

s

00

02

04

06

08

10

12

14

16



(b)






0 200 400 600 800 1000 1200

Num

ber o

f lea

ves a

nd fr

esh

wei

ght (

g)

0

50

100

150

200

250

300

350

Num

ber o

f till

ers

0

1

2

3

4

5

Leaves Fresh weight

Tillers


(a)

Leaves Fresh weight

Tillers

0 200 400 600 800 1000 1200

Num

ber o

f lea

ves a

nd fr

esh

wei

ght (

g)

0

100

200

300

400

Num

ber o

f till

ers

0

1

2

3

4

5

6

7


(b)




50



50


50


4 Discussion


0 200 400 600 800 1000 12000

10

20

30

40

50


After 21 days


IC50

valu

es






0 200 400 600 800 1000 12000

20

40

60

80

100

120

140


After 21 days

IC50

valu

es









The IC50


50


50




50


50


2


2


5 Conclusions




References












































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0 200 400 600 800 1000 1200

Num

ber o

f lea

ves a

nd fr

esh

wei

ght (

g)

0

50

100

150

200

250

300

350

Num

ber o

f till

ers

0

1

2

3

4

5

Leaves Fresh weight

Tillers


(a)

Leaves Fresh weight

Tillers

0 200 400 600 800 1000 1200

Num

ber o

f lea

ves a

nd fr

esh

wei

ght (

g)

0

100

200

300

400

Num

ber o

f till

ers

0

1

2

3

4

5

6

7


(b)




50



50


50


4 Discussion


0 200 400 600 800 1000 12000

10

20

30

40

50


After 21 days


IC50

valu

es






0 200 400 600 800 1000 12000

20

40

60

80

100

120

140


After 21 days

IC50

valu

es









The IC50


50


50




50


50


2


2


5 Conclusions




References












































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0 200 400 600 800 1000 12000

20

40

60

80

100

120

140


After 21 days

IC50

valu

es









The IC50


50


50




50


50


2


2


5 Conclusions




References












































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



50


50


2


2


5 Conclusions




References












































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of