This article was downloaded by: [Aston University]On: 22 August 2014, At: 06:25Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Click for updates

International Journal ofPhytoremediationPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/bijp20

Single and Combined Effects of ExposureConcentration and Duration on BiologicalResponses of Ceratophyllum demersumL. Exposed to Cr SpeciesFatih Duman a & Fatih Dogan Koca aa Erciyes University, Faculty of Sciences, Department of Biology ,TurkeyAccepted author version posted online: 01 Aug 2013.Publishedonline: 10 Mar 2014.

To cite this article: Fatih Duman & Fatih Dogan Koca (2014) Single and Combined Effects ofExposure Concentration and Duration on Biological Responses of Ceratophyllum demersumL. Exposed to Cr Species, International Journal of Phytoremediation, 16:12, 1192-1208, DOI:10.1080/15226514.2013.821450

To link to this article: http://dx.doi.org/10.1080/15226514.2013.821450

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the“Content”) contained in the publications on our platform. However, Taylor & Francis,our agents, and our licensors make no representations or warranties whatsoever as tothe accuracy, completeness, or suitability for any purpose of the Content. Any opinionsand views expressed in this publication are the opinions and views of the authors,and are not the views of or endorsed by Taylor & Francis. The accuracy of the Contentshould not be relied upon and should be independently verified with primary sourcesof information. Taylor and Francis shall not be liable for any losses, actions, claims,proceedings, demands, costs, expenses, damages, and other liabilities whatsoever orhowsoever caused arising directly or indirectly in connection with, in relation to or arisingout of the use of the Content.

This article may be used for research, teaching, and private study purposes. Anysubstantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &

Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

Dow

nloa

ded

by [

Ast

on U

nive

rsity

] at

06:

25 2

2 A

ugus

t 201

4

International Journal of Phytoremediation, 16:1192–1208, 2014Copyright C© Taylor & Francis Group, LLCISSN: 1522-6514 print / 1549-7879 onlineDOI: 10.1080/15226514.2013.821450

SINGLE AND COMBINED EFFECTS OF EXPOSURECONCENTRATION AND DURATION ON BIOLOGICALRESPONSES OF CERATOPHYLLUM DEMERSUM L.EXPOSED TO Cr SPECIES

Fatih Duman and Fatih Dogan KocaErciyes University, Faculty of Sciences, Department of Biology, Turkey

This study aimed to demonstrate the ways in which two chromium species, Cr (III) andCr (VI), can affect various physiological and biochemical parameters in the plant Cer-atophyllum demersum L., and to evaluate the single and combined impact of exposureconcentration and duration. C. demersum was exposed to Cr (III) and Cr (VI) at a varietyof concentrations (1, 2, 5, and 10 mM) and for differing durations (1, 2, 4, and 7 days),after which Cr accumulation, relative growth rate (RGR), malondialdehyde (MDA) content,electrical conductivity (EC), photosynthetic pigmentation, proline content and antioxidantenzyme activities were examined. The single and combined effects of exposure durationand Cr concentration on each parameter were determined using a two-way analysis of vari-ance. For both the Cr (III) and Cr (VI) applications, it was observed that concentrationhad a significant effect on all parameters assessed. However, duration had no statisticallysignificant effect on proline content in the Cr (III) application, or on MDA and proteincontent in the Cr (VI) application. It was determined that concentration exerted greatereffects than duration for both Cr species studied. In addition, the results indicated thatduration and concentration had a synergistic effect on variations of RGR, EC, protein con-tent, and antioxidant enzyme activities in both the Cr (III) and Cr (VI) applications. Theseresults may be useful when planning further phytoremediation and plant biotechnologystudies.

KEY WORDS: Ceratophyllum demersum, biological response, chromium species, exposure

INTRODUCTION

Water pollution has emerged as an important issue in recent years, due to therapid increase in urban settlement, agricultural activities, developing industrialization,and mining practices. Therefore, the protection of bodies of water from the effects ofthis pollution and the purification of contaminated water have become global issues.Chromium (Cr) is the seventh most common metal on earth and is generally used invarious branches of industry, such as the paper, petrochemical, metal, and textile indus-tries, as well as in glass, cement, and asbestos manufacturing (Saha and Orvig 2010).

Address correspondence to Fatih Duman, Erciyes University, Faculty of Sciences, Department of Biology,38039 Kayseri, Turkey. E-mail: fduman@erciyes.edu.tr.

1192

Dow

nloa

ded

by [

Ast

on U

nive

rsity

] at

06:

25 2

2 A

ugus

t 201

4

AQUATIC PLANT RESPONSES TO CHROMIUM 1193

The most common and stable forms of Cr are the trivalent (Cr (III)) and hexavalent (Cr(VI)) species. In addition to the differing physicochemical properties of Cr (III) and Cr(VI), their biochemical reactivity also varies greatly. Both Cr (VI) and Cr (III) are canbe metabolized by plants, and it is known that Cr (VI) has greater toxicity than Cr (III)(Shanker et al. 2005). Cr (VI) is carried from the cell membrane through ion channels,and damages the root membrane, owing to its high oxidation power. In addition, it re-duces the intake of other essential elements (Gardea-Torresdey et al. 2005). Cr (III) isreplaced with Fe (III) and reduces the activities of iron-containing proteins (Shanker et al.2005).

Phytoremediation is an environmentally friendly, safe, cheap, and common methodof remediating contaminated water. The capacity to accumulate Cr may vary among plants;for example, it has been reported that species that are members of the Brassicaceae familyaccumulate Cr more efficiently (Zayed et al. 1998). Moreover, the results of previousstudies indicate that aquatic plants can accumulate substantial amounts of Cr in theirtissues and organs (Vajpayee et al. 2000; Dhir et al. 2009). In addition, Garg and Chandra(1990) determined that Ceratophyllum demersum L., which is a common aquatic plant,is an outstanding Cr accumulator. When Cr enters a plant cell, various physiological andbiochemical changes occur, such as reduction in plant growth and biomass, chlorosis,membrane damage, and alterations in its antioxidant activity (Rai et al. 2004; Ganeshet al. 2008). It is known that the amount of reactive oxygen species (ROS) increasesin plant cells when these cells are exposed to Cr stress, and that in order to reduce theharmful effects of these ROS, plants possess an effective defense system composed ofantioxidant enzymes and antioxidant molecules (Gangwar and Singh 2011). The biologicalresponses of various aquatic and terrestrial plants have been examined following exposureto Cr stress. For example, Paiva et al. (2009) exposed Eichhornia crassipes to 1 and10 mM of Cr (III) (Cr2O3) and Cr (VI) (K2Cr2O7) for 0, 2, and 4 days, respectively,and analyzed Cr accumulation, gas exchange and photosynthetic pigment content. It wasconsequently determined that Cr (VI) negatively affects photosynthetic pigment content andgas exchange, and, moreover, that Cr accumulation in plants is higher when they are exposedto Cr (VI). In a similar study, Dazy et al. (2008) analyzed Cr accumulation, photosyntheticpigmentation, and the antioxidant enzyme activities of the Fontinalis antipyretica plantin a duration- and concentration-dependent manner after its exposure to Cr (III) and Cr(VI). Both Cr (VI) and Cr (III) had effects on the parameters studied, but Cr (VI) wasdetermined as being more harmful than Cr (III). Sinha et al. (2005) exposed the Pistiastratiotes aquatic plant to Cr (VI) (K2Cr2O7) at increasing concentrations (0, 10, 40, 80,and 160 μM) and for increasing durations (48, 96, and 144 hours) and analyzed thebiological response of the plant. It was shown that Cr accumulation increased in a duration-and concentration-dependent manner. A survey of the literature revealed that no studyof the single and combined effects of varied concentrations and durations of Cr speciesapplication on the biological responses of exposed plants has previously been conducted.C. demersum was chosen as the model organism of the present study, since it has noroots, its stem is in full contact with water, it possesses efficient photosynthetic activity,and it is widely distributed (Urnebese and Motajo 2008; Mishra et al. 2009; Aravindand Prasad 2004). This study was conducted to demonstrate how two chromium species,Cr (III) and Cr (VI), can affect various physiological and biochemical parameters in C.demersum and to evaluate the single or combined effect of exposure concentration andduration.

Dow

nloa

ded

by [

Ast

on U

nive

rsity

] at

06:

25 2

2 A

ugus

t 201

4

1194 F. DUMAN AND F. D. KOCA

MATERIAL AND METHODS

Plant Material and Treatment Conditions

Plants of C. demersum were obtained in June of 2010 from local unpolluted ponds(Soysallı Spring, Kayseri, Turkey). Prior to the experiment, containers were disinfected byimmersion in 1% (v/v) NaClO for three to five minutes. Containers were then rinsed threetimes with distilled water (Hou et al. 2007). Before treatment, plants were acclimatized forfive days in laboratory conditions (115 μmol m−2 s−1 light with 14 h photoperiod at 25 ±2◦C) in 10% Hoagland’s solution. The plants that were in good health were selected forsubsequent experiments.

Experimental Design

The experiments conducted in the present study were set-up in triplicate, whereineach replicate constituted approximately 4 g of the evaluated plants. The Cr (III) and Cr (VI)solutions were prepared from CrCl3 and K2Cr2O7, respectively. The C. demersum sampleswere each exposed to four test concentrations (1, 2, 5, and 10 mM) of Cr (III) and Cr (VI)maintained in 10% Hoagland’s solution in separate 400 mL conical flasks. The plants thatwere not exposed to Cr (III) and Cr (VI) served as the control groups of this experiment. Theflasks were placed in a climate chamber under the aforementioned conditions for periodsof 1, 2, 4, and 7 days. The change that occurred in the volume of the solution within theflasks due to evapotranspiration was compensated for by the addition of double distillatedwater. At the end of the exposure experiment, the resultant plant samples were collectedand sieved with a plastic griddle. Each plant was rinsed with deionized water, drained, andthen blotted on paper towels for 2 min.

Chromium Quantification and Calculation of RGRs

The relative growth rate (RGR) of C. demersum was calculated in each treatmentusing the equation:

RGR (%/day) = [ln(W2) − ln(W1)]/t × 100

W1 and W2 are the initial and final fresh weights (g), respectively, and t is the length of theexperimental period (Tanhan et al. 2007).

An aliquot of each sample was dried at 70◦C. Each sample was then digested with10 mL of pure HNO3 using a CEM Mars 5 (CEM Corporation Mathews, NC, USA)microwave digestion system (maximum power: 1200 W, power: 100%, ramp: 20:00 min,pressure: 180 psi, temperature: 210◦C and hold time: 10:00 min). After digestion, thevolume of each sample was adjusted to 50 ml using double deionized water. Determinationsof Cr concentrations in all samples were carried out by inductively coupled plasma massspectroscopy (Agilent, 7500a).Aquatic plant (BCR - 670) was used as reference material,and also all analytical procedures were performed on reference materials. Samples wereanalyzed in triplicate. All chemicals used in this study were analytical reagent grade (Merck,Darmstadt, Germany).

Dow

nloa

ded

by [

Ast

on U

nive

rsity

] at

06:

25 2

2 A

ugus

t 201

4

AQUATIC PLANT RESPONSES TO CHROMIUM 1195

Ion Leakage and Lipid Peroxidation

The ion leakage induced by the Cr species was estimated by measuring the elec-trical conductivity (EC) (Devi and Prasad 1998). Cr-exposed plants were washed withdouble-deionized water. To facilitate maximum ion leakage, 500 mg of plant material wastransferred to 100 mL of deionized water for 24 h. The EC value of the water was thenmeasured with WTW model conductivimeter. MDA concentrations were measured as de-termination of lipid peroxidation. Plant material (500 mg) was homogenized with 3 mL of0.5% TBA (Thiobarbutiric acid) in 20% TCA (Trichloroacetic acid) (w/v). The homogenatewas incubated at 95◦C for 30 min, and ice was used to stop the reaction. The samples werecentrifuged at 10,000 × g for 10 min, and the absorbance of the resulting supernatant wasrecorded at 532 nm and 600 nm. The amount of malondialdehyde (MDA) was calculated(extinction coefficient of 155 mM−1 cm−1) by subtracting the non-specific absorbance at600 nm from the absorbance at 532 nm (Heath and Packer 1968).

Determination of Photosynthetic Pigment Contents

Photosynthetic pigments of treated and untreated plants (100 mg) were extracted in80% chilled acetone in the dark. After centrifugation at 10,000 × g for 10 min, absorbanceof the supernatant was measured at 450, 645, and 663 nm. The chlorophyll content wasestimated by the method of Arnon (1949), and the carotenoid content was estimated aspreviously described (Duxbury and Yentsch 1956).

Determination of Proline and Total Protein Content

Plant samples (500 mg) were homogenized in 1 mL of 100 mM potassium phosphatebuffer (pH 7.0) containing 0.1 mM EDTA and 1% polyvinyl pyrrolidone (PVP, (w/v)) at4◦C. The homogenate was centrifuged at 15,000×g for 15 min at 4◦C. The protein contentwas determined by the method of Lowry et al. (1951) using bovine serum albumin as thestandard protein. The amount of proline was determined according to a modified methodof Bates (1973). Free proline content was extracted from 0.25 g of samples in 3% (w/v)aqueous sulphosalicylic acid and estimated by ninhydrin reagent. Absorbance of the upperphase was recorded at 520 nm against toluene blank.

SOD and CAT Activity

SOD was assayed by the photochemical method described by Giannopolitis and Ries(1977) with some modifications. The reaction mixture contained 20 mM sodium phosphate(pH 7.5), 10 mM methionine, 0.1 mM EDTA, 0.1 mM nitro-blue-tetrazolium (NBT), 5 μMriboflavin and 50 μg mL−1 crude enzyme extract (extracted supernatant of plant sample)in a total volume of 3 ml. One unit SOD activity was defined as the amount of enzymerequired to result in a 50% inhibition of the rate of NBT (ρ-nitro blue tetrazolium chloride)reduction measured at 560 nm (Liang et al. 2003). To measure CAT activity, we performedan extraction step with buffer containing 50 mMTris-HCl (pH 7.0), 0.1 mM EDTA, 1 mMphenyl methyl sulphonyl fluoride (PMSF) and 1% PVP, according to the method of Aebi(1974). The reaction mixture consisted of 2.5 mL of 50 mM sodium phosphate buffer (pH7.0), 300 μL of 20 mM H2O2 and a suitable aliquot of enzyme. The change in absorbance

Dow

nloa

ded

by [

Ast

on U

nive

rsity

] at

06:

25 2

2 A

ugus

t 201

4

1196 F. DUMAN AND F. D. KOCA

was measured at 240 nm (extinction coefficient 0.04 cm2 μmol−1). Enzyme activity wasexpressed in units of protein mg−1.

Statistics

All values are expressed as mean ± standard deviation. Normal distribution for datawas analyzed by The Kolmogorov- Smirnov test and Levene’stest was used to ensurethe normality assumption and the homogeneity of variances. If significant heterogeneityof variance was detected, data were log transformed ln (x+1) and reevaluated. Two-way analysis of variance (two-way ANOVA) was used to assess the significance of theeffects of exposure concentration and duration, as well as of their interaction, on studiedparameters. Post hoc pairwise comparison of sample means was performed with Tukey’shonestly significant difference test. Additionally, we addressed the effect size of associationof each factor to the ANOVA model by calculating the partial eta-squared (η2) value.Correlations between accumulated Cr and studied parameters were also performed. Allstatistical analyses were done with the SPSS 15.0 software package.

RESULTS

Cr Accumulation

In this study, the highest Cr accumulation was found to be in 10 mM-7d (13.89 mgg−1dw) for the Cr (III) application, and 10mM-7d (14.26 mg g−1dw) for the Cr (VI)application (Figure 1 A and B). As it was seen in Table 1, both duration (η2 = 0.958, P <

0.001) and concentration (η2 = 0.978, P < 0.001) have statistically significant effect on theCr (III) application. Furthermore, a significant of concentration - duration interaction wasdetermined on Cr accumulation (η2 = 0.882, P < 0.001). Similarly, both concentration (η2

= 0.897, P < 0.001) and duration (η2 = 0.56, P < 0.001) were determined to be effectiveon the Cr (VI) application. However, no significant duration - concentration interaction wasdetermined on the Cr (VI) application (η2 = 0.328, P > 0.05).

RGR

Although there are no negative values at all for the Cr (III) application (Figure 1 C),negative values were observed in the Cr (VI) application (Figure 1 D). The highest RGRvalue was found to be 8.61 in the 2mM-2d application for Cr (III) and 4.45 for 2 daysof control application for Cr (VI). The lowest RGR values were observed in the 10 mM-7dapplication both for Cr (III) and Cr (VI) (Cr (III):0.37; Cr (VI):-4.26). It was determinedthat both duration and concentration are effective on the Cr (III) and Cr (VI) applicationsand that duration-concentration interaction was also important for both of the applications(Table 1). While no statistical correlation was found between Cr accumulation and RGR(R = –0.222, P > 0.05) for Cr (III), a significant relationship between RGR values and Craccumulation was determined for Cr (VI) (R = –0,795, P < 0.01) (Table 2).

EC

The highest EC value for Cr (III) was observed to be (16.85 mmhos cm−1 g−1fw) inthe 10 mM-10d application, and for Cr (VI) (29.87 mmhos cm−1 g−1fw) in the 10 mM-7d

Dow

nloa

ded

by [

Ast

on U

nive

rsity

] at

06:

25 2

2 A

ugus

t 201

4

AQUATIC PLANT RESPONSES TO CHROMIUM 1197

Figure 1 Cr accumulation, RGR, EC and MDA values of C. demersum exposed to different concentrations ofCr (III) (first column) and Cr (VI) (second column) for various periods. Values represent means. Vertical barsindicate standard error of three separate experiments.

Dow

nloa

ded

by [

Ast

on U

nive

rsity

] at

06:

25 2

2 A

ugus

t 201

4

1198 F. DUMAN AND F. D. KOCA

Table 1 Summary of two-way analysis of variance for analyzed parameters, with exposure concentration andduration as independent variables.

Cr (III) Cr (VI)

d.f. F P Effect size∗ d.f. F P Effect size∗

AccumulationDuration 3 304.481 <0.001 0.958 3 16.985 <0.001 0.560Concentration 4 448.164 <0.001 0.978 4 87.124 <0.001 0.897Duration × Concentration 12 24.809 <0.001 0.882 12 1.630 0.122 0.328RGRDuration 3 54.369 <0.001 0.803 3 13.258 <0.001 0.499Concentration 4 22.334 <0.001 0.691 4 104.009 <0.001 0.912Duration × Concentration 12 6.201 <0.001 0.650 12 2.999 0.004 0.474ECDuration 3 59.497 <0.001 0.817 3 5.876 0.002 0.306Concentration 4 202.235 <0.001 0.953 4 94.457 <0.001 0.904Duration × Concentration 12 11.168 <0.001 0.770 12 2.187 0.032 0.396MDADuration 3 0.722 0.545 0.051 3 19.239 <0.001 0.591Concentration 4 30.358 <0.001 0.752 4 171.651 <0.001 0.945Duration × Concentration 12 1.326 0.243 0.285 12 6.917 <0.001 0.675Chlorophyll aDuration 3 44.358 <0.001 0.769 3 11.057 <0.001 0.453Concentration 4 92.256 <0.001 0.902 4 66.629 <0.001 0.870Duration × Concentration 12 3.710 0.001 0.527 12 1.302 0.255 0.281Chlorophyll bDuration 3 48.118 <0.001 0.783 3 6.913 0.001 0.341Concentration 4 88.629 <0.001 0.899 4 26.320 <0.001 0.725Duration × Concentration 12 8.296 <0.001 0.713 12 0.955 0.506 0.223CarotenoidDuration 3 16.705 <0.001 0.556 3 18.487 <0.001 0.581Concentration 4 32.907 <0.001 0.767 4 66.539 <0.001 0.869Duration × Concentration 12 1.498 0.165 0.310 12 2.019 0.048 0.377ProlineDuration 3 2.878 0.048 0.178 3 1.005 0.400 0.70Concentration 4 34.913 <0.001 0.777 4 188.302 <0.001 0.950Duration × Concentration 12 1.141 0.357 0.255 12 1.473 0.175 0.307ProteinDuration 3 2.218 0.101 0.143 3 7.512 <0.001 0.360Concentration 4 16.862 <0.001 0.628 4 86.340 <0.001 0.896Duration × Concentration 12 6.133 <0.001 0.648 12 7.435 <0.001 0.690SODDuration 3 26.366 <0.001 0.664 3 3.178 0.034 0.192Concentration 4 12.763 <0.001 0.561 4 28.089 <0.001 0.737Duration × Concentration 12 22.405 <0.001 0.870 12 2.750 0.008 0.452CATDuration 3 157.627 <0.001 0.922 3 545.607 <0.001 0.976Concentration 4 23.416 <0.001 0.701 4 415.726 <0.001 0.977Duration × Concentration 12 130.356 <0.001 0.975 12 296.906 <0.001 0.989

Values P < 0.05 are given in bold; d.f., degrees of freedom, ∗Effect size is given in partial eta-squared (η2).

Dow

nloa

ded

by [

Ast

on U

nive

rsity

] at

06:

25 2

2 A

ugus

t 201

4

Tabl

e2

Cor

rela

tions

betw

een

accu

mul

ated

Cr

and

stud

ied

para

met

ers.

RG

RE

CM

DA

Chl

aC

hlb

Car

oten

oid

Prol

ine

Prot

ein

SOD

CA

T

Cr

(III

)C

rac

cum

ula-

tion

−0.2

220.

364(∗

∗ )0.

649(∗

∗ )−0

.883

(∗∗ )

−0.7

80(∗

∗ )−0

.816

(∗∗ )

−0.5

55(∗

∗ )−0

.454

(∗∗ )

0.40

3(∗∗ )

−0.1

50

Cr

(VI)

Cr

accu

mul

a-tio

n−0

.795

(∗∗ )

0.87

4(∗∗ )

0.86

4(∗∗ )

−0.7

96(∗

∗ )−0

.726

(∗∗ )

−0.8

23(∗

∗ )−0

.726

(∗∗ )

−0.3

06(∗

)0.

149

0.48

5(∗∗ )

∗∗C

orre

latio

nis

sign

ifica

ntat

the

0.01

leve

l.∗ C

orre

latio

nis

sign

ifica

ntat

the

0.05

leve

l.

1199

Dow

nloa

ded

by [

Ast

on U

nive

rsity

] at

06:

25 2

2 A

ugus

t 201

4

1200 F. DUMAN AND F. D. KOCA

application (Fig. 1 E and F). It will be seen in Table 1 that there are single and combinedeffects of concentration and duration on EC. However, the synergistic effect for the Cr (III)application was found to be statistically high when compared to the Cr (VI) application.Significant correlations were determined between Cr accumulation and EC values for bothCr (III) (R = 0.364, P < 0.01) and Cr (VI) (R = 0.874, P < 0.01) (Table 2).

MDA

The highest MDA content was observed in the 10 mM-7d application for both Cr(III) and Cr (VI) (24.84 μmol g−1fw for Cr (III); 26.27 μmol g−1fw for Cr (VI)) (Figures 1G and H). Concentration and duration were determined to have a significant effect onMDA content for the Cr (III) application. Additionally, no synergistic effect of durationor combination on MDA content was determined. However, it was seen that duration andcombination have both a single and combined effect in the Cr (VI) application (Table 1).Furthermore, concentration was found to be more effective on MDA content than duration.Correlation between Cr accumulation and MDA values was determined to be significantfor both Cr (III) (R = 0.649, P < 0.01) and Cr (VI) (R = 0.864, P < 0.01) (Table 2).

Photosynthetic Pigments

The lowest level of chlorophyll a was observed in the 2 mM-7d application as 0.17 mgg−1fw for Cr (III) and in the 10 mM-7dapplication as 0,13 mg g−1fw for Cr (VI) (Figure 2A and B). It was determined that duration and concentrations are significantly effectiveon chlorophyll a for both Cr (III) and Cr (IV) and moreover, concentration holds greatersignificance on the amount of chlorophyll a, rather than duration (Table 1). Significantcorrelations were determined between Cr accumulation and chlorophyll a values both forCr (III) (R = –0.883, P < 0.01) and Cr (VI) (R = –0.796, P < 0.01) (Table 2).

The lowest level of chlorophyll b was observed in the 10 mM - 4d application as0.06 mg g−1fw for Cr (III) and in the 10 mM-7d application as 0.05 mg g−1fw for Cr (VI)(Figure 2C and D). It was found that duration and concentration are significantly effectiveon chlorophyll b for both Cr (III) and Cr (VI). In addition, the synergistic effect of durationand concentration for the Cr (III) (η2 = 0.713, P < 0.001) application was observed whilethe same was not observed for Cr (VI) (η2 = 0.223, P > 0.05). Significant correlationswere determined between Cr accumulation and chlorophyll b values for both Cr (III) (R =–0.78, P < 0.01) and Cr (VI) (R = –0.726, P < 0.01) (Table 2).

As it was seen in Figure 2, the lowest cartenoid content was observed in the 10 mM-4d application as 0.05 mg g−1fw for Cr (III) and in the 10 mM-7d application (0,04 mg g−1fw)for Cr (VI) (Figure 2 E and F). Duration and concentration were found to be significantlyeffective on carotenoid for both Cr (III) and Cr (IV). Also, while the synergistic effectof duration and concentration was observed for the Cr (VI) application (η2 = 0.377, P <

0.05), the same was not observed for Cr (III) (η2 = 0.31, P > 0.05) (Table 1). Significantcorrelations were determined between Cr accumulation and cartenoid values for both Cr(III) (R = −0.816, P < 0.01) and Cr (VI) (R = —0.823, P < 0.01) (Table 2).

Proline

The lowest level of proline was observed in the 10 mM-4d application as 0,005 μmolg−1 for Cr (III), and in the 10mM-1d application (0.001 μmol g−1) for Cr (VI) (Figure 2 G

Dow

nloa

ded

by [

Ast

on U

nive

rsity

] at

06:

25 2

2 A

ugus

t 201

4

AQUATIC PLANT RESPONSES TO CHROMIUM 1201

Figure 2 Photosynthetic pigment and proline content of C. demersum exposed to different concentrations of Cr(III) (first column) and Cr (VI) (second column) for various periods. Values represent means. Vertical bars indicatestandard error of three separate experiments.

Dow

nloa

ded

by [

Ast

on U

nive

rsity

] at

06:

25 2

2 A

ugus

t 201

4

1202 F. DUMAN AND F. D. KOCA

and H). According to the findings of this study, both duration (η2 = 0.178, P < 0.05) andconcentration (η2 = 0.777, P < 0.001) are significant for the Cr (III) application while onlyconcentration (η2 = 0.950, P < 0.001) was determined to be significant in the change ofproline content for the Cr (VI) application. No synergistic effect of duration or concentrationwas observed for the Cr (III) and Cr (VI) applications. Significant correlations between Craccumulation and proline values were determined for both the Cr (III) (R = −0.555, P <

0.01)and Cr (VI) (R = −0.726, P < 0.01) applications (Table 2).

Protein

Among all applications, the highest protein content was found in the 2 mM - 4dapplication (18.05 mg g−1fw)for Cr (VI), while the lowest was found in the 1mM-7dapplication(4.64 mg g−1fw) for Cr (III) (Figure 3 A and B). In the Cr (III) application, nosignificant effect of duration on protein content was found; however, the concentration wasobserved to be effective (η2 = 0.628, P < 0.001) (Table 1). For the Cr (VI) application, bothduration and concentration were detected to have not only a single but also a combinedsignificant effect on protein. Significant correlations were determined for both Cr (III) (R= −0.454, P < 0.01) and Cr (VI) (R = −0.306, P < 0.05), between Cr accumulation andprotein content.

SOD and CAT Activity

The highest SOD activity for Cr (VI) was found in the 5 mM-4 d application (41,5units mg−1 protein), while the highest CAT activity for Cr (VI) was found to be in the 5mM-7dapplication (9.29 units mg−1 protein) (Figure 3C, D, E and F). It was determinedthat duration and concentration have not only a single but also a combined effect in both theCr (III) and Cr (VI) applications for SOD and CAT enzymes (Table 1). While a significant(R = 0.403, P < 0.01) positive correlation between Cr accumulation and SOD activity wasfound in the Cr (III) application, no significant relationship between Cr accumulation andCAT activity was detected. Moreover, a positive and significant (R = 0.485, P < 0.01)relation was found between Cr accumulation and CAT activity in the Cr (VI) application,but no statistically significant correlation could be found between Cr accumulation andSOD.

DISCUSSION

C. demersum is an aquatic plant used as a phytoremediator of polluted waters, andprevious studies have determined that this plant is capable of accumulating heavy metals,such as Cd, Cu, Pb, and As (Mishra et al. 2006, 2008, 2009; Devi and Prasad 1998).Several factors are influential in Cr accumulation, such as the external concentration of Cr,as well as the structure and form of the chemical complex formed by Cr (Ksheminska et al.2005). In the present study, it was determined that the C. demersum exposed to Cr (VI)accumulated more Cr than that which was exposed to Cr (III). The reason for this differencemay be explained by the fact that Cr (VI) is up taken with essential ions, such as sulfateand Fe, while Cr (III) uptake does not depend on metabolic energy (Shanker et al. 2005).Similarly, Zayed et al. (1998) determined that vegetables exposed to Cr (VI) accumulatedmore Cr than did vegetables exposed to Cr (III). It is known that Cr accumulation isduration- and concentration-dependent (Dhir et al. 2009). In previous studies, a significant

Dow

nloa

ded

by [

Ast

on U

nive

rsity

] at

06:

25 2

2 A

ugus

t 201

4

AQUATIC PLANT RESPONSES TO CHROMIUM 1203

Figure 3 Protein content and antioxidant enzyme activities of C. demersum exposed to different concentrationsof Cr (III) (first column) and Cr (VI) (second column) for various periods. Values represent means. Vertical barsindicate standard error of three separate experiments.

correlation was determined between accumulated metal concentration in plant and exposedmetal concentration (Mishra et al. 2009; Devi and Prasad 1998). In our study, a significantcorrelation between the application concentration and accumulated Cr was also detected.This indicates that C. demersum has a high capacity for Cr accumulation. We are inagreement with Garg and Chandra (1990), in that we consider that C. demersum may beeffective for the phytoremediation of polluted water. In the present study, the applicationperiod was also determined to have a significant effect on metal accumulation. However,this is the first study to reveal that concentration has a greater effect on Cr accumulationthan does duration, with regard to both the Cr (III) and Cr (VI) applications. In a similarstudy, Duman et al. (2010) determined that concentration exerts a greater effect on As

Dow

nloa

ded

by [

Ast

on U

nive

rsity

] at

06:

25 2

2 A

ugus

t 201

4

1204 F. DUMAN AND F. D. KOCA

accumulation than duration, with regard to As (V) exposure. Duration and concentrationare observed to have a synergistic effect regarding Cr (VI) application, and several factorsmay play a role. However, when the Cr (III) application is considered, it is clear that durationand concentration do not always have a synergistic effect.

Relative Growth Rate (RGR) is an important parameter used to observe the physi-ologic effects of toxic chemicals on plants. It is known that high concentrations of heavymetals reduce RGR values and inhibit plant growth. In their study of Cr accumulation andthe tolerance characteristics of Spartina argentinensis, Redondo-Gomez et al. (2011) deter-mined that there is a negative correlation between external chrome concentration and RGRvalues. In this study, while a negative correlation between accumulated Cr and RGR valueswas shown for the Cr (VI) application, there was no correlation for Cr (III). In addition, forthe Cr (III) application, RGR values were shown to increase in certain conditions of appli-cation in according to control. This may occur because the toxicity of Cr (III) is lower andbecause it is essential for some biochemical reactions (Zayed and Terry 2003). Cedergreen(2008) stated that low-dose toxic chemicals stimulate plant growth, but that higher doseshave a toxic effect. Southam and Erlich (1943) referred to this as the “hormesis effect.”However, the available literature shows that Cr (VI) is regarded as being carcinogenic andtoxic (Shanker et al. 2005). Prado et al. (2010) analyzed the physiologic and biochemicalchanges generated by Salvinia minima against Cr accumulation, on a seasonal basis, anddetermined that Cr accumulation has a greater effect on the RGR values of winter samplesthan on the RGR values of summer samples. Rai et al. (2004) detected that Cr applicationto Ocimum tenuiflorum concentration- and dose-dependently reduces plant biomass, whichis in accordance with our results. In addition, duration had a greater effect on RGR valueswith regard to the Cr (III) application, while concentration was more effective in the Cr(VI) application.

It is known that high concentrations of both Cr (III) and Cr (VI) damage the cell walland membrane, producing ROS (Shanker et al. 2005; Gangwar et al. 2011). A concentration-and duration- dependent increase of EC values indicates the deformation in membrane struc-ture and ion leakage. MDA is an important indicator of lipid peroxidation and productionof free radicals (Ohkawa et al. 1979). Furthermore, an increase in lipid peroxidation isalso an indicator of damaged membrane integration. Sinha et al. (2005) exposed the Pistiastratiotes plant to increasing concentrations (0, 10, 40, 80, and 160 μM) and durations (2, 4,and 6 days) of Cr (VI) to determine lipid peroxidation and antioxidant enzyme activity, andfound that lipid peroxidation increases in a concentration- and duration-dependent manner.Our findings are in agreement with the results of this study with regard to the Cr (VI)application; however in our investigation, concentration had a significant effect with regardto the Cr (III) application, while duration had no significant effect on MDA. Furthermore,MDA concentration-duration interaction was not observed for the Cr (III) application. Itwas also determined that both Cr species enhance lipid peroxidation in C. demersum, whilethe increase had a greater effect with regard to the Cr (VI) application. Paiva et al. (2009)stated that Cr (VI) is more toxic than Cr (III), that Cr (VI) has the capacity to pass throughthe cell membrane due to its negative charge in ion complex as it can be solved in watermore easily and it may disrupt several biochemical reactions upon reaching cytoplasm, andthat Cr (III) shows a toxic effect by blocking ion transportation, mainly as a result of itsaccumulation in the cell membrane. Speranza et al. (2007) stated that while the generaldecision mechanism of Cr (VI) functions as a cellular damage and death disintegratingpolyunsaturatedfattyacid, the lipid reaction formed by Cr (III) is primarily related to ultra-structural changes or organizations, meaning that the primary source of the difference maylie in different mechanisms of action.

Dow

nloa

ded

by [

Ast

on U

nive

rsity

] at

06:

25 2

2 A

ugus

t 201

4

AQUATIC PLANT RESPONSES TO CHROMIUM 1205

Dhir et al. (2009) grew the Salvinia natans aquatic plant in Cr-rich waste water inincreasing concentrations, and determined that Cr stress does not cause any significantchange in pigment concentration (chlorophyll a, b and total chlorophyll). However, in ourstudy, we found that for each of the three photosynthetic pigments (chlorophyll a, andb, and carotenoid), both duration and concentration (not only Cr (III), but also Cr (VI))significantly decreased photosynthetic pigmentation. The reasons for this decrease may beas follows: (1) decrease of Fe content (Vajpayee et al. 2000); (2) inactivation of the enzymesthat possess chlorophyll biosynthesis-related functions (Boonyapookana et al. 2002); or (3)transposition of Mg in the chlorophyll molecule with Cr (Dhir et al. 2009). Paiva et al.(2009) found that total chlorophyll amount decreases when the Eichhornia crassipes aquaticplant is exposed to Cr (VI), and stated that the reason for this may be the decrease in theCO2 exchange rate. Moreover, Shanker (2005) stated that the antenna complex of Crdamages the peripheral part. Vajpayee et al. (2000) determined that a significant amount ofphotosynthetic pigment is reduced even in 10 μM of Cr (VI), which is in accordance withour findings. Moreover, in this study, it was determined that concentration has a greatereffect on each of the three photosynthetic pigments than duration, regarding not only theCr (III), but also the Cr (VI), application. The reason for this occurrence may be explainedby the increase in the adaptation, depending on duration.

Proline generally functions as an osmoprotectant under stress conditions, and pro-tects the cell membrane and biochemical enzymes from free radicals (Torres et al. 2008).It is known that, under conditions of environmental stress, such as heavy metal, drought,and heat, plants enhance their proline content to protect themselves (Alia 1991). Rai et al.(2004) determined that, when exposed to Cr (VI), increase of proline content in the Oci-mum tenuiflorum plant was dependent on concentration increase. However, in our study,we found that although concentration had an effect on proline with regard to both the Cr(III) and Cr (VI) applications, an increase in concentration did not lead to a significantincrease in proline content. We also observed that duration did not have an importanteffect on the proline content of the plants exposed to Cr (VI). In previous studies, pro-line content was reported as having the potential to decrease in plants exposed to highquantities of toxins (Singh et al. 2010; Ozturk et al. 2010). Decreases in proline level aremuch greater with Cr (VI) applications than with Cr (III) applications. This may be theresult of high-toxicity of Cr (VI) and the gradual deformation of proline metabolism overtime.

When plants are exposed to xenobiotics, they endeavor to maintain their lives byproducing proteins as a response to environmental changes, and we know that Cr decreasesprotein content in plants (Ganesh et al. 2008). Sinha et al. (2005) exposed the Pistiastratiotes aquatic plant to Cr in increasing concentrations (10, 40, 80, and 160 μM) anddurations (48, 96, and 144 hours), and found that protein content in the root and leaf ofthe plant increased up to a certain Cr concentration; however, this content decreased inhigh Cr concentrations. We obtained similar results for the Cr (VI) application in our study.The protein content increase in low concentrations may be related to the increase of stressproteins. Conversely, this decrease may be related to damage to several proteins, due tothe increase in ROS; and may also be related to protease, an activity under chrome stress,or protein degradation, which arises as a result of the increase in the activities of othercatabolic enzymes. In addition, Sinha et al. (2005) found a negative relationship betweenaccumulated Cr and protein content, which we also found in our study. We also determinedthat concentration has a greater effect on protein content under Cr exposure, and that theeffect is synergistic. Furthermore, it was also determined that duration has no significanteffect on Cr (III) exposure.

Dow

nloa

ded

by [

Ast

on U

nive

rsity

] at

06:

25 2

2 A

ugus

t 201

4

1206 F. DUMAN AND F. D. KOCA

Plants are equipped with a strong antioxidant defense system against oxidative dam-age. This system includes not only free radical scavengers, such as ascorbates, carotenoidsand thyols, but also antioxidant enzymes, such as SOD and CAT, which play a role in reduc-ing ROS (Dhir et al. 2009). In their study with Fontinalis antipyretica, Dazy et al. (2008)ascertained that the antioxidant enzyme system works effectively in aquatic plants underexposure to both Cr (III) and Cr (VI). These authors also determined that the antioxidantresponse is formed in a “bellshape”, in accordance with concentration and duration. Simi-lar results were also obtained in our study, meaning that SOD and CAT enzyme activitiesincreased for a certain period of time and at a particular concentration, and then decreasedagain. However, such a condition was not observed in antioxidant activities for Cr (III).The reason for the increase may be the effort of trying to manage the oxidative stress. Thedecrease in enzyme activity may result from the deformation in the antioxidant defensesystem, or the attaching of Cr to the active centers of the enzymes. For both enzymes it wasconcluded that enzyme activities were dependent on duration and concentration and thatthe latter had a synergistic effect on enzyme activities.

CONCLUSION

In conclusion, our results suggest that generally concentration is more effective thanduration for both Cr types, with regard to the parameters studied. Duration and concentrationhave a synergistic effect on RGR, EC, and protein content, as well as on SOD and CATenzyme activities. With regard to Cr (III) application, the duration- and concentration-related synergetic effect is present on Cr accumulation; however, no synergetic effectregarding the Cr (VI) application. The results of this study may be useful when planningfuture research in this area.

REFERENCES

Aebi H. 1974. Catalase In: Bergmeyer HU, ed. Methods of enzymatic analysis. Weinheim (Germany):Verlag Chemie. p 773–684.

Alia SPP. 1991. Proline accumulation under heavy metal stress. J Plant Physiol 138:554–558.Aravind P, Prasad MNV. 2004. Zinc protects chloroplasts and associated photochemical func-

tions in cadmium exposed Ceratophyllum demersum L., a freshwater macrophyte. Plant Sci166:1321–1327.

Arnon DI. 1949. Copper enzyme in isolated chloroplast polyphenol oxidase in Beta vulgaris. PlantPhysiol 24:1–15.

Bates LS. 1973. Rapid determination of free proline for water-stress studies. Plant Soil 39:205–207.Boonyapookana B, Upatham ES, Kruatrachue M, Pokethitiyook P, Singhakaew S. 2002. Phytoac-

cumulation and phytotoxicity of cadmium and chromium in duckweed Wolffia globosa. Int JPhytorem 4:87–100.

Cedergreen N. 2008. Is the growth stimulation by low doses of glyphosate sustained over time?Environ Pollut 156:1099–1104.

Dazy M, Beraud E, Cotelle S, Meuxb E, Masfarauda JF, Ferarda JF. 2008. Antioxidant enzymeactivites as affected by trivalent and hexavalent chromium species in Fontinalis antipyreticaHedw. Chemosphere 73:281–290.

Devi SR, Prasad MNV. 1998. Copper toxicity in Ceratophyllum demersum L. (Coontail), a freefloating macrophyte: response of antioxidant enzymes and antioxidants. Plant Sci 138:157–165.

Dhir B, Sharmilla P, Sarahdi P, Nasim SA. 2009. Physiological and antioxidant responses of Salvinanatans exposed to chromium-rich wastewater. Ecotoxicol Environ Safety 72:1790–1797.

Dow

nloa

ded

by [

Ast

on U

nive

rsity

] at

06:

25 2

2 A

ugus

t 201

4

AQUATIC PLANT RESPONSES TO CHROMIUM 1207

Duman F, Ozturk F, Aydin Z. 2010. Biological responses of duckweed (Lemna minor L.) exposedto the inorganic arsenic species As(III) and As(V): effects of concentration and duration ofexposure. Ecotoxicol 19:983–993.

Duxbury AC, Yentsch CS. 1956. Plantkton pigment monograph. J Marine Resour 15:93–101.Ganesh KS, Baskaran L, Rajasekaran S, K Sumathi, ALA, Chidambaram PS. 2008. Chromium stress

induced alterations in biochemical and enzyme metabolism in aquatic and terrestrial plants.Colloids Surf B- Biointerfaces 63:159–163.

Gangwar S, Singh VP. 2011. Indole acetic acid differently changes growth and nitrogen metabolismin Pisum sativum L. seedlings under chromium (VI) phytotoxicity: implication of oxidativestress. Sci Hortic 129:321–328.

Gangwar S, Singh VP, Srivastava PK, Maurya JN. 2011. Modification of chromium (VI) phytotoxicityby exogenous gibberellic acid application in Pisum sativum (L.) seedlings. Acta Physiol Plant33:1385–1397.

Gardea-Torresdey JL, de la Rosa G, Peralta-Videa JR, Montes M, Cruz-Jimenez G, Cano-Aguilera I.2005. Differential uptake and transport of trivalent and hexavalent chromium by tumbleweed(Salsola kali). Arch Environ Contam Toxicol 48:225–232.

Garg P, Chandra P. 1990. Toxicity and accumulation of chromium in Ceratophyllum demersum L.Bull Environ Contam Toxicol 44:473–478.

Giannopolitis N, Ries K. 1977. SOD occurence in higher plants. Plant Physiol 59:309–314.Heath RL, Packer L. 1968. Photoperoxidation in isolated chloroplasts l. Kinetics and stoichiometry

of fatty acid peroxidation. Arch Biochem Biophys 125:189–198.Hou W, Chen X, Song G, Wang Q, Chang CC. 2007. Effects of copper and cadmium on heavy

metal polluted waterbody restoration by duckweed (Lemna minor). Plant Physiol Biochem45:62–69.

Ksheminska H, Fedorowch D, Babyak D, Yanovych D, Kaszycki P, Koloczek H. 2005. Chromium(III) and (VI) tolerance and bioaccumulation in yeast: a survey of cellular chromium contentin selected strains of representative genera. Process Biochem 40:1565–1572.

Liang YC, Chen Q, Liu Q, Zhang WH, Ding RX. 2003. Exogenous silicon (Si) increases antioxidantenzyme activity and reduces lipid peroxidation in roots of salt-stressed barley (Hordeumvulgare L.). J Plant Physiol 160:1157–1164.

Lowry OH, Roenbrough NJ, Farr AL, Randal EJ. 1951. Protein measurement with the folin phenolreagent. J Bio Chem 193:265–275.

Mishra S, Srivastava S, Tripathi RD, Kumar R, Seth CS, Gupta DK. 2006. Lead detoxification bycoontail (Ceratophyllum demersum L.) involves induction of phytochelatins and antioxidantsystem in response to its accumulation. Chemosphere 65:157–165.

Mishra S, Srivastava S, Tripathi RD, Trivedi PK. 2008. Thiol metabolism and antioxidant systemscomplement each other during arsenate detoxification in Ceratophyllum demersum L. AquaticToxicol 86:205–215.

Mishra S, Tripathi RD, Srivastava S. 2009. Thiol metabolism play significant role during cadmiumdetoxification by Ceratophyllum demersum L. Bioresour Technol 100:2155–2161.

Ohkawa H, Ohishi N, Yagi K. 1979. Assay for lipid peroxidation in animal tissues by thiobarbituricacid reaction. Anal Biochem 95:351–358.

Ozturk F, Duman F, Leblebici Z, Temizgul R. 2010. Arsenic accumulation and biological responsesof watercress (Nasturtium officinale R. Br.) exposed to arsenite. Environ Exp Bot 69:167–174.

Paiva LB, de Oliveira JG, Azevedo RA, Ribeiro DR, Gomes da Silva M, Vitoria AP. 2009. Ecophysi-ological responses of water hyacinth exposed to Cr3+ and Cr6+. Environ Exp Bot 65:403–409.

Prado C, Rosa M, Pagano E, Hilal M, Prado FE. 2010. Seasonal variability of physiological and bio-chemical aspects of chromium accumulation in outdoor-grown Salvina minima. Chemosphere81:584–593.

Rai V, Vajpayee P, Singh SN, Mehrotra S. 2004. Effect of chromium accumulation on photosyntheticpigments, oxidative stres defense system, nitrate reduction, proline level and eugenol contentof Ocimum tenuiflorum L. Plant Sci 167:1159–1169.

Dow

nloa

ded

by [

Ast

on U

nive

rsity

] at

06:

25 2

2 A

ugus

t 201

4

1208 F. DUMAN AND F. D. KOCA

Redondo-Gomez S, Mateos-Naranjo E, Vecino-Bueno I, Feldman SR. 2011. Accumulation and toler-ance characteristics of chromium in a cordgrass Cr- hyperaccumulator, Spartina argentinensis.J Haz Mat 185:862–869.

Saha B, Orvig C. 2010. Biosorbents for hexavalent chromium elimination from industrial and mu-nicipal effluents. Coord Chem Rev 254:2959–2972.

Shanker AK, Cervantes C, Loza-Tavera H, Avudainayagam S. 2005. Chromium toxicity in plants.Environ Int 31:739–753.

Singh R, Tripathi RD, Dwivedi S, Trivedi PK, Chakrabarty D. 2010. Lead bioaccumulation potentialof an aquatic macrophyte Najas indica are related to antioxidant system. Bioresour Technol101:3025–3032.

Sinha S, Saxena R, Singh S. 2005. Chromium induced lipid-peroxidation in the plants of Pistasiastratiotes L.: role of antioxidants and antioxidant enzymes. Chemosphere 58:595–604.

Southam CM, Erlich J. 1943. Effects of extract of western red-cedar heartwood on certain wood-decaying fungi in culture. Phytopath 33:517–524.

Speranza A, Ferri P, Battistelli M, Falcieri E, Crinelli R, Scoccianti V. 2007. Both trivalent andhexavalent chromium strongly after in vitro germination and ultrasucture of kiwifruit pollen.Chemosphere 66:1165–1174.

Tanhan P, Kruatrachue M, Pokethitiyook P, Chaiyarat R. 2007. Uptake and accumulation of cadmium,lead and zinc by Siam weed [Chromolaena odorata (L.) King & Robinson]. Chemosphere 68:323–329.

Torres MA, Barros MP, Campos SC, Pinto E, Rajamani S, Sayre RT, Colepicolo P. 2008. Biochemicalbiomarkers in algae and marine pollution: a review. Ecotoxicol Environ Safety 71:1–15.

Urnebese CE, Motajo AF. 2008. Accumulation, tolerance and impact of aluminium, copper and zincon growth and nitrate reductase activity of Ceratophyllum demersum (Hornwort). J EnvironBiol 29:197–200.

Vajpayee P, Tripathi RD, Rai UN, Ali MB, Singh SN. 2000. Chromium (VI) accumulation reduceschlorophyll biosynthesis, nitrate reductase activity and protein content in Nymphaea alba L.Chemosphere 41:1075–1082.

Zayed A, Lytle CM, Qian JH, Terry N. 1998. Chromium accumulation, translocation and chemicalspeciation in vegetable crops. Planta 206:293–299.

Zayed AM, Terry N. 2003. Chromium in the environment: factors affecting biological remediation.Plant Soil 249:139–156.

Dow

nloa

ded

by [

Ast

on U

nive

rsity

] at

06:

25 2

2 A

ugus

t 201

4