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Ecological Engineering 36 (2010) 1277–1284

Contents lists available at ScienceDirect

Ecological Engineering

journa l homepage: www.e lsev ier .com/ locate /eco leng

otential of Typha angustifolia for phytoremediation of heavy metals fromqueous solution of phenol and melanoidin

am Chandra ∗, Sangeeta Yadavnvironmental Microbiology Section, Indian Institute of Toxicology Research (CSIR), Post Office Box No. 80, M.G. Marg, Lucknow 226001, U.P., India

r t i c l e i n f o

rticle history:eceived 9 September 2009eceived in revised form 29 March 2010ccepted 5 June 2010

eywords:eavy metal

a b s t r a c t

Typha angustifolia was evaluated for various heavy metals (Cu, Pb, Ni, Fe, Mn, and Zn) bioremedia-tion potential from aqueous solution containing variable concentrations of phenol (100–800 mg l−1) andmelanoidin (2500–8500 Co–Pt) at 20, 40, and 60 days. The concentration of phenol (200–400 mg l−1) alongwith melanoidin 2500 Co–Pt showed optimum for phytoremediation of tested heavy metals, while, higherconcentrations of melanoidin (5600–8500 Co–Pt) showed toxic effect on T. angustifolia along with phenol.Phenol and melanoidin showed adverse effect on T. angustifolia of up to 20 days incubation, but this leadsto induction of peroxidase and ascorbic acid activity to cope with adverse conditions. Subsequently, as

eroxidasehenolypha angustifoliaetland plant

pollutants were decreased along with plant growth, peroxidase and ascorbic acid also declined. How-ever, with reduction of peroxidase, catalase level was increased. The Cu, Zn, and Ni were accumulated atmaximum in all tested conditions. The TEM observations of T. angustifolia showed clotted deposition ofmetals and shrinkage of cell in root, breakdown of spongy and palisade parenchyma of leaves at higherconcentration of phenol (100 mg l−1) and melanoidin (5500 Co–Pt). Thus, this study concluded that T.angustifolia could be a potential phytoremediator for heavy metals from metal, melanoidin, and phenol

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. Introduction

Heavy metals are a major source of water and soil pollution,enerated either through geogenic activities or industrial wasteischarge (Li et al., 2009). These contaminants have catastrophicffects on human health (Sridhara et al., 2008). Various technolo-ies, including precipitation, reduction, artificial membranes, andon-exchange have been used for toxic metals removal from indus-rial effluents (Qdaisa and Moussa, 2004). But, these methods arexpensive and may generate a huge amount of waste, which leadso disposal problems. Moreover, these methods are not sufficiento remove heavy metals present in low concentration. Therefore,here is an urgent need to develop an innovative process, which canemove heavy metals economically from aqueous solution even at

ow concentration (Volesky, 2000). Recently, it has been advocatedhat phytoremediation is as cheap and eco-friendly technique notust for heavy metals removal but also for various recalcitrant pol-utants such as polychlorinated biphenyls (Smith et al., 2007). It

∗ Corresponding author. Tel.: +91 522 2220107/2614118/2620207;ax: +91 522 2628227/2628471.

E-mail addresses: rc microitrc@yahoo.co.in, ramchandra env@indiatimes.comR. Chandra).

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925-8574/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.ecoleng.2010.06.003

er at optimized condition.© 2010 Elsevier B.V. All rights reserved.

as been reported that wetland plants, i.e. motha (Cyperus malac-ensis), cattail (Typha latifolia) and reed (Phragmites australis), canccumulate heavy metals in their tissues (Ye et al., 2001). Subse-uently, common reed and cattail have been successfully appliedor heavy metals remediation (Cardwell et al., 2002; Ye et al., 2001).. angustifolia has been observed as a widespread, dominant plantpecies, and hyper root accumulator for heavy metals (Cardwell etl., 2002; Deng et al., 2006).

In India there are more than 300 sugarcane molasses-based dis-illeries releasing approximately 3.5 × 1015 l wastewater annually.his wastewater is a major source of aquatic pollution due to highevels of Maillard complex of sugar and amino acid (melanoidin),olor, biological oxygen demand (BOD), chemical oxygen demandCOD), phenolics, total solid (TS), sulfate, nitrogen, and heavy met-ls such as manganese (Mn), copper (Cu), zinc (Zn), nickel (Ni), leadPb) (Chandra et al., 2008a; Kumar and Chandra, 2004). It has beeneported that melanoidins have net negative charges, hence dif-erent heavy metals (Cu2+, Cr3+, Fe3+, Zn2+, and Pb2+, etc.) stronglyind to form large complex molecules with melanoidin (Migo et al.,

997). In addition, oxidative stress arises due to deleterious effectsf reactive oxygen species generated under metal stress, whichan seriously disrupt normal metabolism of the plants. Oxyradicalsan cause lipid peroxidation, inactivation of enzyme, and mem-rane damage leads to cause cellular toxicity in plants (Singh et al.,

1 al Engineering 36 (2010) 1277–1284

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Table 1Experimental details of metal treatment on T. angustifolia in presence of variousconcentrations of phenol and melanoidin.

Pots no. Heavy metals (mg l−1) Phenol(mg l−1)

Melanoidin(Co–Pt)

1st set of experiment (variable concentrations of phenol)ST1 Metal solution as

described in Section 2.1– –

ST2 -do- 100 –ST3 -do- 100 2500ST4 -do- 200 2500ST5 -do- 400 2500ST6 -do- 600 2500ST7 -do- 800 2500

2nd set of experiment (variable concentrations of melanoidin)ST8 -do- – 2500ST9 -do- 100 3000ST10 -do- 100 4000ST11 -do- 100 5500

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278 R. Chandra, S. Yadav / Ecologic

004). To mitigate the oxidative damage, plants developed a com-lex defense antioxidant system, including low molecular weightntioxidants (cysteine, ascorbic acid, and non-protein thiol) as wells enzymes such as superoxide dismutase, catalase, and peroxidaseSingh et al., 2004; Wolf and Hoehamer, 2003). Thus, these antiox-dants play an important role in the cellular defense strategy oflants against oxidative stress, inducing resistance for metals byrotecting labile macromolecules.

The removal of various heavy metals from post-methanatedistillery effluent is still a major environmental problem due toresence of phenol and melanoidin along with other pollutants.ut to date, the effect of phenol and melanoidin on phytoreme-iation of heavy metals is unknown. Therefore, in the presenttudy the phytoremediation capacity of T. angustifolia for heavyetal from aqueous solution containing phenol and melanoidin in

onstructed pots was evaluated, which may lead to proposing anffective phytoremediation technique for removal of heavy met-ls from post-methanated distillery effluent containing phenol andelanoidin in a constructed wetland treatment system for environ-ental safety.

. Materials and methods

.1. Chemicals and reagents

All the metal salts used in this study were purchased fromisco Research Laboratories Pvt. Ltd. (SRL), India. Phenol wasbtained from Sigma, USA. Synthetic melanoidin was preparedy the method of Kumar and Chandra (2006) and diluted withistilled water for preparation of different color unit melanoidin.he selections of heavy metals concentration for phytoremedi-tion study were made on the basis of growth potential of T.ngustifolia in aqueous solution at variable concentrations of phe-ol and melanoidin. The concentrations of metals Cd, 8.00; Cu,8.00; Cr, 2.43; Zn, 26.30; Mn, 20.54; Ni, 16.00; Fe, 296.32, andb, 33.92 mg l−1 were observed to be optimum for the growth of. angustifolia (data not shown). Hence, this concentration waselected for further study.

.2. Experimental setup for study

The study was conducted in thirteen plastic pots, installed inpen natural environment at the Indian Institute of Toxicologyesearch (IITR), Lucknow, India. Pots 1–13 were designated asT1, ST2, ST3, ST4, ST5, ST6, ST7, ST8, ST9, ST10, ST11, ST12, andT13. Each pot, having 45 l capacity (42 cm diameter and 55 cmepth), was filled from bottom to top with coarse gravel (particleize 4 cm), pea gravel (particle size 1 cm), and fine sand (parti-le size 0.1 cm). Each layering was done up to 10 cm. The tenealthy plantlets (rhizome) of T. angustifolia, collected from uncon-aminated site with small amounts of attached native soil, werelanted in each pot with an average area 4.2 cm2/plant. Further,he plantlets were acclimatized in pots with Hoagland’s solution10 l) for 15 days (Chandra et al., 2008b). The water levels in potsere maintained 10 cm above the sand surface through out the

tudy.Treatment applications began in 15 days-acclimatized plants.

o evaluate the effect of phenol and melanoidin concentration oneavy metals phytoremediation, two sets of experiments were con-ucted as detail shown in Table 1. The treatments were replicatedhree times; plant and water samples were collected from pots after0, 40, and 60 days incubation for further study.

i1

(b

ST12 -do- 100 7000ST13 -do- 100 8500

–): Absent.

.3. Samples collection and physicochemical analysis

To observe the physicochemical changes in solution, water sam-les were collected aseptically in clear glass ware periodically fromll the pots at 0, 20, 40, and 60 days of treatments. Suspendedarticulate matter was separated by filtration through 0.45 �mre-weighed Whatman GF/C filters. The pH and nitrate were deter-ined using an ion meter (Orion autoanalyser model-960) by using

heir respective electrode. BOD was measured using 5 days BODest, COD by open reflux method, nitrogen and phenol were esti-

ated by standard methods as described in APHA (2005). The colorf wastewater was measured by visual comparison method no.120 B (APHA, 2005).

.4. Relative growth rate (RGR) and biochemical changes

The plants were harvested after 20, 40, and 60 days of treatmentor biochemical analysis. The relative growth rate (RGR) of plant

aterials was determined using Eq. (1) (Beadle, 1982):

GR = W1 − W0

t1 − t0(1)

here W0 and W1 are dry biomass at the beginning (t0) and at thend of the experimental (t1), respectively.

The RGR will facilitate the assessment of changes in above-round biomass during the experimental period. Plant growthonitoring indicated the feasibility and efficiency of each planted

ot for employing in treatment wetlands under tested conditions.scorbic acid content was determined according to our previousork (Bharagava et al., 2008). To analyze peroxidase and catalase

ctivity, roots of T. angustifolia of both set of experiments, at differ-nt time 20, 40, and 60 days were homogenized in liquid nitrogennd extracted in 2 ml of 0.1 M Na–phosphate buffer pH 7. The result-ng cell homogenate was centrifuged at 7000 × g for 15 min at 4 ◦Cnd the cell free extract assayed for enzyme activity. All the steps inhe preparation of enzyme extract were carried out at 0–4 ◦C. Per-xidase activity was determined at 25 ◦C with a spectrophotometerGBC Cintra-40, Australia) following the formation of tetraguaiacols described by Singh et al. (2006). One unit of peroxidase activ-

ty (U) represents the amount of enzyme catalyzing oxidation ofmmol of guaiacol in 1 min at 25 ◦C.

Catalase was estimated following the method of Singh et al.2008). The reaction mixture contained 0.6 ml of 0.1 M phosphateuffer (pH 7.0), 0.3 ml of 70 mM H2O2 and 0.1 ml of root extract. The

l Engineering 36 (2010) 1277–1284 1279

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R. Chandra, S. Yadav / Ecologica

ctivity was estimated by monitoring the decrease in absorbancet 240 nm due to H2O2 reduction (e = 39.4 M−1 cm−1). The activ-ty was expressed in terms of mmol of H2O2 reduced min−1 g−1 at5 ◦C.

.5. Anatomical study

The root and leaves samples of 60 days treated and untreatedlants were analyzed by light microscopy (LM) and transmissionlectron microscopy (TEM). Root samples of 5 mm length werexcised from 2 cm below the rhizome-root intersection. Leavesamples of 5 mm length were excised from the middle portion ofhe third leaf from the base of the plants. All the samples werexcised and quickly immersed in H2S saturated water at roomemperature for 30 min to precipitate Cd and Zn (Khan et al., 1984).

.5.1. Light microscopy (LM)Plants samples were fixed overnight at 4 ◦C in 3% glutaralde-

yde in 0.1 M sodium cacodylate buffer (pH 6.9), post-fixed forh in 1% osmium tetroxide in the same buffer and then processeds described previously (Rascio et al., 1991). For light microscopy,hin sections (1 �m) were cut with an ultra microtome (Ultracut,eichert-Jung, Wien, Austria) and stained with equal volumes of% toluidine blue and 1% sodium tetraborate, being then examinedPhase Contrast microscope; Nikon; Japan).

.5.2. Transmission electron microscopy (TEM)Root, and leaves segments of approximately 3 mm length were

ollected for transmission electron microscopic. The samples werexed in modified Karnovsky’s fluid (David et al., 1973) bufferedith 0.1 M sodium phosphate buffer at pH 7.4. Fixation was car-

ied out for 10–18 h at 4 ◦C. After fixation, tissues were washed byresh buffer and post-fixed for 2 h in 1% osmium tetroxide in samehosphate buffer. The tissues were dehydrated in graded acetoneolution and embedded in CY 212 araldite. Ultrathin sections ofissue having 60–80 nm thickness were cut using ultra E (Reichertung). Ultra sections were stained with uranyl acetate and lead cit-ate for 10 min before examining the grid in a transmission electronicroscope (Phillips, M-10) operated at 60–80 kV transmission.

.6. Heavy metals analysis

After 20, 40, and 60 days of experiment startup a plant wasooted out from each pots of both sets. The plant samples were care-ully washed with tap water followed by 10% CaCl2 solution. Finally,ll the samples were ringed using deionized water and dried at0 ◦C for >72 h. Further, all samples were ground to a <40 BSS mesh

n a Wiley mill. Thereafter, 5 g dried plant tissues were ashed for 2 hn quartz crucibles at 500 ◦C, treated in 10 ml HNO3 (2%), and cooled.he residue was dissolved in 1 M HCl as per AOAC internationalethods (AOAC, 2002). The heavy metals in pots were also ana-

yzed following the standard method for the examination of waternd wastewater (APHA, 2005). Thereafter, Fe, Pb, Cu, Zn, Mn, and Niere analyzed by using Inductively Coupled Plasma-Atomic Emis-

ion Spectrophotometer (ICP-AES) (IRIS Interepid II XDL: Thermolectron, Waltham, MA, USA). The percent metal accumulation in. angustifolia was measured using formula:

accumulation =[

Fm − Im

Im

]× 100

here Fm is final and Im is initial metal contents in plant.

wc0s

ig. 1. Morphological effect of metals, phenol at variable concentration ofelanoidin (Set II, ST8–ST13 from left to right) on T. angustifolia during metal

ccumulation at 20 (a), 40 (b), and 60 days (c) incubation.

.7. Statistical analysis

The whole experiment was set up in three replicates. All dataere mean (n = 3) of each set. To confirm the variability of data

nd validity of results, all the data were subjected to an analy-is of variance (ANOVA). Turkey’s test (Ott, 1984) using the Graphad software (Graph Pad Software, San Diego, CA) was used fortatistical analysis.

. Results and discussion

.1. Physicochemical characteristics and heavy metalccumulation

Physicochemical changes of metal solution after addition ofncreasing concentration of phenol and melanoidin are shown inables 2 and 3. In 1st set of experiment, constant concentration ofetals and melanoidin along with variable (increasing order) con-

entrations of phenol showed slight alkaline nature (pH 8.1–8.6) ofT1–ST7 solution (Table 2). The increased phenol concentrationsn metal solution along with melanoidin increased the pH, BOD,OD, and color of solution while nitrogen remained almost con-tant in first set of experiment (Table 2). In first set of experimentt was also observed that there was gradual decrease in all thehysicochemical parameters with pace of time and solution wasound growth promoting (Table 2). This might be due to phytore-

ediation potential of T. angustifolia and interaction of metals withhenol and melanoidin.

In 2nd set of experiment there was an increase of melanoidinith fixed concentration of metals and phenol, developed acidic

onditions (pH 3.0–6.5) with high color, COD, BOD, and nitrate atday (Table 3). The T. angustifolia of pots ST8–ST13 at 20 days

howed no adverse effect (Fig. 1a). While, the plants growth was

1280 R. Chandra, S. Yadav / Ecological Engineering 36 (2010) 1277–1284

Table 2Physicochemical analysis of water samples of set I.

Time Parameters Treatment pots

ST1 ST2 ST3 ST4 ST5 ST6 ST7

0 Day

pH 8.00 ± 0.11 8.10 ± 0.11 8.20 ± 0.10 8.30 ± 0.10 8.44 ± 0.10 8.53 ± 0.10 8.61 ± 0.12Color 500 ± 10.12 900 ± 20.34 2400 ± 27.77 2450 ± 34.56 2450 ± 40.56 2500 ± 50.50 2550 ± 50.37COD 2500 ± 100 3200 ± 100 3500 ± 20.08 3600 ± 50.30 3800 ± 60.10 4000 ± 130 4200 ± 133BOD 1200 ± 34.45 1400 ± 20.00 1600 ± 80.56 1700 ± 400 1900 ± 100 2000 ± 50.40 2000 ± 20.45Phenol ND 100 ± 5.23 100 ± 2.24 200 ± 4.54 400 ± 12.05 600 ± 20.02 800 ± 40.04Nitrogen 82.76 ± 3.64 96.00 ± 2.43 92.52 ± 1.57 94.67 ± 3.45 79.00 ± 2.87 74.00 ± 3.43 65.56 ± 1.87Nitrate 9.89 ± 0.34 10.42 ± 0.31 10.86 ± 0.42 11.36 ± 0.32 11.59 ± 0.40 11.97 ± 0.45 12.48 ± 0.36

20 Days

pH 7.77 ± 0.08* 7.84 ± 0.05* 7.85 ± 0.06* 7.87 ± 0.05* 7.83 ± 0.04* 8.00 ± 0.21* 8.00 ± 0.30*

Color 400 ± 20.00* 670 ± 20.43* 1180 ± 20.00* 1550 ± 50.34* 1800 ± 60.12* 2000 ± 50.56* 2180 ± 22.45*

COD 1600 ± 60.47* 2200 ± 40.78* 2300 ± 60.58* 2600 ± 50.00* 2800 ± 70.00* 2900 ± 30.48* 3000 ± 50.68*

BOD 800 ± 50.66* 1000 ± 20.44* 1200 ± 20.67ns 1300 ± 40.57ns 1500 ± 30.74ns 1500 ± 20.48* 1600 ± 20.46*

Phenol ND 80.55 ± 3.05ns 82.00 ± 2.04ns 138 ± 2.07ns 164 ± 4.17* 493 ± 7.20* 689 ± 13.31*

Nitrogen 67.00 ± 0.76* 75.00 ± 0.84* 72.00 ± 0.92* 84.00 ± 2.38* 65.00 ± 2.91ns 66.00 ± 2.52ns 60.00 ± 1.76ns

Nitrate 3.53 ± 0.06* 4.76 ± 0.19* 5.47 ± 0.15* 5.69 ± 0.24* 6.15 ± 0.08* 6.17 ± 0.20ns 6.54 ± 0.04*

40 Days

pH 7.30 ± 0.10* 7.40 ± 0.00* 7.50 ± 0.13* 7.60 ± 0.00* 7.70 ± 0.10ns 7.80 ± 0.00* 7.90 ± 0.00*

Color 300 ± 10.00* 550 ± 10.00* 600 ± 20.00* 650 ± 20.00* 850 ± 12.00* 1800 ± 40.00* 2050 ± 40.00*

COD 1000 ± 50.00* 1100 ± 40.00* 1200 ± 50.00* 1500 ± 20.00* 1600 ± 45.00* 1800 ± 80.00* 2000 ± 80.00*

BOD 500 ± 30.00* 600 ± 20.00* 600 ± 40.12* 700 ± 50.00* 800 ± 30.00* 1000 ± 70.00* 1100 ± 20.00*

Phenol ND 52.00 ± 15.00ns 55.00 ± 20.23ns 97.00 ± 20.00ns 102 ± 5.09ns 477 ± 30.00ns 596 ± 10.00*

Nitrogen 54.00 ± 6.00* 63.00 ± 2.00ns 61.00 ± 3.87ns 70.00 ± 3.00* 56.00 ± 1.50ns 63.00 ± 4.00ns 55.00 ± 2.85ns

Nitrate 3.06 ± 1.20ns 4.15 ± 1.30ns 1.54 ± 1.20* 2.03 ± 1.10* 2.32 ± 1.40ns 5.65 ± 1.80ns 5.95 ± 1.30*

60 Days

pH 7.10 ± 0.00* 7.20 ± 0.00* 7.20 ± 0.00* 7.30 ± 0.00* 7.3 ± 0.00* 7.30 ± 0.00* 7.30 ± 0.00*

Color 265 ± 2.11* 432 ± 0.00* 528 ± 15.00* 440 ± 5.00* 220 ± 10.00* 1675 ± 10.00* 1938 ± 10.00*

COD 800 ± 20.33* 450 ± 10.00* 478 ± 30.14* 561 ± 20.00* 672 ± 20.00* 900 ± 10.00* 1659 ± 10.00ns

BOD 200 ± 13.30* 210 ± 10.00* 220 ± 20.24ns 266 ± 8.00ns 310 ± 10.00* 338 ± 10.00* 360 ± 20.003

Phenol ND 30.00 ± 1.60ns 42.00 ± 18.00ns 62.00 ± 1.82ns 68.00 ± 14.00ns 400 ± 18.00* 579 ± 16.00ns

Nitrogen 42.00 ± 1.12* 51.00 ± 1.44ns 53.00 ± 1.85ns 61.00 ± 2.00* 55.00 ± 8.00ns 59.00 ± 3.89ns 55.00 ± 1.50ns

Nitrate 1.28 ± 1.12* 1.68 ± 0.34ns 4.26 ± 1.33ns 4.36 ± 1.63ns 4.48 ± 0.80ns 2.88 ± 1.50ns 4.14 ± 1.70*

A metaa ticaln

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ll values are mean (n = 3) ± SD in mg l−1 except pH and color is in Co–Pt; ST1, heavynd increasing concentration of phenol (100, 200, 400, 600, and 800 mg l−1). Statissp > 0.05; ND = not detectable.

* p < 0.05.

dversely affected by melanoidin of higher color range 5500–8500o–Pt (ST11, ST12, and ST13) in 2nd set of experiment by develop-

ng necrosis of leaves followed by chlorosis at 40 days of exposureFig. 1b). This might be due to the high metal accumulation at acidicH. Lower pH favors fast accumulation of heavy metals in plantas been reported by Gonzalez et al. (2006). This might be alsoue to cumulative effect of phenol, melanoidin, and other factors,

.e. depletion of O2 and rhizospheric bacterial community distur-ance. However, after 40 days plants acclimatization, there was anmergence of new plantlet from rhizome of the same plants. Thishowed that at initial stage, the plants were shocked by the higheroncentration of pollutants. Eventually, due to innate mechanismf tolerance in plants, there was arrival of new leaves (Fig. 1c).his was also observed that the new plantlets further accumulatedeavy metals as nutrient from the given solution. Consequently,here was reduction in all pollution parameter (e.g. color, BOD, COD,nd phenol) in pots ST1-ST13 at 20, 40, and 60 days of incubationompared to 0 day (Tables 2 and 3).

The most parameters, i.e. color, COD, BOD, phenol, and nitrogeneduced maximum being respectively 86.33, 86.33, 86.25, 82.90,nd 46.88% in set I (ST1–ST7), calculated from Table 2. Tables 2 and 3howed rapid reduction in pollution parameters (color, COD, BOD,nd phenol) in 1st set of experiment as compared to 2nd setf experiment. These observations revealed that high contentsf melanoidin along with constant concentration of phenol andetals were more inhibitor to T. angustifolia for heavy metals phy-

oremediation than aqueous solution containing higher phenol andonstant melanoidin and metals.

Fig. 2 showed the percent accumulation of different heavy met-ls in set I and set II. The percent accumulation of different metals,

cimt

ls; ST2, heavy metals and phenol; ST3–ST7, heavy metals, melanoidin (2500 Co–Pt)significance was evaluated within columns by mean of ANOVA. Significance level

.e. Cu = 22.32, Pb = 9.30, Ni = 11.10, Fe = 2.28, Mn = 5.06, Zn = 15.59%as noted in ST1. However, ST2 containing heavy metals andhenol showed higher accumulation of different metals than ST1xcept Cu (e.g. Cu = 18.67, Pb = 11.58, Ni = 12.90, Fe = 9.29, Mn = 7.09,n = 20.00%). Furthermore, in ST3–ST7 showed decreasing patternf metal accumulation by T. angustifolia (Fig. 2) after 20 days incuba-ion. Decreased heavy metal accumulation by T. angustifolia mighte due to increasing concentration of phenol, where melanoidinlso contributed toxic effect on metabolic activity of plants. Thisbservation was supported by the findings of Singh et al. (2008) forhe inhibitory effect of phenol on heavy metals accumulation byetiver zizanoides. Further, the metal accumulation was increased

n T. angustifolia at 40 and 60 days incubation compared to 20ays. This indicated the growth dependent metal accumulation

n T. angustifolia. The heavy metals accumulation pattern fromot ST1 was in order of Zn > Cu > Ni > Mn > Pb > Fe and in presencef heavy metal and phenol (ST2) the accumulation pattern wasoted Pb > Zn > Cu > Ni > Mn > Fe after 60 days incubation. While, inT3–ST7 the metal accumulation showed variable pattern of metalsemoval from solution.

Pot ST8 (solution containing heavy metals and melanoidin)ccumulated Zn > Cu > Ni > Pb > Mn > Fe after 20 days incubation.elanoidin along with heavy metals (ST8) more adversely affected

he phytoremediation property of T. angustifolia as compared tohenol along with heavy metals (ST2) (Fig. 2). This might be due toegative charge of melanoidin which leads to melanoidin–metal

omplexes formation (Kumar and Chandra, 2004). Further, thencreasing concentrations of melanoidin with constant heavy

etals and phenol (ST9–ST13) inhibited the metal accumula-ion potential of plant after 60 days treatments. But, in all the

R. Chandra, S. Yadav / Ecological Engineering 36 (2010) 1277–1284 1281

Table 3Physicochemical analysis of water samples of set II.

Time Parameters Treatment pots

ST8 ST9 ST10 ST11 ST12 ST13

0 Day

pH 7.21 ± 0.10 7.30 ± 0.10 6.60 ± 0.10 6.50 ± 0.10 6.50 ± 0.20 6.50 ± 0.20Color 2500 ± 50.65 2500 ± 26.74 4000 ± 50.56 5500 ± 67.34 7000 ± 80.23 8500 ± 100COD 5000 ± 150 6000 ± 200 6600 ± 100 7800 ± 200 8000 ± 150 9200 ± 100BOD 2400 ± 76.45 2800 ± 40.20 3200 ± 63.50 3600 ± 40.50 3800 ± 75.40 4200 ± 120Phenol ND 100 ± 2.60 100 ± 3.70 100 ± 4.10 100 ± 5.00 100 ± 5.00Nitrogen 85.34 ± 1.25 68.45 ± 2.44 59.46 ± 2.53 71.17 ± 1.45 65.23 ± 2.41 88.38 ± 1.14Nitrate 4.88 ± 0.35 5.45 ± 0.62 5.26 ± 0.84 6.66 ± 0.64 6.35 ± 0.86 6.65 ± 0.95

20 Days

pH 7.21 ± 0.21ns 7.21 ± 0.14ns 6.70 ± 0.16ns 6.60 ± 0.23ns 6.60 ± 0.28ns 6.70 ± 0.25ns

Color 1400 ± 50.00* 1450 ± 50.00* 1800 ± 83.00* 3800 ± 50.00* 5800 ± 200* 7100 ± 100*

COD 1800 ± 50.00* 2800 ± 100* 3000 ± 100* 3600 ± 64.00* 5200 ± 100* 6600 ± 100*

BOD 1000 ± 87.00* 1400 ± 20.00* 1500 ± 25.00* 1800 ± 30.40* 2500 ± 134* 2600 ± 156*

Phenol ND 82.37 ± 2.63ns 86.27 ± 2.75ns 87.18 ± 1.84ns 89.73 ± 2.36* 92.26 ± 3.21ns

Nitrogen 69.36 ± 1.73* 58.46 ± 1.42* 55.48 ± 1.63ns 63.36 ± 1.72ns 61.48 ± 0.85* 80.75 ± 2.00ns

Nitrate 3.35 ± 0.35ns 4.13 ± 0.21ns 2.16 ± 0.75ns 1.24 ± 0.26* 2.79 ± 0.32* 3.24 ± 0.15*

40 Days

pH 7.00 ± 0.21ns 7.30 ± 0.13* 7.00 ± 0.25ns 6.70 ± 0.22ns 6.60 ± 0.13* 6.60 ± 0.23ns

Color 1100 ± 20.45* 980 ± 30.00ns 1760 ± 40.46ns 3160 ± 50.27* 5200 ± 60.25* 6600 ± 114*

COD 1500 ± 50.21* 2400 ± 60.23* 2800 ± 30.35* 3100 ± 50.36* 4600 ± 40.53* 6500 ± 75.10ns

BOD 700 ± 10.10ns 1000 ± 12.00* 1000 ± 38.36* 1100 ± 46.26* 1200 ± 50.34* 1200 ± 64.00*

Phenol ND 56.03 ± 1.35ns 52.00 ± 1.84ns 56.28 ± 2.01ns 86.58 ± 2.42ns 89.78 ± 1.51ns

Nitrogen 55.00 ± 3.45* 51.00 ± 2.14ns 46.35 ± 2.34* 60.45 ± 1.66ns 60.37 ± 1.95ns 79.17 ± 1.33ns

Nitrate 3.49 ± 0.25ns 3.39 ± 0.26ns 3.44 ± 0.62ns 2.65 ± 0.03ns 1.25 ± 0.03ns 2.57 ± 0.05ns

60 Days

pH 7.01 ± 0.20ns 7.30 ± 0.10ns 7.20 ± 0.11ns 7.20 ± 0.13ns 7.20 ± 0.32ns 7.20 ± 0.31ns

Color 1000 ± 20.00* 900 ± 15.00ns 1680 ± 21.56ns 3080 ± 53.45* 4970 ± 100ns 6545 ± 132ns

COD 1380 ± 50.34ns 2094 ± 60.02ns 2376 ± 53.87* 3081 ± 38.65ns 4528 ± 87.00ns 6440 ± 145ns

BOD 342 ± 10.00* 425 ± 13.40ns 495 ± 18.65* 581 ± 21.50ns 731 ± 16.78* 834 ± 28.45ns

Phenol ND 46.70 ± 4.56ns 38.10 ± 2.18ns 31.01 ± 1.85ns 79.03 ± 1.84ns 81.90 ± 2.12ns

Nitrogen 49.34 ± 1.84ns 43.56 ± 2.13ns 44.78 ± 1.43ns 55.87 ± 1.65ns 56.45 ± 1.34* 78.25 ± 2.14ns

Nitrate 2.40 ± 0.06ns 1.48 ± 0.02ns 1.75 ± 0.05ns 0.68 ± 0.03ns 1.13 ± 0.02ns 1.52 ± 0.02ns

A metai Statisl

eabFdt(aipaio

3

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satam(iTnmd(oase(oibemataS

ll values are mean (n = 3) ± SD in mg l−1 except pH and color is in Co–Pt; ST8, heavyncreasing concentration of melanoidin (3000, 4000, 5500, 7000, and 8500 Co–Pt).evel nsp > 0.05.

* p < 0.05.

xperimental observation Cu and Zn accumulated maximum in T.ngustifolia compared with other heavy metals (Fig. 2). This mighte due to maximum bioavailability of these metals in solution.urther, the metal accumulation in T. angustifolia at 40, and 60ays were higher than 20 days accumulation but higher concen-ration of phenol and melanoidin inhibited metal accumulationFig. 2). Hence, these results supported time dependent heavy met-ls accumulation. Fig. 2a–c showed higher metals accumulationsn set I (heavy metal + melanoidin with variable concentration ofhenol) as compared to set II (heavy metal + phenol with vari-ble concentration of melanoidin). These results suggested thatncreased melanoidin concentration inhibited the growth due tosmotic effect on plant cell and depletion of dissolve oxygen.

.2. Relative growth rate (RGR) and biochemical changes

The RGR can be employed to confirm the health of plants dur-ng treatment period. The RGR values are shown in Fig. 3. RGRf control and ST1 grown T. angustifolia (heavy metal solution)as 0.034, 0.038 g day−1 respectively while it was 0.044 g day−1

or pot ST2 (treated with metals and 100 mg l−1 phenol). Thisndicated T. angustifolia growth is not affected by 100 mg l−1 ofhenol. Later in pot ST3 to ST5 the RGR values ranges between.057 and 0.071 g day−1. This revealed that the provided concen-rations of phenol (400 mg l−1) facilitated the growth of plants.owever, RGR values in ST6, ST7 being respectively 0.039 and

.030 g day−1 this revealed higher concentrations of phenol inhib-

ted the plants growth. Further pot ST8 (treated with metals andelanoidin) RGR value was 0.030 g day−1, which is comparatively

esser than above values. Similarly in pots ST11 to ST13, RGR val-es concomitantly decreases from 0.018 to 0.014 g day−1. Study

wr7sp

ls and melanoidin (2500 Co–Pt); ST9–ST13, heavy metals, phenol (100 mg l−1) andtical significance was evaluated within columns by mean of ANOVA. Significance

howed that heavy metals with higher concentration of phenolnd melanoidin, adversely affected the growth of plant by reducinghe total dry biomass and RGR values. Similarly, observation for T.ngustifolia has been reported earlier for tropical zones receivingunicipal wastewater in constructed wetlands treatment system

Manios et al., 2003). The heavy metals accumulation and growthn presence of phenol and melanoidin indicated the capability of. angustifolia for distillery effluent pollutants (heavy metals, phe-ol, and melanoidin) in a mixed solution. Since, toxic levels of heavyetal enhanced production of reactive oxygen species (ROS), which

amage cell membranes, nucleic acids, and chloroplast pigmentsTewari et al., 2002). Accumulation of ROS may be the consequencef disruption of the balance between their production and thentioxidative system activity, composed of enzymic antioxidantsuch as catalase, peroxidases and superoxide dismutases, and non-nzymic scavengers, e.g. glutathione, carotenoids and ascorbic acidXiao et al., 2008). Under normal circumstances, concentration ofxygen radicals remains low because of the activity of these antiox-dative enzymes. In stress condition, the free radical species maye increased, which will enhance the activities of these detoxifyingnzymes. Hence, to evaluate the combined effects of phenol andelanoidin on enzymic, e.g. catalase, peroxidase and non-enzymic

ctivity, e.g. ascorbic acid in root of T. angustifolia were observed inhis study. Ascorbic acid content in root of T. angustifolia increaseds compared to control during 20, 40, and 60 days incubation exceptT11–ST13 at 60 days incubation (Fig. 4). But, ascorbic acid content

as drastically reduced in pot ST11–ST13 where pH was 6.5, BOD

anging between 3600 and 4200 mg l−1 and COD ranging between800 and 9200 mg l−1, during 60 days incubation in comparison toet I. Similar reduction trend in physicochemical parameters werereviously reported by Chaturvedi et al. (2006). Pots ST1–ST5 of

1282 R. Chandra, S. Yadav / Ecological Engineering 36 (2010) 1277–1284

Fig. 2. Percent accumulation of different heavy metals in T. angustifolia at 20, 40, and 60 dmetals, melanoidin (2500 Co–Pt) and increasing concentration of phenol (100, 200, 400, 60metals, phenol (100 mg l−1) and increasing concentration of melanoidin (3000, 4000, 550

Fig. 3. Dry weight and relative growth rate (RGR) of T. angustifolia at 20, 40, and 60days of treatment.

sbbraatteTt

tTdtdF

ays of treatments. ST1, heavy metals; ST2, heavy metals and phenol; ST3–7, heavy0, and 800 mg l−1); ST8, heavy metals and melanoidin (2500 Co–Pt); ST9–13, heavy0, 7000, and 8500 Co–Pt).

et I and ST8–ST10 of set II up to 60 days of plant growth and incu-ation, ascorbic acid content was gradually increased. This mighte due to plants cope with the oxidative stress induced by freeadicals generated under stress condition of heavy metals, phenolnd melanoidin. However, in pot ST6–ST7 and ST11–ST13 ascorbiccid content was decreased at 60 days incubation as comparisono 40 days incubation. This indicated the decrease in stress pro-ein after long-term incubation (60 days) was probably due to thelevated concentrations of heavy metals and lipid peroxidation.his also indicated the crossing of threshold limit of plant for itsolerance.

The presence of melanoidin along with increased concentra-ion of phenol (ST3-ST5) induced peroxidase activity in root of

. angustifolia as compared to control and ST1 after 20 and 40ays incubation (Fig. 5). This indicated stress environment aroundhe root. But at 60 days of plant growth, peroxidase activity wasecreased along with melanoidin, color, and phenol as shown inig. 5 and Table 2. It was also observed that 57.70–82.90% of phe-

R. Chandra, S. Yadav / Ecological Engineering 36 (2010) 1277–1284 1283

Fig. 4. Ascorbic acid contents in T. angustifolia root. ST1, heavy metals; ST2, heavymetals and phenol; ST3–7, heavy metals, melanoidin (2500 Co–Pt) and increasingconcentration of phenol (100, 200, 400, 600, and 800 mg l−1); ST8, heavy metals andmelanoidin (2500 Co–Pt); ST9–13, heavy metals, phenol (100 mg l−1) and increasingconcentration of melanoidin (3000, 4000, 5500, 7000, and 8500 Co–Pt).

Fig. 5. Peroxidase contents in T. angustifolia root in presence of metal, melanoidin,and phenol. ST1, heavy metals; ST2, heavy metals and phenol; ST3–7, heavy metals,mam5

nga(ac2wtcimsia

3

atmpm

Fig. 6. Catalase contents in T. angustifolia root in presence of metal, melanoidin,and phenol. ST1, heavy metals; ST2, heavy metals and phenol; ST3–7, heavy metals,melanoidin (2500 Co–Pt) and increasing concentration of phenol (100, 200, 400, 600,and 800 mg l−1); ST8, heavy metals and melanoidin (2500 Co–Pt); ST9–13, heavymetals, phenol (100 mg l−1) and increasing concentration of melanoidin (3000, 4000,5500, 7000, and 8500 Co–Pt).

Fig. 7. Light micrograph of T. angustifolia root shows metal deposition (dark stain-isd

goRht

elanoidin (2500 Co–Pt) and increasing concentration of phenol (100, 200, 400, 600,nd 800 mg l−1); ST8, heavy metals and melanoidin (2500 Co–Pt); ST9–13, heavyetals, phenol (100 mg l−1) and increasing concentration of melanoidin (3000, 4000,

500, 7000, and 8500 Co–Pt).

ol disappeared from solution (ST3–ST5) within 60 days of plantrowth. However, the peroxidase activity in root of ST6–ST7 T.ngustifolia decreased compared to ST3–ST5. Similarly, melanidin2500 Co–Pt) in metal solution (ST8) also induced the peroxidasectivity. Further, the presence of phenol along with increasing con-entration of melanoidin (ST9–ST11) induced peroxidase up to0–40 days of growth. But, in ST12 and ST13 peroxidase activityere decreased as compared to ST9–ST11 (Fig. 5). This indicated

he failure of T. angustifolia detoxification mechanism at higheroncentration of melanoidin. But interestingly, the catalase activ-ty decreased in root where peroxidase was higher (Fig. 6). This

ight be due to generation of high H2O2 and reactivity oxygenpecies. The peroxidant activity at cellular level by phenoxyl rad-cals of dietary flavonoids has been previously reported (Galati etl., 2002).

.3. Anatomical changes

The anatomical observation of different parts of T. angustifolia

lso showed physiological and biochemical linked deformities inhe plant tissue (Figs. 7 and 8). T. angustifolia being as root accu-

ulator showed apparent metallic deposition and disruption ofarenchyma cell in pot ST11 as compared to the control under lighticroscopy (Fig. 7a and b). TEM micrographs of T. angustifolia root

cosbr

ng) and disruption of cortex cell (b vs. a; *) and TEM micrograph shows intercellularpace ( ) and nucleus size reduction ( ) (d) in ST11 as compared to control (c)uring metal accumulation in 60 days. Cortex (Ct), phloem (Ph), and xylem (X).

rown in pot ST11 showed shrinkage of cell, resulted in formationf intercellular spaces, and decrease nucleus size (Fig. 7c and d).eduction of nucleus might be due to tolerance failure of plant atigher concentration of melanoidin. Fig. 8a shows the TEM pic-ure of untreated T. angustifolia palisade parenchyma while Fig. 8b,, d has shown damaged trend in spongy tissue and de-shaping

f palisade parenchyma at different time. The deformities of cellhape indicated the disturbance in lignifications of cell wall mighte due to hyper-accumulation for Cu, Zn, and Ni in T. angustifoliaesulted into induced peroxidase activity in 20, 40, and 60 days

1284 R. Chandra, S. Yadav / Ecological Eng

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A

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R

A

A

B

B

C

C

C

C

D

D

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G

K

K

K

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ig. 8. TEM micrograph of T. angustifolia leaves shows gradual change and break-own of cell (b–d; ) in presence of phenol (100 mg l−1), melanoidin (5500 Co–Pt)s compared to control (a) during metal accumulation at different period [20(b),0(c), and 60(d) days]. Arrow showing metals granules deposition.

f plant growth. In Brassica juncea the break down of spongy andalisade parenchyma cells followed by loss of cell shape due toeduced lignifications and hyper-accumulation of Zn, and Cd haseen earlier reported (Sridhar et al., 2005). The parenchyma cellf root which converted to elongate quadrangular might be dueo combined toxic effect of phenol, heavy metals, BOD, COD, and

elanoidin.

. Conclusions

The study concluded that T. angustifolia grown on metal solutionontaining variable concentrations of phenol (200–800 mg l−1),elanoidin (2500–8500 Co–Pt) are well adapted due to increase

evel of antioxidants, which minimized the damage caused by reac-ive oxygen species, high level of color, COD, and BOD. However,oxicity emerges when the concentrations of pollutants exceedhe quenching capacity of T. angustifolia natural protection sys-em. T. angustifolia showed optimum tolerance for various heavy

etal bioremediation in presence of phenol (200–400 mg l−1) andelanoidin (3000–4000 Co–Pt). Hence, it can be concluded that

. angustifolia is effective for heavy metals bioremediation frometal, melanoidin, and phenol containing industrial wastewater

t optimized condition.

cknowledgments

This work was carried out under the CSIR Network ProjectWP-19. The Transmission Electron Microscopy (TEM) was done

rom All India Institute of Medical Sciences (AIIMS), New Delhi,ndia.

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