INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 2, No 2, Copyright 2010 All rights reserved Integrated Publishing Association

Transcription

1 INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 2, No 2, 2011 Copyright 2010 All rights reserved Integrted Publishing Assocition Reserch rticle ISSN Study of Zinc Chloride Induced Chnges in Biochemicl nd Wter Qulity Prmeters with Reference to Borker A. R. 1, Mne A.V. 1, Borker T. M. 1, Pthde G. R. 4,Srtle G. D. 2,3 1 Deprtment of Environmentl Sciences, Fergusson College, Pune, Indi 2 Deprtment of Biochemistry, Shivji University, Kolhpur, Indi 3 Deprtment of Environmentl Biotechnology, Shivji University, Kolhpur, Indi 4 Deprtment of Biotechnology, Fergusson College, Pune, Indi gdsrtle28@gmil.com doi: /ijes ABSTRACT The present investigtion ws imed to ssess the effect of zinc chloride on some biochemicl prmeters nd cpcity of zinc uptke by floting qutic mcrophyte. Initilly the effects of incresing zinc concentrtion on the growth of E. crssipes including root length, lef re nd biomss production ws studied. Zinc ws ccumulted in incresing order in the plnts treted with incresing concentrtion of zinc chloride nd shows mximum efficiency of ccumultion of Zn ++ up to 75 ppm present in the solution. At higher zinc chloride (75 ppm) concentrtion chlorophyll content of E. crssipes is slightly ffected wheres increse in the crotenoid, polyphenol nd proline content ws observed. The effect of zinc concentrtion on ntioxidnt enzyme ctivities such s ctlse nd peroxidse of E. crssipes were lso investigted. Wter qulity nlysis with reference to electricl conductivity, ph, totl dissolved solids (TDS), lklinity, hrdness, sodium nd potssium content were systemticlly investigted. COD s well s BOD content of wter incresed in ll the sets with increse in the concentrtion which might be closely ssocited with the externl ddition of zinc chloride nd definitely due to root exudtes, degrding plnt prts. The mximum vlues of bioconcentrtion fctor (BCF) for Zn ws 545.4, suggesting tht wter hycinth ws moderte ccumultor of Zn nd could be used to tret fresh wters nd industril wstewter contminted with zinc concentrtions. Keywords:, phytoremedition, bioconcentrtion fctor, ctlse, peroxidse, chlorophyll, crotenoid, polyphenol 1. Introduction The 21 st century hs been clled s the century of wter nd the yer 2000 ws declred s the Interntionl Yer of Freshwter by UNEP. However rpid industriliztion nd urbniztion hve resulted in elevted emission of toxic hevy metls entering into the biosphere (Lu et l., 2004). Hevy metls re one of the prominent sources of pollution in industril wstewter nd excessive presence of these metls is cuse of severe helth nd environmentl problems becuse they cnnot be destroyed biologiclly but re only trnsformed from one oxidtion stte or orgnic complex to nother (Grbisu nd Alkort, 2001; Gilbert et l., 2003). Zinc compounds re widely used in industry to mke pint, rubber, dyes, wood preservtives nd ointments. Some mount of zinc relesed into the environment by nturl processes, but most comes from humn ctivities like mining, steel production, col burning, nd wste burning (Andr et l., 2010). It ws estimted tht bout 1,350,000 tones of zinc relesed worldwide nnully (Singh et l., 2003). Received on September 2011 Published on November

2 In recent yers, there hs been incresed globl concern over the deteriorting stte of wter bodies due to hevy metl pollution nd most of the modern technologies used to tret wstewters hve their own implictions, s these technologies re quite costly, concomitntly, posing threts to qutic life (Ri, 2008). The use of plnt species for clening polluted soils nd wters nmed s phytoremedition hs gined incresing ttention since lst decde, s n emerging cheper technology (Srtle et l., 2011). The technology is found 60 to 80% less costly s compred to the conventionl physicl nd chemicl methods used for soil nd wter decontmintion (Dhote nd Dixit, 2009). Severl qutic plnts from green lge to ngiosperms re being identified nd experimented to be used in phytoremedition of chemiclly contminted wter nd wstewter. Tolernce to metls in plnts cn be chieved by sequestering them in tissues or cellulr comprtments (e.g. centrl vcuoles) tht re insensitive to metls (Mne et l., 2010). Severl plnts species, such s Spirodel polyrrhiz, Myriophylhum quticum, Ludwigin plustris, Menth qutic, Nymphe spontne nd Certophyllum demersum hs been studied to determine their potentil in ccumulting hevy metls (Mne et l., 2011). Among the vrious plnt species, qutic mcrophytes ttin the gretest interest in the field of phytoremedition. These plnts cn ccumulte hevy metls up to 100,000 times greter thn the mount in the ssocited wter (Dhote nd Dixit, 2009). Therefore, these mcrophytes hve been used for hevy metl removl from vriety of sources. Among which wter hycinth, is one of the most commonly used plnts in constructed wetlnds becuse of its fst growth rte, lrge uptke of nutrients nd contminnts s well s lrge flt leves nd long spongy petiole prtly submerged in wter nd nchoring in the soil with fibrous roots. This plnt is used for the phytoremedition of hevy metls (including nickel, cdmium, zinc, copper nd iron), pesticides nd severl toxic chemicls from the sewge (Dhote nd Dixit, 2009). The Boston University, University of Florid hs been used E. crssipes for the tretment of sewge. Recently Agunbide et l., (2009) showed the potentil of E. crssipes to serve s phytoremedition plnt in the clening up of hevy metls from contminted costl res. Toxic hevy metl pollution of wter nd soil is mjor environmentl problem, nd most conventionl remedition pproches do not provide cceptble solutions. Wetlnd plnts re being used successfully for the phytoremedition of trce elements in nturl nd constructed wetlnds. This study demonstrtes the phytoremedition potentil of wter hycinth, for the removl of cdmium zinc (Zn). Eeffect of zinc chloride on growth s well s on chlorophylls, crotenoids, polyphenols nd proline of E. crssipes ws studied long with the potentil of wter hycinth for the removl of zinc s n efficient phytoremedition technique. Wter nlysis with reference to electricl conductivity, ph, COD, BOD, lklinity, hrdness, sodium nd potssium content ws lso crried out in order to see the effect of zinc chloride on the wter qulity. The wter hycinth is vilble in Indin wter bodies nd is dmging the qutic ecosystems where it is possible to use it in wstewter tretment by setting wstewter tretment ponds nd by the wy of ecofriendly technique. As low cost, environmentl sustinble option to the conventionl wstewter tretment fcilities for both municipl nd industril wstewter. 2. Mterils nd Method 2.1. Mterils Used in the Study 910

3 Eicchorni crssipes (Mrt.) Solms plnts were collected from Mul-Muth river of Pune city, Indi nd cclimtized in the lbortory condition. The plnts were subjected to vrious concentrtions of zinc chloride i.e. 25, 50 nd 75 ppm long with control set in four plstic tubs of ten-liter of cpcity ech. Uniform sized plnts with similr shoot re nd root length were selected for the study. They were wshed thoroughly under running tp wter, propgted hydroponiclly for twelve dys nd subjected to hevy metl tretments. Whole experiment ws run with the initil concentrtions of zinc chloride in the hydroponics medium nd ll the prmeters were estimted fter twelve dys. The chemicls used in this study were of the highest purity vilble nd of the nlyticl grde Study of the Growth Chrcteristics Ten plnts from ech tretment try were crefully removed nd wshed thoroughly with wter to remove ny dirt nd dust prticles on the surfce of the plnt prts nd blotted to surfce dry. This plnt mteril ws nlyzed for their growth chrcteristics. Vrious growth prmeters nmely root length, verge lef re, fresh weight, dry weight nd moisture content of the rndomly smpled leves were recorded Estimtion of Photosynthetic Pigments Chlorophyll Content The chlorophylls of the mture leves were estimted by following the method of Arnon (1949) with some modifiction. Fresh plnt mteril (1g) ws roughly homogenized in mortr by keeping the temperture t 2 o C in drk condition nd extrction ws crried out using 90% cetone, with the ddition of pinch of mgnesium crbonte, to protect nd stbilize the chlorophylls. This extrct ws filtered through Whtmn No.1 filter pper under suction using Buchner s funnel. The residue ws wshed thoroughly 2-3 times with 90% cetone, collecting ll the wshings in the sme filtrte nd finl volume of the filtrte ws mde to 100 ml with 90% cetone. Absorbnce of chlorophyll nd b ws recorded using double bem UV-Visible spectrophotometer (Elico SL-159, Indi), t 663 nd 645 nm using 90% cetone s blnk. Following formule were used to determine the chlorophyll content. Chlorophyll = X = 12.7 x A x A 645 Chlorophyll b = Y = 22.9 x A x A 663 Totl chlorophyll ( + b) = Z = 8.02 x A x A 645 Chlorophyll Crotenoid Content / b/ totl(mg/ 100 g fresh leves) X/ Y/ Z x volume of extrct x x weight of plnt mteril (g) The crotenoid content of the leves ws determined from the sme extrct used for chlorophyll estimtion, by recording the bsorbnce t 480 nm using spectrophotometer by putting 90% cetone s the control. The crotenoids content were clculted by using the following formul (Kirk nd Allen, 1965). Crotenoids content A480 volume of extrct weight of plnt mteril (g) 911

4 2.4. Polyphenol Content The polyphenol content of the leves ws estimted following the method suggested by Folin nd Denis (1915). 2 ml of cetone extrct used for chlorophyll ws mixed with 10 ml, 20% sodium crbonte nd djust the volume to 35 ml with distilled wter to this mixture 2 ml of Folin-Denis regent ws dded, mixed thoroughly nd finl volume ws djusted to 50 ml with distilled wter. The stndrd tnnic cid solution (0.1 mg ml -1 ) ws used for the preprtion of stndrd polyphenol curve by mesuring the bsorbnce t 660 nm using double bem UV-Visible spectrophotometer Bioconcentrtion Fctor (BCF) The bioconcentrtion fctor provides n index of the bility of plnt to ccumulte the metl with respect to metl concentrtion in substrte. The bioconcentrtion fctor ws clculted s the rtio of the trce element concentrtion in the plnt tissues t hrvest to the concentrtion of the element in the externl environment (Zyed et l., 1998). The BCF ws clculted by using formul BCF = P/E, Where, P represents the trce element concentrtion in plnt tissues (ppm), E represents the residul concentrtion in wter (ppm) or in sediment (ppm dry wt) Zinc (Zn ++ ) Concentrtion Zn ++ concentrtion ws estimted from the dried plnt mteril (shoot nd root combined). 0.5 g oven dried plnt mteril ws cid digested s per the stndrd method suggested by Toth et l. (1948). Plnt mteril ws tken in 150 ml clen beker nd to tht 10 ml concentrted nitric cid ws dded. It ws covered with wtch glss nd kept for n hour till the primry rections subsided. It ws then heted on hot plte until ll the mteril ws completely dissolved. It ws llowed to cool to room temperture nd then 10 ml of perchloric cid (60%) were dded to it nd mixed thoroughly. It ws then heted strongly on the hot plte until the solution becme colorless nd reduced to bout 2-3 ml. While heting, the solution ws not llowed to dry. After cooling, it ws trnsferred quntittively to 100 ml cpcity volumetric flsk, diluted to 100 ml with distilled wter nd kept overnight. Next dy it ws filtered through Whtmn No. 44 filter pper. The filtrte ws stored properly nd used for nlysis of Zn ++ by using Atomic Absorption Spectrophotometer (Perkin-Elmer, 3030A) Wter Qulity Anlysis Following prmeters were determined from the liquid solution of the plstic trys fter twelve dys period. The methods of nlysis were in consistent with the stndrd methods mentioned in Hndbook of Wter Anlysis (Miti, 2004) Electricl Conductivity nd Totl Dissolved Solids Electricl conductivity (EC) nd Totl dissolved solids (TDS) of the wter contining zinc chloride ws determined fter every 4 dys by using ELICO EC-TDS meter (CM 183, Mke- Indi) where the electrode ws directly dipped into the well diluted nd filtered solutions to get direct digitl disply of the result. 912

5 Turbidity nd ph Turbidity of the smple ws mesured by Nephlometric method using CL 52D model of ELICO. Smple ws poured into the cell nd turbidity ws red directly from digitl disply of the instrument. ph of the wter smples ws determined by using ELICO LI 127 ph meter fter every 4 dys of incubtion Hrdness The totl hrdness of the wter smples ws determined by EDTA titrtion methods. In which 50 ml of well mixed smple ws tken in conicl flsk nd ph of solution ws djusted upto 10. A pinch of Eriochrome blck-t ws dded nd titrted with 0.01M EDTA till wine red solution chnges to blue. The volume of EDTA consumed ws noted nd similrly regent blnk with distilled wter ws run to clculte the hrdness of the wter smples Alklinity Alklinity of the smples ws determined of 100 ml smple with the ddition of 2-3 drops of phenolphthlein indictor. Pink solution developed ws titrted with 0.02N H₂ SO₄ till it disppers. Agin the 2-3 drops of methyl ornge were dded to the sme flsk nd titrtion ws continued till ph is down to 4.5 or ornge color chnges to pink. The volume of H₂ SO₄ dded ws noted s for ech time nd the totl lklinity ws clculted. The vlues obtined were expressed in mg l Sodium nd potssium Sodium nd potssium of the wter smples were estimted with the use of Flme photometer (Systronics-128) by prepring stndrd concentrtions of sodium nd potssium. The ir pressure ws kept t 0.5 kg/cm² nd the gs feeder knob ws djusted so s to obtin blue shrp flme Chemicl Oxygen Demnd (COD) COD determintion ws crried out with dichromte reflux method with the ddition of 10 ml of 0.25N potssium dichromte (K 2 Cr 2 O 7 ) nd 30 ml H 2 SO 4 +Ag 2 SO 4 regent in 20 ml diluted smple. The mixture ws refluxed for 2 h nd ws cooled to room temperture. The solution ws then diluted to 150 ml by using distilled wter nd excess K 2 Cr 2 O 7 remined ws titrted with ferrous mmonium sulphte (FAS) using ferroin indictor. The COD ws clculted s follows (Srtle et l., 2009), -1 (A - B) N COD (mg l ) Volume of smple(ml) Where, A is the ml of FAS ws used for blnk; B is the ml of FAS ws used for test smple, N is the normlity of FAS nd 8 is the milliequivlent weight of oxygen Biologicl Oxygen Demnd (BOD) The dilution method ws followed to determine the BOD fter three dys. For the sme dilution wter ws prepred with the ddition of nutrients nmely phosphte buffer, mgnesium sulphte, clcium chloride nd ferric chloride. The diluted smple ws trnsferred to BOD bottles. After determining initil dissolved oxygen (DO), finl DO ws estimted from the bottles kept for incubtion period of three dys. 913

6 2.8 Sttisticl Anlysis Sttisticl nlysis of the dt ws crried out by using Grph Pd softwre. Men, stndrd devition (SD) nd percent vrition ws clculted. 'One Wy Anlysis of Vrince' (ANOVA) ws tested in order to check the sttisticl difference mong the mens. For the sme Tukey-Krmer multiple comprison test of significnce ws crried out which suggested the vrition mong the column mens is significnt or not t different levels of significnce. The dt ws nlyzed for three different levels of significnce bsed on the p vlues s, * Significnt (p = 0.01 to 0.05), ** Very Significnt (p = to 0.01) nd *** Extremely Significnt (p < 0.001) 3. Results nd Discussion 3.1 Growth Anlysis Growth chnges re often first nd most obvious rections of plnts under hevy metl stress (Hgemeyer, 1999). In the present study plnt growth is mesured in terms of root length, lef re nd biomss production Root Length Under the influence of zinc chloride the mximum root length ws observed on 12 th dy in control by 8.5 cm nd minimum root length ws observed by 5.2 cm t 75 ppm concentrtion (Tble 1). The root length ws lso observed to be decresed t 75 ppm of slt from 0 to 12 th dy in ll the sets which is directly relted with the negtive effect of such higher concentrtion of zinc present in the hydroponics. Similrly Scht et l. (1996) reported tht Zn toxicity in plnts ws ssocited with reduced root growth. Tble 1: Effect of zinc chloride on root length (cm) of fter 12 dys of incubtion Durtion Zinc chloride (ppm) Root length (cm) 0 dy 7.1 ± ** ± * ± *** ± th dy 8.1 ± th dy 8.2 ± th dy 8.5 ± ± ** ± * ± ± ** ± ** ± The vlues indictes percentge vrition reltive to control vlues. 5.8*** ± *** ± ±

7 Vlues re men of ten observtions with ±SD. Significntly different from the control t *P<0.05, **P<0.01 ***P< by one-wy ANOVA with Tukey-Krmer multiple comprisons test (for sme dy). It hs been reported tht the non-lethl concentrtion of Zn+ + shows n inhibitory effect on cell division in root tips of Nigell stiv nd Triticum estivum (El-Ghmery et l. 2003). Literture survey showed tht the hevy metls ccumultions in wter hycinth incresed linerly with the solution concentrtion in the order of leves<stems<roots (Strtford et l. 1984). Recently Munns (2003) concluded tht the reduction might be ttributed to the inhibition of hydrolysis of reserved foods nd their trnsloction to the growing shoots. From the present investigtion, it is cler tht the root growth of E. crssipes ws more sensitive nd severely ffected t higher concentrtion of zinc chloride (especilly t 75ppm). It might be due to higher uptke of Zn+ +. From these results, it ppers tht the roots of E. crssipes hve n bility to tolerte moderte levels of zinc concentrtions in the solutions Lef Are It is evident from the results tht the minimum verge lef re ws 9 cm² on 0 dy t 50 ppm concentrtion of zinc chloride nd the mximum verge lef re ws 16.5 cm 2 in control try on 12 th dy of experiment. On 12 th dy the verge lef re ws decresed with the incresing concentrtions, gin showing negtive correltion (Tble 2). The mechnism of plnts behind for growth reduction is not cler to mintin residul growth for short nd long term responses (Munns et l., 2003). The growing grss lef represents n inherent combintion of growth, developmentl nd micro-environmentl zones nd this combintion cn be used to identify signling events, which re involved in the regultion of growth nd growth responses to stress (Fricke et l., 2006). From the present results it is cler tht lef re decresed when exposed to 25, 50 nd 75 ppm concentrtion of zinc chloride. Tble 2: Effect of zinc chloride on verge lef re (cm 2 ) of fter 12 dys of incubtion Durtion Zinc chloride (ppm) Averge lef re (cm 2 ) 0 dy 10.6 ± * ± *** ± * ± th dy 14.1 ± th dy 15.3 ± th dy 16.5 ± ** ± ± ** ± ** ± * ± ** ± *** ± ** ± *** ± The vlues indictes percentge vrition reltive to control vlues. Vlues re men of ten observtions with ±SD. Significntly different from the control t *P<0.05, **P<0.01 ***P< by one-wy ANOVA with Tukey-Krmer multiple comprisons test (for sme dy). 915

8 3.1.3 Moisture Content It is seen from the Tble 3 tht the moisture content ws not much vrible nd ws serilly decresed with the incresed concentrtion of zinc in the solution on 12 th dy. Mximum moisture content ws observed s 87.09% in control on 12 th dy, wheres minimum moisture content ws observed s 74.37% t 75 ppm concentrtion on the sme dy. It ws decresed to 14.61% t 75ppm on 12 th dy represent tht the prolonged exposure hd negtive effect on the plnts. Overll slight fluctutions in the moisture content represent the fvorble nture of this essentil micronutrient especilly t 25 nd 50 ppm concentrtion. Tble 3: Effect of zinc chloride on moisture content of fter 12 dys of incubtion Durtion 0 dy (± 1.21) th dy (± 0.90) th dy (± 0.79) th dy (± 1.56) 0.0 Zinc chloride (ppm) Moisture content (%) 83.12* (±1.02) *± (±0.34) * (± 0.95) (± 0.79) (±1.45) * (± 1.61) (± 1.27) *** (± 2.26) ** (±1.31) ** (± 0.89) ** (± 2.02) ** (± 1.98) The vlues indictes percentge vrition reltive to control vlues. Vlues re men of ten observtions with ±SD. Significntly different from the control t *P<0.05, **P<0.01 ***P< by one-wy ANOVA with Tukey-Krmer multiple comprisons test (for sme dy) Biomss Fresh weight It is evident from the results tht the fresh weight of leves of Eichhorni is sensitive prmeter t vrious concentrtions of zinc chloride. Fresh weight of the leves ws mximum with 0.55 g in control on the 12 th dy while the minimum fresh weight ws recorded by vlue of 0.32 g on 12 th dy t 75 ppm concentrtion (Tble 1). On 12 th dy it showed n increse in the fresh weight from 25 to 50 ppm nd then decresed from 50 to 75 ppm concentrtion. On 12 th dy fresh weight decresed with the increse in the concentrtion s incse observed other growth prmeters. Though E. crssipes showed n increse in fresh weight on 4 th nd 8 th dy t 25, 50 ppm concentrtion over the control, it ws dversely ffected t especilly t 75 ppm (Tble 4). The reduction in shoot biomss production by the plnt my be due to the chlorosis nd necrosis of the leves tht reduce the photosyntheticlly ctive re (DeHerrlde et l., 1998). The decrese in fresh weight of the leves of E. crssipes might be due toxic nture of ZnCl₂ 916

9 nd the suppression of growth under such stress during the erly developmentl stges while the increse in the fresh weight of the plnts t lower concentrtions i.e. t 25 nd 50 ppm ZnCl₂ might be due to fvorble nture of this essentil element in plnt growth. Tble 4: Effect of Zinc Chloride on fresh Weight (g) of leves of fter 12 dys of incubtion Durtion Zinc Chloride (ppm) Fresh weight (g) 0 dy 0.37 (±0.09) (±0.17) (±0.08) (±0.11) th dy 0.49 (±0.19) th dy 0.53 (±0.17) th dy 0.55 (±0.20) (±0.11) (±0.12) (±0.15) (±0.11) (±0.15) (±0.20) (±0.08) (±0.13) (±0.06) The vlues indictes percentge vrition reltive to control vlues. Vlues re men of ten observtions with ±SD. Significntly different from the control t *P<0.05, **P<0.01 ***P< by one-wy ANOVA with Tukey-Krmer multiple comprisons test (for sme dy) Dry Weight Dry weight of the leves ws observed to be mximum by g on 4 th dy t 75 ppm concentrtion, while minimum dry weight ws observed by g t 50 ppm on 0 dy (Tble 2). On 12 th dy the dry weight incresed with the increse in concentrtion showing positive correltion. Leves of E. crssipes showed n increse in dry weight t 75 ppm concentrtion on 4 th nd 8 th nd 12 th dy over the control (Tble 5). Jrunee et l. (2003) observed the inhibition in dry weight of shoots nd roots in Sesbni rostrt. On the other hnd Jin et l. (1990) found tht the ddition of Zn ++ t low concentrtion hd fvorble effect on the growth of wter hycinth, which my be ttributed to the fct tht the plnts utilize Zn ++ s micronutrient for their growth. An increse in dry weight of E. crssipes might be due to ccumultion of zinc in the plnt tissues while decrese in dry weight might be ssocited with the fvorble nture of zinc ions present in the solution Effect of Zinc Chloride on Chlorophylls Content of E. crssipes The effect of zinc chloride (ZnCl 2 ) on chlorophyll content of mture leves of E. crssipes is shown in Figure 1 nd it is evident from the results tht the totl chlorophyll content of the mture leves ws incresed considerbly due to incresing concentrtions of ZnCl 2 upto 50 ppm into the liquid medium but slightly decresed t 75 ppm (0.80%) s compred to the control. Its content ws observed to be mximum (23.92%) by mg g t 50 ppm of zinc chloride concentrtion while it ws minimum (0.80%) by vlue of mg g t 75 ppm of metl slt to the liquid medium. In generl chlorophyll '' ppers to be 917

10 dominnt over chlorophyll 'b' nd ws the rtio of chlorophyll '': chlorophyll 'b' ppers to be not much ffected by zinc. The chl. '': chl. 'b' incresed with mximum in ll three tretments by 28.16% t 50 ppm of zinc chloride. Chlrophyll : b rtio ws slightly decresed t 75 ppm of ZnCl 2 nd in generl it is quite cler tht the higher slt concentrtion (75 ppm) is not much negtively influentil on : b rtio. Our results on reduced levels of chlorophyll pigments t higher levels of metl slt re in consistent with the findings of Mishr et l. (2007). Tble 5: Effect of Zinc Chloride on dry weight (g) of leves of fter 12 dys of incubtion Durtion Zinc Chloride (ppm) 0 dy (±0.03) 0.0 Dry weight (g) (±0.03) (±0.04) (±0.05) th dy (±0.04) (±0.02) (±0.02) (±0.03) th dy (±0.03) (±0.03) (±0.02) th dy (±0.03) (±0.02) (±0.03) (±0.02) The vlues indictes percentge vrition reltive to control vlues. Vlues re men of ten observtions with ±SD. Significntly different from the control t *P<0.05, **P<0.01 ***P< by one-wy ANOVA with Tukey-Krmer multiple comprisons test (for sme dy). Similrly, concentrtion dependent reduction in chlorophyll content over control ws lso observed in the leves of Lycopersicon esculentum (Gub et l., 2007). Severl cses of decresed chlorophyll content owing to metl toxicity hve been reported in the plnt kingdom growing in wetlnd ecosystems (Vlvnidis et l., 2005). The decrese in chlorophyll content in E. crssipes might be due to ccumultion of Zn. From the present investigtion, it is cler tht the totl chlorophyll content in the leves of E. crssipes is incresed t lower concentrtions of ZnCl 2 nd such n increse in the chlorophyll content might be due to the osmotic djustment mechnism developed by the experimentl species Effect of Zinc Chloride on Crotenoid Content of E. crssipes According to Armstrong (1996) crotenoids hve two mjor functions in photosynthesis, they protect chloroplst from photo-oxidtive dmge nd they lso ct s ccessory light hrvesting pigments becuse they bsorb the light energy in the rnge of nm (blue), 918

11 which is not ccessible by the chlorophylls nd pss this excittion energy to chlorophyll molecules. They re lso one of the non-enzymtic ntioxidnts long with vitmin C, vitmin E nd lipoic cid nd ply n importnt role in the protection ginst oxidtive stress (Kojo, 2004). Kenneth et l. (2000) reported tht n increse in crotenoid content ws observed in Cr treted plnts of E. crssipes nd such n increse for the protection from free rdicl formtion is common response to xenobiotics. Totl chlorophyll (mg) 100 g of fresh tissues Chlorophyll '' mg 100 g of fresh tissues Totl chlorophyll Chl-:Chl-b Chl- Chl-b Zinc concentrtion (ppm) Figure 1: Effect of zinc chloride on chlorophyll content of fter 12 dys of incubtion Chlorophyll nd crotenoid degrdtion is the routinely observed response to stress or chiefly in elevted concentrtions of vrious hevy metls (Chen nd Djuric, 2001).The crotenoid content of the leves of E. crssipes under the ZnCl 2 stress is recorded in Tble 6. From the results it is evident tht the crotenoid content of the leves of E. crssipes ws decresed t ll the tretments but the vlues remined below the control. Mximum mount of crotenoid content ws observed with vlue of mg g in the leves of plnts grown t control. From the present investigtion it ppers tht the totl crotenoid content of the leves of E. crssipes ws reduced due to ddition of zinc chloride to the liquid medium nd prove to be negtively influentil indicting the photo-oxidtive dmge while slight increse t 75 ppm but below the control might be to resist the toxic nture of zinc chloride Effect of Zinc Chloride Stress on Levels of Proline Content of E. crssipes Proline occurs widely in higher plnts nd ccumultes in lrger mounts thn other mino cids, regultes the ccumultion of usble nitrogen. Proline production decreses the osmotic potentil of cells, which cn increse the uptke of wter nd its incresed level is n indictor of protein degrdtion (Abrhm et l., 2003). However, proline ccumultion cn lso be ssocited with decrese in its oxidtion, low demnd for protein synthesis nd ccumultion in tissue plnt owing to restriction in its trnsport to other plnt prts (Viegs Chl '': Chl 'b' rtio Chlorophyll 'b' mg 100 g of fresh tissues 919

12 nd Silveir, 1999). Proline ccumultion is considered to be involved in stress resistnce mechnisms (Lutts et l., 1999). It is cler from the results (Tble 6) tht the level of proline in the leves of E. crssipes ws considerbly incresed under the influence of zinc chloride t 50 nd 75 ppm of the slt concentrtion. Mximum increse (20.80%) in proline content ws observed by mg g t 75 ppm concentrtion nd the minimum (-13.87%) proline content ws observed s 7.45 mg g t 25 ppm over the control. From the foregoing discussions it is evident tht in generl proline ccumultes in the plnts exposed to vrious environmentl stresses nd is not n exception to toxicity stress of ZnCl 2 tretment. The incresed levels of proline in the leves of E. crssipes might be ble to mintin osmoregultion, stbiliztion of proteins nd conservtion of nitrogen nd energy for stress developed due to zinc chloride t higher concentrtion Effect of Zinc Chloride Stress on Levels of Polyphenol Content of E. crssipes Very little ttention hs been pid towrds the influence of metls on the polyphenol metbolism in plnts. Prid et l. (2002) were of the opinion tht n ccumultion of polyphenols plyed key role in the plnts towrds stress. From the present study it is cler tht the slight incresed levels of polyphenols t elevted concentrtion of ZnCl 2 might induce ccumultion of secondry metbolites in the experimentl species in order to tolerte higher levels of metl stress. It is evident tht (Tble 6) polyphenol content of the leves of E. crssipes ws lowest by mg g t 25 ppm while lter on gin incresed upto 75 ppm but the vlues remined below the control ( mg g). It ws minimum by vlue of mg g t 25 ppm fter the 12 th dy of the experiment. The slight decrese in polyphenols of the leves of Eichhorni t ll levels of the zinc chloride might be relted to the non-toxic nture of the zinc in growth nd development of the experimentl species nd shows the dpttion of the species to tolerte higher levels of zinc chloride concentrtion. Tble 6: Effect of zinc chloride on crotenoid, proline nd polyphenol content of Eichhorni crssipes fter 12 dys of incubtion Sr. No. Photosynthetic pigments 1 Crotenoid (±2.32) 0.0 Zinc Chloride (ppm) 50.72** (±1.65) ** (±1.59) *** (±2.01) Proline 8.65 (±0.21) *** (±0.26) *** (±0.34) * (±0.44) Polyphenol (±5.64) ** (±4.59) *** (±6.24) *** (±6.15) -3.0 The vlues indictes percentge vrition reltive to control vlues. Vlues re men of three experiments nd expressed in mg g with ±SD. Significntly different from the control t *P<0.05, **P<0.01 ***P< by one-wy ANOVA with Tukey-Krmer multiple comprisons test. 920

13 3.6. Effect of Zinc Chloride on Expression of Antioxidnt Enzymes Activities In n environment of moleculr oxygen (O 2 ), ll living cells re confronted with the rectivity nd toxicity of ctive nd prtilly reduced forms of oxygen nd cn led to the complete destruction of cells (Mittler et l., 2004). In plnts, the links between rective oxygen species (ROS) production nd photosynthetic metbolism re prticulrly importnt. However, n elborte nd highly redundnt plnt ROS network, composed of ntioxidnt enzymes nd ntioxidnts, is responsible for mintining the levels of ROS under tight control (Mittler, 2002). In plnt cell, ntioxidnt enzymes such s peroxidse (POD) nd ctlse (CAT) hve been considered to ct s defensive tem, whose combined purpose is to scvenge rective oxygen species nd protect cells from oxidtive dmge (Mittov et l., 2003). In our study the ctivity of ctlse ws observed to be incresed ( %) in the leves of Eichhorni by t 75 ppm over the control (Tble 7). All the vlues t ll tretments showed perfect positive correltion with seril increse in ctlse ctivity over the control. The increse in ctlse under the influence of zinc chloride might be ssocited with the detoxifiction mechnism probbly cme from n incresed cpcity for oxygen rdicl scvenging nd mintennce of cellulr membrnes which indictes the reltionship between metl tolernce nd ntioxidnt defense system. Similrly induction in the ctlse ctivity by E. crssipes in the presence of hevy metls hs been reported erlier (Odjegb1 nd Fsidi, 2007). Consequently the ctivity of peroxidse ws considerbly incresed over the control nd showed perfect positive correltion with the incresed concentrtion of zinc chloride to the liquid medium. But the ctivity of this enzyme remined below the ctlse ctivity nd showed tht the ctlse is more sensitive to zinc chloride thn peroxidse. The higher ctivity of the peroxidse ws observed t 75 ppm concentrtion (0.358 min. -1 mg -1 protein) which is higher (bout 195%) thn the control set of (0.184 min. -1 mg -1 protein) of E. crssipes (Tble 7). The increse in the ctivity of peroxidse under ZnCl 2 stress my be regrded s n inhibition of stimulted secondry metbolism. It my lso be involved in scvenging the rective oxygen species, prticulrly in plnts grown under stress condition. Recently Odjegb1 nd Fsidi (2007) reported tht Zn hd the lest inducement of peroxidse in E. crssipes nd P. strtiotes while Hg hd the highest inducement of peroxidse ctivity which supports our results. Tble 7: Effect of zinc chloride on ntioxidnt enzyme ctivities of the leves of Eichhorni crssipes fter 12 dys incubtion Sr. Prmeter Zinc Chloride (ppm) No. 1 Ctlse (±0.05) ** (±0.04) *** (±0.08) *** (±0.085) Peroxidse * (±0.02) * (±0.02) ** (±0.03) The vlues indictes percentge vrition reltive to control vlues Vlues re men of three experiments ±SD. Significntly different from the control t *P>0.05, **P<0.01 ***P< by one-wy ANOVA with Tukey Krmer multiple comprisons test. 921

14 3.7. Zinc Accumultion nd Bioconcentrtion Fctor (BCF) in the Leves of E. crssipes The toxic effect of metls on the physiologicl functioning within plnts is connected to their ccumultion in different plnt tissues (Liu et l., 2005). The ccumultion of metls in the roots nd shoots of wter hycinth hs been shown in field studies in which wter hycinth ws used s biologicl monitor in metl pollution (Zrnyik nd Ndpwdz, 1995). Severl cses of ccumultion of hevy metls such s Zn, Cu, Pb, Cd, Ni nd Cr, hve been thoroughly studied in severl wetlnd plnt species, such s E. crssipes, Typh ltifoli, Sprtin lterniflor nd Phrgmites ustrlis (Liu et l., 2005). In the present investigtion, there is n increse in zinc (Zn ++ ) ccumultion in the leves of E. crssipes with the incresing concentrtions of zinc chloride (Tble 8). At the end of experiment, the zinc ions ccumulted in progressive wy with the ddition of zinc chloride to the wter medium. The mximum uptke of zinc (Zn ++ ) by E. crssipes ws 1468 mg/kg t 75 ppm of zinc chloride in the liquid medium while it ws 196 mg/kg in the leves of E. crssipes grown in control set which might hve source from the nturl wter. It ws minimum by 0.62 mg/l in control sets while the highest 4.73 mg/l ws observed t 75 ppm of zinc chloride t the end of experiment nd ws considered s the residul solution. Tble 8: Accumultion of zinc in the leves of Eicchorni crssipes t vrious tretments nd their effect on bioconcentrtion fctor Sr. Zn ++ content Zinc Chloride ppm No. 1 Residul wter (mg/l) 0.62 (±0.002) * (±0.18) ** (±0.16) (±0.24) E. crssipes (mg/kg) 196 (± 0.07) ** (±0.23) *** (±0.19) * (±0.21) Bioconcentrtion Fctor (± 0.09) (± 0.12) (± 0.15) (± 0.11) The vlues indictes percentge vrition reltive to control vlues. Vlues re men of three experiments nd expressed in mg l -1 with ±SD. Significntly different from the control t *P<0.05, **P<0.01 ***P< by one-wy ANOVA with Tukey-Krmer multiple comprisons test Bioconcentrtion fctor (BCF) is useful prmeter to evlute the potentil of the plnts in ccumulting metls nd this vlue ws clculted on dry weight bsis. The chnge in BCF of wter hycinth ws lso studied in the present investigtion to know the cpcity of plnts to concentrte zinc in the leves from the wter t the end of tretment. As fct lrger BCF implies better phytoccumultion cpbility nd Tissues with BCF greter thn 1,000 re considered high, nd less thn 250 low, with those between clssified s moderte (Zyed et l., 1998).The bioconcentrtion fctor ws observed to be high t 25 ppm of zinc chloride by but it ws decresed by nd t 50 nd 75 ppm of slt respectively. In the present study higher nd incresing BCF shows tht the Eichhorni hs good cpcity to ccumulte the Zn ++ through liquid medium fter twelve dys exposure period which is lso comprble with other studies (Tble 9). In the present study the increse in concentrtion of 922

15 zinc in E. crssipes t elevted levels of slt concentrtion might be due to their response to fvor metl tolernce nd involvement in mintennce of plnt growth nd overll development. Tble 9: Bioconcentrtion fctors (BCF) for zinc in vrious plnts used for phytoremedition Sr. Plnt species Bioconcentrtion Reference No. Fctor 1) This study 2) Xiomei et l. (2004) 3) Azoll pinnt (root) 12,000 Sel et l. (1989) 4) Myriophyllum exlbescense 1,640 Frnzin nd McFrlne (1980) 5) Ricciocrpus ntns 3,700-8,800 Cines et l.(1985) 6) Elode nuttlli 3,000 Nkd et l. (1979) 7) Lemn polyrrhiz 44 Jin et l.(1990) 3.9. Effect of Zinc Chloride on Wter Qulity Prmeters Electricl Conductivity (EC) Conductivity is numericl expression of bility of wter to crry n electricl current nd vries with the number nd types of ions, the solution contins. Electricl conductivity is the most common mesure of wter nd is indictive of the bility of n queous solution to crry n electric current. It is fct tht plnts re detrimentlly ffected, both physiclly nd chemiclly, by excess slts in wter nd by high levels of exchngeble sodium in others. It is obvious tht EC of the wter increses with the incresing levels of zinc chloride in the solution. It ws mximum by µs on the 0 dy t 75 ppm concentrtion nd minimum by µs on the 12 th dy t 25 ppm concentrtion. On 12 th dy the EC incresed with the incresing concentrtion nd showed perfect positive correltion (Tble 10). The vrying levels of EC in wter contining different concentrtions of ZnCl₂ might be due the externl dditions of slts nd lso might be due to different binding cpcities of the roots, root excretions nd competition by the roots to get nutrients from the wter. Similr increse in electricl conductivity in rooting medium hs been reported erlier (Gokhle et l., 2008; Mne et l., 2011) Totl Dissolved Solids (TDS) Totl dissolved solids is n expression for the combined content of ll inorgnic nd orgnic substnces contined in liquid which re present in moleculr, ionised or micro-grnulr suspended form. In other words, TDS is simply the sum of the ctions nd nions concentrtion expressed in mg/l. It is obvious tht TDS in the wter increses with the incresing levels of zinc chloride. The mximum TDS content ws observed by mg/l on the 0 dy t 75 ppm concentrtion nd the minimum vlue ws recorded on 12 th dy by Tble 10: Effect of zinc chloride on electricl conductivity (µs) of wter t different incubtion with Durtion Zinc Chloride (ppm) Electricl Conductivity (µs) 0 dy ** 177.0***

16 4 th dy (±1.11) th dy (±0.00) th dy (±3.93) 0.0 (±0.20) (±0.10) (±1.43) ** (±1.55) ** (±2.52) (±0.42) (±0.45) ** (±1.73) *** (±5.13) (±0.40) *** (±1.15) *** (±2.08) *** (±1.53) The vlues indictes percentge vrition reltive to control vlues. Vlues re men of three experiments. EC expressed in µs nd TDS expressed in ppm with ±SD. Significntly different from the control t *P<0.05, **P<0.01 ***P< by one-wy ANOVA with Tukey-Krmer multiple comprisons test. Tble 11: Effect of zinc chloride on totl dissolved solids of wter t different incubtion with Durtion 0 dy 92.1 (±1.66) th dy 88.3 (±0.15) th dy th dy 40.9 (±0.15) 0.0 Zinc Chloride (ppm) Totl Dissolved Solids (ppm) 130.2* (±2.98) ** (±0.23) (±0.25) (±1.00) ** (±0.42) ** (±0.46) *** (±1.15) ** (±1.15) ** (±1.42) *** (±0.58) ** (±0.58) *** (±0.58) The vlues indictes percentge vrition reltive to control vlues. Vlues re men of three experiments. EC expressed in µs nd TDS expressed in ppm with ±SD. Significntly different from the control t *P<0.05, **P<0.01 ***P< by one-wy ANOVA with Tukey-Krmer multiple comprisons test. Vlue of mg/l in control set (Tble 11). On the 12 th dy the TDS content incresed with the increse in concentrtion, showing perfect positive correltion ph ph of the wter is the mesure of the H+ ion ctivity of the wter system. It indictes whether the wter is cidic, neutrl or lkline in nture. The mximum ph ws recorded with 7.18 on 12 th dy in control while it ws minimum by vlue of 6.37 on 4 th dy t 25 ppm concentrtion (Tble 12). In generl ph ws observed to be slightly cid with the ddition of zinc chloride in the hydroponic medium. The chnge in ph my be due to relese 924

17 of some root exudtes in response to the stress so s to dpt itself to the existing environment. Tble 12: Effect of Zinc Chloride on ph nd turbidity of wter t different incubtion with Durtion 0 dy th dy th dy 7.13 (±0.02) th dy Zinc Chloride (ppm) ph 6.74 (±0.00) ** *** (±0.013) *** * *** *** (±0.00) ** (±0.00) ** *** The vlues indictes percentge vrition reltive to control vlues. Vlues re men of three experiments. EC expressed in µs nd TDS expressed in ppm with ±SD. Significntly different from the control t *P<0.05, **P<0.01 ***P< by one-wy ANOVA with Tukey-Krmer multiple comprisons test Turbidity Mximum turbidity ws observed with NTU on 12 th dy t 75 ppm concentrtion of zinc chloride while minimum turbidity ws 0.70 NTU on 0 dy in control solution (Tble 13). 12 th dy the turbidity incresed linerly with increse in concentrtion. It is definite tht the increse in turbidity might be due to the externl ddition of slt nd lso due to the root exudtes relesed to fce the stress condition Hrdness Mximum hrdness ws observed s 104 mg l -1 on 0 dy t 75 ppm concentrtion nd the minimum hrdness ws observed s mg l -1 on 12 th dy in control (Tble 14). Hrdness of wter incresed with the increse in the concentrtion showing perfect positive correltion n such n increse is definitely due to the dded slt concentrtion Alklinity Mximum lklinity ws observed s 60.0 mg l -1 on 12 th dy t 75 ppm concentrtion nd the minimum ws mg l -1 on 12 th dy in control (Tble 14). At the end of experiment lklinity lso incresed similr to tht of hrdness of wter with the increse in the zinc chloride concentrtion. 925

18 Chemicl Oxygen Demnd (COD) Chemicl oxygen Demnd determines the oxygen required for the chemicl oxidtion of most orgnic mtter nd oxidisble inorgnic substnces with the help of strong chemicl oxidnt. Highest vlue for COD ws observed mg l -1 on 0 dy t 75 ppm concentrtion while the minimum COD content ws observed by mg l -1 on 12 th dy in control (Fig. 2). At the end of experiment COD content of wter incresed in ll the sets with increse in the concentrtion. The increse in the COD content is definitely due to the externl ddition of zinc chloride. Tble 13: Effect of Zinc Chloride on ph nd turbidity of wter t different incubtion with Durtion 0 dy 0.70 (±0.08) 0.0 Zinc Chloride (ppm) 2.13** (±0.06) Turbidity 2.50** (±0.07) *** (±0.06) th dy 5.23 (±0.11) ** (±0.10) *** (±0.14) *** (±0.19) th dy 6.07 (±0.18) (±0.25) *** (±0.19) ** (±0.29) th dy 6.53 (±0.06) ** (±0.28) ** (±0.32) ** (±0.34) The vlues indictes percentge vrition reltive to control vlues. Vlues re men of three experiments. EC expressed in µs nd TDS expressed in ppm with ±SD. Significntly different from the control t *P<0.05, **P<0.01 ***P< by one-wy ANOVA with Tukey-Krmer multiple comprisons test. Tble 14: Effect of Zinc Chloride on selected wter qulity prmeters t different incubtion with Dy of Anlysis Hrdness Alklinity Zinc Chloride 0 dy 12 th dy 0 dy 12 th dy (ppm) Control (±1.89) (±1.82) (±1.11) (±1.65)

19 *** (±1.62) ** (±1.25) *** (±1.15) ** (±1.95) * (±1.14) (±1.54) ** (±0.88) ** (±1.58) ** (±1.03) * (±1.04) *** (±0.94) *** (±1.08) 3.45 The vlues indictes percentge vrition reltive to control vlues. Vlues re men of three experiments. EC expressed in µs nd TDS expressed in ppm with ±SD. Significntly different from the control t *P<0.05, **P<0.01 ***P< by one-wy ANOVA with Tukey-Krmer multiple comprisons test. Tble 15: Effect of Zinc Chloride on selected wter qulity prmeters t different incubtion with Dy of Anlysis Sodium Potssium Zinc Chloride 0 dy 12 th dy 0 dy 12 th dy (ppm) Control (±0.15) (±0.12) (±0.15) (±0.12) ** (±0.26) * (±0.27) ** (±0.26) * (±0.27) ** (±0.31) * (±0.28) *** (±0.21) ** (±0.19) ** (±0.31) * (±0.28) *** (±0.21) ** (±0.19) The vlues indictes percentge vrition reltive to control vlues. Vlues re men of three experiments. EC expressed in µs nd TDS expressed in ppm with ±SD. Significntly different from the control t *P<0.05, **P<0.01 ***P< by one-wy ANOVA with Tukey-Krmer multiple comprisons test Biochemicl Oxygen Demnd (BOD) Dissolved oxygen (DO) mesures re vitl for mintining erobic conditions in nturl wters tht receive polluted mtter. Mximum BOD content ws observed s 6.24 mg l -1 on the 12 th dy t 75 ppm concentrtion while minimum BOD content ws observed by 3.69 mgl -1 on 0 dy in control (Fig. 2). It is definite tht the degrding plnt prts nd root exudtes re the min resons behind increse in BOD content t the end of the experiment. 927

20 COD concentrtion (mg l -1 ) BOD concentrtion (mg l -1 ) O dy After 12 dys 0dy After 12 dys COD concentrtion (mg l -1 ) BOD concentrtion (mg l -1 ) 0 Zinc concentrtion (ppm) Figure 2: Effect of Zinc Chloride on BOD nd COD of wter fter 12 dys incubtion with 4. Conclusions The potentil dnger of hevy metls pollution in qutic environments is well known. In the present investigtion E. crssipes shows the mximum efficiency of ccumultion of Zn ++ upto 75 ppm present in the solution. Photosynthetic pigments of E. crssipes were incresed except chlorophyll content which is slightly ffected in t higher zinc chloride concentrtion. The vrying levels of electricl conductivity nd ph in wter contining different concentrtions of ZnCl₂ might be due the externl ddition of slt nd lso due to different binding cpcities of the roots, root excretions nd competition by the roots to get nutrients from the wter. It is definite tht the increse in TDS, COD nd BOD re relted with the incresed concentrtion zinc chloride while sme reson is ssocited with increse in hrdness nd lklinity. The decrese in sodium nd potssium content ws definitely ssocited with the use by the plnt for regultion of norml growth nd development under stress conditions. In the present study, Eichhorni with the BCF vlues of roughly hlf thn recommended, this plnt cn be considered s moderte ccumultor for Zn. Effluents contining these metls t low concentrtion my be treted by continuously pssing them through bed of wter hycinth from constructed wetlnds We lso suggest the need to understnd processes tht ffect metl vilbility, metl uptke nd trnsloction. Moreover better knowledge of these biochemicl mechnisms my led to development of better trnsgenic plnts which will hve better phytoremedition cpcity. Acknowledgements Authors re thnkful to Dr. R. G. Prdeshi, Principl, Fergusson College, Pune for constnt encourgement nd Ms. R. D. Gikwd, Hed, Deprtment of Environmentl Sciences, 0 928

21 Fergusson College, Pune for providing necessry fcilities. Author AVM would like to thnk Dr. P. A. Bnne nd Mr. Mhesh Mhvle, vice-president of Shydri Environmentl Awreness Orgnistion, Kolhpur for vluble suggestions. 5. References 1. Abrhm, E., Rigo, G., Szekely, G., Ngy, R., Koncz, C., Szbdos, L., (2003), Lightdependent induction of proline biosynthesis by bscisic cid nd slt stress is inhibited by brssinosteroid in Arbidopsis. Plnt Moleculr Biology, 51(3), pp Agunbide, F. O., Olu-Owolbi, B. I., Adebowle, K. O., (2009), Phytoremedition potentil of Eichorni crssipes in metl-contminted costl wter, Bioresource Technology, 100 (19), pp Andr, S. S., Srkr, D., Mkris, K. C., Mullens, C. P., Shi, S. V., Bch, S. B. H, (2010), Phytocheltins in vetiver grss upon led exposure in the presence of phosphorus, Plnt nd Soil, specil issue, Arnon. D. I., Copper Enzyme in Isolted Chloroplsts: Polyphenol Oxisdse in Bet vulgris, Plnt Physiology, 24, pp Chen, G., Djuric, Z., (2001), Crotenoids re oxidized but do not protect lipids from peroxidtion in unilmellr liposomes, FEBS Letters, 505, pp De. H. F., Biel, C., Sv, R., Morles, M. A., Torrecills, A., Alrcon, J. J., nchez- Blnco, M. J., (1998), Effect of wter nd slt stresses on the growth, gs exchnge nd wter reltions in Argyrnthemum coronopifolium, Plnt Science, 139, pp Dhote, S., nd Dixit, S., (2009), Wter qulity improvement through mcrophyte- A review Environmentl Monitoring nd Assessment, 152, pp EI Ghmery, A. A., EI-Kholy, M. A., nd Abou EI-Yousser, M. A., (2003), Evlution of cytologicl effects of Zn+ + in reltion to germintion nd root growth of Nigell stiv L. nd Triticum estivum L, Muttion Reserch/Genetic Toxicology nd Environmentl Mutgenesis, 537, pp Folin, O., nd Denis, W., (1915), A clorimetric estimtion of phenol & phenol derivtives in urine, Journl of Biologicl Chemistry, 22, pp Fricke, W., Akhiyrov, G., Wei, W., Alexndersson, E., Miller, A., Kjellbom, P. O., Richrdson, A., Tobis, W., Schreiber, L., Veselov, D., Kudoyrov, G., Volkov, V., (2006), The short-term growth response to slt of the developing brley lef, Journl of Experimentl Botny, 57, pp Grbisu, C., Alkort, I., (2001), Phytoextrction: A cost-effective plnt-bsed technology for the removl of metls from the environment, Bioresource Technology, 77, pp Gub, N., Mhmooduzzfr, T. O., Siddiqui, S., nd Iqbl, M. U., (2007), Lef biochemistry of Lycopersicon esculentum Mill. t different stges of plnt 929