This article was downloaded by: [RMIT University] On: 25 July 2013, At: 09:16 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
International Journal of Phytoremediation Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/bijp20
Cadmium Removal by Lemna minor and Spirodela polyrhiza a
b
c
Devaleena Chaudhuri , Arunabha Majumder , Amal K. Misra & Kaushik Bandyopadhyay a
Jadavpur University, Dept. of Construction Engineering , India
b
Jadavpur University, School of Water Resource Engineering , India
a
c
Jadavpur University, Dept. of Civil Engineering , India Accepted author version posted online: 17 Jul 2013.
To cite this article: International Journal of Phytoremediation (2013): Cadmium Removal by Lemna minor and Spirodela polyrhiza , International Journal of Phytoremediation, DOI: 10.1080/15226514.2013.821446 To link to this article: http://dx.doi.org/10.1080/15226514.2013.821446
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
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 to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and 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 Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial 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
ACCEPTED MANUSCRIPT Cadmium Removal by Lemna minor and Spirodela polyrhiza Devaleena Chaudhuri*1, Arunabha Majumder2, Amal .K. Misra3, Kaushik Bandyopadhyay4 1. *Corresponding author: Research Scholar, Jadavpur University, Dept. of Construction Engineering, India, e mail: [email protected] 2. Emeritus Fellow, Jadavpur University, School of Water Resource Engineering, India, e mail: arunabhamajumder @hotmail.com
Downloaded by [RMIT University] at 09:16 25 July 2013
3. Retired Professor, Jadavpur University, Dept. of Civil Engineering, India 4. Associate professor, Jadavpur University, Dept. of Construction Engineering, India, e mail: [email protected]
ABSTRACT: The present study investigates the ability of two genus of duckweed (Lemna minor and Spirodela polyrhiza) to phytoremediate cadmium from aqueous solution. Duckweed was exposed to six different cadmium concentrations, such as, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/L and the experiment was continued for 22 days. Water samples were collected periodically for estimation of residual cadmium content in aqueous solution. At the end of treatment period plant samples were collected and accumulated cadmium content was measured. Cadmium toxicity was observed through relative growth factor and changes in chlorophyll content. Experimental results showed that Lemna minor and Spirodela polyrhiza were capable of removing 42-78% and 5275% cadmium from media depending upon initial cadmium concentrations. Cadmium was removed following pseudo second order kinetic model. Maximum cadmium accumulation in Lemna minor was 4734.56 mg/kg at 2 mg/L initial cadmium concentration and 7711.00 mg/kg in
1
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Spirodela polyrhiza at 3 mg/L initial cadmium concentration at the end of treatment period. Conversely in both cases maximum bio-concentration factor obtained at lowest initial cadmium concentrations i.e. 0.5 mg/L, were 3295.61 and 4752.00 for Lemna minor and Spirodela polyrhiza respectively. The present study revealed that both Lemna minor and Spirodela polyrhiza was potential cadmium accumulator.
Downloaded by [RMIT University] at 09:16 25 July 2013
Key words: Duckweed, kinetic model, Bio-concentration factor, Hyperaccumulator, Chlorophyll
INTRODUCTION: Pollution of aquatic environment by heavy metal contamination has emerged as a global concern. These toxic heavy metals include lead, mercury, cadmium, nickel, chromium, copper etc. The main problem associated with these heavy metals, is that, unlike organic contaminants they are non biodegradable, persistent in nature and again bio-accumulate in different environmental components especially in living organisms. These heavy metals are released into the environment mainly from different anthropogenic sources like industrial effluents, fuel production, mining, smelting processes, use of agricultural chemicals, small-scale industries, dumping of electronic wastes etc (OECD 2003). Cadmium (Cd) is considered as one of the major toxic heavy metals and its most dangerous property is high mobility in soils, large water solubility and extreme toxicity, even at low concentration (Tkalec et al. 2008). Thus it can pollute all three major environmental compartments i.e. air, soil and water. Cadmium is widely used in electroplating industry, rechargeable batteries, dye industry, plastic, metal alloys etc. It is transported into environment from earth crust through mining activity during extraction of other metals, such as zinc, lead and
2
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT copper (OECD 2003). Cadmium sulphide is known as Cadmium yellow and is used in paints and pigments. Cadmium telluride is a crystalline compound, widely used in solar panels. Cadmium stearate is used as ‘stabilisers’ of plastics such as PVC to prevent them from breaking down in sunlight (Water U.K. 2001). Cadmium serves no constructive role in human body. It may cause severe damage to different organs including the lung, kidney, liver, testis, and even may led to infertility (M. Fleischer et al.
Downloaded by [RMIT University] at 09:16 25 July 2013
1974). The International Agency for Research on Cancer (IARC) has classified cadmium as a Group 2A human carcinogen (Group 2A, limited evidence of carcinogenicity in humans and sufficient evidence of carcinogenicity in animals). These emphasise the extent of cadmium toxicity on human being. Cadmium can be readily uptake by aquatic organisms in its free ionic form. It can accumulate in fish and subsequently can enter into human food chain. In Japan cadmium contaminated water was used for irrigation which led to out broke of itai-itai disease in Japan, the most severe effect caused due to chronic cadmium toxicity (Fleischer et al. 1974). Cadmium discharge limit in inland surface water is 2mg/L (BIS, 2012) and that of public sewer is 1mg/L (BIS). According to WHO (World Health Organisation) as well as BIS (Beauro of Indian Standard) the permissible limit of cadmium in drinking water is 3 µg/L.
Different conventional metal removal techniques like ion exchange, reverse osmosis, precipitation, adsorption, electro-coagulation etc. are used in practice for cadmium removal from aqueous solution. Phytoremediation is an emerging and cost effective eco-friendly technology that uses living green plants for in-situ removal of contaminants from soil and water. The efficiency of phytoremediation varies significantly between species as different mechanisms of
3
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT ion uptake are operative in each species, based on their genetic, morphological, physiological and anatomical characteristics (Rahman & Hasegawa, 2011). Floating macrophytes usually remove metal by applying the process of rhizofiltration. Cadmium removal efficiency of Pistia stratiotes (Mishra & Tripathi, 2008), Salvinia herzogi (Sune et al. 2007), Wolffia globosa (Garg & Chandra, 1993), Eichornia crassipes (Liao and Chang, 2004; Narayan, 2011), Ipomoea
Downloaded by [RMIT University] at 09:16 25 July 2013
aquatic (Wang et al., 2008) etc. have already been demonstrated.
Duckweed has been widely studied for their potential application in metal phytoremediation. Fast growth rate and easy harvest potential made duckweed a good alternative for phytoremediation activities (Zayed et al. 1998). Kara and Kara (2005) reported that Lemna trisulca could remove more than 70% of cadmium after 4 days of treatment within the range of 1 to 7 mg/L of initial cadmium concentration. Lemna minor proved to be a promising nickel accumulator from contaminated waste water (Kara et al. 2003). Chaudhuri et al. (2011) investigated cadmium and nickel removal efficiency of Spirodela polyrhiza and it was reported that the tested plant had greater potential to accumulate cadmium than that of nickel. These findings emphasise success of using different type of duckweed for heavy metal removal. This study aims to determine suitability of two types of commonly available duckweed for phytoremediation of cadmium from aqueous solution. Lemna minor and Spirodela polyrhiza are two types of duckweed selected for this purpose. Suitability of the selected duckweed for cadmium phytoremediation was evaluated through analysis of cadmium removal percentage, cadmium content in plant tissue, bio concentration factor, relative growth factor, and change in chlorophyll content due to cadmium toxicity.
4
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ACCEPTED MANUSCRIPT
MATERIALS AND METHOD Culture media: Culture media was prepared by adding cadmium stock solution to pond water to achieve desired cadmium concentration. Cadmium chloride (CdCl2.H2O) salt of GR grade was used for preparation of cadmium stock solution. Chemical composition of pond water (Table 1) was
Downloaded by [RMIT University] at 09:16 25 July 2013
analysed before using it for preparation of culture media Plant material: Duckweed from a local pond situated 1.1km away towards east from laboratory was collected, washed with tap water to eliminate remains of pond sediments. They were then acclimatized in acid cleaned plastic tubs with corresponding pond water for 7 days. Heavy metal concentration in pond water as well as in the plant sample was analysed before using those for phytoremediation study. After acclimatization, required amount of plant sample was transferred to another plastic tub containing cadmium spiked pond water.
Experimental set up: Each experimental unit comprised metal spiked pond water as working solution and 3.37mg/m 2 of unsterile duckweed, such as Lemna minor and Spirodela polyrhiza. Each plastic vessel used as experimental unit was thoroughly acid (1:1 HNO3) washed before use. The experimental work was carried out on roof top laboratory (Latitude 2233’N and Longitude 8825’E). Everyday evaporative water loss was compensated by adding pond water collected from same pond.
5
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT During the experimental period average temperature was about 23C, average 11 hr (approx) and average relative humidity 62%. The experiment was continued for three weeks.
For each type of plant 6 initial cadmium concentrations were selected i.e. 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/L. A control set up was also used where same amount of plant was cultured in pond water but without addition of any cadmium. Three replicates for each type of experimental set up
Downloaded by [RMIT University] at 09:16 25 July 2013
were taken.
50ml of water sample from experimental units were collected after 2, 5, 10, 15 and 22 days for estimation of residual Cd content in culture media. At the end of experimental period plant samples were harvested from each experimental unit. Wet weight of harvested plant material was recorded after removal of extra water with tissue paper.5g of fresh plant material were kept separately in dark plastic bag in freeze at 4C for chlorophyll estimation. Analysis of heavy metal: Harvested plant biomass (amount left after collecting sample for chlorophyll estimation) was first washed with distilled water. Then they were air dried followed by oven drying at a temperature of 80C for 6 hr and the dry weight of plant samples were measured. Afterwards the dry plant tissues were ground to powder. For metal estimation collected water sample as well as the powdered plant sample were subjected to acid digestion at 105C for 4 hours by adding 1:1 HNO3 and analyzed for cadmium using atomic-absorption spectrophotometer (AAS; VARIAN Spectra AA 50) following the process specified by APHA (2000).
6
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ACCEPTED MANUSCRIPT Chlorophyll estimation: To estimate chlorophyll content of plant sample (APHA 20th Edition), 100mg of duckweed fronds (stored at 4C) were homogenised with 10ml of 90% acetone and kept at 4C overnight in dark. Next day the mixture was centrifuged at 5000 rpm and supernatant was collected. The absorbance of chlorophyll pigments in the collected supernatant was measured at 750, 664 and
Downloaded by [RMIT University] at 09:16 25 July 2013
647 nm wavelength with spectrophotometer.
Removal of Heavy Metals: Removal of metal was calculated on percentage basis using the following equation (Chaudhuri et al., 2011): R(%) = ((C0 - Ct)/C0) * 100
(equ 1)
C0 and Ct were used for denoting residual metal concentration initially and at time t, respectively. Kinetic modelling: In order to determine removal kinetic of cadmium by duckweed, experimental results were fitted to two different types of kinetic model, such as, pseudo first order (equ 2) and pseudo second order kinetic model (equ 3). The pseudo first order equation can be represented as follows: dyt / dt = Kt (yeq – yt)
( equ 2)
yt = Phytoremediation capacity at time t yeq = Phytoremediation capacity at equilibrium K1 = pseudo first order rate constant
7
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT It can be rearranged and integrated by applying boundary conditions yt = 0 at t = 0 and yt = yt at t = t and can be written as Log (yeq - yt) = Log yeq – (K1 t) / 2.303
(equ 3)
A plot of Log (yeq - yt) against t should give a straight line to indicate that the removal reaction follow pseudo first order kinetic. The pseudo second order rate equation can be expressed as :
Downloaded by [RMIT University] at 09:16 25 July 2013
dyt / dt = K2(yeq – yt)2
(equ 4)
where K2 is the pseudo second order rate constant For the boundary conditions yt = 0 at t = 0 and yt = yt at t = t , equation can be integrated and rearranged as t / yt = 1/(K2 yeq2) + (1/yeq) t
(equ 5)
The equation dictates that linear plot of t / yt vs t confirms that the reaction rate follows pseudo second order kinetic model. The rate constant and biosorption at equilibrium can be obtained from the intercept and slop respectively. Optimum kinetic model between pseudo first order and pseudo second order was determined by examine using four different types of error analysis equation (Table 2), those were performed on test results. These equations compared the measured cadmium phytoremediation capacity of duckweed (ymeas) to the calculated cadmium phytoremediation capacity of duckweed (ycal). Kinetic model with least error between measured and calculated data was recommended as the suitable for the present study. Bio Concentration Factor (BCF):
8
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ACCEPTED MANUSCRIPT The Bio Concentration Factor is defined as the concentration of heavy metal in the plant tissue to that in the aquatic environment, expressed as: BCF = (Metal concentration in plant tissue at harvest (mg/kg)) / (Initial metal concentration in external aqueous solution (mg/L)) Calculation of Growth inhibition: Growth rate of control and treated plant (for each concentration) was calculated as follows:
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Growth rate = [(ln (final fresh weight of plant)) - (ln (initial fresh weight of plant))/time] Growth rate was used for the calculation of growth inhibition of duckweed due to cadmium toxicity. Percent inhibition of growth rate for six tested cadmium concentrations was calculated by comparing growth rate of control plant and that of treated plant (Naumann et al. 2007). Data analysis Average value of three replicate in each set of experiment was represented in result section. Mean and Standard deviation were calculated by Origin 6.1. Statistical comparison of the mean, in case of relative growth factor and chlorophyll content, was done by two way analysis of variance (ANOVA) test using Microsoft office excel 2007. Difference was considered to be significant at p ≤ 0.05 level of significance. Correlation coefficient in case of kinetic study was also determined using data analysis pack of Microsoft office excel 2007. RESULTS AND DISCUSSION Morphological changes: Physiological manifestation of cadmium toxicity was almost similar for both the selected duckweed Lemna minor and Spirodela polyrhiza. In case of Lemna minor, fronds gradually became whitish due to cadmium toxicity. On the other hand the fronds of Spirodela polyrhiza
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ACCEPTED MANUSCRIPT turned to brownish colour as an effect of cadmium toxicity. Root detachment was also observed at cadmium concentration above 2mg/L after 2 weeks of treatment. At 2.5 and 3mg/L of initial cadmium concentrations, frond shrinkage was another observable change specially found in case of Lemna minor for longer treatment period. Growth inhibition: Plant growth was inhibited by the presence of cadmium and the percent of inhibition was
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calculated by comparing relative growth factor (RGF) of unexposed and cadmium exposed plant (Figure1). Percent of growth inhibition increased with increase in initial cadmium concentration. It was evident from Figure 1 that Lemna minor was significantly (ANOVA Two way: p
International Journal of Phytoremediation Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/bijp20
Cadmium Removal by Lemna minor and Spirodela polyrhiza a
b
c
Devaleena Chaudhuri , Arunabha Majumder , Amal K. Misra & Kaushik Bandyopadhyay a
Jadavpur University, Dept. of Construction Engineering , India
b
Jadavpur University, School of Water Resource Engineering , India
a
c
Jadavpur University, Dept. of Civil Engineering , India Accepted author version posted online: 17 Jul 2013.
To cite this article: International Journal of Phytoremediation (2013): Cadmium Removal by Lemna minor and Spirodela polyrhiza , International Journal of Phytoremediation, DOI: 10.1080/15226514.2013.821446 To link to this article: http://dx.doi.org/10.1080/15226514.2013.821446
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
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 to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and 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 Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial 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
ACCEPTED MANUSCRIPT Cadmium Removal by Lemna minor and Spirodela polyrhiza Devaleena Chaudhuri*1, Arunabha Majumder2, Amal .K. Misra3, Kaushik Bandyopadhyay4 1. *Corresponding author: Research Scholar, Jadavpur University, Dept. of Construction Engineering, India, e mail: [email protected] 2. Emeritus Fellow, Jadavpur University, School of Water Resource Engineering, India, e mail: arunabhamajumder @hotmail.com
Downloaded by [RMIT University] at 09:16 25 July 2013
3. Retired Professor, Jadavpur University, Dept. of Civil Engineering, India 4. Associate professor, Jadavpur University, Dept. of Construction Engineering, India, e mail: [email protected]
ABSTRACT: The present study investigates the ability of two genus of duckweed (Lemna minor and Spirodela polyrhiza) to phytoremediate cadmium from aqueous solution. Duckweed was exposed to six different cadmium concentrations, such as, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/L and the experiment was continued for 22 days. Water samples were collected periodically for estimation of residual cadmium content in aqueous solution. At the end of treatment period plant samples were collected and accumulated cadmium content was measured. Cadmium toxicity was observed through relative growth factor and changes in chlorophyll content. Experimental results showed that Lemna minor and Spirodela polyrhiza were capable of removing 42-78% and 5275% cadmium from media depending upon initial cadmium concentrations. Cadmium was removed following pseudo second order kinetic model. Maximum cadmium accumulation in Lemna minor was 4734.56 mg/kg at 2 mg/L initial cadmium concentration and 7711.00 mg/kg in
1
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Spirodela polyrhiza at 3 mg/L initial cadmium concentration at the end of treatment period. Conversely in both cases maximum bio-concentration factor obtained at lowest initial cadmium concentrations i.e. 0.5 mg/L, were 3295.61 and 4752.00 for Lemna minor and Spirodela polyrhiza respectively. The present study revealed that both Lemna minor and Spirodela polyrhiza was potential cadmium accumulator.
Downloaded by [RMIT University] at 09:16 25 July 2013
Key words: Duckweed, kinetic model, Bio-concentration factor, Hyperaccumulator, Chlorophyll
INTRODUCTION: Pollution of aquatic environment by heavy metal contamination has emerged as a global concern. These toxic heavy metals include lead, mercury, cadmium, nickel, chromium, copper etc. The main problem associated with these heavy metals, is that, unlike organic contaminants they are non biodegradable, persistent in nature and again bio-accumulate in different environmental components especially in living organisms. These heavy metals are released into the environment mainly from different anthropogenic sources like industrial effluents, fuel production, mining, smelting processes, use of agricultural chemicals, small-scale industries, dumping of electronic wastes etc (OECD 2003). Cadmium (Cd) is considered as one of the major toxic heavy metals and its most dangerous property is high mobility in soils, large water solubility and extreme toxicity, even at low concentration (Tkalec et al. 2008). Thus it can pollute all three major environmental compartments i.e. air, soil and water. Cadmium is widely used in electroplating industry, rechargeable batteries, dye industry, plastic, metal alloys etc. It is transported into environment from earth crust through mining activity during extraction of other metals, such as zinc, lead and
2
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT copper (OECD 2003). Cadmium sulphide is known as Cadmium yellow and is used in paints and pigments. Cadmium telluride is a crystalline compound, widely used in solar panels. Cadmium stearate is used as ‘stabilisers’ of plastics such as PVC to prevent them from breaking down in sunlight (Water U.K. 2001). Cadmium serves no constructive role in human body. It may cause severe damage to different organs including the lung, kidney, liver, testis, and even may led to infertility (M. Fleischer et al.
Downloaded by [RMIT University] at 09:16 25 July 2013
1974). The International Agency for Research on Cancer (IARC) has classified cadmium as a Group 2A human carcinogen (Group 2A, limited evidence of carcinogenicity in humans and sufficient evidence of carcinogenicity in animals). These emphasise the extent of cadmium toxicity on human being. Cadmium can be readily uptake by aquatic organisms in its free ionic form. It can accumulate in fish and subsequently can enter into human food chain. In Japan cadmium contaminated water was used for irrigation which led to out broke of itai-itai disease in Japan, the most severe effect caused due to chronic cadmium toxicity (Fleischer et al. 1974). Cadmium discharge limit in inland surface water is 2mg/L (BIS, 2012) and that of public sewer is 1mg/L (BIS). According to WHO (World Health Organisation) as well as BIS (Beauro of Indian Standard) the permissible limit of cadmium in drinking water is 3 µg/L.
Different conventional metal removal techniques like ion exchange, reverse osmosis, precipitation, adsorption, electro-coagulation etc. are used in practice for cadmium removal from aqueous solution. Phytoremediation is an emerging and cost effective eco-friendly technology that uses living green plants for in-situ removal of contaminants from soil and water. The efficiency of phytoremediation varies significantly between species as different mechanisms of
3
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT ion uptake are operative in each species, based on their genetic, morphological, physiological and anatomical characteristics (Rahman & Hasegawa, 2011). Floating macrophytes usually remove metal by applying the process of rhizofiltration. Cadmium removal efficiency of Pistia stratiotes (Mishra & Tripathi, 2008), Salvinia herzogi (Sune et al. 2007), Wolffia globosa (Garg & Chandra, 1993), Eichornia crassipes (Liao and Chang, 2004; Narayan, 2011), Ipomoea
Downloaded by [RMIT University] at 09:16 25 July 2013
aquatic (Wang et al., 2008) etc. have already been demonstrated.
Duckweed has been widely studied for their potential application in metal phytoremediation. Fast growth rate and easy harvest potential made duckweed a good alternative for phytoremediation activities (Zayed et al. 1998). Kara and Kara (2005) reported that Lemna trisulca could remove more than 70% of cadmium after 4 days of treatment within the range of 1 to 7 mg/L of initial cadmium concentration. Lemna minor proved to be a promising nickel accumulator from contaminated waste water (Kara et al. 2003). Chaudhuri et al. (2011) investigated cadmium and nickel removal efficiency of Spirodela polyrhiza and it was reported that the tested plant had greater potential to accumulate cadmium than that of nickel. These findings emphasise success of using different type of duckweed for heavy metal removal. This study aims to determine suitability of two types of commonly available duckweed for phytoremediation of cadmium from aqueous solution. Lemna minor and Spirodela polyrhiza are two types of duckweed selected for this purpose. Suitability of the selected duckweed for cadmium phytoremediation was evaluated through analysis of cadmium removal percentage, cadmium content in plant tissue, bio concentration factor, relative growth factor, and change in chlorophyll content due to cadmium toxicity.
4
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
MATERIALS AND METHOD Culture media: Culture media was prepared by adding cadmium stock solution to pond water to achieve desired cadmium concentration. Cadmium chloride (CdCl2.H2O) salt of GR grade was used for preparation of cadmium stock solution. Chemical composition of pond water (Table 1) was
Downloaded by [RMIT University] at 09:16 25 July 2013
analysed before using it for preparation of culture media Plant material: Duckweed from a local pond situated 1.1km away towards east from laboratory was collected, washed with tap water to eliminate remains of pond sediments. They were then acclimatized in acid cleaned plastic tubs with corresponding pond water for 7 days. Heavy metal concentration in pond water as well as in the plant sample was analysed before using those for phytoremediation study. After acclimatization, required amount of plant sample was transferred to another plastic tub containing cadmium spiked pond water.
Experimental set up: Each experimental unit comprised metal spiked pond water as working solution and 3.37mg/m 2 of unsterile duckweed, such as Lemna minor and Spirodela polyrhiza. Each plastic vessel used as experimental unit was thoroughly acid (1:1 HNO3) washed before use. The experimental work was carried out on roof top laboratory (Latitude 2233’N and Longitude 8825’E). Everyday evaporative water loss was compensated by adding pond water collected from same pond.
5
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT During the experimental period average temperature was about 23C, average 11 hr (approx) and average relative humidity 62%. The experiment was continued for three weeks.
For each type of plant 6 initial cadmium concentrations were selected i.e. 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/L. A control set up was also used where same amount of plant was cultured in pond water but without addition of any cadmium. Three replicates for each type of experimental set up
Downloaded by [RMIT University] at 09:16 25 July 2013
were taken.
50ml of water sample from experimental units were collected after 2, 5, 10, 15 and 22 days for estimation of residual Cd content in culture media. At the end of experimental period plant samples were harvested from each experimental unit. Wet weight of harvested plant material was recorded after removal of extra water with tissue paper.5g of fresh plant material were kept separately in dark plastic bag in freeze at 4C for chlorophyll estimation. Analysis of heavy metal: Harvested plant biomass (amount left after collecting sample for chlorophyll estimation) was first washed with distilled water. Then they were air dried followed by oven drying at a temperature of 80C for 6 hr and the dry weight of plant samples were measured. Afterwards the dry plant tissues were ground to powder. For metal estimation collected water sample as well as the powdered plant sample were subjected to acid digestion at 105C for 4 hours by adding 1:1 HNO3 and analyzed for cadmium using atomic-absorption spectrophotometer (AAS; VARIAN Spectra AA 50) following the process specified by APHA (2000).
6
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Chlorophyll estimation: To estimate chlorophyll content of plant sample (APHA 20th Edition), 100mg of duckweed fronds (stored at 4C) were homogenised with 10ml of 90% acetone and kept at 4C overnight in dark. Next day the mixture was centrifuged at 5000 rpm and supernatant was collected. The absorbance of chlorophyll pigments in the collected supernatant was measured at 750, 664 and
Downloaded by [RMIT University] at 09:16 25 July 2013
647 nm wavelength with spectrophotometer.
Removal of Heavy Metals: Removal of metal was calculated on percentage basis using the following equation (Chaudhuri et al., 2011): R(%) = ((C0 - Ct)/C0) * 100
(equ 1)
C0 and Ct were used for denoting residual metal concentration initially and at time t, respectively. Kinetic modelling: In order to determine removal kinetic of cadmium by duckweed, experimental results were fitted to two different types of kinetic model, such as, pseudo first order (equ 2) and pseudo second order kinetic model (equ 3). The pseudo first order equation can be represented as follows: dyt / dt = Kt (yeq – yt)
( equ 2)
yt = Phytoremediation capacity at time t yeq = Phytoremediation capacity at equilibrium K1 = pseudo first order rate constant
7
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT It can be rearranged and integrated by applying boundary conditions yt = 0 at t = 0 and yt = yt at t = t and can be written as Log (yeq - yt) = Log yeq – (K1 t) / 2.303
(equ 3)
A plot of Log (yeq - yt) against t should give a straight line to indicate that the removal reaction follow pseudo first order kinetic. The pseudo second order rate equation can be expressed as :
Downloaded by [RMIT University] at 09:16 25 July 2013
dyt / dt = K2(yeq – yt)2
(equ 4)
where K2 is the pseudo second order rate constant For the boundary conditions yt = 0 at t = 0 and yt = yt at t = t , equation can be integrated and rearranged as t / yt = 1/(K2 yeq2) + (1/yeq) t
(equ 5)
The equation dictates that linear plot of t / yt vs t confirms that the reaction rate follows pseudo second order kinetic model. The rate constant and biosorption at equilibrium can be obtained from the intercept and slop respectively. Optimum kinetic model between pseudo first order and pseudo second order was determined by examine using four different types of error analysis equation (Table 2), those were performed on test results. These equations compared the measured cadmium phytoremediation capacity of duckweed (ymeas) to the calculated cadmium phytoremediation capacity of duckweed (ycal). Kinetic model with least error between measured and calculated data was recommended as the suitable for the present study. Bio Concentration Factor (BCF):
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ACCEPTED MANUSCRIPT The Bio Concentration Factor is defined as the concentration of heavy metal in the plant tissue to that in the aquatic environment, expressed as: BCF = (Metal concentration in plant tissue at harvest (mg/kg)) / (Initial metal concentration in external aqueous solution (mg/L)) Calculation of Growth inhibition: Growth rate of control and treated plant (for each concentration) was calculated as follows:
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Growth rate = [(ln (final fresh weight of plant)) - (ln (initial fresh weight of plant))/time] Growth rate was used for the calculation of growth inhibition of duckweed due to cadmium toxicity. Percent inhibition of growth rate for six tested cadmium concentrations was calculated by comparing growth rate of control plant and that of treated plant (Naumann et al. 2007). Data analysis Average value of three replicate in each set of experiment was represented in result section. Mean and Standard deviation were calculated by Origin 6.1. Statistical comparison of the mean, in case of relative growth factor and chlorophyll content, was done by two way analysis of variance (ANOVA) test using Microsoft office excel 2007. Difference was considered to be significant at p ≤ 0.05 level of significance. Correlation coefficient in case of kinetic study was also determined using data analysis pack of Microsoft office excel 2007. RESULTS AND DISCUSSION Morphological changes: Physiological manifestation of cadmium toxicity was almost similar for both the selected duckweed Lemna minor and Spirodela polyrhiza. In case of Lemna minor, fronds gradually became whitish due to cadmium toxicity. On the other hand the fronds of Spirodela polyrhiza
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ACCEPTED MANUSCRIPT turned to brownish colour as an effect of cadmium toxicity. Root detachment was also observed at cadmium concentration above 2mg/L after 2 weeks of treatment. At 2.5 and 3mg/L of initial cadmium concentrations, frond shrinkage was another observable change specially found in case of Lemna minor for longer treatment period. Growth inhibition: Plant growth was inhibited by the presence of cadmium and the percent of inhibition was
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calculated by comparing relative growth factor (RGF) of unexposed and cadmium exposed plant (Figure1). Percent of growth inhibition increased with increase in initial cadmium concentration. It was evident from Figure 1 that Lemna minor was significantly (ANOVA Two way: p
