Efficiency of Phragmites australis and Typha latifolia for heavy metal removal from wastewater

Ecotoxicology and Environmental Safety 112 (2015) 80–86

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Efficiency of Phragmites australis and Typha latifolia for heavy metal removal from wastewater Menka Kumari a, B.D. Tripathi b,n a b

Pollution Ecology Research Laboratory, Department of Botany, Banaras Hindu University, Varanasi 221005, India Centre for Environmental Science and Technology, Banaras Hindu University, Varanasi 221005, India

art ic l e i nf o

a b s t r a c t

Article history: Received 24 May 2014 Received in revised form 15 October 2014 Accepted 22 October 2014

A cost-effective and promising technology has been demonstrated for the removal of copper (Cu), cadmium (Cd), chromium (Cr), nickel (Ni), iron (Fe), lead (Pb) and zinc (Zn) from urban sewage mixed with industrial effluents within 14 days. With the help of P. australis and T. latifolia grown alone and in combination batch experiments were designed to assess the removal of heavy metals from the wastewater collected from 5 sampling stations. The results revealed that P. australis performed better than T. latifolia for Cu, Cd, Cr, Ni, Fe, Pb and Zn removal, while mixing of the plant species further enhanced the removal of Cu to 78.0 71.2%, Cd to 60.0 71.2%, Cr to 68.1 7 0.4%, Ni to 73.87 0.6%, Fe to 80.1 7 0.3%, Pb to 61.071.2% and Zn to 61.07 1.2% for wastewater samples from Raj Ghat. Negative correlation coefficients of Cu, Cd, Cr, Ni, Fe, Pb and Zn concentrations in wastewater with the retention time revealed that there was an increase in the heavy metal removal rate with retention time. P. australis showed higher accumulative capacities for Cu, Cd, Cr, Ni and Fe than T. latifolia. P. australis and T. latifolia grown in combination can be used for the removal of Cu, Cd, Cr, Ni, Fe, Pb and Zn from the urban sewage mixed with industrial effluents within 14 days. & Elsevier Inc. All rights reserved.

Keywords: Adsorption Heavy metal Mixed culture Retention time Wastewater

1. Introduction The release of improperly treated urban sewage and industrial effluents containing heavy metals into rivers has become a serious environmental problem over the world. Consumption of contaminated river water having heavy metals poses a threat to human health. Removal of heavy metals through chemical precipitation, coagulation–flocculation, adsorption, ion exchange, membrane filtration and other advanced oxidation processes require high capital and operating and management costs (Kumari and Tripathi, 2014b). Henceforth, it was imperative to suggest an economic as well as eco-friendly technology to remove these heavy metals and improve the wastewater quality. Aquatic plants are natural absorbers of heavy metals and other nutrients (Mukhopadhyay and Maiti 2010). Removal of heavy metals and other pollutants from wastewater with the help of aquatic plants has been reported as a low cost and effective technology (Rai, 2008). Aquatic plants or constructed wetlands were used extensively in a past few decades for the removal heavy metals and nutrients from wastewater (Allende et al., 2011; Caselles-Osorio and Garcia, 2007; Cui et al., 2011; Hua et al., 2013; n

Corresponding author. Fax: þ91 542 2369139. E-mail addresses: [email protected] (M. Kumari), [email protected] (B.D. Tripathi). http://dx.doi.org/10.1016/j.ecoenv.2014.10.034 0147-6513/& Elsevier Inc. All rights reserved.

Kumari and Tripathi, 2014a,b). Of these aquatic plants, the emergent plants are able to accumulate metals in their tissues several times than their surrounding environment, which might be due to the metal uptake by plant tissues is by adsorption to anionic sites in the cell walls (Sheoran and Sheoran, 2006). Phragmites australis and Typha latifolia are well known hyperaccumulator emergent plants. They are capable to accumulate metals copper, cadmium, chromium, nickel and lead up to 0.1% and iron and zinc up to 1% of the plant dry weight (Kalra, 1998; Sasmaz et al., 2008). In recent years, P. australis and T. latifolia were used for heavy metal removal (Abou-Elela and Hellal, 2012; Bianchi et al., 2011; Bragato et al., 2009; Calheiros et al., 2009; Jacob and Otte, 2004; Laing et al., 2009; Yeh et al., 2009). However, there was a paucity of data regarding the evaluation of role of P. australis and T. latifolia when grown in association in the removal of heavy metals. 1.1. Research AIM The aim of the present study was to evaluate the heavy metals removal efficiency of P. australis and T. latifolia grown alone and in association, which were achieved through the specific objectives: To determine (1) the removal of heavy metals by P. australis and T. latifolia; (2) the heavy metal removal in mixed culture of the two plants; and (3) the relationship between retention time and removal of heavy metals.

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2. Material and methods Untreated urban sewage mixed with industrial effluents were collected from the five sampling stations i.e. Assi Ghat (AG), Harishchandra Ghat (HG), Rajendra Prasad Ghat (RPG), Manikarnika Ghat (MG) and Raj Ghat (RG) along the 10 km stretch of the river Ganga at Varanasi (25˚18′ N, 83˚1′E), India and brought to laboratory for experimental use. In order to minimize the error from each sampling station, 5 samples of wastewater have been collected separately and treated as 5 replicates. Batch experiments were configured during first week of August, September and October (2012) using the same wastewater samples collected in the same period. Physicochemical properties of untreated wastewater from AG, HG, RPG, MG and RG have been shown in Table 1. Wastewater samples collected from RG was found most polluted with the highest concentration of pollution indicators (i.e. biochemical oxygen demand and chemical oxygen demand), nutrients (nitratenitrogen and phosphate-phosphorous) and heavy metals (copper, cadmium, chromium, nickel, iron, lead and zinc), which was followed by MG, RPG, HG and least for AG station (Table 1). 2.1. Experimental set up Four biofiltration units, which consisted of a glass aquarium of 75 L (50 cm length, 50 cm width and 50 cm height) capacity fitted with 20 PVC pipes each of 30 cm length and 5 cm diameter having several pores of 27 mm diameter to enable liquid exchange, were configured (Fig. 1). During present experiment, P. australis and T. latifolia plants of 35.1 72.1 cm length and 45.5 70.5 g fresh weight were selected and placed individually in each PVC pipe. Experimental set 1 consisted of only P. australis kept at a density of 40 plants m  2. Experimental set 2 consisted of only T. latifolia plants kept at a density of 40 plants m  2. In 3rd set, each of P. australis and T. latifolia were kept at a density of 20 plants m  2. Experimental set 4, called as reference set, was kept without any plant and was used to determine the removal of heavy metals through natural precipitation. In order to minimize the error five wastewater samples have been collected separately as replicate samples from each station i.e. AG, HG, RPG, MG and RG station and used for conducting the experiment. 2.2. Operating conditions Plants were acclimatized for 7 days in double distilled water and washed thoroughly with distilled water before being placed

Fig. 1. Experimental layout showing P. australis, T. latifolia and their mixed culture and reference set.

individually in each PVC pipe. Fifty litres of wastewater collected from AG, HG, RPG, MG and RG stations was poured separately in each of the experimental sets and were exposed to 11 h natural sunlight. Evaporational loss was maintained by addition of an equal amount of double distilled water at regular intervals. Since very small quantity of double distilled water was added to make up the volume, it did not show any variation in the pH on scale. 2.3. Analytical procedures From each of the four experimental sets of AG, HG, RPG, MG and RG stations, wastewater samples were collected separately on initial, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th and 14th day of treatment. The samples were analyzed for Temperature (T), pH, dissolved oxygen (DO), biochemical oxygen demand (BOD), chemical oxygen demand (COD), nitrate-N (NO3  -N) and phosphate-P (PO43  -P) following procedures as prescribed in the Standard methods (APHA et al., 2005). For the analysis of copper (Cu), cadmium (Cd), chromium (Cr), nickel (Ni), iron (Fe), lead (Pb) and zinc (Zn), 100 mL of the sample was extracted using 20 mL of extraction mixture (2:1 nitric acid: perchloric acid) until a clear solution was obtained. The extracted solution was then, filtered and diluted to 25 mL with deionized water at 4 °C, after which the metals were analyzed using a flame atomic absorption spectrophotometer (FAAS Perkin Elmer model 2380, USA). The detection limits were 1.5 mg L  1 for Cu, 0.8 mg L  1, 3 mg L  1 for Cr, 1.5 mg L  1 for Ni, 5 mg L  1 for Fe, 15 mg L  1 for Pb and 1.5 mg L  1 for Zn.

Table 1 Physicochemical properties of wastewater collected from Assi Ghat (AG), Harishchandra Ghat (HG), Rajendra Prasad Ghat (RPG), Manikarnika Ghat (MG) and Raj Ghat (RG). Variable Temperature (°C) pH Electrical conductivity (mS cm  1) Dissolved oxygen (mg L  1) Biochemical oxygen demand (mg L  1) Chemical oxygen demand (mg L  1) Nitrate nitrogen (mg L  1) Phosphate phosphorous (mg L  1) Copper (mg L  1) Cadmium (mg L  1) Chromium (mg L  1) Nickel (mg L  1) Iron (mg L  1) Lead (mg L  1) Zinc (mg L  1)

AG 25.63 7 0.26 7.687 0.01 0.2157 0.001 2.157 0.04 112.56 7 0.75 225.54 7 1.09 2.30 7 0.01 5.767 0.01 0.088 7 0.001 0.0577 0.001 0.1177 0.002 0.0657 0.001 0.1327 0.001 0.053 7 0.001 0.1097 0.001

Values are mean of five replicates7 standard error.

HG 25.53 7 0.17 7.677 0.004 0.2167 0.001 1.487 0.007 122.43 7 0.88 250.42 7 1.14 2.38 7 0.01 6.187 0.01 0.095 7 0.002 0.0627 0.001 0.1307 0.002 0.0727 0.001 0.1367 0.002 0.054 7 0.001 0.1087 0.001

RPG 26.447 0.24 7.677 0.02 0.2177 0.001 1.36 7 0.01 134.25 7 0.71 273.02 7 0.84 2.45 7 0.14 6.54 7 0.13 0.0977 0.005 0.0727 0.001 0.1387 0.005 0.080 7 0.001 0.1407 0.001 0.056 7 0.001 0.115 7 0.001

MG 25.36 7 0.23 7.677 0.01 0.2177 0.002 1.25 7 0.005 146.487 0.60 299.667 0.90 2.477 0.01 6.93 7 0.01 0.1017 0.003 0.0747 0.001 0.1417 0.001 0.086 7 0.001 0.1427 0.002 0.0577 0.001 0.1157 0.001

RG 25.197 0.02 7.65 7 0.01 0.222 7 0.002 1.077 0.008 157.54 7 0.58 233.557 0.84 2.52 7 0.02 7.65 7 0.001 0.1107 0.001 0.0797 0.001 0.1427 0.001 0.088 7 0.002 0.1457 0.002 0.0607 0.001 0.1217 0.002

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Accumulation of Cu, Cd, Cr, Ni, Fe, Pb and Zn in the whole plant body for P. australis and T. latifolia was determined and mass balance calculations were conducted using the formula: Net accumulation of heavy metals in all plants harvested from each experimental set (mg) ¼ (Ci-Cf–R) x V, where Ci ¼ average concentration of heavy metal in the wastewater before treatment, Cf ¼average concentration of heavy metal in wastewater after treatment, R¼natural precipitation of heavy metals in unplanted culture, V¼volume of wastewater used in each experimental set (50 mL).

Plants were analyzed initially and at end of the experiment, which washed properly in order to remove debris before analysis. Then, the plant parts were separated, cut into small pieces and dried to a constant mass in a fan forced oven at 78 °C for 24 h. Fine sized powder (0.5 mm diameter) was then obtained from the dried sample with the help of agate and mortar in order to facilitate the analysis of heavy metals. Then with the solution mixture of H2SO4:HNO3:H2O2 (2:3:1), the homogenized sample mixture was digested at a temperature range of 60–70 °C in a flask on hot plate until white fumes appeared, which was further heated till the appearance of a clear solution indicating complete digestion. Then, the solution was cooled and maintained up to 25 mL with double distilled water and stored at 4 °C before analysis (Kalra, 1998). The elemental state of Cu, Cd, Cr, Ni, Fe, Pb and Zn were analyzed by Atomic absorption spectrophotometer (FAAS, Perkins Elmer model 2380, USA) as prescribed by Standard methods (APHA et al., 2005). In order to check accuracy of the data, reagent blanks and standards were analyzed in triplicates. The reproducibility was within 75% in all measurements. In order to check the accuracy of atomic absorption spectrophotometer (AAS, Perkins Elmer model 2380, USA), standards of all 7 heavy metals were prepared by dilution of 1000 mg L  1 certified standard solution or corresponding metal ions with double distilled water and calibrated after every 10 samples following standard procedures (APHA et al., 2005, 311C method). Recoveries in the plant matrices were checked with standard reference materials for each metal. Chemicals and reagents were of analytical grade.

3. Results and discussion 3.1. Role of plants in the removal of heavy metals In order to determine the role selected plant species in the removal of heavy metals, P. australis and T. latifolia were grown individually and in association. A comparison of planted cultures using analysis of variance and Duncan′s multiple range test (at p¼ 0.05) revealed highest mean removal of Cu, Cd, Cr, Ni, Fe, Pb and Zn for mixed culture of P. australis and T. latifolia and lowest for T. latifolia (Table 2). Similar reports of enhancement in removal of organic pollutants and nutrient from dairy wastewater with combination of Eichhornia crassipes, Azolla pinnata and Lemna minor by Tripathi and Upadhyay (2003) support the present findings. Present findings were also supported by similar reports of Marchand et al. (2014), where P. australis and P. arundinacea removed Cu ion from the wastewater. When P. australis and T. latifolia were grown alone or in association, the mechanisms involved in heavy metal removal are metal uptake by these wetland plants, precipitation and co-precipitation as insoluble salts and metal binding to the substrate (Brix, 1994; Raineri et al., 2011). Rhizosphere of P. australis and T. latifolia provided the substrate and supporting media for the growth of microorganisms, which are the main sites of the heavy metals immobilization and uptake by plants (Sekomo et al., 2012; Jacob and Otte, 2004). The possible reason for enhancement in overall effect of metal uptake by P. australis and T. latifolia when grown association might be because the rhizosphere of these plants was increased, which would further augment the cell wall capability to absorb metals through immobilization. Another reason for the enhancement in overall heavy metal removal might be due to the excretion of phytosiderophores by these plants forming complex with free heavy metal ions. Similar findings were reported by Tsednee et al. (2012). The preferential order of removal of heavy metals was Fe4 Zn4 Cu4Ni 4Cr≅Pb 4 Cd in P. australis, Zn 4Cu≅Fe4 Ni4Cr4 Pb 4Cd in T. latifolia alone and Fe4Cu 4Zn 4 Ni4Cr4 Pb≅Cd when P. australis and T. latifolia. Similar order of

2.4. Statistical analyses Before conducting any statistical analysis, normality of the data was checked with Kolmogorov–Sminov (K–S) test. A three-way analysis of variance (ANOVA) was conducted to evaluate the influence of heavy metal concentration, retention time and plant species on the removal of heavy metals. Post hoc test, Duncan multiple range test (DMRT at alpha ¼0.05) was conducted to identify the differences among means. Relationship between retention time and heavy metal concentrations was determined by Spearman rank order correlation. Spearman rank coefficient has been conducted because the two criteria: (1) the variables should be interval or ratio and (2) monotonic relationship exists between them; are fulfilled. Retention time and concentration of heavy metals are interval variables and monotonic relationship exists between them. Percent removal of the heavy metal was calculated using the formula: PR (%) ¼(Ci-Cf) x100/Ci, where, Ci and Cf are the concentration of the heavy metal before and after treatment. The concentration of heavy metals in the wastewater was described as mg L  1, while those in plants were reported as mg kg  1 of dry weight and is mean of the five replicates. Table 2 Comparison of mean removal of heavy metals between locations and plant cultures. Heavy metal (%)

Cu Cd Cr Ni Fe Pb Zn

Region mean values

Plant cultures mean values

AG

HG

RPG

MG

RG

Phr

Ty

Phr þTy

49.57 0.3a 37.8 7 0.3a 44.5 7 0.2a 50.6 7 0.2a 51.2 7 0.2a 40.5 7 0.3a 50.4 7 0.2a

53.8 70.3b 42.13 70.3b 48.4 70.2b 54.2 70.2b 53.3 70.2b 44.0 70.3b 52.9 70.2b

56.6 70.3c 45.5 70.3c 51.4 70.2c 55.7 70.2c 55.5 70.2c 46.1 70.3c 56.0 70.2c

59.4 7 0.3d 45.8 7 0.3d 54.2 7 0.3d 59.17 0.2d 58.2 7 0.2d 50.5 7 0.3d 58.17 0.2d

62.8 7 0.3e 47.3 7 0.3d 56.5 7 0.2e 60.4 7 0.2e 61.6 7 0.2e 51.1 7 0.3d 59.6 7 0.2e

57.0 70.2b 43.3 70.3b 51.2 70.2b 55.8 70.2b 56.1 70.2b 45.7 70.2b 55.1 70.2b

51.4 7 0.2a 39.7 7 0.3a 45.6 7 0.2a 51.17 0.2a 52.6 7 0.2a 40.0 7 0.2a 52.4 7 0.2a

60.9 7 0.2c 48.17 0.3c 58.2 7 0.2c 61.0 7 0.2c 59.3 7 0.2c 53.6 7 0.2c 58.7 7 0.2c

The comparison of mean values between locations and plant cultures are according to analysis of variance and Duncan′s multiple range test. The values followed by same letter are not significantly different at the 0.05 probability level. Phr¼ P. australis, Ty¼ T. latifolia.

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removal i.e. Fe4Cr4 Cu4Cd 4Zn 4Ni using Eichhornia crassipes, Pistia stratiotes, Lemna minor and Spirodela polyrhiza was reported by Upadhyay et al. (2007). In present study, higher Ni and Cr removal from RG wastewater was noted than reported by Dhir and Srivastava (2011) using Salvinia natans from a multi-elemental solution with 56.8% of Ni and 41.4% of Cr removal (Table 2). In present study, Cd and Pb removal from RG wastewater were slightly higher as compared to their removal by algal and duckweed pond as reported by Sekomo et al. (2012), while Zn removal (70.0% ) was similar to the present study (Table 2). The possible reason for higher removal of Cd, Cr, Ni and Pb might be due to the fact that the emergent plants with extensive root system have higher metal uptake capacity than these free floating plants. Khan et al. (2009) has reported similar Fe removal 74.1% as in the present study, while removal of Cu (48.3%), Ni (40.9%) and Pb (50.0%) was considerably low with emergent and free floating plants grown in constructed wetland conditions as compared to the present study (Table 2). The possible reason for higher removal of Cu, Ni and Pb in present study might be due to the higher metal uptake capacity. These findings suggest differential uptake of heavy metals by different plant species. 3.2. Relationship between heavy metal removal and wastewater pH In order to study, the relationship between pH and removal of heavy metals through precipitation and absorption, heavy metal concentration and pH were examined during the experiment. Initially, pH of RG wastewater was slightly alkaline (Table 1) and during the experiment, pH varied from neutral to slightly alkaline (6.82 70.03–7.92 70.02). This might be due to the fact that there is addition of oxygen through dissolution of atmospheric oxygen and photosynthetic activity by the plant. In the present study, the pH varied from neutral to slightly alkaline, which revealed that the possible mechanism of metal removal was mainly due to metal immobilization in rhizosphere and absorption through their roots and partially by precipitation. Similar findings were reported by Barakat (2011), who described the removal of metals through chemical precipitation under alkaline condition (pH 9–11). 3.3. Relationship between retention time and heavy metal removal In order to view, the relationship between metal concentration and retention time, Spearman rank order correlation was determined. In the 3 planted experimental sets of AG, HG, RPG, MG and RG wastewater, significant and negative Spearman rank order correlation coefficients of Cu, Cd, Cr, Ni, Fe, Pb Zn concentrations with the retention time were noted, which confirmed the positive relationship of the retention time with Cu, Cd, Cr, Ni, Fe Pb and Zn percent removal. Significant and negative correlations of retention time with Cu concentration from AG (  0.80, 0.96 and  0.90), HG (  0.92,  0.90 and  0.93), RPG (  0.95, 0.93 and  0.95), MG (  0.92,  0.86 and  0.88) and RG (  0.83,  0.88 and  0.88) wastewater for P. australis, T. latifolia and their mixed culture were noted at p o0.05, respectively. Similarly, the decreasing trend of Cd with retention time was confirmed by their correlation coefficients in P. australis, T. latifolia individual and their mixed cultures of AG (  0.93,  0.85 and  0.89), HG (  0.88,  0.94 and 0.91), RPG ( 0.85,  0.83 and  0.87), MG (  0.92,  0.35 and  0.95) and RG (  0.85,  0.86 and  0.95) wastewater respectively, at p o0.05. Positive relationship of percent removal of Cr with retention time was confirmed by negative and significant correlation coefficients of Cr concentration with retention time when P. australis, T. latifolia when grown individually and in association in AG (  0.89,  0.89 and 0.88), HG ( 0.96,  0.89 and  0.88), RPG (  0.89,  0.91 and  0.85), MG (  0.79,  0.94 and  0.87) and RG (  0.98,  0.98 and  0.94) wastewater respectively, at p o0.05. Ni

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concentration decreased with retention time, which was revealed through negative and significant correlations with retention time for the wastewater samples from AG (  0.93, 0.93 and 0.86), HG (  0.95,  0.93 and  0.91), RPG ( 0.91,  0.97 and 0.90), MG (  0.79,  0.95 and  0.93) and RG (  0.90,  0.91 and  0.93) in P. australis and T. latifolia grown individually and in associations, respectively at p o0.05. Similarly, significant negative correlation coefficient of Fe concentrations with retention time was noted for the wastewater samples from AG (  0.89, 0.86 and  0.89), HG ( 0.83,  0.89 and  0.90), RPG (  0.97,  0.93 and  0.90), MG ( 0.92,  0.89 and 0.98) and RG (  0.86,  0.89 and  0.91) in P. australis and T. latifolia grown individually and in association respectively, at p o0.05. Significant and negative correlation of Pb concentration with retention time was confirmed in P. australis and T. latifolia individual and mixed culture of AG (  0.94,  0.94 and  0.86), HG ( 0.97,  0.94 and  0.86), RPG (  0.96,  0.95 and  0.96), MG (  0.91,  0.83 and  0.93) and RG (  0.91,  0.92 and  0.93) respectively, at po 0.05. Positive relationship of Zn percent removal with retention time was confirmed by significant and negative correlation of Zn concentration with retention time for the wastewater in P. australis and T. latifolia individual and mixed culture of AG ( 0.90,  0.85 and  0.91), HG (  0.92,  0.89 and  0.96), RPG ( 0.86, 0.92 and  0.91), MG (  0.91,  0.97 and  0.94) and RG ( 0.97,  0.87 and  0.89) respectively, at po 0.05. Similar findings were reported by Mishra et al. (2009) for the removal of heavy metals from coal mine effluent. The removal of Cu from AG, HG, RPG, MG and RG wastewater were shown in Fig. 2, which illustrated the decrease in heavy metal concentration with retention time during 14 days of exposure. Similar trends were noted for Cd, Cr, Ni, Fe, Pb and Zn. Similar findings were reported by Kumari and Tripathi (2014b) for the removal of heavy metals by P. australis and T. latifolia from secondary treated wastewater. The possible reason for enhancement in heavy metal removal might be due to continuous uptake of metals by plants. 3.4. Influence of heavy metal concentration in the wastewater on the removal of heavy metals In order to view, the influence of metal concentration on its removal, Cu, Cd, Cr, Ni, Fe, Pb and Zn removal from all five sampling stations were compared. Highest mean values of Cu, Cr, Ni, Fe and Zn were noted in RG wastewater and lowest in AG (Table 2). During present experiment, initial concentration of Cu, Cd, Cr, Ni, Fe, Pb and Zn were highest in wastewater samples collected from RG followed by MG, RPG, HG and AG (Table 1). Similar findings of increase in Pb removal with increase in initial metal concentration for Lemna perpusilla was reported by Tang et al. (2013). Similar findings were also reported by previous researchers (Abou-Elela and Hellal, 2012; Mishra et al., 2009; Sasmaz et al., 2008; Upadhyay et al., 2007). The possible reason for higher removal of heavy metals from RG wastewater might be due to the fact that the higher initial concentration of metals helped in increased rate of metal uptake by the plant species. These findings revealed that the removal of heavy metals from wastewater was dependent on the plant species, heavy metal concentration in the wastewater and retention time. 3.5. Accumulation and mass balance of heavy metals Accumulation of Cu, Cd, Cr, Ni, Fe and Pb was higher in P. australis than the T. latifolia, while T. latifolia showed higher accumulation for Zn. Mass balance calculation at end of the experiment for highly concentrated wastewater collected from RG has been shown in Table 3. Mass balance calculations revealed that the loss of Cu, Cd, Cr, Ni, Fe, Pb and Zn from the wastewater was

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Fig.2. Copper (Cu) from (a) AG; (b) HG; (c) RPG; (d) MG; and (e) RG wastewater by P. australis, T. latifolia and their mixed culture (Phr¼ P. australis; Ty¼ T. latifolia).

equivalent to their net accumulation in plant tissues and loss due to natural precipitation. Natural precipitation occurs through the oxidation of materials in presence of dissolved oxygen. It indicates the deposition of heavy metals in the form of oxidized salts on bottom of the experimental set without any influence of

experimental plants. Net accumulation is the increase in the concentration of the heavy metal over its initial concentration. The decreasing order of total net accumulation of heavy metals in all the 20 plants harvested after 14 days of exposure (mg) was noted as: Fe (70.0) 4Zn (49.52)4 Cu (8.70)4 Cr (5.61) 4 Pb (5.26) 4 Ni

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Table 3 Mass balance calculations of heavy metals in P. australis, T. latifolia and their combination (mass per 14 days). Heavy metal

Plant

Initial concentration (mg)

Concentration after treatment (mg)

Net accumulation in plant (mg)

R (mg)

Cu

Phr þTy Phr Ty

5.487 0.14 5.487 0.14 5.487 0.14

1.277 0.16 1.737 0.17 1.85 7 0.15

2.517 0.39 2.05 7 0.36 1.93 7 0.35

1.7070.28 1.7070.28 1.7070.28

Cd

Phrþ Ty Phr Ty

3.95 7 0.11 3.95 7 0.11 3.95 7 0.11

1.58 7 0.12 1.677 0.22 2.007 0.12

1.737 0.03 1.647 0.34 1.317 0.32

0.64 70.27 0.64 70.27 0.64 70.27

Cr

Phrþ Ty Phr Ty

7.117 0.07 7.117 0.07 7.117 0.07

2.26 7 0.17 2.56 7 0.07 3.08 7 0.06

3.78 7 0.16 3.487 0.08 2.96 7 0.07

1.0770.10 1.0770.10 1.0770.10

Ni

Phrþ Ty Phr Ty

4.417 0.09 4.417 0.09 4.417 0.09

1.167 0.04 1.477 0.10 1.727 0.08

2.54 7 0.08 2.23 7 0.08 1.98 7 0.07

0.71 70.07 0.71 70.07 0.71 70.06

Fe

Phrþ Ty Phr Ty

7.25 7 0.21 7.25 7 0.21 7.25 7 0.21

1.447 0.07 2.02 7 0.16 2.477 0.14

4.28 7 0.14 3.707 0.15 3.25 7 0.20

1.53 70.08 1.53 70.08 1.53 70.08

Pb

Phrþ Ty Phr Ty

3.007 0.09 3.007 0.09 3.007 0.09

0.96 7 0.07 1.107 0.10 1.40 7 0.07

1.62 7 0.08 1.487 0.16 1.187 0.10

0.42 70.07 0.42 70.07 0.42 70.07

Zn

Phr þTy Phr Ty

6.067 0.23 6.067 0.23 6.067 0.23

1.497 0.13 1.81 7 0.14 1.707 0.07

3.487 0.38 3.167 0.48 3.277 0.36

1.09 70.19 1.09 70.19 1.09 70.19

Values are mean of five replicates7 standard error. Phr¼ Phragmites australis; Ty¼Typha latifolia; Phrþ Ty¼Phragmites australis and Typha latifolia; R ¼Removal of heavy metal due to natural degradation in reference set

(3.34) 4 Cd (2.68) when P. australis and T. latifolia were grown in association; Fe (70.73) 4Zn (49.19) 4 Cu (8.18) 4 Cr (5.33)4Pb (3.73) 4 Ni (2.94) 4 Cd (2.58) in P. australis and Fe (68.41) 4Zn (49.83) 4 Cu (8.17) 4 Cr (4.80) 4 Pb (6.24) 4 Ni (2.87) 4 Cd (2.27) in T. latifolia. These findings revealed higher accumulative capacities of P. australis for Fe, Cu, Cr, Ni and Cd, and of T. latifolia for Zn and Pb. The findings also revealed enhancement in the accumulative capacities of the two plant species when grown in combinations, which might be due to the fact that uptake behavior of plants are enhanced when grown in association. Similar heavy metal accumulation i.e. Fe4Zn 4Ni 4Cu4 Mn 4Cr4Pb 4Cd and Fe4Zn 4Ni4 Mn 4Cr4Cu 4Pb 4 Cd in roots and fronds of Azolla caroliniaana (water fern) grown in fly ash effluent (Pandey, 2012) supports the present findings. Possible reason for the highest accumulation of Fe followed by Zn, Cu and Ni might due to the fact that these metals are required as essential micronutrient for plant metabolism. The toxic concentrations (in mg kg  1) for P. australis and T. latifolia as reported by Kalra (1998) was: 20 Cu, 6.5 Cd, 20 Cr, 500 Ni, 500 Fe, 20 Pb and 100 Zn, which were much less than the accumulated metal concentrations in present study (Table 3). P. australis and T. latifolia showed no visible injury during experiment due to accumulation of the heavy metals. This may be due to the fact that P. australis and T. latifolia are well known hyper-accumulators and are capable to accumulate metals copper, cadmium, chromium, nickel and lead up to 0.1% and iron and zinc up to 1% of the plant dry weight (Kalra, 1998; Sasmaz et al., 2008). In unplanted or reference set, up to 31% of heavy metals were removed from Raj Ghat wastewater. These findings might be due to the fact that the heavy metals present in suspended particulate form were adsorbed on the support system of PVC pipes or removed through precipitation. This loss in heavy metal concentrations were regarded as natural precipitation. These findings were supported by similar results of Yeh et al. (2009).

Ni, Fe, Pb and Zn within 14 days even from the most polluted RG wastewater, can be used as an alternative to conventional treatment process. Following conclusions may be drawn from the results obtained during present study: (1) Removal of heavy metals was enhanced when P. australis and T. latifolia were grown in combination than when grown alone, while, P. australis performed better than T. latifolia in monoculture. Rhizosphere of these plants provided the substrate and supporting media for the growth of microorganisms, which are the main sites of the heavy metals immobilization and uptake by plants. When grown in combination the rhizosphere of these plants was increased, which would further augment the cell wall capability to absorb metals through immobilization. (2) There was positive relationship between retention time and removal of Cu, Cd, Cr, Ni, Fe, Pb and Zn in the wastewater used for experiment, which was confirmed by negative and significant correlation coefficients of Cu, Cd, Cr, Ni, Fe, Pb and Zn concentrations with retention time. (3) Mass balance calculations revealed that the loss of heavy metal from wastewater was equivalent to net accumulation in plant harvested and heavy metal removal by natural precipitation. (4) P. australis showed higher accumulative capacities for Cu, Cd, Cr, Ni and Fe, while T. latifolia for Pb and Zn. (5) Positive relationship between initial concentration of heavy metal in the wastewater and their accumulation in the plant species revealed the accumulation of heavy metal depended on the plant species and concentration of the metal in the wastewater. (5) The mixed culture of P. australis and T. latifolia can be used for the removal of Cu, Cd, Cr, Ni, Fe, Pb and Zn from the urban sewage mixed with industrial effluents within 14 days.

4. Conclusions

Authors thank the University Grants Commission, Govt. of India (Ref. no. Bot./2009- 10/Regd.Sept.2009/14/11/09) for financial support and the Head, Department of Botany, Banaras Hindu University for providing laboratory facilities.

Present study demonstrated that the mixed culture of P. australis and T. latifolia that showed the highest removal of Cu, Cd, Cr,

Acknowledgements

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M. Kumari, B.D. Tripathi / Ecotoxicology and Environmental Safety 112 (2015) 80–86

Appendix A. Suplementary Information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2014.10. 034.

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