Environ Earth Sci (2011) 62:1235–1243 DOI 10.1007/s12665-010-0611-6
ORIGINAL ARTICLE
Heavy metals accumulation and ecophysiological effect on Typha angustifolia L. and Cyperus esculentus L. growing in distillery and tannery effluent polluted natural wetland site, Unnao, India Sangeeta Yadav • Ram Chandra
Received: 12 July 2009 / Accepted: 11 June 2010 / Published online: 6 July 2010 Ó Springer-Verlag 2010
Abstract Distillery and tanneries are major source of heavy metals pollution in natural wetland sites in India. Present study deals with the heavy metals accumulation potential of Typha angustifolia and Cyperus esculentus growing in distillery and tannery effluent polluted wetland sites. The metal accumulation pattern in both macrophytes showed direct correlation with the metal content in sediments. Both macrophytes were observed root accumulator for Fe, Cr, Pb, Cu, and Cd. The metal accumulation in T. angustifolia was found higher than C. esculentus, and accumulation pattern was Fe [ Mn [ Cr [ Zn [ Pb [ Cu [ Ni [ Cd. Simultaneously, chlorophyll, protein, cysteine, and ascorbic acid were also induced in T. angustifolia than C. esculentus. In addition, formation of multinucleolus in shoot of T. angustifolia was found an evidence of extra protein synthesis for tolerance under stress conditions. Hence, C. esculentus was observed potential but less tolerance for metals than T. angustifolia. Therefore, these wetland plants could be used for phytoremediation of heavy metals from wastewater. Keywords Cyperus esculentus Metals Phytoremediation Typha angustifolia Wastewater
S. Yadav R. Chandra (&) Environmental Microbiology Section, Indian Institute of Toxicology Research, Council of Scientific Industrial Research (CSIR), Government of India, Post Box No. 80, M. G. Marg, Lucknow 226001, UP, India e-mail:
[email protected];
[email protected]
Introduction Industrial wastes are major source of heavy metals pollution in India due to inadequate wastewater treatment system. Heavy metals are harmful to humans and animals, tending to accumulate in the food chain. Tanneries and distilleries are important source of chromium (Cr), copper (Cu), manganese (Mn), iron (Fe), nickel (Ni), cadmium (Cd), lead (Pb), and zinc (Zn) pollution in the environment (Chandra et al. 2004a, 2008). In addition, mining metallurgical activities, smelting of metal ores and fertilizers have contributed to high level of heavy metal concentrations in the environment (Alloway 1994). The threat that heavy metals poses to human and animal health is aggravated by their long-term persistence in the environment. Several technologies are available to remediate soils that are contaminated by heavy metals. However, many of these technologies are costly (e.g. excavation of contaminated material and chemical/physical treatment) or do not achieve a long-term nor aesthetic solution (Cao et al. 2002; Mulligan et al. 2001). Phytoremediation can provide a costeffective, long-lasting and aesthetic solution for remediation of contaminated sites (Ma et al. 2001). One of the strategies for phytoremediation of metal-contaminated soil is phytoextraction, i.e. through uptake and accumulation of metals into plant shoots, which can then be harvested and removed from the site. Another application of phytoremediation is phytostabilization where plants are used to minimize metal mobility in contaminated soils. However, plants may play an important role in metal removal through absorption, cation exchange, filtration, and chemical changes through root (Dunbabin and Bowner 1992; Wright and Otte 1999). There is evidence that wetland plants such as Typha latifolia and Cyperus malaccensis can accumulate heavy metals in their tissues (Deng et al. 2004; Ye et al.
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2001). Typha and Phragmites sp. have been successfully used for phytoremediation of Pb/Zn mine tailings under waterlogged conditions (Ye et al. 1997a, b). Various other wetland plants are screened from natural wetlands for heavy metals accumulation in their different parts (Cardwell et al. 2002; Demirezen and Aksoy 2004; Deng et al. 2004, 2006; Yoon et al. 2006). Further, the use of wetland plants in constructed wetland ecosystem for remediation of wastewater has recently drawn global attention for the systematic study of potential wetland plants for environmental management (Cheng et al. 2002; Klomjek and Nitisoravut 2005). It is important to use native plants of contaminated site for phytoremediation because these plants are adopted in terms of survival, growth, and reproduction under environmental stresses than those introduced from other environment. There has been a continuing interest for searching native plants that are tolerant for heavy metals. However, few studies have tested the phytoremediation potential of native plants under field conditions (Mcgrath and Zhao 2003; Shu et al. 2002). Heavy metals can cause severe phytotoxicity and may act as powerful factor for the evolution of tolerant plant populations. Therefore, it is possible to identify metal-tolerant plant species from natural vegetation in field sites that are contaminated with various heavy metals. Heavy metals (i.e. Cd, Cu, Cr, Mn, Fe, Pb, Ni, and Zn) are commonly discharged from tanneries and distilleries in India. There are about 290 distilleries and 2,500 tanneries that discharge a huge amount of wastewater with high pH, BOD, chemical oxygen demand (COD), total solid (TS), phosphate, sulfate, and phenol along with heavy metals, Fig. 1 Layout of Unnao industrial wastewater disposal drain
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which are major sources of water and soil pollution. The frequent potential growth of submerged wetland plants such as Typha angustifolia and Cyperus esculentus on industrial wastewater contaminated sites is an indication of heavy metals tolerance (Chaturvedi et al. 2006). It is also a fact that wetland plants such as T. angustifolia, and C. esculentus are resistant to stress factors in the polluted environment and have the capability to accumulate heavy metals in their tissue from contaminated wastewater (Ye et al. 2001). To date, there have been no reports on the comparative heavy metals accumulation pattern and their effect on different parts of these wetland plants. Therefore, the main objectives of the present investigation were (a) to quantify the extent of heavy metals accumulation in T. angustifolia and C. esculentus growing in the contaminated site and to examine relations between metals uptake and metals content of sediment and water, (b) to compare accumulation ratio between T. angustifolia and C. esculentus and distribution of different metals in different parts of plants, and (c) to determine the extent of movement of metals into different tissue and their ecophysiological effects.
Materials and methods Sample collection A cluster of tanneries and one major distillery are located in adjacent part of Unnao city, UP, India (26°320 000 N, 80°300 000 E). Most of the tanneries and distillery discharge
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their partially treated and untreated effluents into a city drain, which outflows into a low lying sink to create a big waterlogged area as natural wetland and damages the surrounding natural flora and fauna. Two native common wetland plants, i.e. T. angustifolia L. and C. esculentus were only able to grow luxuriantly in the waterlogged natural wetland area of the industrial drain. Sampling site is shown in Fig. 1. The upstream flora in wetland area shows dominant growth of T. angustifolia L. while the downstream flora shows luxuriant growth of C. esculentus. This ultimately joins to the Ganga River, the most important river of the country. To determine the concentration of metals in these two potential hyperaccumulant plants, at least ten complete plants of each species were collected randomly from contaminated site and transported to the laboratory in polyethylene bags. Simultaneously, the contiguous soil was separately taken from the depth of 0–20 cm (root growing zone) in a plastic bag and plastic spatula, avoiding any contact with metals. Contaminated water samples were also collected in sterile Jerrycane from contaminated site to determine the heavy metals concentrations. Physicochemical analysis of the effluent The BOD was measured using 5-day BOD test. The COD by open reflux method, total dissolve solids, sulfates, total nitrogen, and phenol were determined by standard methods as described in APHA (2005). The color of wastewater was measured by visual comparison method no. 2120 B (APHA 2005). The pH and a variety of ions were measured using calibrated selective ion electrodes of Cl, K, and Na as per manufacturer’s instruction (Orion autoanalyser model-960, USA). Metal analysis The wetland plants were gently uprooted and washed thoroughly with deionized water to remove sand clinging to the roots and followed by rinsing with a 10 mmol/l CaCl2 solution for surface sterilization of the tissues. The plants were separated into root, shoot, and leaves subsequently oven-dried at 80°C for 7 days to a constant weight. Further, all samples were ground to a \40 BSS mesh in a Wiley mill for homogeneity of the samples. Thereafter, 5 g dried plant tissues were then ashed in the muffle furnace for 6 h at 460°C. Ash was dissolved in 10 ml of 2% HNO3, the solution filtered through 0.45 l glass fiber filter and diluted with deionized water to 15 ml (AOAC 2002). Simultaneously, 1 g sediment was air dried for 2 weeks, and then sieved through 2 mm mesh. Samples were then digested with 10 ml of HNO3. To complete the organic matter oxidation, acid digestion was continued during 24 h by
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adding 0.5 ml of H2O2 (EPA 1996) and the concentrations of Cd, Cu, Cr, Fe, Mn, Ni, Pb, and Zn were measured using an inductively coupled plasma-atomic emission spectrophotometer (ICP-AES) (IRIS Interepid II XDL: Thermo Electron, Waltham, MA, USA). The heavy metals in the effluent were also measured by means of acid digestion, following the standard method for the examination of water and wastewater (APHA 2005). Plant’s accumulation of metals from sediments was measured by ‘‘Bioconcentration factor’’ (BCF), the ratio of metal concentration in the root to sediments. ‘‘Translocation factor’’ (TF) was estimated as the ratio of metal concentration in the shoot to the root. Biochemical parameters analysis The chlorophyll content in leaves of T. angustifolia and C. esculentus was determined by spectrophotometer after extraction in 80% chilled acetone as per Arnon’s (1949) Method. Protein content was analyzed according to Lowry method (Lowry et al. 1951). Lipid peroxidation in the plant tissue was also determined in terms of malondialdehyde (MDA) content, determined by thiobarbituric acid (TBA) reaction (Heath and Packer 1968). Cysteine content was measured by the method of Gaitonde (1967) using Ellman’s reagent (50 ,50 -dithiobis-2-nitrobenzoic acid) by mixing the 0.1 M sodium phosphate buffer (pH 8.0) and samples. Finally, the absorbance was measured at 412 nm (UV 2300 Spectrophotometer, Techcomp, South Korea) after 15 min incubation at room temperature. Ascorbic acid content was estimated as per method of Keller and Scgwager (1977) by taking the supernatant in 4% oxalic acid solution. Anatomical microscopic study Shoot and root samples of 5 mm length were excised from 2 cm above and 2 cm below the shoot–root intersection. The shoot and root samples were prepared for transmission electron microscopy (TEM). The samples were excised and quickly immersed in H2S saturated water as pretreatment for 30 min at room temperature to precipitate Cd and Zn (Khan et al. 1984). TEM Approximately 3-mm long root and shoot segments were used for TEM. The samples were fixed in modified Karnovsky’s fluid buffered with 0.1 M sodium phosphate buffer at pH 7.4 (David et al. 1973). Fixation was done at 4°C temperature for 10–18 h after which the tissues were washed in fresh buffer and post-fixed for 2 h in 1% osmium tetroxide in the same phosphate buffer. The root and shoot tissues were dehydrated in graded acetone
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Table 1 Physicochemical characteristics of distillery and tanneries wastewater before mixing in natural wetland system
Table 3 Accumulation and translocation of heavy metals in T. angustifolia and C. esculentus
Parameters
Values
Metals Bioconcentration factor (BCF)
Color appearance
Dark brown
Discharge permissible limit
T. angustifolia C. esculentus T. angustifolia C. esculentus
_
Color intensity
25,000.00 ± 300
pH BOD
7.30 ± 0.29 1,860.00 ± 43.00
COD
15,900.00 ± 138.00
TS
4,404.98 ± 60.25
TDS
2,233.89 ± 59.33
TSS
1,171.19 ± 18.56
Sulfide
0.72 ± 0.016
Sulfate
2,610 ± 46.10
_
Cd
1.57
1.40
0.27
0.33
_ 40.00
Cr
1.29
0.59
0.21
0.94
Ni
1.33
1.58
0.37
1.05
120.00
Pb
0.81
1.22
0.45
0.31
_
Cu
1.18
1.13
0.53
0.94
_
Fe
1.42
1.10
0.20
0.51
Mn
1.08
1.07
0.29
0.70
Zn
1.17
1.15
1.18
0.50
45.00 0.002 750.00
Phenol
380.00 ± 9.40
0.50
Total nitrogen
275.66 ± 8.03
25.00
Chloride
675.48 ± 14.53
750.00
Magnesium
67.50 ± 1.02
transmission electron microscope (Phillips, M-10) operated at 60–80 kV transmission.
0.20
Potassium
300.00 ± 15.00
_
Sodium
368.00 ± 12.45
200.00
Statistical analysis
Metals Cd Cr
Translocation factor (TF)
0.96 ± 0.01 8.96 ± 0.02
All the data are mean (n = 10). Differentiation between plant potentialities with respect to the mean concentrations of heavy metals in root, shoot and leaves were evaluated using a one-way analysis of variation test (ANOVA). Tukey’s test (Ott 1984) for ANOVA analysis was performed using the Graph Pad software (GraphPad Software, San Diego, CA).
0.01 0.05
Ni
1.79 ± 0.00
0.10
Pb
2.68 ± 0.03
0.05
Cu
2.61 ± 0.00
0.50
Fe
9.36 ± 0.02
2.00
Mn
3.35 ± 0.04
0.20
Zn
2.79 ± 0.04
2.00
All values are mean (n = 10) ± SD in mg/l except color intensity (Co–Pt unit)
solution and embedded in CY 212 araldite. Ultrathin sections of tissue of 60–80 nm thickness were cut using ultra E (Reichert Jung). Ultra sections, obtained using an ultramicrotome were stained with uranyl acetate and lead citrate for 10 min each before examining the grid in a
Results and discussion Physicochemical analysis and metals content in sediments and wastewater The physicochemical characteristics of distillery and tannery wastewater prior to mixing at the wetland plant growing site are shown in Table 1. These values are far
Table 2 Metal contents in wastewater and sediments of natural wetland system Heavy Metals
T. angustifolia growing site Sediment (mg/kg)
Cd Cr Ni
2.387 ± 0.008 177.027 ± 3.30 14.907 ± 0.130
C. esculentus growing site
Water (mg/l)
Sediment/Water
Sediment (mg/kg)
Water (mg/l)
0.011 ± 0.000
217.00
1.058 ± 0.040
0.402 ± 0.016
440.37
96.165 ± 2.560
1.347 ± 0.040
71.39
0.296 ± 0.001
50.36
9.055 ± 0.530
0.194 ± 0.001
46.68
0.021 ± 0.000
Sediment/water 50.38
Pb
29.867 ± 0.586
0.369 ± 0.001
80.94
12.000 ± 0.360
0.790 ± 0.040
15.19
Cu
23.920 ± 0.500
0.192 ± 0.003
124.58
13.465 ± 0.350
0.287 ± 0.000
46.92
Fe
1,872.00 ± 44.981
9.880 ± 0.130
189.47
Mn Zn
193.493 ± 3.060 51.420 ± 0.972
1.958 ± 0.013 0.389 ± 0.010
98.82 132.19
All values are mean (n = 10) ± SD
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1,423.950 ± 20.990 97.006 ± 4.520 35.950 ± 0.850
60.940 ± 1.160
23.37
1.220 ± 0.023 0.547 ± 0.030
79.51 65.72
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4
Cd
Metal (mg/kg)
3 2.5 2 1.5
a
Cu
30
3.5
Metal (mg/kg)
Fig. 2 Heavy metals (mg/kg) content in different parts of T. angustifolia and C. esculentus. All the values are mean of ten replicates ± SD. Letters indicate that they were significantly different at a probability level of 0.05 according to ANOVA test. a P \ 0.05, nsP [ 0.05
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25 20
a
ns
15
a
10 1
a
0.5
5
a
0
0 250
3000
Fe
2500
200
Metal (mg/kg)
Metal (mg/kg)
Cr
150 100
a
ns
50
2000
a
a
500
0
0
500
400
Metal (mg/kg)
a
a
15 10
a
5
Mn
450
Ni
Metal (mg/kg)
a
1000
25 20
a
1500
a
350 300 250 200 150
a
a
100 50
0
0
40
80
Pb
Zn 70
30
60
25 20
a
15 10
a
a
5
Metal (mg/kg)
Metal (mg/kg)
35
50
a
40
a
30
a
20 10
0
0
Root
Shoot
Typha angustifolia
greater than the permissible limit of industrial effluent discharges (EPA 2002). The effluent showed high content of Fe, Cr, and Mn. The iron is released from distillery and chromium from tanneries discharges. Similar reports on physicochemical characteristics and metal contents of tanneries and distillery were previously reported by Chandra et al. (2004a, b, 2008). Luxuriant growth of T. angustifolia and C. esculentus in highly polluted
Leaves
Cyperus esculentus
Root Typha angustifolia
Shoot
Leaves
Cyperus esculentus
conditions shows strong evidence for its tolerance against high pollution load. Earlier reports also showed heavy metals tolerance behavior of T. latifolia, T. angustata L., T. domingensis, and C. corymbosus (Klomjek and Nitisoravut 2005; Ye et al. 2001). The metals content in sediment was noted higher at inlet site of the wetland where T. angustifolia was growing as compared outlet site (downstream) where C. esculentus was growing.
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Environ Earth Sci (2011) 62:1235–1243
b
MDA (µmole/g) & Protein (mg/kg)
a
Typha angustifolia
40
Cyperus esculentus
35
a
30 25
ns
Metal accumulation by wetland plants
20 15
ns
a
10 5 0
MDA
Protein
MDA
Cystein (nmole/g) & Ascorbic acid (mg/kg)
Root
c
900
Protein Leaves
Typha angustifolia
Cyperus esculentus
800 700 600
a
500
a
400 300 200
a
a
100 0
Root
Leaves Cysteine
Root
Leaves
Ascorbic Acid
Fig. 3 The chlorophyll (Chl, a); malondialdehyde and protein (MDA, b); cysteine and ascorbic acid content (c) in T. angustifolia and C. esculentus grown in distillery and tannery effluent-contaminated site. All the values are mean of ten replicates ± SD. Letters indicate that they were significantly different at a probability level of 0.05 according to ANOVA test. aP \ 0.05, nsP [ 0.05
T. angustifolia was growing in the highest polluted area of the wetland, which indicated that T. angustifolia is more tolerant to the polluting metals. However, C. esculentus was growing at downstream of natural wetland system where metals content in sediment was comparatively low (Table 2). The ratio between metal content of
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sediment:metal content of water at T. angustifolia and C. esculentus growing site for different metals were: Cd 217.00, 50.38; Cr 440.37, 71.39; Ni 50.36, 46.68; Pb 80.94, 15.19; Cu 24.58, 46.92; Fe 189.47, 23.37; Mn 98.82, 79.51; Zn 132.19, 65.72 (Table 2). This revealed that sediment contains more metals than wastewater. This might be due to adsorption of metals on the sediments because of continuous addition of distillery and tanneries wastewater to the site. The ability to accumulate metals from sediment to root was also measured as BCF. The BCF was noted ‘[1’ in T. angustifolia and C. esculentus except Pb and Cr, respectively, as shown in Table 3. However, the translocation factor was observed ‘\1’ except Zn and Cr. This revealed that most of metals were absorbed from sediment to root. Further, the translocation of metals from root to shoot was restricted due to protective mechanism of plant.
The accumulation of metals in the T. angustifolia and C. esculentus growing in tannery and distillery effluent polluted sites is shown in Fig. 2. Findings revealed that T. angustifolia and C. esculentus are root accumulators for Cd, Cu, Cr, Fe, and Pb because these metals were found significantly higher in root than shoot or leaves. The order of metal accumulation in root of both plants were Fe [ Cr [ Pb [ Cu [ Cd. This might be due to high plant availability of the substrate metals as well as its limited mobility once taken up by the plants. Besides, higher metal accumulation in root of both wetland plants might be due to strong root development and complexation of metals with sulphydryl group (–SH) of soil constituent resulted into less translocation of metals to upper parts of plants (Sinha and Gupta 2005). It is also possible that metal accumulation in root might be related with co-precipitation in the iron oxyhydrate plaque layers on the root surface of wetland plants (Cardwell et al. 2002; Deng et al. 2004; Ye et al. 1998). Figure 2 shows T. angustifolia is a shoot accumulator for Zn and C. esculentus have accumulated Ni maximum in shoot. T. angustifolia accumulated 70 mg/kg Zn while C. esculentus accumulated Ni 14 mg/kg in their shoots. The higher metal accumulation in T. angustifolia and C. esculentus indicated that internal metal detoxification tolerance mechanisms might exist in these wetland plants (Taylor and Crowder 1983; Demirezen and Aksoy 2004). The accumulation patterns of metals (sum of metals of all parts of wetland plants) in T. angustifolia and C. esculentus have been studied which are as follows: T. angustifolia: Fe [ Mn [ Cr [ Zn [ Pb [ Cu [ Ni [ Cd
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Fig. 4 TEM micrographs of root and shoot of T. angustifolia grown on uncontaminated (a, c) and contaminated site (b, d). Arrow shows multinucleolus formation
Fig. 5 TEM micrographs of C. esculentus root grown on uncontaminated (a) and contaminated site (b). Arrow shows thinning of cell wall
C. esculentus: Fe [ Mn [ Cr [ Zn [ Cu [ Ni [ Pb [ Cd Metal accumulation by T. angustifolia was found higher than C. esculentus as shown in Fig. 2. These observations established that these plants are potential phytoremediators for heavy metals from the distillery and tannery wastewater. However, T. angustifolia showed more tolerance to heavy metals stresses and higher potential to accumulate heavy metals than C. esculentus. This might be due to welldeveloped root–shoot system in T. angustifolia.
Biochemical parameters The total chlorophyll, chlorophyll-a, and chlorophyll-b contents were observed higher in T. angustifolia as compared to C. esculentus (Fig. 3a). T. angustifolia grown in distillery and tannery effluent-contaminated site has shown significant increase (P \ 0.05) in total chlorophyll (28.89%), chlorophyll-a (59.69%), and chlorophyll-b (76.92%) contents as compared to C. esculentus. Metal accumulation pattern for leaves of T. angustifolia and
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C. esculentus was observed as Fe [ Mn [ Cr [ Zn [ Pb [ Cu [ Ni [ Cd. However, the leaves did not show any visual toxicity symptoms. This might be because of the tolerance mechanism of wetland plant for heavy metals. Metals might be essential for the synthesis of chlorophyll directly or indirectly (Chandra et al. 2004a). The necessity of metals for the chlorophyll synthesis up to certain level has also been reported in tomato plant by Singh et al. (2004). However, the higher induction of MDA in C. esculentus than T. angustifolia (Fig. 3b) revealed the lipid peroxidation of cell membrane in leaves and root. Simultaneously, this also indicates the sensitivity of plants. Further, the higher induction of cysteine, ascorbic acid, and protein in T. angustifolia (Fig. 3c) supported an evidence of anti-stress capability of T. angustifolia as reported by Singh et al. (2004). Anatomical observation Anatomical observation through TEM in the root of T. angustifolia did not show any remarkable changes even after higher accumulation of various metals in the roots (Fig. 4a, b). This might be due to tolerance mechanism of plants with higher content of heavy metals for adaptation in adverse conditions. Naturally, all waterlogged soils have higher amounts of plant available Fe, Mn, and Cr due to redox potential. Thus, plants growing in the wetlands uptake more Fe, Mn, and Cr. However, the formation of multinucleolus in the shoot cells revealed the formation of extra protein for the plants protection (Fig. 4c, d). The higher metal accumulation and phytoremediation capability of T. angustifolia was previously reported (Deng et al. 2004). However, the histological view of root tissues of C. esculentus showed thin cell wall as compared to root tissues of C. esculentus collected from uncontaminated site, due to disturbance in lignifications (Fig. 5a, b). This supported the sensitivity of C. esculentus root with heavy metal accumulation. This might also be due to higher accumulation of Fe, Mn along with other metals, and pollutants in natural conditions for long exposure. Similar effects of Cd on lignifications have also been reported by Vitoria et al. (2006). Metals in the tissue can be trapped by negative charges of cell-wall, resulted the metal accumulation in the apoplast (Carrier et al. 2003). The study concluded that T. angustifolia had higher potential for heavy metals accumulation than C. esculentus from distillery and tannery wastewater. Hence, these two plants could be used for the phytoremediation of the heavy metals contaminated swampy lands and wastewater. Acknowledgments We are grateful to Department of Biotechnology (DBT) and NWP-19, Council of Scientific and Industrial Research (CSIR), Government of India for financial assistance. The
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Environ Earth Sci (2011) 62:1235–1243 Transmission Electron Microscopy (TEM) was done at All India Institute of Medical Sciences (AIIMS), New Delhi, India.
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