生物修复与超量富集04

d h y 3 Ipomoea carnea was more effective in extracting cadmium than was Brassica juncea. 1 Abstract Phytoextraction has shown great potential as an alternative technique for the remediation of metal contaminated soils. The objective of this study was to investigate cadmium (Cd) phytoextraction ability of high biomass producing weeds in comparison to indicator plant species. The pot study conducted with 10 to 200 mg Cd kg�1 soil indicated that Ipomoea carnea was more effective in removing Cd from soil than Brassica juncea. Among the five species, B. juncea accumulated maximum Cd, but I. carnea followed by Dhatura innoxia and Phragmytes karka were the most suitable species for phytoextraction of cadmium from soil, if the whole plant or above ground biomass is harvested. In the relatively short time of this experiment, I. carnea produced more than 5 times more biomass in comparison to B. juncea. There were significant differences ( p! 0.05) between the shoot length and shoot mass of control and treated plants. � 2004 Published by Elsevier Ltd. Keywords: Phytoextraction; Cadmium; Bioconcentration factor; High biomass weeds; Transportation index 1. Introduction Cadmium (Cd) is a heavy metal naturally present in soil; it is non-essential and highly toxic to most organisms, having toxicity 2 to 20 times higher than many other heavy metals (Vassilev et al., 1998). Cadmium is the fourth most toxic metal to vascular plants (Jones, 1993; Oberlunder and Roth, 1978). It is placed in seventh position in the top ten priority hazardous substances list as provided by the American Agency for Toxic Substance and Disease Registry (Kamnev and Lelie, 2000), and therefore is considered a very serious pollutant. Total Cd levels exceeding 8 mg kg�1, or soluble (bioavailable) levels exceeding 0.001 mg kg�1, are considered toxic to plants (Kabata- Pendius and Pendius, 1992; Bohn et al., 1985). The primary risk pathway associated with Cd contaminated soils has been identified as the soil–plant–human pathway and the consumption of the crop or byproducts grown on these soils leads to its biomagnification in the food chain (Page et al., 1982). Cadmium content in soil has dramatically increased from anthropogenic sources including smelters, agricultural applications of fertilizer, and sewage sludge. Since Cd in soil is available for plant A comparative study of ca accumulator an Moyukh Ghos Biomass and Waste Management Laboratory, School of Energ Devi Ahilya University, In Received 12 April 200 Environmental Pollution * Corresponding author. Tel.: C91 731 2460309; fax: C91 731 2467378. E-mail address: sp_singh@excite.com (S.P. Singh). 0269-7491/$ - see front matter � 2004 Published by Elsevier Ltd. doi:10.1016/j.envpol.2004.05.015 mium phytoextraction by d weed species , S.P. Singh* and Environmental Studies, Faculty of Engineering Sciences, dore 452017, M.P., India ; accepted 21 May 2004 33 (2005) 365–371 www.elsevier.com/locate/envpol uptake and subsequent human uptake, Cd in the environment poses a significant health risk. Plants can extract Cd from the soil and transport it via the xylem into shoots and leaves where it accumulates. n Cadmium absorption occurs because of the chemical similarity to zinc (Blaylock et al., 1997). It has been shown that pollutants can be removed from a contami- nated site by harvesting the plant biomass containing the pollutant; this is referred as phytoextraction (Chaney, 1983). The two major factors that determine the total amount of metal extracted by plants are: (a) the concentration of the pollutants in dry biomass; and (b) the total biomass produced by the plant. Plants used for phytoextraction should be fast growing, deep rooted, easily propagated and accumulate the target metal. Ideally the species should have a high bioconcentration factor (BCF), which is defined as the plant/soil metal concentration. As per their ability to absorb, accumulate, and tolerate metal within their tissues, plants exhibit three major responses and can be classified into three categories: hyperaccumulators, indicators and excluders (Wagner and Yeargan, 1986; Alloway, 1995). Plants with extreme levels of metal tolerance are called as hyperaccumulators. In 1983, Chaney reported that several hyperaccumulator species have a high BCF, but due to very low biomass the amount of phytoextraction is less; additionally, harvest- ing of these species commercially is difficult as per the present agronomic practices (Chaney et al., 1995). Indi- cator plants such as Brassica juncea (Indian mustard) (Wagner and Yeargan, 1986; Alloway, 1995), in com- parison to hyperaccumulators, have a lower metal bioaccumulation but have at least 10 times the biomass production, so that the actual amount of extraction is higher. Indicators regulate metal uptake so that the internal concentration reflects the external levels. Excluders maintain low and constant metal concen- tration in their shoots. High biomass species like willow and poplar fall under this category and accumulate small amount of metal (9–167 mg g�1 by willow; 6–75 mg g�1 by poplar) per unit of dry weight. Excluders produce more biomass, up to 30 tones per hectare (Robinson et al., 2000). However, a very long time is needed for re- mediation by excluder plants; a period of 12 years was calculated for removal of 0.6 mg kg�1 of cadmium, based on realistic willow tree biomass production rates and experimentally-determined cadmium uptake rates (Greger and Landberg, 1999). The aim of this study was to investigate the phytoextractive potential of high biomass producing, commonly found weed species that can grow in both dry land and marshy conditions. An efficient method for testing the potential of commonly found weed species would be to grow an accumulator plant, such as B. juncea in comparison to hardy species like Ipomoea carnea, Phragmytes karka, Dhatura innoxia, Cassia tora, and Lantana camara. Use of edible species increases the risk of heavy metals being introduced into the food chain. Species like B. juncea are accumulators and are edible; to 366 M. Ghosh, S.P. Singh / Environme restrict the passage of heavy metals into the food chain, non-edible species were chosen and the phytoextraction was compared with respect to total biomass and Cd uptake. I. carnea, P. karka, D. innoxia, C. tora and L. camara were chosen, but only the results of the first three were reported. Although they may have a lower accumulation capacity, their potential of high biomass and ability to grow in deficient conditions makes them ideal candidates for this study. 2. Material and methods 2.1. Experimental set up Pot culture experiments were conducted using soil treated (spiked) with cadmium nitrate [Cd(NO3)2] solution and for comparison, an unamended control. The solution was uniformly mixed with air-dried soil sieved to!2 mm and placed in pots (8 kg); the final soil concentration of Cd was 10, 20, 50, 100 and 200 mg kg�1 ( ppm), respectively. The soil used was Typic Chromus- terts with Semectite as the dominant mineral (Ramesh et al., 1998). The soil pH was 8G 0.2, organic carbon 5.5G 0.3 g kg�1, CaCO3 70G 6 g kg �1, and clay 590 g kg�1. Soil pH was measured in double distilled water using a solid to liquid ratio 1:2.5, after 2.5 h of equilibrium. Seeds or propagules were procured from the naturally growing plants. Twenty seeds/propagules were sown in the soil to germinate, but only six uniform plants were allowed to grow in each pot, at a uniform distance apart. Six replicates of each treatment were used, and the pots were placed in a net house shaded with transparent polythene sheet to protect from rainwater leaching. Plants were grown under natural light and ambient temperature, in order to keep all plants under conditions as similar as possible. No artificial fertilizers or soil amendments were added to the soil during the course of the experiment. 2.2. Plant growth and harvesting Plants were harvested after 15, 30, 60 and 90 days without damaging the roots. They were rinsed in distilled water to remove dust and soil mineral particles and were separated into leaves, stems and roots. Shoot and root length and dry biomass (oven dried at 85 �C for 36 h) of the different plant parts were measured. 2.3. Analysis of plant mass Dried samples were homogenized using a Wiley mill before analysis. Cadmium analysis was performed after digesting the samples in an acidic mixture of nitric and perchloric acid (HNO3:HClO4; APHA method, 1992) with an atomic absorption spectrophotometer (GVC- tal Pollution 133 (2005) 365–371 902). Cadmium uptake, depicted by a bioconcentration Table 1 20 18.57G 1.3** 16.13G 1.2** * * 50 12.70G 1.1** 12.05G 1.1* 100 12.17G 0.7** 10.73G 0.6* 200 NG NG Values are meanG SD of six replicates. Significantly different *(p! 0.05) and **(p! 0.005) to control plant. NG= no growth. 65.83G 5.88* 84.75G 5.75** 90.67G 7.01* 55.33G 7.76** 81.13G 7.45** 75.83G 4.26** 53.50G 6.32** 83.13G 5.69** 56.83G 6.05** NG NG NG Effect of cadmium on shoot length (cm) after 90 days of growth Cd conc. in soil (mg kg�1) Brassica campestris Brassica juncea Dhatura innoxia Ipomoea carnea Phragmytes karka Control 24.5G 2.3 22.2G 2.1 73.01G 6.39 108.13G 5.44 99.83G 6.7 10 20.01G 1.8* 20.92G 1.6* 67.83G 6.65 86.38G 7.93** 88.67G 8.96* The transportation index (Ti) gives the leaf/root cadmium concentration and depicts the ability of the plant to translocate the metal species from roots to leaves at different concentrations Ti ¼ Cadmium in leaves ðmg kg �1Þ Cadmium in roots ðmg kg�1Þ!100 2.4. Data analysis Statistical comparison of means was done by Student’s t-test and an analysis of variance (ANOVA) test. Differences were considered to be significant at p% 0.05 and highly significant at p% 0.005, level of significance. 3. Results and discussion Indicating the toxic level, the selected plants failed to grow at 200 mg Cd kg�1 soil, but they are capable of extraction up to or little above 100 mg Cd kg�1 soil. With an increase in metal concentration, the lower leaves of D. innoxia showed necrosis (death and disintegration of cells) and the upper leaves showed epinasty followed by wilting. Ernest et al. (1992) have proposed that leaf fall is a metal detoxification mechanism. Cadmium treatment of Dhatura resulted in an early completion of the plant’s life cycle; reproductive growth was enhanced as it showed flowering (20 days), which was earlier than the control plants. Cadmium-treated P. karka and I. carnea did not show any deformation or deficiency. Our results and B. juncea showed little shoot length reduction with increase in Cd. 3.1. Evaluation of plant growth The final biomass decreased with increase in soil Cd, after 90 days of growth in comparison to control plants (Table 2). Stem biomass, which is useful for energy generation, was maximum in I. carnea (w60% of the total mass) followed by P. karkaOD. innoxiaO B. junceaO B. campestris. In Dhatura, the percentage of stem biomass decreased and relative foliar biomass increased with increase in soil Cd, showing that Cd increase did not inhibit foliage biomass. In accumulator species such as B. juncea, foliage biomass constituted 60–70%, and in B. campestris it was 74–80% of the total biomass, respectively. Cadmium suppresses transpira- tion (Leita et al., 1993), with the resistance in water flow attributed to hydroactive stomatal closure, accompanied by a decrease in stomatal conductance (Marchiol et al., 1996). This was observed in the form of wilting in Dhatura and Brassica species, which presumably affects metabolism and reduces photosynthesis. As Cd is transported with sap flow, a greater content in the leaves is observed (Marchiol et al., 1996). The higher Cd content in leaves of D. innoxia, I. carnea and P. karka, corroborates these observations. Cadmium uptake by plants is bound or complexed in the conducting system where cell walls and other apoplastic structures act as filters, limiting movement in the symplast and aerial parts (Marchiol et al., 1996). The root systems of the treated plant species, especially factor (BCF), provides an index of the ability of the plant to accumulate a particular metal with respect to its concentration in the soil substrate (Zayed et al., 1998). It is calculated as follows: in Table 1 show that there was a statistically significant ( p! 0.05) reduction in shoot lengths of all the species tested; shoot length reduction was highest in shoots ofD. innoxiaO I. carnea and P. karka; whereas B. campestris BCF¼ Metal concentration in plant tissue at harvest ðmg kg �1Þ Initial concentration of the metal added in substrate ðmg kg�1Þ 367M. Ghosh, S.P. Singh / Environmental Pollution 133 (2005) 365–371 e B j 2 2 3 3 4 u the wilting of leaves. There was a 55–65% reduction in root biomass of the treated plants, and Cd accumulation in the roots exceeded that in the upper parts, similar to the results of Breckle et al. (1989). 3.2. Plant metal uptake Studies on Cd speciation in soil solution (Bingham et al., 1986) show that cadmium mainly occurs as the free metal ion Cd2C, and that ion exchange mechanisms have a dominating influence on metal concentration in soil solution when other cations increase in concen- tration (Lorenz, 1994). Cadmium bioavailability depends on soil pH, redox potential, and rhizosphere chemistry. The accumulation and extraction of Cd by plants, calculated on a dry weight basis, is reported only for the maturation level (90 days). Comparative Cd concen- tration in the leaves, stems and roots of the studied plants are presented in Table 3. The highest Cd concentration was observed in the roots of B. juncea (81.9 mg Cd kg�1), Table 3 Comparison of the cadmium accumulation in leaves, stems and roots aft Total Cd conc. in soil (mg kg�1) Soil pH Plant part Brassica campestris 10 8.2 Leaf 9.20G 1.0 bc Stem 10.4G 1.2 b Root 33G 2.5 a 20 8.0 Leaf 19.2G 0.8 c Stem 23.8G 0.9 b Root 42.8G 1.8 a 50 8.0 Leaf 30.4G 1.0 b Stem 27.0G 1.2 c Root 55G 2.0 a 100 7.8 Leaf 34G 1.1 b Stem 34.4G 0.9 b Root 64.5G 3.1 a Units in mg Cd kg�1G standard deviation associated with the mean val significantly different (p= 0.05). 3.3. Cadmium extraction Because of their high biomass production I. carnea, P. karka and D. innoxia have excellent potential for phytoextraction. Maximum extraction was shown by I. carnea (401 mg plant�1) followed by D. innoxia (201 mg plant�1), although their BCF was lower than accumulator species. Fig. 1 shows the extraction by the different plant organs and by the whole plant after 90 days. The phytoextraction ratio of B. campestris, B. juncea, D. innoxia, I. carnea and P. karka was 1:1.1:2.6:5.2:2.5 at 100 mg Cd kg�1 soil. In both indicator species, B. juncea and B. campestris, 60–70% of Cd extraction was observed in the leaves. In I. carnea, D. innoxia and P. karka, stem biomass contributed to 24–70% of the Cd extraction, with the stem consisting of 44–64% of the total biomass throughout the treatments. The total extraction by roots at 100 mg Cd kg�1 soil showed a sharp drop in these species. r 90 days rassica uncea Dhatura innoxia Ipomoea carnea Phragmytes karka 12.5G 0.2 b 13.5G 0.4 a 7.4G 0.1 b 12.5G 0.2 b 13.2G 0.8 b 7.5G 0.3 c 5.2G 0.1 c 5.1G 0.3 c 42G 2.1 a 11.5G 0.1 b 28G 4.4 a 23G 2.3 a 2.47G 1.1 c 17.5G 0.4 a 10.4G 0.2 b 16.5G 0.3 b 7.83G 0.6 b 10G 0.2 c 9.6G 0.1 c 8.6G 0.5 c 49.6G 3.4 a 11.5G 0.1 b 37G 1.5 a 32G 4.5 a 5.57G 2.2 b 24.75G 1.7 c 15.3G 0.7 c 24.6G 1.6 b 4.30G 1.9 b 12G 0.6 b 23G 1.2 b 15.6G 0.3 c 69.6G 2.0 a 38.5G 1.9 a 45G 3.8 a 41G 3.0 a 43.2G 0.7 c 28.5G 1.8 bc 20.1G 2.0 c 39.5G 2.8 b 9.78G 1.5 b 29.85G 2.4 b 37.8G 1.4 b 23.1G 1.4 c 81.9G 4.4 c 37G 1.7 a 51G 5.2 a 53G 5.0 a e (n= 3). Values among the plant parts having the same letter are not B. campestris, B. juncea and D. innoxia were visibly damaged at 100 mg Cd kg�1 soil. Furthermore, their capacity to conduct water was impaired as indicated by which was 1.89 times and 1.6 times more than the concentration in leaves and stems, respectively. The highest concentration of Cd in both leaves and stems was observed in B. juncea at 20, 50 and 100 mg Cd kg�1 soil. In Brassica, minimum leaf uptake was observed between 31 and 60 days, a dilution whichmay be due to additional biomass and lower translocation. However, at later stages the accumulation increased indicating that Cd was translocated to leaves until the end of the growth period. Therefore, leaf Cd concentration was more than that of the stem. Similar phenomenon was observed in P. karka, whereas inD. innoxia and I. carnea the increase in substrate Cd resulted in a decrease in foliar Cd with respect to time, and most of the accumulation took place in stems. At the latter stages of growth, translocation was reduced (61 to 90 days) and most of the metal accumulated in roots. Table 2 Dry biomass (g plant�1) (average of 6 replicates) grown in soils with different cadmium concentrations for 90 days Total soil Cd conc. (mg kg�1) Brassica campestris Brassica juncea Dhatura innoxia Ipomoea carnea Phragmytes karka Control 3.28 3.31 12.32 19.59 11.46 10 3.01* 2.92* 9.18** 17.24** 9.52** 20 2.57** 2.13** 7.77** 16.04** 10.95* 50 2.70** 2.05** 7.95** 11.14** 7.93** 100 2.17** 1.73** 6.40** 11.10** 5.88** 200 NG NG NG NG NG Significantly different *(p! 0.05) and **(p! 0.005) to control plant. NG= no growth. 368 M. Ghosh, S.P. Singh / Environmental Pollution 133 (2005) 365–371 M. Ghosh, S.P. Singh / Environme 0 20 40 60 80 100 120 10 20 50 100 10 20 50 100 10 20 50 100 10 20 50 100 Brassica campestris C on ce nt ra tio n µ g C d / p la nt p ar t Leaves Stem Whole PlantRoots (a) Brassica juncea 0 20 40 60 80 100 120 C on ce nt ra tio n µ g C d / p la nt p ar t 10 20 50 100 10 20 50 100 10 20 50 100 10 20 50 100 Leaves Stem Whole PlantRoots (b) C on ce nt ra tio n µ g C d / p la nt p ar t 10 20 50 100 10 20 50 100 10 20 50 100 10 20 50 100 Leaves Stem Whole PlantRoots 0 20 40 60 80 100 120 140 160 180 200 220 Dhatura innoxia(c) C on ce nt ra tio n µ g C d / p la nt p ar t 10 20 50 100 10 20 50 100 10 20 50 100 10 20 50 100 Leaves Stem Whole PlantRoots 0 50 100 150 200 250 300 350 400 450 Ipomoea carnea (d) C on ce nt ra tio n µ g C d / p la nt p ar t 10 20 50 100 10 20 50 100 10 20 50 100 10 20 50 100 Leaves Stem Whole PlantRoots 0 20 40 60 80 100 120 140 160 180 200 220 Phragmutes karka(e) Dhatura innoxia Ipomoea carnea Phragmytes karka I. carnea showed a maximum Cd extraction at maturity with the extraction exceeding all the other plants and treatments. This was due to a much higher biomass at lower Cd uptake. In field conditions the biomass produced is bound to be more, and if the plant uptake is equivalent to that which has been achieved in these experiments then I. carnea, D. innoxia and P. karka could be used to extract soils contaminated with 100 mg kg�1 total Cd. Phytoextraction of Cd can be further enhanced by coppice harvesting the biomass at regular intervals (i.e. 90 days), particularly with I. carnea and P. karka. Only D. innoxia requires sowing. Coppice harvesting will also help in avoiding recycling of biomass bound metals. 3.4. Transportation index (Ti) The leaf/root cadmium concentration index (Ti) is shown for each plant species in Table 4. Maximum transport was observed in D. innoxia at 20 mg kg�1 Cd, with a Ti maximum variation ranging from 1.17 to 0.64. For other plants it was !1, indicating that the root Cd concentration was always high. At 100 mg Cd kg�1 soil, the order of Ti was D. innoxiaO P. karkaO B. campestrisZ B. junceaO I. carnea. In the case of I. carnea and P. karka, the large difference between root and leaf concentrations indicates an important restriction of the internal transport of Cd from roots towards stems and leaves, resulting in higher root concentrations rath