- Email: [email protected]
Nitrilotriacetate- and citric acid-assisted phytoextraction of cadmium by Indian mustard (Brassica juncea (L.) Czernj, Brassicaceae)
Chemosphere 59 (2005) 1249–1255 www.elsevier.com/locate/chemosphere
Nitrilotriacetate- and citric acid-assisted phytoextraction of cadmium by Indian mustard (Brassica juncea (L.) Czernj, Brassicaceae) M.F. Quartacci a, A.J.M. Baker b, F. Navari-Izzo a
a,*
Dipartimento di Chimica e Biotecnologie Agrarie, Universita` di Pisa, Via del Borghetto 80, 56124 Pisa, Italy b School of Botany, The University of Melbourne, Parkville, VIC 3010, Australia Received 1 July 2004; received in revised form 4 November 2004; accepted 17 November 2004
Abstract In a pot experiment the effects of nitrilotriacetate (NTA) and citric acid applications on Cd extractibility from soil as well as on its uptake and accumulation by Indian mustard (Brassica juncea) were investigated. Plants were grown in a sandy soil with added CdS at four levels ranging from 50 to 200 mg Cd kg 1 soil. After 30 days of growth, pots were amended with NTA or citric acid at 10 and 20 mmol kg 1. Control pots were not treated with chelates. Harvest of plants was performed immediately before and one week after chelate addition. Soil water-, NH4NO3- and EDTAextractable Cd fractions increased constantly with both increasing soil metal application and chelate concentration. Shoot dry weights did not suffer significant reductions with increasing Cd addition to the soil except for both NTA treatments in which at 200 mg Cd kg 1 a 30% decrease in dry matter was observed. Generally, following NTA and citric acid amendments, Cd concentration in shoots increased with soil Cd level. However, due to Cd toxicity, at the highest metal application rate both NTA treatments lowered Cd concentration in the above-ground parts. Compared to the control, at 10 mmol kg 1 citric acid did not change Cd concentration in shoots, whereas NTA-treated plants showed an about 2-fold increase. The addition of chelates at 20 mmol kg 1 further enhanced Cd concentration in shoots up to 718 and 560 lg g 1 dry weight in the NTA and citrate treatments, respectively. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Brassica juncea; Chelates; Desorption; Cadmium
1. Introduction Slight to moderately elevated Cd concentrations are observed in many agricultural soils due to long-term use of phosphatic fertilisers, sewage sludge application
* Corresponding author. Tel.: +39 050 971921; fax: +39 050 598614. E-mail address: [email protected] (F. Navari-Izzo).
as well as smelter dust deposition. Furthermore, increased Cd levels are also found in the surface soils near the metal processing industry (Vassilev et al., 2002). The high mobility of this metal in the soil–plant system allows its easy entry into the food chain, where it may provoke both human diseases and toxic effects on animals, microorganisms and plants (Wagner, 1994). There is growing evidence that metal phytoextraction is a promising approach applicable to slightly or moderately contaminated soils as an alternative to ex situ
0045-6535/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2004.11.053
1250
M.F. Quartacci et al. / Chemosphere 59 (2005) 1249–1255
decontamination techniques, which are very expensive and unacceptable from an ecological point of view (Baker et al., 1994). However, the slow desorption of metals in soils has been a major impediment to the successful phytoremediation of contaminated sites (Salt et al., 1995; Ebbs and Kochian, 1998; Kulli et al., 1999). In order to overcome the low dry biomass of many hyperaccumulator plants which could be used as soil remediators as well as the low phytoavailability of some metals, a combination of high biomass-producing species and chemically-assisted phytoextraction has been developed (Salt et al., 1998). Biomass crops compensate for a lower metal accumulation by high above-ground dry matter yields. Among them, Indian mustard (Brassica juncea (L.) Czernj), has been identified as a species able to take up and accumulate into its above-ground parts metals such as Cd, Cu, Ni, Zn, Pb and Se (Haag-Kerwer et al., 1999). Chelates have been shown to desorb metals from the soil matrix into the soil solution, and thereby facilitate metal transport into the xylem, so increasing translocation from roots to shoots of several fast-growing, high biomass-producing plants (Huang et al., 1997). Among chelates, EDTA has been found to be the most efficient in increasing water-soluble metal concentrations (Blaylock et al., 1997; Wu et al., 1999), but its use has been seen to potentiate toxic effects on plants and to enhance both leaching of metals to groundwater (Cre`man et al., 2001) and promotion of off-site migration (Cooper et al., 1999; Kulli et al., 1999) leading, in some cases, to a lower uptake of metals by plants (Jiang et al., 2003). Due to the above-cited potential risks, chelates that combine high biodegradability, low phytotoxicity and chelating strength such as nitrilotriacetate (NTA) and citric acid were proposed in the chemically-assisted phytoextraction of some metals (Mench and Martin, 1991; Wenger et al., 2003). In spite of its expected positive properties, few studies have been performed with NTA as the ligand to assist phytoextraction of metals. Indeed, NTA was found to degrade as fast as citric acid in soils and is also rapidly degraded in anaerobic conditions (Ward, 1986). Kulli et al. (1999) observed that Cd, Cu and Zn concentrations in the above-ground plant biomass were 4–24 times greater than in the untreated plants. However, the reduction in growth following relatively high NTA applications resulted in total uptakes of metals that never exceeded more than 2.5-fold that of controls. In a field experiment, NTA application increased the solubility of Cd by a factor of 58, but metal accumulation in Indian mustard was enhanced only by a factor of 2 (Kayser et al., 2000). Low molecular weight (MW) organic acids are natural products in root exudates and influence ion solubility and uptake by their metal chelating/complexing proper-
ties as well as by their indirect effects on microbial activity and rhizosphere physical properties (Wu et al., 2003). In durum wheat cultivars it was found that Cd accumulation was proportional to the concentrations of low MW organic acids present in the rhizosphere (Cies´lin´ski et al., 1998). In addition, citric acid reduced the toxicity of Cd to radish, and stimulated its translocation from roots to shoots converting the metal into more easily transported forms (Chen et al., 2003). In the present pot experiment we compared the efficiency of two chelating agents, NTA and citrate, on Cd uptake by Indian mustard. Four levels of soil Cd and two chelate application rates were used in an attempt to elucidate the potential role of these biodegradable chelating agents in the availability, uptake and accumulation of Cd.
2. Materials and methods 2.1. Soil preparation and plant material Surface soil (0–20 cm) was collected at the ÔE. AvanziÕ Agricultural Farm of the University of Pisa and was airdried, gently ground to pass through a 2-mm sieve, homogenised and stored dry for subsequent analyses. Soil characterisation was done according to Italian Standard Methods (Mi.P.A.F., 2000). Physical and chemical properties of the soil were as follows: clay 10.3%; silt 6.4%; sand 83.3%; pH (H2O) 5.8; organic matter 13.3 g kg 1; cation exchange capacity (CEC) 5.62 meq 100 g 1; total N 1.47 g kg 1; exchangeable P 92.5 mg kg 1; exchangeable K 95.0 mg kg 1. Soil samples were amended with CdS in order to provide the metal in a form with limited solubility, dependent on the reaction of the metal with the soil. Analytical Reagent grade solid CdS was thoroughly mixed with the soil to achieve Cd concentrations of 50, 100, 150 and 200 mg kg 1 dry soil. The soils were then allowed to equilibrate for a period of two weeks undergoing three cycles of saturation with water and air-drying before being remixed and finally planted. The soil was then used to fill 22 cm diam. plastic pots (650 g soil per pot) and moistened with distilled water to approximately 80% field capacity. Basal fertilisers (150, 75 and 95 mg kg 1 N, P and K, supplied as NH4NO3 and KH2PO4) were also applied. After one week pots were planted with B. juncea (L.) Czernj (Brassicaceae) cv. 426308 seeds, at a density of 10 seeds per pot and germinated in a growth chamber at 400 lmol m 2 s 1 photon flux density, 16-h photoperiod, temperatures of 22/20 °C (day/night) and 75% relative humidity. Following seedling emergence, the pots were thinned to two plants per pot. The design of the experiment was three replicates of each treatment in a randomised block design. When soil moisture content
M.F. Quartacci et al. / Chemosphere 59 (2005) 1249–1255
decreased to 25% field capacity, pots were watered with 150 ml distilled water. After 30 days of growth (at the onset of flowering), plants were distributed into five groups and NTA and citric acid treatments, both at the 10 (NTA-10 and cit-10) and 20 (NTA-20 and cit20) mmol kg 1 soil additions. One set of plants was not amended with any chelate (NA), and kept as a further control in addition to those sampled immediately before amendment (BA). Chelates were applied to the soil surface as solutions. Following chelate application, soils were irrigated on a daily basis. Collection trays were placed under all pots to retain any leachate, which was re-applied immediately to the soil in the pot. Plants were harvested before and one week after treatment by cutting stems 1 cm above the soil surface. The above-ground parts were washed carefully with distilled water to remove any soil splash and oven-dried at 110 °C for 24 h. Dried material was then digested with conc. HNO3 and the digest analysed for Cd using a Perkin–Elmer 373 atomic absorption spectrophotometer (AAS). 2.2. Extractable Cd in the soils Soils were sampled from the pots immediately before chelate applications (BA) and after plant harvest (NA + NTA and citrate treatments), and analysed for: (i) water-soluble Cd by adding distilled water to give a 1:1 (w/v) soil:solution ratio; (ii) NH4NO3-extractable Cd by adding 1 M NH4NO3 to give a 1:2.5 (w/v) soil:solution ratio; and (iii) EDTA-extractable Cd by adding 0.05 M EDTA (pH 7.0) to give a 1:5 (w/v) soil:solution ratio (Jiang et al., 2003). After shaking for 60 min, tubes were centrifuged at 17 400g for 10 min and the supernatants collected after filtering through a Whatman No. 41 filter paper. Analysis of the filtrate for Cd was performed by AAS.
1251
3. Results The ability of EDTA, NTA and citrate to desorb Cd from the metal-contaminated soils is illustrated in Fig. 1. In general, the amount of Cd extracted increased at an almost linear fashion both with increasing metal application to the soil and chelate concentration, independently of the extractant used. Among the chelates employed, EDTA was the more effective in desorbing Cd, whilst the amount of Cd extracted by citrate at both concentrations was the lowest, showing only an average 3-fold increase in comparison to water. The desorption of Cd by NTA-10 resulted in a metal concentration about 3-fold higher than cit-10, whereas NTA-20 caused a further increase in Cd extractability (Fig. 1). In comparison to the unamended soils (before and one week after chelate treatment) application of chelating agents to soils resulted in higher water-extractable Cd fraction only in the NTA treatments and increased with Cd application rate (Fig. 2a). The concentration of NH4NO3-extractable Cd increased with Cd application following both NTA and citrate additions (Fig. 2b), NTA being more effective than citric acid in extracting the metal. Chelate treatments increased EDTAextractable Cd at each metal application compared to the unamended soils (Fig. 2c). For NTA-10 and NTA20 treatments Cd extraction increased almost proportionately up to 150 mg Cd kg 1, remaining unchanged at the highest Cd application rate. In both citrate-treated soils, EDTA-extractable Cd doubled from 50 to 100 mg kg 1 soil, remaining constant thereafter. Dry weights of shoots showed a general but not significant reduction with increasing Cd concentration in the soil and chelate application rate (Fig. 3a and b), with the exception of the two NTA treatments where, at the highest Cd application rate, an about 30% decrease in
2.3. Desorption of Cd from soils by chelates 40
Soil extractable Cd (mg kg-1)
The ability of chelates to desorb Cd from the contaminated soils was examined using a consecutive solubilisation approach with four extraction steps (Cooper et al., 1999). Chelate-extracting solutions were prepared by dissolving citric acid in water and the free acid forms of NTA and EDTA in NaOH at pH 7.0 in a background matrix of 10 mM CaCl2. For each treatment Cd desorption was measured by placing 3 g soil in a centrifuge tube and adding 30 ml extractant. Tubes were then capped, shaken for 16 h, and centrifuged at 17 400g for 10 min. The supernatant was collected and the residue soil samples were re-suspended in 30 ml extractant. The above steps were repeated three times for a total of four consecutive extractions. Analysis of Cd in the combined supernatants was performed by AAS.
50 mg Cd kg-1 soil 100 150 200
35 30 25 20 15 10 5 0
H 2O
EDTA-10 EDTA-20 NTA-10
NTA-20
cit-10
cit-20
Fig. 1. Effects of EDTA, NTA and citric acid applied to the soil before sowing on the solubilisation of Cd. Chelates were added at 10 and 20 mmol kg 1 soil. Bars denote standard error of the mean (n = 3).
1252
M.F. Quartacci et al. / Chemosphere 59 (2005) 1249–1255
50 mg Cd kg-1 soil 100 150 200
2.00
(a)
dry weight (g plant-1)
Cd desorbed (mg kg-1)
16
12
8
4
1.50
(a)
50 mg Cd kg-1 soil 100 150 200
1.00
0.50
0.00
(b)
0
Cd desorbed (mg kg-1)
25 20 15 10 5
1.00
0.50
0.00 BA
0 50
Cd desorbed (mg kg-1)
1.50
dry weight (g plant-1)
(b)
30
(c)
40 30
NA
NTA
citric acid
Fig. 3. Influence of NTA and citric acid applied to the soil at 10 (a) and 20 (b) mmol kg 1 one week before harvest on shoot dry weight of Indian mustard. BA, untreated pots immediately before amendments; NA, untreated pots after harvest (one week-old). Bars denote standard error of the mean (n = 3).
20 10 0 NA
BA
NTA-10
NTA-20
cit-10
cit-20
Fig. 2. Influence of NTA and citric acid applied to the soil at 10 and 20 mmol kg 1 one week before harvest of Indian mustard on (a) H2O-extractable, (b) NH4NO3-extractable, and (c) EDTA-extractable Cd in the soil after harvest. BA, untreated pots immediately before amendments; NA, untreated pots after harvest (one week-old). Bars denote standard error of the mean (n = 3).
dry matter yield was observed in comparison to plants grown at 150 mg Cd kg 1. At both chelate application rates shoots generally accumulated Cd with increasing soil metal level (Fig. 4a and b), with the exception of the NTA-10 and NTA-20 treatments, which caused a decrease in shoot Cd accumulation at the highest Cd concentration. At 10 mmol kg 1 (Fig. 4a), Cd concentration in tissues was higher in the NTA-treated plants than in the citrate-treated ones (on average 33%), reaching a value of 415 lg g 1 dry weight in the 150 mg Cd treatment. In comparison to the untreated shoots, citric acid resulted in a higher Cd concentration only starting from the
150 mg Cd concentration treatment, reaching 290 lg g 1 dry weight at the highest metal application rate. The addition of chelates at 20 mmol kg 1 enhanced Cd accumulation in shoots (Fig. 4b). Indeed, Cd concentration in shoots showed average increases of 31 and 57% in the NTA and citrate treatments, respectively. As a consequence, the accumulation of Cd in the shoots increased up to 719 and 600 lg g 1 dry weight in the soils amended with 150 and 200 mg kg 1 Cd respectively. Due to the slight differences in plant dry matter yield resulting from the different treatments, shoot Cd uptake showed a trend similar to that of Cd concentration in tissues, NTA being more effective than citrate up to 150 mg Cd kg 1 in the treatment at 10 mmol kg 1 (Fig. 5a and b).
4. Discussion As illustrated by comparative experiments (Hong and Pintauro, 1996; Krishnamurti et al., 1997), in soils Cd complexation with various chelating agents typically follows the order EDTA and related synthetic chelators > NTA > citric acid, even though several factors such as soil physico-chemical characteristics and pH may have a profound influence on metal desorption
M.F. Quartacci et al. / Chemosphere 59 (2005) 1249–1255
50 mg Cd kg-1 soil 100 150 200
500
(a) Cd uptake (mg plant-1)
mg Cd g-1 DW
400
300
200
100
0
Cd uptake (mg plant-1)
mg Cd g-1 DW
600 450 300 150
50 mg Cd kg -1 soil 100 150 200
(a)
300 200 100 0 750
(b)
750
400
1253
(b)
600 450 300 150
0 BA
NA
NTA
citric acid
0 BA
NA
NTA
citric acid
Fig. 4. Influence of NTA and citric acid applied to the soil at 10 (a) and 20 (b) mmol kg 1 one week before harvest on Cd concentration in the shoots of Indian mustard. BA, untreated pots immediately before amendments; NA, untreated pots after harvest (one week-old). Bars denote standard error of the mean (n = 3).
Fig. 5. Influence of NTA and citric acid applied to the soil at 10 (a) and 20 (b) mmol kg 1 one week before harvest on Cd uptake by shoots of Indian mustard. BA, untreated pots before amendments; NA, untreated pots after harvest (one weekold). Bars denote standard error of the mean (n = 3).
and dissolution by chelates. This study also confirmed that in our soils, EDTA is a more effective chelator than NTA and citrate in complexing Cd (Fig. 1) and in making it available for plant uptake. However, the decrease in shoot metal concentration in both the NTA-10 and NTA-20 plants at the highest soil Cd level (Fig. 4a and b) is an indication that the amount of available Cd in the soil exceeded Indian mustardÕs capacity to accumulate it in the above-ground tissues. As a consequence, the environmental problems potentially deriving from a further increase in soil-available Cd following EDTA addition (Fig. 1) should limit the use of this complexing agent to slightly contaminated soils. The availability of elements for leaching and plant uptake depends, not only on their solubility, but on their speciation in the soil solution. In this study, Cd was added as the sulphide, a form that is only sparingly soluble in soils and that undergoes little or no oxidation (Bataillard et al., 2003), remaining mainly adsorbed to particles even though some CdS may be present in pore spaces as individual crystal particles. In our experiment the retention effects were partially reduced by the low organic matter and clay contents. Thus, in contrast to more soluble chemical forms often used in other workerÕs studies, the highest CdS applications were not detrimental to untreated plants as shown by the absence
of visual toxic symptoms and by their unchanged shoot dry matter yields (Fig. 3a and b). On the contrary, the decline in dry biomass (Fig. 3a and b) and shoot metal concentration (Fig. 4a and b) shown at the highest Cd application rate by plants treated with 10 and 20 mmol kg 1 NTA, indicates that metal phytotoxicity did occur due to the desorption and dissolution effects by NTA. This observation correlates well with the highest water- and NH4NO3-extractable Cd concentrations detected in the same highly Cd-polluted soils (Fig. 2a and b). It is known that Cd does not interfere directly with cellular oxygen metabolism but may cause injury to plant cells by oxidative stress (Navari-Izzo and Quartacci, 2001; Milone et al., 2003). It has been proposed that Cd exposure results in a severe depletion of glutathione due to its increased consumption for phytochelatin production. This, in turn, leads to insufficient cell redox balance and thereafter to an enhanced oxyradicals production due to a general decrease in defense enzyme activities which are not able to control the overoxidative production (Schu¨tzendu¨bel and Polle, 2002). In the present study, the positive relationship between Cd concentration in the shoots and that in the soil solution following both chelate treatments up to 150 mg Cd kg 1 (regression coefficient always >0.85), is an indication that the anti-oxidative defense systems of Indian
1254
M.F. Quartacci et al. / Chemosphere 59 (2005) 1249–1255
mustard are able to detoxify the metal and allow its accumulation in the aboveground tissues. Krishnamurti et al. (1997) demonstrated that various low MW organic acids were able to influence the rate of Cd release from different soils and increase the solubility of Cd in bulk soil through the formation of soluble Cdorganic acid complexes. Preliminary experiments performed in our laboratory on the polluted soils showed that, among several low MW organic acids tested, citric acid had the greatest capacity to extract Cd in comparison with malic, oxalic and succinic acids (data not reported here). Notwithstanding, citrate is a less effective complexing agent than NTA (Fig. 1); when applied at 20 mmol kg 1 to soils amended up to 150 mg Cd kg 1 it resulted in similar or enhanced shoot Cd uptake in comparison to NTA (Fig. 5a and b). This could be related to a change in the chemical forms of Cd following citric acid application, with transformation of more toxic forms into less toxic forms (Chen et al., 2003) that did not reduce plant growth at high soil amendment levels. In accordance with previous findings (Kayser et al., 2000; Nigam et al., 2001; Wu et al., 2003), treatment with citric acid had no effect on the soil solution pH (data not shown). Thus, in our experiment soil acidification cannot be responsible for the increased Cd concentration following citric acid application. Other mechanisms, such as surface complexation with subsequent complex dissociation and/or cation exchange (through ammonium), could be involved in Cd mobilisation (Blay et al., 1997). The results of this study indicate that Indian mustard did not exhibit Cd hyperaccumulation, with maximum shoot concentration of 600 mg kg 1 dry weight (Fig. 5b) in the cit-20 treated plants at the highest soil Cd level at which slight phytotoxicity occurred. However, the treatments with NTA and citric acid, enhancing the bioavailability of Cd could be useful in making the metal available at concentrations compatible from both environmental and phytotoxicity perspectives. Compared to untreated (NA) soils, shoot Cd concentrations following 20 mmol kg 1 NTA and citrate additions were enhanced on average by a factor of 2.6 and 2.3, respectively. These values are of the same magnitude previously observed in pot and field experiments for Indian mustard and other herbaceous species treated with NTA at a range of concentrations (Kulli et al., 1999; Kayser et al., 2000), and in corn after citric acid application (Nigam et al., 2001). Even though NTA is a stronger complexing agent than low MW organic acids and, as a consequence, more effective in removing Cd at the lowest chelate application rate (10 mmol kg 1 soil), the use of citric acid in contrast, may be advantageous in highly polluted soils where phytotoxic effects due to enhanced soluble Cd concentrations can reduce plant yield and impair Cd detoxification mechanisms.
Acknowledgement Seeds of Brassica juncea cv. 426308 were kindly provided by the North Central Regional Plant Introduction Station (NCRPIS), Iowa State University, USA. This study was funded by the MIUR (Cofinanziamento 2001) and the University of Pisa (Fondi di Ateneo 2003).
References Baker, A.J.M., McGrath, S.P., Sidoli, C.M.D., Reeves, R.D., 1994. The possibility of in situ heavy metal decontamination of polluted soils using crops of metal-accumulating plants. Resources Conservation and Recycling 11, 41–49. Bataillard, P., Cambier, P., Picot, C., 2003. Short-term transformations of lead and cadmium compounds in soil after contamination. European Journal of Soil Science 54, 365– 376. Blay, K., Fischer, K., Mo¨lter, K., Filser, J., Kettrup, A., 1997. Extraction of a copper contaminated soil material using an aminoacid-containing residue hydrolysate. I. Cu-elution dynamics and binding-specific release. Zeitschrift fur Pflanzenernahrung und Bodenkunde 160, 393–400. Blaylock, M.J., Salt, D.E., Dushenkov, S., Zakharova, O., Gussman, C., Kapulnik, Y., Ensley, B.D., Raskin, I., 1997. Enhanced accumulation of Pb in Indian mustard by soilapplied chelating agents. Environmental Science and Technology 31, 860–865. Chen, Y.X., Lin, Q., Luo, Y.M., He, Y.F., Zhen, S.J., Yu, Y.L., Tian, G.M., Wong, M.H., 2003. The role of citric acid on the phytoremediation of heavy metal contaminated soil. Chemosphere 50, 807–811. Cies´lin´ski, G., Van Rees, K.C.J., Szmigielska, A.M., Krishnamurti, G.S.R., Huang, P.M., 1998. Low-molecularweight organic acids in rhizosphere soils of durum wheat and their effect on cadmium bioaccumulation. Plant Soil 203, 109–117. Cooper, E.M., Sims, J.T., Cunningham, S.D., Huang, J.W., Berti, W.R., 1999. Chelate-assisted phytoextraction of lead from contaminated soils. Journal of Environmental Quality 28, 1709–1719. Cre`man, H., Velikonja-Bolta, Sˇ., Vodnik, D., Kos, B., Lesˇtan, D., 2001. EDTA enhanced heavy metal phytoextraction: metal accumulation, leaching and toxicity. Plant Soil 235, 105–114. Ebbs, S.D., Kochian, L.V., 1998. Phytoremediation of zinc by oat (Avena sativa), barley (Hordeum vulgare) and Indian mustard (Brassica juncea). Environmental Science and Technology 32, 802–806. Haag-Kerwer, A., Scha¨fer, H.J., Heiss, S., Walter, C., Rausch, T., 1999. Cadmium exposure in Brassica juncea causes a decline in transpiration rate and leaf expansion without effect on photosynthesis. Journal of Experimental Botany 50, 1827–1835. Hong, J., Pintauro, P.N., 1996. Desorption–complexation– dissolution characteristics of adsorbed cadmium from kaolin by chelators. Water Air Soil Pollution 86, 35–50. Huang, J.W., Chen, J., Berti, W.R., Cunningham, S.D., 1997. Phytoremediation of lead contaminated soils: role of syn-
M.F. Quartacci et al. / Chemosphere 59 (2005) 1249–1255 thetic chelates in lead phytoextraction. Environmental Science Technology 3, 800–805. Jiang, X.J., Luo, Y.M., Zhao, Q.G., Baker, A.J.M., Christie, P., Wong, M.H., 2003. Soil Cd availability to Indian mustard and environmental risk following EDTA addition to Cd-contaminated soil. Chemosphere 50, 813–818. Kayser, A., Wenger, K., Keller, A., Attinger, W., Felix, H., Gupta, S.K., Schulin, R., 2000. Enhancement of phytoextraction of Zn, Cd, and Cu from calcareous soil: the use of NTA and sulfur amendments. Environmental Science and Technology 34, 1778–1783. Krishnamurti, G.S.R., Cielinski, G., Huang, P.M., van Rees, K.C.J., 1997. Kinetics of cadmium release from soils as influenced by organic acid: implementation in cadmium availability. Journal of Environmental Quality 26, 271–277. Kulli, B., Balmer, M., Krebs, R., Lothenbach, B., Geiger, G., Schulin, R., 1999. The influence of nitrilotriacetate on heavy metal uptake of lettuce and ryegrass. Journal of Environmental Quality 28, 1699–1705. Mench, M., Martin, E., 1991. Mobilization of cadmium and other metals from two soils by root exudates of Zea mays L., Nicotiana tabacum L., and Nicotiana rustica L. Plant Soil 132, 187–196. Milone, M.T., Sgherri, C., Cljisters, H., Navari-Izzo, F., 2003. Antioxidative responses of wheat treated with realistic concentration of cadmium. Environmental and Experimental Botany 50, 265–276. Mi.P.A.F (Ministero delle Politiche Agricole e Forestali), 2000. Metodi di analisi chimica del suolo. Franco Angeli Editore, Milan. Navari-Izzo, F., Quartacci, M.F., 2001. Phytoremediation of metals. Tolerance mechanisms against oxidative stress. Minerva Biotecnologica 13, 73–83. Nigam, R., Srivastava, S., Prakash, S., Srivastava, M.M., 2001. Cadmium mobilisation and plant availability—the impact
1255
of organic acid commonly exuded from roots. Plant Soil 230, 107–113. Salt, D.E., Smith, R.D., Raskin, I., 1998. Phytoremediation. Annual Review of Plant Physiology and Plant Molecular Biology 49, 643–668. Salt, D.E., Blaylock, M., Kuma, P.B.A.N., Dushenkov, V., Ensley, B.D., Chet, I., Raskin, I., 1995. Phytoremediation: a novel strategy for the removal of toxic metals from the environment using plants. Biotechnology 13, 468– 478. Schu¨tzendu¨bel, A., Polle, A., 2002. Plant responses to abiotic stresses: heavy metal-induced oxidative stress and protection by mycorrhization. Journal of Experimental Botany 53, 1351–1365. Vassilev, A., Vangronsveld, J., Yordanov, I., 2002. Cadmium phytoextraction: present state, biological backgrounds and research needs. Bulgarian Journal of Plant Physiology 28, 68–95. Ward, T.E., 1986. Aerobic and anaerobic biodegradation of nitrilotriacetate in subsurface soils. Ecotoxicology Environmental Safety 11, 112–125. Wagner, G.J., 1994. Accumulation of cadmium in crop plants and its consequences to human health. Advances in Agronomy 51, 173–212. Wenger, K., Gupta, S.K., Furrer, G., Schulin, R., 2003. The role of nitrilotriacetate in copper uptake by tobacco. Journal of Environmental Quality 32, 1669–1676. Wu, J., Hsu, F.C., Cunningham, S.D., 1999. Chelate-assisted Pb phytoextraction: Pb availability, uptake and translocation constraints. Environmental Science and Technology 33, 1898–1904. Wu, L.H., Luo, Y.M., Christie, P., Wong, M.H., 2003. Effects of EDTA and low molecular weight organic acids on soil solution properties of a heavy metal polluted soil. Chemosphere 50, 819–822.
Nitrilotriacetate- and citric acid-assisted phytoextraction of cadmium by Indian mustard (Brassica juncea (L.) Czernj, Brassicaceae) M.F. Quartacci a, A.J.M. Baker b, F. Navari-Izzo a
a,*
Dipartimento di Chimica e Biotecnologie Agrarie, Universita` di Pisa, Via del Borghetto 80, 56124 Pisa, Italy b School of Botany, The University of Melbourne, Parkville, VIC 3010, Australia Received 1 July 2004; received in revised form 4 November 2004; accepted 17 November 2004
Abstract In a pot experiment the effects of nitrilotriacetate (NTA) and citric acid applications on Cd extractibility from soil as well as on its uptake and accumulation by Indian mustard (Brassica juncea) were investigated. Plants were grown in a sandy soil with added CdS at four levels ranging from 50 to 200 mg Cd kg 1 soil. After 30 days of growth, pots were amended with NTA or citric acid at 10 and 20 mmol kg 1. Control pots were not treated with chelates. Harvest of plants was performed immediately before and one week after chelate addition. Soil water-, NH4NO3- and EDTAextractable Cd fractions increased constantly with both increasing soil metal application and chelate concentration. Shoot dry weights did not suffer significant reductions with increasing Cd addition to the soil except for both NTA treatments in which at 200 mg Cd kg 1 a 30% decrease in dry matter was observed. Generally, following NTA and citric acid amendments, Cd concentration in shoots increased with soil Cd level. However, due to Cd toxicity, at the highest metal application rate both NTA treatments lowered Cd concentration in the above-ground parts. Compared to the control, at 10 mmol kg 1 citric acid did not change Cd concentration in shoots, whereas NTA-treated plants showed an about 2-fold increase. The addition of chelates at 20 mmol kg 1 further enhanced Cd concentration in shoots up to 718 and 560 lg g 1 dry weight in the NTA and citrate treatments, respectively. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Brassica juncea; Chelates; Desorption; Cadmium
1. Introduction Slight to moderately elevated Cd concentrations are observed in many agricultural soils due to long-term use of phosphatic fertilisers, sewage sludge application
* Corresponding author. Tel.: +39 050 971921; fax: +39 050 598614. E-mail address: [email protected] (F. Navari-Izzo).
as well as smelter dust deposition. Furthermore, increased Cd levels are also found in the surface soils near the metal processing industry (Vassilev et al., 2002). The high mobility of this metal in the soil–plant system allows its easy entry into the food chain, where it may provoke both human diseases and toxic effects on animals, microorganisms and plants (Wagner, 1994). There is growing evidence that metal phytoextraction is a promising approach applicable to slightly or moderately contaminated soils as an alternative to ex situ
0045-6535/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2004.11.053
1250
M.F. Quartacci et al. / Chemosphere 59 (2005) 1249–1255
decontamination techniques, which are very expensive and unacceptable from an ecological point of view (Baker et al., 1994). However, the slow desorption of metals in soils has been a major impediment to the successful phytoremediation of contaminated sites (Salt et al., 1995; Ebbs and Kochian, 1998; Kulli et al., 1999). In order to overcome the low dry biomass of many hyperaccumulator plants which could be used as soil remediators as well as the low phytoavailability of some metals, a combination of high biomass-producing species and chemically-assisted phytoextraction has been developed (Salt et al., 1998). Biomass crops compensate for a lower metal accumulation by high above-ground dry matter yields. Among them, Indian mustard (Brassica juncea (L.) Czernj), has been identified as a species able to take up and accumulate into its above-ground parts metals such as Cd, Cu, Ni, Zn, Pb and Se (Haag-Kerwer et al., 1999). Chelates have been shown to desorb metals from the soil matrix into the soil solution, and thereby facilitate metal transport into the xylem, so increasing translocation from roots to shoots of several fast-growing, high biomass-producing plants (Huang et al., 1997). Among chelates, EDTA has been found to be the most efficient in increasing water-soluble metal concentrations (Blaylock et al., 1997; Wu et al., 1999), but its use has been seen to potentiate toxic effects on plants and to enhance both leaching of metals to groundwater (Cre`man et al., 2001) and promotion of off-site migration (Cooper et al., 1999; Kulli et al., 1999) leading, in some cases, to a lower uptake of metals by plants (Jiang et al., 2003). Due to the above-cited potential risks, chelates that combine high biodegradability, low phytotoxicity and chelating strength such as nitrilotriacetate (NTA) and citric acid were proposed in the chemically-assisted phytoextraction of some metals (Mench and Martin, 1991; Wenger et al., 2003). In spite of its expected positive properties, few studies have been performed with NTA as the ligand to assist phytoextraction of metals. Indeed, NTA was found to degrade as fast as citric acid in soils and is also rapidly degraded in anaerobic conditions (Ward, 1986). Kulli et al. (1999) observed that Cd, Cu and Zn concentrations in the above-ground plant biomass were 4–24 times greater than in the untreated plants. However, the reduction in growth following relatively high NTA applications resulted in total uptakes of metals that never exceeded more than 2.5-fold that of controls. In a field experiment, NTA application increased the solubility of Cd by a factor of 58, but metal accumulation in Indian mustard was enhanced only by a factor of 2 (Kayser et al., 2000). Low molecular weight (MW) organic acids are natural products in root exudates and influence ion solubility and uptake by their metal chelating/complexing proper-
ties as well as by their indirect effects on microbial activity and rhizosphere physical properties (Wu et al., 2003). In durum wheat cultivars it was found that Cd accumulation was proportional to the concentrations of low MW organic acids present in the rhizosphere (Cies´lin´ski et al., 1998). In addition, citric acid reduced the toxicity of Cd to radish, and stimulated its translocation from roots to shoots converting the metal into more easily transported forms (Chen et al., 2003). In the present pot experiment we compared the efficiency of two chelating agents, NTA and citrate, on Cd uptake by Indian mustard. Four levels of soil Cd and two chelate application rates were used in an attempt to elucidate the potential role of these biodegradable chelating agents in the availability, uptake and accumulation of Cd.
2. Materials and methods 2.1. Soil preparation and plant material Surface soil (0–20 cm) was collected at the ÔE. AvanziÕ Agricultural Farm of the University of Pisa and was airdried, gently ground to pass through a 2-mm sieve, homogenised and stored dry for subsequent analyses. Soil characterisation was done according to Italian Standard Methods (Mi.P.A.F., 2000). Physical and chemical properties of the soil were as follows: clay 10.3%; silt 6.4%; sand 83.3%; pH (H2O) 5.8; organic matter 13.3 g kg 1; cation exchange capacity (CEC) 5.62 meq 100 g 1; total N 1.47 g kg 1; exchangeable P 92.5 mg kg 1; exchangeable K 95.0 mg kg 1. Soil samples were amended with CdS in order to provide the metal in a form with limited solubility, dependent on the reaction of the metal with the soil. Analytical Reagent grade solid CdS was thoroughly mixed with the soil to achieve Cd concentrations of 50, 100, 150 and 200 mg kg 1 dry soil. The soils were then allowed to equilibrate for a period of two weeks undergoing three cycles of saturation with water and air-drying before being remixed and finally planted. The soil was then used to fill 22 cm diam. plastic pots (650 g soil per pot) and moistened with distilled water to approximately 80% field capacity. Basal fertilisers (150, 75 and 95 mg kg 1 N, P and K, supplied as NH4NO3 and KH2PO4) were also applied. After one week pots were planted with B. juncea (L.) Czernj (Brassicaceae) cv. 426308 seeds, at a density of 10 seeds per pot and germinated in a growth chamber at 400 lmol m 2 s 1 photon flux density, 16-h photoperiod, temperatures of 22/20 °C (day/night) and 75% relative humidity. Following seedling emergence, the pots were thinned to two plants per pot. The design of the experiment was three replicates of each treatment in a randomised block design. When soil moisture content
M.F. Quartacci et al. / Chemosphere 59 (2005) 1249–1255
decreased to 25% field capacity, pots were watered with 150 ml distilled water. After 30 days of growth (at the onset of flowering), plants were distributed into five groups and NTA and citric acid treatments, both at the 10 (NTA-10 and cit-10) and 20 (NTA-20 and cit20) mmol kg 1 soil additions. One set of plants was not amended with any chelate (NA), and kept as a further control in addition to those sampled immediately before amendment (BA). Chelates were applied to the soil surface as solutions. Following chelate application, soils were irrigated on a daily basis. Collection trays were placed under all pots to retain any leachate, which was re-applied immediately to the soil in the pot. Plants were harvested before and one week after treatment by cutting stems 1 cm above the soil surface. The above-ground parts were washed carefully with distilled water to remove any soil splash and oven-dried at 110 °C for 24 h. Dried material was then digested with conc. HNO3 and the digest analysed for Cd using a Perkin–Elmer 373 atomic absorption spectrophotometer (AAS). 2.2. Extractable Cd in the soils Soils were sampled from the pots immediately before chelate applications (BA) and after plant harvest (NA + NTA and citrate treatments), and analysed for: (i) water-soluble Cd by adding distilled water to give a 1:1 (w/v) soil:solution ratio; (ii) NH4NO3-extractable Cd by adding 1 M NH4NO3 to give a 1:2.5 (w/v) soil:solution ratio; and (iii) EDTA-extractable Cd by adding 0.05 M EDTA (pH 7.0) to give a 1:5 (w/v) soil:solution ratio (Jiang et al., 2003). After shaking for 60 min, tubes were centrifuged at 17 400g for 10 min and the supernatants collected after filtering through a Whatman No. 41 filter paper. Analysis of the filtrate for Cd was performed by AAS.
1251
3. Results The ability of EDTA, NTA and citrate to desorb Cd from the metal-contaminated soils is illustrated in Fig. 1. In general, the amount of Cd extracted increased at an almost linear fashion both with increasing metal application to the soil and chelate concentration, independently of the extractant used. Among the chelates employed, EDTA was the more effective in desorbing Cd, whilst the amount of Cd extracted by citrate at both concentrations was the lowest, showing only an average 3-fold increase in comparison to water. The desorption of Cd by NTA-10 resulted in a metal concentration about 3-fold higher than cit-10, whereas NTA-20 caused a further increase in Cd extractability (Fig. 1). In comparison to the unamended soils (before and one week after chelate treatment) application of chelating agents to soils resulted in higher water-extractable Cd fraction only in the NTA treatments and increased with Cd application rate (Fig. 2a). The concentration of NH4NO3-extractable Cd increased with Cd application following both NTA and citrate additions (Fig. 2b), NTA being more effective than citric acid in extracting the metal. Chelate treatments increased EDTAextractable Cd at each metal application compared to the unamended soils (Fig. 2c). For NTA-10 and NTA20 treatments Cd extraction increased almost proportionately up to 150 mg Cd kg 1, remaining unchanged at the highest Cd application rate. In both citrate-treated soils, EDTA-extractable Cd doubled from 50 to 100 mg kg 1 soil, remaining constant thereafter. Dry weights of shoots showed a general but not significant reduction with increasing Cd concentration in the soil and chelate application rate (Fig. 3a and b), with the exception of the two NTA treatments where, at the highest Cd application rate, an about 30% decrease in
2.3. Desorption of Cd from soils by chelates 40
Soil extractable Cd (mg kg-1)
The ability of chelates to desorb Cd from the contaminated soils was examined using a consecutive solubilisation approach with four extraction steps (Cooper et al., 1999). Chelate-extracting solutions were prepared by dissolving citric acid in water and the free acid forms of NTA and EDTA in NaOH at pH 7.0 in a background matrix of 10 mM CaCl2. For each treatment Cd desorption was measured by placing 3 g soil in a centrifuge tube and adding 30 ml extractant. Tubes were then capped, shaken for 16 h, and centrifuged at 17 400g for 10 min. The supernatant was collected and the residue soil samples were re-suspended in 30 ml extractant. The above steps were repeated three times for a total of four consecutive extractions. Analysis of Cd in the combined supernatants was performed by AAS.
50 mg Cd kg-1 soil 100 150 200
35 30 25 20 15 10 5 0
H 2O
EDTA-10 EDTA-20 NTA-10
NTA-20
cit-10
cit-20
Fig. 1. Effects of EDTA, NTA and citric acid applied to the soil before sowing on the solubilisation of Cd. Chelates were added at 10 and 20 mmol kg 1 soil. Bars denote standard error of the mean (n = 3).
1252
M.F. Quartacci et al. / Chemosphere 59 (2005) 1249–1255
50 mg Cd kg-1 soil 100 150 200
2.00
(a)
dry weight (g plant-1)
Cd desorbed (mg kg-1)
16
12
8
4
1.50
(a)
50 mg Cd kg-1 soil 100 150 200
1.00
0.50
0.00
(b)
0
Cd desorbed (mg kg-1)
25 20 15 10 5
1.00
0.50
0.00 BA
0 50
Cd desorbed (mg kg-1)
1.50
dry weight (g plant-1)
(b)
30
(c)
40 30
NA
NTA
citric acid
Fig. 3. Influence of NTA and citric acid applied to the soil at 10 (a) and 20 (b) mmol kg 1 one week before harvest on shoot dry weight of Indian mustard. BA, untreated pots immediately before amendments; NA, untreated pots after harvest (one week-old). Bars denote standard error of the mean (n = 3).
20 10 0 NA
BA
NTA-10
NTA-20
cit-10
cit-20
Fig. 2. Influence of NTA and citric acid applied to the soil at 10 and 20 mmol kg 1 one week before harvest of Indian mustard on (a) H2O-extractable, (b) NH4NO3-extractable, and (c) EDTA-extractable Cd in the soil after harvest. BA, untreated pots immediately before amendments; NA, untreated pots after harvest (one week-old). Bars denote standard error of the mean (n = 3).
dry matter yield was observed in comparison to plants grown at 150 mg Cd kg 1. At both chelate application rates shoots generally accumulated Cd with increasing soil metal level (Fig. 4a and b), with the exception of the NTA-10 and NTA-20 treatments, which caused a decrease in shoot Cd accumulation at the highest Cd concentration. At 10 mmol kg 1 (Fig. 4a), Cd concentration in tissues was higher in the NTA-treated plants than in the citrate-treated ones (on average 33%), reaching a value of 415 lg g 1 dry weight in the 150 mg Cd treatment. In comparison to the untreated shoots, citric acid resulted in a higher Cd concentration only starting from the
150 mg Cd concentration treatment, reaching 290 lg g 1 dry weight at the highest metal application rate. The addition of chelates at 20 mmol kg 1 enhanced Cd accumulation in shoots (Fig. 4b). Indeed, Cd concentration in shoots showed average increases of 31 and 57% in the NTA and citrate treatments, respectively. As a consequence, the accumulation of Cd in the shoots increased up to 719 and 600 lg g 1 dry weight in the soils amended with 150 and 200 mg kg 1 Cd respectively. Due to the slight differences in plant dry matter yield resulting from the different treatments, shoot Cd uptake showed a trend similar to that of Cd concentration in tissues, NTA being more effective than citrate up to 150 mg Cd kg 1 in the treatment at 10 mmol kg 1 (Fig. 5a and b).
4. Discussion As illustrated by comparative experiments (Hong and Pintauro, 1996; Krishnamurti et al., 1997), in soils Cd complexation with various chelating agents typically follows the order EDTA and related synthetic chelators > NTA > citric acid, even though several factors such as soil physico-chemical characteristics and pH may have a profound influence on metal desorption
M.F. Quartacci et al. / Chemosphere 59 (2005) 1249–1255
50 mg Cd kg-1 soil 100 150 200
500
(a) Cd uptake (mg plant-1)
mg Cd g-1 DW
400
300
200
100
0
Cd uptake (mg plant-1)
mg Cd g-1 DW
600 450 300 150
50 mg Cd kg -1 soil 100 150 200
(a)
300 200 100 0 750
(b)
750
400
1253
(b)
600 450 300 150
0 BA
NA
NTA
citric acid
0 BA
NA
NTA
citric acid
Fig. 4. Influence of NTA and citric acid applied to the soil at 10 (a) and 20 (b) mmol kg 1 one week before harvest on Cd concentration in the shoots of Indian mustard. BA, untreated pots immediately before amendments; NA, untreated pots after harvest (one week-old). Bars denote standard error of the mean (n = 3).
Fig. 5. Influence of NTA and citric acid applied to the soil at 10 (a) and 20 (b) mmol kg 1 one week before harvest on Cd uptake by shoots of Indian mustard. BA, untreated pots before amendments; NA, untreated pots after harvest (one weekold). Bars denote standard error of the mean (n = 3).
and dissolution by chelates. This study also confirmed that in our soils, EDTA is a more effective chelator than NTA and citrate in complexing Cd (Fig. 1) and in making it available for plant uptake. However, the decrease in shoot metal concentration in both the NTA-10 and NTA-20 plants at the highest soil Cd level (Fig. 4a and b) is an indication that the amount of available Cd in the soil exceeded Indian mustardÕs capacity to accumulate it in the above-ground tissues. As a consequence, the environmental problems potentially deriving from a further increase in soil-available Cd following EDTA addition (Fig. 1) should limit the use of this complexing agent to slightly contaminated soils. The availability of elements for leaching and plant uptake depends, not only on their solubility, but on their speciation in the soil solution. In this study, Cd was added as the sulphide, a form that is only sparingly soluble in soils and that undergoes little or no oxidation (Bataillard et al., 2003), remaining mainly adsorbed to particles even though some CdS may be present in pore spaces as individual crystal particles. In our experiment the retention effects were partially reduced by the low organic matter and clay contents. Thus, in contrast to more soluble chemical forms often used in other workerÕs studies, the highest CdS applications were not detrimental to untreated plants as shown by the absence
of visual toxic symptoms and by their unchanged shoot dry matter yields (Fig. 3a and b). On the contrary, the decline in dry biomass (Fig. 3a and b) and shoot metal concentration (Fig. 4a and b) shown at the highest Cd application rate by plants treated with 10 and 20 mmol kg 1 NTA, indicates that metal phytotoxicity did occur due to the desorption and dissolution effects by NTA. This observation correlates well with the highest water- and NH4NO3-extractable Cd concentrations detected in the same highly Cd-polluted soils (Fig. 2a and b). It is known that Cd does not interfere directly with cellular oxygen metabolism but may cause injury to plant cells by oxidative stress (Navari-Izzo and Quartacci, 2001; Milone et al., 2003). It has been proposed that Cd exposure results in a severe depletion of glutathione due to its increased consumption for phytochelatin production. This, in turn, leads to insufficient cell redox balance and thereafter to an enhanced oxyradicals production due to a general decrease in defense enzyme activities which are not able to control the overoxidative production (Schu¨tzendu¨bel and Polle, 2002). In the present study, the positive relationship between Cd concentration in the shoots and that in the soil solution following both chelate treatments up to 150 mg Cd kg 1 (regression coefficient always >0.85), is an indication that the anti-oxidative defense systems of Indian
1254
M.F. Quartacci et al. / Chemosphere 59 (2005) 1249–1255
mustard are able to detoxify the metal and allow its accumulation in the aboveground tissues. Krishnamurti et al. (1997) demonstrated that various low MW organic acids were able to influence the rate of Cd release from different soils and increase the solubility of Cd in bulk soil through the formation of soluble Cdorganic acid complexes. Preliminary experiments performed in our laboratory on the polluted soils showed that, among several low MW organic acids tested, citric acid had the greatest capacity to extract Cd in comparison with malic, oxalic and succinic acids (data not reported here). Notwithstanding, citrate is a less effective complexing agent than NTA (Fig. 1); when applied at 20 mmol kg 1 to soils amended up to 150 mg Cd kg 1 it resulted in similar or enhanced shoot Cd uptake in comparison to NTA (Fig. 5a and b). This could be related to a change in the chemical forms of Cd following citric acid application, with transformation of more toxic forms into less toxic forms (Chen et al., 2003) that did not reduce plant growth at high soil amendment levels. In accordance with previous findings (Kayser et al., 2000; Nigam et al., 2001; Wu et al., 2003), treatment with citric acid had no effect on the soil solution pH (data not shown). Thus, in our experiment soil acidification cannot be responsible for the increased Cd concentration following citric acid application. Other mechanisms, such as surface complexation with subsequent complex dissociation and/or cation exchange (through ammonium), could be involved in Cd mobilisation (Blay et al., 1997). The results of this study indicate that Indian mustard did not exhibit Cd hyperaccumulation, with maximum shoot concentration of 600 mg kg 1 dry weight (Fig. 5b) in the cit-20 treated plants at the highest soil Cd level at which slight phytotoxicity occurred. However, the treatments with NTA and citric acid, enhancing the bioavailability of Cd could be useful in making the metal available at concentrations compatible from both environmental and phytotoxicity perspectives. Compared to untreated (NA) soils, shoot Cd concentrations following 20 mmol kg 1 NTA and citrate additions were enhanced on average by a factor of 2.6 and 2.3, respectively. These values are of the same magnitude previously observed in pot and field experiments for Indian mustard and other herbaceous species treated with NTA at a range of concentrations (Kulli et al., 1999; Kayser et al., 2000), and in corn after citric acid application (Nigam et al., 2001). Even though NTA is a stronger complexing agent than low MW organic acids and, as a consequence, more effective in removing Cd at the lowest chelate application rate (10 mmol kg 1 soil), the use of citric acid in contrast, may be advantageous in highly polluted soils where phytotoxic effects due to enhanced soluble Cd concentrations can reduce plant yield and impair Cd detoxification mechanisms.
Acknowledgement Seeds of Brassica juncea cv. 426308 were kindly provided by the North Central Regional Plant Introduction Station (NCRPIS), Iowa State University, USA. This study was funded by the MIUR (Cofinanziamento 2001) and the University of Pisa (Fondi di Ateneo 2003).
References Baker, A.J.M., McGrath, S.P., Sidoli, C.M.D., Reeves, R.D., 1994. The possibility of in situ heavy metal decontamination of polluted soils using crops of metal-accumulating plants. Resources Conservation and Recycling 11, 41–49. Bataillard, P., Cambier, P., Picot, C., 2003. Short-term transformations of lead and cadmium compounds in soil after contamination. European Journal of Soil Science 54, 365– 376. Blay, K., Fischer, K., Mo¨lter, K., Filser, J., Kettrup, A., 1997. Extraction of a copper contaminated soil material using an aminoacid-containing residue hydrolysate. I. Cu-elution dynamics and binding-specific release. Zeitschrift fur Pflanzenernahrung und Bodenkunde 160, 393–400. Blaylock, M.J., Salt, D.E., Dushenkov, S., Zakharova, O., Gussman, C., Kapulnik, Y., Ensley, B.D., Raskin, I., 1997. Enhanced accumulation of Pb in Indian mustard by soilapplied chelating agents. Environmental Science and Technology 31, 860–865. Chen, Y.X., Lin, Q., Luo, Y.M., He, Y.F., Zhen, S.J., Yu, Y.L., Tian, G.M., Wong, M.H., 2003. The role of citric acid on the phytoremediation of heavy metal contaminated soil. Chemosphere 50, 807–811. Cies´lin´ski, G., Van Rees, K.C.J., Szmigielska, A.M., Krishnamurti, G.S.R., Huang, P.M., 1998. Low-molecularweight organic acids in rhizosphere soils of durum wheat and their effect on cadmium bioaccumulation. Plant Soil 203, 109–117. Cooper, E.M., Sims, J.T., Cunningham, S.D., Huang, J.W., Berti, W.R., 1999. Chelate-assisted phytoextraction of lead from contaminated soils. Journal of Environmental Quality 28, 1709–1719. Cre`man, H., Velikonja-Bolta, Sˇ., Vodnik, D., Kos, B., Lesˇtan, D., 2001. EDTA enhanced heavy metal phytoextraction: metal accumulation, leaching and toxicity. Plant Soil 235, 105–114. Ebbs, S.D., Kochian, L.V., 1998. Phytoremediation of zinc by oat (Avena sativa), barley (Hordeum vulgare) and Indian mustard (Brassica juncea). Environmental Science and Technology 32, 802–806. Haag-Kerwer, A., Scha¨fer, H.J., Heiss, S., Walter, C., Rausch, T., 1999. Cadmium exposure in Brassica juncea causes a decline in transpiration rate and leaf expansion without effect on photosynthesis. Journal of Experimental Botany 50, 1827–1835. Hong, J., Pintauro, P.N., 1996. Desorption–complexation– dissolution characteristics of adsorbed cadmium from kaolin by chelators. Water Air Soil Pollution 86, 35–50. Huang, J.W., Chen, J., Berti, W.R., Cunningham, S.D., 1997. Phytoremediation of lead contaminated soils: role of syn-
M.F. Quartacci et al. / Chemosphere 59 (2005) 1249–1255 thetic chelates in lead phytoextraction. Environmental Science Technology 3, 800–805. Jiang, X.J., Luo, Y.M., Zhao, Q.G., Baker, A.J.M., Christie, P., Wong, M.H., 2003. Soil Cd availability to Indian mustard and environmental risk following EDTA addition to Cd-contaminated soil. Chemosphere 50, 813–818. Kayser, A., Wenger, K., Keller, A., Attinger, W., Felix, H., Gupta, S.K., Schulin, R., 2000. Enhancement of phytoextraction of Zn, Cd, and Cu from calcareous soil: the use of NTA and sulfur amendments. Environmental Science and Technology 34, 1778–1783. Krishnamurti, G.S.R., Cielinski, G., Huang, P.M., van Rees, K.C.J., 1997. Kinetics of cadmium release from soils as influenced by organic acid: implementation in cadmium availability. Journal of Environmental Quality 26, 271–277. Kulli, B., Balmer, M., Krebs, R., Lothenbach, B., Geiger, G., Schulin, R., 1999. The influence of nitrilotriacetate on heavy metal uptake of lettuce and ryegrass. Journal of Environmental Quality 28, 1699–1705. Mench, M., Martin, E., 1991. Mobilization of cadmium and other metals from two soils by root exudates of Zea mays L., Nicotiana tabacum L., and Nicotiana rustica L. Plant Soil 132, 187–196. Milone, M.T., Sgherri, C., Cljisters, H., Navari-Izzo, F., 2003. Antioxidative responses of wheat treated with realistic concentration of cadmium. Environmental and Experimental Botany 50, 265–276. Mi.P.A.F (Ministero delle Politiche Agricole e Forestali), 2000. Metodi di analisi chimica del suolo. Franco Angeli Editore, Milan. Navari-Izzo, F., Quartacci, M.F., 2001. Phytoremediation of metals. Tolerance mechanisms against oxidative stress. Minerva Biotecnologica 13, 73–83. Nigam, R., Srivastava, S., Prakash, S., Srivastava, M.M., 2001. Cadmium mobilisation and plant availability—the impact
1255
of organic acid commonly exuded from roots. Plant Soil 230, 107–113. Salt, D.E., Smith, R.D., Raskin, I., 1998. Phytoremediation. Annual Review of Plant Physiology and Plant Molecular Biology 49, 643–668. Salt, D.E., Blaylock, M., Kuma, P.B.A.N., Dushenkov, V., Ensley, B.D., Chet, I., Raskin, I., 1995. Phytoremediation: a novel strategy for the removal of toxic metals from the environment using plants. Biotechnology 13, 468– 478. Schu¨tzendu¨bel, A., Polle, A., 2002. Plant responses to abiotic stresses: heavy metal-induced oxidative stress and protection by mycorrhization. Journal of Experimental Botany 53, 1351–1365. Vassilev, A., Vangronsveld, J., Yordanov, I., 2002. Cadmium phytoextraction: present state, biological backgrounds and research needs. Bulgarian Journal of Plant Physiology 28, 68–95. Ward, T.E., 1986. Aerobic and anaerobic biodegradation of nitrilotriacetate in subsurface soils. Ecotoxicology Environmental Safety 11, 112–125. Wagner, G.J., 1994. Accumulation of cadmium in crop plants and its consequences to human health. Advances in Agronomy 51, 173–212. Wenger, K., Gupta, S.K., Furrer, G., Schulin, R., 2003. The role of nitrilotriacetate in copper uptake by tobacco. Journal of Environmental Quality 32, 1669–1676. Wu, J., Hsu, F.C., Cunningham, S.D., 1999. Chelate-assisted Pb phytoextraction: Pb availability, uptake and translocation constraints. Environmental Science and Technology 33, 1898–1904. Wu, L.H., Luo, Y.M., Christie, P., Wong, M.H., 2003. Effects of EDTA and low molecular weight organic acids on soil solution properties of a heavy metal polluted soil. Chemosphere 50, 819–822.