EDTA and HEDTA printed - Các phức EDTA và HEDTA, vai trò của chúng trong môi trường

Các phức EDTA và HEDTA, vai trò của chúng trong môi trường

EDTA and HEDTA eects on Cd, Cr, and Ni uptake by Helianthus annuus Hong Chen, Teresa Cutright * Department of Civil Engineering, The University of Akron, Akron, OH 44325-3905, USA Received 26 October 2000; received in revised form 16 January 2001; accepted 18 January 2001 Abstract Phytoremediation has shown great potential as an alternative treatment for the remediation of heavy-metal-con- taminated soils and groundwater. However, the lack of a clear understanding pertaining to metal uptake/translocation mechanisms, enhancement amendments, and external eects on phytoremediation has hindered its full-scale applica- tion. The objective of this research was to investigate the ability of synthetic chelators for enhancing the phytoreme- diation of cadmium-, chromium- and nickel-contaminated soil. Ethylenediaminetriacetic acid (EDTA) and N -(2-hydroxyethyl)-ethylenediaminetriacetic acid (HEDTA) were applied to the soil at various dosages to elevate metal mobility. Uptake into and translocation within Helianthus annuus was determined. It was found that EDTA at a rate of 0.5 g/kg signi®cantly increased the shoot concentrations of Cd and Ni from 34 and 15 to 115 and 117 mg/kg, re- spectively. The total removal eciency for EDTA was 59 lg/plant. HEDTA at the same application rate resulted in a total metal uptake of 42 lg/plant. These research demonstrated that chelator enhancement is plant- and metal-speci®c and is subjective to inhibition when multiple heavy metals are present. Results also showed that chelator toxicity re- duced the plant's biomass, thereby decreasing the amount of metal accumulation. Ó 2001 Elsevier Science Ltd. All rights reserved. Keywords: Chelators; EDTA; HEDTA; Helianthus annuus; Phytoremediation; Metals 1. Introduction Phytoremediation is a process that uses living green plants for the in situ risk reduction for contaminated soil, sludge, sediments, and groundwater through con- taminant removal, degradation, or containment (Anonymous, 1998). It has been shown to be more ad- vantageous than conventional technologies for remedi- ating heavy-metal-contaminated soils. The advantages include large-scale application; growing plants is rela- tively inexpensive; plants provide an aesthetic value to the landscape of contaminated sites, and concentrated hazardous wastes require smaller disposal facilities, and the potential exists to recover metals from the biomass (Saxena and KrishnaRaj, 1999). At sites contaminated with heavy metals, phyto- remediation can be applied as dierent strategies based on the speci®c site condition. They may include phy- toextraction, where metals are transported from the soil into the harvestable shoots (Salt and Blaylock, 1995), rhizo®ltration, where plant roots or seedlings grown in aerated water precipitate and concentrate toxic metals (Raskin et al., 1997), phytovolatilization, in which plants extract volatile metals (e.g., Hg and Se) from soil and volatilize them from the foliage (Salt and Blaylock, 1995), and phytostabilization, in which metal-tolerant plants are used to reduce the mobility of heavy metals (Raskin et al., 1997). For sites contaminated with both heavy metals and toxic organics, phytoremediation is Chemosphere 45 (2001) 21±28 www.elsevier.com/locate/chemosphere * Corresponding author. Tel.: +1-330-972-4935; fax: +1-330- 972-6020. E-mail address: tcutright@uakron.edu (T. Cutright). 0045-6535/01/$ - see front matter Ó 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 045-6535(01)00031-5 still applicable (Saxena and KrishnaRaj, 1999) because the rhizosphere association between plants and soil mi- croorganisms can be utilized to degrade or transform complex organic±metal mixtures. This process has been called phytotransformation or phytodegradation. All plants have the potential to absorb a wide variety of metals from the soil. For the most part, plants tend to only absorb those metals that are essential for their survival and growth. The most remarkable exception to this general rule is a small group of plants that can tolerate, uptake, and translocate high levels of certain heavy metals that would be toxic to any other known organism. Such plants are termed ``hyperaccumula- tors''. According to Brown et al. (1995), hyperaccu- mulator species are those plants whose leaves may contain >100 mg/kg Cd, >1000 mg/kg Ni and Cu, or >10 000 mg/kg Zn and Mn (dry weight) when grown in metal-rich soils. With this extraordinary ability, these plants can be used in future environmental remediation activities, however, full-scale applications have yet to be achieved. One important reason for this lies with the lack of thorough knowledge on the biological processes involved in metal acquisition, transport, and shoot accumulation. Salt et al. (1998) proposed that in the process of acquiring metal ions from soil, plants have evolved several strategies for increasing the metal bioavailability due to the high binding capacity for metallic micronu- trients by soil particles. The ®rst strategy is the plants' ability to produce metal-chelating compounds (phytos- iderophores) such as mugenic and avenic acids to mobilize metal compounds from soil (Vonwiren et al., 1996). The second approach involves the solubilization of metals by exuding protons from roots to acidify the rhizosphere soil (Crowley et al., 1991). Alloway (1995) further suggested that the roots possess a signi®cant CEC due to the presence of carboxyl groups, which might help to move ions through the outer part of the root to the plasmalemma where active absorption occurs. In addition to natural plant adaptations, the addi- tion of synthetic chelators, soil acidi®ers, or commer- cial nutrients can enhance phytoremediation. Several studies have documented the success of pH adjustments for mobilizing metals (Salt et al., 1998; Entry et al., 1996; Chaney et al., 1997; Huang et al., 1997). Al- though soil acidi®cation increased metal mobility, it decreased the microbial activity of the surrounding area (Cornish et al., 1995; Salt et al., 1998; Chen, 2000). Only the addition of synthetic chelators has been shown to increase both the metal mobility within the soil as well as the uptake (and translocation) through the plant tissue without being irreversibly toxic to mi- crobial activity. For instance, Huang and Cunningham (1996) tested N -(2-hydroxyethyl)-ethylenediaminetri- acetic acid (HEDTA) on Pb accumulation enhance- ment and found that 1 week after transplanting, the shoot Pb concentration was increased from 40 to 10 600 mg/kg. In addition to shoot concentration, the shoot to root Pb content was increased from 0.2 to 1.2. Blaylock (1997) showed that chelator supplements in- creased the uptake of Pb, Cd, Cu, Ni, and Zn. Huang et al. (1997) further reported that among the ®ve che- lators, Ethylenediaminetriacetic acid (EDTA) was the most ecient in increasing shoot Pb concentration in both pea and corn, followed by HEDTA. They found the order of the eectiveness in increasing Pb accu- mulation to be EDTA > HEDTA > diethylenetrinitril- opentacetic acid (DTPA) > ethylenegluatarotriacetic acid (EGTA) > Ethylenediaminedinitrilodiac acid (EDDTA). Dushenkov et al. (1999) demonstrated that to increase 137 Cs bioavailability, of the 20 amendments tested ammonium salts had the greatest eect. Although chelators may increase the eectiveness of phytoremediation by means of increasing the removable metal concentrations, not all studies agree. Robinson et al. (1999) reported that in their study, neither calcium and magnesium carbonates, nor the addition of syn- thetic chelating agents were eective in increasing metal uptake by Berkheya coddii on serpentine soils. Bennett et al. (1998) also found that their attempt to enhance nickel uptake in B. coddii by adding EDTA and citric acid to the substrates actually caused a decrease in nickel uptake, despite causing an increase in the concentration of soluble nickel. The objective of this research was to investigate the ability of synthetic chelators to enhance the phyto- remediation of cadmium-, chromium- and nickel-con- taminated soil. The preferred source (EDTA or HEDTA) and dosage required to elevate metal mobility and subsequent uptake and translocation within the plant tissues were determined. 2. Experimental methods 2.1. Soil sources and characterization An agricultural soil was collected from a clean resi- dential garden center in northeastern Ohio. The soil was air-dried under room temperature and mixed daily until an 8% water content was reached. Soil was characterized for soil texture, soil pH, ®eld capacity, cation-exchange capacity (CEC), organic matter content (OM), and contaminant background concentrations. Soil texture, pH, and ®eld capacity were measured by the procedures described by Tan (1995). CEC was determined by the method proposed by Gillman (Nedelkoska and Doran, 2000). OM and TOC were analyzed with a Shimadzu total organic carbon analyzer (TOC-5000) equipped with a solid sample module (SSM-5000A). The back- ground concentrations of total sorbed Cd, Cr, and Ni 22 H. Chen, T. Cutright / Chemosphere 45 (2001) 21±28 were determined with EPA method 3050 (Dramer et al., 1996) (Table 1). 2.2. Soil preparation To initiate the experiments, air-dried soil was weighed and loaded into 21 Al pans (0.32 m  0.25 m  0.04 m). Each pan contained 1.5 kg soil DW per pan. The soil was then rehydrated with a standard nu- trient solution containing 250 mg N (NH 4 NO 3 ,60mg Mg (MgSO 4 , 109 mg P (KH 2 PO 4 , and 207 mg K (KH 2 PO 4 +K 2 SO 4  per kg soil DW (Senden et al., 1990). Two days later, the appropriate metal solution was spiked into the soil in Al pans and mixed thor- oughly. For the ®rst experimental set, the solution contained 50 ppm Cd 2 , (as CdCl 2 Á 2.5H 2 O), 50 ppm Cr 3 (CrCl 3 Á 6H 2 O and 50 ppm Ni 2 (NiSO 4 Á 6H 2 O) for a total metal concentration of 150 mg/kg. The metal concentration corresponds to the individual metal ele- ment content and not the overall compound. The second experimental set had a reduced concentration of 30 mg/ kg for each metal. After the metal solution was added, the soil was allowed to equilibrate for a period of 10 days in the greenhouse. The equilibration involved un- dergoing three cycles of saturation with DI water and air drying, before being remixed and vegetated (Muller and Kordel, 1993). At day 12, pans were amended with ei- ther EDTA or HEDTA (Sigma Chemical) at a concen- tration of 1 or 2 g/kg. Chelator selection was based on the previous work by Huang et al. (1997). A control and blank pan were also prepared with supplemental nutrients and/or metals and were subjected to the saturation cycles as outlined above. The control contained non-metal-spiked-vegetated soil for the in- tention of obtaining data related to background activi- ties, such as the plant accumulation of background heavy metals and biomass growth in uncontaminated soil. The four blanks consisted of the same levels of spiked metal concentrations as treatments with an ex- ception that no plants were grown in the soil. The pur- pose of the blanks was to determine the vegetation eect on metal mobility in the contaminated soil and to ensure accuracy and precision in the analyses. 2.3. Cultivar source and seedling preparation Cultivar selection was based on the plant's ability to achieve hyperaccumulator status for at least one metal. The dwarf sunspot sun¯ower, Helianthus annuus, has been proven to be eective at removing heavy metals and is capable of extracting higher than average amounts of several radionuclides (Cooney, 1996; Gal- lego et al., 1996; Dushenkov et al., 1997; Gouthu et al., 1997; Sun and Shi, 1998; Chen, 2000; Zavoda et al., 2001). Seeds of H. annuus were obtained from USDA/ARS Plant Introduction Station of Iowa State University. They were initially sown in commercial potting soil (SCHULTZ Professional Potting Soil Plus, SCHULTZ Company) in a greenhouse illuminated with natural light. Supplementary light was provided for maintaining 15-h photo-period daily. Greenhouse temperature was 28°C in the daytime and 15°C at night. After 2 weeks of growth in the potting soil, seedlings with similar biomass were transferred to the metal-spiked soil and the ex- periment was initiated. Nine seedlings were used per pan. Unless otherwise speci®ed, seedlings were harvested 4 weeks later. 2.4. Plant harvest and analysis During harvest, plants were gently removed from soil and washed until free of soil. Roots, leaves, and stems were further separated with scissors and dried in a convection oven at 70°C for 3 days (Page, 1982). Tissues were milled with mortar and pestle and digested fol- lowing the procedure outlined by Zheljazkov and Er- ickson (1996). One g of milled plant matter was soaked in 20 ml of concentrated nitric acid. After 6 h, the mixture was boiled to 50% of its original volume. Then, 4 ml of perchloric acid was added and the mixture ref- luxed for 90 min. The solution was ®nally diluted with DI water to 25 ml of total volume and analyzed with ¯ame atomic absorption spectroscopy (Buck 200 AA). 2.5. Analysis of total metal and mobile metal fractions in the soil For this manuscript, the mobile metal fraction is de®ned as the fraction that is not tightly bound to soil and is mobile without the addition of chelators. The Table 1 Physical and chemical characteristics of agricultural soil used in this study Soil properties Agricultural soil Sand (%) 16.62 Æ 0.87 a Silt (%) 42.10 Æ 0.69 Clay (%) 41.28 Æ 1.13 Soil texture Silt clay loam Soil pH (1:1 soil/1 M KCl ratio) 5.51 Æ 0.21 Field capacity (%) 22.56 Æ 0.49 CEC (meq/100 g) 9.56 Æ 0.19 TOC (%) 1.53 Æ 0.10 OM (%) 3.06 Æ 0.07 Background Cd 2 level (mg/kg soil DW) 2.35 Æ 0.25 Background Cr 3 level (mg/kg soil DW) 8.05 Æ 1.57 Background Ni 2 level (mg/kg soil DW) 11.60 Æ 1.67 a Mean Æ SE (each analysis was performed in duplicate). H. Chen, T. Cutright / Chemosphere 45 (2001) 21±28 23 total metal concentration is the summation of the bound and mobile fractions. In order to dierentiate between the mobile and sorbed fractions, two dierent extraction methods were used. The concentration of the total Cd, Cr, and Ni was determined via an EPA acid digestion method 3050 (Carter, 1993). Approximately 10 ml of 1:1 HNO 3 was added to 2 g of air-dried soil (<1 mm) in a 500-ml ball-shaped ¯ask and heated at 95°C for 15 min. Five ml concentrated HNO 3 was added and the solution was re¯uxed for an additional 30 min at 95°C. This was repeated once and the ®nal solution obtained was reduced to 5 ml. Once cooled, approximately 25 ml of 30% H 2 O 2 was added to the solution in 1-ml increments, followed by the addition of 5 ml of concentrated HCl. The digestate was ®ltered through a Whatman â No. 42 ®lter paper and the solu- tion was diluted to 50 ml with DI water. The solution was analyzed by FAAS (Buck 200 AA). To extract the mobile metal fraction in the soil, a procedure proposed by Maiz et al. (1997) was followed. Two grams of air-dried soil sample was transferred into a capped 40 ml heavy-duty PRYEX centrifuge tube, mixed with 20 ml 0.01 M CaCl 2 solution, and agitated in a rotary shaker at 200 rpm for 2 h. After 2 h, the soil suspension was centrifuged at 2500 rpm for 15 min and the supernatant was collected for FAAS analysis. 2.6. Statistical analysis The experiments were designed as a two-stage nested design with two types of chelators as the primary factors. For each factor, two dierent concentrations were used. The dierence between speci®c pairs of means was identi®ed by Student±Newmans±Keuls (SNK) test P < 0: 05. Statistical analysis of the data was performed by using SigmaStat 2.0 (SPSS Science, Chicago, IL). 2.7. Results and discussion 2.7.1. Chelator eect on plant growth Adding HEDTA and EDTA led to a severe yield reduction in the biomass across the treatments. In the ®rst experimental set with higher metal concentration and chelator (1 and 2 g/kg) doses, plants appeared to be chlorotic and showed signs of wilting 1 day after the experiment was initiated. Within 1 week, all plants were dead. Therefore, the metal concentration was lowered to 30 mg/kg per metal and the chelator additions low- ered to 1.0 and 0.5 g/kg for the next set of experiments. Lowering both metal concentrations and chelator ad- ditions extended plant growth to some degree but a large number of plants still died within 2 weeks. Plants grown in 0.5 g/kg EDTA-treated soil exhibited better growth rate and higher biomass was obtained. This was supported by the visual observations where more than half of the plants grown in the soil amended with 0.5 g/kg EDTA maintained vigorous growth through- out 4 weeks. However, growth was still severely retarded in comparison to non-chelator treatments. For example, plants subjected to 30 mg/kg per metal with- out chelators had less than a 10% reduction in biomass, and none of the plants died. Furthermore, control pans containing only chelator additions (i.e., no metals pre- sent) did not exhibit a severe biomass reduction. Therefore, the severe reduction in growth was attrib- uted to the combination of heavy metal concentration and chelator addition. As compared with the control plants, the average shoot biomass of the treatment plants decreased by more than 75% for the 150-ppm contaminated soil (Fig. 1(a)) and more than 50% for the 90-ppm soil (Fig. 1(b)). Plants in HEDTA-amended soil exhibited approxi- mately the trend in biomass reduction. This indicated that the levels of HEDTA added or the metal±HEDTA compounds formed in soil were already too high and therefore, toxic to the plants. Addition of EDTA ap- peared to be less toxic to plants compared to HEDTA as shown by a higher biomass. However, the yields be- tween EDTA and HEDTA were not statistically dier- ent. A possible reason was due to the dierent toxicity of the two chelators and/or their metal±chelator com- pounds formed. As a whole, this study demonstrated that synthetic chelator addition had a signi®cant adverse eect on plant growth. Fig. 1. Eect of adding chelators on shoot biomass of 9 plants grown in heavy-metal-contaminated soil. (a) Cd, Cr, and Ni at 50 mg/kg of each, (b) Cd, Cr, Ni spiked at 30 mg/kg of each. Bars marked with (*) are statistically dierent with the control P < 0:05. Error bars represent ÆSE of (n  3). 24 H. Chen, T. Cutright / Chemosphere 45 (2001) 21±28 2.7.2. Eects of chelators on mobile fractions of Cd, Cr, and Ni in soil As anticipated, chelator addition signi®cantly in- creased the mobile fractions of Cd, Cr, and Ni as com- pared with control (Fig. 2). Cr had the greatest increase as its mobile fraction was raised by approximately 40- fold in HEDTA-treated soil and 60-fold in EDTA- amended soil (Fig. 2(b)). Cd and Ni were also increased by more than 4- and 2-fold, respectively (Figs. 2(a) and (c)). The mobile fractions of Cd and Ni were shown to increase with increasing levels of HEDTA and EDTA added to the soil (Fig. 2). For chromium, however, in- creasing of mobile fraction was more strongly dependent on chelator species than on chelator concentration. Since its mobile fraction did not increase when chelator levels were increased to 1.0 g/kg, it may indicate that the chelator level of 0.5 g/kg was high enough to elevate the bioavailable Cr to the maximum level. Compared with HEDTA, EDTA had approximately the same capability to increase the mobile fraction of Cd and Ni while it was more ecient at mobilizing Cr than HEDTA. The general order of bioavailable metal concentrations as a result of chelator addition in each treatment was Cr > Cd  Ni. While enhanced metal mobility can increase the up- take into plants, the potential for movement into the groundwater is also increased. An increase in metal migration to the groundwater would have a detrimental impact on the environment. Therefore, care should be taken when selecting the ®nal chelator addition for ®eld applications. The dosage must be high enough to mo- bilize the metals to the root zone without being too high to cause toxicity or elevated groundwater concentra- tions. 2.7.3. Impact of chelator amendments on metal accumu- lation in plants Fig. 3 contains the shoot and/or root tissue accu- mulation for each metal that resulted from the dierent chelator amendments. Adding chelators signi®cantly Fig. 2. Eects of HEDTA and EDTA additions on the mobile fractions of (a) Cd, (b) Cr, and (c) Ni in soil for individual metal concentrations of 30 mg/kg. For comparing chelator treatments with the control, bars marked with a (*) are statistically dif- ferent (P < 0:05). For comparing dierent chelator treatments, the mean value followed by dierent capital letters are statis- tically dierent (P < 0:05). Error bars represent ÆSE of n  3. Fig. 3. Eect of adding HEDTA and EDTA on the tissue concentrations of the dwarf sunspot sun¯ower. (a) Cd, (b) Cr, and (c) Ni with individual spiked concentration of 30 mg/Kg. For a given plant tissue, bars denoted with (*) are statistically dierent from the control. Error bars represent ÆSE of n  3. H. Chen, T. Cutright / Chemosphere 45 (2001) 21±28 25 enhanced shoot concentrations of Cd and Ni (Figs. 3(a) and (c)). The shoot content of Cd and Ni were increased by more than 2-fold and 4-fold as a result of the in- creased mobile fractions of Cd and Ni in soil. In contrast to shoot concentrations, root levels of Cd and Ni were decreased by a small fraction as compared to the con- trol. Therefore, they may be translocated to the shoot to a greater extent than the non-chelated complexes. As a result, the root concentrations of Cd and Ni were slightly lowered. Fig. 3(b) indicates that chelator additions, regardless of the source or concentration, did not increase the shoot concentration of Cr. This was surprising since the mobile fraction of Cr surged from 0.47 to over 15 mg/kg as shown in Fig. 2(b). In contrast, the root content of Cr was enhanced. The root concentration increase was ap- parently due to the increase of bioavailable Cr in soil. The Cr-chelator compound may have dierent physio- chemical properties as compared with Cd- and Ni- complexes, therefore, it could not be translocated to shoots. Analysis of Fig. 3 indicated that the 0.5 g/kg HEDTA dose had the best performance in enhancing the concentrations of Cd and Ni in shoot and the concentration of Cr in root tissue. However, it should be noted that the enhanced high tissue concentration as a result of chemical amendment might not necessarily produce a high removal eciency for the target metal contaminant since biomass change is another deter- mining factor. Some researchers (Huang and Cunningham, 1996; Blaylock, 1997; Huang et al., 1997) have reported that chelators such as HEDTA and EDTA may enhance the shoot concentration of Pb by more than 100-fold. However, in this study, these chelators demonstrated only limited capability to improve the shoot accumula- tions of Cd, Cr, and Ni. This is because most of the current chelator studies focus on single metals like Pb. Therefore inhibition from other metals would not im- pede uptake and translocation. Moreover, dierent plant species have been used in their studies. As a result, it is believed that chelator enhancement is plant- and metal-speci®c and is also subject to the interaction and subsequent inhibitory eects when multiple heavy metals are present. 2.7.4. Eect of chelator addition on total metal accumu- lation As compared with the control, the addition of che- lators decreased heavy metal accumulation by plants (Fig. 4). This was due to the severe biomass reduction. If phytoremediation enhancement with chelators is go- ing to succeed, a strategy that may protect plant bio- mass from heavy loss is necessary. In this study, EDTA at 0.5 g/kg appeared to be the best addition of the four treatments with a total removal rate of 59 lg/plant (535 lg/pan), even though it caused a decrease in compari- son with the control which was 103 lg/plant (927 lg/ pan). HEDTA at the application rate of 0.5 g/kg had the highest metal concentration increase, yet it only resulted in a total metal uptake of 42 lg/plant (376.7 lg/pan). These results indicate that the 0.5 g/kg chelator dosage may be still be too high. 2.8. Conclusions and recommendations EDTA and HEDTA both signi®cantly enhanced the metal concentration in plant tissues, however, they re- sulted in a severe biomass loss of more than 50%. As a result, the total amount of metals removed by plants was decreased. The study also determined that the eect of synthetic chelators on phytoremediation is subject to the in¯uence of multiple metal interactions and speci®c plant species. With decreasing biomass aside, the che- lator additions resulted in bioavailable metal order of Cr > Cd  Ni. For this study, the 0.5 g/kg EDTA application achieved the best results. However, at this application rate the use of chelators may not be economically Fig. 4. Eect of adding HEDTA and EDTA on the total metals accumulated by nine plants. (a) total Cd accumulation, (b) total Cr accumulation and, (c) total Ni accumulated. Error bars represent ÆSE of n  3. 26 H. Chen, T. Cutright / Chemosphere 45 (2001) 21±28 competitive with other technologies. Future studies will focus on identifying the lowest, cost-eective chelator addition that will enhance metal mobility and uptake without posing a detrimental impact on groundwater quality. 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Essential Oil Res. 8, 259±274. 28 H. Chen, T. Cutright / Chemosphere 45 (2001) 21±28 . chromium- and nickel-con- taminated soil. The preferred source (EDTA or HEDTA) and dosage required to elevate metal mobility and subsequent uptake and translocation. in HEDTA- treated soil and 60-fold in EDTA- amended soil (Fig. 2(b)). Cd and Ni were also increased by more than 4- and 2-fold, respectively (Figs. 2(a) and (c)). The