Environ. Sci. Technol. 2000, 34, 1778-1783
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Enhancement of Phytoextraction of Zn, Cd, and Cu from Calcareous Soil: The Use of NTA and Sulfur Amendments A . K A Y S E R , * ,† K . W E N G E R , ‡ A . K E L L E R , † W. ATTINGER,† H. R. FELIX,§ S. K. GUPTA,‡ AND R. SCHULIN† Swiss Federal Institute of Technology, Institute of Terrestrial Ecology, Grabenstrasse 3, CH-8952 Schlieren, Switzerland, Institute of Environmental Protection and Agriculture, CH-3003 Bern, Switzerland, and Metallophag GmbH, Bu ¨ ndtenstrasse 20, CH-4419 Lupsingen, Switzerland
In a field experiment we investigated the efficiency of two hyperaccumulating species, four agricultural crop plants, and one woody crop, at phytoextraction of Zn, Cd, and Cu from a polluted calcareous soil. In addition, we examined the possibility to enhance the phytoextraction of these metals by application of nitrilotriacetate (NTA) and elemental sulfur (S8) to the soil. Metal uptake by hyperaccumulating species was higher than that by crop species but was generally low in all treatments compared to results reported in the literature, maybe as a result of lower total and available soil metal concentrations. Soil amended with either S8 or NTA increased the solubility (NaNO3-extraction) of Zn, Cd, and Cu ions by factors of 21, 58, and 9, respectively, but plant accumulation of these metals was only increased by a factor of 2-3. As a result, even the highest metal removal rates achieved in this study were still far from what would be required to make this technique practicable for the remediation of the Dornach field site. To extract for example 50% of the total Cu, Zn, or Cd present in this soil within 10 years, plant metal concentrations of 10.000 mg kg-1 Cu or 10.000 mg kg-1 Zn or 45 mg kg-1 Cd would be required at a biomass production of 7.8 t ha-1, or 10t ha-1, or 10t ha-1, respectively, assuming a linear decrease in soil metals.
Introduction Pollution of soils by heavy metals is of considerable concern with respect to health risks, phytotoxicity to plants, longterm effects on soil fertility, and depreciation of land. A particular problem is the wide-spread low- to medium-level pollution of agricultural and other cultivated land. Conventional cleanup technology, in turn, is generally too costly, and often harmful to desirable soil properties (i.e. texture, organic matter), to be used for the restoration of such sites. As a consequence, there is a need which exists for more effective in situ methods for the remediation of metalcontaminated sites. In recent years, a number of authors * Corresponding author phone: ++41-1-6336011; fax: ++41-16331122; e-mail:
[email protected]. † Swiss Federal Institute of Technology, Institute of Terrestrial Ecology. ‡ Institute of Environmental Protection and Agriculture. § Metallophag GmbH. 1778
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have proposed the use of metal-accumulating plants to remove metals from soil (1-4). Early phytoremediation research focused on “hyperaccumulating” plant genera (e.g. Thlaspi (5) or Alyssum (6)). These plants possess an ability to take up abnormally high amounts of heavy metals. For example, under certain circumstances (i.e. when grown on heavily polluted mine soils), Zn in T. caerulescens can account for up to 3% of the aboveground biomass (7). Unfortunately, biomass production in most hyperaccumulator species is low, thereby limiting heavy metal removal from soil, since total metal extraction is the product of biomass and tissue concentrations. As an alternative, it has been suggested to use high biomass species such as oat (Avena sativa), tobacco (Nicotiana tabacum), or maize (Zea mays) (8-10) with improved genetic potential to accumulate metals, or to improve plant husbandry and soil management practices to enhance metal uptake. Plant breeding and selection is under way to improve metal uptake capabilities in these plants (11). Regardless of the plants used, availability of heavy metals to plant roots is considered a key factor limiting the efficiency of phytoextraction (12). Phytoavailability of metals is strongly controlled by soil factors such as pH, cation exchange capacity, or organic matter content, any of which may limit successful soil remediation. To increase metal availability, the use of complexing agents such as DTPA or EDTA has been proposed (13, 14). These synthetic chelators can desorb heavy metals from the soil matrix to form water soluble metal complexes. In addition to complexation of metals, the lowering of soil pH has been proposed as an enhancement tool in phytoextraction (15). This assay is of special interest for neutral or slightly alkaline soils contaminated with Zn and Cd, since these metals are readily solubilized at moderate degrees of acidity. Restrictions apply, however, to both the use of complexing agents and artificial soil acidification. Many synthetic chelators, e.g. EDTA, show a low degree of biodegradability (16). In situ application of such chelators bears the risk of water pollution by uncontrolled metal solubilization and leaching. Similar restrictions apply for artificial soil acidification. In addition, the amount of acidifying agents required may become prohibitive on well buffered, in particular carbonate rich, soils. Some of the restrictions that apply to chelate-assisted phytoextraction may be overcome by usage of easily biodegradable chelating agents, such as Nitrilotriacetate (NTA) (17), and injection to the root zone of metal-accumulating plants rather than surface application. Minimum risk acidification, on the other hand, may be achieved by the application of elemental sulfur (S8), which, in soils, is gradually oxidized by sulfur-oxidizing bacteria (18). In the present study, we (a) compared the efficiency of four agricultural crop plants and tree species as well as two hyperaccumulator plant species and (b) investigated the potential to increase metal uptake by these plants through application of low doses of NTA and usage of elemental sulfur.
Material and Methods Site Description. The experimental site was located in the village of Dornach, NW Switzerland (approximately 300 m above sea level). The site has been heavily contaminated with Cu, Zn, and Cd by about a century of particulate emissions from a brass smelter located 300 m west. Dornach has a temperate climate, with a mean annual temperature of 10 °C, and an average annual rainfall of 800 mm. The soil 10.1021/es990697s CCC: $19.00
2000 American Chemical Society Published on Web 03/28/2000
TABLE 1: Selected Soil Properties and Heavy Metal Distributiona pH
CaCO3
Corg
clay
silt
0-10 -20 -30 -40 -50 -60
7.3 ( 0.1 7.3 ( 0.1 7.3 ( 0.1 7.3 ( 0.1 7.4 ( 0.1 7.6 ( 0.1
14 ( 3 13 ( 3 17 ( 7 19( 13 19 ( 12 20 ( 19
6.0 ( 0.4 6.0 ( 1.0 4.2 ( 2.0 3.3 ( 2.9 2.2 ( 1.6 1.7 ( 1.3
38 ( 1 33 ( 2 32 ( 6 31 ( 8 32 ( 8 32 ( 5
39( 9 35 ( 1 33 ( 4 36 ( 10 48 ( 11 45 ( 11
a
sand
CECpot (mequiv 100 g-1)
Cdtot
Zntot
Cutot
23 ( 8 32 ( 2 35 ( 11 33 ( 18 20 ( 19 24 ( 16
31 ( 9 29 ( 7 25 ( 5 25 ( 7 20 ( 5 20 ( 5
2.5 ( 0.3 2.1 ( 0.7 2.2 ( 0.6 2.0 ( 0.8 1.4 ( 0.2 1.1 ( 0.1
673 ( 115 360 ( 165 732 ( 243 389 ( 259 122 ( 53 44 ( 1
516 ( 57 568 ( 91 521 ( 178 322 ( 137 69 ( 36 21 ( 1
(%)
depth (cm)
(mg kg-1)
(µg kg-1) Cdsol
Znsol
Cusol
2.2 ( 0.7 90 ( 20 700 ( 110 2.4 ( 0.8 110 ( 30 800 ( 80 2.7 ( 0.6 140 ( 70 720 ( 230 2.6 ( 1.3 120 ( 80 610 ( 290 1.0 ( 0.1 30 ( 10 180 ( 100 1.0 ( 0.1 30 ( 10 120 ( 40
Mean and standard deviation of samples taken from three soil profiles surrounding the Dornach field site.
had been classified as Calcaric Fluvisol (19) but has since been disturbed by road construction. Selected soil characteristics and heavy metal contents are summarized in Table 1. Experimental Design. Seven plant species were grown on plots of 2.5 × 2.5 m, with four replicates per species. Plants were cultivated on the same plots in two consecutive years. To ensure comparable growing periods, experiments were conducted between the beginning of May and the first week of September each year. During the first year, no additional solubilization treatments were administered. In the second year, plots were subdivided into four subplots of 1.25 × 1.25 m, and soil amendments were added separately to three of these subplots. The remaining subplot was kept as a control. Soil amendments used were elemental sulfur (S8) (36 mol m-2) and two dosages of NTA (4.2 and 8.4 mmol per injection treatment, respectively). Sulfur was sieved to 0.05).
b
Treatments within the same plant and metal with letters in common are not significantly different
To permit direct comparison of the effects of solubilization treatments on plant metal uptake, we evaluated the results in terms of a relative treatment effect (RTE) following Krebs (24) (eq 1).
RTE )
T1998 C1997 * T1997 C1998
(1)
The RTE relates the heavy metal concentration of a plant grown on a particular subplot in the year of treatment (T1998) to that of the first year (T1997), in which no treatments were applied. The ratio C1997:C1998 between the heavy metal concentrations measured in plants grown on adjacent control subplots in the corresponding years (C1997 and C1998, respectively) represents a correction term to account for differences in heavy metal uptake that occurred between experimental years as well as the spatial variability within the experimental site. All plant species responded to the increase in NaNO3extractable heavy metals with an increase in metal uptake (Figure 1). Table 2 shows the means and standard deviations of the RTE for the four replicate subplots of each plantamendment combination. In terms of average RTE, calculated as the mean RTE for all species, the NTA-2-treatment was the most effective, for all three metals. Average RTE values of Cd, Zn, and Cu for the NTA-2 treatment were 1.9, 1.9, and 2.4, respectively. Variability of RTE between species was generally small in this treatment. Cu uptake in S. viminalis increased the most, but in this particular case, variability between replicates was relatively high. In one plot, the RTE reached a value of 8. NTA-1 was less effective than NTA-2, but enhanced uptake was also observed for all metals. For the NTA-1 treatment, average RTE values were 1.3, 1.5, and 1.6 for Cd, Zn, and Cu, respectively. The strongest effects were found in N. tabacum and S. viminalis, but, again, variability between S. viminalis replicates was high. RTE for S8 amended subplots varied between the effects of the two NTA treatments with respect to Cd and Zn but had no effect on Cu uptake. Average RTE was 1.7 for Cd, 1.8 for Zn, and 1.0 for Cu, respectively. In general, metal uptake was increased most in S. viminalis and N. tabacum, as in the NTA treatments. Total Removal of Heavy Metals. Mean total heavy metal phytoextraction in the second experimental year, when sulfur and NTA amendments were applied, is shown in Figure 3. With the exception of Cd removal by H. annuus, which reached a maximum of the mean of four replicates on S8amended subplots, maximum metal removal was achieved through NTA-2 application. The best results for Cd phyto-
FIGURE 3. Total removal of heavy metals by plants grown with and without soil amendments in second experimental year. Error bars denote standard deviation of subplot averages (n ) 4). extraction were achieved using N. tabacum, whereas H. annuus and Z. mays were best for Zn removal, and H. annuus removaled the most Cu from soil. Even without treatment, the removal of metals by the crop plants was in the same order of magnitude or higher than that by the hyperaccumulator species.
Discussion Plant Growth and Uptake of Heavy Metals without Solubilization Treatment. Growth of the hyperaccumulating species was affected by high mortality, resulting in poor soil coverage in both years. Development of the surviving individuals, however, appeared normal, and plants reached growing heights comparable to values reported in the VOL. 34, NO. 9, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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literature (25). Nevertheless, even when extrapolated to full soil coverage, yields were much lower than reported by other authors. In our study the extrapolated dry weight of T. caerulescens ranges from 0.6 to 1.0 t ha-1 (actual mean of yield 0.49 t ha-1) as compared to 4.6 to 9.9 t ha-1 reported in the literature (26). Biomass production in most crop plants was normal or even high relative to tabulated average yields for Swiss agriculture (27). The cultivars of H. annuus used were probably among the best suited for the conditions of our site. Biomass production of H. annuus almost tripled that given in the above survey (27). Yields in S. viminalis were low in both experimental years, but much faster growth can be antizipated in subsequent years, once the plants are fully established, as was demonstrated in energy production studies in Sweden (28). In all plants, Zn and Cd uptake were considerably higher than that observed for agricultural crops and garden plants grown on the same field in earlier years (29), whereas Cu uptake was in the same range. The relatively low metal concentrations found in T. caerulescens and A. murale are in good agreement with results from an earlier experiment near our site (12), where concentrations of 2545 and 675 mg kg-1 of Zn were recorded in T. caerulescens and A. murale, respectively. Accumulation of Zn and Cd by these species was much lower in our case than reported in the literature for plants grown in nutrient solutions or on more heavily contaminated and probably more acidic soils or mine spoils. This was especially evident in the hyperaccumulator species. As an example, T. caerulescens has been reported to take up more than 25 000 mg kg-1 Zn (26, 27, 30) and 250 mg kg-1 Cd (31). Other authors found Cd concentrations of more than 47 mg kg-1 in Z. mays (32) grown in nutrient solution. The relatively poor metal uptake by the plants in this study may be attributed to limitations in metal availability in this particular soil. Given the high pH values, only minor fractions of the total heavy metals present in soil were soluble (Table 1). Metal uptake was increased significantly when solubilization agents were applied to the soil (see below). Metal Solubilization in Soil. Both NTA treatments increased the amount of Zn, Cd, and Cu readily extracted from the treated soil without decreasing soil pH. This indicates that the heavy metals formed soluble complexes rather than responding to a pH effect. The solubilization effects by NTA were not as effective as observed in an earlier greenhouse study, where the same soil was treated with NTA pulses of 1.33 mmol kg-1 of soil at comparable time intervals between treatments (17). In that greenhouse study, NaNO3extractable Zn and Cu concentrations were 12 and 24 mg kg-1 of soil after NTA treatment, respectively. In contrast, in the current study the NTA-2 (2 mmol kg-1 soil) treatment caused the mean NaNO3-extractable Zn and Cu to be 4 and 9 mg kg-1 soil, respectively. The differing results may be attributed to different experimental conditions. In our field experiment, NTA was injected directly into the root zone of the plants, whereas soil was sampled as a mixed sample from an entire subplot. Hence, the actual solubilization of metals in the root zone was certainly less uniform than in greenhouse pot studies, where also the roots tend to be more homogeneously distributed (17). On the S8-treated plots, a small decrease was observed in soil pH, determined from bulk samples. Since carbonate content is high and by far sufficient to buffer H+ input through S8 application in the Dornach soil, this may indicate that buffering is limited kinetically. Despite the very small decrease in soil pH, S8 application significantly increased Zn and Cd mobility. High doses of S8 were required in comparison to NTA treatments to achieve a similar solubilization of Zn and Cd. Furthermore, no effect was observed on Cu solubilization. This is in agreement with the chemical speciation of Cu in 1782
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soils, the availability of which usually increases only when pH drops below 5.5. In our study, the lowest pH value recorded was 6.2. Plant Uptake of Heavy Metals after Solubilization Treatment. While NaNO3-extractable soil concentrations of Cd, Zn, and Cu were increased by the soil amendments, they did not translate into an equivalent increase in metal uptake by the plants. In no case did the solubilization treatment lead to a more than an 8-fold increase in plant tissue concentrations of any metal. Average RTE values between 1.6 and 2.2 show a roughly 100% increase of metal uptake compared to the controls. Much higher tissue concentrations (i.e. Cd concentrations of 16.3, 2.8, and 14.2 mg kg-1 dry matter) had been observed in our preliminary greenhouse studies, when N. tabacum, Zea mays, and B. juncea were grown on the same soil in pots. The different effects of NTA on metal solubility in soil and on plant uptake may partially be attributed to a short duration of the mobilizing effect and the fact that our soil sampling was carried out shortly after application, when the effect was still maximum. In a preliminary greenhouse study (33) the effect of NTA on metal mobility decreased sharply within 3 weeks of application. This was consistent with a laboratory incubation experiment, where NTA was found to degrade rapidly in soil environments (34). Shorter application intervals of NTA may therefore result in higher metal uptake by the plants. Another reason may be metal toxicity to root growth and function whereby metabolically active growing roots avoid solubilization zones in the field study. Sampling of roots of B. juncea and S. viminalis indicated a tendency that roots of untreated plants were more abundant in the top 20 cm. However, this was not statistically significant (35). Metal uptake may also have been limited by physiological barriers (i.e. specific uptake systems), inhibiting the uptake of synthetic chelates by plants, but considerable debate exists on that issue. While some authors reported that the use of chelating substances such as DTPA or EDTA did not enhance (36), or even decrease (37), heavy metal uptake, others observed impressive increases after EDTA and DTPA application (38) and suggest that synthetic chelates enhance translocation of certain metals from roots to shoots (39). It appears that further investigations are needed to clarify this subject and identify the regulatory mechanisms involved. Total Removal of Heavy Metals. Because the solubilization treatment had no negative effect on yield of the crop species, increased metal accumulation by the plants directly translated into equivalent increases in metal removal. Since differences in yield between plant species were higher than species differences in metal accumulation, crop plants were more effective in extracting heavy metals from soil than the hyperaccumulator species. However, even the highest metal removal rates achieved in this study were still far from what would be required to make this technique practicable for the remediation of the Dornach field site. To extract 50% of the total heavy metal present in this soil within 10 yearss assuming a linear decrease in soil metal concentrationsa plant would be required to take up 10.000 mg kg-1 of Cu and produce a biomass of 7.8t ha-1; 10.000 mg kg-1 of Zn with a biomass of 10t ha-1; and 45 mg kg-1 of Cd with a biomass of 10t ha-1. Other tissue concentration:biomass combinations resulting in equal metal removal would also be desirable. None of the crops or the hyperaccumulator plant species examined in this study removes metals in accordance with their reported potentials. While the crop species showed maximum biomass production, metal uptake was only 1030% of that reported in the literature (8, 9, 32, 40, 41). Metal accumulation by the hyperaccumulator species was considerably below that reported for plants grown on natural sites, greenhouse, and hydroponic solution studies (5, 37). In addition, biomass production was small compared to the
crop species. Little has been published on the biomass of hyperaccumulator species grown under field conditions. Some data suggest that yields of T. caerulescens and N. tabacum may be in the same range (26). Although numerous pilot studies conducted under greenhouse or climate chamber conditions indicate that metal extraction by both the crop and hyperaccumulating species hold potential for removing metals from contaminated soil (1, 3, 8, 10, 30), this field study shows that this may not apply to all kinds of soils. The results indicate that limitations in phytoavailability of metals can be a major obstacle, especially in soils high in pH. Thus, if efficient and environmentally safe methods of metal solubilization were developed, phytoremediation potential could possibly be greatly enhanced.
Acknowledgments This work was supported by the Swiss National Science Foundation, Swiss Priority Program Environment (Grant 044749). We gratefully thank A. Baker, N. Billenkamp, M. Greger, Phytotech Inc., Samen Mauser, and UfA for providing seeds and seedlings and wood cuttings. Our gratitude goes to J. D. Joslin for his patience proofreading the manuscript as well as to A. Gru¨nwald and K. Barmettler for their analytical work.
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Received for review June 21, 1999. Revised manuscript received December 2, 1999. Accepted February 1, 2000. ES990697S
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