The ability of Helianthus annuus L. and Brassica juncea to uptake and translocate natural uranium and 226Ra under different milieu conditions

Chemosphere 74 (2009) 293–300

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The ability of Helianthus annuus L. and Brassica juncea to uptake and translocate natural uranium and 226Ra under different milieu conditions F. Vera Tomé a,*, P. Blanco Rodríguez b, J.C. Lozano c a

Departamento de Física Aplicada, Facultad de Ciencias, Universidad de Extremadura, 06071 Badajoz, Spain Departamento de Física, Facultad de Ciencias, Universidad de Extremadura, 06071 Badajoz, Spain c Laboratorio de Radiactividad Ambiental, Facultad de Ciencias, Universidad de Salamanca, 37008 Salamanca, Spain b

a r t i c l e

i n f o

Article history: Received 21 May 2008 Received in revised form 3 September 2008 Accepted 3 September 2008 Available online 10 October 2008 Keywords: Phytoremediation Natural radionuclides pH Phosphates EDTA Citrate

a b s t r a c t Seedlings of Helianthus annuus L. (HA) and Brassica juncea (BJ) were used to test the effect of the pH, the presence of phosphates, and the addition of ethylene-diamine-tetraacetic acid (EDTA) or citrate on the uptake and the translocation of uranium isotopes (238U, 235U, and 234U) and 226Ra. The results indicated that the presence of phosphates generally reduces the uptake and transfer of uranium from the roots to the shoots of HA. In the case of BJ, while phosphate enhanced the retention of uranium by roots, the translocation was poorer. Likewise, for 226Ra, the best translocation was in the absence of phosphates for both species. The addition of citrate increased the translocation of uranium for both species, but had no clear effect on the transfer of 226Ra. The effect of EDTA was much more moderate both for uranium and for 226Ra, and for both plant species. Only noticeable was a slightly better uptake of 226Ra by BJ at neutral pH, although the translocation was lower. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction The determination of the activity concentration of the natural uranium isotopes (234U, 235U, and 238U) and 226Ra in different compartments of ecological importance has long been a topic of concern (Gerzabek et al., 1998; Bernhard, 2005). Several studies have described the distribution and mobility of these radionuclides in the environment (IAEA, 1982; Vera Tomé et al., 2002), and their transfer between natural compartments (IAEA, 1994; Baker and Toque, 2005). Especially important is the transfer from soil to plants as a first step in the pathway of incorporation of radioactive species to humans (Bunzl and Trautmannsheimer, 1999; Ehlken and Kirchner, 2002), and hence for the estimation of radiological risks (IAEA, 1994; Begonia et al., 1998). The use of plants for the decontamination of environmental systems is another area of interest (Prasad and Freitas, 2003; Arthur et al., 2005). These techniques are collectively termed phytoremediation, which is a promising cost-effective alternative to more conventional methods for decontamination (Raskin et al., 1997; Garbisu and Alkorta, 2001). Soils and waters have frequently been the object of phytoremediation applications (Chen and Cutright, 2001; Lee et al., 2002a). In both cases, seedlings of terrestrial plants have been the most studied (Dushenkov et al., 1995; Lasat, 2002).

* Corresponding author. Tel.: +34 924 289524; fax: +34 924 289651. E-mail address: [email protected] (F. Vera Tomé). 0045-6535/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2008.09.002

The selection of an appropriate plant species for decontamination is a crucial step (Dushenkov et al., 1995; Huang et al., 1998), but the protocols followed for screening between candidate species can result in very complex procedures (Mkandawire and Dudel, 2005). From the literature one gathers that Helianthus annuus L. (HA) (Chen and Cutright, 2001; Meers et al., 2005) and Brassica juncea (BJ) (Huang et al., 1998; Prasad and Freitas, 2003) are two promising candidates for phytoremediation. Both have been studied in hydroponics trials (Lee et al., 2002a) at the laboratory scale (Chen and Cutright, 2001) and at the field scale (Vanek et al., 2002) for the decontamination of soils and water containing low to moderate levels of heavy metals (Chen and Cutright, 2001), artificial radionuclides (Lee et al., 2002a,b), and natural radionuclides (Vera Tomé et al., 2008). Uranium has been widely studied, alone or together with other heavy metals (Huang et al., 1998; Gramss et al., 2004). However, 226 Ra has been less widely studied in the context of phytoremediation (Soudek et al., 2004; Vandenhove and Van Hees, 2007), although numerous studies have been devoted to the determination of its transfer from soil to plants (Soudek et al., 2007). Here, our main objective was to compare the uptake process in HA and BJ seedlings for natural uranium and 226Ra from a hydroponics medium containing known activities of these radionuclides. The aim is to assess the potential of the two species for the elimination of uranium and 226Ra under defined chemical conditions in order to study the differential behaviour of the plants

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F. Vera Tomé et al. / Chemosphere 74 (2009) 293–300

under stress. Mkandawire et al. (2006) studied the effects of stress on fronds of Lemna gibba in the presence of phosphate and uranium in solution. This was done in the present experiments by changing the pH, and by introducing phosphate into the solution (Mkandawire et al., 2005). Another set of trials consisted of conditioning the solution by the addition of the chelating agents citric acid and ethylene-diamine-tetraacetic acid (EDTA), widely used for the amendment of soils (Shahandeh and Hossner, 2002; Lesage et al., 2005). The importance of pH for element speciation in solution and therefore for the plant uptake process is well known (Prasad and Freitas, 2003; Niu et al., 2007). The concentration of phosphate was selected to introduce a stress factor, since its excess can indirectly have, through the formation of complexes and precipitation, negative effects on many plant species by reducing the availability of essential elements. Additionally, chelating agents were added in order to represent a situation frequently found after the amendment of soils (Kirpichtchikova et al., 2006). In particular, in the chemical conditioning of soils, chelating agents are used with two main aims: the first, to enhance the passage of toxins from the soil matrix to the soil solution; the second, to improve the bioavailability to plants of those chemicals (Schmidt, 2003; Evangelou et al., 2007). Once applied, and depending on the amounts used in the amendment, chelating agents normally appear in the soil solution (Lombi et al., 2001), sometimes at high concentrations, and occasionally reaching groundwaters together with the metal-chelates (Alkorta et al., 2004). Again their presence in such concentrations can also have negative effects on plants (Evangelou et al., 2007). 2. Materials and methods 2.1. Seedling growth Seedlings of HA and BJ were grown hydroponically in 250 mL wide-mouth polypropylene vials containing 200 mL of nutrient solution with (in g L 1) 0.708 Ca(NO3)2
4H2O, 0.492 MgSO4
7H2O, 0.17 KNO3, 0.272 KH2PO4, 0.0083 FeCl3, 0.0025 H3BO3, 0.0015 MnCl2
4H2O, 0.0001 ZnCl2, 0.00005 CuCl2
2H20, and 0.00005 Na2MoO4
2H2O (Jones, 1997). After 4 wk, the nutrient solution was removed, and 200 mL of fresh nutrient solution containing the standard solutions of natural uranium and 226Ra was added. Additional details can be found elsewhere (Vera Tomé et al., 2008). The experimental protocols were similar for both natural uranium and 226Ra. About 6 Bq (2.5  10 4 g) of natural uranium, or 4 Bq (1.1  10 10 g) of 226Ra (CIEMAT, Madrid, Spain) were added to each nutrient solution. 2.2. Experimental design 2.2.1. Influence of pH and phosphates In order to determine the influence of the pH and phosphates on the uptake of natural uranium and 226Ra, we designed an experiment with variable pH values of 5.0, 6.7, and 8.0. The pH was adjusted with addition of either HNO3 or KOH. For each pH value, the experiment was performed with and without phosphates in the nutrient solution. Three replicates were considered in all the cases. Hence, eighteen seedlings were grown for each radionuclide studied and for each species selected (HA and BJ). The seedlings were grown in a tracer-free pH 6.7 solution for 4 wk. The solution was then replaced with a fresh nutrient solution containing the tracer of natural uranium or 226Ra. The pH value was adjusted as noted above to 5.0, 6.7, or 8.0. The seedlings were allowed to grow for one more week before harvest (Vandenhove and Van Hees, 2004).

2.2.2. Influence of EDTA and citrate The reagents EDTA and citrate were added to the nutrient solution as salts, in order to avoid any drastic influence on the pH value. Values were adjusted to either 5.0 or 6.7. The pH 8.0 condition was not used because it induced poor growth in seedlings in combination with a chelant. Since the degradation of citrate can produce changes in pH (Ebbs et al., 1998a; Chang et al., 2005), the pH was monitored daily and readjusted when necessary. Concentrations of 5 mmol kg 1 of EDTA and 20 mmol kg 1 of citrate have been used by other workers (Huang et al., 1998; Chen and Cutright, 2001). Hence, we initially selected concentrations of 5 mM for EDTA, and 20 mM for citrate. However, laboratory trials of phytotoxicity indicated that this EDTA concentration had a negative effect on the seedlings. Therefore the EDTA concentration was reduced. Concentrations of 4 mM for EDTA and 20 mM for citrate were finally selected. Five replicates were considered in each case. As in the previous experiment, the seedlings were grown for 4 wk in a tracer-free solution, and then the solution was replaced with 200 mL of fresh nutrient solution with the chelating agent and spiked with added natural uranium or 226Ra. The seedlings were allowed to grow for 1 wk more before harvest. Fifteen seedlings of each species (HA and BJ) were grown for each radionuclide and each pH value: five without agent (control), five with EDTA, and five with citrate. 2.3. Radiochemical methods and measurement techniques After the total dissolution of the samples by acid digestion under pressure, the activity concentrations of the uranium and radium isotopes in both shoots and roots were determined by alpha-spectrometry. For uranium isotopes, the radiochemical method followed until the preparation of the high-resolution alpha sources was based on the chemical separation by tri-n-butyl phosphate (Sam et al., 1998). Finally, the sample was electrodeposited (Vera Tomé et al., 1994). The method used for the determination of radium was based on chemical purification by precipitation of PbSO4/BaSO4, and the subsequent source preparation by microprecipitation of Ba(Ra)SO4 (Blanco et al., 2002). The radiotracers used for the determination of the radiochemical yields of uranium and radium were 232U and 225Ra, respectively, added at the start of the radiochemical procedure. 2.4. Evaluation of the speciation The Geochem code version 2.0 (Parker et al., 1995; Ebbs et al., 1998b) was used to evaluate changes in the speciation of uranium and radium with pH, the presence or absence of phosphate, and the chelating agents tested (Table 1). However, this evaluation has to be considered with caution, because the code does not include the participation of the plant. Moreover, the high concentrations of the anions phosphate, citrate, and EDTA used here likely also mask the effects of the exudates from the roots. Plant exudates can contribute to changing the pH, the Eh, and the predominant chemical species of the toxins of concern (Pivetz, 2001; Niu et al., 2007). Even so, we used the simulation results as a first approximation to the chemical mechanisms in the near-root zone. 3. Results and discussion The results of natural uranium will hereafter be expressed as activity of the isotope 238U. Although natural uranium was used as spiker in every experiment, the isotope 238U practically confers all the mass to the element. Moreover, in our analyses the activity ratio between 238U and 234U was always in agreement with the natural equilibrium, so that any conclusion obtained from the destiny of 238U can be equally applied to 234U. The third isotope

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F. Vera Tomé et al. / Chemosphere 74 (2009) 293–300 Table 1 Speciation of uranium and radium at different pH values, as obtained by using the Geochem code Element

Amendment applied to the basic nutrient solution

Uranium

Phosphate

Phosphate + citrate 20 mM Phosphate + EDTA 4 mM Radium

Phosphate

Phosphate + citrate 20 mM

Phosphate + EDTA 4 mM

Species

Percentage of total element amount pH = 5.0

UO22+ UO2
CO3 UO2
(CO3)22 UO2
(CO3)34 UO2
SO4 UO2
(SO4)22 UO2
OH+ (UO2)2
(OH)22+ (UO2)3
(OH)5+ UO2
(CO3)22 UO2
(CO3)34 UO2
H2
(PO4)22 UO2
H
Citr UO2
H2
(PO4)22 Ra2+ Ra
SO4 Ra
NO3+ Ra
(NO3)2 Ra2+ Ra
SO4 Ra
H
PO4 Ra
H2
PO4+ Ra
EDTA2 Ra
NO3+ Ra
(NO3)2 Ra2+ Ra
SO4 Ra
H
PO4 Ra
H2
PO4+ Ra
Citr Ra
H
Citr Ra
H2
Citr+ Ra
NO3+ Ra
(NO3)2 Ra2+ Ra
SO4 Ra
H
PO4 Ra
H2
PO4+ Ra
EDTA2Ra
H
EDTA Ra
NO3+

of uranium, 235U, is considerably less important in the context of this study because it appears in the natural isotopic mix only as a minor contributor whether in mass units or in activity. The parameters to analyze are the transfer factor (TF), defined as the ratio of the activity concentration in the plant (Bq kg 1) to the activity concentration in the substrate (in our study Bq L 1) ( ICRU, 2001), and the translocation ratio (TR), defined as the ratio of the activity concentrations in shoots and roots (dimensionless) (ICRU, 2001). The results of replicated series were compared by Student’s ttest using as null hypothesis the identical mean of the populations, and considering a 95% confidence level. 3.1. Uranium uptake When the nutrient solution was initially free of phosphate and chelating agents, HA was more efficient than BJ at transferring uranium from the solution to the roots of the seedling at all the pH’s tested. For HA, the lowest root-TF was at pH 6.7 and the highest at pH 8 (Fig. 1a). This behaviour is in agreement with that reported by Dushenkov et al. (1997) at pH 5 and 7. Probably, at acidic pH, the free uranyl ion is available to be adsorbed by ion exchange onto the charged walls of the roots, or even assimilated directly. Ebbs et al. (1998b) describe this behaviour in Pisum sativum. At pH 6.7, the prevalent form of uranium is a neutral complex with car-

33 48 <1 11 <1 4 1 <1

pH = 6.7

2 73 25

<1

<1

<1

100 100 100

100 100 100

88 9 3 <1 87 9

88 9 3 <1 86 10 <1 <1

<1 3 <1 37 2 <1 36 23 <1 <1 <1 75 8 <1 7 7 2

pH = 8.0

<1 33 65 1 <1

3 <1 15 <1 <1 <1 83 1

<1 <1 100

87 9 3 <1 86 11 <1 <1 3 <1

<1 61 6 <1 <1 30 <1 2

bonate (Table 1) which should remain in solution. When the pH rises to 8, one expects the appearance of precipitates [Fe(OH)3, CaCO3] (Parker et al., 1995) which could coprecipitate or occlude the uranium and fix it onto the wall of the roots. The results indicate that the carbonate complexes of uranium could be directly assimilated by HA roots. Similar results have been observed in the roots of Phaseolus vulgaris (Laroche et al., 2005). For BJ, the tendency observed for root-TF with pH, with the highest value at pH 6.7, is surprising (Fig. 1b). As will be seen below, similar behaviour was found for radium. This suggests that the two radionuclides simultaneously participate in chemical compounds that are involved in the retention by the roots. At the pH values studied, uranium and radium speciate very differently in the solution (Table 1). This leads one to think that there may be exudation of a ligand that can promote the conjoint precipitation (or coprecipitation) of radium and uranium. Phosphate could play this role. When there are phosphates in solution, the TF values for 238U in roots and shoots of HA systematically decreased (Fig. 1a). This result was observed at every pH, although the values followed different trends to those observed without phosphate in solution. The 238 U root-TF of HA decreased as the pH became more alkaline, with a tenfold reduction from pH 5 to 8. The formation of a precipitate of uranium with phosphates should reduce the availability of uranium in solution. This could explain the behaviour observed for

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F. Vera Tomé et al. / Chemosphere 74 (2009) 293–300

a

Helianthus annuus L.

c

Helianthus annuus L.

8

30

6

U TF (L·kg )

-1

2 0

Shoots Roots

238

238

-1

U TF (L·kg )

20 4

1000

10 Shoots 0 Roots 1000

2000 3000

2000

4000 3000 5000 Without P With P 6

Brassica5 juncea 6,7

8

Brassica juncea

b

pH 5 pH 6,7

d

4 3

4

U TF (L·kg )

0

-1

Shoots Roots

238

238

-1

U TF (L·kg )

2 2

1000

1 0

Shoots Roots

500 1000

2000

1500 2000

3000 2500

5.0

6.7

8.0

Control

EDTA

Citrate

pH Fig. 1. 238U uptake to shoots and roots in the presence and absence of phosphates at three different pH values. (a) HA, and (b) BJ. Error bars denote the standard error of the mean (n = 3). Also shown is the effect of adding EDTA and citrate at two pH values. (c) HA, and (d) BJ. Error bars denote the standard error of the mean (n = 5).

the root-TF of uranium by HA: as the pH increased, the formation of precipitate also increased, and therefore the availability of uranium decreased. This tendency was not so clear for BJ (Fig. 1b), where for neutral and alkaline conditions, the root-TF values were statistically indistinguishable. Contrary to the behaviour observed for HA, the root-TF of 238U to BJ was higher when there was phosphate in the solution. This result was the case for all three pHs values tested, but was more evident at pH 5. At this pH value, the root-TF of 238U in the presence of phosphates was 25 times greater than without phosphates. These results indicate that the process of 238 U uptake by the roots was different for the two species tested. If there is really the formation of a precipitate, it seems that BJ is able to retain this precipitate on its roots more efficiently than HA. The addition of EDTA to the nutrient solution does not change the uranium speciation, which remains as a phosphate complex (Table 1). However, the influence on other elements in the solution can indirectly modify the ultimate speciation of uranium. If there is phosphate in solution, one expects the uranium to form a complex with phosphate, which can evolve to precipitation in the absence of other ligands. The addition of chelating agents can change this evolution, avoiding the precipitation.

The results obtained for HA showed that, at pH 5, the use of EDTA did not improve the root-TF of 238U to HA relative to the control samples, but the citrate did (Fig. 1c). By modifying the pH to the neutral region (pH 6.7), certain changes were observed for 238 U. The root-TF of 238U increased in the presence of both chelating agents relative to the control seedlings. The treatment with EDTA did not change significantly the root-TF relative to the value found at pH 5, whereas the addition of citrate led to a worse rootTF. With respect to BJ, at pH 5, the highest root-TF value of 238U was observed for the control seedlings (Fig. 1d). Concentrations of 4 mM EDTA or 20 mM citrate caused a decrease in the root-TF for BJ, especially in the presence of citrate for which the root-TF did not exceed a value of 300 L kg 1. When the pH was adjusted to 6.7, the root-TF obtained after the treatment with EDTA was statistically indistinguishable from that at pH 5. In the case of citrate, there was a decline relative to the value obtained at pH 5. The results observed for the root-TF in the presence of EDTA were in agreement with the hypothesis that, in the presence of phosphate and without EDTA, the adsorption of a fraction of uranium onto the root surface could occur by precipitation. This

F. Vera Tomé et al. / Chemosphere 74 (2009) 293–300

would explain why no decline in root-TF was observed with pH in the presence of phosphate when there was EDTA in the solution. The presence of EDTA, by avoiding the precipitation, would maintain the root-TF value statistically indistinguishable from the control seedlings at pH 5. The addition of citrate to the solution had different effects on the root-TF of uranium for the two plant species. In the case of HA, the root-TF values were greater than those of the control seedlings at both pH values. However the presence of citrate in solution clearly diminished the root-TF for BJ (Fig. 1d). The simulation results (Table 1) indicate that, in the presence of citrate, uranium is completely sequestered by citrate to form a very stable chelate (Shahandeh and Hossner, 2002). At the same time, this chelating agent greatly reduces the concentration of free-ion forms of several other elements in the nutrient solution, especially at neutral pH. For instance, Ca(II) and Mg(II) pass from 11% and 14%, respectively as free ions at pH 5, to 3% and 3.5% at pH 6.7. Likewise, Fe(III) is also sequestered by citrate. Evangelou et al. (2007) indicate a possible destruction of the natural barriers in the roots of HA by citrate excess, which can allow the uptake of other trace elements including uranium. Shahandeh and Hossner (2002) also find better retention of uranium in the roots of HA compared to BJ with the use of citrate. Other workers (Chang et al., 2005) report an enhancement of root-TF for uranium with HA and BJ from the action of citrate at pH 5, although this result corresponds to transfer from soils. Once uranium is adsorbed onto or assimilated by the roots, it must be translocated to the shoots. The element on or in the roots forms a reserve for its uptake by the plant. Some workers (Begonia et al., 1998) have studied the translocation of lead in BJ, and found that, besides the dependence on the concentration of the source (soil-solution or water), the plant’s uptake is greatest when there is an almost saturated capacity in its roots. According to the Free Ion Activity Model (Fortin et al., 2004; Hullenbusch et al., 2005), there is only a single chemical form of this element once it has crossed the root surface. When an element is not essential for the plant, a constant TR value should be expected, whereas, when an element is essential, the behaviour expected is a constant concentration in the shoots, regardless of the root concentration. However, if the concentration of the element in the roots is not completely available for uptake by the plant so that only a fraction is available, it could occur that a constant TR value is observed for an essential element. For both species, when there were no phosphates in solution, the TR values were statistically indistinguishable for all the pH values tested (Table 2). Only at pH 8 for the HA seedlings was the TR value

Table 2 238 U and

226

Ra translocation factors for HA and BJ at different pH values pH = 5.0

238

HA

BJ

226

pH = 6.7

pH = 8.0

U Without phosphates With phosphates With EDTA With citrate Without phosphates With phosphates With EDTA With citrate

3.2  10 1.5  10 3.2  10 8.7  10 7.9  10 1.8  10 7.5  10 0.011

Without phosphates With phosphates With EDTA With citrate Without phosphates With phosphates With EDTA With citrate

0.033 8.5  10 2.9  10 3.0  10 0.33 0.23 0.11 0.84

3 3 3 3 3 4 4

2.7  10 4.4  10 6.9  10 0.018 8.7  10 4.5  10 6.6  10 0.011

3 3

1.5  10 0.020

3

9.4  10 1.5  10

3

3

3 4

4

4

Ra HA

BJ

3 3 3

0.062 8.7  10 7.2  10 4.2  10 0.20 0.23 0.019 0.023

3

0.24 8.4  10

3 3

0.68 0.09

3

297

slightly less than for the other two pH values. However, in the presence of phosphates, the HA shoot-TF remained constant at all three pH values tested (Fig. 1a). These results seem to indicate that the uranium may behave as an essential element. In the presence of phosphates, the BJ had its lowest shoot-TF at pH 8. If the roots can be considered a nutrient reservoir, the reduction in shoot-TF at pH 8 could be due to the uranium in the roots being in a chemical form less available for its transport through the seedling. This chemical form could correspond to a precipitate which is found adsorbed to the root surface. The TR values obtained for both plant species in the presence of EDTA were statistically indistinguishable from the control seedlings at both pH’s tested, with a practically constant TR value. These results are only in partial agreement with those reported by Shahandeh and Hossner (2002) who found a slightly greater efficiency in the uranium transfer to the roots of BJ relative to HA, and a clearly greater efficiency to the shoots. The effects of citrate presence on the shoot-TF of uranium was a clear improvement relative to the control seedlings, for both HA and BJ. The best transfer to the shoots corresponded to HA, which is not in agreement with other workers’ results (Huang et al., 1998; Shahandeh and Hossner, 2002; Chang et al., 2005). If these results of the amendment with citrate are compared with the results without phosphate in solution at every pH value, the transfer of uranium to the shoots is again enhanced in both plant species. This is in agreement with the results of Ebbs et al. (1998b) using hydroponics and also of Chang et al. (2005) using soil as substrate, who found much greater transfers of uranium to the above-ground parts of the plant in the presence of this chelating agent. Chang et al. (2005) proposed that the citrate plays an important role in that the uranium chelate passes into the root tissues, as against the other theory that the uranium is transported as the free uranyl ion (Vassil et al., 1998).

3.2. Radium uptake In HA growing in a phosphate-free solution, as was the case with 238U, the pattern of the transfer of 226Ra to the roots of HA was clearly different from that obtained when there was phosphate present (Fig. 2a). Only at pH 8 was the root-TF lower than the value found with phosphate. There was a systematic decrease of the 226Ra transfer to HA roots as the pH increased. The highest TF value of 226Ra to the roots of HA seedlings was obtained under acidic conditions (twice as great as the corresponding value in the absence of phosphate). The explanation of the systematic decrease of the root-TF with pH could be the effect of the cation exchange capacity for soils with great amounts of Ca, as was proposed by Vandenhove and Van Hees (2007). It is expected that, as Ra2+ is a more basic cation than Ca2+, the latter should have a greater tendency to bind to the negatively charged sites in the root surface, with the effect increasing with increasing pH. This behaviour is based on radium’s larger ion radius, which makes it difficult for Ra2+ to substitute Ca2+ in the adsorption sites. In the seedlings of BJ, the behaviour of the root-TF without phosphates was similar to that for uranium (Fig. 2b). As was discussed above, this result indicates that the exudation of a ligand by the plant could promote coprecipitation of radium and uranium. When there was phosphate in solution, the root-TF of 226Ra to HA showed a more regular pattern with pH than 238U (Figs. 1a and 2a). The root-TF values were statistically indistinguishable for the three pH values. However, for BJ the highest value of the root-TF of 226Ra was observed at alkaline pH (an average value of 968 L kg 1) (Fig. 2b). The precipitation of radium with phosphates is coherent with the BJ root-TF results. As in the case of uranium,

298

F. Vera Tomé et al. / Chemosphere 74 (2009) 293–300

Helianthus annuus L.

8

Without P With P

6

20

4

-1

Ra TF (L·kg )

30

10 0 200

c

10

Shoots Roots

226

226

-1

Ra TF (L·kg )

40

Helianthus annuus L.

a

50

400 600

2 Shoots 0 Roots 500 1000

800 1500

1000 1200

2000

1400 140 120

Without P With P

Brassica 5 juncea 6,7 pH

8

120

b

Brassica junceaEDTA Control

pH 5 pH 6,7 Citrate

100

100

d

80

80

Ra TF (L·kg-1)

40 20 0

Shoots Roots

200

40 20 0

Shoots Roots

226

226

-1

Ra TF (L·kg )

60 60

200

400 600

400 800 1000

600 5.0

6.7

8.0

pH

Control

EDTA

Citrate

Fig. 2. 226Ra uptake to shoots and roots in the presence and absence of phosphates at three different pH values. (a) HA, and (b) BJ. Error bars denote the standard error of the mean (n = 3). Also shown is the effect of adding EDTA and citrate at two pH values. (c) HA, and (d) BJ. Error bars denote the standard error of the mean (n = 5).

the BJ must be able to retain this precipitate on its roots more efficiently than HA. With the addition of EDTA or citrate to the nutrient solution, the root-TF of 226Ra to HA showed a more regular pattern than 238U under acidic conditions, with values statistically indistinguishable for the two agents with respect to the control seedlings (Fig. 2c). With the change of pH to neutral conditions, the treatment with EDTA had hardly any effect on the transfer of 226Ra to the roots of HA, as also was the case with the control seedlings. However, now the addition of citrate improved this transfer somewhat. Relative to the control seedlings and at the pH values tested, the root-TF of 226Ra to BJ did not increase in the presence of citrate (Fig. 2d). The addition of EDTA kept the transfer of 226Ra to the roots of BJ close to the control level at pH 5, but enhanced the transfer under neutral conditions. Radium’s different behaviour in the presence of EDTA or citrate is explained by its different speciation due to the addition of the chelating agents (Table 1). When there is citrate in the solution, the free ion abundance falls from about 37% at pH 5 to 15% at pH 6.7. In the presence of EDTA, the same pH change produces an even greater reduction in the free ion abundance, from 67% to 11%.

For both plant species, the concentration of radium in solution is less as the pH rises. However, the results for HA seem to indicate better assimilation of the citrate complex of radium than of the free ion, whereas for BJ the decline in the free-ion abundance does seem to affect the root-TF value. When there are no phosphates in solution, the almost constant value of the shoot-TF observed for radium in HA indicates that radium behaves as an essential element (probably because of its similarity to calcium). The TR values (Table 2) indicate that BJ translocates radium to the shoots better than HA. This behaviour was also observed in the presence of phosphates. It seems that the channels for the assimilation of radium function more efficiently in BJ than in HA, just the opposite of the case for uranium. In the presence of EDTA, the translocation was less than in the control seedlings. This suggests a low availability of the Ra–EDTA complex for the plant, probably due to the size of the chelate or to steric difficulties in its formation because of the Ra ion radius compared to Ca. The presence of citrate in solution did not enhance the shoot-TF values (in general they declined). It can be concluded that Ra–citrate is not as good a form for translocation as it is for retention in the root tract. Plants themselves produce changes in

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the chemical form assimilated by the roots (Epstein et al., 1999), so that a plant’s natural exudation of citrate could be understood as a mechanism of immobilizing unwanted elements, as could be the case with radium in the present study. 4. Summary This comparative study of HA and BJ in eliminating uranium and radium has revealed the two species to have different ranges of utility depending on the chemical environment. In the absence of chelating agents, HA was better at eliminating uranium through the roots if phosphate is absent, while, if phosphate is present, BJ was better, which is an important result for rhizofiltration applications. In the presence of phosphate, while the use of chelating agents such as citrate or EDTA can be useful to improve the elimination of uranium by the roots of HA, they do not improve the elimination via the aerial part of the plant (the shoots). However, the presence of citrate and EDTA enhances the recovery of uranium by the shoots of BJ, with BJ seedlings being therefore the more recommendable for phytoextraction. In the absence of chelating agents, HA was better at eliminating radium through the roots under acidic and neutral conditions if phosphate is absent. Under the same conditions, BJ is more appropriate than HA for phytoextraction because of better transfers to the harvested part of the seedlings, especially if phosphate is present. In the presence of phosphate, the use of chelating agents such as citrate or EDTA does not improve the elimination of 226Ra either by HA or by BJ, so that their addition does not seem advisable if the decontamination of radium is the goal of phytoextraction. In this context, HA and BJ give very similar recoveries in the shoots, with their most appropriate application being in the absence of phosphate. HA perhaps yields slightly better results, and at the same time it has a more regular behaviour with the variation of pH. Acknowledgements Thanks are due to the Ministerio de Educación y Ciencia, Plan Nacional de I+D+I (2004-2007) (CTM2005-02910/TECNO project) and the Fondo Social Europeo de Desarrollo Regional (FEDER), for financial support. We also acknowledge financial support from the Empresa Nacional de Resíduos Radiactivos (ENRESA), the Spanish National Agency for Radioactive Management (project 0078000102). References Alkorta, I., Hernández-Allica, J., Becerril, J.M., Amezaga, I., Albizu, I., Onaindia, M., Garbisu, C., 2004. Chelate-enhanced phytoremediation of soils polluted with heavy metals. Rev. Environ. Sci. Biotechnol. 3, 55–70. Arthur, E.L., Rice, P.J., Rice, P.J., Anderson, T.A., Baladi, S.M., Henderson, K.L.D., Coats, J.R., 2005. Phytoremediation – An overview. Crit. Rev. Plant Sci. 24, 109– 122. Baker, A.C., Toque, C., 2005. A review of the potential for radium from luminising activities to migrate in the environment. J. Radiol. Prot. 25, 127–140. Begonia, G.B., Davis, C.D., Begonia, M.F.T., Gray, C.N., 1998. Growth responses of Indian mustard [Brassica juncea (L.) Czern.] and its phytoextraction of lead from a contaminated soil. B. Bull. Environ. Contam. Toxicol. 61, 38–43. Bernhard, G., 2005. Speciation of uranium in environmental relevant compartments. Landbauforschung Völkenrode 55, 139–148. Blanco, P., Lozano, J.C., Vera Tomé, F., 2002. On the use of 225Ra as yield tracer and Ba(Ra)SO4 microprecipitation in 226Ra determination by a-spectrometry. Appl. Radiat. Isotopes 57, 785–790. Bunzl, K., Trautmannsheimer, M., 1999. Transfer of 238U, 226Ra and 210Pb from slagcontaminated soils to vegetables under field conditions. Sci. Total Environ. 231, 91–99. Chang, P., Kim, K.-W., Yoshida, S., Kim, S.-Y., 2005. Uranium accumulation of crop plants enhanced by citric acid. Environ. Geochem. Hlth. 27, 529–538. Chen, Y., Cutright, T., 2001. EDTA and HEDTA effects on Cd, Cr, and Ni uptake by Helianthus annuus. Chemosphere 45, 21–28.

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