Journal of Environmental Radioactivity 70 (2003) 139–154www.elsevier.com/locate/jenvrad

Technetium species induced in maize asmeasured by phosphorimager

M. Simonoff a,∗, T.V. Khijniak a,b, C. Sergeanta,M.H. Vesvresa, M.S. Pravikoffa, E. Leclerc-Cessacc,

G. Echevarriad, S. Denysc,d

a CNRS-UMR 5084—Chimie Nucleaire Analytique et Bioenvironnementale, Le Haut Vigneau, B.P. 120,33175 Gradignan Cedex, France

b Institute of Microbiology, Russian Academy of Sciences, 7/2 Prospect 60-letiya Oktyabrya,Moscow 117811, Russia

c ANDRA, Parc de la Croix Blanche, 17, rue Jean Monnet, 92298 Chatenay-Malabry Cedex, Franced Laboratoire Sols et Environnement, UMR 1120 ENSAIA-INPL/INRA-2, Avenue de la Foret de la

Haye-B.P. 172, 54505 Vandoeuvre-les-Nancy Cedex, France

Received 1 June 2002; accepted 1 March 2003

Abstract

Leaves of plants have the ability to accumulate the long-lived fission product99Tc. In thepresent work, an attempt was made to separate and characterize technetium species formed inmaize grown on soil contaminated with TcVII O4

� solution. Data obtained from selective extrac-tion, a Phosphorimager and liquid scintillation were employed. 2003 Elsevier Science Ltd. All rights reserved.

Keywords: Technetium; Speciation; Chromatography; Radionuclide; Bioaccumulation; Maize; Nuclearwaste

1. Introduction

Technetium (Tc) has only radioactive isotopes. Nuclide99Tc ( half-life, 2.13.105

yr, β- emitter with Eβ�max = 292 keV) is a fission product of235U and239Pu and is the

most important isotope from an environmental point of view. The Tc content of soil

∗ Corresponding author. Fax:+33-557-120-900.E-mail address: simonoff@cenbg.in2p3.fr (M. Simonoff).

0265-931X/03/$ - see front matter 2003 Elsevier Science Ltd. All rights reserved.doi:10.1016/S0265-931X(03)00123-1

140 M. Simonoff et al. / J. Environ. Radioactivity 70 (2003) 139–154

is anthropogenic (nuclear power generation, nuclear weapon testing programs,nuclear medicine). Soil plays a key role in the transfer of radionuclides to plants.Two processes are involved, the bioavailability in the soil and the plant uptake.The cumulated Tc in soil is now about 500 mBq.m�2 (Holm et al., 2000). Higherconcentrations in soil and plants can be found in contaminated areas (Chernobylexclusion zone: 1.1 to 14.8 Bq.kg�1 dry soil) (Uchida et al., 1999, 2000).

Tc has 9 oxidation states ranging from –I to VII, the most stable ones being zero,VII and IV. Under oxidizing conditions, the most stable species is pertechnetate99TcO4

� (+ VII). Under reducing conditions in anoxic soils, 99TcO4� can be reduced

to the less mobile form, technetium dioxide 99TcO2 (+IV) (Saas et al., 1981). Thisform, possibly sorbed on the soil through complexation with organic matter, is veryslowly reoxidized (Van Loon et al., 1986). The reduced forms of Tc (dioxide, TcS2)have low solubilities and are less phytoavailable than TcO4

� (Yanagisawa and Mara-matsu, 1997; Yanagisawa et al., 1997). In aerated environments, and especially incultivated soils, the oxidized form, 99TcO4

�, is poorly sorbed and very mobile(Wildung et al., 1986; Sheppard and Hawkins, 1991). In such conditions, Kd values(soil to solution distribution coefficients) for 99Tc are very low. However, a decreasein bioavailability of 99Tc over time has been reported in aerated soils and attributedto changes in chemical state due to local reducing conditions and immobilization bymicroorganisms (Henrot, 1989; Macaskie, 1991; Lloyd et al., 1999; Khijniak et al.,2003). Thus Tc sorption on the soil constituents might be influenced by severalfactors, such as redox conditions, organic matter content and microflora.

Plants take up 99Tc in the pertechnetate (TcO4�) form and root absorption involves

passive as well as active processes that are common to other anions such as sulfate,phosphate, selenate, and molybdate (Cataldo et al., 1983, 1986). Pertechnetate isxylem mobile and rapidly transferred to aerial parts (Myttenaere et al., 1986; Echev-arria et al., 1995). It is accumulated in leaves and especially in chlorophyll-containingcells, where part of it may be reduced and incorporated into amino-acid-N in thechloroplasts (Cataldo et al., 1989; Degenkolb et al., 1994). Plant-to-soil concentrationratios reported by many authors vary widely, depending on plant species and plantpart (Vandecasteele et al., 1982; Myttenaere et al., 1986; Echevarria et al., 1995).Contaminated above ground parts may return to the soil through plant residues,becoming a secondary source of 99Tc to the following crop (Dehut et al., 1989).

TcO4� and NO3

� ions compete for transport in the plant (Krijger et al., 2000;Echevarria et al., 1997, 1998). In tomato plants, TcO4

� and NO3� use the same

transporter (Krijger et al., 2000). Tc would be mostly located in chlorophyllianorgans where it would be reduced by photosynthetic processes (Myttenaere et al.,1986). When Tc is taken up by roots, it moves to aerial parts, mostly to leaves,without accumulation in stalk (Shinonaga et al., 1998). Accumulation in seeds islow. In the case of wheat, 95% of the Tc is accumulated in leaves and less than 1%in seeds (Echevarria et al., 1997). In rye-grass, Tc accumulation in the leaves canreach 78% of the input (Echevarria, 1996).

Tc speciation in plant organs is difficult to study and oxidation of species boundto Tc under the TcO4

� is thought to occur during the experimental procedures. How-ever, Tc has been shown to reach leaves as TcO4

� using xylem flow and to be bound

141M. Simonoff et al. / J. Environ. Radioactivity 70 (2003) 139–154

to organic compounds (Krijger et al., 1999; Lembrechts et al., 1986). About 10 Tcorganic compounds have been identified in spinach leaves, more than 80–90% havinga molecular mass lower than 6000 g mol�1 (Lembrechts et al., 1986). In soya beanleaves, most of the Tc is bound to polypeptides with molecular weights between4000 and 10,000 Da (Vandecasteele et al., 1982). In leaves, Tc metabolism wouldbe associated with sulphur; in vitro Tc would replace sulphur in cystein-type aminoacids via cystein synthetase (Cataldo et al., 1989). Other studies confirm the affinityof Tc for sulphur-containing groups like cystein and glutathione. The results of stud-ies performed so far on speciation of Tc in plants such as spinach (Harms et al.,1999), tomato (Krijger et al., 1999) and maple leaf (Garten and Lomax, 1989) suggesta metabolic transformation of TcVIIO4

� to reduced Tc forms, so-called Tc X, notidentified but generally considered to be low molecular mass Tc complexes (Tc-cystein, Tc-glutathione) and Tc proteins.

In the present work, several extraction techniques have been used to separate dif-ferent proteins resulting from Tc incorporation in maize cultivated on soil corescontaminated by 99TcO4

�. By using both Phosphorimager and liquid scintillationmethods, information concerning Tc species in maize has been obtained and indicatesthe simultaneous presence of several valences, as well as Tc in the free state andbound to protein.

2. Material and methods

2.1. Reagents

All reagents were of analytical grade and purchased from Sigma-Aldrich.

2.2. Culture

Maize was cultivated in a lysimeter with undisturbed soil cores (Denys et al.,2002). A nutrient solution was applied one day before seeding, at a rate of 50 kg.N.ha�1 (NH4NO3, KNO3), 125 kg.P.ha�1 (P2O5) and 175 kg.K.ha�1 (KNO3) perlysimeter; 100 kg.N.ha�1 (NH4NO3) per lysimeter was incorporated 20 days afterseeding. Ten seeds of maize (Zea mays L., DEA, Pionner ) were sown per lysimeterand five plants were removed from each lysimeter 10 days after germination. At 44days, 2100 kBq of 99Tc, (as NH4TcO4 , Perkin Life Science, Belgium), was addedto the soil at the surface of the lysimeters. Maize was harvested 135 days afterseeding and leaves from one plant were blotted together and crushed.

2.3. Extraction

Extraction techniques from Garten and Lomax (1989); Farago (1994) and Harmset al. (1999) were used to separate the Tc species from the maize homogenate.

Maize powder (0.5 g) was homogenized in 25 ml of HEPES buffer (70 mmol l�1

N-(2-hydroxyethyl)-piperazine-N�–(2 ethanesulphonic acid) and 8 mmol l�1 NaCl,

142 M. Simonoff et al. / J. Environ. Radioactivity 70 (2003) 139–154

pH 7.0) using a Potter–Elvehjem homogenizer at 0–5 °C. Homogenate was dividedinto five aliquots of 5 ml in order to apply several extraction techniques to differen-tiate between classes of Tc species (Harms et al., 1999).

Homogenate I was centrifuged (20 min, 15,000 g, 4 °C) ; supernatant S1 (samples110, 210) contained free technetium, T, and proteic and non proteic fractions(NP1 + NP2 + HP1 + HP2 + T). The pellet was resuspended in 1 M NaOH ; aftercentrifugation, a pellet PP1, a supernatant SP1 were obtained (samples 111, 211).

Homogenate II was treated by ammonium sulphate until it reached a final concen-tration of 2.1 mol l –1. After centrifugation, supernatant S2 (samples 120, 220) con-tained fractions NP1 + NP2 + T. Acetone was added to a final concentration of 80%w/w. After centrifugation, supernatant containing NP1 + T was obtained (samples122, 222), corresponding to non-protein Tc species and TcO4

� soluble in acetone.Pellet P2 was added to water in order to extract proteins HP1 + HP2 (samples 121–

221), then centrifuged.Homogenate III was treated by acetone (final concentration 80% w/w) to precipi-

tate insoluble Tc species, highly polar non-protein Tc species and hydrophilic protein(I + NP2 + HP2). After centrifugation, the supernatant contains non-protein Tc speciessoluble in acetone, Tc bound to hydrophobic protein, TcO4

� and non-retained NP1

+ HP1 + T.Homogenate IV was submitted to diethyl ether for extraction of non-polar Tc spec-

ies.Homogenate V was treated by 4% w/w 8-hydroxyquinoline (H-8-Q) in chloroform

to extract labile TcV species (Hwang et al., 1984).Radioactive Tc was measured by liquid scintillation in aliquots of supernatant and

in pellets in order to evaluate the distribution of classes of Tc species. Speciationwas determined with a phosphorimager on supernatant fractions after paper chroma-tography.

2.4. Spectrophotometric determination of the extracted fractions

UV-visible spectra were recorded in digital form, using a Beckman DU-50 Seriesdouble-beam diode array UV-visible spectrophotometer in the range 250–700 nm.Two series of extractions were carried out (the second eight months after the first)using maize leaves cultivated in presence of Tc. The fractions were examined andcompared with those obtained from maize not exposed to Tc.

2.5. Chromatographic separation and identification of Tc

Tc in solution was assayed by autoradiography with a Phosphor Imager (BioRad, France).

This technique is routinely used to detect and quantify radioactive labels used incellular and molecular biological studies.

Different valences of Tc were separated on Whatmann 3MM chromatographypaper, with 0.3 N HCl as the mobile phase (Shukla, 1966). The chromatographypaper was impregnated with 5–100 µl aliquots. After separation, air dried chromatog-

143M. Simonoff et al. / J. Environ. Radioactivity 70 (2003) 139–154

raphs were exposed to a phosphor storage screen for 24–28 h. Chromatographs werevisualized and quantified with the phosphorimager technique (Lloyd and Macas-kie, 1996).

2.6. Extraction techniques for determination of inorganic Tc species

For comparison with chromatographic Tc determinations, several extraction tech-niques were used to separate inorganic Tc species with valencies VII and V. Toextract Tc VII, 1 ml of phenylarsonium chloride (Ph)4AsCl in chloroform (0.05 M)was added to an equal volume of sample (Shwochau, 1994, 2000). After 1 minagitation, the phases were separated and the amounts of Tc in the organic and aque-ous phases were determined. To extract the Tc V species, 4% (w/w) 8-hydroxyquino-line in chloroform (Hwang et al., 1984) was added to the media. After 5–10 minagitation, the phases were separated and the amounts of Tc in the organic and aque-ous phases were determined. In all cases, the radioactivity of each phase (organicor aqueous) was determined with a Beckman liquid scintillation analyser and InstaGel cocktail (Packard Bioscience Company).

2.7. Preparation of standards (reduced Tc and Tc complexes with organicligands)

For Tc species identification ( mostly Tc IV and Tc V), TcVIIO4� was reduced to

lower valences. Tetravalent Tc IV was prepared by two methods: the reduction ofpertechnetate by hot concentrated hydrochloric acid, or by SnCl2.

The TcIV cysteine and TcV cysteine complexes, as well as TcIV glutathion andTcV glutathione, have been prepared by reduction of TcVIIO4� with cystein or gluta-thione at a low pH (Johannsen et al. 1978; Harms et al., 1999). To an aliquot of0.65 ml containing 99TcO4

� (0.25 mmol l�1) in trifluoroacetic acid (TFA, 10 mmoll�1) and NH4OH (4 mmol L�1), we added 18 mg (0.11 mmol) L-cystein, HCl. Thesolution (pH 1.0) was heated for 5 min in a water bath at 55 °C. The solutionimmediately turned yellow, then red after 24 h storage at room temperature. Thesame reduction was carried out with glutathione to prepare the Tc-glutathione com-plex (55 µmol glutathione was added to a 0.33 ml aliquot containing TcO4

� (0.25mmol l�1) in TFA (10 mmol l�1) and NH4OH (4 mmol l�1)). Once again, the solutionturned yellow.

3. Results and discussion

3.1. Tc standards

The conditions employed for preparing standards with SnCl2 as reducing agent inpresence of hydrochloric acid are summarized in Table 1. Fig. 1 presents the corre-sponding phosphorimager chromatograms for the reaction at time zero and 65 h later.With the given concentrations of SnCl2 and HCl, the presence of one, two or three

144 M. Simonoff et al. / J. Environ. Radioactivity 70 (2003) 139–154

Table 1Reduction of Tc VII by SnCl2 in acid medium. The amount of Tc in each reaction is 63.6 µg

N Final acid Amount of SnCl2 Speciation of Tc After 65 hconcentration (mol (mg) zero timel�1)

A 6.0 600.0 VII et IV VII and IVB 1.5 150.0 IV IVC 0.15 15.0 VII, V, IV VII, V, IVD 0.050 5.0 VII, V, IV VE 0.025 2.5 VII, V, IV VF 0.005 0.5 V V

Fig. 1. Tc standards, prepared with SnCl2 in hydrochloric acid. Reduction of Tc by SnCl2 in HCl, fol-lowed by chromatography in 0.3 N HCl with Phosphorimager. Tc (V) (Rf = 0) and Tc (IV) (Rf = 0.9)were identified in the reactive medium. Lines 1 to 7, time zero. Lines 8 to 14, after 65 h. Lines 1, 8,TcO4

�; Lines 2, 9, sample A; Lines 3, 10, sample B; Lines 4, 11, sample C; Lines 5, 12, sample D;Lines 6, 13, sample E; Lines 7, 14, sample F.

spots corresponding to the three Tc valencies can be observed. The Rf values are:Tc VII Rf = 0.7, Tc V Rf = 0.0, Tc IV Rf = 0.9.

We verified by spectrophotometry UV-vis that solutions prepared in conditions ofTable 1 showed absorbance peaks associated with Tc VII (290 nm), Tc V (418nm), Tc IV (512 nm) and quantitatively correlated (Paquette and Lawrence, 1985;Vichot, 2001).

The intermediate species of Tc observed during reduction of pertechnetate by con-centrated hydrochloric acid are strongly adsorbed on cellulose, especially in hydro-chloric acid at concentrations below 1.0 N. A chromatographic method whichexploits this property was developed for the separation of the intermediate speciesTc V from the other more stable valencies Tc IV and Tc VII. Shukla (1966) testedseveral paper and hydrochloric acid concentrations, the best result for separation ofpeaks was obtained with Whatman 3M paper (pure cellulose) and 0.3 N HCl. Other

145M. Simonoff et al. / J. Environ. Radioactivity 70 (2003) 139–154

mobile phases (acetone, acetonitrile, NaCl) and other chromatographic supports weretested, without obtaining better results for the separation of the Tc valencies.

3.2. Extraction

The liquid phases obtained after various stages of the extraction protocol for pow-dered maize are identified by three digits, x y z, where

� x = 1 or 2 identifies the extraction series. The second was realized 8 months afterthe first, on the same powdered maize sample.

� y = 1 or 2 identifies the starting homogenate (1 indicates Hepes medium andhomogenate I; 2 indicates ammonium sulfate medium and homogenate II).

� z = 0,1,2 indicates the stage of the chemical treatment of each homogenate.

Two series of analyses (referred to as 110, 111, 120, 121, 122 for the first seriesand 210, 211, 220, 221, 222 for the second) were obtained. They were carried outon the same ground, dry maize sample at 8-month intervals, using the chemicalprotocol indicated in Fig. 2, followed by chromatography using a Phosphorimagerand by analyses using UV-visible spectrophotometry.

The homogenate I produced samples 110 and 210 in a Hepes medium, and samples111 and 121 after NaOH treatment. The homogenate II led to samples 120 and 220in an ammonium sulfate mixture, to 121 and 221 after water addition and to 122and 222 after 80% acetone treatment.

The radiochromatograms and UV-visible spectra were similar for the two series.Moreover, for the first series, measurements were performed immediately afterextraction, then again several months later at the same time as for the second series,after keeping the solutions at –18 °C. The intensity and position of the peaks on

Fig. 2. Extraction scheme for differentiation between Tc species in a maize leaf homogenate. P denotesprecipitate, S supernatant. Double letters indicate an additional step resulting in a new pellet and super-natant.

146 M. Simonoff et al. / J. Environ. Radioactivity 70 (2003) 139–154

radiochromatograms were the same for the freshly prepared samples as for thosekept several months at –18 °C, showing the stability of the Tc compounds at that tem-perature.

3.3. Tc-total balance in maize tissue

The radioactivity measured by liquid scintillation in precipitates P1 and PP2 per-mits an estimate of the amounts of insoluble Tc species they obtain. The amountsof Tc bound to hydrophilic and to hydrophobic proteins, respectively, can be esti-mated from P2 and the supernatant SP2. The supernatant S2, obtained after centrifug-ation in ammonium sulfate medium, and SS2, obtained after treatment with 80%acetone (which extracts the non-protein Tc species soluble in acetone), plus the radio-activity of PS2, allows the distribution (relative amounts) of NP1 and NP2 (Tc boundto non-protein species) and of free TcO4

� (species T) to be obtained.Table 2 shows the relative amounts of species containing Tc in maize. The pre-

cision of the measurements is about 5%, which results from cumulated errors inthe measurement of volumes, including aliquots for liquid scintillation, and weightsof precipitates.

After centrifugation of the homogenate I, 66% of the total Tc was found in thesupernatant and the remainder in the pellet (32%) (insoluble Tc species I). Thisagrees very well with results obtained elsewhere for maize (Zea mays L., 31% forTc in the pellet) and quite similar to results obtained for soya bean (39%)(Vandecasteele et al., 1982).

Most of the Tc species in the pellet after centrifugation of homogenate I (speciesinsoluble I) were extracted into 1 mol l�1 NaOH, suggesting, in this case, that Tcwas bound to hemicellulose (hemicellulose contains poorly defined polysaccharidesof the cell wall, which are soluble in alkaline solution). This speciation of Tc hasbeen observed previously on spinach (Harms et al., 1999; Garten and Lomax, 1989).

After treatment of homogenate II by ammonium sulphate, the supernatant con-tained NP1 + NP2 + T and 51% of the total radioactivity. If submitted to acetoneextraction, the supernatant contained soluble Tc species (non-protein Tc species NP1

and T), and the pellet highly polar non-protein Tc species NP2. Tc species that pre-cipitated in ammonium sulphate (48%) were both insoluble and bound to hydro-

Table 2Distribution of species containing Tc

Species Relative amounts (%) Description

I 32.0 Insoluble Tc speciesNP1 4.5 Non-protein Tc species soluble in 80% w/w acetoneNP2 10.6 Highly polar non-protein Tc speciesHP1 2.3 Tc bound to hydrophobic proteinHP2 13.3 Tc bound to hydrophilic proteinT 35.5 TcO4

�

147M. Simonoff et al. / J. Environ. Radioactivity 70 (2003) 139–154

phobic or hydrophilic proteins. A linear correlation with Tc-protein precipitation wasfound on increasing (NH4)2SO4 concentration from 20% to 100%. This agreed with alarge protein distribution since no narrow peak was associated with particular species.

Acetone addition to homogenate III precipitated hydrophilic Tc species andextracted NP1 (non-protein Tc species soluble in acetone), HP1 (Tc bound to hydro-phobic protein) and free TcO4

�. No very hydrophobic Tc species was observed,since no radioactivity was extracted in diethyl ether. The attempt to extract labileTcV species from homogenate V by H-8-Q in chloroform (Hwang et al., 1984) failed,since all the Tc stayed in the aqueous phase, as previously observed for spinach byHarms et al. (1999) and by Lambrechts et al. (1986).

3.4. Spectrophotometric results for maize cultivated in presence and in absence ofTc

The UV-visible absorbance spectra for extractions from maize cultivated with orwithout Tc were identical (Fig. 3), showing that the presence of Tc did not stronglyperturb the distribution of the constituents of maize.

Fig. 3. Spectrophotometric analyses of the fractions obtained from maize leaf homogenates: 110/210,111/211, homogenate I in HEPES; 120/220, 121/221, 122/222, from homogenate II in ammonium sulfate.

148 M. Simonoff et al. / J. Environ. Radioactivity 70 (2003) 139–154

3.5. Tc speciation and phosphorimager results

3.5.1. Samples 110 and 210 (HEPES)The results obtained by Phosphorimager after paper chromatography with 0.3 M

HCl as mobile phase are shown in Figs. 4–7. Fig. 4 shows the radiochromatogramobtained with homogenate I (maize leaf in HEPES medium) after centrifugation.The TcVIIO4

� standard on Whatman paper gave spot 1 (Rf 0.7). The 50 µl aliquotsof solutions 110 or 210 on the base line shows the presence of small quantities ofTc V (Rf = 0.0), spot 4, and that of Tc IV (Rf = 0.9) in the upper part of frame 2.Also, the existence of the intense spot 3 (Rf � 0.7) could correspond to Tc VIIand/or Tc IV bound to proteins, which would cause a slower migration with regardto the expected Rf. The extraction of the aqueous phase by phenylarsonium in chloro-form, which mainly extracts Tc VII, confirmed that most of the Tc was present inoxidation state VII (spot 8–9), while the result for the aqueous phase shows theabsence of Tc in states IV, VII and V (spots 5, 6, 7).

Fig. 4. Radiochromatograms obtained with phosphorimager for samples 110 and 210 (hemolysate inHEPES). 1: TcVIIO4

�-standard (Rf = 0.7); 2.3.4: sample 110 or 210 (50 µl) deposited at base line onWhatman paper and the associated radioactive spots obtained after chromatography; 5.6.7: aqueous phase(50 µl) after treatment by phenylarsonium in chloroform; 8.9: organic phase (50 µl) after treatment byphenylarsonium in chloroform; bg: background.

149M. Simonoff et al. / J. Environ. Radioactivity 70 (2003) 139–154

Fig. 5. Radiochromatograms obtained with Phosphorimager for samples 120 and 220 (hemolysate inammonium sulfate 2.1 M). 1: TcVIIO4

�-standard (Rf = 0.7); 2.3: sample 120 or 220 (50 µl) deposited atbase line on Whatman paper; 4.5: aqueous phase (50 µl) after treatment by phenylarsonium in chloroform;6.7: organic phase (50 µl) after treatment by phenylarsonium in chloroform; bg: background.

3.5.2. Samples 111 and 211 (sodium hydroxide)After NaOH addition to precipitate P1 containing insoluble species of Tc, the

supernatant did not contain species associated with Tc or free TcVIIO4�, as shown

by the absence of any spot except for the control standard, TcVIIO4�.

3.5.3. Samples 120 and 220 (ammonium sulfate)Fig. 5 shows the radiochromatogram obtained for homogenate II (maize leaves in

ammonium sulfate medium) after centrifugation. Spot 1 (Rf = 0.7) corresponds to abase line deposit of 10 µl of standard solution of TcVIIO4

� on Whatman paper.Similarly, spots 2 and 3 correspond to deposits of 50 µl of samples 120 or 220. Thecorresponding radiochromatogram indicates the presence of small amounts of Tc V(spot 3). The intense spot 2, corresponding to an Rf lower than 0.7, was probablyTc in the VII or IV oxidation state, bound to proteins. The extraction by phenylar-sonium showed that it consisted essentially of Tc VII (spot 6).

150 M. Simonoff et al. / J. Environ. Radioactivity 70 (2003) 139–154

Fig. 6. Radiochromatograms obtained with phosphorimager for samples 122 and 222 (acetone 80 %).TcIVO4

� standard on the left of the figure.

3.5.4. Samples 121 and 221 (water)Only species containing Tc in the VII oxidation state seemed to be present.

3.5.5. Samples 122 and 222 (acetone)From supernatants 120/220, acetone (final concentration 80% w/w) precipitated

highly polar non-protein Tc species so that, after centrifugation, the supernatant SS2

(122/222) contained only the T fraction (TcO�4 = 35.5 %) and the NP1 fraction (non-

protein Tc species soluble in 80% acetone, i.e 4.5 %), thus explaining why only onespot corresponding to Rf = 0.7 (Tc VII) can be observed on the radiochromatogram(Fig. 6).

3.5.6. Tc cystein complexesThe reaction mixture obtained by reduction of pertechnetate in presence of cystein

was analysed by chromatography, showing one, two or three peaks correspondingto the reduction, as a function of time, of Tc VII to Tc V and then to Tc IV (Fig. 7).

Controls of TcVIIO4� and standards of TcIV and TcV (prepared by reduction in

concentrated HCl, or diluted HCl in presence of SnCl2) are shown on lines 1, 2 and 4.

151M. Simonoff et al. / J. Environ. Radioactivity 70 (2003) 139–154

Fig. 7. Reduction of TcO4� with L-cysteine at pH 1.0 resulting in the formation of TcV cystein and

TcIV cystein complexes as function of incubation time: 1, standard TcVIIO4� (Rf = 0.7); 2, standard TcIV

(Rf = 0.9); 3, time 2 h: TcVII decreases and yellow TcV cystein appears; 4, standard TcV prepared fromTcVIIO4

� by reduction with SnCl2 in HCl and containing some TcIV; 5, time 6 h: TcV cystein presentwith TcIV cystein and some TcVII; 6, time 20 h: essentially red TcIV cystein.

When TcVII is reduced in presence of the cystein ligand, the solution turned yellowimmediately. The radiochromatogram corresponding to time 2 h (line 2) showed aspot at Rf = 0.0, due presumably to TcV cystein. The solution became red after 24h of storage at room temperature and gave a chromatographic spot at Rf = 0.9 (line5), due presumably to Tc IV-cystein. In presence of the glutathione ligand, a yellowTc-glutathione complex was observed immediatly and the reduction of Tc VII to TcV and Tc IV glutathione with time was observed at the same Rf as that with cystein.

3.6. Tc speciation in homogenates

The positions of the chromatographic spots obtained from homogenates I and II(Figs. 4 and 5) correspond to the Rf values for TcV, TcIV and to values less than0.7 (TcVII). It is not possible to be certain that Tc was bound to ligands like cysteinor glutathione or to proteins.

The radiochromatogram for samples 110/210 in HEPES clearly showed a spot forRf � 0.9, presumably due to a mixture of Tc IV-cystein or Tc IV-glutathione only,and Tc IV bound to a larger ligand ( slowing the migration to Rf = 0.9). An attemptwas made to obtain more information with EXAFS experiments at ROBL (ESRFGrenoble), but the specific activity of Tc in leaves of maize (1000 kBq kg�1 ) wasinsufficient (under the detection limit) to obtain a signal for Tc. We project newexperiments with maize exposed to higher concentrations of Tc.

The phosphorimager-based technique used to detect Tc species in plant homogen-ate solution by autoradiography offered several advantages compared to other liquid-based speciation methods (HPLC, capillary electrophoresis, reversed-phase ion pair

152 M. Simonoff et al. / J. Environ. Radioactivity 70 (2003) 139–154

chromatography). The samples were fixed on paper chromatographs and sealed in aplastic film to avoid contamination and to simplify radioactive waste management.The phosphor screen is several hundred times more sensitive than X-ray film auto-radiography and can be routinely used for α, β and γ emitters (Lloyd and Macaskie,1996). This technique offers large possibilities for environment and nuclear wastestudies in giving rapid access to speciation of actinides and fission products in sol-ution or in several materials, after appropriate treatment to extract the radionuclideand its ligands without modifying the valences.

4. Conclusions

The combination of selective extraction and the use of a phosphorimager hasshown the presence in maize leaves of Tc VII and of reduced Tc, presumably com-plexed by ligands such as amino acids or with both low and high molecular weightTc-proteins. The next step will be to combine other chromatographic tools, massspectrometry and EXAFS to try to elucidate the nature of the Tc ligand.

Acknowledgements

We are very grateful to ANDRA for funding this study and to Dr MacKenziePeers for his help.

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