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Detection and analysis of the microdistribution of uranium in the gills of freshwater Corbicula fluminea by SIMS technique
Nuclear Instruments and Methods in Physics Research B 267 (2009) 1931–1935
Contents lists available at ScienceDirect
Nuclear Instruments and Methods in Physics Research B journal homepage: www.elsevier.com/locate/nimb
Detection and analysis of the microdistribution of uranium in the gills of freshwater Corbicula fluminea by SIMS technique C. Tessier a,*, D. Suhard a, O. Simon b, M. Floriani b, F. Rebière a, J.-R. Jourdain a a
IRSN, Direction de la RadioProtection de l’Homme, Service de Dosimétrie Interne, Laboratoire de RadioChimie, IRSN, BP 17, F-92262 Fontenay aux Roses Cedex, France IRSN, Direction de l’Environnement et de l’Intervention, Service d’Etude du Comportement des Radionucléides dans les écosystèmes, Laboratoire de Radioécologie et d’écotoxicologie, IRSN, BP F6 Cadarache, France b
a r t i c l e
i n f o
Article history: Received 18 December 2008 Received in revised form 11 March 2009 Available online 28 March 2009 Keywords: SIMS Contamination Uranium Corbicula fluminea Imaging
a b s t r a c t The microdistribution of uranium in the gills of freshwater bivalve Corbicula fluminea following chronic direct exposure to this radioelement has been investigated using the SIMS technique. Different exposure levels and exposure durations have been studied. The SIMS mass spectra and 238U+ ion images produced with a SIMS CAMECA 4F-E7 show an U accumulation with the lower aqueous U concentration (20 lg/L) and the influence of the exposure levels on the bioaccumulation capacities. Furthermore, the ionic images display a heterogeneous distribution of uranium within the gill structure whatever the exposure conditions are. This study, in keeping with the ENVIRHOM French research program, was led to the conclusion that ion microscopy is an appropriate analytical method for trace elements and can give elemental cartography in a biological tissue section. Ó 2009 Elsevier B.V. All rights reserved.
1. Introduction The ENVIRHOM research French program supported by the Institute for Radioprotection and Nuclear Safety (IRSN) is intended to improve assessment of the risks to the general public and the ecosystems associated with chronic exposure to low levels of radioactive contaminants. The main objectives are to study the speciation, transfer, biokinetic and accumulation processes of radionuclides and also the biological effects correlated with this exposure on the human model (rats and mice) and on environmental organisms (algae, molluscs, crustaceans, fish, plants, etc.). Uranium has been the first element studied as part of this program. Chronic exposure of uranium may occur naturally in contaminated areas (underground water) or as a result of human activity (nuclear fuel cycle, agricultural use, military use of depleted uranium). Concerning the environmental aspect of the program, several biological responses have been studied in various living organisms to establish concentration-effects relationships: feeding strategies, ventilation of molluscs, reproduction, growth rate of living organisms and the effects on the ecosystems [1–3]. A thermodynamic equilibrium model of uranium in aqueous systems has been described to interpret the experimental data obtained on the bioavailability of uranium (VI) [4–6]. Simon and Garnier-Laplace
* Corresponding author. E-mail address: [email protected] (C. Tessier). 0168-583X/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2009.03.096
[7,8] have studied the bioaccumulation of uranium in the freshwater bivalve Corbicula fluminea. They have shown a pH influence and a correlation between the exposure conditions (exposure durations and treatments) and the distribution of this actinide in the bivalve organs. The aim of this paper is to present a study of the microdistribution of uranium in gills of the bivalve C. fluminea after chronic direct exposure of this radionuclide, by SIMS (Secondary Ion Mass Spectrometry) microscopy. The SIMS technique introduced in the early sixties by Castaing and Slodzian [9] is used for the chemical characterization of solid surfaces mainly in geochemistry, microelectronics and material sciences. However, few years ago, the study and analysis of biological samples by ion microscopy were developed in the biomedical [10–15], nuclear [16] and botanic fields [17–19]. In the literature, only a few papers describe uranium detection by this technique in biological samples [20,21]. In particular, Markich et al. [21] used SIMS to measure metal ratios such as U/Ca in freshwater bivalve shells (Velesunio angasi), showing that these clams can be used over their lifetime as archival indicators of metal pollution in surface waters of the Finniss River (in tropical northern Australia near a copper–uranium mine). Moreover, the isotope ratio of this long-lived radionuclide at very low concentration levels has been measured by SIMS technique for environmental monitoring in geoscience, cosmochemistry, and planetary and nuclear sciences [22–33].
1932
C. Tessier et al. / Nuclear Instruments and Methods in Physics Research B 267 (2009) 1931–1935
2. Materials and methods
Table 2 Treatment conditions of contaminated Corbicula.
2.1. SIMS microscopy
Total U concentration in water (lg/L)
Exposure duration (days)
pH
500 20 20 20
10 10 40 90
7 7 7 7
The aim of SIMS microscopy is the elemental and isotopic analysis of a solid surface by an ion beam coupled with a mass spectrometer. The principle of this technique is based upon the sputtering of a few atomic layers from the surface of a sample, induced by a bombardment of focused primary ions of sufficiently high energy (some keV). A more detailed description of the physical phenomenon is provided in the literature [9,34,35]. The SIMS analysis were performed on a CAMECA IMS 4F-E7 instrument. For this study, O2+ beam bombardment was used to enhance the ionisation field of electropositive species such as uranium. In this scanning microscope, the primary beam is focused into a small spot (around 0.5 lm), which scans the sample surface. The collected secondary ions can be measured with an electron multiplier and also sequentially converted into an image. Mass resolution can reach M/DM = 10,000 and the lateral resolution of the imaging is only 0.5 lm. The experimental conditions for this work are indicated in Table 1. For each area analysed, mass spectra at around the mass of isotope 238 of uranium, and ion images were obtained. 40 Ca+ images give the histological structure of the bivalve gills and 238 + U images show uranium fixation within the structures. 2.2. Corbicula fluminea exposure conditions The experimental exposure conditions of bivalves, C. fluminea, have been widely described by Simon and Garnier-Laplace [7,8]. The freshwater bivalves, collected from Lake Sanguinet (Gironde, France), underwent an acclimatisation phase for at least 1 month under laboratory conditions (ambient temperature: 19–20 °C; artificial water in mg/L: Ca2+ = 11.5, Mg2+ = 8, Na+ = 11.6, K+ = 6.2, Cl = 13.5, NO3 = 6.3, SO4 = 8.1, HCO3 = 71, photoperiod: 12 h/ 12 h) in a storage tank containing quartz sand. The organisms were fed with an algae suspension (Chlamydomonas reinhardii). After the acclimatisation period they were exposed to aqueous uranium under laboratory conditions and they were not fed until the end of the experiments. The experimental system consists of two connected tanks: the first one maintains a constant pH and a constant U concentration in water and the second tank contains sand, aerated water and the bivalves. The uranium used for the contamination is a solution of uranyl nitrate. Two levels of exposure were used [U]water = 500 lg/L and 20 lg/L, with different exposure durations. Table 2 indicates the main parameters of each treatment. C. fluminea controls were prepared in the same way without the addition of uranium to the water. 2.3. Preparation of the biological samples for SIMS analysis
Epon mixture. Finally, they were embedded in pure EPON-type resin. Serial thin sections (0.5 lm) embedded in resin were cut and laid on polished ultra pure gold holders for SIMS analysis (to avoid relief effects and minimize charge effects) or on glass slides for histological controls with an optical microscope. 3. Results and discussion The paired gills consist of two plate-like flaps (the outer demibranch and inner demibranch). They are attached to the two sides of the visceral mass and to the proximal part of the foot, and hang inside the mantle cavity on each side of the foot. Each demibranch contains an ascending limb and a descending limb (Fig. 1). The gill structure consists mainly of filaments. All the filaments are parallel and are arranged in series connected by interlamellar septa (Fig. 2). Around the gills filaments, we distinguish cilia. The function of the gills is to transport water and gather food. The water passes between the gill filaments, which keep the respiratory exchanges steady. The ciliary and mucous cells draw, collect and transfer the food particles to the palps, the mouth and the digestive tract [37–39]. 3.1. Corbicula fluminea controls The morphology of the analysed gills filaments of the bivalves is shown in an optical microscope image and a 40Ca+ ion image (Fig. 3). In the SIMS image, the hot colours represent the highest Ca concentrations. Under these SIMS experimental conditions, the mass spectra of the gills of the clams that were not exposed to uranium, recorded at around the mass of isotope 238 of uranium at a low mass resolution (M/DM = 300), do not show the presence of a significant peak at mass 238 (Fig. 4). This result suggests that natural uranium is not detected by SIMS and no polyatomic ions are superimposed on the element of interest at a low mass resolution. In this case, working at high mass resolution is not essential, which will improve secondary ion transmission and therefore also the detection limits.
After the exposure phase, the gills of the clams were collected and underwent a classic chemical fixation procedure. They were fixed in a solution containing 6% glutaraldehyde in a sodium cacodylate buffer one day at 4 °C, then dehydrated in various propylene oxide and ethanol baths and permeated with a propylene oxide/
Table 1 Experimental analysis conditions by SIMS technique. SIMS experimental conditions
1
2
Primary ion beam Primary beam energy Primary beam intensity Secondary beam energy Primary beam raster Mass resolution
O2+ 12.5 keV 7 10 9 A 4.5 keV 200 200 lm2 M/DM = 300
O2+ 12.5 keV 2 10 9 A 4.5 keV 100 100 lm2 M/DM = 300
Fig. 1. Schematic representation of bivalve branchial [36].
C. Tessier et al. / Nuclear Instruments and Methods in Physics Research B 267 (2009) 1931–1935
Fig. 2. Optical microscope cross-section of gills.
Fig. 3. (a) Optical microscope cross-section of gills; and (b) ionic image40Ca+ (image field 100 lm 100 lm).
1933
ent exposure durations from 10 days to 90 days were studied using the SIMS method. The analysis of SIMS mass spectra reveals uranium accumulation in the gills since 10 days exposure period at masses of 238, corresponding to the 238U+ ion (Fig. 5). The Table 3 presents all the results we obtained in function exposure conditions: the intensity peaks at 238 mass according to the exposure conditions. We can observe a more important uranium fixation in the case of the high-level treatment (500 lg/L). Moreover, the uranium bioaccumulation gets rapidly after the beginning of the contamination; the exposure period does not seem to increase the uranium absorption level in the gill structure. Previous analysis of total tissues by ICP-OES (Inductively Coupled Plasma-Optical emission Spectrometry) confirm the uranium retention [7,8]. These experiments were performed in order to test the influence of exposure conditions parameters on the bioaccumulation process, the pH, uranium concentration in water and exposure duration. A significant pH effect was observed on the bioaccumulation rate (10X; from pH 8.1 to 7); the decrease of the pH contributes to the modification of the uranium carbonato-complexes speciation. Furthermore, for a given pH and same exposure duration, the retention is depending upon the aqueous concentration of uranium. An increase in the exposure level (from 100 lg/L to 1500 l/L) leads to an increase bioaccumulation in the gills. These experiments also have showed for the low aqueous U concentration (100 lg/L) a link between the accumulation and the exposure duration with a linear increase until day 21 and a plateau tendency from day 21 to day 42. However, an important individual variation was observed relative to the uranium concentration measured in the gills for the short exposure durations. This present work using the analytical ion microscopy completes the previous results with the study of a very low exposure level (20 lg/L). Furthermore, the SIMS images display a heterogeneous uranium distribution along the filaments and the interlamellar septa in the form of patches of a few micrometers in diameter (Fig. 6). This microdistribution profile is obtained whatever the exposure conditions may be (Fig. 7). At the present time we are studying the bioaccumulation and the transport mechanisms of uranium within these organisms.
Fig. 4. Mass spectrum recorded at mass around 238, showing no uranium isotopes in this biological control sample.
3.2. Uranium distribution in the gills of exposed clams The demibranches of the C. fluminea contaminated with two aqueous uranium concentrations (500 lg/L and 20 lg/L) for differ-
Fig. 5. Mass spectrum of gills Corbicula 20 lg/L, 10 days contamination: around 238.
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C. Tessier et al. / Nuclear Instruments and Methods in Physics Research B 267 (2009) 1931–1935
Table 3 238 + U results according to treatment conditions.
238
+
U (cts/s)
Control
20 lg/L 10 days
20 lg/L 40 days
20 lg/L 90 days
500 lg/L 10 days
<1
70–90
50–80
90
400
Fig. 6. Ionic images of gills Corbicula 20 lg/L, 90 days contamination: (a)
40
Ca+; (b)
238
In this study, a freshwater bivalve larva was also examined by SIMS technique, since the demibranch of one of the molluscs contaminated with 500 lg U/L for 10 days contained embryos. Fig. 8 represents the 40Ca+ and 238U+ ion images of a larva. These
Fig. 7. Ionic images of Corbicula gills 500 lg/L 10 days: (a)
40
Ca+ and (b)
U+ and (c)
238
U+ (red) superposed to
40
Ca+ (blue) (image field 200 lm 200 lm).
very interesting results show also an heterogeneous uranium distribution within the larva structure, indicating that the embryos had already assimilated a substantial quantity of this element.
238
U+ (red) superposed to
40
Ca+ (blue) (image field 100 lm 100 lm).
C. Tessier et al. / Nuclear Instruments and Methods in Physics Research B 267 (2009) 1931–1935
Fig. 8. Ionic images of larvae Corbicula 500 lg/L, 10 days contamination: (a) 40Ca+; and (b) 238U+ (red) superposed to 40Ca+ (blue) (image field 100 lm 100 lm).
4. Conclusion As part of the ENVIRHOM research program funded by the Institute for Radioprotection and Nuclear Safety, the study described in this paper presents uranium microdistribution in the gills of freshwater bivalves C. fluminea after internal contamination, analysed by the SIMS technique. The aim of this research program is to study the biological effects on ecosystems and the public of the bioaccumulation of radionuclides in situations of chronic exposure. This method of analysis and imaging using an ion beam coupled with a mass spectrometer has displayed the uranium sites of bioaccumulation in the gill structures at micrometer scale. These data confirm and complete the previous studies realised by ICP-OES. Indeed mass spectra and ion images (40Ca+ and 238U+) were produced on thin cross-sections of demibranches of C. fluminea submitted to various exposure conditions (different uranium concentrations in water and different contamination durations). We can observe uranium distribution profiles rapidly after the beginning of the contamination even with a very low exposure level in water (20 lg/ L). These cartographies will be a tool to explain and interpret the mechanisms of radionuclides transport in the ecosystems. In the future, other clams will be examined, in particular to analyse the supposed correlation between precipitates of iron and uranium distribution. Vertebrate animals, the zebrafishs Danio rerio affected by uranium exposure will investigated using the SIMS technique. We will take, more particularly, an interest in reproductive system and embryonic development [40]. References [1] E. Fournier, D. Tran, F. Denison, J.C. Massabuau, J. Garnier-Laplace, Environ. Toxicol. Chem. 23 (5) (2004) 1108. [2] D. Tran, J.C. Massabuau, J.C. Garnier-Laplace, Environ. Toxicol. Chem. 23 (3) (2004) 739.
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[3] D. Tran, J.C. Massabuau, J.P. Bourdineaud, J. Garnier-Laplace, Environ. Toxicol. Chem. 24 (9) (2005) 2278. [4] F. Denison, J. Garnier-Laplace, Geochim. Cosmochim. Acta 69 (9) (2005) 2183. [5] F. Denison, Thèse Chimie, Environnement et Santé, Université Aix-Marseille, Thèse IRSN 2004/43-FE, Clamart, 2004. [6] C. Fortin, L. Dutel, J. Garnier-Laplace, Environ. Toxicol. Chem. 23 (4) (2004) 974. [7] O. Simon, J. Garnier-Laplace, Aquat. Toxicol. 68 (2004) 95. [8] O. Simon, J. Garnier-Laplace, Aquat. Toxicol. 74 (2004) 372. [9] R. Castaing, G. Slodzian, J. Microsc. 1 (1962) 395. [10] P. Galle, J.P. Berry, in: R. Feder, J.Wm. McGovan, D.G. Shinozaki (Eds.), Examining the Submicron Word, Plenum Publishing Co., 1986. [11] R.W. Linton, J.G. Goldsmith, Biol. Cell 74 (1992) 147. [12] J. Clerc, C. Fourré, P. Fragu, Cell. Biol. Int. 21 (10) (1997) 1. [13] E. Kahn, C. Tessier, G. Lizard, A. Petiet, Anal. Quant. Cytol. Histol. 24 (5) (2002) 295. [14] J.L. Guerquin-Kern, T.D. Wu, C. Quintana, A. Croisy, Biochim. Biophys. Acta 1724 (2005) 228. [15] S. Chandra, D.R. Lerey II, Int. J. Mass Spectrom. 260 (2007) 90. [16] E. Hidié, A. Petiet, K. Bourahla, N. Colas-Linhart, G. Slodzian, R. Dennebouy, P. Galle, Cell. Mol. Biol. 47 (3) (2001) 403. [17] N. Grignon, S. Halpern, J. Jeusset, C. Briançon, P. Fragu, J. Microsc. 186 (1) (1997) 51. [18] N. Grignon, J.J. Vidùar, F. Hillion, B. Jaillard, in: A. Benninghoven, P. Bertrand, H.-N. Migeon, H.W. Werner (Eds.), Proceedings of the 12th International Conference on Secondary Ion Mass Spectrometry, Elsevier Science, 2000. [19] N. Blair, K.E. Prince, R.D. Faulkner, A.R. Till, Scanning 28 (5) (2006) 259. [20] A. Amaral, P. Galle, F. Escaig, C. Cossonnet, M.H. Henge-Napoli, E. Ansoborlo, L. Zhang, J. Alloy Comp. 271–273 (1998) 19. [21] S.J. Markich, R.A. Jeffree, P.T. Burke, Environ. Sci. Technol. 36 (5) (2002) 821. [22] J.L.A.M. Kramer, A.C. Kik, R.D. Vis, Nucl. Instr. and Meth. Phys. Res. B 104 (1995) 494. [23] M. Betti, G. Tamborini, L. Koch, Anal. Chem. 71 (1999) 2616. [24] L. Ottolini, R. Oberti, Anal. Chem. 72 (2000) 3731. [25] N. Erdmann, M. Betti, O. Stetzer, G. Tamborini, J.V. Kratz, N. Trautmann, J. van Geel, Spectrochim. Acta B 55 (2000) 1565. [26] G. Tamborini, M. Betti, Mikrochim. Acta 132 (2000) 411. [27] B. Wallenius, G. Tamborini, L. Koch, Radiochim. Acta 89 (2001) 55. [28] P.R. Danesi, A. Markowicz, E. Chinea-Cano, W. Burkart, B. Salbu, D. Donohue, F. Ruedenauer, M. Hedberg, S. Vogt, P. Zahradnik, A. Ciurapinski, J. Environ. Radioact. 64 (2003) 143. [29] C. Pomiès, B. Hamelin, J. Lancelot, R. Blomqvist, Appl. Geochem. 19 (2004). [30] G. Tamborini, Microchim. Acta 145 (2004) 237. [31] S. Török, J. Osan, L. Vincze, S. Kurunczi, G. Tamborini, M. Betti, Spectrochim. Acta B 59 (2004) 689. [32] B. Bingen, H. Austrheim, M.J. Whitehouse, W.J. Davis, Contrib. Miner. Petrol 147 (2004) 671. [33] F. Esaka, K.T. Esaka, C.G. Lee, M. Magara, S. Sakurai, S. Usuda, K. Watanabe, Talanta 71 (2007) 1011. [34] G. Slodzian, Physica 9 (1964) 594. [35] A. Benninghoven, F.G. Rüdenauer, H.W. Werner, Chem. Anal., Vol. 86, John Wiley, New York, 1987. [36] M. Le Pennec, L. Barillé, H. Grizel, in: H. Grizel (Ed.), An Atlas of Histology and Cytology of Marine Bivalve Molluscs, France, 2003. [37] E. Fournier, Bioaccumulation du Sélénium et Effets Induits Chez le Bivalve Filtreur Corbicula fluminea, IRSN-2005/58. [38] M. Byrne, H. Phelps, T. Church, V. Adair, P. Selvakumaraswamy, J. Potts, Hydrobiologia 418 (2000) 185. [39] A.V. Korniushin, M. Glaubrecht, Acta Zoologica (Stockholm) 84 (2003) 239. [40] S. Bourrachot, O. Simon, R. Gilbin, Aquat. Toxicol. 90 (2008) 29.
Contents lists available at ScienceDirect
Nuclear Instruments and Methods in Physics Research B journal homepage: www.elsevier.com/locate/nimb
Detection and analysis of the microdistribution of uranium in the gills of freshwater Corbicula fluminea by SIMS technique C. Tessier a,*, D. Suhard a, O. Simon b, M. Floriani b, F. Rebière a, J.-R. Jourdain a a
IRSN, Direction de la RadioProtection de l’Homme, Service de Dosimétrie Interne, Laboratoire de RadioChimie, IRSN, BP 17, F-92262 Fontenay aux Roses Cedex, France IRSN, Direction de l’Environnement et de l’Intervention, Service d’Etude du Comportement des Radionucléides dans les écosystèmes, Laboratoire de Radioécologie et d’écotoxicologie, IRSN, BP F6 Cadarache, France b
a r t i c l e
i n f o
Article history: Received 18 December 2008 Received in revised form 11 March 2009 Available online 28 March 2009 Keywords: SIMS Contamination Uranium Corbicula fluminea Imaging
a b s t r a c t The microdistribution of uranium in the gills of freshwater bivalve Corbicula fluminea following chronic direct exposure to this radioelement has been investigated using the SIMS technique. Different exposure levels and exposure durations have been studied. The SIMS mass spectra and 238U+ ion images produced with a SIMS CAMECA 4F-E7 show an U accumulation with the lower aqueous U concentration (20 lg/L) and the influence of the exposure levels on the bioaccumulation capacities. Furthermore, the ionic images display a heterogeneous distribution of uranium within the gill structure whatever the exposure conditions are. This study, in keeping with the ENVIRHOM French research program, was led to the conclusion that ion microscopy is an appropriate analytical method for trace elements and can give elemental cartography in a biological tissue section. Ó 2009 Elsevier B.V. All rights reserved.
1. Introduction The ENVIRHOM research French program supported by the Institute for Radioprotection and Nuclear Safety (IRSN) is intended to improve assessment of the risks to the general public and the ecosystems associated with chronic exposure to low levels of radioactive contaminants. The main objectives are to study the speciation, transfer, biokinetic and accumulation processes of radionuclides and also the biological effects correlated with this exposure on the human model (rats and mice) and on environmental organisms (algae, molluscs, crustaceans, fish, plants, etc.). Uranium has been the first element studied as part of this program. Chronic exposure of uranium may occur naturally in contaminated areas (underground water) or as a result of human activity (nuclear fuel cycle, agricultural use, military use of depleted uranium). Concerning the environmental aspect of the program, several biological responses have been studied in various living organisms to establish concentration-effects relationships: feeding strategies, ventilation of molluscs, reproduction, growth rate of living organisms and the effects on the ecosystems [1–3]. A thermodynamic equilibrium model of uranium in aqueous systems has been described to interpret the experimental data obtained on the bioavailability of uranium (VI) [4–6]. Simon and Garnier-Laplace
* Corresponding author. E-mail address: [email protected] (C. Tessier). 0168-583X/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2009.03.096
[7,8] have studied the bioaccumulation of uranium in the freshwater bivalve Corbicula fluminea. They have shown a pH influence and a correlation between the exposure conditions (exposure durations and treatments) and the distribution of this actinide in the bivalve organs. The aim of this paper is to present a study of the microdistribution of uranium in gills of the bivalve C. fluminea after chronic direct exposure of this radionuclide, by SIMS (Secondary Ion Mass Spectrometry) microscopy. The SIMS technique introduced in the early sixties by Castaing and Slodzian [9] is used for the chemical characterization of solid surfaces mainly in geochemistry, microelectronics and material sciences. However, few years ago, the study and analysis of biological samples by ion microscopy were developed in the biomedical [10–15], nuclear [16] and botanic fields [17–19]. In the literature, only a few papers describe uranium detection by this technique in biological samples [20,21]. In particular, Markich et al. [21] used SIMS to measure metal ratios such as U/Ca in freshwater bivalve shells (Velesunio angasi), showing that these clams can be used over their lifetime as archival indicators of metal pollution in surface waters of the Finniss River (in tropical northern Australia near a copper–uranium mine). Moreover, the isotope ratio of this long-lived radionuclide at very low concentration levels has been measured by SIMS technique for environmental monitoring in geoscience, cosmochemistry, and planetary and nuclear sciences [22–33].
1932
C. Tessier et al. / Nuclear Instruments and Methods in Physics Research B 267 (2009) 1931–1935
2. Materials and methods
Table 2 Treatment conditions of contaminated Corbicula.
2.1. SIMS microscopy
Total U concentration in water (lg/L)
Exposure duration (days)
pH
500 20 20 20
10 10 40 90
7 7 7 7
The aim of SIMS microscopy is the elemental and isotopic analysis of a solid surface by an ion beam coupled with a mass spectrometer. The principle of this technique is based upon the sputtering of a few atomic layers from the surface of a sample, induced by a bombardment of focused primary ions of sufficiently high energy (some keV). A more detailed description of the physical phenomenon is provided in the literature [9,34,35]. The SIMS analysis were performed on a CAMECA IMS 4F-E7 instrument. For this study, O2+ beam bombardment was used to enhance the ionisation field of electropositive species such as uranium. In this scanning microscope, the primary beam is focused into a small spot (around 0.5 lm), which scans the sample surface. The collected secondary ions can be measured with an electron multiplier and also sequentially converted into an image. Mass resolution can reach M/DM = 10,000 and the lateral resolution of the imaging is only 0.5 lm. The experimental conditions for this work are indicated in Table 1. For each area analysed, mass spectra at around the mass of isotope 238 of uranium, and ion images were obtained. 40 Ca+ images give the histological structure of the bivalve gills and 238 + U images show uranium fixation within the structures. 2.2. Corbicula fluminea exposure conditions The experimental exposure conditions of bivalves, C. fluminea, have been widely described by Simon and Garnier-Laplace [7,8]. The freshwater bivalves, collected from Lake Sanguinet (Gironde, France), underwent an acclimatisation phase for at least 1 month under laboratory conditions (ambient temperature: 19–20 °C; artificial water in mg/L: Ca2+ = 11.5, Mg2+ = 8, Na+ = 11.6, K+ = 6.2, Cl = 13.5, NO3 = 6.3, SO4 = 8.1, HCO3 = 71, photoperiod: 12 h/ 12 h) in a storage tank containing quartz sand. The organisms were fed with an algae suspension (Chlamydomonas reinhardii). After the acclimatisation period they were exposed to aqueous uranium under laboratory conditions and they were not fed until the end of the experiments. The experimental system consists of two connected tanks: the first one maintains a constant pH and a constant U concentration in water and the second tank contains sand, aerated water and the bivalves. The uranium used for the contamination is a solution of uranyl nitrate. Two levels of exposure were used [U]water = 500 lg/L and 20 lg/L, with different exposure durations. Table 2 indicates the main parameters of each treatment. C. fluminea controls were prepared in the same way without the addition of uranium to the water. 2.3. Preparation of the biological samples for SIMS analysis
Epon mixture. Finally, they were embedded in pure EPON-type resin. Serial thin sections (0.5 lm) embedded in resin were cut and laid on polished ultra pure gold holders for SIMS analysis (to avoid relief effects and minimize charge effects) or on glass slides for histological controls with an optical microscope. 3. Results and discussion The paired gills consist of two plate-like flaps (the outer demibranch and inner demibranch). They are attached to the two sides of the visceral mass and to the proximal part of the foot, and hang inside the mantle cavity on each side of the foot. Each demibranch contains an ascending limb and a descending limb (Fig. 1). The gill structure consists mainly of filaments. All the filaments are parallel and are arranged in series connected by interlamellar septa (Fig. 2). Around the gills filaments, we distinguish cilia. The function of the gills is to transport water and gather food. The water passes between the gill filaments, which keep the respiratory exchanges steady. The ciliary and mucous cells draw, collect and transfer the food particles to the palps, the mouth and the digestive tract [37–39]. 3.1. Corbicula fluminea controls The morphology of the analysed gills filaments of the bivalves is shown in an optical microscope image and a 40Ca+ ion image (Fig. 3). In the SIMS image, the hot colours represent the highest Ca concentrations. Under these SIMS experimental conditions, the mass spectra of the gills of the clams that were not exposed to uranium, recorded at around the mass of isotope 238 of uranium at a low mass resolution (M/DM = 300), do not show the presence of a significant peak at mass 238 (Fig. 4). This result suggests that natural uranium is not detected by SIMS and no polyatomic ions are superimposed on the element of interest at a low mass resolution. In this case, working at high mass resolution is not essential, which will improve secondary ion transmission and therefore also the detection limits.
After the exposure phase, the gills of the clams were collected and underwent a classic chemical fixation procedure. They were fixed in a solution containing 6% glutaraldehyde in a sodium cacodylate buffer one day at 4 °C, then dehydrated in various propylene oxide and ethanol baths and permeated with a propylene oxide/
Table 1 Experimental analysis conditions by SIMS technique. SIMS experimental conditions
1
2
Primary ion beam Primary beam energy Primary beam intensity Secondary beam energy Primary beam raster Mass resolution
O2+ 12.5 keV 7 10 9 A 4.5 keV 200 200 lm2 M/DM = 300
O2+ 12.5 keV 2 10 9 A 4.5 keV 100 100 lm2 M/DM = 300
Fig. 1. Schematic representation of bivalve branchial [36].
C. Tessier et al. / Nuclear Instruments and Methods in Physics Research B 267 (2009) 1931–1935
Fig. 2. Optical microscope cross-section of gills.
Fig. 3. (a) Optical microscope cross-section of gills; and (b) ionic image40Ca+ (image field 100 lm 100 lm).
1933
ent exposure durations from 10 days to 90 days were studied using the SIMS method. The analysis of SIMS mass spectra reveals uranium accumulation in the gills since 10 days exposure period at masses of 238, corresponding to the 238U+ ion (Fig. 5). The Table 3 presents all the results we obtained in function exposure conditions: the intensity peaks at 238 mass according to the exposure conditions. We can observe a more important uranium fixation in the case of the high-level treatment (500 lg/L). Moreover, the uranium bioaccumulation gets rapidly after the beginning of the contamination; the exposure period does not seem to increase the uranium absorption level in the gill structure. Previous analysis of total tissues by ICP-OES (Inductively Coupled Plasma-Optical emission Spectrometry) confirm the uranium retention [7,8]. These experiments were performed in order to test the influence of exposure conditions parameters on the bioaccumulation process, the pH, uranium concentration in water and exposure duration. A significant pH effect was observed on the bioaccumulation rate (10X; from pH 8.1 to 7); the decrease of the pH contributes to the modification of the uranium carbonato-complexes speciation. Furthermore, for a given pH and same exposure duration, the retention is depending upon the aqueous concentration of uranium. An increase in the exposure level (from 100 lg/L to 1500 l/L) leads to an increase bioaccumulation in the gills. These experiments also have showed for the low aqueous U concentration (100 lg/L) a link between the accumulation and the exposure duration with a linear increase until day 21 and a plateau tendency from day 21 to day 42. However, an important individual variation was observed relative to the uranium concentration measured in the gills for the short exposure durations. This present work using the analytical ion microscopy completes the previous results with the study of a very low exposure level (20 lg/L). Furthermore, the SIMS images display a heterogeneous uranium distribution along the filaments and the interlamellar septa in the form of patches of a few micrometers in diameter (Fig. 6). This microdistribution profile is obtained whatever the exposure conditions may be (Fig. 7). At the present time we are studying the bioaccumulation and the transport mechanisms of uranium within these organisms.
Fig. 4. Mass spectrum recorded at mass around 238, showing no uranium isotopes in this biological control sample.
3.2. Uranium distribution in the gills of exposed clams The demibranches of the C. fluminea contaminated with two aqueous uranium concentrations (500 lg/L and 20 lg/L) for differ-
Fig. 5. Mass spectrum of gills Corbicula 20 lg/L, 10 days contamination: around 238.
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C. Tessier et al. / Nuclear Instruments and Methods in Physics Research B 267 (2009) 1931–1935
Table 3 238 + U results according to treatment conditions.
238
+
U (cts/s)
Control
20 lg/L 10 days
20 lg/L 40 days
20 lg/L 90 days
500 lg/L 10 days
<1
70–90
50–80
90
400
Fig. 6. Ionic images of gills Corbicula 20 lg/L, 90 days contamination: (a)
40
Ca+; (b)
238
In this study, a freshwater bivalve larva was also examined by SIMS technique, since the demibranch of one of the molluscs contaminated with 500 lg U/L for 10 days contained embryos. Fig. 8 represents the 40Ca+ and 238U+ ion images of a larva. These
Fig. 7. Ionic images of Corbicula gills 500 lg/L 10 days: (a)
40
Ca+ and (b)
U+ and (c)
238
U+ (red) superposed to
40
Ca+ (blue) (image field 200 lm 200 lm).
very interesting results show also an heterogeneous uranium distribution within the larva structure, indicating that the embryos had already assimilated a substantial quantity of this element.
238
U+ (red) superposed to
40
Ca+ (blue) (image field 100 lm 100 lm).
C. Tessier et al. / Nuclear Instruments and Methods in Physics Research B 267 (2009) 1931–1935
Fig. 8. Ionic images of larvae Corbicula 500 lg/L, 10 days contamination: (a) 40Ca+; and (b) 238U+ (red) superposed to 40Ca+ (blue) (image field 100 lm 100 lm).
4. Conclusion As part of the ENVIRHOM research program funded by the Institute for Radioprotection and Nuclear Safety, the study described in this paper presents uranium microdistribution in the gills of freshwater bivalves C. fluminea after internal contamination, analysed by the SIMS technique. The aim of this research program is to study the biological effects on ecosystems and the public of the bioaccumulation of radionuclides in situations of chronic exposure. This method of analysis and imaging using an ion beam coupled with a mass spectrometer has displayed the uranium sites of bioaccumulation in the gill structures at micrometer scale. These data confirm and complete the previous studies realised by ICP-OES. Indeed mass spectra and ion images (40Ca+ and 238U+) were produced on thin cross-sections of demibranches of C. fluminea submitted to various exposure conditions (different uranium concentrations in water and different contamination durations). We can observe uranium distribution profiles rapidly after the beginning of the contamination even with a very low exposure level in water (20 lg/ L). These cartographies will be a tool to explain and interpret the mechanisms of radionuclides transport in the ecosystems. In the future, other clams will be examined, in particular to analyse the supposed correlation between precipitates of iron and uranium distribution. Vertebrate animals, the zebrafishs Danio rerio affected by uranium exposure will investigated using the SIMS technique. We will take, more particularly, an interest in reproductive system and embryonic development [40]. References [1] E. Fournier, D. Tran, F. Denison, J.C. Massabuau, J. Garnier-Laplace, Environ. Toxicol. Chem. 23 (5) (2004) 1108. [2] D. Tran, J.C. Massabuau, J.C. Garnier-Laplace, Environ. Toxicol. Chem. 23 (3) (2004) 739.
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