Detection of radionuclides in the shell of the freshwater molluscs Michael A. Zuykov1, Stanislav I. Shabalev1, Viktor P. Tishkov1, Andrey I. Smagin2, Yulia V. Plotkina3, Maxim R. Pavlov3, Marina I. Orlova4,Thomas Servais5 1 Laboratory of Applied Mineralogy and Radiogeochemistry, V.G. Khlopin Radium Institute, 28, 2nd Murinskiy Ave., 194021 St. Petersburg, Russia. 2 PA “Mayak”, 31, Lenina pr., 456780 Ozersk, Russia.3Laboratory of Isotopic Geology, Institute of Precambrian Geology and Geochronology RAS, 2, Makarova emb., 199034 St. Petersburg, Russia. 4Zoological Institute, RAS, 1, University emb., 199034 St. Petersburg, Russia.5 Université Lille 1,Laboratoire Géosystèmes (UMR 8157 CNRS), UFR des Sciences de la Terre - bâtiment SN5, 59655 Villeneuve d'Ascq cedex, France. INTRODUCTION Carbonates of biological origin which, in particular, forming skeletal elements of nonhuman water organisms (e.g., mollusc shells) have mainly been under discussion as a simple tool for measurement of bulk activities of radionuclides, whereas little is known about the mechanism of incorporation and the structural response of biogenic carbonates to radionuclides. It is also important to note, that geochemical interpretations of biogenic carbonates are more difficult than those of non-biogenic origin, because chemical element incorporation into organic-mineral matrix is also subjected to the biological control during growth of skeletal elements. On the other hand, high sensitivity of spectrometric technique makes easily the measurement of radionuclides concentrations in bio-indicators. But, relatively low concentrations of radionuclides in skeletal elements make difficult to apply traditional methods (e.g. SEM microprobe analysis) for study of detailed characters of distribution of radionuclides in biogenic carbonates. There are published reports on successful application of new generation of electron microscopy technique for characterization of radionuclides distribution within the skeleton and soft tissues of hydrobionts (e.g., see TEM-EDX in Floriani, 2005) and details of shell biomineralization (e.g., see XANES spectroscopy in Gilbert et al. 2005). Unfortunately, that equipment is not easily accessible for researchers, yet. There are also safety and administrative restrictions for transportation and study of radioactive samples derived from nuclear factories or nuclear waste depositories where the most important (highly polluted) samples are occurred. Cooperation between geochemical and hydrobiological laboratories in Russia and France was established to carry out complex study of mollusc shells collected from the localities contaminated with radionuclides in Russia and Ukraine, and shells obtained after laboratory controlled experiments. Inevitably, the main focus was on the technological water reservoirs of PA “Mayak” (Chelyabinsk region, Russia), which gives the opportunity to study shells with high concentration of radionuclides that were growing in natural, permanently radioactive environment which can be characterized as low level radioactive waste. At present, our research program involves six sets of samples, which differ in radionuclides composition (or its non-radioactive analogues). These are: 1) pure (control) shells; 2) shells doped with 241Am; 3) shells doped with 137Cs, 85Sr, and 241Am; 4) shells doped with 137Cs and

90Sr; 5) shells doped with 137Cs, 90Sr, and 60Co; 6) shells doped with non radioactive Eu (imitator of Am). The studied molluscs are characterized by calcareous, aragonitic shell mineralization identifiable from XRD analysis. There are considerable difficulties with synthetic aragonite growth in controlled environment. Thus, the samples of artificial calcite (pure, Eu-doped and 241Am-doped) synthesized during our previous work (Zamoryanskaya et

al. 2006) with use slightly modified method by Gruzensky (1966) have been studied for comparison. The main objective of this report was to provide a data on measuring of radionuclides in molluscs shell from polluted and controlled environments, and to describe their cathodoluminescence response to study possible application of сathodoluminescent analysis for detection of radionuclides in carbonates of biogenic origin. In the French laboratory the samples of mollusc shells and artificial crystalline calcite, pure and doped with non-radioactive Eu has been examined. In addition, all studied samples were analyzed with electron probe microanalyser to determine trace-elements that may have influence on Cathodoluminescent emission in carbonates (Jimenez-Berrocoso et al. 2004); and by X-ray diffractometer for study of phases composition. MATERIALS AND METHODS

(a) (b)

Figure 1. Maps showing location of sampling sites: (a) in Sweden, Russia and Ukraine (1 – Mälaren Lake, 2 – Gulf of Finland, 3 – Udomlya Lake (Kalinin NPP), 4 – Cooling Pond of Chernobyl NPP, 5 – Techa Cascade of Reservoires, PA “Mayak”); (b) near PA “Mayak” (Chelyabinsk region, Russia). Table 1. Predominately distributed radionuclides concentrations (in kBq/l) in water where studied molluscs were collected. 1 data by authors; 2 data from Smagin (2007).

Localities Sampling year 137Cs 90Sr 60Co Mälaren Lake, Sweden no data 1Gulf of Finland (eastern part, NW Russia) 2006 0.00003 0.00001 — 1Cooling-reservoir of Kalinin NPP 2006 0.00001 — — 1Cooling-pond of Chernobyl NPP 2005 0.003 0.004 — 2PA “Mayak” - Water reservoir # 4 2006 0.4 5 0.03 2PA “Mayak” - Water reservoir # 10 2006 0.03 3 0.002 Collection of molluscs Freshwater bivalve (Colletopterum sp., Dreissena polymorpha, D.bugensis, Pseudanodonta sp.) and gastropod (Lymnaea auricularia, Lymnaea stagnalis, Lymnaea fragilis, Planorbis planorbis) molluscs were manually collected from the site on the bank of the five natural and artificial lakes located in Sweden (in 2006), Russia (in 2000-2007) and Ukraine (in 2005), as

shown in Figure 1. These include reservoirs: (i) with natural radioactivity level (control points); (ii) with probable low content of radioactive material; and (iii) with high level of radioactive contamination (Table 1). Laboratory controlled experiments The experiments with artificial contamination of molluscs by 137Cs, 85Sr and 241Am were performed in 2001 in the Laboratory of Applied Mineralogy and Radiogeochemistry at Khlopin Radium Institute (Table 2). Radionuclides were added into the water medium of aquariums both individually and in mixtures (like those in polluted environment). Freshwater bivalve Dreissena polymorpha (collected in the eastern part of the Gulf of Finland, Fig.1) and gastropod Potamopyrgus antipodarum (laboratory population) were used in experiments. The laboratory facilities include glass aquariums with brackish water (salinity, 4 0/00, temperature 20±2°C) and aquaria equipments which were placed in glove box. During all days of experiment radionuclides or water were not added to aquariums, and animals did not receive food. In order to create the material for comparative study, one experiment with Eu3+ and D.polymopha under analytical conditions similar to the Experiment 2 (see in Table 2) but only with exposure time of 25 days was carried out. Table 2. Details of Radionuclide Uptake Experiments.

Total activity of radionuclide used (Bq)* Organisms

How radionuclides were added into experimental aquariums 241Am 137Cs 85Sr

Dreissena polymorpha in mixture 2·106 38·106 3·106 Experiment 1 Nine molluscs of middle size were placed into 2,4-L aquarium. After 8, 10 and 26

days, the content of radionuclides in molluscs (shell and soft tissues) was measured. Dreissena polymorpha Individual 6·108 — —

Experiment 2 Nine molluscs (three molluscs of small, middle and large size) were placed into 2,5-L aquarium. After 1, 11 and 25 days, the content of radionuclides in molluscs (shell and soft tissues) was measured. Potamopyrgus antipodarum Individual 6.7·105 — —

Experiment 3 Nine molluscs were placed into 0,12-L box. After 8, 10, 29 and 68 days, the content of radionuclides in whole molluscs (shell plus soft tissues) was measured.

*- bulk activity of radionuclide in each experimental aquarium

Analytical procedures A total of about 90 specimens of molluscs shell (conjoined valves) were obtained after laboratory experiments and about 150 specimens have been collected from controlled and polluted environment. 25 molluscs shell of different species in various growth, various locations or various exposure time (in case of laboratory experiments) have been selected for study with SEM, Microprobe and Cathodoluminescent analyser. The samples preparation procedure for these methods included the following activities: cuting of the shell in different directions, forming of analytical sample (up to 9 sections in one sample), covering the sample by dental plastic (Protacril-M), polishing a plastic tablet, coating the tablet with carbon. In addition, three tablets with samples of artificial calcite (pure, Eu-doped, 241Am-doped) were made. All sets of samples were examined with a scanning electron microscope ABT-55 (Akashi) with microprobe system Link AN 10000/85 S and with Cold Cathodoluminescent apparatus (Mini-CL) in the Institute of Precambrian Geology and Geochronology (St. Petersburg). Operating conditions included an accelerating voltage of 25 kV at 0.9 nA beam current, with beam diameter of approximately 2.5 µm. Analytical conditions were a beam

current of 0.9 nA for all elements, an accelerating voltage of 25 kV and a spot diameter of 2.5 µm. Moreover, pure and Eu-doped shells of Dreissena polymorpha and artificial calcite were analysed by electron probe microanalyser (Quanta 200) to determine content of main chemical elements at LP3, University of Lille. Analytical conditions were a beam current of 3 nA for all elements, an accelerating voltage of 20 kV and a spot diameter of 6 µm.

Figure 2. Photo (SEM) of microstructure of the shell of Dreissena polymorpha in thin section. Remaining fragments of molluscs shell left preparation of plastic tablets were used for determination of radionuclides content in radioisotopic laboratory of the Khlopin Radium Institute in a well calibrated geometry. 137Cs, 90Sr, 60Co predominating radionuclides that were determined in shells collected from polluted environment. 241Am and 85Sr have been determined in materials obtained after laboratory experiments. For 90Sr, selected samples have been weighed and then dissolved in concentrated acid (6M HCl). Spectrometric equipment includes gamma-spectrometer ORTEC with Ge detectors GEM-30185 and GWL-90-15, and alfa-beta radiometer LB-770 (BERTHOLD). In table 3 data on radionuclides concentrations in studied molluscs shell and their cathodoluminescent response are summarised.

Figure 3. Thin sections of the molluscs shell in plastic polished samples: Dreissena polymorpha collected from pure water (1), and ones obtained after laboratory experiment with 241Am (2). D. bugensis collected near Chernobyl NPP (3). Pseudanodonta sp. (4), Lymnaea stagnalis (5) and Planorbis planorbis (6) collected near PA “Mayak”. A – pictures in SEM; B – Cathodoluminescent image.

Table 3. Concentrations (in Bq/g) of radionuclides in selected studied molluscs shell and their cathodoluminescent response. * – data on shell plus soft body together.

Concentration of radionuclides in Bq/g Taxon/ Samples

Localities

137Cs 90Sr 85Sr 60Co 241Am

Eu

CL res-ponse

Gulf of Finland 0.1 0.4 Mälaren Lake 0.2 Kalinin NPP 0.005

7000 30000 20000 135000

NO

+ 78000

Dreissena polymorpha Laboratory

Experiments

123000 YES

Dreissena bugensis Chernobyl NPP 3 NO

B I V A L V I A

Pseudanodonta sp. 34 7020 1.5 Lymnaea stagnalis 1.23 14173 YES

Lymnaea auricularia 15 4315 3.5 NO

Planorbis planorbis

PA “Mayak”

12.3 2743 0.7 YES

Lymnaea fragilis Chernobyl NPP 0.10 150

G A S T R O P O D A

Potamopyrgus antipodarum

Laboratory Experiments

3000000*

Artificial calcite crystal +

NO

RESULTS AND CONCLUSIONS 1. Cathodoluminescent images of three (among six) studied sets of samples of molluscs shell are characterized by light bands of high intensity which are oriented in parallel to the shell surface, which correspond to different structural layers (Figs. 2, 3). After comparison of selected CL images and ones obtained by SEM (Fig. 2) and optical microscopy we can only proposed, that the most intensive zones of luminescence correspond to the boundaries between structural layers.

2. Distribution of luminescent zones through various parts of the shell (from umbonal area to anterior margin) was studied in numerous specimens of D.polymorpha obtained after laboratory experiments with 241Am. There are no serious variations in CL images through the shell.

3. There is no dependence between taxonomical affiliation of molluscs, chemical composition, radionuclide composition or their concentration, and cathodoluminescent response of the shells have been observed within investigated sets of samples (Table 3).

4. There is a difference in intensity of luminescence among studied sets of samples (Fig.3). The molluscs shell from technological water reservoirs of PA “Mayak” are characterized by highest intensity of light bands.

5. Unfortunately, the available analytical equipment does not show difference in chemical composition between studied shells through low detection limit that is 0.5 and 0.2 wt % for French and Russian equipment, respectively. The only traditional components of molluscs

shell like Na, Mg, Sr and some other impurities in trace concentration have been detected. However, as it is known from published data, various contaminants (e.g. heavy metals, radionuclides another than 137Cs, 90Sr or 60Co) are also occurred in water objects near PA “Mayak” and Chernobyl NPP which may have influence on Cathodoluminescent emission of shells from these areas.

6. Cathodoluminescent images of artificial calcite crystals doped with Eu3+ are different from ones of molluscs shell, where presence of Eu has been supported by data of microprobe analysis. Zamoryanskaya et al. (2006) reported that the Cathodoluminescent emission of Americium was not confirmed for all samples of calcite crystals which were grown in laboratory condition. Different concentration of Am in studied samples has been considered as a one of the reasons for that.

7. Further investigations of all sets of samples discussed in present report by autoradiography with control by high resolution quantitative analysis of trace-elements are needed to increase the accuracy of data of CL analysis. ACKNOWLEDGEMENT Authors thank Dr. Leonid Popov for helpful comments; Dr. Alexander Naseka and Dr. Yurii Shibetsky for assistance in the field work on PA “Mayak” and Chernobyl NPP. The authors gratefully acknowledge the Russian Foundation for Basic Research (Projects # 03-05-65095, 05-05-65091, 08-05-00957) for funding this project. Visits between laboratories by M.I. Orlova, T. Servais and M.A. Zuykov have been supported by grant NATO (CLG. 982397). M. A. Zuykov personally thanks ALSAM foundation (USA). REFERNCES Floriani, M., 2005. Subcellular localization of radionuclides by transmission electron microscopy: Application

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Zamoryanskaya M.V., Burakov B.E., Kolesnikova E.V., Zuykov M.A. 2006. Cathodoluminescence study of americium incorporation into calcite single crystals. Material Research Society, Symposium Proceedings "Scientific Basis for Nuclear Waste Management”, (Pierre Van Iseghem ed.), vol. 932, 919-924.

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