M. Iammarino, D. dell’Oro, N. Bortone, A. E. Chiaravalle* 3rd EAVLD Congress on veterinary diagnostics, 12th – 15th October 2014, Pisa - Italy STRONTIUM-90 ACCUMULATION IN ANIMAL BONES: RADIOCHEMICAL DETERMINATION BY LIQUID SCINTILLATION COUNTING (LSC) M. Iammarino, D. dell’Oro, N. Bortone, A. E. Chiaravalle* Centro di Referenza Nazionale per la Ricerca della Radioattività nel Settore Zootecnico- Veterinario, Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata, Italy *izsfgchimica@tiscali.it INTRODUCTION 90Sr is a radiotoxic isotope, characterized by long physical and biological half-life. It is produced during nuclear fission processes and then it decays to its radioactive daughter nuclide 90Y, emitting high energy (546 keV) β particles [1]. Animal feeding in polluted soils and with contaminated water is the largest source of exposure to 90Sr, which may be absorbed by following metabolic pathways similar to those of Ca. Once 90Sr enters the blood, it may be accumulated in bones where it is retained, so it is very important to control the radiocontamination level in the environment and in food. A direct correlation between the activity of 90Sr in soil sample and site altitude was demonstrated [2] and 90Sr content increases also with the age of the animal. The radiochemical determination of 90Sr represents a challenge for Organisms in charge of analytical controls. The most complex analytical step is the separation of 90Sr from other alkaline earth elements, such as Ca and Ba. In this work, a radiochemical procedure by ultra low level liquid scintillation counting (LSC) for the monitoring of 90Sr in animal bone samples, after achieving 90Y secular equilibrium condition, was implemented and validated. The validation parameters, such as trueness, precision, counting efficiency, and measurement uncertainty were determined using samples fortified with known activities of 90Sr (at 10 and 100 Bq kg-1). In order to verify the method applicability for routinely controls, it was then employed for the analyses of 10 cow bone samples. MATERIALS & METHODS Dissolution in 250 ml of HNO3 + 0.5 ml HF Add 50 ml of Oxalic Acid 8 % at pH 2,5 Lanthanides and Actinides, Y, La, Ce, Th, U, Pu Add Y stable carrier Precipitation of interferences as sulfides and filtration Add Sr stable carrier Sample ashing 1000 °C Extracion with 20% HDEHP at pH 1.0 Dissolution in HNO3 + H2O2 Precipitation of strontium oxalate at pH 4.5 liquid Organic Inorganic Add Pb and Bi stable carrier and 50 mg of Na2S Extracion with 5% HDEHP at pH 1.0 L/L extraction with HNO3 3N Ca, 89Sr, 90Sr, 134Cs, 137Cs, 40K, 140Ba Add 50 mL of Oxalic Acid 8 % at pH 2,5 Precipitation of Y oxalate and dissolution in HNO3. Add HCl and scintillation cocktail Counting in LSC; T= 1000 min Strontium was separated from the bone sample using the method described in figure 1. 50 g of bone sample was ashed at 1000 °C, dissolved in 8M HNO3 +0.5 ml of 50% HF. The mixture was treated by leaching at 320°C and filtered. Oxalic acid (~20 g) and sodium acetate (~7 g) were added and strontium oxalate was precipitated at pH 4.5. The precipitate was filtered and dissolved in 35% H2O2 and 8M HNO3 and then dried. The residue was dissolved in 0.1 M HCl; then the interferences (210Pb e 210Bi) were removed as sulphides and the solution was placed in a separatory funnel with 200 ml of 20% Bis-(2-ethylhexyl) phosphate (HDEHP) in toluene and mixed. The acid phase (200 mL) was maintained at 25°C for two weeks until achievement of 90Sr/90Y secular equilibrium. The solution at pH 1.0 was placed in a separatory funnel with 200 ml of 5% HDEHP in toluene and mixed vigorously. Y forms strong complexes with HDEHP and so it can be selectively precipitated as oxalate at pH of 2,5. Y-oxalate was then dissolved in HCl and 12 mL of scintillation cocktail (Optima Gold AB - Perkin Elmer) were added for counting by LSC. In order to verify an acceptable recovery of Sr and Y from samples, spectrometric quantifications of these stable elements, previously added to the samples, were obtained by ICP/MS analyses and then used to calculate the final 90Sr activity. METHOD PERFORMANCES & RESULTS Methods performances such as trueness, precision, counting efficiency, detection limit and decision threshold were carefully determined by adopting an intra-laboratory validation scheme. The validation parameters were determined using cow, pork and chicken bone samples fortified with known activities of 90Sr (10 Bq kg-1) and two replicates of each species were carried out. Decision threshold and detection limit were also calculated in compliance with ISO 11929:2010 by analysing six blank samples. The counting efficiency (ε) was achieved by analysing three 90Sr solutions with an activity of 1.0 Bq, previously treated in order to separate the Yttrium as Yttrium oxalate according to described procedure. Method precision and trueness were demonstrated evaluating CV% values and mean recovery equal to 12% and 98% respectively (Tab.1). As shown in figure 2, no interfering radionuclides were observed in analysed spectra. Detection limit and decision threshold correspond to 8 and 3 mBq kg-1 respectively (α=β=0.05). Quantitative analyses on real samples were carried out by the above described method on 10 cow bone samples. In all samples 90Sr was present in appreciable concentrations between 5.22 and 20.61 Bq kg-1, as shown in Table 2. Figure 1: Sample preparation Bone Samples Cow Pork Chicken N.1 Spiked sample at 10 Bq kg-1 9.39 ± 2.98 9.27 ± 2.94 11.36 ± 3.61 N.2 Spiked sample at 10 Bq kg-1 10.97 ± 3.48 8.03 ± 2.55 10.02 ± 3.18 Recovery 102% 87% 107% CV% 11% 10% 9% Cow bone sample 90Sr Activity ± Measurement Uncertainty (Bq kg-1) Sample N.1 6,79 ± 2,19 Sample N.2 5,87 ± 1,87 Sample N.3 5,44 ± 1,75 Sample N.4 8,59± 2,78 Sample N.5 5,22 ± 1,68 Sample N.6 20,61 ± 6,59 Sample N.7 8,11 ± 2,59 Sample N.8 10,19 ± 3,27 Sample N.9 5,73 ± 1,83 Sample N.10 6,39 ± 2,07 Tab. 1. Results of spiked samples of different species Tab. 2. 90Sr in cow bone samples DISCUSSION & CONCLUSIONS The method developed for the determination of 90Sr in bone samples represents an efficient and reliable confirmatory method. This new analytical procedure allows the selective extraction of the desired radionuclide, without interferences. LSC provides information about the shape of the β-spectra with high counting efficiency (89%). In particular, considering that analyzed samples are coming from significant farm animals, this method is an important improvement in food safety controls and it is also suitable in radiocontamination surveillance programs. REFERENCES Fig. 2. Spectrum of a cow bone sample spiked with 90Sr at a level of 100 Bq kg-1 (red) and a blank cow bone sample (green) 1. Stamoulis KC et al. 2007. J. Environ Radioact, 93:144-156. 2. Wallova G et al. 2012. J. Environ Radioact, 107:44-50.