Agricultural and Food Science in Finland, Vol. 11 (2002): 137–141 137 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 11 (2002): 137–141. © Agricultural and Food Science in Finland Manuscript received May 2001 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 11 (2002): 137–141. The effect of clinoptilolite on 137Cs binding in broiler chickens Gordana Vitorovic, Branislava Slavata and Katarina Stosic Faculty of Veterinary Medicine, Department of Radiology, Belgrade, SR Yugoslavia, e-mail: vitor@afrodita.rcub.bg.ac.yu Verica Mladenovic Military Technical Institute, Belgrade, SR Yugoslavia Dusko Vitorovic Faculty of Agriculture, Department of Animal Science, Belgrade, SR Yugoslavia The objective of this study was to evaluate the 137Cs binding capacity of clinoptilolite. In the first in vitro experiment we investigated sorption of 137Cs to natural and modified forms of clinoptilolite in highly acid solution, prepared to be similar to that of the gut of pigs (pH = 2–3) at 37ºC. In the second in vivo experiment 137Cs binding to a modified form of clinoptilolite was studied in orally contami- nated broiler chickens. 137Cs sorption in the high acidity solution depended on clinoptilolite concen- tration and varied between 30–85% of the initial activity. In the chickens, three hours after 137Cs administration, there was 67% and 63% lower accumulation of 137Cs in meat and edible organs (re- spectively) and seven hours after 137Cs administration, there was 69% and 49% lower accumulation of 137Cs in meat and edible organs (respectively) compared to the controls with no clinoptilolite add- ed in food. Natural and modified forms of clinoptilolite have been shown to high sorption efficiency towards 137Cs ions and could be recommended as possible radiocaesium binders in domestic animals. Key words: clinoptilolite, 137Cs, sorption, broilers Introduction Contamination of the environment with radio- active substances has become one of the most important problems in modern civilization. Therefore, there is a need for constant and sys- tematic control of radionuclide concentrations in the biosphere. Radioactive contamination of the environment as a consequence of the Cher- nobyl nuclear accident has led to contamination of vegetation and feedstuffs for animal nutrition with biologically significant radionuclides, 90Sr, 131I and 137Cs. Prevention of contamination via the oral ingestion of these radionuclides is a pri- ority in radioprotection. In animal production, there is a need to ensure that contamination of animal products is below intervention levels. mailto:vitor@afrodita.rcub.bg.ac.yu 138 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vitorovic, G. et al. The effect of clinoptilolite on 137Cs binding in broiler chickens Howard et al. (2001) reviewed the current avail- ability of countermeasures for preventing or reducing contamination of animal products by radioisotopes of iodine, caesium and stron- tium. Reducing contamination via the gut of do- mestic animals and the consequent health risk for humans, requires the development of meth- ods which use binding substances to bind radio- caesium in the digestive tract of animals and eliminate it through faeces. After the first nu- clear accidents, initial investigations were con- ducted on 137Cs sorption. Potassium was added into the feed to stimulate radiocaesium elimina- tion from the organism (Williams and Patrick 1957). Beresford et al. (1989) used bentonite as a radiocaesium binder in sheep and achieved a reduction of between 20–80%. Currently, the best caesium binding effects have been achieved by Prussian Blue compounds, the most common- ly used compound being AFCF - ammonium fer- ric hexacyanoferrate (Arnaud et al. 1988, Hove and Ekern 1988, Unsworth et al. 1989, Hove and Hansen 1993, Paasikallio et al. 2000). Reduc- tion of 90–98% was achieved in cows with 3 g of AFCF daily, calves and pigs 2 g of AFCF dai- ly (Giese 1988) and in broilers (Vitorovic 1993) with 0.2 g of AFCF daily per chick. Philippo et al. (1988), Pethes et al. (1988) and Jandl and Novosad (1995) demonstrated that zeolite (a clinoptilolite clay mineral) can be used as a pos- sible binder for 137Cs in the gut of domestic ani- mals. Zeolites are crystalline, hydrated aluminosil- icates that have an indefinite three-dimensional structure. Their characteristic is the ability to exchange water and some of its constitutional cations without significant changes in their struc- ture. Natural zeolites are used as catalysts in many reactions, as molecular sieves and for cat- ion exchange (Ming and Mumpton 1989). Con- sidering this, zeolites have wide use as animal feed additives with the ability for mycotoxine adsorption (Rodrigues 1991, Harvey et al. 1993, Amon and Dobesic 1994, Krilov et al.1994). Clinoptilolite could be also used in agriculture to reduce 137Cs deposition in soil because of its ability to bind 137Cs (Paul and Jones 1995, Camp- bell and Davies 1997, Jones et al. 1999). The objective of the present study was to evaluate the 137Cs binding capacity of a natural white form of clinoptilolite and different modi- fied clinoptilolite forms in in vitro and in vivo conditions. Material and methods In the in vitro experiment, natural clinoptilolite from Zlatokop mine was used. The investigated zeolite had above 80% clinoptilolite mainly in Ca(II) form. Silicate analysis was used for Si and Al determination, atomic absorbtion for Na, K, Ca, Mg determination and the Kjeldhal method for NH4 + ion. The cation exchange (Na, K, Ca and Mg) capacity was above 130 cmol kg-1. In the first stage of this experiment, 100 mg and 300 mg of clinoptilolite were treated with 100 ml electrolytic solution of HCl, NaCl and KCl, to which 1440 Bq 137Cs was added. The acidity of the solutions was initially adjusted to be pH 2–3, with NaOH, which resembles the acidity condition within the gut of pigs. Sorption was performed in a water bath at 37ºC with slow shaking. Contact times were 2, 4, 6 and 8 hours and three replicates were taken for each period of time. After sorption, the samples were centri- fuged and remaining activity measured in the separated liquid fraction. In the second stage of this experiment, we compared 137Cs binding ca- pacity of natural and mono-ionic, sodium (Na) and ammonium (NH4) forms of clinoptilolite. The hydrated samples of natural clinoptilolite were treated with 3M NH4Cl and 3M NaCl solu- tions to prepare the mono-ionic forms of clinop- tilolite. 500 mg of the sample was equilibrated with 50 ml electrolytic solution to which 1440 Bq 137Cs was added under the same solution com- position and acidity as in the first stage of the experiment. The contact time was 6 hours at 37ºC after which the remaining activity of 137Cs in solution was measured, as before. 139 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 11 (2002): 137–141. For the in vivo experiment, broiler chickens (six weeks of age) were fed with their normal diet and each chick was orally given a 3 ml CsCl solution with a total activity of 3120 Bq. In ad- dition, the experimental group was given a 6 ml aqueous solution of clinoptilolite modified with NaCl called Minazel (1.6 g Minazel per chick which is approximately 1–1.5% in the diet). Prin- ciples of laboratory animal keeping were in ac- cordance with national laws for animal protec- tion and welfare. Birds of each group were sac- rificed 3 and 7 hours (five chicks per group) af- ter the contamination was administered. Samples of total musculature and edible organs (heart, gizzard and liver) were taken from each chick. A HP Ge detector with high-energy resolution (1.85 KeV – 1.33 MeV) was used for gamma spectrometery measurement of activity of the samples. The measurement time was 40,000 s. The effect of clinoptilolite treatments on 137Cs binding was evaluated by analysis of variance and differences between treatments were tested using t-test. Results and Discussion Administration of the lower clinoptilolite con- centration (100 mg) reduced by half the initial 137Cs activities (52%) in solution during 6 hours of incubation. Prolongation of the contact time gave no further decreasing in solution activity (48.6% for 8 hours). Using a higher clinoptilo- lite amount (300 mg), the equilibrium of Cs ions was also established after 6 hours, but 75% (74.7% in 6 hours and 74.2% in 8 hours) of ini- tial 137Cs activities was sorbed. The higher clinoptilolite administration therefore have a higher sorption efficiency (between 67.6–74.7%) than that of the lower amount (30.4–48.6 %). The activities of solution depending of different con- tact times and clinoptilolite concentrations are given in Table 1. Natural and prepared forms of clinoptilolite (Na- and NH4- clinoptilolites, 500 mg) showed similar and significant effects on 137Cs sorbtion, sorbing 84–85% of initial activity (Table 2). The higher 137Cs sorption efficiency of clinoptilolite in this experiment compared to the results in the first stage was the result of using higher clinop- tilolite concentrations. Philippo et al. (1988) es- tablished that clinoptilolite and hexacyanocobalt ferrate are the most effective adsorbers of radi- ocaesium in vitro. When used in vivo they sig- nificantly reduced the absorption of radiocaesi- um by sheep fed contaminated herbage. In our in vivo experiment, 137Cs binding effi- ciency was evaluated for the modified clinopti- lolite form - Minazel, in broiler chickens. Three hours after administering 137Cs contamination and the mineral, significanly (P < 0.01) lower 137Cs activity concentration was obtained in the Table 1. Clinoptilolite efficency in sorption of Cs137 from a solution, with an activity of 1440 Bq. Time of 137Cs activity in solution (Bq) Sorbed activity contact 100 mg clinoptilolite 300 mg clinoptilolite (% of starting activity) (hours) Mean SD Mean SD 100 mg 300 mg clinoptilolite clinoptilolite 2 1001.0 15.0 466.0** 10.0 30.4 67.6 4 894.0 4.0 383.0** 8.0 37.8 78.4 6 748.0 15.0 364.0** 11.0 48.0 74.7 8 739.0 12.0 371.0** 10.0 48.6 74.2 SD = standard deviation ** = statistically significant differences (P < 0.01) between treatment of clinoptilolite concentration. 140 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vitorovic, G. et al. The effect of clinoptilolite on 137Cs binding in broiler chickens meat of broilers treated with Minazel than in the control group. Compared to the control group, the reduction of 137Cs accumulation in the treat- ed group was 67%. Seven hours after contami- nation and mineral administration, the decrease due to 137Cs binder was 69%. Pethes et al. (1988) found that accumulation of radiocaesium was significantly decreased by adding 6% zeolite to broiler feed. The accumulation of 137Cs in zeo- lite treated groups ranged 62–70% of the non- treated groups. Jandl and Novosad (1995) inves- tigated in vivo reduction of radiocaesium in sheep using modified clinoptilolite (ZEOFe) and non-modified clinoptilolite (ZEO). More than ten times lower sorption efficinecy has been ob- served with ZEO comparison to ZEOFe. In our investigation, the modified clinoptilolite Mina- zel, showed significant protection effect in edi- ble organs (heart, gizzard and liver) of broilers contaminated with 137Cs. Three hours after con- taminaton and mineral administration there was about 63% lower accumulation of 137Cs in edi- ble organs of the group treated with Minazel compared to controls; after seven hours it was 49%. Considering that these are preliminary results in our investigation of the ability of clinoptilo- lite to bind 137Cs in the digestive organs of do- mestic animals, it is still too early to give sug- gestions for its practical application and to com- pare it with other 137Cs binders (bentonite, ver- miculite, AFCF). It is also too early to evaluate animal products obtained using zeolite as 137Cs binder in human nutrition especially because in this investigation the degree of oral 137Cs con- tamination of chickens (3150 Bq per day) was far above potential radioactive contamination of chick feeds in accidental situations (like the one produced by Chernobyl). It was used to obtain a clear data on the efficiency of clinoptilolite in 137Cs binding in the digestive tract of chickens. In our further investigations more attention will be given to radioactive contamination of animal feeds in practical conditions with long-term ob- servations in the ability of clinoptilolite to bind 137Cs in orally contaminated animals. Table 2. Efficiency of different forms of clinoptilolite in 137Cs sorption from a solution, with an activity of 1440 Bq. Forms of 137Cs activity (Bq) Sorbed activity clinoptilolite Mean SD (% of starting activity) Natural 230.0 11.0 84.0 Sodium (Na-) 215.0 11.0 85.1 Ammonium (NH 4 -) 215.0 7.0 85.3 SD = standard deviation Fig. 1. Efficiency of Minazel in re- ducing 137Cs accumulation in the meat and edible organs of broiler chickens, orally contaminated with 3150 Bq 137Cs per chick, ab- breviation C is for “control” and M for “Minazel” (means and standard errors). 141 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 11 (2002): 137–141. Conclusion Natural clinoptilolite and modified forms of clinoptilolite have a high sorption efficiency to- wards 137Cs ions. In Serbia, there are deposits of this particular mineral of high quality. Because of that, and its low price, it could be recommend- ed as a possible radiocaesium binder in domes- tic animals. Additional in vivo studies are need- ed to evaluate optimal clinoptilolite concentra- tions for different species, breed and categories of domestic animals. Acknowledgements. We thank Mrs Jelena Perovic, from Patentcommerce, Belgrade, official distributor of clinopti- lolite and Minazel, for providing help in this investigation. References Amon, M. & Dobesic, M. 1994. Influence of local clinop- tilolite in the broiler chickens production. Proceeding of the 8th International Congress of Animal Hygiene. St. Paul, Minnesota, USA. p. 210–217. Arnaud, M.J., Clement, C., Getaz, F., Tanhauser, F., Schonegge, R., Blum, J. & Giese, W. 1988. 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Title Introduction Material and methods Results and Discussion Conclusion References