Agricultural and Food Science in Finland The effect of ammonium ferric hexacyanoferrate on reducing radiocaesium transfer from grass silage to sheep Arja Paasikallio Agricultural Research Centre of Finland, Resource Management Research, FIN-31600 Jokioinen, Finland, e-mail: arja.paasikallio@mtt.fi Riitta Sormunen-Cristian and Seija Jaakkola Agricultural Research Centre of Finland, Animal Production Research, FIN-31600 Jokioinen, Finland Matti Kaikkonen Department of Basic Veterinary Sciences, PO Box 57, FIN-00014 University of Helsinki, Finland A study was carried out to examine the effect of ammonium ferric hexacyanoferrate (AFCF) on the transfer of radiocaesium from grass silage to the tissues of male lambs. During ensiling, a formic acid based additive and AFCF were sprayed on grass contaminated with 134 Cs and the mixture was allowed to incubate for 45 days. A dose of 21 mg AFCF d -1 , fed to sheep offered contaminated silage for four- teen days, reduced 134 Cs transfer to muscle by 45% compared to that of control sheep. An equivalent dose of AFCF administered in a capsule reduced transfer by only 3%. In another experiment, AFCF intake of 50, 100 and 150 mg d -1 for ten days reduced 134 Cs transfer to sheep muscle by 75, 82 and 86%, respectively. In control lambs, of average live weight 38 and 47 kg, the feed to muscle 134 Cs transfer coefficient averaged 0.15 d kg -1 , but equilibrium between tissue and feed 134 Cs had probably not been reached due to the short feeding period. Increasing doses of AFCF from 0 to 150 mg d -1 increased the faecal/urinary 134 Cs ratio from 2 to 42. Key words: AFCF, excretion, preservation, radiocaesium, sheep, silage, transfer Introduction Different materials have been administered to ruminants for reducing radiocaesium transfer to meat and milk products. These include bento- nite (Andersson 1989, Beresford et al. 1989, Mitchell et al. 1989), zeolite (Phillippo et al. 1988, Unsworth et al. 1989), vermiculite (Haz- zard 1969), kaolin (Giese 1989), stable caesium (Oughton et al. 1991) and crude fibre (Johnson et al. 1968). Organic complexes such as ferric hexacyanoferrates, commonly referred to as 135 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. 9 (2000): 135–147. �Agricultural and Food Science in Finland Manuscript received September 1999 mailto:arja.paasikallio@mtt.fi Prussian Blue (PB) compounds are, however, the most effective caesium binders on a unit weight basis (Giese 1989, Unsworth et al. 1989). PB compounds reduce the intestinal ab- sorption of radiocaesium, and are most effective when administered with contaminated feed. PB compounds react with radiocaesium in the gut to form a complex that is excreted in the faeces. Furthermore, they have been reported to en- hance tissue excretion of radiocaesium into the gut (Hove et al. 1990, Nielsen et al. 1990, Åh- man 1996). Ammonium ferric hexacyanoferrate (AFCF) is the most commonly used PB com- pound and binds radiocaesium in the gut in ex- change for an ammonium ion. AFCF has been used as a feed supplement in silage and concen- trates for housed ruminants and in salt licks and boli for grazing animals (Unsworth et al. 1989, Pearce et al. 1989, Hove et al. 1990, Voigt 1993, Hansen et al. 1996, IAEA 1997). In general, studies have only considered the effects of AFCF mixed with silage some time prior to feeding (Arnaud et al. 1988, Giese 1989, Un- sworth et al. 1989, Vreman et al. 1992), while information concerning the use of AFCF at grass harvesting and its effects during and after storage in silo is limited. In the 1960s, studies were carried out on the toxicity of PB on animals and humans (Nigrovic 1963, Madhus et al. 1966). After radioactive emissions following the Chernobyl accident, toxicological as well as other studies on PB were revived (Arnaud et al. 1988, Giese 1988, Nielsen et al. 1990, Pearce 1994). The results showed that the consumption of milk and meat from PB treated animals could be considered safe with respect to human health. So far, the ap- plication of AFCF to soil directly or via the fae- ces does not seem to have any negative effects on soil-plant environments (Vandenhove et al. 1997, 1998, 2000). However, long-term and more comprehensive studies are lacking (Jones et al. 1999). Following the Chernobyl accident, PB com- pounds were officially approved for use as a feed additive in Russia, Ukraine, Belarus, Nor- way, Germany and Austria (IAEA 1997). The EC directive allows the use of AFCF as a feed additive in the EU countries, but its use should be authorized at the national level (Commission Directive 1996). PB compounds have also been used successfully with human casualties of the Goiânia radiation accident in Brazil (Lipsztein et al. 1991, Melo et al. 1994). This study was carried out to examine the ef- fect of low levels of AFCF, mixed in contami- nated grass during ensiling, on the transfer of 134 Cs to ovine tissues. The hypothesis tested was that, in situations of radioactive fallout, AFCF could be mixed with contaminated grass at har- vesting together with an acid based ensiling ad- ditive. Material and methods Preparing 134Cs-AFCF-silage Ryegrass was grown in pots in peat soil con- taminated with 134 Cs. Grass was harvested after 45 days. One kilogram of contaminated ryegrass was placed on a plastic sheet and thoroughly mixed with 6 kg of uncontaminated chopped and prewilted timothy-meadow fescue grass. The purpose of prewilting was to minimize si- lage effluent production. Grass was treated with a formic acid based additive (AIV2-solution containing 80% formic acid and 2% orthophos- phoric acid) at a rate of 5 ml kg -1 . Additive di- luted with water (50%) was sprayed on grass prior to ensiling. In addition, AFCF (ammonium ferric hexacyanoferrate, NH 4 Fe 3+ [Fe 2+ (CN) 6 ], containing 33% NH 4 Cl) as a water solution was also applied to grass. ACFC was applied at a rate of 0 and 100 mg kg -1 and 0, 250, 500 and 750 mg kg -1 of grass in experiments 1 and 2, respec- tively. Once applied, grass (7 kg) was ensiled in laboratory silos (volume of cylinder 15.4 dm 3 , diameter 14 cm). Each silo was fitted with a drainage system for collecting and monitoring silage effluent production. After filling, each silo was tightly sealed and weighted with con- 136 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 Paasikallio, A. et al. Effect of AFCF on reducing 1 3 4 Cs transfer from grass silage to sheep crete blocks and water bags to a pressure of 585 kg m -2 . Preservation lasted for about 45 days. After opening the silos, silage was sampled and divided into daily doses (0.2 kg/sheep) and fro- zen in plastic bags for later use. Fermentation quality of both 134 Cs contaminated and uncon- taminated silages was good. Animals and experimental design Male crossbred Finnish Landrace lambs, mean age 171 (experiment 1) and 214 days (experi- ment 2) and live weight of 38 and 47 kg, respec- tively, were used in the study (Table 1). Lambs were allocated according to live weight into two blocks of three animals (experiment 1) and into three blocks of four animals (experiment 2). Lambs within each block were exposed to dif- ferent experimental treatments. The animals were individually housed in metabolic cages. Before the experiments started, the animals were allowed 7 days to become accustomed to the cages. In experiment 1, silage contaminated with 134 Cs was given daily to all sheep for four- teen days. AFCF was given either with contami- nated silage or in gelatine capsules. Daily in- takes of AFCF were 0, 21 and 21 mg d -1 for con- trol, treated silages and AFCF capsules (two sheep each), respectively. In experiment 2, con- taminated silage with different AFCF doses was given to twelve sheep for ten days. The intake of AFCF in silage were 0, 50, 100 and 150 mg d -1 for three sheep each (Table 1). Experiments 1 and 2 were conducted in September 1995 and October 1996, respectively. Feeding and sampling In both experiments, the sheep received a daily ration fed in the mornings, consisting of 3 kg of uncontaminated farm silage, 0.3 kg of barley and 0.2 kg of 134 Cs contaminated silage (with or without AFCF). When AFCF (21 mg d -1 ) was given in a capsule, the capsule was administered immediately after the morning feed (experiment 1). The daily feed intake of each animal was re- corded in both experiments. Water was avail- able ad libitum with consumption recorded for all experimental animals. Urine and faeces were collected separately and output was measured daily. Sub-samples of urine and faeces were stored frozen prior to 134 Cs determinations. At the end of both experiments, the animals were slaughtered and sampled. In experiment 1, dor- sal and femural muscle, heart, liver, kidney, whole blood and digesta samples were col- 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. 9 (2000): 135–147. Table 1. Some aspects of experimental conditions (daily AFCF doses, 134 Cs intake and sheep pa- rameters). lected. For the second experiment, samples of neck and femural muscle, diaphragm, heart, liver and kidney were collected. In all cases, samples were stored frozen and subsequently monitored for 134 Cs. Sample analysis For activity measurement animal tissues were cut into small pieces. Visible fat was removed from samples of muscle and kidney. The 134 Cs activity concentration of contaminated grass si- lage, urine, faeces and animal tissues was deter- mined using a low-background semiconductor spectrometer coupled to a germanium detector. Activity concentrations of 134 Cs are presented on a fresh weight (FW) basis. Transfer coefficients of 134 Cs (d kg -1 ) from feed to tissues was calcu- lated as 134 Cs activity concentration in tissues (Bq kg -1 ) per 134 Cs intake (Bq d -1 ). Statistical methods In both experiments, the effect of AFCF treat- ments on tissue 134 Cs activity concentration was evaluated by Analysis of Variance for repeated measures using the MIXED PROCEDURE within SAS (SAS 1992). Repeated measure- ments within each animal were found to be highly correlated, a factor which was taken into account by using a compound symmetry covari- ance structure assigned on the basis of Akaike’s and Scharz’s Bayesian information criteria (Wolfinger 1996). The statistical model (Gum- peretz and Brownie 1993) used to assess the ef- fect of treatments was: Y ijk = � + A i + B j + e ij + T k + (AT) jk + (BT) ik + h ijk where Y ijk is the observed response (e.g. 134 Cs activity concentration), � is the intercept, A i the fixed effect from the ith treatment, B j the ran- dom block effect. e ij is a random effect that rep- resents the error associated with the ijth cell. T k and (AT) jk represent the fixed effect of tissue and treatment-tissue interactions, respectively, while (BT) ik is the random effect of block-tissue interaction and h ijk are error terms. Due to a low activity concentration in blood (experiment 1), the effect of different AFCF treatments on 134 Cs activity concentration in blood was analyzed separately by Analysis of Variance for a randomized complete block de- sign as follows: Y ij = � + A i + B j + e ij where Y ij , �, A i , B j and e ij are equivalent to those in the previously described model. Differences between treatments and between tissues were tested using orthogonal contrasts. In experiment 2, linear and quadratic effects of AFCF treatment were studied. Prior to analysis, data concerning 134 Cs activity concentration was transformed into natural logarithms to the con- stancy of error variance. Assumptions of both models were checked using graphical methods i.e. box-plot for normality and plots of residuals to ensure constancy of error. Results Experiment 1 The sheep consumed their whole feed ration. When AFCF (21 mg d -1 ) was administered to sheep as a capsule for fourteen days, the 134 Cs activity concentration of tissues except the liver did not differ from that of the controls. When an equivalent dose of AFCF was administered in silage, the average transfer of 134 Cs to tissues was reduced to 50% of that of controls (Table 2). Activity concentrations of 134 Cs differed sig- nificantly (P<0.05) between tissues in controls, being highest in kidneys and lowest in muscle. The feed to muscle transfer coefficient for 134 Cs in control sheep was twice as high as in animals treated with AFCF administered in silage (Table 3). 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 Paasikallio, A. et al. Effect of AFCF on reducing 1 3 4 Cs transfer from grass silage to sheep 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. 9 (2000): 135–147. Table 2. Activity concentration (least square mean with 95% confidence interval) and reduction (% of control) of 134 Cs in sheep tissues after administering 134 Cs contaminated silage daily, without ammonium ferric hexacyanofer- rate (AFCF) (control) and with 21 mg d -1 of AFCF given as a capsule or in silage fed for 14 days (experiment 1). Table 3. Transfer coefficients of 134 Cs (muscle/silage) for sheep administered daily with different doses of ammonium ferric hexacyanoferrate (AFCF). Tissue 134 Cs in sheep Significance Control AFCF 21 mg d -1 (capsule) AFCF 21 mg d -1 (silage) (Bq kg -1 ) (Bq kg -1 ) (%) (Bq kg -1 ) (%) P1 P2 P3 Muscle 961 942 3 522 46 0.89 < 0.005 < 0.005 (770, 1199) (755, 1176) (418, 651) Heart 1163 960 17 559 52 0.18 < 0.005 0.01 (932, 1452) (769, 1198) (448, 697) Liver 1483 918 38 598 60 0.01 < 0.001 0.02 (1189, 1851) (736, 1146) (479, 746) Kidney 2032 1773 13 1011 50 0.33 < 0.005 < 0.005 (1628, 2536) (1420, 2212) (810, 1262) Whole blood 127 84 34 54 57 0.33 0.12 0.31 (47, 339) (31, 225) (20, 146) (capsule), P2 = control vs. AFCF (silage), P3 = AFCF (capsule) vs. AFCF (silage) Number of sheep per treatment = 2, muscle is a mean of dorsal and femural muscles, P1 = control vs. AFCF Gastrointestinal tract contents had higher 1 3 4 Cs activity concentrations in AFCF (si- lage)-treated sheep than in the other treatment groups. In general, the 134 Cs level of digesta tended to increase towards the posterior part of gastrointestinal tract being highest in the distal part of the large intestine (Table 4). Excretion of 134 Cs expressed as a % of 134 Cs intake was greater in faeces than urine (Table 5). Urinary 134 Cs excretion was significantly (P< 0.001) higher in sheep fed the control diet. Fae- cal 134 Cs excretion increased continuosly before reaching a plateau after 5–6 days (Fig. 1). Experiment 2 During the first day two sheep refused to con- sume silage. After this, the sheep consumed all given feed. Tissue 134 Cs activity concentrations were clearly higher in control animals than in those treated with AFCF, therefore controls were not included in further comparisons be- tween treatments. Caesium 134 activity concen- tration of tissues except the liver differed sig- nificantly (P<0.05) between the three AFCF- treatments. When AFCF was given to sheep at 50, 100 and 150 mg d -1 in silage for ten days, 134 Cs transfer to muscle reduced by 75, 82 and 86% relative to control values, respectively (Ta- ble 6). In control animals, the 134 Cs activity con- centration in kidneys was significantly (P< 0.001) higher than that determined in all other tissues. The feed to muscle transfer coefficient of 134 Cs was 0.16 d kg -1 in controls, while for AFCF-treated sheep, a value around 0.03 d kg -1 was observed (Table 3). Faecal excretion of 134 Cs was clearly lower, and urinary excretion higher, in control sheep than in AFCF-treated sheep (Fig. 2). There were, however, only negligible differences in 134 Cs excretion between AFCF treatments (Ta- ble 5). Daily water intake of individual animals varied from 0.07 to 1.81 l d -1 . Feed and water in- takes were not affected by the AFCF-treatments. 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 Paasikallio, A. et al. Effect of AFCF on reducing 1 3 4 Cs transfer from grass silage to sheep Table 4. Activity concentration of 134 Cs in digesta after administering 134 Cs contaminated silage daily, without am- monium ferric hexacyanoferrate (AFCF) (control) and AFCF given as a capsule or in silage fed for 14 days (experi- ment 1). 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. 9 (2000): 135–147. a) b) Fig. 1. Effect of ammonium ferric hexacyanoferrate (AFCF) (21 mg d -1 ) administered in a capsule or in silage on 134 Cs faecal (a) and urinary (b) excretion (% of intake) during fourteen days. (experiment 1) Fig. 2. Effect of different doses of ammonium ferric hexacyanoferrate (AFCF) (50, 100 and 150 mg d -1 ) on 134 Cs fae- cal (a) and urinary (b) excretion (% of intake) during ten days. (experiment 2) a) (% ) (% ) (% ) (% ) b) 142 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 Paasikallio, A. et al. Effect of AFCF on reducing 1 3 4 Cs transfer from grass silage to sheep Table 6. Activity concentration (least square mean with 95% confidence interval) and reduction (% of control) of 134 Cs in sheep tissues after administering 134 Cs contaminated silage daily, without ammonium ferric hexacyanoferrate (AFCF) (control) and with different doses of AFCF given in silage for 10 days (experiment 2). Table 5. Mean 134 Cs excretion (% of intake) in faeces and urine and the faecal/urinary ratio (F/U) of sheep administered 134 Cs contaminated silage daily with different doses of ammonium ferric hexa- cyanoferrate (AFCF). Discussion Reduction of 134Cs In this study, the lowest AFCF dose of 21 mg d -1 fed in silage, reduced the final 134 Cs activity con- centration in ovine muscle by 46%, which was further decreased by 86% at the highest dose of 150 mg d -1 . According to Pearce et al. (1989), a bolus providing AFCF 20–24 mg d -1 reduced the 137 Cs level of sheep muscle by 42% and in ani- mals receiving 200 mg d -1 the reduction was 85%. Intakes of AFCF from salt licks or boli of between 25–300 mg d -1 , have reduced the trans- fer of 137 Cs to sheep tissue by 50–90% (Hove 1993). In reindeer, an AFCF dose of 500 mg d -1 prevented the absorption of 137 Cs almost com- pletely (Åhman 1996). A comparison of the cur- rent results with those documented in the litera- ture, indicates that incubation with silage did not reduce the efficacy of AFCF to inhibit radio- caesium transfer to sheep tissues. In this respect, the use of AFCF would be suitable for field con- ditions. The AFCF doses used in the present study were considerably lower than those gener- ally recommended for small ruminants (1–2 g d -1 ) (Giese 1988, 1989). Silage AFCF contents, as high as 500 mg kg -1 , would not exceed recom- mended AFCF daily doses in sheep fed that si- lage 3 kg d -1 . In experiment 1, AFCF given in silage, was more effective in reducing tissue 134 Cs levels than the same dose of AFCF administrated via capsules. Administered in silage, AFCF may have bound 134 Cs during ensiling, whilst when AFCF is given in capsule form, 134 Cs probably has a greater chance of being transferred to tis- sues before AFCF is released into the rumen. This hypothesis was supported by digesta activ- ity concentrations, since 134 Cs activity concen- trations in the rumen-abomasum content of ani- mals receiving AFCF in silage was higher than in capsule fed animals. Radiocaesium activity concentrations of control sheep was signifi- cantly higher in renal than in other tissues, which is in accordance with other studies (How- ard and Lindley 1985, Howard et al. 1989). Transfer coefficients In control animals, the transfer coefficient for radiocaesium from feed to muscle averaged 0.15 d kg -1 . The value was rather low compared, particularly, to those of post-Chernobyl studies (Howard et al. 1987, 1989, Andersson 1989, Beresford et al. 1989, Howard 1989). The low value was probably due to the short feeding pe- riods (10 and 14 days) used in the current study. The transfer coefficients for radiocaesium are valid only when the radiocaesium level of tissue has equilibrated with intake,which in sheep is at- tained between 20 and 30 days post-contam- ination (Howard et al. 1989, Pearce et al. 1989). Excretion of 134Cs A plateau for faecal 134 Cs excretion seems to be reached after 5–6 days from the beginning of the experiment (large day-to-day variation). This was a rather short period compared to the find- ings of Vandecasteele et al. (1989) who reported a faecal excretion plateau after 20 days in preg- nant ewes. In control sheep, faecal 134 Cs excre- tion was twice that in urine. Mean faecal 134 Cs excretion accounted for 38% of intake, which was somewhat lower than values (50%) re- ported by Beresford et al. (1989) and Vandecas- teele et al. (1989). However, the contamination period in their studies was much longer (34 and 76 days, respectively) than in the present experi- ment. The administration of AFCF mixed in si- lage increased faecal 134 Cs excretion considera- bly in this study, and was attributed to AFCF in- hibition of 134 Cs intestinal absorption. 143 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. 9 (2000): 135–147. Conclusion Adding AFCF to contaminated silage considera- bly reduced 134 Cs transfer to sheep tissues. Incu- bation in silage did not reduce the efficacy of AFCF as a Cs-binder. In this respect, use of AFCF in the field would be possible. However, additional studies are needed to develop spray- ing technics for AFCF application during ensil- ing in combination with formic acid or enzyme inoculant silage additives under field condi- tions. Furthermore, long term studies are re- quired to validate 134 Cs transfer coefficients, since the feeding period was too short to reach equilibrium between tissue and feed 134 Cs in the current study. Acknowledgements. We thank Ms Päivi Vähämäki, Ms Taina Lilja and Ms Helvi Kananen for their invaluable assistance. We also thank Mr Lauri Jauhiainen for sta- tistical expertise. References Åhman, B. 1996. Effect of bentonite and ammonium- ferric(III)-hexacyanoferrate(II) on uptake and elimi- nation of radiocaesium in reindeer. Journal of Envi- ronmental Radioactivity 31: 29–50. Andersson, I. 1989. Transfer of 137Cs from feed to lambs’ meat and the influence of feeding bentonite. Swedish Journal of Agricultural Research 19: 85–92. Arnaud, M.J., Clement, C., Getaz, F., Tannhauser, F., Schoenegge, R., Blum, J. & Giese, W. 1988. Syn- thesis, effectiveness and metabolic fate in cows of the caesium complexing compound ammonium fer- ric hexacyanoferrate labelled with 14C. Journal of Dairy Research 55: 1–13. Beresford, N.A., Lamb, C.S., Mayes, R.W., Howard, B.J. & Colgrove, P.M. 1989. The effect of treating pas- tures with bentonite on the transfer of 137Cs from grazed herbage to sheep. Journal of Environmental Radioactivity 9: 251–264. Commission Directive 1996. Commission Directive 96/66/EC of 14 October 1996. Official Journal of the European Communities No. L 272/32–35. Giese, W.W. 1988. Ammonium-ferric-cyano-ferrate(II) (AFCF) as an effective antidote against radiocae- sium burdens in domestic animals and animal de- rived foods. British Veterinary Journal 144: 363– 369. – 1989. Countermeasures for reducing the transfer of radiocesium to animal derived foods. The Science of the Total Environment 85: 317–327. Gumperetz, M.L. & Brownie, C. 1993.Repeated meas- ures in randomized block and split-plot experi- ments. Canadian Journal of Forest Research 23: 625-–639. Hansen, H.S., Hove, K. & Barvik, K. 1996. The effect of sustained release boli with ammoniumiron(III)-hex- acyanoferrate(II) on radiocesium accumulation in sheep grazing contaminated pasture. Health Phys- ics 71: 705–712. Hazzard, D.G. 1969. Percent cesium-134 and strontium -85 in milk, urine, and feces of goats on normal and verxite-containing diets. Journal of Dairy Science 52: 990–994. Hove, K. 1993. Chemical methods for reduction of the transfer of radionuclides to farm animals in semi- natural environments. The Science of the Total En- vironment 137: 235–248. –, Hansen, H.S. & Strand, P. 1990. Experience with the use of caesium binders to reduce radiocaesium con- tamination of grazing animals. In: Environmental Contamination Following a Major Nuclear Accident 2. Proceedings of a Symposium of FAO, IAEA, UNEP, WHO. Vienna. p. 181–189. Howard, B.J. 1989. A comparison of radiocaesium transfer coefficients for sheep milk and muscle de- rived from both field and laboratory studies. The Sci- ence of the Total Environment 85: 189–198. –, Beresford, N.A., Burrow, L., Shaw, P.V. & Curtis, E.J.C. 1987. A comparison of caesium 137 and 134 activity in sheep remaining on upland areas con- taminated by Chernobyl fallout with those removed to less active lowland pasture. Journal of the Soci- ety for Radiological Protection 7: 71–73. – & Lindley, D.K. 1985. Aspects of the uptake of radio- nuclides by sheep grazing on an estuarine salt- marsh. 2. Radionuclides in sheep tissues. Journal of Environmental Radioactivity 2: 199–213. –, Mayes, R.W., Beresford, N.A. & Lamb, C.S. 1989. Transfer of radiocesium from different environ- mental sources to ewes and suckling lambs. Health Physics 57: 579–586. IAEA 1997. The use of Prussian Blue to reduce radio- caesium contamination of milk and meat produced on territories affected by the Chernobyl accident. IAEA-TECDOC-926. Report of United Nations Pro- ject E 11. International Atomic Energy Agency. Vi- enna. 80 p. 144 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 Paasikallio, A. et al. Effect of AFCF on reducing 1 3 4 Cs transfer from grass silage to sheep Johnson, J.E., Ward, G.M., Firestone, E. & Knox, K.L. 1968. Metabolism of radioactive cesium (134Cs and 137Cs) and potassium by dairy cattle as influenced by high and low forage diets. Journal of Nutrition 94: 282–288. Jones, D.R., Paul, L. & Mitchell, N.G. 1999. Effects of ameliorative measures on the radiocaesium transfer to upland vegetation in the UK. Journal of Environ- mental Radioactivity 44: 55–69. Lipsztein, J.L., Bertelli, L., Oliveira, C.A.N. & Dantas, B.M. 1991. Studies of Cs retention in the human body related to body parameters and Prussian Blue administration. Health Physics 60: 57–61. Madhus, K., Strömme, A., Bohne, F. & Nigrovic, V. 1966. Diminution of radiocaesium body-burden in dogs and human beings by Prussian Blue. Interna- tional Journal of Radiation Biology 10: 519–520. Melo, D.R., Lipsztein, J.L., de Oliveira, A.N. & Bertelli, L. 1994. 137Cs internal contamination involving a Brazil- ian accident, and the efficacy of Prussian Blue treat- ment. Health Physics 66: 245–252. Mitchell, N.G., Coughtrey, P.J., Beetham, C.J., Hughes, J.G., Clench, S.F. & Walters, B. 1989. Transfer of caesium from silage to cows milk: observations and models. The Science of the Total Environment 85: 307–316. Nielsen, P., Dresow, B., Fischer, R. & Heinrich, H.C. 1990. Bioavailability of iron and cyanide from oral potassium ferric hexacyanoferrate(II) in humans. Archives of Toxicology 64: 420–422. Nigrovic, V. 1963. Enhancement of the excretion of ra- diocaesium in rats by ferric cyanoferrate (II). Inter- national Journal of Radiation Biology 7: 307–309. Oughton, D.H., Day, J.P., Howard, B.J., Beresford, N.A., Lamb, C.S., Mayes, R.W., Preston, T. & East, B.W. 1991. Caesium dosing reduces uptake of ra- diocesium by sheep. Journal of Environmental Ra- dioactivity 14: 105–121. Pearce, J. 1994. Studies of any toxicological effects of Prussian Blue compounds in mammals- a review. Food and Chemical Toxicology 32: 577–582. –, Unsworth, E.F., McMurray, C.H., Moss, B.W., Lo- gan, E., Rice, D. & Hove, K. 1989. The effects of Prussian Blue provided by indwelling rumen boli on the tissue retention of dietary radiocaesium by sheep. The Science of the Total Environment 85: 349–355. Phillippo, M., Gvozdanovic, S., Gvozdanovic, D., Ches- ters, J.K., Paterson, E. & Mills, C.F. 1988. Reduc- tion of radiocaesium absorption by sheep consum- ing feed contaminated with fallout from Chernobyl. Veterinary Record 122: 560–563. SAS, 1992. SAS/STAT Software: Changes and En- hancements, Release 6.07, SAS Technical Report P-229, Statistical Analysis Systems Institute Inc., Cary, NY. 620 p. Unsworth, E.F., Pearce, J., McMurray, C.M., Moss, B.V., Gordon, F.J. & Rice, D. 1989. Investigations of the use of clay minerals and Prussian Blue in reduc- ing the transfer of dietary radiocaesium to milk. The Science of the Total Environment 85: 339–347. Vandecasteele, C.M., Van Hees, M., Culot, J.P. & Vank- erkom, J. 1989. Radiocaesium metabolism in preg- nant ewes and their progeny. The Science of the To- tal Environment 85: 213–223. Vandenhove, H., Van Hees, M., De Brouwer, S. & Vande- casteele, C. 1997. Effects of ammonium-ferric (III)-hexacyano-ferrate(II) and faeces addition on yield and soil-plant transfer of radiocaesium to rye- grass. Journal of Environmental Radioactivity 37: 235–246. –, Van Hees, M. & Vandecasteele, C.M. 1998. Effec- tiveness of immediate and delayed AFCF applica- tion in reducing radiocaesium transfer to ryegrass. Journal of Environmental Radioactivity 41: 47–63. –, Van Hees, M. & Vandecasteele, C. 2000. Potential side effects of ammonium-ferric-hexacyano-ferrate application: enhanced radiostrontium transfer and free cyanide release. Journal of Environmental Ra- dioactivity 47: 149–155. Voigt, G. 1993. Chemical methods to reduce the radio- active contamination of animals and their products in agricultural ecosystems. The Science of the Total Environment 137: 205–225. Vreman, K.,van den Hoek, J., van der Struijs, T.D.B. 1992. Administration of ammonium ferric hexacy- anoferrate strongly reduces radiocaesium contami- nation of cows’ milk. Netherlands Milk and Dairy Journal 46: 81–88. Wolfinger, R. 1996. Heterogeneous variance-covariance structures for repeated measures. Journal of Agricul- tural, Biological, and Environmental Statistics 1: 205–230. 145 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. 9 (2000): 135–147. SELOSTUS Lihan 134Cs-aktiivisuuspitoisuuden vähentäminen ferriheksasyanoferraatin avulla Arja Paasikallio, Riitta Sormunen-Cristian, Seija Jaakkola ja Matti Kaikkonen Maatalouden tutkimuskeskus ja Helsingin yliopisto Radiocesiumin kulkeutumista märehtijöihin on voitu vähentää syöttämällä eläimille erilaisia cesiumia si- tovia mineraaleja kuten esimerkiksi bentoniittia, zeo- liittia, vermikuliittia ja kaoliinia sekä kuitupitoista rehua. Ferriheksasyanoferraatit (preussinsininen, PB-yhdisteet) ovat edellisiä huomattavasti tehok- kaampia cesiumsitojia. Ammoniumferriheksasyano- ferraatti (AFCF) on PB-yhdisteistä tehokkaimpia ja eniten tutkittu. AFCF sitoo itseensä radiocesiumia vaihtaen sen ammoniumioniin. Koska PB ei imeydy ruoansulatuskanavasta kudoksiin, poistuu radiocesi- um eläimestä PB-yhdisteeseen sitoutuneena sonnan mukana. Paras tulos saavutetaan kun cesiumsitojaa syötetään samanaikaisesti aktiivisen rehun kanssa. Joissakin tapauksissa sitojan on myös todettu poista- van elimistössä jo olevaa radiocesiumia. Sisätiloissa PB:tä tavallisesti syötetään eläimille kerran pari päi- vässä sekoittamalla sitä pieneen määrään rehua. Lai- tumella sitojaa voidaan helpoimmin antaa lisäämällä sitä nuolukiveen tai bolukseen. Lukuisten tutkimus- ten perusteella PB-yhdisteitä voidaan pitää vaaratto- mina eläimille ja ihmisille, sillä yhdisteiden ei ole to- dettu havaittavissa määrin hajoavan ja imeytyvän ku- doksiin ja maitoon. Tutkimuksen tarkoituksena oli selvittää pienten AFCF-määrien, joita muhitettiin aktiivisessa säilöre- hussa sen kypsymisen ajan, vaikutusta radiocesiumin kulkeutumiseen lampaan kudoksiin. Muhittamisen vaikutusta cesiumsitojan tehokkuuteen ei ole aikai- semmin selvitetty. Tutkimuksen taustalla oli ajatus, että laskeumatilanteessa AFCF voitaisiin ruiskuttaa, kuten säilöntäainekin, saastuneeseen ruohoon jo pel- lolla sadonkorjuun yhteydessä. AFCF:n vaikutusta tutkittiin puolivuotiailla päs- sikaritsoilla, joita pidettiin aineenvaihduntahäkeissä. Raiheinää kasvatettiin astiakokeissa turvemaassa, jo- hon oli lisätty 134 Cs:a. Näin saatua aktiivista raiheinää lisättiin silputtuun pellolla kasvatettuun raiheinään, ja seokseen ruiskutettiin säilöntäainetta sekä veteen sekoitettua AFCF:ia. Säilörehun valmistuttua se jaet- tiin päiväannoksiin. Lampaille syötettiin AFCF:ia päivittäin neljäntoista päivän ajan 21 mg kapselissa ja 21 mg rehussa (koe 1) ja päivittäin kymmenen päi- vän ajan 50, 100 ja 150 mg rehussa (koe 2). Kontrolli- lampaat eivät saaneet AFCF:ia. Kudosten 134 Cs- aktiivisuuspitoisuus oli yleensä korkein munuaisissa ja matalin lihaksessa. Jo pienet- kin AFCF-määrät vähensivät selvästi lampaan lihan 134 Cs-aktiivisuuspitoisuutta. Rehun mukana annettu päivittäinen 21 mg AFCF-annos vähensi radioce- siumin kulkeutumista lampaan lihakseen 45 % kont- rollieläimeen verrattuna. Sama AFCF-määrä annettu- na kapselissa vähensi kulkeutumista 3 %. Eläinten saadessa päivittäin aktiivisen rehun mukana 50, 100 ja 150 mg AFCF:ia väheni radiocesiumin kulkeutu- minen lihakseen 75, 82 ja 86 %. Tässä tutkimuksessa käytetyt AFCF-määrät olivat pienille märehtijöille suositeltuja annoksia (1–2 g päivässä) huomattavasti pienempiä. Lihas/rehu-siirtokertoimet laskettiin jakamalla lihaksen 134 Cs-aktiivisuuspitoisuus (Bq kg -1 ) päivit- täisen rehuannoksen sisältämällä 134 Cs-määrällä (Bq d -1 ). Kokeessa 1 siirtokertoimet olivat 0,14 (kontrol- li), 0,14 (kapseli) ja 0,07 d kg -1 (rehu). Kokeessa 2 kontrollieläinten siirtokerroin oli 0,16 ja muiden kes- kimäärin 0,03 d kg -1 . Kontrollieläinten siirtokertoi- met olivat pienempiä kuin useissa Tshernobylin jäl- keen suoritetuissa tutkimuksissa, minkä katsottiin johtuvan tämän tutkimuksen kokeiden lyhytaikaisuu- desta. Eräiden selvitysten mukaan tasapaino kudos- ten radiocesiumpitoisuuden ja radiocesiumin jatku- van saannin välillä saavutetaan lampailla vasta noin 20–30 päivän kuluttua. Radiocesiumin erittyminen sonnan mukana oli kontrollilampailla selvästi vähäisempää ja virtsan mukana runsaampaa kuin AFCF:ia saaneilla lampail- la. Kun rehussa annetun AFCF:n päiväannosta nos- tettiin 0:sta 21 mg:aan, radiocesiumin keskimääräi- nen erittyminen sonnan mukana oli vastaavasti 41 ja 59 % ja virtsan mukana 16 ja 6 % radiocesiumin saan- nista (koe 1). Kun AFCF-annosta nostettiin 0:sta 150 mg:aan, vastaavat radiocesiumin erittymismäärät oli- vat sonnassa 34 ja 69 % ja virtsassa 17 ja 2 % (koe 2). Tutkimuksessa saatiin alustavaa tietoa aktiivises- sa säilörehussa muhineen AFCF:n vaikutuksesta lampaan lihan radiocesiumtasoon. AFCF:n muhitta- 146 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 Paasikallio, A. et al. Effect of AFCF on reducing 1 3 4 Cs transfer from grass silage to sheep minen useita viikkoja säilörehussa ei vähentänyt sen tehoa cesiumsitojana; pienetkin AFCF-määrät vä- hensivät huomattavasti radiocesiumin kulkeutumista eläimen kudoksiin. Ainakin tältä osin AFCF:n käyttö pellolla olisi mahdollista. AFCF:n ruiskutusteknii- kan kehittämiseksi tarvitaan lisätutkimuksia. Lisäksi lihas/rehu- siirtokertoimien luotettavuuden tarkista- minen vaatisi pitempiaikaisia ruokintakokeita kuin mitä tässä tutkimuksessa oli mahdollista tehdä. 147 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. 9 (2000): 135–147. Title Introduction Material and methods Results Discussion Conclusion References SELOSTUS