Hexacyanoferrates and bentonite as binders of radiocaesium for reindeer

The effects of varying doses of caesium binders (Bentonite and several forms of iron-hexacyanoferrates) on radiocaesium accumulation in red blood cells and on radiocaesium transfer to urine and faeces were studied in feeding experiments with reindeer calves. The caesium binders were added to a ration of lichen (containing 9.5 kBq of 1 3 4 Cs+ 1 3 7 Cs originating from the Chernobyl accident) and fed together with a pelleted reindeer feed (RF-71) for 42 days. A 50% reduction in red blood cell radiocaesium concentration was obtained with a daily dose of 1 mg/kg body weigth of ammoniumironhexacyanoferrate (AFCF) and with 500 mg/kg of bentonite. Three mg/kg of A F C F or 2 g/kg of bentonite reduced both urinary excretion and R B C concentrations with more than 80%. It is concluded that iron-hexacyanoferrates, as a result of their high caesium binding capacity, are particularly useful as caesium binders for free ranging ruminants like the reindeer. Rangifer, 11 (2): 43-48


Introduction
After the Chernobyl nuclear accident radiocaesium content of meat from reindeer in parts of Scandinavia increased far above the intervention levels (6 kBq/kg in Norway).Decontamination of live animals was required before marketing of reindeer meat.Feeding schemes utilizing uncontaminated feeds were immediately implemented, and caesium binders of the clay mineral type (zeolites and bentonite) were tested experimentally and used in practical situations (Ahman 1988, Ahman et al.;1990.;Hove & Ekern, 1988).From 1989 ammoniumiron-hexacyanoferrate (AFCF) replaced bentonite as the caesium binder in reindeer feeds in Norway.The major benefit of AFCF was a very high efficiency and specificity in binding radiocaesium and a high chemical stability in the gastrointestinal tract of ruminants (Arnaude£^/.., 1988).Our study was undertaken to establish the relationship between daily doses of the caesium binders and the accumulation of radiocaesium in animals fed lichen contaminated by fallout from the Chernobyl nuclear accident.An abstract of parts of the work was presented earlier (Hove et al, 1990a).

Animals and feeding
Male reindeer calves born in May 1986 were used for the experiment which lasted from November 1986 until April 1987.The animals were obtained from semidomestic herds in Southern Norway, and had been accustomed to handling during a period of feeding before the experiments started.During the experimental period calves were kept indoors (10°C) and alternated every two weeks between metabolism cages allowing collection of faeces and urine and tie stalls on the floor where collection of excreta was not possible.The calves weighed 45-55 kg at the start of the experiment, and most animals lost 1-3 kg during the study.
Lichen (mainly Cladina alpestris and Cetraria spp.) with a radiocaesium concentration of 30 ±4 kBq/kg dry matter (DM) of 134 Cs+ 137 Cs was obtained from a heavily contaminated area in Vågå, S. Norway.The lichen was stored frozen.Material for a feeding period of about two months was thawed when required, and contaminating soil and other non-lichen constituents were sorted out.The lichen was mixed manually, weighed into portions in plastic bags, and again stored frozen.The calves received 0.67 kg of lichen (0.32 kg DM) daily, giving a radiocaesium intake of 9.5 kBq/d.In addition 1 kg of the pelleted reindeer feed RF-71+4% sodium bicarbonate added as a buffer (Jacobsen & Skjenneberg, 1979) was given.

Samples
Collection of faeces and urine was done twice weekly during the two weeks when the calves were in metabolism cages.Urine was weighed and 5 ml used for radiometry.The whole collection of faeces was dried, ground, and a 3-5 g sample was used for radiometry.Blood samples were taken twice week-ly from the jugular vein.Plasma was separated, and 5 ml of the red cellfraction (RBC) transferred to counting vials.The hematocrit of the red cell fraction was used to correct for plasma dilution when the radiocaesium concentration of RBC was calculated.

Experimental
Two series of experiments were carried out.
In Series 1 the accumulation of radiocaesium with time was studied in 4 calves to gain knowledge on the length of the period required to reach steady state conditions in urine and RBC radiocaesium concentrations.Lichen was fed for 72 days.The results showed that more than 90% of plateau levels of urinary radiocaesium excretion and about 75% of plateau levels of RBC radiocaesium concentrations were reached after 42 days.This time period was therefore considered satisfactory for the testing of the caesium binders in series 2. Two of the calves were from day 42 given 300 mg/d of AFCF to assess the effect on the caesium binder in animals already loaded with radiocaesium.
In Series 2 the effects of different doses of caesium binders on the accumulation of radiocesium were studied.Hexacyanoferrates included Ammoniumiron(III)-hexacyanoferrate(II) from Riedel de Haen, Seelze, Germany, an insoluble prussian blue preparation (iron(III)-iron(III)-hexacyanoferrate(II), FeFCFl) from British Drug Houses, UK, and a similar compound (FeFCF2) produced as a pigment by WEB Kali Chemie, Berlin, Germany.AFCF was given in daily doses of 50, 150, 300, 700 and 1500 mg/d.The other hexacyanoferrates were only tested in the 300 mg/d dose.Finely ground, feed grade Wyoming bentonite was given in daily doses of 25, 50 and 100 g.
The animals were fed contaminated lichen and the caesium binder for a period of 42 days.Two calves were used per dose.The caesium binders were sprinkled onto the lichens.The hexacyanoferrates and the 25 g dose of bentonite were readily eaten, but the higher doses of bentonite were consumed more reluctantly.Feeding of 100 g of bentonite resulted in clogging of bentonite to the hairs of the muzzle and face of the calves.

Radiometry
Samples of urine, faeces and red blood cells were counted for 134 Cs and 137 Cs in a LKB ultro-gamma counter in vials containing 5 or 10 ml of sample.The level of detection for radiocaesium in RBC and urine was between 20-50 Bq/L with the counting times used.

Calculations
The build up of radiocaesium concentrations in RBC, urine and faeces C(t) with time (t) could be described by the formula C(t) = P(l-e^) [1) where P is the estimated equilibrium concentration and k is the rate constant for the exponential term.The time required to reach 50% of plateau levels (halftime for the exponential term) was calculated as t/2 = ln(2)/k.

Results
Series 1. Radiocesium excretion in faeces and urine reached 90-100% of their plateau levels within 6 weeks (Fig. 1).Estimated plateau levels for urinary excretion was 15% of the daily ingested dose of radiocaesium.Fitting equation (l) to the measurements of urinary radiocaesium concentrations resulted in a halftime of 11 d (Fig. 1).Radiocaesium excretion via faeces reached a plateau of approximately 75% of the daily ingested dose, and the build up of radiocaesium activity was described by equation 1 with a halftime of 6 d (Fig. 1).Loss of radiocaesium in faeces and urine accounted for about 90% of the daily ingested activity during the last weeks of the experiment, indicating that some accumulation of radiocaesium in muscle cells and other intracellular compartments were still taking place.RBC radiocaesium had attained about 80% of the equilibrium level at 6 weeks, and continued to increase for 2-4 more weeks (Fig. 1).Estimated plateau for RBC activity was 5.5% of the daily dose per liter of cells, and the half time for the exponential describing the build up was 21 days.
When AFCF (300 mg/d) was added to the diet, faecal excretion of radiocaesium increased immediately to 110% of the ingested dose, demonstrating that a reduction in body load took place at the same time as the absorption decreased.After 4 weeks of feeding 300 mg/d AFCF faecal excretion decreased, probably as a result of a depletion of the body load of radiocaesium.Urinary excretion of radiocaesium declined by 85-90% compared to the animals which not were fed AFCF, and RBC concentrations of radiocaesium were reduced by about 90% in 3-4 weeks (Fig. 1).
Series 2. A dose-dependant reduction both in urinary radiocaesium excretion (Fig. 2, Table 1) and in RBC concentrations (Table 1) was observed when bentonite was added to the diet in increasing amounts.Average reduction in urinary excretion ranged Curves with broken lines represent the fitted exponential formulas for the build up of radiocaesium concentrations.
from 47% with the 25 g dose to 85% when 100 g bentonite was fed.RBC concentrations of radiocaesium during the last 3 weeks of the 6 week observation period were reduced with 35, 75 and 80% re-  The curve for the control animals (0 g) was constructed from observations in series 1 of four animals sampled at different times.Each data point is the average of observations from two animals.
Mean RBC radiocaesium concentrations day 21-42 were reduced by 60% with 50 mg/d of AFCF increasing to 87% and to 93% with the doses of 150 to 1500 mg/d (Table 1).Radiocaesium concentrations in RBC were 59 and 57 Bq/L when FeFCFl and FeFCF2 were fed.These values were very close to the individual averages of 40-50 Bq/L observed for the 150 and 300 mg doses of AFCF (Table 1).The same dose dependent reductions were found in urinary excretion of radiocaesium (Table 1).In urine the reduction was significantly greater for the two highest dose levels than for the 150 and 300 mg/d doses.No difference in the level of reduction was detected between the three different hexacyanoferrate preparations when tested at the 300 mg/d dose level.

Discussion
The experimental period for testing the efficiency of the caesium binders was 42 days.Analysis of the kinetics of radiocaesium excretion showed that an equilibrium in the excretion of radiocaesium in urine and faeces had probably been attained during the period from 21-42 d.Blood radiocaesium activity was however still increasing (Fig. 1).From other studies (Pedersen, Staaland & Hove, unpublished) it can be inferred that also the meat would accumulate radiocaesium after 42 days of feeding of contaminated lichen.In calculating the levels of reduction for the various treatments more precise estimates could probably have been reached by pro-longation of the experimental periods, but considerably longer feeding periods would have been neccessary to obtain equilibrium in slow turnover pools like muscle cells.
Hexacyanoferrates have for long been recognized as effective caesium binders reducing radiocaesium accumulation in a wide variety of animals including rats (Nigrovic, 1963), pigs (Giese., 1970;Rudnicki, 1988) and several ruminant species like the goat (Havlicek etal, 1967;Hove etal, 1990b), cow (Arnaud et al, 1988;Giese, 1988) and reindeer (Hove etal, 1988;Staaland etal, 1990).The early studies with prussian blue used the iron(III) and the potassium complexes of the hexacyanoferrates.Giese & Hantzsch (1968) and later Nielsen et al (1987) reported a somewhat superior efficiency for the ammonium salt when compared to the alkali hexacyanoferrates in vitro.Only AFCF has been used in Norway after the Chernobyl accident.
The doses of AFCF used in animal experiments have recently been reviewed by Giese (1989).In dairy cows doses of 3 g/d reduced milk radiocaesium with 80-90% and meat radiocaesium with 75%.In small ruminants like sheep and goats 1-2 g/d (20-40 mg/kg) were recommended by the manufacturer.Our experiments showed a significant reduction in radiocaesium transfer with as little as 50 mg/d (1 mg/kg body weigth).A dose dependent reduction in urinary radiocaesium excretion was observed up to 700 mg/d, while the the RBC reduction was maximal already at the 150 mg (3 mg/kg ) dose level (Table 1).In agreement with this Mathiesen et al (1990) reported that 250 mg/d AFCF prevented accumulation of radiocaesium.This effect of AFCF in dose levels much lower than those recommended is clearly beneficial when the caesium binder is used for free-ranging animals where daily feeding of concentrates is impractical.The efficacy of the preparation in small doses has enabled the use of AFCF as a countermeasure for grazing animals in the form of salt licks impregnated with 2.5% AFCF and in sustained release boli (Hove et al, 1990a,b).
No controlled comparison seem however to have been performed on the action of the different chemical forms of the hexacyanoferrate binders in ruminants.The special features of the ruminant forestomachs with a large liquid volume and a long retention time which allow good contact between radiocaesium and the binder may allow for differences in the relative effect of the binders compared to monogastric species.The amount of caesium recycling to the rumen with saliva is also very large (Staaland et al, 1990).In our study no significant difference between the effect of the studied forms of the hexacyanoferrates could be found at the 300 mg/d dose level (Table 1).This dose level was however not the most sensitive, since the level of reduction of radiocaesium in urine was already 80-90%, and further studies should be carried out with lower daily doses of the various hexacyanoferrate forms to adress this question.
Bentonite has been used for reduction of the transfer of radiocaesium from feed to animal products in both reindeer and domestic ruminants after the Chernobyl accident.In reindeer Ahman et al (1990) reported about 80% reduction in whole blood 137 Cs levels when feeding reindeer either 23 or 46 g bentonite per day during a 21 day feeding trial.Simultaneously the urinary excretion was reduced by almost 90%.Such levels of reduction was only obtained with the 100 g dose levels in the present study.An explanation to this may be that Ahman et al (1990) used a concentrate feed with bentonite which may have prevented the clogging of the preparate which was observed when the bentonite was fed as a powder.Differences in the caesium binding capacity of the bentonite preparations could however not be precluded.
The dose dependent reduction observed in radiocaesium transfer in the present study agrees well with results in dairy cows.Hove & Ekern (1988) observed 33, 77 and 68 % reduction in the radiocaesium transfer to milk when 250, 500 or 750 g bentonite was fed with contaminated hay, and Mitchell et al (1989) reported 37 and 67% reductions in radiocaesium transfer to milk on the two lowest dose levels.Results from comparable dose/response trials appear not to be available for sheep, but reductions of 80% or more after feeding daily doses of about 50g was reported both by Van den Hoek (1976) and Anderson (1989), and Daburon et al (1991) reported 60 and 85% reduction when sheep were fed 30 or 60 g/d of vermiculite respectively.
Caesium binders are added to the feed which is given to reindeer, sheep and cows during the special feeding programs undertaken to reduce the radiocaesium content of the meat to below the intervention limit.This addition is chosen to assure a minimal absorption from soil or from lichen fed to maintain appetite and optimal rumen function.Judged from studies in other ruminant species increased rates of radiocaesium loss from the body would hardly be expected in contaminated animals fed uncontaminated feeds (Bailer, 1988;Hove & Ekern, 1988;Philippo etal, 1988).The present study shows that hexacyanoferrates will reduce radio-caesium accumulation by about 50% even in closes as low as 1 mg/kg body weigth.This high efficiency makes the hexacyanoferrates particularily well suited for reducing radiocaesium levels in grazing reindeer and other ruminants.

Fig. 2 .
Fig.2.Urinary radiocaesium excretion from animals fed 9.5 kBq/d of radiocaesium in contaminated lichen and different amounts of bentonite (Series 2, mean of two animals per dose level of bentonite).The curve for the control animals (0 g) was constructed from observations in series 1 of four animals sampled at different times.Each data point is the average of observations from two animals.

Table 1 .
Radiocaesium excretion in urine of reindeer calves fed radiocaesium and given different daily doses of hexacyanoferrates or bentonite.The values given are averages of daily excretions of radiocaesium day 11-42 for urine and averages of values day 21-42 for red blood cells (RBC).The percent reduction was calculated relative to urinary radiocaesium excretion in the control animals.