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Tissue and subcellular distributions of 210Po in the crustacean Saduria entomon inhabiting the southern Baltic Sea
Journal of Environmental Radioactivity 49 (2000) 195}199
Tissue and subcellular distributions of 210Po in the crustacean Saduria entomon inhabiting the southern Baltic Sea Piotr Stepnowski!,*, Bogdan Skwarzec" !Faculty of Chemistry, University of Gdan& sk, 80-952 ;Gdan& sk, ul. Sobieskiego 18/19, Poland "Institute of Oceanology, Polish Academy of Sciences, 81-712 Sopot, ul. Powstan& co& w Warszawy 55, Poland Received 1 December 1998; received in revised form 15 July 1999; accepted 25 August 1999
Abstract The paper presents the results of 210Po determinations, and 210Po/210Pb activity ratios, in the tissues and organs of the Baltic crustacean Saduria entomon. Additionally, the subcellular localisation of 210Po in the hepatopancreas of this organism has been studied using centrifugation followed by determination of the 210Po concentrations in isolated fractions. The 210Po concentrations fall within the range 0.63}543.5 Bq kg~1 (dry wt). The highest values of 210Po and 210Po/210Pb activity ratio (10.6) were found, as expected, in the hepatopancreas. The majority of the 210Po in the hepatopancreatic cells was found in the cytosol fraction (60% of the total 210Po). ( 2000 Elsevier Science Ltd. All rights reserved. Keywords: Polonium, 210Po; Crustacean; Baltic; Subcellular distribution
1. Introduction In the marine environment polonium-210 (t "138.4 d) is largely produced from 1@2 the decay of 210Pb deposited from the atmosphere. A small amount of 210Po in the seawater originates from the atmospheric deposition of polonium itself. 210Po provides a major contribution (90%) to the natural radiation dose from alpha-emitting radionuclides to most marine organisms (Cherry & Shannon, 1974). The available data on 210Po and 210Pb concentrations in marine organisms show inhomogeneous
* Corresponding author. E-mail address: [email protected] (P. Stepnowski) 0265-931X/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 5 - 9 3 1 X ( 9 9 ) 0 0 0 9 3 - 4
196
P. Stepnowski, B. Skwarzec / J. Environ. Radioactivity 49 (2000) 195}199
distributions between tissues. This is well demonstrated in the case of marine invertebrates where the concentrations of the nuclides in the hepatopancreas are consistently greater than those in other tissues. This phenomenon appears to constitute one of the highest known natural radiation dose domains in the biosphere (Cherry & Heyraud, 1982; Cherry, Heyraud & Higgo, 1983; Heyraud, Cherry & Dowdle, 1987; Carvalho, 1988; Carvalho & Fowler, 1993). The hepatopancreas of marine invertebrates is known to play an important role in the accumulation of stable elements, e.g. Ag, Cd, Cu and Zn, where it is likely that sulphur-containing proteins such as the metallothioneins play a crucial role in the binding processes (Cherry et al., 1983). In this study we present data for the 210Po and 210Pb activity concentrations measured in the organs and tissues of the crustacean Saduria entomon inhabiting the southern Baltic. These results also include the data for subcellular fractions derived from the hepatopancreas of these crustaceans.
2. Materials and methods The specimens of Saduria entomon were collected from the Gulf of GdanH sk and the S"upsk Narrow in the Southern Baltic Sea during the cruise of r/v `Oceaniaa in 1996. The organisms (n"160) were dissected into the following parts: hepatopancreas, alimentary tract, gonads, muscles, hemolymph and chitinous shell. The hepatopancreas was homogenised in bu!er of 10 mM tris-HCl #0.25 M sucrose solution #0.15 M KCl. The homogenate was then centrifuged to yield basic cellular fractions (nuclei at 714g, mitochondria at 15100g, microsomes at 105 000g and residue * cytosol) (Graham & Rickwood, 1997). The protein content of each fraction was measured using the Coomasie blue binding assay (Spector, 1978). The biological material and cellular fractions were oven-dried at 603C for two days and then digested using concentrated nitric acid with a 209Po spike added as a yield tracer. The dry residue was then dissolved in 0.5 M HCl. After adding ascorbic acid, the polonium in the samples was spontaneously deposited onto silver discs (Flynn, 1968; Skwarzec, 1997). Measurements were made on silicon surface-barrier detector (TENNELEC TC 256, USA), and recovery was not less than 90%. The activity of 210Pb was calculated indirectly by measuring the 210Po which has originated from the radioactive decay of its grandparent 210Pb after an ingrowth period of one year (Takizawa, Zhao, Yamamoto, Abe & Ueno, 1990).
3. Results and discussion The results for the 210Po concentrations and 210Po/210Pb activity ratios in the dissected organs and the whole body of the crustacean Saduria entomon are given in Table 1. The presented results refer to one pooled sample from 160 specimens. The data show that there is a signi"cant disproportion of polonium activity concentration in the tissues and organs examined. The highest 210Po concentration was found in the
P. Stepnowski, B. Skwarzec / J. Environ. Radioactivity 49 (2000) 195}199
197
Table 1 210Po activity concentration and 210Po/210Pb activity ratios in organs and tissues of the crustacean Saduria entomon Organ, tissue
Activity ratio 210Po/210Pb
210Po concentration $1SD Bq.kg!1 (dry wt)
Wet : dry ratio
Hepatopancreas Alimentary tract Muscles Gonads Head Chitinous shell Haemolymph Whole body!
10.6 6.4 3.6 3.2 4.4 0.2 0.2 4.8
543.5$9.9 353.6$12.5 52.4$2.0 12.1$1.4 8.6$0.7 0.6$0.1 11.1$3.7 29.5$0.8
2.4 6.0 2.9 2.1 2.5 4.4 8.3 4.1
!total soft tissue#shell.
hepatopancreas and was 543.5 Bq kg~1 (dry wt). In comparison, the mean concentration of 210Po measured in the Saduria entomon hepatopancreas 10 years ago (Skwarzec & Falkowski, 1988) was 586.6 Bq kgv1 (dry wt). Therefore, it is concluded that over the last decade there has been little change in the 210Po bioaccumulation process in this southern Baltic invertebrate. The 210Po/210Pb activity ratios re#ect the chemical properties of both radionuclides. They are in the range of 0.2}10.6, where the lowest values are common for hard tissues (210Pb a$nity to mineral structures) and higher ratios are characteristic of internal organs (210Po absorption on organic matter). However, the 210Po/210Pb activity ratio in the hepatopancreas is the highest value obtained and this organ also illustrates a rather high 210Pb concentration (51.5 Bq kg~1 dry wt) relative to the tissues and organs examined. It should be noted that, apart from hard tissues, low 210Po/210Pb ratios were also measured for haemolymph due to a high speci"c uptake of 210Pb (70.3 Bq kg~1 dry wt) in that #uid. This may be an additional cause of the relatively high accumulation of radiolead in the hepatopancreas, where haemocyanin (the basic component of haemolymph) is biosynthesised (Senkbeil & Wriston, 1981). Obviously, at this stage of the study, this observation remains speculative, but it is likely that 210Pb could form a complex with haemocyanin due to possible interactions (exchange) with the copper ions which are ligated within the structure of the protein (Lerch, 1994). Fig. 1 and Table 2 illustrate the percentage of 210Po and of protein in the basic cellular fractions. The highest value was measured for cytosol, in which 60% of the total 210Po content of the hepatopancreas is localised. Other fractions are characterised by a rather non-uniform distribution of 210Po with mitochondria, microsomes and nuclei containing 14, 7 and 18% of the total cell content, respectively. It is noticeable that the trends of 210Po content and protein content are similar; their concentrations decrease in the following order: cytosol, nuclei, mitochondria and microsomes. The data from our experiment di!er from results obtained in previous studies on the intracellular sites of 210Po in the invertebrate hepatopancreas. In a study on the South African lobster Jasus lalandii, Cherry, Dowdle and Todd (1979)
198
P. Stepnowski, B. Skwarzec / J. Environ. Radioactivity 49 (2000) 195}199
Fig. 1. Percentage distribution of 210Po and protein in subcellular fractions of hepatopancreatic cells of Saduria entomon. Table 2 210Po and protein contents and 210Po/protein ratios in subcellular fractions of hepatopancreatic cells of Saduria entomon Fraction
210Po (mBq)
Protein (mg)
210Po /Protein (mBq mg~1)
Cytosol Nuclei Mitochondria Microsomes
31.0 8.4 8.3 3.7
174.1 88.4 66.5 62.5
0.18 0.09 0.12 0.06
reported that the maximum 210Po concentration was evident in the 105 000g pellet which corresponds to the microsomal fraction. Furthermore, during experiments with the same species, it was found that the majority of 210Po in microsomes is likely to be associated with a component of high molecular weight (Heyraud et al., 1987). There are, however, reported data concerning 210Po localisation in rat liver. These reports suggest an a$nity of 210Po for moieties of cytosol proteins (Lanzola, Allegrini & Taylor, 1973) and identify one of these proteins as metallothionein (Aposhian & Bruce, 1991). The data for 210Po subcellular localisation in this experiment are of a preliminary character. Our future investigations of the bioaccumulation of 210Po in Saduria entomon should include separations of the cytoplasmic and microsomal pellet components followed by a study of the 210Po concentration in isolated subfractions. Additionally, more detailed studies should be undertaken to investigate the role of haemolymph in the transport of 210Pb within the body of Saduria entomon.
P. Stepnowski, B. Skwarzec / J. Environ. Radioactivity 49 (2000) 195}199
199
Acknowledgements The authors would like to thank the Polish State Committee for Scienti"c Research (KBN) for the "nancial support of this work under Grant DS./8000-4-00-26-8.
References Aposhian, H. V., & Bruce, D. C. (1991). Binding of polonium-210 to liver metallothionein. Radiation Research, 126, 379}382. Carvalho, F. P. (1988). 210Po in marine organisms: A wide range of natural radiation dose domains. Radiation Protection Dosimetry, 24, 113}117. Carvalho, F. P., & Fowler, S. W. (1993). An experimental study on bioaccumulation and turnover of polonium-210 and lead-210 in marine shrimp. Marine Ecology Progress Series, 102, 125}133. Cherry, R. D., & Heyraud, M. (1982). Evidence of high natural radiation doses in certain mid-water oceanic organisms. Science, 218, 54}56. Cherry, R. D., & Shannon, L. V. (1974). The alpha radioactivity of marine organisms. Atomic Energy Reviews, Vienna, 12, 3}45. Cherry, R. D., Heyraud, M., & Higgo, J. J. W. (1983). Polonium-210: its relative enrichment in the hepatopancreas of marine invertebrates. Marine Ecology Progress Series, 13, 229}236. Cherry, R. D., Dowdle, E. B. D., & Todd, G. (1979). Intracellular sites of natural 210Po in the lobster Jasus lalandii. South African Journal of Science, 75, 39}40. Flynn, W. W. (1968). The determination of low levels of polonium-210 in environmental materials. Analyt. Chimica Acta, 43, 221}227. Graham, J.M. & Rickwood, D. (1997). Subcellular fractionation, a practical approach. Oxford, UK: Oxford University Press. Heyraud, M., Cherry, R. D., & Dowdle, E. B. (1987). The subcellular localization of natural 210Po in the hepatopancreas of the rock lobster (Jasus lalandii). Journal of Environmental Radioactivity, 5, 249}260. Lanzola, E. E., Allegrini, M. E., & Taylor, D. M. (1973). The binding of polonium-210 to rat tissues. Radiation Research, 56, 370}384. Lerch, K. (1994). Copper proteins with dinuclear active sites. In R. B. King, Encyclopedia of Inorganic Chemistry (pp. 850}869). Chichester: Wiley. Senkbeil, E. G., & Wriston, J. C. (1981). Hemocyanin synthesis in the American lobster Homarus americanus. Comparative Biochemistry Physiology, 68B, 163}171. Skwarzec, B. (1997). Radiochemical methods for determination of polonium, radiolead, uranium and plutonium in environmental samples. Chem. Anal., 42, 107}115. Skwarzec, B., & Falkowski, L. (1988). Accumulation of 210Po in Baltic invertebrates. Journal of Environmental Radioactivity, 8, 99}109. Spector, T. (1978). Re"nement of the coomasie blue method of protein quantitation. Analytical Biochemistry, 86, 142}146. Takizawa, Y., Zhao, L., Yamamoto, M., Abe, T., & Ueno, K. (1990). Determination of 210Pb and 210Po in human tissues of Japanese. Journal of Radioanalytical Nuclear Chemistry, 138, 145}152.
Tissue and subcellular distributions of 210Po in the crustacean Saduria entomon inhabiting the southern Baltic Sea Piotr Stepnowski!,*, Bogdan Skwarzec" !Faculty of Chemistry, University of Gdan& sk, 80-952 ;Gdan& sk, ul. Sobieskiego 18/19, Poland "Institute of Oceanology, Polish Academy of Sciences, 81-712 Sopot, ul. Powstan& co& w Warszawy 55, Poland Received 1 December 1998; received in revised form 15 July 1999; accepted 25 August 1999
Abstract The paper presents the results of 210Po determinations, and 210Po/210Pb activity ratios, in the tissues and organs of the Baltic crustacean Saduria entomon. Additionally, the subcellular localisation of 210Po in the hepatopancreas of this organism has been studied using centrifugation followed by determination of the 210Po concentrations in isolated fractions. The 210Po concentrations fall within the range 0.63}543.5 Bq kg~1 (dry wt). The highest values of 210Po and 210Po/210Pb activity ratio (10.6) were found, as expected, in the hepatopancreas. The majority of the 210Po in the hepatopancreatic cells was found in the cytosol fraction (60% of the total 210Po). ( 2000 Elsevier Science Ltd. All rights reserved. Keywords: Polonium, 210Po; Crustacean; Baltic; Subcellular distribution
1. Introduction In the marine environment polonium-210 (t "138.4 d) is largely produced from 1@2 the decay of 210Pb deposited from the atmosphere. A small amount of 210Po in the seawater originates from the atmospheric deposition of polonium itself. 210Po provides a major contribution (90%) to the natural radiation dose from alpha-emitting radionuclides to most marine organisms (Cherry & Shannon, 1974). The available data on 210Po and 210Pb concentrations in marine organisms show inhomogeneous
* Corresponding author. E-mail address: [email protected] (P. Stepnowski) 0265-931X/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 5 - 9 3 1 X ( 9 9 ) 0 0 0 9 3 - 4
196
P. Stepnowski, B. Skwarzec / J. Environ. Radioactivity 49 (2000) 195}199
distributions between tissues. This is well demonstrated in the case of marine invertebrates where the concentrations of the nuclides in the hepatopancreas are consistently greater than those in other tissues. This phenomenon appears to constitute one of the highest known natural radiation dose domains in the biosphere (Cherry & Heyraud, 1982; Cherry, Heyraud & Higgo, 1983; Heyraud, Cherry & Dowdle, 1987; Carvalho, 1988; Carvalho & Fowler, 1993). The hepatopancreas of marine invertebrates is known to play an important role in the accumulation of stable elements, e.g. Ag, Cd, Cu and Zn, where it is likely that sulphur-containing proteins such as the metallothioneins play a crucial role in the binding processes (Cherry et al., 1983). In this study we present data for the 210Po and 210Pb activity concentrations measured in the organs and tissues of the crustacean Saduria entomon inhabiting the southern Baltic. These results also include the data for subcellular fractions derived from the hepatopancreas of these crustaceans.
2. Materials and methods The specimens of Saduria entomon were collected from the Gulf of GdanH sk and the S"upsk Narrow in the Southern Baltic Sea during the cruise of r/v `Oceaniaa in 1996. The organisms (n"160) were dissected into the following parts: hepatopancreas, alimentary tract, gonads, muscles, hemolymph and chitinous shell. The hepatopancreas was homogenised in bu!er of 10 mM tris-HCl #0.25 M sucrose solution #0.15 M KCl. The homogenate was then centrifuged to yield basic cellular fractions (nuclei at 714g, mitochondria at 15100g, microsomes at 105 000g and residue * cytosol) (Graham & Rickwood, 1997). The protein content of each fraction was measured using the Coomasie blue binding assay (Spector, 1978). The biological material and cellular fractions were oven-dried at 603C for two days and then digested using concentrated nitric acid with a 209Po spike added as a yield tracer. The dry residue was then dissolved in 0.5 M HCl. After adding ascorbic acid, the polonium in the samples was spontaneously deposited onto silver discs (Flynn, 1968; Skwarzec, 1997). Measurements were made on silicon surface-barrier detector (TENNELEC TC 256, USA), and recovery was not less than 90%. The activity of 210Pb was calculated indirectly by measuring the 210Po which has originated from the radioactive decay of its grandparent 210Pb after an ingrowth period of one year (Takizawa, Zhao, Yamamoto, Abe & Ueno, 1990).
3. Results and discussion The results for the 210Po concentrations and 210Po/210Pb activity ratios in the dissected organs and the whole body of the crustacean Saduria entomon are given in Table 1. The presented results refer to one pooled sample from 160 specimens. The data show that there is a signi"cant disproportion of polonium activity concentration in the tissues and organs examined. The highest 210Po concentration was found in the
P. Stepnowski, B. Skwarzec / J. Environ. Radioactivity 49 (2000) 195}199
197
Table 1 210Po activity concentration and 210Po/210Pb activity ratios in organs and tissues of the crustacean Saduria entomon Organ, tissue
Activity ratio 210Po/210Pb
210Po concentration $1SD Bq.kg!1 (dry wt)
Wet : dry ratio
Hepatopancreas Alimentary tract Muscles Gonads Head Chitinous shell Haemolymph Whole body!
10.6 6.4 3.6 3.2 4.4 0.2 0.2 4.8
543.5$9.9 353.6$12.5 52.4$2.0 12.1$1.4 8.6$0.7 0.6$0.1 11.1$3.7 29.5$0.8
2.4 6.0 2.9 2.1 2.5 4.4 8.3 4.1
!total soft tissue#shell.
hepatopancreas and was 543.5 Bq kg~1 (dry wt). In comparison, the mean concentration of 210Po measured in the Saduria entomon hepatopancreas 10 years ago (Skwarzec & Falkowski, 1988) was 586.6 Bq kgv1 (dry wt). Therefore, it is concluded that over the last decade there has been little change in the 210Po bioaccumulation process in this southern Baltic invertebrate. The 210Po/210Pb activity ratios re#ect the chemical properties of both radionuclides. They are in the range of 0.2}10.6, where the lowest values are common for hard tissues (210Pb a$nity to mineral structures) and higher ratios are characteristic of internal organs (210Po absorption on organic matter). However, the 210Po/210Pb activity ratio in the hepatopancreas is the highest value obtained and this organ also illustrates a rather high 210Pb concentration (51.5 Bq kg~1 dry wt) relative to the tissues and organs examined. It should be noted that, apart from hard tissues, low 210Po/210Pb ratios were also measured for haemolymph due to a high speci"c uptake of 210Pb (70.3 Bq kg~1 dry wt) in that #uid. This may be an additional cause of the relatively high accumulation of radiolead in the hepatopancreas, where haemocyanin (the basic component of haemolymph) is biosynthesised (Senkbeil & Wriston, 1981). Obviously, at this stage of the study, this observation remains speculative, but it is likely that 210Pb could form a complex with haemocyanin due to possible interactions (exchange) with the copper ions which are ligated within the structure of the protein (Lerch, 1994). Fig. 1 and Table 2 illustrate the percentage of 210Po and of protein in the basic cellular fractions. The highest value was measured for cytosol, in which 60% of the total 210Po content of the hepatopancreas is localised. Other fractions are characterised by a rather non-uniform distribution of 210Po with mitochondria, microsomes and nuclei containing 14, 7 and 18% of the total cell content, respectively. It is noticeable that the trends of 210Po content and protein content are similar; their concentrations decrease in the following order: cytosol, nuclei, mitochondria and microsomes. The data from our experiment di!er from results obtained in previous studies on the intracellular sites of 210Po in the invertebrate hepatopancreas. In a study on the South African lobster Jasus lalandii, Cherry, Dowdle and Todd (1979)
198
P. Stepnowski, B. Skwarzec / J. Environ. Radioactivity 49 (2000) 195}199
Fig. 1. Percentage distribution of 210Po and protein in subcellular fractions of hepatopancreatic cells of Saduria entomon. Table 2 210Po and protein contents and 210Po/protein ratios in subcellular fractions of hepatopancreatic cells of Saduria entomon Fraction
210Po (mBq)
Protein (mg)
210Po /Protein (mBq mg~1)
Cytosol Nuclei Mitochondria Microsomes
31.0 8.4 8.3 3.7
174.1 88.4 66.5 62.5
0.18 0.09 0.12 0.06
reported that the maximum 210Po concentration was evident in the 105 000g pellet which corresponds to the microsomal fraction. Furthermore, during experiments with the same species, it was found that the majority of 210Po in microsomes is likely to be associated with a component of high molecular weight (Heyraud et al., 1987). There are, however, reported data concerning 210Po localisation in rat liver. These reports suggest an a$nity of 210Po for moieties of cytosol proteins (Lanzola, Allegrini & Taylor, 1973) and identify one of these proteins as metallothionein (Aposhian & Bruce, 1991). The data for 210Po subcellular localisation in this experiment are of a preliminary character. Our future investigations of the bioaccumulation of 210Po in Saduria entomon should include separations of the cytoplasmic and microsomal pellet components followed by a study of the 210Po concentration in isolated subfractions. Additionally, more detailed studies should be undertaken to investigate the role of haemolymph in the transport of 210Pb within the body of Saduria entomon.
P. Stepnowski, B. Skwarzec / J. Environ. Radioactivity 49 (2000) 195}199
199
Acknowledgements The authors would like to thank the Polish State Committee for Scienti"c Research (KBN) for the "nancial support of this work under Grant DS./8000-4-00-26-8.
References Aposhian, H. V., & Bruce, D. C. (1991). Binding of polonium-210 to liver metallothionein. Radiation Research, 126, 379}382. Carvalho, F. P. (1988). 210Po in marine organisms: A wide range of natural radiation dose domains. Radiation Protection Dosimetry, 24, 113}117. Carvalho, F. P., & Fowler, S. W. (1993). An experimental study on bioaccumulation and turnover of polonium-210 and lead-210 in marine shrimp. Marine Ecology Progress Series, 102, 125}133. Cherry, R. D., & Heyraud, M. (1982). Evidence of high natural radiation doses in certain mid-water oceanic organisms. Science, 218, 54}56. Cherry, R. D., & Shannon, L. V. (1974). The alpha radioactivity of marine organisms. Atomic Energy Reviews, Vienna, 12, 3}45. Cherry, R. D., Heyraud, M., & Higgo, J. J. W. (1983). Polonium-210: its relative enrichment in the hepatopancreas of marine invertebrates. Marine Ecology Progress Series, 13, 229}236. Cherry, R. D., Dowdle, E. B. D., & Todd, G. (1979). Intracellular sites of natural 210Po in the lobster Jasus lalandii. South African Journal of Science, 75, 39}40. Flynn, W. W. (1968). The determination of low levels of polonium-210 in environmental materials. Analyt. Chimica Acta, 43, 221}227. Graham, J.M. & Rickwood, D. (1997). Subcellular fractionation, a practical approach. Oxford, UK: Oxford University Press. Heyraud, M., Cherry, R. D., & Dowdle, E. B. (1987). The subcellular localization of natural 210Po in the hepatopancreas of the rock lobster (Jasus lalandii). Journal of Environmental Radioactivity, 5, 249}260. Lanzola, E. E., Allegrini, M. E., & Taylor, D. M. (1973). The binding of polonium-210 to rat tissues. Radiation Research, 56, 370}384. Lerch, K. (1994). Copper proteins with dinuclear active sites. In R. B. King, Encyclopedia of Inorganic Chemistry (pp. 850}869). Chichester: Wiley. Senkbeil, E. G., & Wriston, J. C. (1981). Hemocyanin synthesis in the American lobster Homarus americanus. Comparative Biochemistry Physiology, 68B, 163}171. Skwarzec, B. (1997). Radiochemical methods for determination of polonium, radiolead, uranium and plutonium in environmental samples. Chem. Anal., 42, 107}115. Skwarzec, B., & Falkowski, L. (1988). Accumulation of 210Po in Baltic invertebrates. Journal of Environmental Radioactivity, 8, 99}109. Spector, T. (1978). Re"nement of the coomasie blue method of protein quantitation. Analytical Biochemistry, 86, 142}146. Takizawa, Y., Zhao, L., Yamamoto, M., Abe, T., & Ueno, K. (1990). Determination of 210Pb and 210Po in human tissues of Japanese. Journal of Radioanalytical Nuclear Chemistry, 138, 145}152.