Cultured material was extracted from the inner cavity of the DC and individual colonies were picked up for the preparation of the cytospin slides and Pappenheim staining for identification of the cell types. Obtained data indicated that BM of the animals of the 1st group was affected by the IR. Cell aggregate numbers were on average 24 colonies and 48 clusters in the cultures of the 1st day after exposure. Almost no colony-forming activity was observed in the culture of 7th and 30th day after exposure, that was indicative of significant suppression of the bone marrow function by the IR. At the same time in the culture of bone marrow cells that were obtained from the animals, which received MGC prior to exposure, the average colony count was 32 with 86 clusters at the 1st day after exposure, 45 colonies and 112 clusters on the 7th day after exposure, and 80 colonies and 136 clusters on the 30th day after exposure.

These results may indicate that MGC is able to protect the population of the bone marrow stem cell population for the influence on the IR and stimulate recovery after irradiation at

Fig. 33. Granulocyte-macrophage colony of mouse bone marrow culture of Balb/c,

Fig. 34. Granulocyte cluster of mouse bone marrow culture of Balb/c, irradiated with a dose

Cultured material was extracted from the inner cavity of the DC and individual colonies were picked up for the preparation of the cytospin slides and Pappenheim staining for identification of the cell types. Obtained data indicated that BM of the animals of the 1st group was affected by the IR. Cell aggregate numbers were on average 24 colonies and 48 clusters in the cultures of the 1st day after exposure. Almost no colony-forming activity was observed in the culture of 7th and 30th day after exposure, that was indicative of significant suppression of the bone marrow function by the IR. At the same time in the culture of bone marrow cells that were obtained from the animals, which received MGC prior to exposure, the average colony count was 32 with 86 clusters at the 1st day after exposure, 45 colonies and 112 clusters on the 7th day

These results may indicate that MGC is able to protect the population of the bone marrow stem cell population for the influence on the IR and stimulate recovery after irradiation at

after exposure, and 80 colonies and 136 clusters on the 30th day after exposure.

irradiated with a dose of 5.0 Sv. Inverted microscope, increase 200.

of 5.0 Sv. Inverted microscope, increase 400.

dose comparable to the LD50/30. This conclusion can also be supported by increase of the quantity of the CFU in comparison to the 1st group of animals that have not received MGC. Implantation of the normal BM into the organism of the irradiated recipients at the 1st day after exposure after culturing resulted in formation of 114 colonies and 386 clusters on average. In the cultures of the 7th and 30th days such proliferation activity was observed, that it was not possible to determine individual colonies or clusters. There is indicative of a significant stimulation of the release of the compensatory signal substances by the radioresistant stromal cells of the BM, which highly stimulated the recovery of the radiationdamaged BM of the recipient. Normal bone marrow cells implanted into the irradiated mice treated with MGC yielded in 84 colonies and 192 clusters in the cultures of the 1st day after exposure, 52 colonies and 548 clusters on the 7th day, and 106 colonies and 302 clusters in the 30th day post exposure cultures (Figure 35).

Fig. 35. Determination of the CFU activity of the bone marrow derived stem cells: Group 1A irradiated bone marrow culture in normal recipients; Group 2A - irradiated BM protected with MGC in normal recipients; Group 1C - normal bone marrow culture in irradiated recipients; Group 2C - normal bone marrow culture in irradiated recipients treated with MGC.

Such decrease in the CFU activity is indicative of less apparent stimulation of the compensatory factors release as a result of the radiation exposure due to protective effects of the MGC on the bone marrow cells so that less factors are required for reparation and therefore less factors are available for stimulation of the CFU activity in the DC.

Results of the *in vivo* culture of the bone marrow cells indicated that colony-forming activity of the hematopoietic progenitor cells of the animals non-treated with MGC prior to radiation exposure was significantly lower if compared to the control while treatment of the animals with MGC had increased the functional activity of the bone marrow cells.

Therefore upon irradiation exposure quantity of the hematopoietic stem cells is decreased and as a result, stromal component of the BM starts to secrete large quantities of cytokines

Radiobiological Characterization Environment Around Object "Shelter" 261

state, an other part – as "hot" particles, the interrelation between there forms being unstable and change under the influence of biotic and abiotic environmental factors. As rule in this conditions accumulation of radionuclides in plants which occurs mainly at the expense of their water-soluble and exchangeable forms, reflects rather complicated transitional processes in the soil, the rate and direction of these ones is determined by biological activity of all component of the plant rhizosphere inhabited layer of the soil. After Chernobyl accident already during 25 year a lot of "hot" particles transferred into fine dispersive conditions, which easy movements in outdoors where captured by biota which could characterize by help of transfer coefficient (TC) radionuclide ongoing. The transfer coefficient is ratio specific activities (kBq/kg) of plant to specific activity of soil (kBq/kg) where its grow that characterize go over a radionuclide from soil to vegetative plant on experimental plot. Necessary mentioned that the TC not constant and it differed on depend of parts of plant were determinate. For radionuclide 241Аm observed the value TC a lot of plants and mushrooms several order less than for isotopes 137Cs, 90Sr. Especially for matured seed the value of the TC observed less than for other vegetative parts of the plant [Rashydov N., Berezhna V., Kutsokon N. 2007, Rashydov N.M., Kutsokon N.K. 2008, Rashydov N.M., Berezhna V.V., Grodzinsky D.M. 2009, Rashydov N., Berezhna V. 2010, Rashydov N.M. 2010, Rashydov N.M. 2011]. To study of the TC peculiarity modification is reason elucidation of our field research in alienation

Contamination of plant in natural experimental fields at the alienation zone of Chernobyl significant added by flying dust with very small size radioactivity particles less than "hot particles" in environment. The results received for plants soybean (content: 137Cs - 3.6 kBq/kg and 90Sr – 11.84 kBq/kg) and flax (content: 137Cs - 0.78 kBq/kg and 90Sr – 3.55 kBq/kg) which grown in Chistogalovka (specific activity of soil is 20.65 kBq/kg and 5.18 kBq/kg for radionuclide 137Cs and 90Sr, accordingly) and Chernobyl confirmed this hypothesis. The value TC for above mentioned seeds specimens collected from plant which grow on Chistogalovka was approximately 22.3 (soybean) and 6.63 (flax) times (for isotope 137Cs) and 13.97 and 4.71 (for isotope 90Sr) times higher by comparison with control variants which grown in Chernobyl where specific activity was 1.41 kBq/kg for radionuclide 137Cs

The peculiarity distribution in controlled laboratory conditions the radionuclide 241Аm in *Arabidopsis thaliana* plant on high level first layer leaves, in petiole and in carry out fascicles of the leaves significantly that go into this isotope from root system to top of plant very slow and membrane of cells played as discrimination barrier in this processes as mentioned in our previously investigations [Rashydov N.M., Berezhna V.V., Grodzinsky D.M. 2009].

In laboratory conditions for autoradiography investigation purpose the seedlings *Arabidopsis thaliana* were aseptically grown in hard agar cultured medium containing 241AmCl3 in concentration with specific activity 50 kBq/kg. After 25 days some leaves and top of stems of plants witch had not direct contact with medium were carefully cut off so that to avoid contact with medium. Selected parts of plants *Arabidopsis thaliana* settled down on the microscopic glass slides and dried a few days. During this process they were gluing to the slides themselves. The slides with parts of plants were coated with photo emulsion LM-1 in gel (Amersham – Biosciences UK) and exposure during time 20 days at temperature +40 C. After development the samples of slides were observed of the track of α-particles from radionuclide 241Аm with light microscope. A lot of datum confirmed that the coefficient

Chernobyl zone around object "Shelter".

and 0.55 kBq/kg for isotope 90Sr, correspondingly.

and growth factors, which are able to stimulate HSC to active proliferation [Goldberg E.D., Dygai A.M. 2001]. Clearly, in animals irradiated at doses of 5.0 Sv, production of growth factors was blocked by excessive amounts of free radicals which are formed when passing through biological tissue of ionizing particles [Timofeev-Resovskii N.V. 1963]. At the same time, free radicals can quickly neutralized by the MGC in mice receiving radioprotector. Improved products of growth factors caused the hyperproduction of bone marrow stem cells. On the 7th day after exposure in the bone marrow culture of the irradiated animals, a deep depression in the CFU activity was observed. Finally pretreatment of animals with MGC resulted in the significant increase of the CFU activity. These data indicates that in the BM of irradiated animals the pool of later mononuclear cells was rapidly exhausted that resulted in the decrease of the CFU activity [Bilko N.M., Klimenko S.V., Velichko E.A. 1999]. In the group of animals pretreated with MGC prior to exposure, CFU activity in the BM increased during the experiment. On the 30th day post exposure, CFU activity of the bone marrow stem cells remained low and in the animals that have not received MGC, and in the animals treated with this radioprotector these values were significantly higher.

Obtained data may be indicative of the gradual recovery of the BM by implementation of the so called "golden reserve" of the stem cells that during radiation exposure were positioned in the crypts of the stroma and therefore remained undamaged [Tsyb A.F., Budagov R.S., Zamulaeva A.I. 2005]. Such significant effects of the MGC on the numbers of colonies and clusters in the DC cultures indicates decrease quantity of the stimulating bystander signals and strong radioprotective properties of the MGC, however the exact mechanism of action remains to be fully elucidated.

Concluding on the obtained experimental data it is clear that MGC is a strong radiation protector that helps to avoid consequences of the LD50/30 dose of irradiation at the level of HSC by affecting quantity of available growth stimulating factors that are commonly associated with radiation-induced damage to the BM.

#### **2.4 Influence radionuclide fallout to plant grown around object "Shelter" in Chernobyl alienation zone**

Contaminated of the wide territories in Ukraine not only with radionucludes 137Cs and 90Sr, and with fission products of uranium and transuranium elements is an essential consequence of the accident at the IV block of Chernobyl Nuclear Power Plant that is classified as a global ecological catastrophe. The biota behaviors and adapt in this areas captured dose from radionuclide with long half-value period decay isotopes. As dose related amount of the isotopes 137Cs and 90Sr during long time after accident were decreased. But only the amount of the radioactive isotope 241Am depend of time is increasing exactly in environmental alienation zone of Chernobyl. Radionuclide 241Am as -emitter is a daughter product of 241Pu isotope appeared after β-decay. The activity in environment of the isotope 241Am is increasing with during time owing to β-decay of the 241Pu isotope. The biota behavior in this areas captured dose from radionuclide with long half-value period decay isotopes. The peculiarity of radionuclides contamination associated with the Chernobyl accident is verified of physical and chemical forms of radioactivity elements through out into the environment [Rashydov N.M. 1999, Rashydov N.M., Konoplyova A.A., Grodzinsky D.M. 2004, Rashydov N.M., Kutsokon N.K. 2005, Rashydov N.M., Grodzinsky D., Berezhna V. 2006]. A part of the radioactivity isotopes is registered in water soluble droplets-liquid

and growth factors, which are able to stimulate HSC to active proliferation [Goldberg E.D., Dygai A.M. 2001]. Clearly, in animals irradiated at doses of 5.0 Sv, production of growth factors was blocked by excessive amounts of free radicals which are formed when passing through biological tissue of ionizing particles [Timofeev-Resovskii N.V. 1963]. At the same time, free radicals can quickly neutralized by the MGC in mice receiving radioprotector. Improved products of growth factors caused the hyperproduction of bone marrow stem cells. On the 7th day after exposure in the bone marrow culture of the irradiated animals, a deep depression in the CFU activity was observed. Finally pretreatment of animals with MGC resulted in the significant increase of the CFU activity. These data indicates that in the BM of irradiated animals the pool of later mononuclear cells was rapidly exhausted that resulted in the decrease of the CFU activity [Bilko N.M., Klimenko S.V., Velichko E.A. 1999]. In the group of animals pretreated with MGC prior to exposure, CFU activity in the BM increased during the experiment. On the 30th day post exposure, CFU activity of the bone marrow stem cells remained low and in the animals that have not received MGC, and in the

animals treated with this radioprotector these values were significantly higher.

mechanism of action remains to be fully elucidated.

associated with radiation-induced damage to the BM.

**alienation zone**

Obtained data may be indicative of the gradual recovery of the BM by implementation of the so called "golden reserve" of the stem cells that during radiation exposure were positioned in the crypts of the stroma and therefore remained undamaged [Tsyb A.F., Budagov R.S., Zamulaeva A.I. 2005]. Such significant effects of the MGC on the numbers of colonies and clusters in the DC cultures indicates decrease quantity of the stimulating bystander signals and strong radioprotective properties of the MGC, however the exact

Concluding on the obtained experimental data it is clear that MGC is a strong radiation protector that helps to avoid consequences of the LD50/30 dose of irradiation at the level of HSC by affecting quantity of available growth stimulating factors that are commonly

**2.4 Influence radionuclide fallout to plant grown around object "Shelter" in Chernobyl**

Contaminated of the wide territories in Ukraine not only with radionucludes 137Cs and 90Sr, and with fission products of uranium and transuranium elements is an essential consequence of the accident at the IV block of Chernobyl Nuclear Power Plant that is classified as a global ecological catastrophe. The biota behaviors and adapt in this areas captured dose from radionuclide with long half-value period decay isotopes. As dose related amount of the isotopes 137Cs and 90Sr during long time after accident were decreased. But only the amount of the radioactive isotope 241Am depend of time is increasing exactly in environmental alienation zone of Chernobyl. Radionuclide 241Am as -emitter is a daughter product of 241Pu isotope appeared after β-decay. The activity in environment of the isotope 241Am is increasing with during time owing to β-decay of the 241Pu isotope. The biota behavior in this areas captured dose from radionuclide with long half-value period decay isotopes. The peculiarity of radionuclides contamination associated with the Chernobyl accident is verified of physical and chemical forms of radioactivity elements through out into the environment [Rashydov N.M. 1999, Rashydov N.M., Konoplyova A.A., Grodzinsky D.M. 2004, Rashydov N.M., Kutsokon N.K. 2005, Rashydov N.M., Grodzinsky D., Berezhna V. 2006]. A part of the radioactivity isotopes is registered in water soluble droplets-liquid state, an other part – as "hot" particles, the interrelation between there forms being unstable and change under the influence of biotic and abiotic environmental factors. As rule in this conditions accumulation of radionuclides in plants which occurs mainly at the expense of their water-soluble and exchangeable forms, reflects rather complicated transitional processes in the soil, the rate and direction of these ones is determined by biological activity of all component of the plant rhizosphere inhabited layer of the soil. After Chernobyl accident already during 25 year a lot of "hot" particles transferred into fine dispersive conditions, which easy movements in outdoors where captured by biota which could characterize by help of transfer coefficient (TC) radionuclide ongoing. The transfer coefficient is ratio specific activities (kBq/kg) of plant to specific activity of soil (kBq/kg) where its grow that characterize go over a radionuclide from soil to vegetative plant on experimental plot. Necessary mentioned that the TC not constant and it differed on depend of parts of plant were determinate. For radionuclide 241Аm observed the value TC a lot of plants and mushrooms several order less than for isotopes 137Cs, 90Sr. Especially for matured seed the value of the TC observed less than for other vegetative parts of the plant [Rashydov N., Berezhna V., Kutsokon N. 2007, Rashydov N.M., Kutsokon N.K. 2008, Rashydov N.M., Berezhna V.V., Grodzinsky D.M. 2009, Rashydov N., Berezhna V. 2010, Rashydov N.M. 2010, Rashydov N.M. 2011]. To study of the TC peculiarity modification is reason elucidation of our field research in alienation Chernobyl zone around object "Shelter".

Contamination of plant in natural experimental fields at the alienation zone of Chernobyl significant added by flying dust with very small size radioactivity particles less than "hot particles" in environment. The results received for plants soybean (content: 137Cs - 3.6 kBq/kg and 90Sr – 11.84 kBq/kg) and flax (content: 137Cs - 0.78 kBq/kg and 90Sr – 3.55 kBq/kg) which grown in Chistogalovka (specific activity of soil is 20.65 kBq/kg and 5.18 kBq/kg for radionuclide 137Cs and 90Sr, accordingly) and Chernobyl confirmed this hypothesis. The value TC for above mentioned seeds specimens collected from plant which grow on Chistogalovka was approximately 22.3 (soybean) and 6.63 (flax) times (for isotope 137Cs) and 13.97 and 4.71 (for isotope 90Sr) times higher by comparison with control variants which grown in Chernobyl where specific activity was 1.41 kBq/kg for radionuclide 137Cs and 0.55 kBq/kg for isotope 90Sr, correspondingly.

The peculiarity distribution in controlled laboratory conditions the radionuclide 241Аm in *Arabidopsis thaliana* plant on high level first layer leaves, in petiole and in carry out fascicles of the leaves significantly that go into this isotope from root system to top of plant very slow and membrane of cells played as discrimination barrier in this processes as mentioned in our previously investigations [Rashydov N.M., Berezhna V.V., Grodzinsky D.M. 2009].

In laboratory conditions for autoradiography investigation purpose the seedlings *Arabidopsis thaliana* were aseptically grown in hard agar cultured medium containing 241AmCl3 in concentration with specific activity 50 kBq/kg. After 25 days some leaves and top of stems of plants witch had not direct contact with medium were carefully cut off so that to avoid contact with medium. Selected parts of plants *Arabidopsis thaliana* settled down on the microscopic glass slides and dried a few days. During this process they were gluing to the slides themselves. The slides with parts of plants were coated with photo emulsion LM-1 in gel (Amersham – Biosciences UK) and exposure during time 20 days at temperature +40 C. After development the samples of slides were observed of the track of α-particles from radionuclide 241Аm with light microscope. A lot of datum confirmed that the coefficient

Radiobiological Characterization Environment Around Object "Shelter" 263

Huner N.P.A., 2009]. The generative phase growth of plant include appear the floral organ with flower and filling seed event is involve important question: Why do plant this sensitivity processes controlled in shot during flowering spring and/or summer under stress factors? The flax plant flowering under chronic irradiation is a complex event that involves genome destabilizations as well as posttranslational regulation, signal transduction and epigenetic regulation metabolism inside living cell [Kutsokon N.K., Rashydov N.M., Grodzinsky D.M. 2007, Kutsokon N., Rashydov N.M. & Grodzinsky D. 2003, Kutsokon N., Rashydov N.M. & Berezhna V. 2004, Kutsokon N., Lazarenko L.M. & Bezrukov V.F. 2004]. For shed light this problem we carry out especially experiment by help of pretreatment by melanin-glucan complex flax seeds which growth under chronic irradiation in Chernobyl zone. As well known the melanin-glucan complex has high gene protective response against of chronic IR in a wide range of doses due to capturing free radical regulation and influencing on epigenetic changes in cells. The curve bloom rate of flax plant depend of during of term observe and treatment by the melanin-glucan

Fig. 37. Flowers per day depend of observed time and approach of treatment flax plants: with melanin-glucan complex plus chronic irradiation (+mel), irradiated by only chronic radiation (), with melanin-glucan complex (mel) and control variant (con) without any treatments.

On base above given curves we were calculated for all variants for the experiment curve parameters that characterized the altitude rate bloom and the time of flower appear necessary for realization of the half of peak flowering. The height of the altitude curve of bloom H (%) and half height of the peak flowering term L (days) of first and second bloom peak of flax plants which grown under different specific chronic irradiation with rate 2.60.3 mcSv/h and 25.40.4 mcSv/h to versus of control variants depend of melanin-glucan

complex treatments shown at tables 1 and 2.

complex under influenced chronic irradiation shown in figure 37.

uptake very small for radionuclide 241Am and this element maldistribution by organs and tissues. We observed that accumulation the radionuclide of 241Am depended of carry out fascicles system of the leaves and localization of the layer leaves not far from length root collar of plant which grow in laboratory conditions. The first layer leaves were taken up high-level amount radionuclide 241Аm. As result the capture dose also may tissues of plant distribute no uniform. It is known that mineral nutrients are transported apoplastically, i.e. in the wall system outside the plasma membrane, or symplastically, i.e. in the cytoplasm from cell to cell deal with through plasmodesmata. The nutrient elements that penetrate into the cytoplast can also be shuttled into the vacuole via various mechanisms depending of biological function in cell life behaviors for mentioned isotope.

For field experiments we use plant white blow (*Erophila verna (L.) Bess.)* for autoradiography investigation from Chistogalovka and Yaniv contaminated soil sites the distribution radionuclide essential differs in spite of above-mentioned experiment. On the top shoot apex leaves and flower observed a lot of tracks of the particles α- and β- decays [Rashydov N.M., Berezhna V.V. 2010] (Figure 36).

Our experimental data confirms that radioactivity fallout in environment essentially differed important amendment of the TC. Thus extra-root nutrition that included micro- or nano- size "hot" particles had essential role of plant behavior in environment. But for plants that harvested from contaminated sites distribution of the radionuclide 241Am by tissue and organs essentially differed from plants which grown in laboratory conditions.

Fig. 36. Appear inside radioactivity of petal of bud (a) and also tracks of α-particles on surface of sepal (b) flower of the plants white blow (*Erophila verna (L.) Bess.*).

Contamination with radionuclide in natural experimental fields significant added tracks elementary particles from flying in air very small dust such as nano- and micro-size with radioactivity similarly "hot" particles in environment by help foliar pathway uptake into top leaves and aboveground apical apex of plants, especially around the object "Shelter".

#### **2.5 Flowering flax plant under chronic irradiation**

Flowering plant and seed development have long held the interest of general biologists because they represent a critical sensitivity changes phases in the pattern of shoot development and have significant consequences in creating yield seed [Hopkins W.G.,

uptake very small for radionuclide 241Am and this element maldistribution by organs and tissues. We observed that accumulation the radionuclide of 241Am depended of carry out fascicles system of the leaves and localization of the layer leaves not far from length root collar of plant which grow in laboratory conditions. The first layer leaves were taken up high-level amount radionuclide 241Аm. As result the capture dose also may tissues of plant distribute no uniform. It is known that mineral nutrients are transported apoplastically, i.e. in the wall system outside the plasma membrane, or symplastically, i.e. in the cytoplasm from cell to cell deal with through plasmodesmata. The nutrient elements that penetrate into the cytoplast can also be shuttled into the vacuole via various mechanisms depending of

For field experiments we use plant white blow (*Erophila verna (L.) Bess.)* for autoradiography investigation from Chistogalovka and Yaniv contaminated soil sites the distribution radionuclide essential differs in spite of above-mentioned experiment. On the top shoot apex leaves and flower observed a lot of tracks of the particles α- and β- decays [Rashydov

Our experimental data confirms that radioactivity fallout in environment essentially differed important amendment of the TC. Thus extra-root nutrition that included micro- or nano- size "hot" particles had essential role of plant behavior in environment. But for plants that harvested from contaminated sites distribution of the radionuclide 241Am by tissue and

(a) (b)

Contamination with radionuclide in natural experimental fields significant added tracks elementary particles from flying in air very small dust such as nano- and micro-size with radioactivity similarly "hot" particles in environment by help foliar pathway uptake into top leaves and aboveground apical apex of plants, especially around the object "Shelter".

Flowering plant and seed development have long held the interest of general biologists because they represent a critical sensitivity changes phases in the pattern of shoot development and have significant consequences in creating yield seed [Hopkins W.G.,

Fig. 36. Appear inside radioactivity of petal of bud (a) and also tracks of α-particles on

surface of sepal (b) flower of the plants white blow (*Erophila verna (L.) Bess.*).

**2.5 Flowering flax plant under chronic irradiation**

organs essentially differed from plants which grown in laboratory conditions.

biological function in cell life behaviors for mentioned isotope.

N.M., Berezhna V.V. 2010] (Figure 36).

Huner N.P.A., 2009]. The generative phase growth of plant include appear the floral organ with flower and filling seed event is involve important question: Why do plant this sensitivity processes controlled in shot during flowering spring and/or summer under stress factors? The flax plant flowering under chronic irradiation is a complex event that involves genome destabilizations as well as posttranslational regulation, signal transduction and epigenetic regulation metabolism inside living cell [Kutsokon N.K., Rashydov N.M., Grodzinsky D.M. 2007, Kutsokon N., Rashydov N.M. & Grodzinsky D. 2003, Kutsokon N., Rashydov N.M. & Berezhna V. 2004, Kutsokon N., Lazarenko L.M. & Bezrukov V.F. 2004]. For shed light this problem we carry out especially experiment by help of pretreatment by melanin-glucan complex flax seeds which growth under chronic irradiation in Chernobyl zone. As well known the melanin-glucan complex has high gene protective response against of chronic IR in a wide range of doses due to capturing free radical regulation and influencing on epigenetic changes in cells. The curve bloom rate of flax plant depend of during of term observe and treatment by the melanin-glucan complex under influenced chronic irradiation shown in figure 37.

Fig. 37. Flowers per day depend of observed time and approach of treatment flax plants: with melanin-glucan complex plus chronic irradiation (+mel), irradiated by only chronic radiation (), with melanin-glucan complex (mel) and control variant (con) without any treatments.

On base above given curves we were calculated for all variants for the experiment curve parameters that characterized the altitude rate bloom and the time of flower appear necessary for realization of the half of peak flowering. The height of the altitude curve of bloom H (%) and half height of the peak flowering term L (days) of first and second bloom peak of flax plants which grown under different specific chronic irradiation with rate 2.60.3 mcSv/h and 25.40.4 mcSv/h to versus of control variants depend of melanin-glucan complex treatments shown at tables 1 and 2.

Radiobiological Characterization Environment Around Object "Shelter" 265

excited appear sterility flowers but for flax was appeared yield in second peak bloom a lot of fertile seeds in generally with germination rate proximally 97%. However the average numbers of seeds per seed box or per infructescence of plant flax grown in several

As known epigenetic events regulate the activities of gene which take part in vegetative and generative phases of growth plant by help of processes methylation of DNA, acetylation, phosphorylation of the histone tails in chromatin fiber. The heritability of DNA methylation, which often occurs in the early and late stages of vegetative development of the ontogenesis plant, allows cells to keep irrelevant genes silenced in successive generations of embryo cell. However some genes – such as plant genes that govern cell dormancy and spring time flowering – require silenced genes to be reactivated. Evidence is beginning to emerge those different classes by help of micro ribonucleic acid (miRNA) and on noncoding RNAs regulate these protein synthesis [Danchenko, M., Škultéty, L. & Berezhna, V.V. 2008, Danchenko M, Skultety L. & Rashydov N.M. 2009, Danchenko M, Klubicova K. & Skultety L. 2011]. Thus the seedlings since germination until flowering, filling seed and matured seed during all ontogenesis term under chronic irradiation perceived some signal transduction events which

accumulate permanently in plants as epigenetic changes transient heritability.

Quality and qualitative changes in the structure of the radiation factor of object "Shelter" occurred over twenty-five years of its existence. Inside the "Shelter" the spontaneous destruction of the fuel-containing materials in IV Unit of the ChNPP which arose out of nuclear fuel in the acute phase of the disaster under the influence of intense ionizing radiation and high temperatures was developing. As a result of a new type of radioactive aerosol particles of nanometer size were appeared as called fuel hot particles. Accumulation of radionuclides with long half-life, in particular, radioactivity isotopes of strontium and plutonium and their decay products in the tissues of plant and animal organisms is especially dangerous. The fuel-containing materials are dissolved under the action of microorganisms and a new compound of radioactivity isotopes with organic matter was appeared. They are potentially more mobile and more ecologically dangerous around and inside "Shelter" environments. The radiation fields with different intensity inside object "Shelter" have contributed to increasing the mutation rate and breeding of radiationresistant microorganisms with new aggressive properties. A lot of number particles of micrometer and nanometer size which consisting the cells of microorganisms and spores in the investigated samples of aerosols originated from "Shelter" premises. It is known that the light roof of sarcophagus has a large number of defects and some of these microorganisms, especially in the form of spores, may potentially carry away outside of the object "Shelter". The hot particles of fuel contain gamma-, beta- and alpha- irradiators, such as radioisotopes of strontium and plutonium and their decay products continue irradiating organism late times even after termination of introduce to them. When IR from similarly radioactivity isotopes interaction with living matter they pass it relatively high energy. It is being spent on the excitation of atoms or ions and changes the chemical properties of matter and

conditions do not differed from control variant significantly.

additional radicals may be appeared in living cells.

**3. Conclusion**

Table 1. The altitude curve of bloom H (%) and half height time L (days) of first and second flowering peak flax plants which grown under chronic irradiation with specific rate of radiation 2.60.3 mcSv/h and control variants depend of melanin-glucan complex treatments

Table 2. The altitude curve of bloom H (%) and half height time L (days) of first and second flowering peak flax plant which grown under chronic irradiation with specific rate of radiation 25.40.4 mcSv/h and control variants depend of melanin-glucan complex treatments

As shown from figure 37 after 10-15 days since started flowering period the percent of flax flower per day increased until maximal magnitude and the curve passed first peak. During 10-12 day bloom probable decrease until zero and keeping in spontaneous level a few days. But after that during 10-12 days probable of bloom increased and on the curve appear second late flowering peak. For treatment melanin-glucan complex reveal more quantity flower release during second bloom period depend of chronic irradiation: yield proximally 3.3% under chronic irradiation with specific dose 2.6 mcSv/h, in case specific dose 25.4 mcSv/h it increased until 5.5%, accordingly. Increasing the second peak inflorescence appear for both curves variant treatment with melanin-glucan complex at dose 2.6 mcSv/h as well as under chronic irradiation specific dose 25.4 mcSv/h deal with involve this subtract in epigenetic changes in genome regulations flowering process during ontogenesis of flax plants. Necessary mentioned that the bloom of second peak for several plant usually excited appear sterility flowers but for flax was appeared yield in second peak bloom a lot of fertile seeds in generally with germination rate proximally 97%. However the average numbers of seeds per seed box or per infructescence of plant flax grown in several conditions do not differed from control variant significantly.

As known epigenetic events regulate the activities of gene which take part in vegetative and generative phases of growth plant by help of processes methylation of DNA, acetylation, phosphorylation of the histone tails in chromatin fiber. The heritability of DNA methylation, which often occurs in the early and late stages of vegetative development of the ontogenesis plant, allows cells to keep irrelevant genes silenced in successive generations of embryo cell. However some genes – such as plant genes that govern cell dormancy and spring time flowering – require silenced genes to be reactivated. Evidence is beginning to emerge those different classes by help of micro ribonucleic acid (miRNA) and on noncoding RNAs regulate these protein synthesis [Danchenko, M., Škultéty, L. & Berezhna, V.V. 2008, Danchenko M, Skultety L. & Rashydov N.M. 2009, Danchenko M, Klubicova K. & Skultety L. 2011]. Thus the seedlings since germination until flowering, filling seed and matured seed during all ontogenesis term under chronic irradiation perceived some signal transduction events which accumulate permanently in plants as epigenetic changes transient heritability.

#### **3. Conclusion**

264 Nuclear Power Plants

**glucan complex** 7.2 <sup>14</sup> 0.91 3.3 <sup>15</sup> 0.96

 **- irradiated** 9.4 12 0.95 2.1 11 0.96

**complex** 9.2 <sup>13</sup> 0.89 1.5 <sup>12</sup> 0.94

**Control** 9.2 12 0.92 1.5 12 0.96

Table 1. The altitude curve of bloom H (%) and half height time L (days) of first and second flowering peak flax plants which grown under chronic irradiation with specific rate of radiation 2.60.3 mcSv/h and control variants depend of melanin-glucan complex treatments

**glucan complex** 6.6 <sup>9</sup> 0.91 5.5 <sup>8</sup> 0.96

 **- irradiated** 8.4 10 0.89 3.0 8 0.96

**complex** 7.2 <sup>12</sup> 0.87 2.4 <sup>7</sup> 0.74

**Control** 7.6 10 0.92 3.7 8 0.94

Table 2. The altitude curve of bloom H (%) and half height time L (days) of first and second flowering peak flax plant which grown under chronic irradiation with specific rate of radiation

As shown from figure 37 after 10-15 days since started flowering period the percent of flax flower per day increased until maximal magnitude and the curve passed first peak. During 10-12 day bloom probable decrease until zero and keeping in spontaneous level a few days. But after that during 10-12 days probable of bloom increased and on the curve appear second late flowering peak. For treatment melanin-glucan complex reveal more quantity flower release during second bloom period depend of chronic irradiation: yield proximally 3.3% under chronic irradiation with specific dose 2.6 mcSv/h, in case specific dose 25.4 mcSv/h it increased until 5.5%, accordingly. Increasing the second peak inflorescence appear for both curves variant treatment with melanin-glucan complex at dose 2.6 mcSv/h as well as under chronic irradiation specific dose 25.4 mcSv/h deal with involve this subtract in epigenetic changes in genome regulations flowering process during ontogenesis of flax plants. Necessary mentioned that the bloom of second peak for several plant usually

25.40.4 mcSv/h and control variants depend of melanin-glucan complex treatments

**First flowering peak Second flowering peak**

**First flowering peak Second flowering peak**

**H, % L, days R2 H, % L, days R2**

**H, % L, days R2 H, % L, days R2**

**Variants**

 **+ melanin-**

**melanin-glucan**

**Variants**

 **+ melanin-**

**melanin-glucan**

Quality and qualitative changes in the structure of the radiation factor of object "Shelter" occurred over twenty-five years of its existence. Inside the "Shelter" the spontaneous destruction of the fuel-containing materials in IV Unit of the ChNPP which arose out of nuclear fuel in the acute phase of the disaster under the influence of intense ionizing radiation and high temperatures was developing. As a result of a new type of radioactive aerosol particles of nanometer size were appeared as called fuel hot particles. Accumulation of radionuclides with long half-life, in particular, radioactivity isotopes of strontium and plutonium and their decay products in the tissues of plant and animal organisms is especially dangerous. The fuel-containing materials are dissolved under the action of microorganisms and a new compound of radioactivity isotopes with organic matter was appeared. They are potentially more mobile and more ecologically dangerous around and inside "Shelter" environments. The radiation fields with different intensity inside object "Shelter" have contributed to increasing the mutation rate and breeding of radiationresistant microorganisms with new aggressive properties. A lot of number particles of micrometer and nanometer size which consisting the cells of microorganisms and spores in the investigated samples of aerosols originated from "Shelter" premises. It is known that the light roof of sarcophagus has a large number of defects and some of these microorganisms, especially in the form of spores, may potentially carry away outside of the object "Shelter".

The hot particles of fuel contain gamma-, beta- and alpha- irradiators, such as radioisotopes of strontium and plutonium and their decay products continue irradiating organism late times even after termination of introduce to them. When IR from similarly radioactivity isotopes interaction with living matter they pass it relatively high energy. It is being spent on the excitation of atoms or ions and changes the chemical properties of matter and additional radicals may be appeared in living cells.

Radiobiological Characterization Environment Around Object "Shelter" 267

The main outcome of the 25-year study of morbidity in different categories of exposed persons in connection with the Chernobyl catastrophe is a significant increase in primary morbidity is not associated with tumor pathology. An additional mechanism of radiobiological effects in mammals, which can have a direct influence on the biomedical effects of radiation, was described. This is a phenomenon of production by irradiated cells of signals that cause lesions similar to radiation damage in no irradiated neighbor cells as "bystander effect" (BSE) was named. We used index of SSB DNA as indicator to assess the effects of ionizing radiation. It was shown that the living medium obtained after three hours stay of IrCs on the first day after exposure at dose 5.0 Sv for 16 hour is able to induce an additional level of SSB DNA in different types of cells from not-irradiated mice Balb/c - in lymphocytes, hepatocytes, bone marrow hematopoietic cells, splenocytes and astroglial cells. Identification BSE in liver tissue is an additional argument in favor of accepting the

At the same time it was shown that the transmission "bystander" signals within the irradiated BALB/c mice were observed for at least one month after exposure. We also found a correlation between high rate of LD50/30 and low induction SSB DNA in non-irradiated cells after placing them in the living environment of cells from irradiated animals. In mice with lower level of LD50/30 (Balb/c) induction of SSB in cellular DNA non-irradiated lymphocytes after exposure in living environment of the irradiated cells in various periods after of irradiation was higher in comparison with similar index of C57Bl/6 mice with higher level of LD50/30. We observed BSF in mice of two lines with different modes and doses: the external influence of gamma-ray dose, 5.0 Sv (for 16 hours), and external chronic exposure for 231 days with a cumulative dose of 0.29 Sv, with internal radiation for 74 days

The reactive oxygen species (ROS) play an important role in the mechanisms of signal transmission to "bystander" cells. Reinforced ROS production in non-irradiated cells incubated in medium from serum, irradiated gamma-particles, or from supernatant of the suspension of irradiated cells was shown by many authors. Irradiated cells produce a spectrum of "bystander" signals - cytokines, fragments of DNA (from apoptotic cells) or other factors of protein nature. These factors cause a change in oxidative metabolism and gene expression profiles in irradiated cells, and induce enhanced production of highly reactive oxygen species. In addition to chemical modification of DNA nucleotides, the formation of radicals can also lead to changes in the higher levels of organization structure

Therefore the melanin-glucan complex (MGC) from higher mushroom basidiomycetes - *Fomes fomentarius*, was used as an antioxidant to confirm the involvement of molecules of free-radical nature to the transmission of signals from irradiated cells to non-irradiated cells after external influence of gamma-rays (the model of the single and chronic exposure of mice). Antioxidants, photo-and radio-protective properties are a direct consequence of the free-radical structure of melanin providing the opportunity to participate in electronic exchange of redox in cell and radical recombination processes around DNA damages. We have shown that intra peritoneal injection of MGC, which has powerful antioxidant properties, to mice prior to irradiation at a dose procedure 5.0 Sv decreases from BSE in all kinds of testing cells and increase the number of colony forming units in bone marrow

reality of the existence of diseases of hepato-biliary system of radiation origin.

with the accumulation of activity in the mouse body about 18 kBq.

of the molecule - the secondary, tertiary and quaternary.

As rule the linear non-threshold concept suggests that DNA damage induced by low doses of radiation do not contribute significantly to increased risk of disease because a significant amount of endogenous genome damage occurs and is restored in cells constantly. The main difference between DNA damage induced by IR, from the endogenous damaged - it's clustering and the complexity of their chemical nature. The proportion of complex, critical for the fate of the cell damage is much higher when exposed to chronic IR. Especially the massive clustering of DNA damage occurs when the ionization tracks pass along chromatin fiber. At the same time it is known that the probability of occurrence of endogenous clustered DNA damage is extremely low. Accumulation a lot of endogenous damages does not occur because in the cells are constantly functioning mechanisms of reparation, specifically targeted at removing various types damages. Low doses of IR can not activate the G2/M chekpoynt-arrest and DNA repair are not activated when the number of DSB DNA damage and crosslinks between the fibers under 10 - 20 per cell.

There are four outcomes for the cell fate, if the cellular radiation damage is not adequately was repaired. The cell may die or will delay it or keep playing with the viability of new qualities or mutations as the basis for the development of remote descendants.

The energy dependence of the mechanisms of apoptosis is disabled after irradiation, and the cell dies with loss of cell membrane integrity and its release of macromolecular components, such as intracellular enzymes etc., into the intercellular space. These substances cause an immune response in the form of inflammation as leukocyte infiltration of the affected tissue, interstitial fluid accumulation and subsequent induction of specific immune responses (specifically sensible T-lymphocyte and autoantibody) to the unmasked and recognized by lymphocytes of intracellular components.

We detected a significant increase in the rate of ALT levels in chronically irradiated by low doses mice Balb/c compared with non-irradiated. Increased serum levels of this enzyme are considered a sign of inflammation in the liver because a large amount of ALT is released from the damaged cells of this body. Positive changes of serum levels of AuAB to liverspecific protein (LSP) that is a mixture of antigenic determinants of the substrate from the membranes of hepatocytes are also determined in the process of dose accumulation at irradiated animals. Because of their labiality, some proteins that are part of the LSP in particular the asialoglycoprotein receptor under certain conditions including under the influence of small doses of radiation may acquire properties of auto antigens.

Thus chronic exposure to low doses of ionizing radiation of low intensity may lead to the abolition of immune tolerance, not only in the liver but also in other organs. As is known in turn increased the activity of autoimmunity is a favorable condition for the transfer of persistent infections in the active state and for stimulation of vegetation as saprophyte and pathogenic microorganisms.

It is known that the IR increases the level of signal transduction in the infected cell and can activate the promoter of certain genes of the virus and subsequent synthesis of the corresponding proteins. As a result of these events, the cell does not die, as is the case with an active infection, and changes its functional properties. In this changing antigenic repertoire of cell membranes was described. Antigens become available for immune recognition. As a result by an autoimmune reaction and changes the pathogenesis of a wide range of infectious diseases to cancer stimulated.

As rule the linear non-threshold concept suggests that DNA damage induced by low doses of radiation do not contribute significantly to increased risk of disease because a significant amount of endogenous genome damage occurs and is restored in cells constantly. The main difference between DNA damage induced by IR, from the endogenous damaged - it's clustering and the complexity of their chemical nature. The proportion of complex, critical for the fate of the cell damage is much higher when exposed to chronic IR. Especially the massive clustering of DNA damage occurs when the ionization tracks pass along chromatin fiber. At the same time it is known that the probability of occurrence of endogenous clustered DNA damage is extremely low. Accumulation a lot of endogenous damages does not occur because in the cells are constantly functioning mechanisms of reparation, specifically targeted at removing various types damages. Low doses of IR can not activate the G2/M chekpoynt-arrest and DNA repair are not activated when the number of DSB

There are four outcomes for the cell fate, if the cellular radiation damage is not adequately was repaired. The cell may die or will delay it or keep playing with the viability of new

The energy dependence of the mechanisms of apoptosis is disabled after irradiation, and the cell dies with loss of cell membrane integrity and its release of macromolecular components, such as intracellular enzymes etc., into the intercellular space. These substances cause an immune response in the form of inflammation as leukocyte infiltration of the affected tissue, interstitial fluid accumulation and subsequent induction of specific immune responses (specifically sensible T-lymphocyte and autoantibody) to the unmasked and recognized by

We detected a significant increase in the rate of ALT levels in chronically irradiated by low doses mice Balb/c compared with non-irradiated. Increased serum levels of this enzyme are considered a sign of inflammation in the liver because a large amount of ALT is released from the damaged cells of this body. Positive changes of serum levels of AuAB to liverspecific protein (LSP) that is a mixture of antigenic determinants of the substrate from the membranes of hepatocytes are also determined in the process of dose accumulation at irradiated animals. Because of their labiality, some proteins that are part of the LSP in particular the asialoglycoprotein receptor under certain conditions including under the

Thus chronic exposure to low doses of ionizing radiation of low intensity may lead to the abolition of immune tolerance, not only in the liver but also in other organs. As is known in turn increased the activity of autoimmunity is a favorable condition for the transfer of persistent infections in the active state and for stimulation of vegetation as saprophyte and

It is known that the IR increases the level of signal transduction in the infected cell and can activate the promoter of certain genes of the virus and subsequent synthesis of the corresponding proteins. As a result of these events, the cell does not die, as is the case with an active infection, and changes its functional properties. In this changing antigenic repertoire of cell membranes was described. Antigens become available for immune recognition. As a result by an autoimmune reaction and changes the pathogenesis of a wide

DNA damage and crosslinks between the fibers under 10 - 20 per cell.

lymphocytes of intracellular components.

pathogenic microorganisms.

range of infectious diseases to cancer stimulated.

qualities or mutations as the basis for the development of remote descendants.

influence of small doses of radiation may acquire properties of auto antigens.

The main outcome of the 25-year study of morbidity in different categories of exposed persons in connection with the Chernobyl catastrophe is a significant increase in primary morbidity is not associated with tumor pathology. An additional mechanism of radiobiological effects in mammals, which can have a direct influence on the biomedical effects of radiation, was described. This is a phenomenon of production by irradiated cells of signals that cause lesions similar to radiation damage in no irradiated neighbor cells as "bystander effect" (BSE) was named. We used index of SSB DNA as indicator to assess the effects of ionizing radiation. It was shown that the living medium obtained after three hours stay of IrCs on the first day after exposure at dose 5.0 Sv for 16 hour is able to induce an additional level of SSB DNA in different types of cells from not-irradiated mice Balb/c - in lymphocytes, hepatocytes, bone marrow hematopoietic cells, splenocytes and astroglial cells. Identification BSE in liver tissue is an additional argument in favor of accepting the reality of the existence of diseases of hepato-biliary system of radiation origin.

At the same time it was shown that the transmission "bystander" signals within the irradiated BALB/c mice were observed for at least one month after exposure. We also found a correlation between high rate of LD50/30 and low induction SSB DNA in non-irradiated cells after placing them in the living environment of cells from irradiated animals. In mice with lower level of LD50/30 (Balb/c) induction of SSB in cellular DNA non-irradiated lymphocytes after exposure in living environment of the irradiated cells in various periods after of irradiation was higher in comparison with similar index of C57Bl/6 mice with higher level of LD50/30. We observed BSF in mice of two lines with different modes and doses: the external influence of gamma-ray dose, 5.0 Sv (for 16 hours), and external chronic exposure for 231 days with a cumulative dose of 0.29 Sv, with internal radiation for 74 days with the accumulation of activity in the mouse body about 18 kBq.

The reactive oxygen species (ROS) play an important role in the mechanisms of signal transmission to "bystander" cells. Reinforced ROS production in non-irradiated cells incubated in medium from serum, irradiated gamma-particles, or from supernatant of the suspension of irradiated cells was shown by many authors. Irradiated cells produce a spectrum of "bystander" signals - cytokines, fragments of DNA (from apoptotic cells) or other factors of protein nature. These factors cause a change in oxidative metabolism and gene expression profiles in irradiated cells, and induce enhanced production of highly reactive oxygen species. In addition to chemical modification of DNA nucleotides, the formation of radicals can also lead to changes in the higher levels of organization structure of the molecule - the secondary, tertiary and quaternary.

Therefore the melanin-glucan complex (MGC) from higher mushroom basidiomycetes - *Fomes fomentarius*, was used as an antioxidant to confirm the involvement of molecules of free-radical nature to the transmission of signals from irradiated cells to non-irradiated cells after external influence of gamma-rays (the model of the single and chronic exposure of mice). Antioxidants, photo-and radio-protective properties are a direct consequence of the free-radical structure of melanin providing the opportunity to participate in electronic exchange of redox in cell and radical recombination processes around DNA damages. We have shown that intra peritoneal injection of MGC, which has powerful antioxidant properties, to mice prior to irradiation at a dose procedure 5.0 Sv decreases from BSE in all kinds of testing cells and increase the number of colony forming units in bone marrow

Radiobiological Characterization Environment Around Object "Shelter" 269

phosphorylation of the histone tails in chromatin fiber of chromosome. However some genes – such as plant genes that govern cell dormancy and spring time flowering – require silenced genes to be reactivated. The heritability of DNA methylation, which often occurs in the early and late stages of vegetative development of the ontogenesis plant, allows cells to keep irrelevant genes silenced in successive generations of embryo cell. Evidence is beginning to emerge those different classes by help of micro ribonucleic acid (miRNA) and

Our experiments carry out with chronic irradiation revealed that in mammalian and plants cells excided not only direct damage DNA or appear mutation event as well as were observed complex responsible processes including genome instability, signal transduction, bystander effect between sells and epigenetic changes several profiles protein synthesis depend of activity or silent some genes which adjusted by help of

The authors thanks to deputy director of the Nuclear Research Institute of National Academy of Sciences of Ukraine Dr. Tryshyn V.V. help us with maintenance radioactivity

Chernobyl, object "Shelter", radioactive isotopes, "hot" particles with micro- and nano-sizes, radioactivity aerosol, embryogenesis, signal system, autoradiography, single strand break (SSB) DNA, chronic irradiation, bystander effect, autoimmunity, alanine aminotransferase (ALT), liver-specific lipoprotein (LSP), radiation hepatitis, bone marrow, hematopoietic stem

Anderson S, Bankier A.T., Barrell B.G., de Bruijn M.H., Coulson A.R., Drouin J, Eperon I.C.,

Ashmarin I.P., Freidlin V.P*.* History and the practical aspects of a new understanding of

Azzam E.I., Little J.B. The Radiation-induced Bystander effect: Evidence and Significance.

Azzam, E.I., de Toldeo, S.M., Little, J.B. Stress signaling from irradiated to non-irradiated

Ballot E., Homberg J. C., Johanet C. Antibodies to soluble liver antigen: an additional

cells// Current Cancer Drug Targets. 2004. Vol.4 N 1. P. 53-64.

Nierlich D.P., Roe B.A., Sanger F, Schreier P.H., Smith A.J., Staden R, Young I.G. Sequence and organization of the human mitochondrial genome// Nature. 1981

the role of autoimmunity. Abstracts 1st Moscow International Conference "Natural autoimmunity in health and disease. " Moscow. 15 - 17 September, 2005,

In: Bystander effects and the dose response. Belle News-letter/ Biological Effects of

marker in type 1 auto-immune hepatitis // J. Hepatology, – 2000. – 33, – P. 208 –

measure of several contaminated samples by radionuclides from Chernobyl zone.

cells, medical consequence, melanin-glucan complex, flowering of flax, epigenetic.

on noncoding RNAs regulate these protein synthesis.

treatment melanin-glucan complex.

Apr 9; 290(5806): p.457-65.

Low level Exposure. 2003. V.11.N 3 P .2 – 6.

**4. Acknowledgment**

**5. Keywords**

**6. References**

245p.

215.

of irradiated mice. These results may serve as an argument in favor of the hypothesis about the important role of free radicals, molecules in the implementation of the phenomenon of BSE. Concluding on the obtained experimental data it is clear that melanin-glucan complex is a strong radiation protector that helps to avoid consequences of the LD50/30 dose of irradiation at the level of HSC by affecting quantity of available growth stimulating factors that are commonly associated with radiation-induced damage to the bone marrow.

In the experiments on vegetative plant test-system evidence that under influence chronic irradiation were formed radiobiological reactions living organisms which include several level functions and structure organization processes since metabolic pathways and cellular systems until genetically and population changes. We observed that accumulation the radionuclide of 241Am depended of carry out fascicles system of the leaves and localization of the layer leaves not far from length root collar of plant which grow in laboratory conditions. The first layer leaves were taken up high-level amount radionuclide 241Аm. The peculiarity distribution radionuclide 241Аm in Arabidopsis thaliana plant on high level layer leaves, in petiole and in carry out fascicles of the leaves significant that go into this radioactivity isotope from root system to top of plant very slow and membrane of cells played as discrimination barrier in this processes as mentioned in our previously investigations. It is known that mineral nutrients are transported apoplastically, i.e. in the wall system outside the plasma membrane, or symplastically, i.e. in the cell wall system outside the plasma membrane, or symplastically, i.e. in the cytoplasm from cell to cell deal with through plasmodesmata. Elements that penetrate into the cytoplast can also be shuttled into the vacuole via various mechanisms depending of element. But for plants that harvested from contaminated sites distribution of the radionuclide 241Am by tissue and organs essentially differed from plants which grown in laboratory conditions. When we observed plant white blow (*Erophila verna (L.) Bess*.) for autoradiography investigation from Chistogalovka and Yaniv contaminated soil sites the distribution tracks of radionuclide essential differ in compare of above mentioned experiment. Contamination with radionuclide in natural experimental fields significant added tracks elementary particles from flying in air very small dust such as nano- and micro-size with radioactivity less than "hot" particles in environment by help foliar pathway into top leaves and apex of plant. Existence of nano- and micro- size dust as radioactive aerosols with specific properties around object "Shelter " circulate an environment represents potential risk radiobiological consequences of plant that grow in alienation Chernobyl 30-km zone.

The chronic irradiation around object "Shelter" was including instability genome, which carried out genetic and somatic changers in late heritable on perennials as well as in several generation annual plants. Proteomics analysis observes; bloom, filling and maturing seeds evidence that there is induce epigenetic changes of genome that excited late genetic changers on progenies and somatic changes of flax plant. Treatment by the melanin-glucan complex from fungus *F. fomentarius* before sowing seed procedure increase second peak on flowering curve depend of ontogenesis flax under influence chronic irradiation.

It is known that under chronic irradiation the epigenetic events regulate the activities or inactivate of genes by help of several mechanisms which may be include miPNA inter action deal with process that take part in vegetative and generative phases (bloom, filling and maturing seed) of growth plant by help of processes methylation of DNA, acetylation, phosphorylation of the histone tails in chromatin fiber of chromosome. However some genes – such as plant genes that govern cell dormancy and spring time flowering – require silenced genes to be reactivated. The heritability of DNA methylation, which often occurs in the early and late stages of vegetative development of the ontogenesis plant, allows cells to keep irrelevant genes silenced in successive generations of embryo cell. Evidence is beginning to emerge those different classes by help of micro ribonucleic acid (miRNA) and on noncoding RNAs regulate these protein synthesis.

Our experiments carry out with chronic irradiation revealed that in mammalian and plants cells excided not only direct damage DNA or appear mutation event as well as were observed complex responsible processes including genome instability, signal transduction, bystander effect between sells and epigenetic changes several profiles protein synthesis depend of activity or silent some genes which adjusted by help of treatment melanin-glucan complex.

#### **4. Acknowledgment**

The authors thanks to deputy director of the Nuclear Research Institute of National Academy of Sciences of Ukraine Dr. Tryshyn V.V. help us with maintenance radioactivity measure of several contaminated samples by radionuclides from Chernobyl zone.

#### **5. Keywords**

268 Nuclear Power Plants

of irradiated mice. These results may serve as an argument in favor of the hypothesis about the important role of free radicals, molecules in the implementation of the phenomenon of BSE. Concluding on the obtained experimental data it is clear that melanin-glucan complex is a strong radiation protector that helps to avoid consequences of the LD50/30 dose of irradiation at the level of HSC by affecting quantity of available growth stimulating factors that are commonly associated with radiation-induced damage

In the experiments on vegetative plant test-system evidence that under influence chronic irradiation were formed radiobiological reactions living organisms which include several level functions and structure organization processes since metabolic pathways and cellular systems until genetically and population changes. We observed that accumulation the radionuclide of 241Am depended of carry out fascicles system of the leaves and localization of the layer leaves not far from length root collar of plant which grow in laboratory conditions. The first layer leaves were taken up high-level amount radionuclide 241Аm. The peculiarity distribution radionuclide 241Аm in Arabidopsis thaliana plant on high level layer leaves, in petiole and in carry out fascicles of the leaves significant that go into this radioactivity isotope from root system to top of plant very slow and membrane of cells played as discrimination barrier in this processes as mentioned in our previously investigations. It is known that mineral nutrients are transported apoplastically, i.e. in the wall system outside the plasma membrane, or symplastically, i.e. in the cell wall system outside the plasma membrane, or symplastically, i.e. in the cytoplasm from cell to cell deal with through plasmodesmata. Elements that penetrate into the cytoplast can also be shuttled into the vacuole via various mechanisms depending of element. But for plants that harvested from contaminated sites distribution of the radionuclide 241Am by tissue and organs essentially differed from plants which grown in laboratory conditions. When we observed plant white blow (*Erophila verna (L.) Bess*.) for autoradiography investigation from Chistogalovka and Yaniv contaminated soil sites the distribution tracks of radionuclide essential differ in compare of above mentioned experiment. Contamination with radionuclide in natural experimental fields significant added tracks elementary particles from flying in air very small dust such as nano- and micro-size with radioactivity less than "hot" particles in environment by help foliar pathway into top leaves and apex of plant. Existence of nano- and micro- size dust as radioactive aerosols with specific properties around object "Shelter " circulate an environment represents potential risk radiobiological

consequences of plant that grow in alienation Chernobyl 30-km zone.

flowering curve depend of ontogenesis flax under influence chronic irradiation.

The chronic irradiation around object "Shelter" was including instability genome, which carried out genetic and somatic changers in late heritable on perennials as well as in several generation annual plants. Proteomics analysis observes; bloom, filling and maturing seeds evidence that there is induce epigenetic changes of genome that excited late genetic changers on progenies and somatic changes of flax plant. Treatment by the melanin-glucan complex from fungus *F. fomentarius* before sowing seed procedure increase second peak on

It is known that under chronic irradiation the epigenetic events regulate the activities or inactivate of genes by help of several mechanisms which may be include miPNA inter action deal with process that take part in vegetative and generative phases (bloom, filling and maturing seed) of growth plant by help of processes methylation of DNA, acetylation,

to the bone marrow.

Chernobyl, object "Shelter", radioactive isotopes, "hot" particles with micro- and nano-sizes, radioactivity aerosol, embryogenesis, signal system, autoradiography, single strand break (SSB) DNA, chronic irradiation, bystander effect, autoimmunity, alanine aminotransferase (ALT), liver-specific lipoprotein (LSP), radiation hepatitis, bone marrow, hematopoietic stem cells, medical consequence, melanin-glucan complex, flowering of flax, epigenetic.

#### **6. References**

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**10**

*Brazil*

**Radiochemical Separation of Nickel for**

For legal and regulatory purposes, the International Atomic Energy Agency (IAEA, 1994) defines radioactive waste as "waste that contains or is contaminated with radionuclides at concentrations or radioactivity levels greater than clearance levels as established by the regulatory body¨. The radioactive wastes are residues that have been produced by human nuclear activity and for which no future use is foreseen. Besides the nuclear power plants, the nuclear weapons testing, medical uses and various research studies involve a large number of radionuclides. In particular the nuclear accidents such as Three Mile Island Nuclear Power Station, where some gas and water were vented to the environment around the reactor, Chernobyl Nuclear Power Plant, the effects of the disaster were very widespread and Fukushima II Nuclear Power Plant have also released a large amount of

In the case of radioactive wastes each country has its own classification, in general we can identify three types of wastes, that are, Low Level Waste (LLW), the LLW wastes contain primarily short lived radionuclides which refer to half-lives shorter than or equal to 30-year half-life, Intermediate Level Waste (ILW), radioactive non-fuel waste, containing sufficient quantities of long-lived radionuclides which refer to half-lives greater than 30 years. And a third one that is High Level Waste (HLW), arise from the reprocessing of spent fuel from nuclear power reactors to recover uranium and plutonium, containing fission products that are high radioactive, heat generating and long-lived. We would like to call attention to the fact that the waste classification LLW, ILW, HLW used here is only one of several alternative

Identification and characterization of radioactive wastes is a technical challenge because of their importance in choosing the appropriate permanent storage mode or further processing. Characterization definition of nuclear waste by IAEA (IAEA, 2003) is ¨the determination of the physical, chemical and radiological properties of the waste to establish the need for further adjustment, treatment, conditioning, or its suitability for further handling, processing, storage or disposal. Thus, it involves a collection of data that pertains to specific waste properties as well as processing parameters and quality assurance, some of

**1. Introduction**

radionuclides to environment.

schemes; we adopted the simplest one.

**59Ni and 63Ni Activity Determination**

**in Nuclear Waste Samples**

Aluísio Sousa Reis, Júnior, Eliane S. C. Temba, Geraldo F. Kastner and Roberto P. G. Monteiro *Centro de Desenvolvimento da Tecnologia Nuclear – (CDTN)*

## **Radiochemical Separation of Nickel for 59Ni and 63Ni Activity Determination in Nuclear Waste Samples**

Aluísio Sousa Reis, Júnior, Eliane S. C. Temba, Geraldo F. Kastner and Roberto P. G. Monteiro *Centro de Desenvolvimento da Tecnologia Nuclear – (CDTN) Brazil*

#### **1. Introduction**

278 Nuclear Power Plants

Zhdanova N.N., Zakharchenko V.A., Tugay T.I., Yu.V. Karpenko, L.T. Nakonechnaya, A.K.

Zedman, A.J.W., Vossenaar E.R. Autoantibodies to circulated (poly) peptides: a key

P.1. 2005. P. 78-86.

37. P. 295-299.

Pavlichenko, V.A. Zheltonozhsky, A.V. Zhydkov, O.F. Seniuk. Fungal damage of "Shelter" // Problems of safety of nuclear power Plants and of Chornobyl. Issue.3.

diagnostic and prognostic marker for rheumathoid arthritis. Autoimmunity. 2004.

For legal and regulatory purposes, the International Atomic Energy Agency (IAEA, 1994) defines radioactive waste as "waste that contains or is contaminated with radionuclides at concentrations or radioactivity levels greater than clearance levels as established by the regulatory body¨. The radioactive wastes are residues that have been produced by human nuclear activity and for which no future use is foreseen. Besides the nuclear power plants, the nuclear weapons testing, medical uses and various research studies involve a large number of radionuclides. In particular the nuclear accidents such as Three Mile Island Nuclear Power Station, where some gas and water were vented to the environment around the reactor, Chernobyl Nuclear Power Plant, the effects of the disaster were very widespread and Fukushima II Nuclear Power Plant have also released a large amount of radionuclides to environment.

In the case of radioactive wastes each country has its own classification, in general we can identify three types of wastes, that are, Low Level Waste (LLW), the LLW wastes contain primarily short lived radionuclides which refer to half-lives shorter than or equal to 30-year half-life, Intermediate Level Waste (ILW), radioactive non-fuel waste, containing sufficient quantities of long-lived radionuclides which refer to half-lives greater than 30 years. And a third one that is High Level Waste (HLW), arise from the reprocessing of spent fuel from nuclear power reactors to recover uranium and plutonium, containing fission products that are high radioactive, heat generating and long-lived. We would like to call attention to the fact that the waste classification LLW, ILW, HLW used here is only one of several alternative schemes; we adopted the simplest one.

Identification and characterization of radioactive wastes is a technical challenge because of their importance in choosing the appropriate permanent storage mode or further processing. Characterization definition of nuclear waste by IAEA (IAEA, 2003) is ¨the determination of the physical, chemical and radiological properties of the waste to establish the need for further adjustment, treatment, conditioning, or its suitability for further handling, processing, storage or disposal. Thus, it involves a collection of data that pertains to specific waste properties as well as processing parameters and quality assurance, some of

Radiochemical Separation of Nickel for 59Ni

**2.1 The semiconductors detectors**

normally used for determination of isotopes of elements.

and 63Ni Activity Determination in Nuclear Waste Samples 281

spectrometric methods can be also used for the determination of radionuclides once they are

There are several types of detectors that can be used for the measurement of ionizing radiation. In the specific case of 59Ni and 63Ni, the more common radiation detection systems are ultra low energy gamma detection and liquid scintillation detection on the basis of charge carriers (holes and electrons) and liquid scintillation phenomena, respectively. Furthermore, for sequential analysis of these radionuclides alpha spectrometry can be

The most recent class of detector developed is the *solid-state semiconductor detector*. In these detectors, radiation is measured by means of the number of charge carriers set free in the detector, which is arranged between two electrodes. Ionizing radiation produces free electrons and holes. The number of electron-hole pairs is proportional to the energy transmitted by the radiation to the semiconductor. As a result, a number of electrons are transferred from the valence band to the conduction band, and an equal number of holes are created in the valence band. Under the influence of an electric field, electrons and holes travel to the electrodes, where they result in a pulse that can be measured in an outer circuit. Solid state detectors are fabricated from a variety of materials including: germanium, silicon,

Germanium detectors are mostly used for spectrometry in nuclear physics and chemistry. The Ultra Low Energy Germanium (Ultra-LEGe) detectors extends the performance range of germanium detectors down to a few hundred electron volts, providing resolution, peak shape, and peak-to-background ratios once thought to be unattainable with semiconductor detectors. According to it specification this detector offers excellent performance over a wide range of detector sizes. The resolution, for example, of a 100 mm2 Ultra-LEGe is less than 150

Radionuclides commonly emit gamma rays in the energy range from a few keV to ~10 MeV, corresponding to the typical energy levels in nuclei with reasonably long lifetimes. The boundary between gamma rays and X rays is somewhat blurred, as X rays typically refer to the high energy electromagnetic emission of atoms, which may extend to over 100 keV, whereas the lowest energy emissions of nuclei are typically termed gamma rays, even though their energies may be below 20 keV. Therefore, 59Ni that decays by electron capture with emission of 6.9 keV X-rays is suitable to be detected by low energy gamma

Beta emitting radionuclides are normally measured by a gas ionization detector or liquid scintillation counting (LSC). In LSC the scintillation takes place in a solution, the cocktails contain two basic components, the solvent and the scintillator(s). This allows close contact between the isotope atoms and the scintillator what becomes an advantage in measuring low-energy electron emitters due to the absence of attenuation. Once the solvent must act as an efficient collector of energy, and it must conduct that energy to the scintillator molecules

applied to alpha emitters associated and presents in ILW and LLW samples.

cadmium telluride, mercuric iodide, and cadmium zinc telluride.

eV in terms of full-width-half-maximum (FWHM) at 5.9 keV.

spectroscopy using Ultra Low Energy Germanium detectors.

**2.2 The liquid scintillation counting**

which include the following: thermal, mechanical, physical, biological, chemical and radioactivity properties (IAEA, 2007).

Testing and analyzes to demonstrate the radioactive content and the quality of final waste forms and waste packages are key components of this knowledge and control and are essential to accurate characterization of the waste. Physical characterization involves inspection of the waste to determine its physical state (solid, liquid or gaseous), size and weight, compactability, volatility and solubility, including closed waste packages which can be done using a variety of techniques, such as radiography (X-ray). Chemical waste characterization involves the determination of the chemical components and properties of the waste that is, potential chemical hazard, corrosion resistance, organic content, reactivity. This is most often done by chemical analysis of a waste sample. The radioactive inventory of various materials needs to be assessed for the classification of the nuclear waste. Radiological waste characterization involves detecting the presence of individual radionuclides and its properties such as half-life, intensity of penetrating radiation, activity and concentration and quantifying their inventories in the waste. This can be done by a variety of techniques, such as radiometric methods, mass spectrometric methods depending on the waste form, radionuclides involved and level of detail/accuracy required.

Furthermore, for developing a scaling factor (IAEA, 2009) to be applicable to the assessment of the radioactive inventory of the wastes with various matrices, it is indispensable to prepare a database compiled with a large numbers of information related to the radioactive inventory of long lived alpha and beta emitting nuclides which are difficult to measure (DTM) and gamma emitting nuclides which are easy to measure (ETM). It is necessary to develop analytical techniques for the DTM nuclides.

The aim of this work was to develop a sensitive analytical procedure for simultaneous determination of radionuclides difficult to measure. Between them is the 59Ni and 63Ni determination in low and intermediate level wastes from Brazilian Nuclear Power Plants – Eletrobrás Termonuclear according to an analytical protocol developed based on sequential separation of different radionuclides presents in the waste matrices (Reis et al, 2011). Sources for 59Ni are austenitic steel in the reactor and activation of nickel dissolved in the coolant and in corrosion particles deposited on the core. The content of nickel in stainless steel is around to 10% and in Inconel in the range of 50–75%. Furthermore, nickel is found as an impurity in Zircaloy, ~ 40 ppm, and in reactor fuel, ~ 20 ppm (Lingren et al, 2007).

#### **2. The radiometric detection and techniques for 59Ni and 63Ni**

Radioactive wastes are residues with different radionuclide compositions, placing, therefore considerable demands by measurement techniques used in their characterization. All radioisotopes, at some stage, require quantitation of the isotope, which is done by measuring the intensity of radiation emitted for the three main types of ionizing radiation. Radioactive isotopes of elements are normally determined by their characteristic radiation, i. e., by radiometric methods. Radiometric determination is performed by instrumental analysis using sophisticated methods such as liquid scintillation counters that allow beta spectrometry, alpha spectrometry with semiconductor detectors and high resolution gamma spectrometry for high and low energy gamma emitting nuclides. Besides, mass spectrometric methods can be also used for the determination of radionuclides once they are normally used for determination of isotopes of elements.

There are several types of detectors that can be used for the measurement of ionizing radiation. In the specific case of 59Ni and 63Ni, the more common radiation detection systems are ultra low energy gamma detection and liquid scintillation detection on the basis of charge carriers (holes and electrons) and liquid scintillation phenomena, respectively. Furthermore, for sequential analysis of these radionuclides alpha spectrometry can be applied to alpha emitters associated and presents in ILW and LLW samples.

#### **2.1 The semiconductors detectors**

280 Nuclear Power Plants

which include the following: thermal, mechanical, physical, biological, chemical and

Testing and analyzes to demonstrate the radioactive content and the quality of final waste forms and waste packages are key components of this knowledge and control and are essential to accurate characterization of the waste. Physical characterization involves inspection of the waste to determine its physical state (solid, liquid or gaseous), size and weight, compactability, volatility and solubility, including closed waste packages which can be done using a variety of techniques, such as radiography (X-ray). Chemical waste characterization involves the determination of the chemical components and properties of the waste that is, potential chemical hazard, corrosion resistance, organic content, reactivity. This is most often done by chemical analysis of a waste sample. The radioactive inventory of various materials needs to be assessed for the classification of the nuclear waste. Radiological waste characterization involves detecting the presence of individual radionuclides and its properties such as half-life, intensity of penetrating radiation, activity and concentration and quantifying their inventories in the waste. This can be done by a variety of techniques, such as radiometric methods, mass spectrometric methods depending on the waste form, radionuclides involved and level of

Furthermore, for developing a scaling factor (IAEA, 2009) to be applicable to the assessment of the radioactive inventory of the wastes with various matrices, it is indispensable to prepare a database compiled with a large numbers of information related to the radioactive inventory of long lived alpha and beta emitting nuclides which are difficult to measure (DTM) and gamma emitting nuclides which are easy to measure (ETM). It is necessary to

The aim of this work was to develop a sensitive analytical procedure for simultaneous determination of radionuclides difficult to measure. Between them is the 59Ni and 63Ni determination in low and intermediate level wastes from Brazilian Nuclear Power Plants – Eletrobrás Termonuclear according to an analytical protocol developed based on sequential separation of different radionuclides presents in the waste matrices (Reis et al, 2011). Sources for 59Ni are austenitic steel in the reactor and activation of nickel dissolved in the coolant and in corrosion particles deposited on the core. The content of nickel in stainless steel is around to 10% and in Inconel in the range of 50–75%. Furthermore, nickel is found as

an impurity in Zircaloy, ~ 40 ppm, and in reactor fuel, ~ 20 ppm (Lingren et al, 2007).

Radioactive wastes are residues with different radionuclide compositions, placing, therefore considerable demands by measurement techniques used in their characterization. All radioisotopes, at some stage, require quantitation of the isotope, which is done by measuring the intensity of radiation emitted for the three main types of ionizing radiation. Radioactive isotopes of elements are normally determined by their characteristic radiation, i. e., by radiometric methods. Radiometric determination is performed by instrumental analysis using sophisticated methods such as liquid scintillation counters that allow beta spectrometry, alpha spectrometry with semiconductor detectors and high resolution gamma spectrometry for high and low energy gamma emitting nuclides. Besides, mass

**2. The radiometric detection and techniques for 59Ni and 63Ni**

radioactivity properties (IAEA, 2007).

detail/accuracy required.

develop analytical techniques for the DTM nuclides.

The most recent class of detector developed is the *solid-state semiconductor detector*. In these detectors, radiation is measured by means of the number of charge carriers set free in the detector, which is arranged between two electrodes. Ionizing radiation produces free electrons and holes. The number of electron-hole pairs is proportional to the energy transmitted by the radiation to the semiconductor. As a result, a number of electrons are transferred from the valence band to the conduction band, and an equal number of holes are created in the valence band. Under the influence of an electric field, electrons and holes travel to the electrodes, where they result in a pulse that can be measured in an outer circuit. Solid state detectors are fabricated from a variety of materials including: germanium, silicon, cadmium telluride, mercuric iodide, and cadmium zinc telluride.

Germanium detectors are mostly used for spectrometry in nuclear physics and chemistry. The Ultra Low Energy Germanium (Ultra-LEGe) detectors extends the performance range of germanium detectors down to a few hundred electron volts, providing resolution, peak shape, and peak-to-background ratios once thought to be unattainable with semiconductor detectors. According to it specification this detector offers excellent performance over a wide range of detector sizes. The resolution, for example, of a 100 mm2 Ultra-LEGe is less than 150 eV in terms of full-width-half-maximum (FWHM) at 5.9 keV.

Radionuclides commonly emit gamma rays in the energy range from a few keV to ~10 MeV, corresponding to the typical energy levels in nuclei with reasonably long lifetimes. The boundary between gamma rays and X rays is somewhat blurred, as X rays typically refer to the high energy electromagnetic emission of atoms, which may extend to over 100 keV, whereas the lowest energy emissions of nuclei are typically termed gamma rays, even though their energies may be below 20 keV. Therefore, 59Ni that decays by electron capture with emission of 6.9 keV X-rays is suitable to be detected by low energy gamma spectroscopy using Ultra Low Energy Germanium detectors.

#### **2.2 The liquid scintillation counting**

Beta emitting radionuclides are normally measured by a gas ionization detector or liquid scintillation counting (LSC). In LSC the scintillation takes place in a solution, the cocktails contain two basic components, the solvent and the scintillator(s). This allows close contact between the isotope atoms and the scintillator what becomes an advantage in measuring low-energy electron emitters due to the absence of attenuation. Once the solvent must act as an efficient collector of energy, and it must conduct that energy to the scintillator molecules

Radiochemical Separation of Nickel for 59Ni

alpha spectrometry.

**3.1 Radiochemical separation**

asymmetric shape rather than the Gaussian shape.

they cannot be chemically separated and analyzed.

**3. Radiochemical for radionuclides difficult to measure**

and 63Ni Activity Determination in Nuclear Waste Samples 283

which the peak areas can be determined is compromised because the peaks tend to have an

The alpha particle energies of many isotopes differ by as little as 10 to 20 keV (Canberra, n.d.). The relatively small difference in alpha particle energy between some alpha emitters makes it difficult to spectrometrically separate the peaks once this is near the resolution of the silicon detectors used in alpha spectrometers. If two of these alpha particle energies are so close, they cannot be spectrometrically separated and if they are chemically the same,

Resolution is the ability of the spectrometry system to differentiate between two different alpha particles and its quantitative measure is the FWHM. Besides, a FWHM of about 15 keV can be achieved with electroplated sources because they have very little mass to slow down the alpha particles. For this reason it is essential that a thin source to be prepared in

The methods for separating, collecting, and detecting radionuclides are similar to ordinary analytical procedures and employ many of the chemical and physical principles that apply to their nonradioactive isotopes. One of the differences is interesting from the viewpoint of methodology. Substance separation in analytical chemistry in the majority of cases is not an end in itself. In radiochemistry, separation is most often an end in itself, for example, when a radionuclide is purified of other radioactive elements (Zolotov, 2005). Techniques used for separation include co-precipitation, liquid-liquid extraction, ion exchange and extraction

In order to account for the inevitable loss of the sample during separation, a specific isotope or tracer is added to the sample. A tracer represents the addition to an aliquot of sample a known quantity of a radioactive isotope that is different from that of the isotope of interest but expected to behave in the same way. Sample results are normally corrected based on tracer recovery. The percent of tracer lost in the chemical processes is equal to the percent of sample lost, assuming the tracer is homogeneously mixed with the sample and is brought into chemical equilibrium with the sample. Radiochemical analysis frequently requires the radiochemist to separate and determine radionuclides that are present at extremely small quantities. The amount can be in the picomole range or less, at concentrations in the order of 10-15 to 10-11 molar (United States Environmental Protection Agency, 2004). The use of a material that is different in isotopic make-up to the analyte and that raises the effective concentration of the material to the macro level is referred to as a carrier, a substance that

Radiochemical waste characterization is the identification of radionuclides contained in a package of nuclear waste and the determination of their concentration. The problem the waste producers have to cope with comes from the fact that those nuclides which are mainly (pure) β- or α-emitters cannot be measured by direct methods such as γ-scanning. In the waste packages produced by a nuclear power reactor the radionuclides may be originated as fission products from the nuclear fuel, activation products and transmutation nuclides, Table 1.

chromatography. In some cases, two or more of these techniques are combined.

has a similar crystalline structure that can incorporate the desired element.

instead of dissipating the energy by some other mechanism (National Diagnostics, 2004). Liquid scintillation cocktails absorb the energy emitted by radioisotopes and re-emit it as flashes of light. A β particle, passing through a scintillation cocktail, leaves a trail of energized solvent molecules. These excited solvent molecules transfer their energy to scintillator molecules, which give off light. With LSC the short path length of soft β emissions is not an obstacle to detection. LSC can thus be used for the measurement of both high and low energy emitters.

A pulse height spectrum is a representation of the average kinetic energy associated with the decay of a particular isotope. When an isotope decays it liberates an electron or beta particle and a neutrino that have the energy associated shared between the two particles. As a result of that the resulting beta particles have a continuous distribution of energies from 0 to maximum decay energy (Emax). The amount of light energy given off is proportional to the amount of energy associated with the beta particle. Therefore, the beta decay shows a continuous energy distribution and beta particle spectrometry becomes an analytical thecnique in which it is difficult to identify individual contributions in the spectrum beta. The determination of various beta emitters such as 3H, 14C, 63Ni, 55Fe, 90Sr requires chemical separation of the individual radionuclides from the matrix and from the other radionuclides before couting.

The isotope 63Ni is an artificial radionuclide. It is a pure β emitter with a half-life of 100 years. The maximum energy of the emitted β-radiation is 67 keV. No γ radiation is observed. Except 59Ni with a half-life of 7.6 x 104 years all nickel radionuclides have very short halflifes. They range between 18 seconds and 54.6 hours. Therefore they don´t disturb a measurement of 63Ni. Besides, LSC has a high couting efficiency for 63Ni, about 70%., i. e., the ratio cpm/dpm, counts per minute to disintegration per minute expressed as a percentage, in other words, the percentage of emission events that produce a detectable pulse of photons, making the technique widely used for the determination of 63Ni.

#### **2.3 The alpha spectrometry**

The sequential analyses determine in addition to 59Ni and 63Ni others DTM´s present in the nuclear waste including alpha emitters. Therefore, alpha spectrometry is one complementary technique for the nuclear waste characterization either ILW or LLW.

In this technique to achieve results with good quality, the sample must be converted into a chemically isolated, thin layered and uniform source. The preparation of an alpha sample contains three basic steps: preliminary treatment, chemical separation and source preparation

Alpha-emitting radioisotopes spontaneously produce alpha particles at characteristic energies usually between about 4 and 6 MeV. Alpha particles (or 4He nuclei) are heavy charged, large and slow particles and loses some of its energy each time it produces an ion (its positive charge pulls electrons away from atoms in its path), finally acquiring two electrons from an atom at the end of its path to become a complete helium atom. These attenuation characteristics, which manifest themselves both within the sample and with any materials between the sample and the active detector volume, cause a characteristic tailing in the alpha peak. When tailing occurs (it is also called "spill down"), the accuracy with

instead of dissipating the energy by some other mechanism (National Diagnostics, 2004). Liquid scintillation cocktails absorb the energy emitted by radioisotopes and re-emit it as flashes of light. A β particle, passing through a scintillation cocktail, leaves a trail of energized solvent molecules. These excited solvent molecules transfer their energy to scintillator molecules, which give off light. With LSC the short path length of soft β emissions is not an obstacle to detection. LSC can thus be used for the measurement of both

A pulse height spectrum is a representation of the average kinetic energy associated with the decay of a particular isotope. When an isotope decays it liberates an electron or beta particle and a neutrino that have the energy associated shared between the two particles. As a result of that the resulting beta particles have a continuous distribution of energies from 0 to maximum decay energy (Emax). The amount of light energy given off is proportional to the amount of energy associated with the beta particle. Therefore, the beta decay shows a continuous energy distribution and beta particle spectrometry becomes an analytical thecnique in which it is difficult to identify individual contributions in the spectrum beta. The determination of various beta emitters such as 3H, 14C, 63Ni, 55Fe, 90Sr requires chemical separation of the individual radionuclides from the matrix and from the

The isotope 63Ni is an artificial radionuclide. It is a pure β emitter with a half-life of 100 years. The maximum energy of the emitted β-radiation is 67 keV. No γ radiation is observed. Except 59Ni with a half-life of 7.6 x 104 years all nickel radionuclides have very short halflifes. They range between 18 seconds and 54.6 hours. Therefore they don´t disturb a measurement of 63Ni. Besides, LSC has a high couting efficiency for 63Ni, about 70%., i. e., the ratio cpm/dpm, counts per minute to disintegration per minute expressed as a percentage, in other words, the percentage of emission events that produce a detectable

The sequential analyses determine in addition to 59Ni and 63Ni others DTM´s present in the nuclear waste including alpha emitters. Therefore, alpha spectrometry is one

In this technique to achieve results with good quality, the sample must be converted into a chemically isolated, thin layered and uniform source. The preparation of an alpha sample contains three basic steps: preliminary treatment, chemical separation and source

Alpha-emitting radioisotopes spontaneously produce alpha particles at characteristic energies usually between about 4 and 6 MeV. Alpha particles (or 4He nuclei) are heavy charged, large and slow particles and loses some of its energy each time it produces an ion (its positive charge pulls electrons away from atoms in its path), finally acquiring two electrons from an atom at the end of its path to become a complete helium atom. These attenuation characteristics, which manifest themselves both within the sample and with any materials between the sample and the active detector volume, cause a characteristic tailing in the alpha peak. When tailing occurs (it is also called "spill down"), the accuracy with

pulse of photons, making the technique widely used for the determination of 63Ni.

complementary technique for the nuclear waste characterization either ILW or LLW.

high and low energy emitters.

other radionuclides before couting.

**2.3 The alpha spectrometry**

preparation

which the peak areas can be determined is compromised because the peaks tend to have an asymmetric shape rather than the Gaussian shape.

The alpha particle energies of many isotopes differ by as little as 10 to 20 keV (Canberra, n.d.). The relatively small difference in alpha particle energy between some alpha emitters makes it difficult to spectrometrically separate the peaks once this is near the resolution of the silicon detectors used in alpha spectrometers. If two of these alpha particle energies are so close, they cannot be spectrometrically separated and if they are chemically the same, they cannot be chemically separated and analyzed.

Resolution is the ability of the spectrometry system to differentiate between two different alpha particles and its quantitative measure is the FWHM. Besides, a FWHM of about 15 keV can be achieved with electroplated sources because they have very little mass to slow down the alpha particles. For this reason it is essential that a thin source to be prepared in alpha spectrometry.

#### **3. Radiochemical for radionuclides difficult to measure**

#### **3.1 Radiochemical separation**

The methods for separating, collecting, and detecting radionuclides are similar to ordinary analytical procedures and employ many of the chemical and physical principles that apply to their nonradioactive isotopes. One of the differences is interesting from the viewpoint of methodology. Substance separation in analytical chemistry in the majority of cases is not an end in itself. In radiochemistry, separation is most often an end in itself, for example, when a radionuclide is purified of other radioactive elements (Zolotov, 2005). Techniques used for separation include co-precipitation, liquid-liquid extraction, ion exchange and extraction chromatography. In some cases, two or more of these techniques are combined.

In order to account for the inevitable loss of the sample during separation, a specific isotope or tracer is added to the sample. A tracer represents the addition to an aliquot of sample a known quantity of a radioactive isotope that is different from that of the isotope of interest but expected to behave in the same way. Sample results are normally corrected based on tracer recovery. The percent of tracer lost in the chemical processes is equal to the percent of sample lost, assuming the tracer is homogeneously mixed with the sample and is brought into chemical equilibrium with the sample. Radiochemical analysis frequently requires the radiochemist to separate and determine radionuclides that are present at extremely small quantities. The amount can be in the picomole range or less, at concentrations in the order of 10-15 to 10-11 molar (United States Environmental Protection Agency, 2004). The use of a material that is different in isotopic make-up to the analyte and that raises the effective concentration of the material to the macro level is referred to as a carrier, a substance that has a similar crystalline structure that can incorporate the desired element.

Radiochemical waste characterization is the identification of radionuclides contained in a package of nuclear waste and the determination of their concentration. The problem the waste producers have to cope with comes from the fact that those nuclides which are mainly (pure) β- or α-emitters cannot be measured by direct methods such as γ-scanning. In the waste packages produced by a nuclear power reactor the radionuclides may be originated as fission products from the nuclear fuel, activation products and transmutation nuclides, Table 1.