Analele tiinifice ale Universitii Alexandru Ioan Cuza, Seciunea Genetic i Biologie Molecular, TOM XIV, 2013

SOME ASPECTS OF ANTIOXIDANT DEFENSE MECHANISMS IN WOOD SPECIES EXPOSED TO ANTHROPIC POLLUTION IN SUCEAVA COUNTY
MARIUS VIOREL ONICIUC1, ELENA TUTU1, COJOCARU SABINA IOANA1, ELENA CIORNEA1*
Keywords: pollution, sulphur, copper, barite, catalase, peroxidase, angiosperms, gymnosperms. Abstract: Increased production of reactive oxygen species in plant tissues caused by unfavorable environmental conditions is early response to different stresses and may provide cells with resistance against their formation. The subject of this paper is determination of catalase and peroxidase levels, components of the antioxidant defense mechanism, in various types of woody plants in order to study the effect of pollution by sulphur and copper exploitation in mining areas on the antioxidant enzymes activity in the leaf material taken from different Gymnosperm species as Picea abies L.Karst., Larix decidua Mill. and Angyosperms like Salix ssp alba L., Populus tremula, Betula verrucosa Ehrh. i Fagus sylvatica L. For this purpose, the measurement of catalase activity was performed using the Sinha method (Artenie Vl. et al., 2008), the determination of peroxidase level was carried out on the basis of orthodianisidine method (Cojocaru D.C., 2009) and the determination of soluble proteins on Bradford method (Cojocaru et al.,2009). The results obtained lead to the conclusion that both catalase and peroxidase are effective biomarkers of pollution with sulfur, copper ores and barite but especially the acclimatization of species studied in conditions of chronic exposure.

INTRODUCTION
Pollution impact on woody plants is due to the generation of reactive oxygen species and induction of socalled oxidative stress (Schtzendbel and Polle, 2002); their counteracting beeing made by the various enzymatic and non-enzymatic antioxidant systems (Smirnoff, 1995, Navarri-Izzoand and Rascio, 2010, Singh Gill and Tuteja, 2010), the beginning of stress in plants requiring a reorganization of cellular metabolism in this ensemble, for their acclimatization to stress. The incipient stages of the response (the so-called alarm stage), involve rapid induction on the specific signaling paths to stress and to a strong oxidative stress, while the later stages (the acclimatization stage), are associated with the de novo biosynthesis of proteins with protective functions against stress (chaperones, antioxidant enzymes) and other compounds (carotenoids, tocopherols, osmoprotectans -proline), followed by the followed by the activation of degrading processes of these protective compounds and a stabilizations of the new cell homeostasis during the recovery period (Kosova et al., 2011). Starting from the hypothesis that a significant increase of enzymatic production integrated in foliar defensive system in some genotypes of wood plant population may result from increase of respective varieties resistance to anthropogenic pollution factors, we can conclude therefore, that these enzymes can be used as biomarkers of oxidative stress, and also in evaluating the degree of acclimatization of the species in mining areas studied.

MATERIAL AND METHODS

The investigations were performed on leaf material collected in May 2011 from Gymnosperms species of the conifers family such as Picea abies L.Karst., Larix decidua ssp Mill. and Angiosperms such as Salix alba L., Populus tremula and Betula verrucosa Ehrh. under the influence of pollution (Climani and Tarnia), and from species located in the control areas considered unpolluted Rduti, Suceava County. The determination of the catalase activity was made using the spectrophotometric Sinha method (Artenie et al., 2007), the peroxidase activity was detected by the Gudkova, L. and Degtiari, G. method (Artenie et al., 2007) with ortho-dianisidine, while the total soluble proteins were dosed using the Bradford method (Cojocaru et al., 2009). For each biochemical determination were used tree parallel samples, the presented data representing the arithmetic mean of the obtained results.

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Marius Viorel Oniciuc et al Some aspects of antioxidant defense mechanisms in wood species exposed to anthropic pollution in Suceava County

RESULTS AND DISCUSSION The results regarding the catalase and peroxidase specific activity to Gymnosperms and Angiosperms species from polluted and unpolluted areas of Suceava are presented graphically in Figures 1-2. Some studies in the literature highlight the fact that the catalase activity in needles peroxisomes of Picea abies decreases with age, being higher in the growth conditions of plants in the presence of ozone (Morral et al., 1990), and can also be concluded that, in terms of pollution of air and soil with sulfur, the catalase activity can be used as a biomarker, higher than the one found in Picea abies specimens grown in unpolluted areas (15.731 UC/mg proteins detected in Climani Mountains, compared to 7.001 UC/mg proteins recorded in the needles taken from Rduti). A quantitative approach on the data obtained about the antioxidant defense in leaf tissues of Populus tremula specimens of Climani Mountains, shows a three times higher activity of catalase (17.182 UC/mg proteins) compared to those provided by the foliar limb of the species harvested from the control zone (5.054 UC/mg proteins). According to other investigations, Populus sp. is one of the species in which catalase shows a high sensitivity to pollution (Stobrawa and Lorenc-Pluciska, 2007), the decreased fluid regime potentiating the formation of superoxide radicals, which results in a sustained activity of the superoxidedismutase and implicitly, in a catalase activity that prevents the accumulation of hydrogen peroxide.
30

2 1,8

25

1,6 1,4 PU/mg protein 1,2 1 0,8 0,6 0,4 0,2

CU/mg protein

20

15

10

0 Picea abies Populus tremula Calimani Salix spp alba Betula verucosa Larix decidua

0 Picea abies Populus tremula Calimani Salix spp alba Betula verucosa Radauti Larix decidua

Tarnita

Radauti

Tarnita

Fig.1.The specific activity of catalase in Gymnosperm and Angiosperm species in polluted and unpolluted areas of Suceava County

Fig.2.The specific activity of peroxidase in Gymnosperms and Angiosperms species in polluted and unpolluted areas of Suceava County

The existence of a strong oxidative stress in this area is confirmed by the very high activity of peroxidase (1.822 UP/mg proteins in samples from Climani), which indicates the high sensitivity of this species to pollutants. Moreover in the leaf device of Populus tremula, the oxidoreductase activity was the highest among all species in the areas monitored in our studies (fig. 2), which allows us to use it as a biomarker in the investigations of this genus. Salix alba is considered to be a resistant species, moderately affected by gaseous pollutants and is commonly used to restore damaged ecosystems (Pulford and Dickinson, 2005) together with Populus sp. (Komives and Gullner, 2006, Olejniczak et al., 2012). In the leaves of these species, this antiperoxidative enzyme catalase, had registered a dominant level compared to that of other studied species (29.192 UC/mg proteins).

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Analele tiinifice ale Universitii Alexandru Ioan Cuza, Seciunea Genetic i Biologie Molecular, TOM XIV, 2013

Immediately after this value, Betula verrucosa confirms the fact that hydrogen peroxide acts as signal molecule under abiotic stress factors, which together with the excess sulfur present in the environment, forces a successful acclimatization with bioremediation effect at this species 28.222 UC/mg proteins. Larix decidua, in its turn, is known in the literature as being a species that shows a high susceptibility to SO2 pollution, its sensitivity becoming extreme as the plant ages (Davis and Wilhour, 1976). From our findings, the catalase activity in this species of conifers was higher than that of Picea abies (16.642 UC/mg proteins), which proves the high antioxidant adaptability of Larix decidua, confirming a higher resistance than other conifers to stress induced by anthropogenic sulfur, since the contamination deposits on the permanently green needles of the coniferous trees and not on larch (Lines, 1984 cited by Chalupa, 1991). The activity of copper and barite ore processing works of Tarnia, even though stopped in 2007, focused on copper ores exploited trough underground mining (dominant minerals in copper zinc ores are: chalcocite, chalcopyrite, blende (ZnS), pyrite (FeS2), pyrotine (FeS), and baritine. The impact the two sections activity was felt in the same area, with the same receptors of the noxious pollutants, having the same waste facilities. The dominant pollutants are the polymetallic particulate matters, sulfur oxides, nitrogen oxides and hydrogen sulfide, The analyzes for copper and zinc indicators shows constant exceeds for these parameters, while the zinc indicator shows a steady increase after the closing of the mining operations (Ionce, 2010). The synergistic action of a toxic metal with a key metal for the cellular activity has as a consequence the reduction of the cellular damage, while the combinations of metals and phytochelatin toxicity can act against the excess toxicity of the elements found in the environment (Bertrand and Poirier, 2005, Grill et al., 2007). This could explain, probably, why at the analytic approach of the catalase activity on the foliar samples taken from different wood species located in the polluted areas from the Tarnita region, the values obtained after their quantitative measuring, the level of the oxidative stress at these species is smaller than that of other mining areas (Climani), having medium values, some of them quite similar to those found in the control area (Rduti). The effective defense capabilities of plants to the negative action of the oxidative stress inducted by the presence of metallous and non-metallous (baritine) particles in the environment is different. In Larix decidua species was registered the existence of a maximum level of catalase activity 19.62 UC/mg proteins, while in the leaves of birch (Betula verrucosa) a relatively high value was detected -18.35 UC /mg proteins. The accumulation of hydrogen peroxide in the leaf material of Populus tremula induces an increase in catalase activity to a level of 15.966 UC/mg proteins, and in Salix alba was detected the presence of a relatively high oxidative stress in the presence of iron-contaminated soil (Wahsha et al., 2011), the species in question being considered more tolerant toenvironmental pollution with pyrite, which recommends it as a useful in the phytoremediation of contaminated sites. Minimal level of activity of the oxidoreductase in Picea abies 8.262 UC/mg protein probably is due to processes in the rhizosphere (Vamerali et al., 2009), the absorption of iron being made in the roots, metallothioneins, ferritin and other chellators being capable to create a protection against damage induced by excess iron, copper, zinc and barite. In interpreting of the one should take into account the fact that, it is possible that under stressful conditions, in the leaves of species with low resistant to anthropogenic pollution might be a misbalance balance between the production of reactive oxygen species and antioxidative defense enzyme activity. This assumption relies on the fact that the production of antioxidant in its turn affected by stressful conditions (Bowler et al., 1992), for in some cases enzyme activity

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Marius Viorel Oniciuc et al Some aspects of antioxidant defense mechanisms in wood species exposed to anthropic pollution in Suceava County

rise and in others decreases, facts that depend on the intensity of stress factors and chronic pollution, of the receptivity of plant organism, individual development stage, the climatic conditions and, especially, the genetic background. The great versatility of peroxidase is its predominant characteristic, and, therefore, there is no major physiological process that would function without this enzyme, that has a wide range of isoenzymes and large physiological implications: auxins oxidation (Tognetti et al., 2012), involvmnent in the ethylene biosynthesis (Gaspar et al., 1982), lignin metabolism (Diaz et al., 2001, Chen et al., 2002, Rodrguez Dorantes and Guerrero Ziga, 2012), the hydroxylation of proline to hydroxyproline (Ishikawa et al., 2006), the mechanisms of plant resistance (Cheng, 2003). Consequently, in the interpretation of the data obtained in regard to peroxidase activity in the leaf samples collected from species of Picea abies, Salix alba, Larix decidua, Betula verrucosa and Populus tremula in areas with a different nature of pollution (metalliferous, nonmetalliferous, radioactive) or from places considered to have no mining residual wastes, one should take into account all these aspects, and not only the participation of this oxidoreductase to antioxidant defense. In concordance with the data from the literature, the peroxidase is considered to be an indicator of anthropogenic sulfur accumulation in the environment (Keler, 1976, Horsman and Welburn, 1977 quoted by Khan and Malhotra, 1982), the activity of this oxidoreductase being higher in the aging leaf tissues of Betula and Pinus sp. compared to the younger ones, their possible role in their senescence being attributed to peroxidase, because it has the ability to oxidize indolil-acetic acid, a growth hormone and, in agreement with other studies, the senescence is proportional directly to the accumulation of hydrogen peroxide at cells. The chronic exposure to anthropogenic sulfur was followed by a strong induction of the peroxidase activity at Populus tremula, the foliar material sampled from the Climani Mountains showing a quantitative level of de 1.822 UP/ mg proteins. It is known SO2 is capable of altering the biochemistry of sulfur in cells and to rise the internal basin of the active osmotic material, affecting its metabolism. At certain plants, the start of the growth of the peroxidase activity caused by high quantities of manmade sulfur at a certain exposure can coincide with the apparition of the foliar necrosis, despite the fact that a close bio-monitoring of certain Angiosperm species had shown that the activity of the oxidoreductase can be an early biological marker capable of giving an alert before the apparition of visible symptoms, when sometimes it can be too late (Tripathi and Gautam, 2007). In this case, given the chronic exposure to pollutant, it is very likely that the high activity of the enzyme can be due to an adaptive mechanism of the plant to stressful conditions that has a well-regulated homeostasis. The poplar is well known as being an excellent choice for bio-recovery, as it offers multiple cycles of decontamination (Bittsnszky et al., 2005). The samples taken from Salix spp. alba individuals from the Climani Mountains, showed in their turn a high activity of the peroxidase 1.736 UP/mg proteins, which is nothing unusual considering the fact that normally, the activity of the peroxidase rises with together with the growing quantity of sulfur of the plants exposed to chronic pollution, an eventual tolerance phenomenon in this could be related to the activity of the SOD and of peroxidase in the vegetal cell (Jager et al., 1985, Agrawal et al., 1986 cited by Abrol and Ahmad, 2003). In regard to the activity of this peroxidase in the foliar material sampled from Betula verrucosa and Larix decidua, the results are somehow surprising, both species showing enzymatic activity values close to those registered in the control area (0.258 UP/mg proteins for

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Analele tiinifice ale Universitii Alexandru Ioan Cuza, Seciunea Genetic i Biologie Molecular, TOM XIV, 2013

birch, respectively 0.146 UP/mg proteins for larch). The investigations made on Larix deciduas showed that a growth of the peroxidase on the course of the growing season is in close connection with the synthesis of new izoenzymes of the same oxidoreductase (Grill et al., 1980). Taking into account the fact that the intervention of the peroxidase takes place in the condition in which the quantity of H2O2 is small and that the activity of the enzyme is much more diminished in the condition of the acidification of the reaction environment, it cannot be excluded the valability of this explanation for the obtain results, even more if we account for the high activity of the catalase at this species, this oxidoreductase acting complementary with the peroxidase. Analyzing the samples taken from the wood species from Tarnia it can be observed that the greatest sensibility for the accumulation of the hydrogen peroxide in the vegetal cell under the pressure of the metals found in the environment was found at Populus tremula - 1.754 UP/mg proteines, Salix alba -1.58 UP/mg proteins and Picea abies - 1.446 UP/mg proteins. In the Rdui area, considered to be non-polluted, the accumulation of small hydrogen peroxide quantities was different from one species to another, having a higher intensity at Populus tremula (1.79 UP/mg proteins), Picea abies (1.693 UP/mg proteins) i Salix alba (0,676 UP/mg proteins). CONCLUSIONS In this stage of our investigations, proposing a so-called scale of resistance for Angiosperm and Gymnosperm species to the aggression of anthropic pollution has a rather quasi guide character. A biochemical monitoring on long term is necessary in order to follow the effects of chronic exposure to heavy metals and barite, but also the supplementation of test that can offer a larger picture in regard to the implication of contaminating factors on the foliar mechanisms of woodland plants. REFERENCES
Abrol, Y.P. and Ahmad, A. (2003): Sulphur in plants, Springer, page 285. Artenie, Vl., Ungureanu E., Negura, A.M. (2008): Metode de investigare a metabolismului glucidic i lipidic manual de lucrri practice, Editura Pim, Iai. Bertrand, M. and Poirier, I. (2005): Photosynthetic organisms and excess of metals, Photosynthetica, vol.43, no.3, pages 345-353. Bittsnszky A, Kmives T, Gullner G, Gyulai G, Kiss J, Heszky L, Radimszky L, Rennenberg H. (2005): Ability of transgenic poplars with elevated glutathione content to tolerate zinc (2+) stress, Environ Int., vol. 31, no. 2: 251-254. Bowler, C.,Van Montagu, M., Inze, D. (1992): Superoxide dismutase and stress tolerance. Annu. Rev. Plant. Physiol., 43: 83 116. Chalupta, V. (1991): Larch (Larix decidua Mill. )in Trees III, Biotechnology Agriculture and Forestry 16 (ed. Bajaj, Y.P.S.), Springer, pages 446-447. Chen, E.L., Chen, Y.A., Chen, L.M. and Liu, Z.H. (2002): Effect of copper on peroxidase activity and lignin content in Raphanus sativus. Plant Physiology and Biochemistry, 40: 439-444. Cheng, S. (2003): Effects of heavy metals on plants and resistance mechanisms, Environ. Sci. & Pollut. Res., vol. 10, no. 4, pages 2 5 6 - 2 6 4. Cojocaru D. C., Toma O., Sabina Ioana Cojocaru, Elena Ciornea (2009): Practicum de biochimia proteinelor i acizilor nucleici, Ed. Tehnopress, Iai, ISBN 978-973-702-640-8, 261 p. Davis, D.D. and Wilhour, R.G. (1976): Susceptibility of woody plants to sulphur dioxide and photochemical oxidants:a literature review, U.S. Environmental Protection Agency, Office of Research and Development, Corvallis Environmental Research Laboratory, pages 34, 41.

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Marius Viorel Oniciuc et al Some aspects of antioxidant defense mechanisms in wood species exposed to anthropic pollution in Suceava County Diaz, J., Bernal, A., Pomar, F., Merino, F. (2001): Induction of shikimate dehydrogenase 862 and peroxidase in pepper (Capsicum annuum L.) seedlings in response to copper stress and its relation to lignification. Plant Science, vol. 161, pages 179-188. Gaspar, T., Penel, Cl., Thorpe, T. and Greppin, H. (1982): Peroxidases. A survey of their biochemical and physiological roles in higher plants, pages 44-48. Grill, D., Esterbauer, H. and Birkner, M. (1980): Untersuchungen fiber die Peroxidaseaktivit/it in L/irchennadeln. Beitr. Bol. Pflanzen, 55: 67-76. Grill, E., Mishra, S., Srivastava, S. and Tripathi, R.D. (2007): Role of phytochelatins in phytoremediation of heavy metals, Environmental Bioremediation Technologies, pages 101-146. Ionce, A. (2010): Impactul sistemic al activitii de preparare a substanelor minerale utile n judeul Suceava, Tez de doctorat. Ishikawa, T., Dowdle, J., Smirnoff, N. (2006): Progress in manipulating ascorbic acid biosynthesis and accumulation in plants. Physiologia Plantarum, vol.126: 343355. Khan, A.A. and Malhotra, S.S. (1982): Peroxidase activity as a indicator of SO2 injury in Jack Pine and White Birch, Biochem.Physiol.Phlanzen., vol. 177: 648-650. Komives, T. and Gullner, G. (2006): Dendroremediation: The Use of Trees in Cleaning up Polluted Soils, Phytoremediation Rhizoremediation, pages 23-31. Kosova, K., Vtmvs, P., Prasil, I.T. and Renaut, J. (2011): Plant proteome changes under abiotic stress Contribution of proteomics studies to understanding plant stress response, Journal of Proteomics, 74 (8): 13011322. Morr, D.J., Selldn, G., Ojanper,K., Sandelius, A.S., Egger, A., Morr, D.M., Chalko, C.M. and Chalko, R.A. (1990): Peroxisome proliferation in Norway spruce induced by ozone, Protoplasma, 155 (1-3): 58-65. Navarri-Izzoand, F. and Rascio, N. (2010): Heavy Metal Pollution Damage and Defense Strategies in Plants, in Handbook of Plant and Crop Stress, Third Edition, Edited by Mohammad Pessarakli, CRC Press 2010, pages 635674. Olejniczak, A., Cyganiuk, A., Kuciska, A. and ukaszewicz, J.P. (2012): Energetic Willow (Salix viminalis) Unconventional Applications, in Sustainable Growth and Applications in Renewable Energy Sources, pages 181-208. Pulford, I.D. and Dickinson, M.N. (2005): Phytoremediation technologies using trees - in Trace Elements in the Environment (Eds. Prassad, M.N.V. and Naidu, R.), CRC Press, New York, pages. 375-395. Rodrguez Dorantes, A. and Guerrero Ziga, L.A. (2012): Phenoloxidases activity in root system and their importance in the phytoremediation of organic contaminants, Journal of Environmental Chemistry and Ecotoxicology, vol. 4, no.3, pages. 35-40. Schtzendbel, A. and Polle A. (2002): Plant responses to abiotic stresses: Heavy metal-induced oxidative stress and protection by mycorrhization. J. Exp Bot., 53: 1351-65. Singh Gill, S. and Tuteja, N. (2010): Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants, Plant Physiology and Biochemistry, 48: 909-930. Smirnoff, N. (1995): Antioxidant systems and plant response to the environment. In:Environment and plant Metabolism (Smirnoff, N. ed.). Bios Scientific Publishers, pages 217-243. Stobrawa, C. and Lorenc-Pluciska, G. (2007): Changes in antioxidant enzyme activity in the fine roots of black poplar (Populus nigra L.) and cottonwood (Populus deltoides Bartr. ex Marsch) in a heavy-metal-polluted environment, Plant and Soil, vol. 298, no. 1-2, pages 57-68. Tognetti, V.B., Mhlenbock, P. and Van Breusegem, F. (2012): Stress homeostasis the redox and auxin perspective, Plant, Cell & Environment, Special Issue: Special Issue on Redox Signaling, vol. 35, Issue 2, pages 321333. Tripathi, A.K. and Gautam, M. (2007): Biochemical parameters of plants as indicators of air pollution, J. Environ. Biol., vol. 28, no.1, pages 127-32. Vamerali, T., Bandiera, M., Coletto, L., Zanetti, F., Dickinson, N.M. and Mosca, G. (2009): Phytoremediation trials on metal- and arsenic-contaminated pyrite wastes (Torviscosa, Italy), Environ Pollut. 157(3): 887-94. Wahsha, M., Bini, C., Fontana, S., Washa, A. and Zilioli, D. (2011): Toxicity assessment of contaminated soils from a mining area in Northeast Italy by using lipid peroxidation assay, Journal of Geochemical Exploration, 113: 112117 1) Alexandru Ioan Cuza University of Jassy, Romania * ciornea@uaic.ro

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Analele tiinifice ale Universitii Alexandru Ioan Cuza, Seciunea Genetic i Biologie Molecular, TOM XIV, 2013

EVALUATING THE PHYSIOLOGICAL AND BIOCHEMICAL RESPONSE OF CERTAIN FOREST SPECIES IN THE CONDITION OF ANTHROPOGENIC POLLUTION IN CRUCEA, SUCEAVA COUNTY
MARIUS VIOREL ONICIUC1, ELENA TUTU1, DUMITRU COJOCARU1, ELENA CIORNEA1*
Keywords: uranium pollution, chlorophyll, carotens, dehydrogenase, catalase, peroxidase Abstract: Most economic activities that constitute the progress generator of an unprecedented development of the human society had led to a multiple and sever impact on the ecosystems. The scale in which this anthropogenic impact manifests itself can vary greatly, being conditioned by the general and local dynamics of the pollution generating processes. For this reason, there had been made some necessary investigations in order to evaluate its dimensions. Considered to be polluting for the forest ecosystem and the surrounding area, the target of this study had been the uranium mine at Crucea, located in Suceava Country. Taking into account the fact that depending on the quantity of the polluting material substances to which the species are exposed, different physiological and biochemical effects appear such as the reduction of the photosynthesis due to the degradation of the chlorophyll, an increase in the respiratory frequency, changes in the metabolism of the proteins and the enzymatic activity, the following physiological and biochemical indices where measured: catalase, peroxidase and the assimilating pigments that are included in the photoreceptors of the two photosystems integrated in the photosynthesis process, chlorophyll a and chlorophyll b. Moreover, the chlorophyll a/ chlorophyll b ratio, the caretonoid pigments and the relation between the chlorophyll and the carotenes where taken into account while a determination was made on the activity of the Krebs cycle dehydrogenases. Researches had been made on foliar material sampled from different species of gymnosperms and angiosperms and on soil samples taken in the contaminated area (Crucea) as well as in the control area (Putna, Suceava County).

INTRODUCTION
The 25 year exploitation of uranium at Crucea created three deposits, two of them situated on the territory of Crucea and another in Ostra, Botuana, Suceava County, having as a consequence the contamination of the area. This is produced, especially by the sterile deposits, by the ores with low uranium content and of the mine waters, all of them leading to an increase in the radioactivity of the soil. It is worth to keep in mind the fact that due to the traditional methods of exploitation that rely on moving large amounts of mineralized rocks and sterile, the mining operations created over 30 solid radioactive dumping sites in the CruceaBotuana area (Bistria Mountains) (Petrescu, L. et al., 2010), that are disposed in the vicinity of the mining facilities (Petrescu, L. and Bilal, E., 2007). This facts make the Crucea site to have the highest environmental radioactivity, even more higher as a series of uranium radionuclides had been found in high amount in the river sediments as well as in the surface waters (Requia, K., 2001). Very little information is available regarding the radiotoxicity and the chemotoxicity of the uranium on plants at cell level. However, the phenomenon of bioaccumulation of the radionuclides produces trough uranium fusion in tissues had been already mentioned, this process leading to potential lethal and sub lethal effects on the organisms composing an ecosystem, because the auto isotopes are part of the flux between the system and between the two systems (Hossner, L.R. et al., 1998; Auerbach, S.I., 2006). According to the existing data in the literature, the sub lethal effects of the uranium on plants can manifest themselves at radicular level, stopping growth trough death of the leaves at foliar levels, lesions on the stem and on the petiole but also trough a abnormal number of chromosomes, reflected trough their shrinking in the cellular nuclei (Ripley, E.A. et al., 1996). The dispersion of the uranium, thorium and strontium in the sediments of this mining area had been studied (Petrescu, L. and Bilal, E., 2006), the bioavailability of those actinides in the soil decreasing in the following order: Th(21,91%) > U(6,20%) > Sr(3,01%). Picea abies i Abies alba had been found to be coniferous that accumulate large amounts of uranium, with the greatest concentrations in roots, followed by the followed by branches and leaves (needles); while the strontium and thorium are concentrated in roots, the strontium is more concentrated in the needles and the branches of the evergreen gymnosperms than in their roots. The assimilation of these elements is done in a passive manner, as they are not necessary to the physiological and biochemical processes of the plants, as Picea abies and Abies alba are two species capable of being excellent bio-accumulators on the long term, thus diminishing the amount of mobile natural radioactive elements. Starting from these data, the mode in which these bioaccumulation is reflected in the photosynthesis process and in the metabolically mechanisms of the leaf, had constituted, together with the study of the microbial activity in the soil, the objective of the present research.

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Marius Viorel Oniciuc et al Evaluating the physiological and biochemical response of certain forest species in the condition of anthropogenic pollution in Crucea, Suceava County

MATERIALS AND METHODS

The investigations had been made on foliar material sampled in May 2011 from gymnosperms of the coniferous family such as Picea abies L.Karst., Larix decidua Mill. and angiosperms like Salix ssp alba L., Populus tremula, Betula verrucosa Ehrh. and Fagus sylvatica, that where under the influence of pollution (Crucea) as well as from species that were found in the control area that was considered to be unpolluted (Putna, Suceava County). The determination of the activity of the catalase was made using the spectrophotometric Sinha method (Artenie et al., 2007), while the activity of the peroxides was evidentiated by means of the photocolorimetric Gudkova, L. and Degtiari, G method (Artenie et al., 2007) with ortho-dianisidine. The content of assimilating pigments (chlorophyll a, chlorophyll b) and caretonoid pigments was dosed using the colorimetric method after their extraction with acetone, while the quantitative measuring of the activity of dehydrogenases was made through the colorimetric method with triphenyltetrazolium (Cojocaru, D., 2009). The activity of the dehydrogenases in soil was determined using Casida method with some modifications (Dragan-Bularda, M., 2000).

RESULTS AND DISCUSSION The radionuclides are capable of producing oxidative stress and redox cell disequilibrium, as it can be seen in fig. nr. 1. The cumulated influence of uranium, thorium, strontium on different forest species that grow in Crucea, confirm the fact that the spruce (Picea abies) is a species with a high phytoremediation capacity, taking into account the high level of enzymatic activity (343.124 CU), through which the accumulation of the hydrogen peroxide at the level of the foliar apparatus is slowed. In the leaf fragments taken from Salix alba it had been seen a lower activity than that found in the spruce samples (320.0125 CU), but much higher than that recorded at the same species in areas considered uncontaminated (83.158 CU), while in the foliar samples taken from Populus tremula it was also found a strong influence on the activity of this oxidoreductase (286.038 CU) compared to that found in the control areas (75.14 CU). The fact that the ions of the actinides present in the soil at Crucea intensifies the oxidative stress at foliar level is also shown by the consistent notations at Betula verrucosa (276.22 CU) and Larix decidua (212.04 CU), a fact confirmed by the values found in the control area at the birch (49.0125 CU) and larch (37.855 CU). A close analysis of the results regarding the activity of the peroxidase, as it is shown in fig. nr. 1, show that uranium had a strong inducting effect on this enzyme in the case of Populus tremula (15.16 PU), a possible explanation being the fact that the enzyme, acting complementary with the catalase, its running his activity at a lower concentration of hydrogen peroxide, while, as it is well known, catalase acts at higher concentrations. So, taking into account the fact that in the control area (Putna) the activity of the peroxidase is much smaller (4.6 PU), although we are inclined to affirm that Populus tremula is a species sensitive to pollution with radioactive metals and the peroxidase can be considered a veritable biochemical marker (the activity of the enzyme is high at all species taken into account when compared to that seen in the samples taken from the uncontaminated account), we consider that further investigations are necessary, in order to monitor the activity of both enzymes in regard to seasons and years, before we can be sure about anything. In any case, the reduction of the activity of the oxidative stress enzymes is, considering to certain authors, a clear marker for the significant reduction of the plant development in the conditions of a rich-uranium environment, our researches certifying the conclusion of some authors that the presence of the uranium has as a consequence an enrichment of the antioxidative defense mechanisms (Vandenhove, H., A. et al., 2005). Altough we could be inclined to say that the radioactive metals present in the mining area from Crucea Botuana act synergic as catalyzers in the antioxidant defensive and also taking

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Analele tiinifice ale Universitii Alexandru Ioan Cuza, Seciunea Genetic i Biologie Molecular, TOM XIV, 2013

into account the bioaccumulating capacity of the spruce (770 ppm uranium per total in plant) according to Petrescu, L. and Bilal, E., 2006, but also the phytostabilisation effects of certain plants that reduce the biodisponibility and the migration beyond contaminated sites containing preferentially the uranium and thorium at a higher level than in the nearby soil without harmful effects, we cannot eliminate the hypothesis that the other forest species (Salix alba sp, Betulla verrucosa, Populus tremula i Larix decidua) might have the same accumulative properties. The retention capacity varies with plant type, with its tissues (for example, the concentration of uranium in the tissues of the coniferous that belong to the Crucea ecosystem decreases in the following order: roots > body > branches > needles), with the type of radioactive wastes (the bioavailability of the metals in soil decreases in the following order: Th (21.91%) > U (6.20%) > Sr (3.01%) (Popescu, L. and Bilal, E., 2006).
400 350 300 250 CU 200 150 100 50 0 Populus tremula Betula verucosa Populus tremula Picea abies Picea abies Salix spp alba Salix spp alba Betula verucosa Larix decidua Larix decidua 16 14 12 10 8 6 4 2 0 PU

Crucea Catalase

Putna Peroxidase

Fig.1. The activity of the catalase and peroxidase at different species of gymnosperms and angiosperms in the areas polluted with uranium (Crucea) and areas considered unpolluted (Putna)

At a careful analysis of the graphic results regarding the activity of the Krebs cycle dehydrogenases in the foliar material of the forest species taken into account, as it is shown in fig. 2 and 3 it can be observed that the uranium and the other actinides present in the medium are capable of inducing a much more intense energetic metabolism than in the its absence in the leaves taken from the species located in the control area. It is known that the isocitrate dehydrogenase with peroxisomal localization is involved in the cellular senescence (del Rio, L.A. et al., 1998). As senescence is a process that can also be caused by pollution, the NADPH present in the peroxisomes is regulated by this enzyme, a fact that also confers to it a central role in the supplying of the cell with NADPH, necessary in the antioxidative protection of the mitochondria, while being in the same time the reducing power of certain biosynthetic processes and of some metabolically situations in which the activity of the pentosophospates pathway is diminished or blocked (Mller, I. and Rasmusson, G., 1998, Chen, R. et al., 1988, cited by Corpas, J.C. et al., 1999). It cannot be ruled out that the radioactive elements present in the environment might induce a strong activity of this enzyme, together with the mitochondrial isoenzyme Populus tremula (39.993 g formazane/g. vegetal mat.), Salix alba

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Marius Viorel Oniciuc et al Evaluating the physiological and biochemical response of certain forest species in the condition of anthropogenic pollution in Crucea, Suceava County

(24.216 g formazane/g. vegetal mat.), Fagus sylvatica (21.746 g formazane/g. vegetal mat.) and Betula verrucosa (20.7345 g formazane/g. vegetal mat.), in order to maintain in the citosol and mitochondria the necessary concentration of NADPH and NADH that might contribute to the diminishing of the oxidative stress and to the lengthening of the vegetal cell`s viability. The biochemical determinations had shown the fact that Salix alba, Populus tremula, Betula verrucosa i Picea abies from areas contaminated with uranium had a high quantity of ketoglutarate that necessitates a high activity of the corresponding enzyme. The possibility that the substrate resulted from the oxidative decarboxilation of the isocitrate might have entered in the connected anabolic pathways cannot be excluded, considering the much smaller activity of the enzyme compared to the isocitrate dehidrogenase at Populus tremula (19.444 g formazane/g. vegetal mat.) i Fagus sylvatica (11.876 g formazane/g. vegetal mat).
40 g formazan/g biological matter 35 30 25 20 15 10 5 0
a ab P ic e a sa ies ul a dua al ba va tic tre m rruco deci ss p s syl ulus la v e Sal ix Lari x Fagu P op Be tu

Isocitrate-DH

Malate - DH

Succinate - DH

-Keto-glutarate - DH

Fig.2. The activity of the Krebs cycle dehydrogenases at different species of angiosperms and gymnosperms from the area contaminated with uranium, thorium and strontium (Crucea)

Regarding the activity of the succinate dehidrogenase, it can be seen that in the foliar samples of Fagus sylvatica and Larix deciduas, its activity is much higher than that of the ketoglutarate dehidrogenase (16.950 g formazane/g. vegetal mat. and 6.080 g formazan/g. vegetal mat). A possible explanation might be that the uranium stimulates the production of asparagines and aspartic acid, and these accumulate in plants under stressful conditions (Heuer, B., 1999), while they can be utilized for furnishing energy in the tricarboxillic acids cycle where they enter trough the junction with the succinate, once their role in the vegetal cell is finished. At all forest species discussed in this study, with the exception of Betula verrucosa, the activity of the malate dehydrogenase was higher than that of succinate dehydrogenase and ketoglutarate dehydrogenase. It is known the fact that in the case of other metallic pollutants (Zn, Cd, for example), at the tolerant ecotypes, the malate plays a significant role in the removal of the metallic particles from the cytoplasm (Lazr, Gh., 2002), so it cannot be excluded such a comportment in the case of the uranium, but also, the thorium and cesium present at Crucea.

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9 g formazan/g biological matter 8 7 6 5 4 3 2 1 0

a ies ul a ti ca osa al ba cidu a ab ylva tre m rruc ss p x de Pic e us s la v e ulus Lari Salix Fag P op Be tu

Isocitrate - DH

Malate - DH

Succinate - DH

-ketoglutarate - DH

Fig.3. The activity of the Krebs cycle dehydrogenases at different species of gymnosperms and angiosperms in the area considered not polluted (Putna)

Another hypothesis takes into account the fact that the reducing of the malate and implicitly, of the malate dehydrogenase might take place in the same time with an unexpected rise of the photosynthesis rate (Nunes-Nesi, A. et al., cited by Finkemeyer, I. and Sweetlove L., J., 2009). That is why, in order to confirm these new hypothesis, further studies are necessary. After Bogoev et al. (2010), following the supplementary investigations necessitated by the absence of a known impact of uranium on indicator plants, the pigment content from the leafs taken from forest species in contaminated areas does not differ significantly from that in the control area, the quantity of chlorophyll a and chlorophyll b from the sampled foliar material being put by the author on certain perturbation of the hydric regime in the area and on the texture of the soil rather than on uranium toxicity. In the researches made by us, there can be observed in an identical manner, the lack of a visible impact of uranium on the quantity of a and b chlorophyll at all species taken into account, being no visible differences between the species from the contaminated area and those from the control zone. In regard to the carotenoid pigments, their concentration was smaller at Betula verrucosa in the foliar fragments taken at Crucea.

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Chlorophyll a 20 mg/g biological matter 18 16 14 12 10 8 6 4 2 0 Populus tremula Betula verucosa

Chlorophyll b

Carotens

Populus tremula

Picea abies

Picea abies

Salix spp alba

Salix spp alba

Betula verucosa

Larix decidua

Crucea

Putna

Fig.4. The content of a chlorophyll, b chlorophyll and carotens at different species of gymnosperms and angiosperms from areas polluted with uranium, thorium and strontium (Crucea) and unpolluted areas (Putna) Tabel 1- The ratio of the assimilating pigments at different forest species in areas contaminated with uranium thorium and strontium (Crucea) and unpolluted areas (Putna) Area Species Ca/Cb ratio Pc/(Ca+Cb) ratio Picea abies 1.85 0.188 Populus tremula 1.392 0.133 Crucea Salix alba 1.311 0.163 Betula verrucosa 1.773 0.283 Larix decidua 1.921 0.206 Populus tremula 1.662 0.168 Salix alba 1.448 0.141 Putna Betula verrucosa 1.046 0.130 Larix decidua 1.857 0.263 Fagus sylvatica 1.902 0.101

Some researches regarding the impact of uranium show that not all negative effects are due to the singular activity of the radio nuclides on vegetation, soil and microbial community, but also due to the chemical toxicity of a complex made up by uranium, heavy metals, non-metallic ores and compounds used in the preparing of the uranium (for example sulfuric acid, kerosene, etc.) (Bogoev et al., 2010). The bacterial communities constitute a veritable biological basis for evaluating the impact of mining exploitation (Islam et al., 2011), uranium being one of the elements capable of generating constant pressure on those, some authors suggesting even the possible presence of a redox uranium cycle at this level (Mondani et al., 2011). The role of the bacteria in the circuit of the uranium in nature is very well known. Gad, G.M. (2010), Kenarova A. et al. (2010) say that certain bacterias show tolerance towards uranium, while other have the ability to accumulate and transform it for example, the reducing of Fe III by certain microbial stems can lead to the release of the radon from the remains of uranium mines (Landa, E.R and Gray, J.R., 1995). The negative effects on the bacterial communities located in the soil are registered only at the level of their activity, not in their numerical presence; a fact conformed also by other studies (Rastogy et al., 2010). At the level of activity, the pollution of the environment with uranium and other chemical compounds is reflected well in the level of the enzymatic activity of the

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dehydrogenases complex; in the opinion of Bogoev et al. (2010), these enzymes can be used as sensible indicators in evaluating the adverse effects of uranium on the natural communities.
18 16 g formazane/g dry soil 14 12 10 8 6 4 2 0
e-D itrat I soc H i nate Succ - DH H DH te - D ntial Mal a Pote al D H Ac tu

depth (15 - 30 cm)

surface (0 - 15 cm)

Fig.5. The activity of the Krebs cycle dehydrogenases in the soil samples taken from the area contaminated with uranium, thorium and strontium (Crucea)

Indicator of the intensity of soil degradation in areas polluted with radioactive metals, but also a good marker of the activity of the microbial community in whole, indifferent of the bacterial or fungal species involved, the dehydrogenases reflect many types the functioning pattern of the intercellular enzymes involved in the metabolic reactions that imply metabolic energy transfer. That is why, at a first analysis of the results from the graph exposed in 5 and 6 fig. it can easily be seen that unlike the microbial consortiums from the soil of Putna, the microbial community in the radioactively polluted area from Crucea prefers a higher dehydrogenasic activity for the first two stages of the Krebs cycle, a fact that proves their essential role in the initial stages of the organic material oxidation at soil level, both in-depth as well as on the surface. The fumarate constitutes a substrate little used by the microbial constituents of the soil, given the fact that the activity of the flavindehydrogenase that catalyses the conversion of the succinic acid to fumaric acid is very small in comparison with the activity of the isocitrate-dehydrogenase and oxoglutarate-dehydrogenase at the entire level of the in-depth microbial consortium (1.882 g formazane/g. dried soil compared to 14.740 g formazan/g. dried soil, respectively 15.432 g formazane/g. dried soil). Not the same thing can be said about the malate, the enzyme that catabolises it having a higher level then that of the succinatedehydrogenase in-depht (3.746 g formazane/g. dried soil), while at the surface the level is the most reduced compared to all enzymes of the Krebs cycle (1.574 g formazane/g. dried soil). What attracts is the fact that at surface, the activity of the -ketoglutarate dehydrogenase is higher, the substrate metabolized by this enzyme entering the Krebs cycle trough other metabolic pathways.

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Marius Viorel Oniciuc et al Evaluating the physiological and biochemical response of certain forest species in the condition of anthropogenic pollution in Crucea, Suceava County

18 16 g formazane/g dry soil 14 12 10 8 6 4 2 0

-D itrat e Isoc H - DH inate Succ H DH te - D ntial Mala Pote al D H Ac tu

depth (15 - 30 cm)

surface (0 - 15 cm)

Fig.6. The activity of the Krebs cycle dehydrogenases in the soil samples taken from the uncontaminated area (Putna)

The rising activity of dehydrogenases from surface to in-depth both in the polluted area as well as in the control area, is related, probably, to the changing of the aeration of the soil. The commuting of the microorganisms metabolism from aerobic to anaerobic, due to the exhaustion of the oxygen, is accompanied by a lowering of the redox potential, the alteration of the pH and the rising concentration of reduced mineral forms (for example, the reducing of Fe (III) liberates radon that accumulates) and as a consequence of this biochemical transformation, there is an increase in the activity of the dehydrogenases in the soil (Brzeziska M. et al., 1998). Its worth noticing that in the soil samples considered unpolluted, the succinate dehydrogenase had, indepth, a higher intensity of its activity than in the polluted area, a fact that determines us to conclude that either one of the three radioactive elements present in the area is capable of inhibiting this respiratory enzyme, either their synergy or that of the wastes remaining in environment due to the mining exploitations might be responsible for this effect. It is easy to conclude that the obtained data cannot be generalized; they are having an more rough character, being necessary to certify the hypotheses that stood at the basis of the justification of the obtained results. There are necessary repeated biochemical determinations, both seasonal and annual, but also supplementary morphological and physiological analyses. CONCLUSIONS The presence of actinides in the environment is seconded by a strong oxidative stress at all the species taken into account, this fact being reflected by a visibly higher activity of the catalase and peroxidase, compared to the levels registered at the species in the control area. Both in the polluted area as well as in the control area, the Krebs cycle had a balanced evolution, the activity of the Krebs cycle dehydrogenases being higher at the forest species located in the polluted area; it noticeable the high activity of the isocitrate dehydrogenase in the foliar material taken from Populus tremula. There was no visible impact of the uranium and other actinides on the quantities of a and b chlorophyll at the species taken into account, there were no significant differences between

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the species from the contaminated areas and those from the control area; was registered only a lower level of the caretenoid pigment at Betula verrucosa. In the radioactive soil, the dehydrogenasic activity of the in-depth microbial consortium was significantly higher in the case of isocitrate dehydrogenase and -ketoglutaratedehidrogenase, while the succinate dehydrogenase and the malate dehydrogenase had lower levels, compared to those found in the samples taken from the surface where the dehydrogenasic activity at all soil probes was visibly lower than that from the in-depth ones. REFERENCES
Artenie, V., Ungureanu, E., Negur, A. M., (2008): Metode de investigare a metabolismului glucidic i lipidic manual de lucrri practice. Editura Pim, Iai. Auerbach, S.I., (2006): Radiation Ecology, in Encyclopedia of Environmental Science and Engineering, Fifth Edition, Volumes One and Two, Edited by James R . Pfafflin and Edward N., Ziegler, CRC Press, pages 10411046. Bogoev, V., Kenarova, A., Traykov, I., Tzonev, R., Tzekova, R., Stoyanova, T., Boteva, S. and Parleva, N., (2010): Natural communities of uranium mining impacted area in the vicinity of the Sekonos Village, Biotechnology & Biotechnological Equipment, volume 24, no.2, pages 240-246. Brzeziska, M., Stpniewska, Z. and Stpniewski W., (1998): Soil oxygen status and dehydrogenase activity. Soil Biology & Biochemistry, volume 30, pages 1783-1790. Cojocaru, D.C., (2009): Enzimologie practic, Editura Tehnopress, Iai. DelRo, L.A., Pastori, G.M.,Palma, J.M.,Sandalio, L.M., Sevilla, F., Corpas, F.J.,Jimnez, A., Lpez-Huertas, E. and Hernndez, J.A., (1998): The activated oxygen role of peroxisomes in senescence. Plant Physiol., vol. 116, pages 11951200. Drgan-Bularda, M., (2000): Microbiologie general lucrri practice, Univ. Babe-Bolyai, Cluj-Napoca Finkemeyer, I., Sweetlove L.J., (2009): The role of malate in plant homeostasis, F1000 Biol. Reports, pages 1:47 Gadd, G.M., (2010): Metals, minerals and microbes: geomicrobiology and bioremediation, Microbiology, volume. 156 no. 3, pages 609-643. Hossner, L.R., Loeppert, R.H., Newton, R.J., Szaniszlo, P.J., Moses- Attrep, J., (1998): Literature review: phytoaccumulation of chromium, uranium and plutonium in plant systems, Amarillo National Resources Center for Plutonium. Islam, E., Dhal, P.K., Kazy, S.K, and Sar., P., (2011): Molecular analysis of bacterial communities in uranium ores and surrounding soils from Banduhurang open cast uranium mine, India: A comparative study., J. Environ. Sci. Health. A Tox. Hazard Subst. Environ. Eng., vol.46, no.3, pages 271-80. Kenarova, A., Radeva, G., Danova, I., Boteva, S. and Dimitrova, I., (2010): Soil bacterial abundance and diversity of uranium impacted area in North Western Pirin Mountain, Biotechnolog. & Biotechnolog. EQ. 24, Special Editon online, Second Balkan Conference on Biology, 21-23 May 2010, Plovdiv, 50 years University Plovdiv. Landa, E.R., Gray, J.R., (1995): US Geological Survey research on the environmental fate of uranium mining and milling wastes, Enviromental Geology, volume 26, no. 1, pages 19-31. Lazr, Gh., (2002): Mecanisme de toleran a plantelor la metale, Constantin Brncui University, Engineering Faculty, Universitys Days, 8th International Conference, Tg. Jiu, may, 24-26. Mondani, L., Benzerara, K., Carrire, M., Christen, R., Mamindy-Pajany, Y., Fvrier, L., Marmier, N., Achouak, W., Nardoux, P., Berthomieu, C., Chapon, V., (2011): Influence of uranium on bacterial communities: a comparison of natural uranium-rich soils with controls. PLoS One., volume 6, no.10, e25771. Petrescu, L., Bilal, E., (2006): Natural actinides studies in conifers grown on uranium mining dumps (the East Carpathians, Romnia), Carpathians Journal Of Earth and Environmental Sciences, vl. 1, no.1, pages 63-80. Petrescu L., Bilal E., (2007): Environmental impact assessment of a uranium mine, East Carpathians, Romania: Metal distribution and partitioning of U and Th. Carpathian, Journal of Earth and Environmental Sciences, vol. 2, no. 1, pages 39-50. Petrescu, L., Bilal, E. and Iatan, L.E., (2010): The impact of a uranium mining site on the stream sediments (Crucea mine, Romnia), Scientific Annals of the Schol of Geology, vol. 100, pages 121-126. Rastogi, G., Osman, S., Vaishampayan, P.A., Andersen, G.L., Stetler, L.D., Sani, R.K., (2010): Microbial diversity in uranium mining-impacted soils as revealed by high-density 16S microarray and clone library, Microb. Ecol., volume 59, no.1, pages 94-108. Requia, K., (2001): Enironmental Performance Reviews Series, Romnia, No.13 Economic Comission for Europe, Comitee on Environmental Policy, United Nations, New-York and Geneva, pages 139-152. Ripley, E. A., Redmann, R. E., Crowder, A. A., (1996): The Environmental Effects of Mining. St. Lucie Press, Florida.

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Marius Viorel Oniciuc et al Evaluating the physiological and biochemical response of certain forest species in the condition of anthropogenic pollution in Crucea, Suceava County Vandenhove, H., Cuypers, A., Van Hees, M. and Wannijn, J., (2005): Effect of uranium and cadmium uptake on oxidative stress reactions for Phaseolus vulgaris. Uranium in the Environment: Mining Impact and Consequences, Eds. B.J. Merkel and A. Hasche-Berger, Springer-Verlag, Berlin-Heidelberg., pages 175-182. 1) Alexandru Ioan Cuza University of Jassy, Romania * ciornea@uaic.ro

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