Analele tiinifice ale Universitii Alexandru Ioan Cuza, Seciunea Genetic i Biologie Molecular, TOM XIII, 2012

STUDIES REGARDING THE INFLUENCE OF CERTAIN INORGANIC POLLUTANTS ON THE QUALITY OF THE SOIL FROM MINING AREAS IN SUCEAVA COUNTY
ELENA TUTU 1, ONICIUC MARIUS VIOREL1, ELENA CIORNEA1*
Keywords: Climani Mountains, Tarnia, mining exploitations, soil pollution, sulphur, copper, zinc, barite, microorganisms, dehydrogenases. Abstract: The correct functioning of the soil is assured by the biologic activity of the microorganisms at this level and the enzymatic activity reflects the metabolic processes that take place in various environmental conditions, being also a very good indicator for both natural and anthropic disturbances, presenting a quick response to the changes induced by these in the soil ecosystem. The precise identification of the effects caused by sulphur exploitation and its contamination with heavy metals on certain microbiological parameters (the activity of the isocitrate-dehydrogenase, ketoglutarate-dehydrogenase, succinate-dehydrogenase, malate-dehydrogenase) is the main purpose of this study. The experiment was made on probes sampled from the Climani Mountains, Tarnia and Rdui (the last is considered to be unpolluted area), both in-depth as well as on the soil surface. The method that was used is based mainly on the capacity of the dehydrogenases to transfer the excess hydrogen from different substrates to 2,3,5-triphenil-tetrazolium chloride, that can be reduced and goes into red colored triphenil-formazane, the intensity of the formazane`s color being determined spectrophotometrically. The analysis of the obtained results show that the level of activity of the microorganisms in the soil, expressed trough the global dehydrogenasic activity of the microbial consortium, can be considered as an indicator to the situation of the examined ecosystem, reflected by the disponibility of the organic matter on soil surface and by the intensity of the transformation and circulation process of the organic matter.

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INTRODUCTION MATERIALS AND METHODS
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The degradation of the aquatic and terrestrial ecosystems due to mining activity trough with the copper and zinc concentrates together with pyrite had accumulated at Tarnia, Suceava County, constitutes at the present hour one of the most sever environmental problem, due to the fact that unlike the organic pollutants, the metals cannot be degraded, their concentration in the upper layers of the soil causing turbulences in their homeostasis. In their turn, the barite in preparation activity that takes place in the same area, trough which oxides such as SiO2, Al2O3 and Fe2O3 are liberated into the air (Ionce, A., 2010) also represents a component that further enhances the destructive effects on the ecosystems. Also, the mining activities that took place for 30 years in the Climani Mountains, had as a result the direct emission into the air of certain emissions such as sulphur dioxide, sulphur hydrogen and solid particles, creating the conditions for acid precipitations to appear, with direct destructive effects on the ecological equilibrium of the different biocenosis. Trough the depositing of the mining wastes, trough the infiltration of waste-waters and toxic deposits driven directly on soil or from air and precipitations, the soil is polluted, while certain ecological unbalances appear, sometimes very hard to heal (Dioiu, V. and Oean, V. 2007). The most effective indicators of the degree of deterioration are the microorganisms and their enzymes, these playing, thus, a very important role in the ecology of the soil, being involved in its physicochemical properties, in its fertility and health. The soil enzymes, having different localizations, are associated with various biotic and abiotic components, such the difference between the inter-cellular and extra-cellular activities are very important, as the last is not directly correlated with the activity of the microbial consortiums, or at least, is not as sensible to all the physiological and environmental variations that affect the microorganisms (Burns, 1982 and Nannipieri, 1994 quoted by Ciardi, C., 1998). Dehydrogenases are strictly intra-cellular enzymes, representing the reflection of the basal respiration of a microbial community, supposing the collaboration of certain enzymes that catalyses the transfer of hydrogen and electrons from one composite to another (Nannipieri, P. et al., 2003). On this principle, of using synthetic electronic acceptors, are based, like in other soil biochemistry tests, the methods of quantitative dosing of the dehydrogenasic activity in lab conditions used in this present study, in which we try to see the impact of mining activities in certain areas of the Suceava County on the soil biocenosis and its degree of deterioration.

The soil probes were sampled in May 2012, from areas located near the Climani Mining Exploatation, ale the Tarnia plant for preparing non-ferrous copper and zinc ores, but also from areas considered to be non-polluted (Rdui).

Elena Tutu et al Studies regarding the influence of certain inorganic pollutants on the quality of the soil from mining areas in Suceava county Because the superficial layer had proved to be dry in certain polluted areas, the probes were sampled from two different depths: from the surface (0 15 cm) and from in-depth (15-30 cm). The activity of the dehydrogenases was determined trough the reduction of the 2,3,5-triphenil-tetrazolium chloride using the Casida et al., 1964 method (Drgan-Bularda, M., 2000). After 24 hr at 37C, the triphenyl formazan (TPF) released was extracted with methanol and assayed at 540 nm in an UV spectrophotometer. The unit of dehydrogenase activity was g TPF/g dry soil.

RESULTS AND DISCUSSION It is a known fact that the key microbial enzymes from the superior layers of the terrestrial crust are actively participating not only in processes such as de degradation of the celluloses and other plant residues, but also in the transformation suffered at this level by certain elements such as sulphur (Rusel,S., 2005 quoted by Kukarski, J. et al., 2009). In accord to the data obtained by numerous scientists, according to the results obtained by us, the dehydrogenases are sensible parameters to use in the evaluation of the sulphur`s toxicity on the soil (Dowarah, J. et al., 2009, Levik, V. et al., 2007).

The analysis of the data registered trough the investigations of the activity of Krebs cycle dehydrogenases, of potential and actual dehydrogenases, as are reproduced in fig. 1, show enzyme behaviors dependent on the soil pollution, as well as on the depth from which the samples where taken. An important aspect that one must take into account is the fact that the four enzymes, translate, despite of the low levels of the dehydrogenases activity, an image of a balanced Krebs cycle, that could be the expression of the activity of certain teams of consortium microorganism`s that act synchronously on distinct metabolic pathways or a prevalence of a certain microbial group that follows this path. In accord with the volcanic mother-rock and its cold climatic conditions, the superior coating of the Climani Mountains is predominantly acid, while in-depth, the supplementation with water is much better and the soil is richer in humus (Erzberger, P. et. al., 2012). The global metabolism of the surface microbiota is characterized by a dehydrogenasic activity with very low values, in an anthropical environment (Climani) the proportion between the potential and actual dehydrogenases being much higher than in the natural environment (Rdui). The temperature and water content of the soil act in a synergic manner at this level together with the pH of the

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30 25 20 15 10 5 0 g formazane/g dry soil
itrat I soc e-D H DH te ci na S uc ate Mal - DH l DH nti a Pote DH ual Ac t

depth (15 - 30 cm)

surface (0 - 15 cm)

Fig.1 The activity of dehydrogenases in the probes sampled from the soil polluted with anthropic sulphur (Climani Mountains)

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Analele tiinifice ale Universitii Alexandru Ioan Cuza, Seciunea Genetic i Biologie Molecular, TOM XIII, 2012

soil, being capable of strongly influencing the redox status of the soil, which in its turn affect the activity of the dehydrogenases (Brzezinska, M. et. al., 1998). Moreover, it is well known that the activity of this enzyme is much smaller in the soil with no vegetation (Bastida et al. quoted by Karaka, A. et al., 2011), in the sulphur circuit going at this level, the bacterias having an overwhelming role (Tang, K. et al., 2009). It was proven that these are much more effective than the fungi that prefer the rhizospheres, the actinomycetes choosing for soils with non-acidic pH (pH>5). This process is taking place in areas with trees with resistance to pollution, where indepth, the radicular exudates are releasing the carbon sources necessary for the mycorrhizal organisms, a fact that also happens in this case. It cannot be ruled out that for this reason, the activity of the dehydrogenases from the upper layer is lower, given the fact that until the month of May, the organic material existing at this level was already decomposed and used in different metabolic processes by plants and the microbiota of the soil. The level of isocitrate - dehydrogenase was shown in our analyses to be, directly proportional with the depth, being two times higher in depth than at surface (4.753 g formazane/g. vegetal mat. between 0-15 cm and, 9.469 g formazane/g. vegetal mat. between 1530 cm), being in the same time, the highest compared to the other dehydrogenases of the studied microbial consortium`s Krebs cycle. The lower amplitude of the oxoglutarate - dehydrogenase suggest the joining of certain anabolic pathways to the cycle of the tricarboxilic acids that form amino acids, in both soil samples taken at Climani (2.802 g formazane/g. vegetal mat. at terrestrial surface and double in-depth 5.833 g formazane/g. vegetal mat.). The succinate is the substrate preferred by the microorganisms from this area, because the metabolically implication of the succinate - dehydrogenase in the energetic circuit is more intense than that of the malate - dehydrogenase, in both levels of the microbiocenosis from the Climani Mountains 2.641 g formazane/g. vegetal mat., 7.512 g formazane/g. vegetal mat., respectively, 3.277 g formazane/g. vegetal mat. and 8.877 g formazane/g. vegetal mat). The role of the precipitations in the diminished dehydrogenasic activity in the exterior must be taken into account, these being capable to clean the atmosphere of noxes, diminishing their concentration in the air but redirecting it towards the vegetation, surface waters and soil. Moreover, through its manifestation process, the precipitations have a great degree of erosion power, accelerating the washing of mining dumps and uncovered terrains (Ionce, A., 2010), a phenomenon that could had come together with a permanent diminishing of the bacterial inoculums from the surface of the Climani Mountains and implicitly, a more reduced enzymatic activity at this level. It is necessary to take into account when interpreting these results also the moment of the sampling of the soil probes, that means, to take into account the seasonal changes of the enzymatic activity, this being closely related to numerous other factors such as the soil type (andosoil and podsoil in the Climani Mountains), the degree of soil aeration, the level of humidity and its temperature, on the type of microorganisms, on the influx of organic matter. A visibly insignificant activity of the biological processes in the soil was seen throughout the whole year in the Climani Mountains by Erzberger, P. et al. (2012), the changes in the bio-dynamic of the terrestrial crust during summer taking an ascending trend, that permit to fundament the thesis according to which the soil dehydrogenases have a higher activity during this season. Because of this, we consider that the obtained data represent only a stage of research, and more systematic tests are necessary, both seasonal and multi-annual in order to produce a thorough analysis of the influence of sulphur pollution on the soil microbiota.

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Elena Tutu et al Studies regarding the influence of certain inorganic pollutants on the quality of the soil from mining areas in Suceava county

In conformity with Kizilkaya, R. et al. (2004), Oliveira A. and Pampulha, M.A. (2006), Nadgorska-Socha et al. (2006), the dehydrogenases can be seen as veritable biological markers of long term soil contamination with heavy metals, the researches signaling a significant negative correlation between the total content of heavy metals and the soil characteristics. Trough their physicochemical properties and their acting mechanisms, the metals present in the environment can decisively influence on long and short terms the properties of the soil. The soil is capable of immobilizing heavy metals in the condition of a growth of a pH that plays a decisive role unless its top value is slightly alkaline. However, in Tarnia the soils had been found to have a pH situated in the range of acids (Dioiu, V. and Oean, V., 2007). The study regarding the character of the interaction of heavy metals that pollute the Tarnia area with the microbial complex of the soil had permitted us to observe the fact that the actual dehydrogenase activity, the potential dehydrogenase activity and the ratio between those represent good indicators of an intense contamination of the area.

This fact is explainable, because besides the contamination resulted from the mining exploitation itself, a rise of the soils acidification together with a rise in the content of sulfates over the intervention limit, and values even over alert limit for copper and zinc, is due to the high amount of precipitations fallen in the year 2006 that had caused a series of damages at the decantation pond, that triggered a series of dump slides downstream (Dioiu, V. and Oean, V., 2007). In acid soils (pH 4.2-6.6) Zn has a very high mobility, while Cu is practically immobile, and in the soils with pH from neuter to alkaline (pH 6.7-7.8) (Fuller quoted by Aydinalp, C. and Marinova, S., 2003), Zn has a moderate mobility. This fact has a great importance in the unfolding of the enzymatic activity of the microbial consortium at soil level, because the excess heavy metals stationed at this level are capable to get fixed on the cell surface and alter their net charging load, being toxic to most bacteria trough the inhibition of the basic cellular functions, closely related to the energetic metabolism. Because of this, in the present study, the activity of the Krebs cycle dehydrogenases is low, being due probably to the activity of fungi that developed diverse strategies to resist metal toxicity, in the bibliographical data being mentioned the fact that

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18 16 14 12 10 8 6 4 2 0 g formazane/g dry soil
e-D itrat Isoc H DH te ci na S uc Mal - DH ate H al D enti P ot ual Ac t DH

depth (15 - 30 cm)

surface (0 - 15 cm)

Fig.2 The activity of some dehydrogenases in the probes sampled from the soil contaminated with Cu-Zn and barite ores (Tarnia, Suceava County)

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Analele tiinifice ale Universitii Alexandru Ioan Cuza, Seciunea Genetic i Biologie Molecular, TOM XIII, 2012

they had a higher tolerance towards heavy metals compared to the actinomycetes (who are the most sensible) and the bacteria. Besides the pH, other soil properties such as the capacity for cation changes, the content of organic matter, the content in iron oxides, aluminum, manganese and potential redox, determines the soils capacity to retain and immobilize heavy metals. These heavy the tendency to form complexes together with the organic materials from the soil (humic and fulvic acids) (Stevenson, F.J., 1983) the organic matter having thus, an essential role not only in the forming of these complexes, but also in their maintenance in changeable form, influencing thus, the bacterial activity in the soil and implicitly, the level of the enzymatic activities, the mobile metals being accessible to plants, while the less mobile ones, from less changeable complexes being accessible to the microbial community in the soil. The total oxidative activity of the microbial biomass involved in the energetic metabolism has in the soil probes sampled from the surface at Tarnia, a lower amplitude compared to the levels noted at higher depth, with two notable exceptions: the activity of ketoglutarate - dehydrogenase (0.950 g formazane/g. dried soil at surface compared to 0.895 g formazane/g. dried soil in-depth) and that of succinate - dehydrogenase, almost double at surface compared to in-depth (2.024 g formazane/g. dried soil to 1.339 g formazane/g. dried soil). For the isocitrate - dehydrogenase was registered a level of activity at 3.296 g formazane/g. dried soil in-depth, while the surface sample had only 2.901 g formazane/g. dried soil, while in the case of malate - dehydrogenase 2.604 g formazane/g. dried soil in depth and 2.043 g formazane/g. dried soil at the surface.
12 10 8 6 4 2 0

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g formazane/g dry soil
e-D itrat 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. 3 The activity of certain dehydrogenases in the probes sampled from the soil considered to be non-contaminated (Rdui)

The enzymes had been, also, active in soil probes considered non-contaminated, sampled from Rdui, where the dehydrogenasic activity is not comparable to that found in polluted areas, the amplitudes of manifestation being different (vastly superior to those from the areas under the incidence of contamination), having only the dept-related pattern. CONCLUSIONS Both in the Climani Mountains, as well as in Tarnia, the anthropic activities had provoked major disruptions of the soil quality. The soil is very affected by the presence of heavy metals or sulphur in concentrations which are much higher than the normal admitted limits, a fact that is

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Elena Tutu et al Studies regarding the influence of certain inorganic pollutants on the quality of the soil from mining areas in Suceava county

reflected at this level in the activity of the dehydrogenases belonging to the microbial consortiums. The ratio between the potential and real dehydrogenase is very high in the soil samples considered to be biological material under the influence of contamination, the enzymes being very good indicators of soil health, indicating the impact of anthropogenic activities on the associations of microorganisms. The oxireductases involved in the energetic metabolism (isocitrate - dehydrogenase, ketoglutarate - dehydrogenase, succinate - dehydrogenase, malate - dehydrogenase) are active at in all the investigated experimental combinations; their manifestations severely influenced by the presence of pollutants in the environment; the dehydrogenasic activity is dependent on the type of pollution, it was observed a much greater deterioration of the soil quality at Tarnia. Distinct behaviors had been registered for soil probes sampled at different depths, the enzymes having higher amplitudes in the in-depth probes compared to those from the surface.

1. Aydinalp, C., Marinova, S., (2003): Distribution and Forms of Heavy Metals in Some Agricultural Soils, Polish Journal of Environmental Studies, vol. 12, no. 5, pages 629-633 2. Brzezinska, M., Stepniewska, Z., Stepniewski, W., (1998): Soil oxygen status and dehydrogenase activity. Soil Biol Biochem., volume 30, pages 17831790. 3. Ciardi, C., (1998): Intracellular and extracellular urease and dehydrogenase activities of soils, Agrochimica, vol. XLII, no. 3-4, pages 118-125. 4. Dioiu, V., Oean, V., (2007): Modificri ale calitii solului produse prin activiti miniere n judeul Suceava, Analele Universitii tefan cel Mare Suceava, Seciunea Geografie., anul XVI, pages 19-202. 5. Dowarah, J, Deka Boruah, H.P., Gogoi, J., Pathak, N., Saikia, N., Handique, A.K., (2009): Eco-restoration of a high-sulphur coal mine overburden dumping site in northeast India: A case study, J. Earth Syst. Sci., vol. 118, no. 5, pages 597608. 6. Drgan-Bularda, M., (2000): Microbiologie General. Lucrri practice, Universitatea Babe-Bolyai, ClujNapoca. 7. Erzberger, P., Hhn, M., Pcs, T., (2012): Contribution to the bryoflora of Climani Mountains in the Eastern Carpathians, Romania, I., Acta Biologica Plantarum Agriensis, vol. 2, pages 73-95. 8. Ionce, A., (2010): Impactul sistemic al activitii de preparare a substanelor minerale utile n judeul Suceava, Alexandru Ioan Cuza University, PhD Thesis 9. Karaka, A., Cetin, S.C., Turgay, O.C., Kizilkaya, R., (2011): Soil enzymes as indication of soil quality, in Soil Enzymology, Shukla, G. And Varma, A. Editors, page 132. 10. Kizilkaya, R., Askin, T., Bayrakli, B., Salam, M., (2004): Microbiological characteristics of soils contaminated with heavy metals. Eur. J. Soil Biol., vol. 40, pages 95-102. 11. Levyk, V., Maryskevych, O., Brzeziska, M., Wodarczyk, T., (2007): Dehydrogenase activity of technogenic soils of former sulphur mines (Yavoriv and Nemyriv, Ukraine), Int. Agrophysics, vol. 21, pages 255-260. 12. Nadgorska-Socha, A., Lukasik, I., Ciepal, R., Pomierny, S., (2006): Activity of selected enzymes in soils loaded with varied levels of heavy metals. Acta. Agrophysica, vol. 8, pages 713-725. 13. Nannipieri, P., Ascher, J., Ceccherini, M.T., Landi, L., Pietramellara, G., Renella, G., (2003): .Microbial diversity and soil functions, European Journal of Soil Science, vol. 54, pages 655670. 14. Oliveira., A., Pampulha, M.E., (2006): Effects of long-term heavy metal contamination on soil microbial characteristics. J. Bioscience and Bioengineering, vol.102, no.3, pages 157-161. 15. Stevenson, F.J., (1982): Humus chemistry. John Wiley & Sons, New York, NY, 1982 16. Tang, K.., Baskaran, V., Nemati, M., (2009): Bacteria of the sulphurcycle: An overview of microbiology, biokinetics and their role in petroleum and mining industries, Biochemical Engineering Journal, vol. 44, issue 1, pages 7394. 1) Alexandru Ioan Cuza University of Jassy, Romania * ciornea@uaic.ro

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