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ENZYME ACTIVITIES IN SOIL AT INCREASING METAL (CU, NI, PB) DOSES AND TIME-DEPENDENCE IN A MODELL EXPERIMENT

Aim of this paper is to examine the effect of spiked copper (Cu), nickel (Ni) and lead (Pb) metal salts on the dehydrogenase (oxydo-reductase) and phosphatase (hydrolase) enzyme activities in a characteristic Hungarian soil, the pseudomycelliar chernozem. Pot-experiment was performed with a soil, originating from a spot of the Hungarian soil-information-monitoring (TIM) system of Bicserd. The added metal salts were used in water soluble forms and incorporated uniformly to the soil. Soils were treated with increasing metal concentrations to give the following metal amounts: 0, 50, 200, and 800 kg.ha -1 . Enzyme activities of the soil were analysed at the 0 th , 7 th , 14 th , and 28 th days after the metal addition. The laboratory modelexperiment has been set up in three replicates. Effects of metal salts were largely dependent on the chemical and physical properties of pseudomycelliar chernozem soil, the applied heavy metal-types, the doses of used metals and the elapsed time after the pollution. Considering the different metals, the copper prowed to be the most toxic one on the studied enzyme activities, whereas the lead induced those. By comparison with copper the nickel affected a smaller decrease in the soil microbial activity. The dehydrogenase, oxydo-reductase enzyme was found to be more sensitive parameter in comparison with the phosphatase, hydrolase enzyme among the studied condition. Studied enzymes and used methods are suggested, as fast and rather reliable tools for estimating the soilresilience capacities at heavy metal pollution.


INTRODUCTION
Soil is the most important renewable natural resource. Protection of the soils in the Earth is one of the inevitable task nowadays and might be the key-issue of the sustainable agricultural practices [1]. For this reason on the basis of the UNESCO decision, 2015 is the annual year of the soils in the World. It is rather evident for nowadays, that soil-functioning is largely dependent on the soil biota, among them the soilmicroorganisms. The activity of rarely measured microbial parts is resulted from the interactions among soil-physical-chemical-and biological parameters. Metal pollutions can alter those soil-biological activities, and can reduce the soil-fertility and function. The use of soil biological markers related to microbial activity, for instance microbial biomass, enzyme activities are the most wide-spread [2] [3].Those parameters can be correlated with the soil quality and function. The soil-enzymology with other methods can contribute to better understanding of substance circulation in soil. Many studies reported about the effects influences the enzyme activities [4]. Metal contaminations might influence on the quantity indicators of the soil-biological activities and might affecting on the enzyme activities, as well. Since the enzyme activities of the soils are determined by the organisms living in the soil, alterations in the enzyme activities could indicate the biological activity and the real functioning of the soil. Thus, the current biological activity of soils could represent therefore the values of soil-quality, among them the status of metal pollutions. Severity of metal pollution might be highly dependent on the binding form and availability of metals in the soils. The physico-chemical properties of metals and their affinity to the soil-particles are known to be crucial parameters [5]. In case of copper (Cu) for instance the form fixed to the organic substances is known to be the largest. Binded of lead (Pb) to the Fe and Mn oxids is also considerable. In case of the nickel (Ni) on the other hand, the exchangeable form is known to be the biggest. The effect of increasing heavy metal doses were studied and characterised by the indigenous microbial enzyme activities in a main Hungarian soil, by an in vitro model-experiment. The phosphatase and dehydrogenase activities were representing the hydrolase and oxido-reductase types of enzymes in the soil. The study was concentrating on the interrelations between the physical-chemical soil characteristics, the studied soil-biological parameters and the metal contaminations in a characteristic soil in Hungary.

Main characteristics of the pseudomycelliar chernozem soil
A characteristic Hungarian soil-type was used in the in vitro model-experiment. It is the pseudomycelliar chernozem from Bicsérd. The most frequent physical and chemical parameters of the soil are shown in the Table 1.

Experimental conditions
The water-soluble salt of the copper-sulphate (CuSO 4 *5H 2 O, concentration = 39,29 g/100cm 3 ), nickelchlorid (NiCl 2 *6H 2 O, concentration = 40,50 g/100cm 3 ) and lead-acetate ((CH 3 -COO) 2 Pb*3H 2 O, concentration = 36,62 g/100cm 3 ) were added to the tested soil in vitro. The test-soil was treated with increasing metal concentrations: 0, 50, 200 and 800 kg.ha -1 . Enzyme activities of the pseudomycelliar chernozem were analysed on the 0 th , 7 th , 14 th , and 28 th days after the metal addition. The experiment was carried out in three replicates. The heavy metal content of soils depend strongly on the physical type of the soil. Pseudomycelliar chernozem is a loamy soil, and the average Cu-content is 18 mg.kg -1 , the Ni-content is 20 mg.kg -1 , Pbcontent is 16 mg.kg -1 on the basis of Hungarian Soil Monitoring Survey [6]. The soil pH influenced largely the damaging effect of heavy metals. Therefore the pH of metal-solutions have been set up on the neutral pH with KOH. The own pH (KCl) of pseudomycelliar chernozem is 7.04 and the pH (H 2 O) 7.84. Organic substances (humus content 2,05 %) increase the mobility of heavy metals in a pH neutral environment.

Methods used in the study
The total substrate decomposition of soil-phosphatases has been determined by the method elaborated through Tabatabai and Bremner (1969) [7]. PNP (P-nitro-phenil-phosphate) P-nitro-phenol The soil-dehydrogenase activity characterizes accuratly the intensity of decomposition -processes of soilmicroorganisms [8], and has been specified by the Hungarian standard [9].

2.4The applied apparatus
The phosphatase and dehydrogenase enzyme activities has been assessed by a semi-automatic spectrophotometer (Type JASCO -530PC UV/VIS). Data were analysed with statistical program using method of min. square error.  Three metals used in the experiment resulted different changes in the activity of soil-microorganisms. The copper was toxic on dehydrogenase and phosphatase enzyme activities in all cases, while the lead increased the two enzyme activities in 90% of variations of exposition times and treatments. The nickel effected a similar, but smaller change in the microbial activity of soil. Increasing doses of Cu 2+ and Ni 2+ extended the dehydrogenase and phosphatase activity-changes, but proportionally to the amount of metals. In the event of Pb 2+ dose-dependent has been found only by dehydrogenase activity. The oxido-reductase-type dehydrogenase enzyme system proved to be more sensitive than the hydrolasetype phosphatase enzyme system in case of soils with large adsorption capacity.

CONCLUSIONS
The change of the enzyme activities of psedudomycelliar chernozem was very sensitive to the environmental conditions, and much faster to detect, as the quantitative change in the number of the microorganisms. The method of the determination of enzyme activities in case of metal pollutions were suitable to estimate the soil-toxicity. These experiments help us to establish a model for the natural ability of self-cleaning of soils and to detect the efficiency of remediation of the metal pollution.