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Şule Ataman 1 , Sevinç Arcak 2 1 Republic of Turkey, Ministry of Environment, Ankara 2 Department of Soil Science, Faculty of Agriculture, University Ankara, Ankara ABSTRACT Soil sample was treated with different levels of sewage sludge (20,40,80 and 160 t ha-1) urease, alkaline phosphatase activity and CO2 evolution were assayed after 1,7,12,28,42,56,70,105 and 140 days of incubation. Results showed that when soil was treated with the sewage sludge, urease activity was often inhibited at the lower dosage rate (20 t ha-1), but was enhanced substantially with the higher application rates (40 t ha-1 and 80 t ha-1). Urease activity in the sewage sludge amended soils decreased at various times of incubation. Inhibation of the enzyme activity (urease and alkaline phosphatase) was attributed to the presence of heavy metals in the sludge. The increased activity of urease and alkaline phosphatase in the sludge amended soil at the highest application rate. Enhanced urease activity was believed to be due to the additional source of organic matter and nutrients supplied by the sludge which stimulated microbial activity and subsequent urease synthesis. The amount of CO2 evolved decreased with the incubation time. These results were found statistically significant at 0.01 level. Soil enzyme activities are often used as indices at microbial growth and activity in soil. Quantitative information concerning which soil enzymes most accurately reflect microbial growth and activity is lacking. INTRODUCTION Current interest in assessing the quality of soil resources has been triggered by increasing awarenes of soil as a component of the earth's biosphere. Soil has a role not only in the production of food and fibre but also in the maintenance of environmental quality. Thus, it is critical to define and evaluate the quality of soil resources. Conceptually,soil quality is defined as the capacity of soil to function within ecosystem boundaries to sustain biological productivity, to maintain quality of the environment and to promote plant and animal health (Doran and Parkin, 1994). Soil biology is a significant component of soil quality and is the catalytic agent responsible for many of the transformations occuring in soil, most notably the reactions involved in nutrient cycling. Thus, it is meaningful to evaluate the biological aspects of soil quality within the context of overall system function (Karlen et al., 1997). There is limited information available on how sewage sludge application can influence soil microbial and bio-chemical characteristics with respect to maintaining soil quality. The effects of heavy metals on the soil microbial community, with emphasis on specific microbial activities, have been reported (Brookes et al., 1986; Reber, 1992) Generally, the application of low metal sludges had beneficial effects on microbial biomass, organic C and on the soil microbial activity, whereas, higher heavy metal contamination of soil resulted in considerable decrease in biomass C (Fliebbach et al., 1994; Knight et al., 1997). Kandeler et al. (1996) demonstrated that microbial biomass and enzyme activities decreased with increasing heavy metal pollution using salts of heavy metals but the amount of decrease differed among the enzymes. They also observed that heavy metal pollution severely decresed the funcional diversity of the soil microbial community. However, Sastre et al., (1996) showed that sewage sludge applications at recommended rates increased microbial activity in soil. It appears from various studies that soil and sewage sludge tie up the heavy metals making them unavailable to plants and soil. Thus, metals contained in sludge have less effect t han equivelent loadings as inorganic salts. The availability of metals in sludge depends upon the concentration of heavy metals present in the sewage sludge and the nature of the sludge itself. Nevertheless, scientific evidence to support the selection of sludge application limits is still inadequate, particularly as the sensitivity of soil microorganisms to relatively modest metal contamination has only recently been reported (Lorenz et al., 1992). Thus, an integrative concept based on soil microbial and biochemical components that would predict nutrient availability from sewage sludge and its toxicity at different rates and frequencies of application is still lacking. Although the effects of trace elements on bio-chemical tranformations have been studied extensively, very little information is available on the relative effects of sewage sludges on these tranformations. We have determined the effects of sewage sludges containing high levels of hevamy metals on urease alkaline phospatase activity and CO2 evolution in soils. MATERIALS and METHOD The soil used was surface samples (0-20cm). Before use soil sample was siewed (< 2 mm). The chemical and physical properties of the soil and sludge used in this study (Table 1) were determined as described by Page (1982). The sewage sludge sample was collected from Ankara Wastewater Treatment Plants. Treatment processes, sewage flow and BOD (biological oxygen demand) are reported in Table 2. The sample was dried at 65 OC, ground (<1mm) and then stored in poliethylene bags at room temperature.The soil was amended with the sewage sludge (20,40,80 and 160 t ha-1) and throughly mixed. Each treatment was replicated three times giving a total of 135 pots. The sewage sludge amended and unamended soils (control samples) were incubated for 140 days at 28 ± 2 OC in 500 g capacity plastic pots and soil moisture maintained gravimetrically at 60% water-holding capacity throughout study period. Soil samples were taken at 1, 7, 12, 28, 42, 56, 70, 105 and 140 for analytical purposes. Soil respiration (mg CO2 100 g-1 24 h) was determined by titration of the NaOH- solution with 0.1 M HCl in an excess of BaCl2.The method of Hoffman and Teicher (1957) was used to assay urease activity. Phosphatase activity was determined by the method of Hofmann and Hoffman (1966). At various times up 140 days of incubation, triplicate samples were assayed for urease, phosphatase enzyme activity and soil respiration. 
 RESULTS and DISCUSSION The source and selected general characteristics of the sewage sludge studied are shown in Table 2. The sludge was anaerobically digested with a mixture of primary and waste activated sludge typically entering the digester. Some additional comments on sludge treatment are shown in Table 2. The chemical characteristics of the sludge are presented in Table 1. Anaerobically digested sludges are devoid of NO-3 when samples are collected directly from the digester. Since several of the anaerobically digested sludges contained > 500 mg NO3--N kg-1, it is obvious that the sludge samples were slowly dried allowing nitrification occur. Similarly, the NH+4-N contents are likely a reflection of NH3- volatization losses which occurred during air or owen drying of the sludges. The organic N levels in the sludges ranged from 0.50 to 6.81 % N with a mean of 2.18 % (SD=1.45 %) Urease and alkaline phosphatase activities increased with high application dosage rates (Figure 1 and 2). And this changings was found statistically significant. (p<0.01). Increasing in CO2 evolution with incubation time was found statistically significant (p<0.05) (Figure 3).   Shaw and Raval (1961) reported that metal ion inhibition was non-competitive and postulated that the inhibation involved the reaction of metal ions with sulfhydryl groups in the catalytic site of urease. In our study, inhibation of urease activity was probably due to several metals and not solely to one metal component. In most cases, heavy metal pollution has little effect on CO2 evolution at low levels of contamination, but with higher doses the soil respiration rate decreases. Bond et al. (1976) and Chaney et al.(1978) also found increased respiration rates at low amendment levels of sewage sludge, but they used rather short incubation times. Soil respiration rate is easy to measure and appears to be a sensitive measurement with which to detect heavy metal pollution, especially under standardized conditions. (Bääth, 1989). CO2 evolution in the sewage sludge amended soil (except highest application rates) increased with time but began to decline after 14 days (Figure 3). The decreased CO2 evolution in the amended soil when treated with the highest application rate. Carbon and nitrogen turnover in soils occurs through heterotrophic microorganisms and lower concentration of heavy metal has not influenced their catabolic activities. However, at higher concentrations more soluble forms of heavy metal seems to have exposed microbes to free metal, therefore, resulting in adverse effects on mineralization processes. The presence of clay, organic matter, hydrous oxides and phosphatas seem to have influenced the metal mobility through adsorbtion or chelation on clay minerals, exchangeable organic residues and oxide fractions. It was observed that CO2 evalution increased at the 20 t ha-1 dosage in all incubation periods and it decrease with the application dosage rate. It was observed that CO2 evolution decreased with incubation time. Incerasing of CO2 evolution is because of the inhibation of the active microorganisms in soil system (Tyler, 1974). Several measurements of enzyme activities have been used in relation to heavy metal pollution in soil. The activity of phosphatase appears to be a good indicator of pollution. Urease appears in many cases to be equally or even more sensitive to heavy metal pollution as phosphatase.  Reddy et al, (1987) found that dehydrogenase and phosphatase enzyme activity was inhibited in all their experimental sludge soils and was related to their heavy metal concentrations. Increasing rates of sludge application reduced urease activity in some soils, but increased it in others. The decline in enzyme activities found in several investigations is probably mainly an effect of a decreased enzyme synthesis associated with inhibated microbial growth t han to direct enzyme inhibation by the metals. Assays of potential enzyme activity are important in estimating the effects of metal pollution on the soil environment. It is difficult, however, to establish whether low enzymatic activity is because of metal inhibition or from low concentrations of the enzyme, resulting from impeded microbial growth and enzyme synthesis (Tyler, 1974). Nevertheless, soil enzymatic activity is believed to be a sensitive indicator of the effect of environmental factors on microbial functions. (Dick, 1994). Thus, because of their role in nutrient cycling, enzymes like arylsulfatase, acid phosphatase and alkaline phosphatase are suggested to be good indicators of potentially beneficial or harmful effects on the ecosystem. In the present investigation, sludge application had relatively little effect on any of the enzymes studied, potential soil enzyme activities were generally increased or not affected by the sludge application. Similar result were observed for the potential activities of acid and alkaline phosphatase (Banerjee et al, 1997). REFERENCES Bääth E. (1989). Effects of heavy metals in soil on microbial processes and populations (A review) Water, Air and Soil Pollution 47:335-379. Banerjee M.R. Burton D.L. Depoe S. (1997). Impact of sewage sludge application on soil biological characteristics. Agriculture, Ecosystems and Environment 66, 241-249. Bond H., Lighthart R., Shimabuku R., Russel L. (1976). 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(1957). Das Enzyme Sistem Unserer Kultur Boden 7, Protecsan 11. Zeitschift Für Pfalanzone-Nahrung Und Bodenkunde, 77-1227, B. Hofmann E., Hoffman G. (1966). Die Bestimmung Der Biologischen Tatigkeit In Boden Mit Enzymethoden. Reprinted From Advance In Enzymology And Related Subject Of Biochemistry, V. 28, 365-390. Hughes R.B., Sidney A.K., Stubbins S.E. (1969). Inhibition of urease by metal ions. Enzymologia 36, 332-334. Isermayer H. (1952). Eine Einfache Methode Zur Bestimmung Der Bodenatmung Und Karbonate In Boden. Z. Pflanzenernaehrung, Düngüg Und Bodenkunde, 56, 26-8. Kandeler E., Kampichler C., Horak O. (1996). Influence of heavy metals on the functional diversity of soil microbial communities. Biol. Fertil. Soils 23, 299-306. Karlen D.L., Mausbach M.J. Doran J.W., Cline, R.G., Harris, R.F., Schuman, G.E. (1997). Soil quality: a concept, definition, and framework for evaluation. Soil Sci.Soc.Am.J.61, 4-10. Knight B.P., McGrath S.P., Chaudri A.M. (1977). 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