www.elsevier.com/locate/apsoil

Applied Soil Ecology 30 (2005) 192–202

Effects of N-enriched sewage sludge on soil enzyme activities

Rıdvan Kızılkaya *, Betul Bayraklı

Ondokuz Mayıs University, Faculty of Agriculture, Department of Soil Science, 55139 Samsun, Turkey

Received 16 July 2004; received in revised form 21 February 2005; accepted 21 February 2005

Abstract

Sewage sludge is increasingly used as an organic amendment to soil, especially to soil containing little organic matter.

However, little is known about utility of this organic amendment with N-enriched or adjusted C:N ratios in soil. We studied the

effects of adding of different doses (0, 100, 200 and 300 t ha�1) and C:N ratios (3:1, 6:1 and 9:1) of sewage sludge on enzyme

activities (b-glucosidase, alkaline phosphatase, arylsulphatase and urease) in a clay loam soil at 25 8C and 60% soil water

holding capacity. Nitrogen was added in the form of (NH4)2 SO4 solution to the sludge to reduce the C:N ratio from 9:1 to 6:1 and

3:1. The addition of different doses and C:N ratios of the sludge caused a rapid and significant in the enzymatic activities in soils,

this increase was specially noticeable in soil treated with high doses of the sludge. In general, enzymatic activities in sludge-

amended soils tended to decrease with the incubation time. All activities reached peak values at 30 days incubation and then

gradually decreased up to 90 days of incubation. Sewage sludges also the increased available metal (Cu, Ni, Pb and Zn) contents

in the soils. However, the presence of available soil metals due to the addition of the sludge at all doses and C:N ratios did

negatively affect all enzymatic activities in the soils. This experiment indicated that all doses and C:N ratios of sewage sludge

applied to soil would have harmful effects on enzymatic activity. Some heavy metals found in sewage sludge may negatively

influence soil enzyme activities during the decomposition of the sludge.

# 2005 Elsevier B.V. All rights reserved.

Keywords: Sewage sludge; Soil; C:N ratio; Urease; Alkaline phosphatase; Arylsulphatase; b-Glucosidase activity

1. Introduction

Interest in the disposal of sewage sludges, which

contain a range of valuable nutrients (N, P, Fe, Ca, Mg)

and various other macro- and micro-nutrients essential

for plant growth, on agricultural land has increased

during the last decade (Singh and Pandeya, 1998). One

* Corresponding author. Tel.: +90 362 457 60 20;

fax: +90 362 457 60 34.

E-mail address: ridvank@omu.edu.tr (R. Kızılkaya).

0929-1393/$ – see front matter # 2005 Elsevier B.V. All rights reserved

doi:10.1016/j.apsoil.2005.02.009

potential use for sewage sludge as a farm fertiliser and/

or soil conditioner is to assist with the growth of arable

land and to help improve and maintain the structure of

the soil by increasing soil aeration and the water

holding capacity of the soil (Pagliai et al., 1981).

Previous studies have demonstrated favorable plant

yield responses to the application of sewage sludge

(King and Morris, 1972). In contrast, the effects of

sewage sludge on biological process in soil have been

questioned by some authors (Knight et al., 1997;

Banerjee et al., 1997).

.

R. Kızılkaya, B. Bayraklı / Applied Soil Ecology 30 (2005) 192–202 193

Most papers concerned with the results of sewage

sludge studies deal with the influence of sewage

sludge on soil biological and enzymatic activity.

Knight et al. (1997) observed a decrease of soil

biological activity such as microbial biomass and

enzyme activities, due to sewage sludge application.

Conversely, Sastre et al. (1996) and Banerjee et al.

(1997) found that the sewage sludge amendment

increased soil microbial activity, soil respiration and

enzyme activities. These differences might be a result

of the heavy metal content of sewage sludge and of the

decomposition rate of the sludge (Tam and Wong,

1990). Sewage sludges are a by-product of sewage

treatment and contain not only nutrients and organic

matter, but also contaminants such as heavy metals

and synthetic organics discharged into the sewers.

Because most heavy metals remain in the soil for a

very long time, any additions should be considered

permanent additions to the total quantity in the soil.

Considerable attention has been focused on the N

content of sewage sludges (Stewart et al., 1975). With

increased cost and shortage of nitrogenous fertilizer,

there is increased emphasis on using sludge for its

nutrient content rather than for disposal. The N content

generally is considered the limiting factor which

determines the application rate of sewage sludges to

agricultural land (Mile and Graveland, 1972).

Increased crop yields have been obtained with the

use of sludges (Hinesly et al., 1972). In some cases

supplemental addition of N further increased yields

(Coker, 1966). Thus low C:N ratio is caused by

additional nitrogen of the sludge. In addition,

numerous studies suggested that different concentra-

tions of nitrogen might predominate in different

sewage sludges (Coker, 1966; Mile and Graveland,

1972; Hinesly et al., 1972). So, the behaviour and

effects of N after incorporation into the soil may not be

similar for different C/N ratios of sludges.

Studies of enzyme activities provide information on

the biochemical processes occurring in soil. There is

growing evidence that soil biological parameters may

be potential and sensitive indicators of soil ecological

stress or restoration. Measurements of several enzy-

matic activities have been used to establish indices soil

biological fertility (Dick and Tabatabai, 1992). In the

present study, soil enzymes representative of main

nutrient cycles (C, N, P, S) were selected. Glucosidases

are widely distributed in nature and their hydrolysis

products as low molecular weight sugars are important

source of energy for soil microorganisms. b-glucosi-

dase catalyzes the hydrolysis of b-D-glucopyranoside

and is one of the three or more enzymes involved in the

saccharification of cellulose (Bandick and Dick, 1999;

Turner et al., 2002).Urease is involved in the hydrolysis

of urea to carbon dioxide and ammonia, which can be

assimilated by microbes and plants. It acts on carbon–

nitrogen (C–N) bonds other than the peptide linkage

(Bremner and Mulvaney, 1978; Karaca et al., 2002).

Phosphatase is an enzyme of great agronomic value

because it hydroles compounds of organic phosphorus

and transforms them into different forms of inorganic

phosphorus, which are assimilable by plants (Amador

et al., 1997). Variations in phosphatase activity apart

from indicating changes in the quantity and quality of a

soil’s phosphorated substrates, are also a good indicator

of its biological state (Pascual et al., 1998, 2002).

Arylsulphatase is the enzyme involved in the hydrolsis

of arylsulphate esters by fission of the oxygen–sulphur

(O–S) bond. This enzyme is believed to be involved in

the mineralisation of ester sulphate in soils (Tabatabai,

1994). Also, it may be an indirect indicator of fungi as

only fungi (not bacteria) contain ester sulphate, the

substrate of arylsulphatase (Bandick and Dick, 1999).

Soil enzymes are biological catalysts of specific

reactions and these reactions depend on a variety of

factors such as pH, temperature and the presence (or

absence) of inhibitors (Burns, 1978). Climate, type of

amendment, cultivation techniques, crop type and

edaphic properties also effect enzyme catalyzed

reactions. Process as related to the degradation of

organic material may be followed through the action of

hydrolases (Pascual et al., 1998). Nannipieri et al.

(1990) pointed out that enzymatic activities are

substrate specific and related to specific reactions.

For this reason it is difficult to obtain an overall picture

of soil status from one enzymatic activity value. The

simultaneous measurement of various enzymes, on the

other hand, seems to be useful to evaluate soil

biochemical activity and related processes (Pascual

et al., 1998).

Indeed, enzyme activities have been shown to be

more sensitive total carbon concentration to soil

management practices. Application of organic wastes,

such as sewage sludge, as a source of organic matter is

a common practice, especially to soils containing little

organic matter, to maintain or improve soil quality and

R. Kızılkaya, B. Bayraklı / Applied Soil Ecology 30 (2005) 192–202194

Table 1

Total and available metal contents of the soil and the sewage sludge

Total (mg g�1) Available (mg g�1)

Soil Sewage sludge Soil Sewage sludge

Cu 33.7 214.5 1.16 14.4

Ni 46.5 75.8 0.41 1.5

Pb 38.6 180.4 0.36 5.7

Zn 59.1 435.9 0.35 59.4

soil enzymatic activities (Giusquiani et al., 1995).

However, the use of sewage sludge can lead to

problems pertaining to their metal content and their

successive application result in metal accumulation in

soil. In addition, the practice can affect the availability

of metal in soil (Sloam et al., 1997). Moreover, sewage

sludge treatments increase the activity and diversity of

soil microbial populations and their tolerance of

metals (Barkay et al., 1985).

The objective of this study was to determine the

effects of different application rates (0, 100, 200 and

300 t ha�1) and adjusted C/N ratios (3, 6 and 9) of

anaerobically digested sewage sludge on available

metal (Cu, Ni, Pb and Zn) contents and enzyme

activities (b-glucosidase, urease, alkaline phosphatase

and arylsulphatase) in an incubated clay loam soil for

90 days at 25 8C and 60% soil water holding capacity.

2. Materials and methods

2.1. Soil and sludge description

Surface soils (0–20 cm) were taken from the

Experimental Farm of the Agricultural Faculty of

University of Ondokuz Mayıs, Samsun, Turkey. The

soil had been developed from basalt and contained

31.2% clay, 36.2% silt and 32.6% sand. The pH in

water was 7.1, the oxidizable organic matter content

1.98% and the soil C/N ratio was 28:1. The soil was

bulked, all stones, visible roots and fauna removed,

sieved to less than 2 mm and stored at 5 8C until used.

Sewage sludge was obtained from the wastewater

facility set up by the Ankara Wastewater Treatment

Plants, Ankara, Turkey. The sludge was anaerobically

digested with a mixture of primary and waste activated

sludge typically entering the digester. The sewage

sludge was composed of approximately 37% by weight

of oxidable organic matter. The organic fraction

comprised 21% C and 2.3% N while the inorganic

fraction contains 2.3% Fe2O3, 11.5% CaO, 1.3%MgO,

0.2% Na2O and 4.4% Al2O3 by weight. Other elements

of agronomic interest were also analyzed: the sludge

contained 1.3% P and 0.2% K by weight. The sewage

sludge in this experiment was digested and air-dried and

sieved to less than 1 mmand stored in polyethylene bags

at 5 8C until used. The content of the some metal of

interest in the sludge and soil are given in Table 1.

2.2. Soil treatment and incubation

Based on the hypothesis that lowering C/N ratio

enhances C mineralisation was conducted to study the

effect of C:N ratio on enzyme activities in the soil.

Nitrogen was added in the form of (NH4)2 SO4

solution to the sewage sludge to reduce the C:N ratio

from 9:1 to 6:1 and 3:1. The soil samples (250 g air-

dried soil) were placed in 500 ml cylindrical plastic

pots. The sewage sludge was thoroughly mixed with

the soil at a rate equivalent to 0, 100, 200 and

300 t ha�1 on a dried weight basis.

The sewage sludge-amended soils were then

moistened up to 60% soil water holding capacity and

incubated for 90 days at 25 � 0.5 8C. The moisture

contentwasmaintained throughout the experiment. Sub

samples were removed at time intervals of 7, 15, 30, 45,

60 and 90 days to determine the changes in enzyme

activities.

2.3. Experimental design

Soil without sewage sludge addition was used as a

control. A randomized complete plot design with three

replicates per treatment and soil was used. The

experiment was performed with the following 10

treatments:

(1) c

ontrol

(2) +

100 t ha�1 sewage sludge (C/N ratio 3:1)

(3) +

200 t ha�1 sewage sludge (C/N ratio 3:1)

(4) +

300 t ha�1 sewage sludge (C/N ratio 3:1)

(5) +

100 t ha�1 sewage sludge (C/N ratio 6:1)

(6) +

200 t ha�1 sewage sludge (C/N ratio 6:1)

(7) +

300 t ha�1 sewage sludge (C/N ratio 9:1)

(8) +

100 t ha�1 sewage sludge (C/N ratio 6:1)

(9) +

200 t ha�1 sewage sludge (C/N ratio 6:1)

(10) +

300 t ha�1 sewage sludge (C/N ratio 9:1)

R. Kızılkaya, B. Bayraklı / Applied Soil Ecology 30 (2005) 192–202 195

2.4. Measurement of soil enzymatic activities

b-Glucosidase (EC 3.2.1.21) activity (GA) was

measured according to Eivazi and Tabatabai (1988).

0.25 ml toluene, 4 ml Tris (hydroximethyl) amino-

methane buffer (pH 12) and 1 ml of 0.05 M p-

nitrophenyl b-D-glucopyranoside solution were added

to the 1 g sample and the samples were incubated for

1 h at 37 8C. The formation of p-nitrophenol was

determined spectrophotometrically 410 nm and

results were expressed as mg p-nitrophenol g�1 dry

sample.

Urease (EC 3.5.1.5) activity (UA) was measured by

the method of Hoffmann and Teicher (1961). 0.25 ml

toluene, 0.75 ml citrate buffer (pH 6.7) and 1 ml of

10% urea substrate solution were added to the 1 g

sample and the samples were incubated for 3 h at

37 8C. The formation of ammonium was determined

spectrophotometrically at 578 nm and results were

expressed as mg N g�1 dry sample.

Alkaline phosphatase (EC 3.1.3.1) activity (APA)

was determined according to Tabatabai and Bremner

(1969). 0.25 ml toluene, 4 ml phosphate buffer (pH

8.0) and 1 ml of 0.115 M p-nitrophenyl phosphate

(disodium salt hexahydrate) solution were added

to the 1 g sample and the samples were incubated for

1 h at 37 8C. The formation of p-nitrophenol was

determined spectrophotometrically at 410 nm and

results were expressed as mg p-nitrophenol g�1 dry

sample.

Arylsulphatase (EC 3.1.6.1) activity (ASA) was

measured according to Tabatabai and Bremner (1970).

0.25 ml toluene, 4 ml acetate buffer (pH 5.5) and 1 ml

of 0.115 M p-nitrophenyl sulphate (potassium salt)

solution were added to the 1 g sample and the samples

were incubated for 1 h at 37 8C. The formation of

p-nitrophenol was determined spectrophotometrically

410 nm and results were expressed as mg p-

nitrophenol g�1 dry sample.

All determinations of enzymatic activities were

performed in triplicate, and all values reported are

averages of the three determinations expressed on an

oven-dried soil basis (105 8C).

2.5. Measurement of soil metal contents

Available metals in soils were determined by

extraction with diethylene triamine penta acetic acid

(DTPA) solution (0.005 M DTPA + 0.01 M

CaCI2 + 0.1 M TEA buffered at pH 7.3) and analyzed

by atomic absorption spectrophotometer (Lindsay and

Norvell, 1978).

2.6. Statistical analysis

Means and standard deviation of triplicates were

determined and all the figures presented include

standard errors of the data. Analysis of variance

(three-way ANOVA) was carried out using three

factors randomized complete plot design (C/N

ratios � doses of sewage sludge � incubation period).

The means were compared using least significant

difference (LSD) test, with a significance level of

P < 0.01. All statistical calculations were performed

using MSTAT and SPSS 11.0 computer programs.

3. Results and discussion

3.1. Metal contents of the sewage sludge

The total Cu, Ni, Pb and Zn concentrations in

the sewage sludge were 214.5, 75.8, 180.4 and

435.9 mg g�1, respectively. The concentrations were

similar to those reported by Arcak et al. (2000),

Karaca and Haktanır (2000) and Kızılkaya (2004) for

same sewage sludge treatment works. However, the

concentrations were relatively low when compared to

sewage sludge originating from industrialized cities in

Europe (Kelly et al., 1999). The concentrations of Cu,

Ni, Pb and Zn in the soil sampled are shown in Table 1.

3.2. Available metal concentrations

The changes of metal (Cu, Ni, Pb and Zn)

availability in sewage sludge-amended soils during

the incubation are shown in Fig. 1.

Different C/N ratios of sewage sludge application

significantly affected the levels and distribution of

available (DTPA-extractable) Cu, Ni, Pb and Zn in the

soil, when compared with the control treatment. Fig. 1

shows that the Cu, Ni, Pb and Zn concentrations of the

low C/N ratio and high doses of sewage sludge

application were higher than those of other treatments

at all sampling times. In general sewage sludge

treatments with initial high C/N ratios had low metal

R. Kızılkaya, B. Bayraklı / Applied Soil Ecology 30 (2005) 192–202196

Fig. 1. Changes in available metals (Cu, Ni, Pb and Zn) of soil amended with sewage sludge during the incubation period. Vertical bars indicate

standard error of mean of three replicates at 95% confidence level.

concentrations at all sludge doses. This situation might

be related availability of metals such as initial C/N

ratios, doses of sewage sludge and chemical condi-

tions (organic matter, pH, etc.) in soil. Organic wastes

as sewage sludge and their C/N ratios are the most

important factors that control the availability of heavy

metals in soil (Doelman and Haanstra, 1984). The

concentration of Cu, Ni, Pb and Zn to the soil can be

attributed to the high affinity of the metals to organic

matter (Tyler and McBride, 1982; McGrath and Lane,

1989). The study, although based on one soil type, was

in agreement with the results by McGrath et al. (1994)

and Nyamangara and Mzezewa (1999) who reported

that generally metal concentrations relatively

increased in sewage sludge treated soil compared to

the non-treated soil.

3.3. Enzyme activities

Considerable variations in all enzyme activities,

GA, APA, ASA and UA were found for the different

doses and C:N ratios of sewage sludge at different

sampling times. Statistically significant variations

were found in all enzyme activities at various

application rates and C/N ratios of the sewage sludge.

Enzyme activities were also affected by incubation

period. The analysis of variance of the results obtained

in our experiment on the periodic sampling times with

sewage sludge showed that all factors (sewage sludge

doses, C/N ratios and incubation periods) significantly

influenced all enzyme activities (Table 2). After

sewage sludge addition a rapid and significant

increase in enzymatic activities was observed in

R. Kızılkaya, B. Bayraklı / Applied Soil Ecology 30 (2005) 192–202 197

Table 2

Results of ANOVA

Variables GA APA ASA UA

F-value LSDa = 0.01 F-value LSDa = 0.01 F-value LSDa = 0.01 F-value LSDa = 0.01

C:N ratio (CN) 58.37*** 1.608 22.04*** 2.151 0.61ns 1.314 33.56*** 1.027

Sewage sludge doses (DS) 453.56*** 1.856 90.52*** 2.484 4.21** 1.518 445.41*** 1.186

Incubation period (IN) 221.43*** 2.274 315.57*** 3.042 172.00*** 1.453 583.37*** 1.453

CN � DS 8.56*** 3.125 8.96*** 3.255 0.22ns 2.629 8.92*** 2.054

CN � IN 2.23* 3.938 3.65** 5.270 4.66*** 3.220 14.55*** 2.516

DS � IN 53.41*** 4.547 53.06*** 6.085 52.11*** 3.718 150.48*** 2.905

CN � DS � IN 0.85ns 7.876 1.54ns 10.539 0.75ns 6.439 3.26*** 5.032

ns: Not significant.* P < 0.05.** P < 0.01.*** P < 0.01.

sludge-amended soils followed by a progressive

decrease in the GA, UA, APA and ASA in soils

amended with the sludge. At the end of the incubation,

the GA, UA, APA and ASA measured in treated soils

were statistically different from those measured in the

control soils.

Among the C:N ratios and application doses of

sludge, soils amended with high C:N ratio and high

doses of sludge presented the highest values of GA

(Fig. 2). The GA is closely involved in the C cycle and

plays an important role in the hydrolytic processes that

take place during the organic matter breakdown

(Bandick and Dick, 1999; Turner et al., 2002). The GA

in soils treated with sludges can be attributed to high

levels of GA in the material itself and the stimulation

of microbial activity (Martens et al., 1992).

Alkaline phosphatase activity showed a similar

trend to that of ASA in both soils: an increase in the

first 30 days of incubation followed by a pronounced

decrease in activities (Figs. 3 and 4). The APA and

ASA plays an essential role in the mineralisation of

organic P and organic S in soil, respectively (Speir and

Ross, 1978). The APA and ASA of low C:N ratio and

high doses of sludge treatments were higher than those

of other treatments. Thus indicated that there were

higher amounts of readily available substrates in the

substrates in the sewage sludge initially. Organic

matter in sludge usually contains high amounts of

APA and ASA substrates (Garcıa et al., 1993).

Urease activity (UA) exhibited the same behaviour

as those of the APA and ASA. However, the UA

increased rapidly and reached a maximum activity

after 15 days for all treatments; then the UA decreased

significantly until the end of the incubation period

(Fig. 5). The UA is involved in the hydrolysis C–N

bonds of some amides and urea (Bremner and

Mulvaney, 1978). The highest values of UA were

observed in soil amended with low C:N ratio and high

doses of sludge, probably due to the UA is a

constitutive-intracellular enzyme and consequently

it increases or decreases proportional to microbial

biomass (Nannipieri et al., 1979).

Nitrogen can have different effects on decomposi-

tion rates depending on the stage of decomposition.

The initial stage of litter decomposition will often be

N limited, and consequently, addition of N will

enhance the degradation rate. Several studies (Cotrufo

et al., 1995; Recous et al., 1995) have demonstrated

that low C/N ratio is an indicative of a high

decomposition rate.

In general, GA, UA, APA and ASA in sludge-

amended soils decreased dramatically of 30 days of

incubation at all C:N ratios and doses of sludge.

Taking into account the evolution of the enzyme

activities during the incubation period, two different

stages can be distinguished: a first stage lasting 30

days during which a great activation of the enzymes as

a result of adding the different doses and C:N ratios of

sewage sludge was recorded, and a second stage

lasting until the end of the incubation in which a major

decrease in the enzyme activities in soils treated with

the sludge was recorded. The first stage depends on

microbial growth and available substrate for enzymes

(Garcıa et al., 1993) stimulated by addition of sewage

sludge to soil. Thus, increased microbial activity may

have synthesized enzymes in soil (Bremner and

R. Kızılkaya, B. Bayraklı / Applied Soil Ecology 30 (2005) 192–202198

Fig. 2. Changes in b-glucosidase activity (GA) of soil amended

with sewage sludge during the incubation period. Vertical bars

indicate standard error of mean of three replicates at 95% confidence

level.

ig. 3. Changes in alkaline phosphatase activity (APA) of soil

mended with sewage sludge during the incubation period. Vertical

ars indicate standard error of mean of three replicates at 95%

onfidence level.

F

a

b

c

R. Kızılkaya, B. Bayraklı / Applied Soil Ecology 30 (2005) 192–202 199

Fig. 4. Changes in arylsulphatase activity (ASA) of soil amended

with sewage sludge during the incubation period. Vertical bars

indicate standard error of mean of three replicates at 95% confidence

level.

Fig. 5. Changes in urease activity (UA) of soil amended with

sewage sludge during the incubation period. Vertical bars indicate

standard error of mean of three replicates at 95% confidence

level.

R. Kızılkaya, B. Bayraklı / Applied Soil Ecology 30 (2005) 192–202200

Mulvaney, 1978; Bandick and Dick, 1999). The

second stage depends on contents and availability of

sludge borne metals.

In general, enzymatic activity diminished with

increasing available concentration of metals (Tyler,

1974; Baath, 1989; Kızılkaya et al., 2004). In this

study, sewage sludge application significantly affected

the levels of available Cu, Ni, Pb and Zn concentra-

tions in soil, when compared with control (Fig. 1).

Similarly, several studies demonstrated that enzyme

activities decreased with increasing metal concentra-

tions using sewage sludge (Madojon et al., 2001;

Karaca et al., 2002; Moreno et al., 2003).

Nannipieri (1994) and Kandeler et al. (2000)

reported that the effect of sludge borne metals on

enzyme activities is very complex and is due to: (1) the

metal reacting with sulphydral groups of enzymes

causing inhibition or inactivation of the enzymatic

activity; (2) metals indirectly affecting soil enzymatic

activities by altering the microbial community which

synthesizes enzymes; or (3) a combination of these

factors. Assays of soil 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 occur because of

metal inhibition, or from low concentrations of the

enzyme resulting from impeded microbial growth and

enzyme synthesis (Tyler, 1974). The metal suscept-

ibility of enzyme activities was reported by Kandeler

et al. (1996), Kuperman and Carreiro (1997) and

Kunito et al. (2001). The inhibition of enzyme

activities by metals in soils is principally attributed

to an indirect effect (suppression of microbial

population and cellular activities) and/or a direct

effect (inactivation of extracellular enzymes due to

binding with metals). Also, Geiger et al. (1998a, b)

reported the direct inactivation of an extracellular

enzyme by metals due to their binding to some amino

acids in the enzyme. Because GA, APA and ASA are

present extracellularily in the soils (Bremner and

Mulvaney, 1978; Bandick and Dick, 1999) the

suppression might be attributable to the fact that

metal suppressed the number of microorganisms

producing the enzymes and the microbial activity

and that the extracellular enzymes were directly

inactivated by metals. The results described here on

the suppression of GA, UA, APA and ASA would

indicate the disruption of soil function by the metal

contamination in the soils. Nevertheless, soil enzy-

matic activity is believed to be a sensitive indicator of

the effect of environmental factors on microbial

functions (Dick, 1992). Thus, because of their role in

nutrient cycling, enzymes like GA, UA, APA and ASA

are suggested to be good indicators of potentially

beneficial or harmful effects on the soil.

The negative effect of metals on enzymatic

activities might have been masked in our study by

the positive effect of the sewage sludge (Moreno et al.,

1999). In addition, a metal fraction might be adsorbed

on the organic colloids added with the sludge. The

rapid decomposition of organic matter which occurs

after the application of composts to soil, increases the

proportion of available metals as a result of miner-

alisation of organically complexed (OM–) metals

(Dudley et al., 1986). The OM– heavy metal fractions,

which are readily available for plant uptake, occur in

organic matter and soil solutions. This would prevent

the heavy metal from interacting directly with the

active sites of enzyme, thus affecting the enzyme’s

activity (Doelman and Haanstra, 1984).

4. Conclusions

At present, many risk assessments and regulations

for the application of sewage sludge in many countries

are based on total and available metal concentrations

in soil (McGrath et al., 1994). In this study,

considerable effort has been devoted to estimating

the fate of enzymatic activity in the sludge-amended

soils. A good N balance is critical factor in any land

application program. High amounts of N in the initial

sludge application or N fertilization in the sludge-

amended soil can result in enzyme inhibition.

However, our present study suggests that measure-

ments of only available metal (Cu, Ni, Pb and Zn)

contents and hydrolytic enzyme activities (GA, UA,

APA and ASA) cannot provide a presence prediction

of toxicity other extracellular and intracellular

enzymes in soils amended with sewage sludge,

indicating that their scientific validity is questioned.

Hence, further studies are needed to explore the

relevancy in using enzyme activity to reflect different

types of sewage sludge and soil. This would not only

overcome problems of enzyme inhibition but also

would reduce a major area of public concern such as

R. Kızılkaya, B. Bayraklı / Applied Soil Ecology 30 (2005) 192–202 201

nitrate leaching, heavy metal and pathogen contam-

ination, plant uptake of sludge borne metals and soil

fertility and health.

Acknowledgements

The authors thank Dr. Cafer Turkmen of Ankara

University for his assistance with sewage sludge

collection. This work was supported by the OMU

Center of Scientific Research (project number Z-385).

References

Amador, J.A., Glucksman, A.M., Lyons, J.B., Gorres, J.H., 1997.

Spatial distribution of soil phosphatase activity within a riparian

forest. Soil Sci. 162, 808–825.

Arcak, S., Karaca, A., Haktanır, K., 2000. Investigations on

sewage sludges: chemical composition and effects on some

chemical properties of soil. In: Proceedings of International

Symposium on Desertification (ISD), Konya, Turkey, 13–17

June.

Baath, E., 1989. Effects of heavy metals in soil on microbial

processes and populations (a review). Water Air Soil Pollut.

47, 335–379.

Bandick, A.K., Dick, R.P., 1999. Field management effects on soil

enzyme activities. Soil Biol. Biochem. 31, 1471–1479.

Banerjee, M.R., Burton, D.L., Depoe, S., 1997. Impact of sewage

sludge application on soil biological characteristics. Agric.

Ecosyst. Environ. 66, 241–249.

Barkay, T., Tripp, S.C., Olson, B.H., 1985. Effect of metal rich

sewage sludge application on the bacterial communities of

grassland. Appl. Environ. Microb. 49, 333–337.

Bremner, J.M., Mulvaney, R.L., 1978. Urease activity in soils. In:

Burns, R.G. (Ed.), Soil Enzymes. Academic Press, New York,

pp. 149–196.

Burns, R.G., 1978. Enzyme activity in soil. Some theoretical and

practical considerations. In: Burns, R.G. (Ed.), Soil Enzymes.

Academic Press, New York, pp. 295–339.

Coker, E.G., 1966. The value of liquid digested sewage sludge II.

Experiments on rye grass in south-east England, comparing

sludgewith fertilizer supplying equivalent nitrogen, phosphorus,

potassium, and water. J. Agric. Sci. 67, 99–103.

Cotrufo, M.F., Ineson, P., Roberts, J.D., 1995. Decomposition of

birch leaf litters with varying C-to-N ratios. Soil Biol. Biochem.

27, 1219–1221.

Dick, R.P., 1992. A review: long-term effects of agricultural systems

on soil biochemical and microbiological parameters. Agric.

Ecosyst. Environ. 40, 25–36.

Dick, W.A., Tabatabai, M.A., 1992. Potential uses of soil enzymes.

In: Meeting, F.B. (Ed.), Soil Microbial Ecology: Applications in

Agricultural and Environmental Management. Marcel Dekker,

New York, pp. 95–127.

Doelman, P., Haanstra, L., 1984. Short-term and long-term effects of

cadmium, chromium, copper, nickel, lead and zinc on soil

microbial respiration in relation to abiotic soil factors. Plant

Soil 79, 317–327.

Dudley, L.M., McNeal, B.L., Baham, J.E., 1986. Time-dependent

changes in soluble organics, copper, nickel, and zinc from

sludge-amended soils. J. Environ. Qual. 15, 188–192.

Eivazi, F., Tabatabai, M.A., 1988. Glucosidases and galactosidases

in soil. Soil Biol. Biochem. 20, 601–606.

Garcıa, C., Hernandez, T., Costa, C., Ceccanti, B., Masciandaro, G.,

Ciardi, C., 1993. A study of biochemical parameters of com-

posted and freshmunicipal wastes. Bioresource Technol. 44, 17–

23.

Geiger, G., Livingston, M.P., Funk, F., Schulin, R., 1998a. b-

Glucosidase activity in the presence of copper and geothite.

Eur. J. Soil Sci. 49, 17–23.

Geiger, G., Brandl, H., Furrer, G., Schulin, R., 1998b. The effect of

copper on the activity of cellulase and b-glucosidase in the

presence of montmorillonite or Al-montmorillonite. Soil Biol.

Biochem. 30, 1537–1544.

Giusquiani, P.L., Pagliai, M., Gigliotti, G., Businelli, D., Benetti, A.,

1995. Urban waste compost: effects of physical, chemical and

biochemical soil properties. J. Environ. Qual. 24, 175–182.

Hinesly, T.D., Jones, R.L., Ziegler, E.L., 1972. Effects on corn

applications of heated anaerobically digested sludge. Compost

Sci. 13, 26–30.

Hoffmann, G.G., Teicher, K., 1961. Ein Kolorimetrisches Verfahren

zur Bestimmung der Urease Aktivitat in Boden. Z. Pflanzener-

nahr. Bodenk. 91, 55–63.

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.

Kandeler, E., Tscherko, D., Bruce, K.D., Stemmer, M., Hobbs, P.J.,

Bardgett, R.D., Amelung, W., 2000. Structure and function of

the soil microbial community in microhabitats of a heavy metal

polluted soil. Biol. Fertil. Soils 32, 390–400.

Karaca, A., Haktanır, K., 2000. Arıtma camurlarının toprakta alın-

abilir kursun ve dehidrogenaz enzim aktivitesi uzerine etkileri.

A. U. Z. F. Tarım Bilimleri Dergisi 6 (3), 13–19.

Karaca, A., Naseby, D.C., Lynch, J.M., 2002. Effect of cadmium

contamination with sewage sludge and phosphate fertiliser

amendments on soil enzyme activities, microbial structure

and available cadmium. Biol. Fertil. Soils 35, 428–434.

Kelly, J.J., Max Haggblomb, M., Tate III, R.L., 1999. Effects of the

land application of sewage sludge on soil heavy metal concen-

trations and soil microbial communities. Soil Biol. Biochem. 31,

1467–1470.

Kızılkaya, R., 2004. Cu and Zn accumulation in earthworm L.

terrestris L. in sewage sludge-amended soil and fractions of

Cu and Zn in casts and surrounding soil. Ecol. Eng. 22, 141–151.

Kızılkaya, R., Askın, T., Bayraklı, B., Saglam, M., 2004. Micro-

biological characteristics of soils contaminated with heavy

metals. Eur. J. Soil Biol. 40, 95–102.

King, L.D., Morris, H.D., 1972. Land disposal of liquid

sewage sludge. 2. The effects on soil pH, manganese, zinc,

and growth and chemical composition of rye. J. Environ. Qual. 1,

325–329.

R. Kızılkaya, B. Bayraklı / Applied Soil Ecology 30 (2005) 192–202202

Knight, B.P., McGrath, M.J., Doran, J.W., Cline, R.G., Harris, R.F.,

Schuman, G.E., 1997. Biomass carbon measurements and sub-

strate utilization patterns of microbial populations from soils

amended with cadmium, copper, or zinc. Appl. Environ. Microb.

63, 39–43.

Kunito, T., Saeki, K., Goto, S., Hayashi, H., Oyaizu, H., Matsumoto,

S., 2001. Copper and zinc fractions affecting microorganisms in

long-term sludge-amended soils. Bioresource Technol. 79, 135–

146.

Kuperman, M.M., Carreiro, R.G., 1997. Soil heavy metal concen-

trations, microbial biomass and enzyme activities in a contami-

nated grassland ecosystem. Soil Biol. Biochem. 29, 179–190.

Lindsay, W.L., Norvell, W.A., 1978. Development of a DTPA soil

test for zinc, iron, manganese and copper. Soil Sci. Soc. Am. J.

42, 421–428.

Madojon, E., Burgos, P., Lopez, R., Cabrera, F., 2001. Soil enzy-

matic response to addition of heavy metals with organic resi-

dues. Biol. Fertil. Soils 34, 144–150.

Martens, D.A., Johanson, J.B., Frankenberger, W.T., 1992. Produc-

tion and persistence of soil enzyme with repeated addition of

organic residues. Soil Sci. 153, 53–61.

McGrath, S.P., Lane, P.W., 1989. An explanation for the apparent

losses of metals in long-term field experiment with sewage

sludge. Environ. Pollut. 60, 235–256.

McGrath, S.P., Chang, A.C., Page, A.L., Witter, E., 1994. Land

application of sewage sludge: scientific perspectives of heavy

metal loading limits in Europe and the US. Environ. Rev. 2, 108–

118.

Mile, R.A., Graveland, D.N., 1972. Sewage sludge as a fertilizer.

Can. J. Soil Sci. 52, 270–273.

Moreno, J.L., Hernandez, T., Garcıa, C., 1999. Effects of a cadmium

contaminated sewage sludge compost on dynamics of organic

matter and microbial activity in an arid soil. Biol. Fertil. Soils 28,

230–237.

Moreno, J.L., Garcıa, C., Hernandez, T., 2003. Toxic effect of

cadmium and nickel on soil enzymes and the influence of adding

sewage sludge. Eur. J. Soil Sci. 54, 377–386.

Nannipieri, P., 1994. The potential use of soil enzymes as

indicators of productivity, sustainability and pollution. In:

Pankhurst, C.E., Double, B.M., Gupta, V.V.S.R., Grace, P.R.

(Eds.), Soil Biota. Management in Sustainable Farming Sys-

tems. CSIRO, East Melbourne, pp. 238–244.

Nannipieri, P., Greco, S., Ceccanti, B., 1990. Ecological significance

of the biological activity in soil. In: Bollag, J.M., Stotzky, G.

(Eds.), Soil Biochemistry, vol. 6. Marcel Dekker, New York, pp.

293–355.

Nannipieri, P., Pedrazzini, F., Arcara, P.G., Piovanelli, C., 1979.

Changes in amino acids, enzyme activities, and biomass during

soil microbial growth. Soil Sci. 127, 26–34.

Nyamangara, J., Mzezewa, J., 1999. The effect of long-term sewage

sludge application on Zn, Cu, Ni and Pb levels in a clay loam soil

under pasture grass in Zimbabwe. Agric. Ecosys. Environ. 73,

199–204.

Pagliai, M., Guidi, G., La Marca, M., Giachetti, M., Lucamante, G.,

1981. Effects of sewage sludges and composts on soil porosity

and aggregation. J. Environ. Qual. 10, 556–561.

Pascual, J.A., Hernandez, T., Garcia, C., Ayuso, M., 1998. Enzy-

matic activities in an arid soil amended with urban organic

wastes: laboratory experiment. Bioresource Technol. 64, 131–

138.

Pascual, J.A., Moreno, J.L., Hernandez, T., Garcıa, C., 2002.

Persistence of immobilised and total rease and phosphatase

activities in a soil amended with organic wastes. Bioresource

Technol. 82, 73–78.

Recous, S., Robin, D., Darwis, D., Mary, B., 1995. Soil inorganic N

availability: effect on maize residue decomposition. Soil Biol.

Biochem. 27, 1529–1538.

Sastre, I., Vicente, M.A., Lobo, M.C., 1996. Influence of the

application of sewage sludges on soil microbial activity. Bior-

esource Technol. 57, 19–23.

Singh, A.K., Pandeya, S.B., 1998. Modelling uptake cadmium

by plants in sludge-treated soils. Bioresource Technol. 66,

51–58.

Sloam, J.J., Doedg, R.H., Dolan, M.S., Linden, D.R., 1997. Long-

term effects of biosolid applications on heavy metal. Bioavail-

ability in agriculturaal soils. J. Environ. Qual. 26, 966–974.

Speir, T.W., Ross, D.J., 1978. Soil phosphatase and sulphatase. In:

Burns, R.G. (Ed.), Soil Enzymes. Academic Press, New York,

pp. 197–250.

Stewart, N.E., Beauchamp, E.G., Corke, C.T., Webber, L.R., 1975.

Nitrate nitrogen distribution in corn land following applications

of digested sewage sludge. Can. J. Soil Sci. 55, 287–294.

Tabatabai, M.A., 1994. Soil enzymes. In: Mickelson, S.H., Bighan,

J.M. (Eds.), Methods of Soil Analysis. Part 2. Microbiological

and Biochemical Properties. Soil Science Society of America,

Madison, pp. 775–826.

Tabatabai, M.A., Bremner, J.M., 1969. Use of p-nitrophenyl phos-

phate for assay of soil phosphatase activity. Soil Biol. Biochem.

1, 301–307.

Tabatabai, M.A., Bremner, J.M., 1970. Arylsulphatase activity of

soils. Soil Sci. Soc. Am. Proc. 34, 225–229.

Tam, N.F.Y., Wong, Y.S., 1990. Respiration studies on the decom-

position of organic waste-amended colliery spoil. Agric. Eco-

syst. Environ. 32, 25–38.

Turner, B.L., Hopkins, D.W., Haygarth, P.M., Ostle, N., 2002. b-

Glucosidase activity in pasture soils. Appl. Soil Ecol. 20, 157–

162.

Tyler, G., 1974. Heavy metal pollution and soil enzymatic activity.

Plant Soil 41, 303–311.

Tyler, L.D., McBride, M.B., 1982. Mobility and extractability of

cadmium, copper, nickel, and mineral soil columns. Soil Sci.

134, 198–225.