Agriculture, Ecosystems and Environment 86 (2001) 145–153

Use of microbial parameters to assess treatment methods of

municipal sewage sludge applied to grey forest soils of Tatarstan
S.Yu. Selivanovskaya a,∗ , V.Z. Latypova a , S.N. Kiyamova b , F.K. Alimova b
a Applied Ecology Department, Kazan State University, Kremlyevskaya Str. 18, 420008 Kazan, Tatarstan, Russia
bMicrobiology Department, Kazan State University, Kremlyevskaya Str. 18, 420008 Kazan, Tatarstan, Russia
Received 29 March 1999; received in revised form 5 October 1999; accepted 9 September 2000

Abstract
The benefits and limitation of sewage sludge application as an organic fertilizer need to be investigated for various procedures
of sludge treatment. For this purpose, three types of municipal sewage sludge of the city of Kazan (Tatarstan, Russia), i.e.,
anaerobically digested, composted and untreated, were added to field plots of a grey forest soil (Haplic Greyzem) and planted
to spring barley (Hordeum distichum L.). Soil microbial biomass carbon, respiration and N2 -fixing activity as indicators of
soil fertility as well as plant yield and the metal content in soils and plants were measured to determine the most suitable type
of sludge as organo-mineral fertilizer.
As shown, sludge additions resulted in significant increase of metal contents in the soil, although the maximum metal
concentrations found remained lower than the current critical limits established for Russia. The application of composted
sludge to soils was followed by the increase in microbial biomass (about 1.9–4.4-fold), basal respiration (about 2.3–6.3-fold)
and in N2 -fixing activity (about 2.1–35-fold) in comparison with the parameters found for control soil without any sludge
addition. The application of anaerobically digested sludge had no significant effects on microbial biomass and activity. In the
case of untreated sludge application, a significant decrease in N2 -fixing activity was noted. Beneficial effects on microbial
biomass and activity were greater in plots that had received composted sludge. In the absence of any detrimental effect on crop
quality, this study lends support to using this type of sludge as the organo-mineral fertilizer for grey forest soil of Tatarstan.
© 2001 Elsevier Science B.V. All rights reserved.
Keywords: Sewage sludge; Soil microbial biomass; Respiration; Heavy metals; Nitrogen fixation; Tatarstan

1. Introduction sludge contains plant nutrients and organic matter, it

The agricultural practices used to increase produc-
tivity have often exerted adverse effects on soil quality
and fertility. Recently, the use of sewage sludge as
an organic fertilizer has become a common practice.
It is considered a viable alternative to the landfill
or incineration as a disposal method. Since sewage
∗ Corresponding author.
E-mail address: svetlana.selivanovskaya@ksu.ru
(S.Yu. Selivanovskaya).
may be beneficial to soils and their productivity. It
provides a useful organic amendment to improve soil
properties, i.e., soil structure and nutrient content.
However, sewage sludge can often contain consider-
able amounts of heavy metals and toxic organics. Due
to their persistence and potential toxicity, they may
pose a risk to ecosystems.
Much research has been performed on the use of the
sewage sludge as crop fertilizers (Van den Berg, 1993;
Hue, 1995; Merzlaya et al., 1995; Gardiner et al.,
1995; Selivanovskaya et al., 1997; Selivanovskaya

0167-8809/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 7 - 8 8 0 9 ( 0 0 ) 0 0 2 8 1 - 4
146 S.Yu. Selivanovskaya et al. / Agriculture, Ecosystems and Environment 86 (2001) 145–153
and Latypova, 1999). With regard to the toxic com-
ponents of the sewage sludge, the contamination of
the soil by heavy metals from sewage sludge and the
fate of heavy metals in the plants have been the main
areas of concern (Hue, 1995; Tsadilas et al., 1995;
Kasatikov et al., 1997). One area of current research
interest is the impact of complex toxicants of sewage
sludge on soil microflora, which constitute the living
pool of soil organic matter and is responsible for an
essential part of major nutrient cycles (C, N, P). Soil
microorganisms have a key role in soil biochemical
processes as well as of the transformation of com-
monly used agrochemicals.
Soil microflora is primarily affected when environ-
mental conditions are changed (Van den Berg, 1993;
Brookes, 1995). Soil microbial assays are used for
estimation of the effect of various synthetic prod-
ucts or environmental pollutants (pesticides, fertiliz-
ers, heavy metals, etc.) on soil fertility and agricultural
productivity (Brendecke et al., 1993; McGrath, 1994;
Hani et al., 1996). According to Chander and Brookes
(1991, 1993), microbial parameters are useful indica-
tors of changing soil conditions caused by chemical
pollution long before any detectable change in the soil
organic C content. It might be expected that the micro-
bial activity in soil would increase with the application
of organic matter in sewage sludge. On the other hand,
negative effects on the microflora by the simultaneous
enrichment of inorganic and organic pollutants have
been reported (Brendecke et al., 1993; Fließbach et al.,
1994; McGrath, 1994; Hani et al., 1996).
Soil respiration, the soil microbial biomass and the
fixation of atmospheric nitrogen are three of the most
commonly used parameters for the study of soil mi-
croflora (Anderson and Domsch, 1978, 1990, 1993;
Fließbach et al., 1994; McGrath, 1994; Leita et al.,
1995; Hani et al., 1996). In recent years, several in-
vestigators have proposed derived indexes, i.e., basal
respiration per unit weight of biomass and the ratio of
basal respiration to induced respiration of the soil, to
gain better understanding of the relationships between
the function of the microbial community and the envi-
ronmental conditions (Anderson and Domsch, 1990;
Chander and Brookes, 1993; Valsecchi et al., 1995).
However, little information is available regarding the
effect of sewage sludge on soil microbial activity in
field conditions (Brendecke et al., 1993; McGrath,
1994; McGrath et al., 1995; Hani et al., 1996). No
research has been conducted on the grey forest soil
of Tatarstan in central region of Russia.
Anaerobic digesting and composting are the most
common methods of sludge treatment before its ap-
plication to agricultural soil as fertilizer. Anaerobic
digesting involves sludge incubation in the absence of
air for at least 15 days at 35–55◦ C. In the composting
process, sludge is commonly mixed with a bulking
agent, e.g., wood chips, bark, cereal hulls or straw,
before the mixture is allowed to decompose aerobi-
cally for several weeks. Biological treatment before
the disposal could change the matrix of the sludge
which might result in a stronger binding of the toxic
compounds in the matrix or stimulate their decompo-
sition (Van den Berg, 1993; Hue, 1995; Garcia et al.,
1995; Voutsa et al., 1996). Both treatment processes,
i.e., anaerobic digestion and aerobic composting,
make it possible to stabilize the organic matter, to
decrease the water content of the sediments, and to
reduce the amount of pathogens as well as the avail-
ability of mobile forms of heavy metals due to their
immobilization. An immobilization of toxic metal
forms can also be achieved due to the admixture of
various bulking agents. The selection of the most
suitable type of sludge, i.e., anaerobically digested,
composted or untreated sludge, for soil fertilization
involves comparison of their effect on soil microflora.
The amount of sewage sludge accumulated in the
water treatment plant of the city of Kazan, Russia, is
estimated to be 3.6 Mg of dry matter annually and is
constantly increasing. The storage of vast quantities
of untreated sludge is an important environmental
problem which calls for an effective solution. In this
respect, land application of the accumulated amounts
of sludge could be an inexpensive and attractive way
of its utilization. In this study, the optimal technology
of sludge processing was investigated using correla-
tions among the microbial biomass, respiratory activ-
ity and N2 -fixing activity of soil with metal contents
of soils and plants as well as crop yields.
2. Materials and methods
2.1. Experimental site
The study was carried out at the Biological Sta-
tion of Kazan State University, Zaimische, Tatarstan,
 S.Yu. Selivanovskaya et al. / Agriculture, Ecosystems and Environment 86 (2001) 145–153 147

Table 1
Chemical properties of three types of sewage sludge used
Parameter Untreated sludge Anaerobically digested sludge Composted sludge

Corg (g kg−1 ) 480 ± 91a 420 ± 79 320 ± 61

Ntotal (mg kg−1 ) 20000 ± 5200 16000 ± 4160 9000 ± 2340
Kextractable (g kg−1 ) 361 ± 50 314 ± 44 195 ± 27
Pextractable (g kg−1 ) 348 ± 52 384 ± 58 267 ± 37
Ptotal (%) 0.66 ± 0.13 0.71 ± 0.14 0.4 ± 0.08
pH (0.1 M KCl) 4.8 ± 0.3 4.9 ± 0.3 4.9 ± 0.3
pH (H2 O) 5.1 ± 0.4 5.1 ± 0.4 5.2 ± 0.4
Total metal content (mg kg−1 )
Cu 320.2 ± 35.2 375.1 ± 41.3 160.8 ± 17.7
Cr 1550.3 ± 162.8 1650.1 ± 173.3 650.2 ± 68.2
Pb 67.4 ± 6.4 65.3 ± 6.2 23.8 ± 2.3
Cd 55.9 ± 13.9 66.0 ± 16.5 15.3 ± 3.8
Ni 140.9 ± 11.3 160.6 ± 12.8 61.7 ± 4.9
Zn 68.2 ± 10.9 77.5 ± 12.4 40.4 ± 6.5
a Mean ± standard error.

Russia. Quadruplicate field plots were established on
the grey forest soils (Haplic Greyzem) in a randomized
block design. The plots which measured 2.5 m×3.5 m
were planted to “Rachat” spring barley (Hordeum
distichum L.). Three types of sewage sludge, i.e.,
anaerobically digested, composted and untreated
sludges, were obtained from the municipal wastewater
treatment plant of the city of Kazan, Tatarstan, Russia.
The characteristics of the sewage sludge are shown
in Table 1. The anaerobically digested and untreated
sludges were added to the plots at a rate of 10 Mg ha−1 ,
while the composted sewage sludge was added at a rate
of 30 Mg ha−1 on a dry matter basis. The composted
sewage sludge was prepared from the untreated sludge,
peat and a mixture of wood shaving and sawdust taken
in the ratio 1:1:1 on a dry solids basis. The materials
were fermented for 6 weeks followed by 7 months
maturation. There was no sludge addition to the con-
trol plots. The application rates were calculated taking
into account the maximum allowable concentration of
heavy metals in soil. The sewage sludge was added
to the soils to the depth of 0.1 m and mixed by hand.
All the plants in each plot were harvested by hand
and weighed. Grains were separated from steams and
leaves. The grain samples were oven dried at 80◦ C for
48 h, ground with agate mill prior to analysis. All the
samples were used for the heavy metal analysis within
24 h after sampling.
2.2. Soil sampling and analysis
Soil samples were taken on the 1st, 3rd, 7th, 15th,
30th, 60th and 90th day after sludge application
starting from 3 May 1997. There were 9 days with
precipitation during the sampling period.
Five soil samples were collected from each of the
four replicates at a depth of 0–0.1 m. The samples were
pooled, the mass of pooled samples of each replicate
was about 0.5 kg. All visible plant debris and soil fauna
were removed manually. The soils were then sieved
(2 mm), mixed to ensure homogeneity and stored at
4◦ C prior to analysis.
The water-holding capacity of each soil was deter-
mined and the moisture content was adjusted to 60%
for microbial analysis. Subsamples for the determi-
nation of total heavy metals were air-dried before
analysis.
2.3. Analytical methods
Chemical characteristics of the soil and the sewage
sludge were determined according to the procedures
in State Standards of the Soviet Union (GOST No.
26204-84, 1984; GOST No. 26713-85, 1985; GOST
No. 26715-85, 1985; GOST No. 26717-85, 1985;
GOST No. 26213-91, 1992) issued by the State
Committee of Soviet Union for Standardization,
148 S.Yu. Selivanovskaya et al. / Agriculture, Ecosystems and Environment 86 (2001) 145–153
Moscow, Russia. Soil and sludge pH were measured
potentiometrically in water (1:1) and 1.0 M KCl
(1:25). Water content (moisture) was determined as
the weight loss after drying wet samples at 105◦ C
for 24 h. Organic C was determined by wet oxi-
dation with 0.167 M K2 Cr2 O7 and by subsequent
titration with 0.1 M (NH4 )2 Fe(SO4 )2 ·6H2 O. Total N
was determined by the Kjeldahl method. Total P was
determined after digestion with concentrated H2 SO4
and H2 O2 followed by the subsequent photometric
measurement of the optic density of P–Mo complex
formed. Available P and K amounts were determined
after extraction of 1 g dry soil in 25 ml 0.05 M acetic
acid by shaking for 60 min.
Total content of acid extractable heavy metals was
determined in 1 g samples of soil, sewage sludge
and ashes of the plants using the wet oxidation with
concentrated HNO3 and 3% H2 O2 . The metals were
determined by atomic absorption spectroscopy with
an Analyst-300 (Perkin-Elmer).
2.4. Microbial parameters
Soil microbial parameters were estimated on the
moist soil after incubation for 10 days in the dark at
25◦ C to eliminate the effects of sampling, sieving and
storage.
Soil microbial biomass (Cmicr ) was determined by
fumigation of the samples with C2 H5 OH-free CHCl3
and extraction with 0.5 M K2 SO4 . The extracted C
content was determined by bichromate oxidation in
accordance with ISO 14240-2 (1997).
Basal respiration (CO2 evolution without substrate
addition) was determined by placing subsamples of
moist soil (equivalent to 1 g of oven-dried soil) in
15 ml vessels. The CO2 released was determined
after 1 h incubation in the dark at 27◦ C using gas
chromatograph “Chrom 5” (Prague, Czechoslovakia)
equipped with a silica gel L column 2.5 m × 4 mm.
Temperatures during gas chromatography operation
were as follows: evaporator, 70◦ C; detector, 70◦ C;
column, 60◦ C. The heat conductivity detector was
operated with 80 mA current.
The metabolic quotient (qCO2 ) was calculated
by dividing CO2 –C released from the sample in 1 h
exposure by the biomass C content.
The N2 -fixing activity was determined by the acety-
lene reduction method (Zvagincev, 1991). Subsamples
of moist soil corresponding to 2.5 g oven-dry soil were
placed in 15 ml vessels, then 0.25 ml of 20% glucose
solution was added and the vessels were incubated for
24 h at 27◦ C. Thereafter, the vessels were hermetically
sealed and 1 ml of acetylene was injected. The quan-
tity of ethylene formed in 1 h incubation in the dark
and at 27◦ C was estimated by gas chromatography
(“Chrom 5”, Prague, Czechoslovakia) using a silica
gel ACK column 2.5 m × 4 mm. The conditions of gas
chromatography were as follows: gas-carrier, helium;
flow rate, 30 ml/min; hydrogen flow rate, 30 ml/min;
air flow rate, 300 ml/min; temperature of evaporator,
70◦ C; temperature of thermostat of the column, 60◦ C;
temperature of detector, 70◦ C.
2.5. Statistical analysis
All results were expressed on an air-dry soil basis
and based on five replicates. Statistical analyses were
performed using an analysis of variance (ANOVA) for
a randomized complete block design with α = 0.05.
The means were compared using Fisher’s protected
least significant difference at α = 0.05. All the tables
presented include standard errors of the data.
3. Results and discussions
3.1. Metal contents and crop yields
Metals concentrations of soil samples were
determined on the 1st, 3rd, 7th, 15th, 30th, 60th and
90th day after the application of each type of sewage
sludge to the soils. The mean metal concentrations for
the entire study period are presented in Table 2. No
significant differences (P < 0.05) in the metal con-
tents between the means of the three amended soils
were found. This was expected because the account
of sludge application was based on metal contents in
each type of the sludge added. Significant differences
(P < 0.05) were observed between the means of
metal contents in the treated and untreated (control)
soil. However, even in the sewage sludge amended
from the plots, the concentrations of individual heavy
metal were below the current limits established for
Russia and European countries (Fließbach et al., 1994;
McGrath, 1994; McGrath et al., 1995; SanPiN No.
2.1.7.573-96, 1997). Although Fließbach et al. (1994),
 S.Yu. Selivanovskaya et al. / Agriculture, Ecosystems and Environment 86 (2001) 145–153 149

Table 2
Chemical properties of a grey forest soil in Tatarstan in relation to type of sewage sludge used
Parameter Soils amended with Control soil

Untreated sludge Anaerobically digested sludge Composted sludge

Corg (g kg−1 ) 12.8 ± 2.4 aa 12.6 ± 2.4 a 13.3 ± 2.5 a 12.2 ± 2.3 a
Ntotal (mg kg−1 ) 1100 ± 320 a 1140 ± 285 a 1250 ± 324 a 1100 ± 286 a
Kextractable (g kg−1 ) 94 ± 13 a 90 ± 12 a 96 ± 13 a 91 ± 13 a
Pextractable (g kg−1 ) 138 ± 21 a 132 ± 20 a 144 ± 22 a 125 ± 17 a
pH (H2 O) 7.1 ± 0.4 a 7.1 ± 0.4 a 7.15 ± 0.3 a 7.2 ± 0.35 a
Total metal content (mg kg−1 )
Cu 39.1 ± 4.3 a 29.2 ± 3.2 b 42.5 ± 4.7 a 31.7 ± 3.5 b
Cr 109.5 ± 11.5 a 100.3 ± 10.5 a 122.9 ± 12.9 a 62.6 ± 6.6 b
Pb 17.4 ± 1.6 a 19.6 ± 1.8 a 18.7 ± 1.7 a 12.1 ± 1.1 b
Cd 1.7 ± 0.42 a 1.2 ± 0.28 a 1.5 ± 0.37 a 0.8 ± 0.2 b
Ni 40.1 ± 3.2 a 32.1 ± 2.6 a 38.4 ± 3.1 a 24.8 ± 1.9 b
Zn 58.5 ± 9.4 a 47.8 ± 7.6 a 51.5 ± 8.2 a 32.8 ± 5.2 b
a Mean ± standard error. Values followed by the same letter within each row are not significantly different at 5% level by Fisher’s

range test.

McGrath (1994), McGrath et al. (1995), Hani et al.
(1996) and Giller et al. (1998) reported deleterious
effects occurred even when the metal concentrations
in soil remained below the limits mentioned.
The addition of sludge (untreated, anaerobically
digested and composted) to the soil produced sig-
nificantly higher barley yields (P < 0.05) than that
observed in the control without any amendment
(3120, 3240, 3390 and 2150 kg ha−1 , respectively)
(Table 3). The metal contents in the barley grain
were generally lower in the control compared to the
amended soils. However, metal contents in the barley
grain grown from the amended soils did not exceed
the current limits established for Russia (SanPiN No.
2.1.7.573-96, 1997) and, hence, were not considered
critical.
3.2. Microbial biomass
One of the most commonly used parameters in soil
biology is the microbial biomass. As shown in Table 4,
sludge application generally increased soil microbial
biomass carbon. On the first day after sludge appli-
cation, the maximum values of this parameter were
found to be 4241, 4202 and 4788 mg Cmicr kg−1 for
soils amended with untreated, anaerobically digested

Table 3
Barley grain yields and metal contents for grain grown on sludge amended grey forest soil
Parameter Soils amended with Control soil

Untreated sludge Anaerobically digested sludge Composted sludge

Yield (kg ha−1 ) 3120 ± 420 aa 3240 ± 431 a 3390 ± 458 a 2150 ± 358 b
Total metal content (mg kg−1 )
Cu 23.1 ± 4.6 a 18 ± 3.6 a 21.6 ± 4.3 a 16.1 ± 3.2 a
Cr 0.4 ± 0.08 a 0.3 ± 0.06 a 0.3 ± 0.06 a 0.3 ± 0.06 a
Pb 2.8 ± 0.6 a 2.6 ± 0.06 a 2.2 ± 0.4 a 2.0 ± 0.4 a
Cd 0.07 ± 0.015 a 0.065 ± 0.012 a 0.09 ± 0.02 a 0.06 ± 0.012 a
Ni 0.38 ± 0.08 a 0.33 ± 0.07 a 0.39 ± 0.08 a 0.3 ± 0.05 a
Zn 14.2 ± 3.1 a 15.8 ± 3.6 a 18.5 ± 4.2 a 11.4 ± 2.7 a
a Mean ± standard error. Values followed by the same letter within each row are not significantly different at 5% level by Fisher’s

range test.
150 S.Yu. Selivanovskaya et al. / Agriculture, Ecosystems and Environment 86 (2001) 145–153
Table 4
Changes in microbial biomass C (mg Cmicr kg−1 ) in samples of unamended grey forest soil (control) and in samples after application of
untreated, anaerobically digested and composted sludge
Time, day after Soils amended with
sludge application Untreated sludge Anaerobically digested sludge
1 4020 ± 840 aa 3960 ± 2140 a
3 1120 ± 160 a 1220 ± 230 a
7 990 ± 400 a 580 ± 92 b
15 871 ± 131 a 720 ± 115 a
30 856 ± 86 a 910 ± 264 a
60 711 ± 156 a 420 ± 109 b
90 1190 ± 350 a 1590 ± 350 ab
a Mean ± standard error. Values followed by the same letter within each row are not significantly different at 5% level by Fisher’s
range test.
and composted sewage sludge, respectively. The ap-
propriate value for the control soil was found to be
2043 mg Cmicr kg−1 . This can be attributed to the in-
corporation of easily biodegradable organic matter.
Over time, the differences between the mean values of
microbial biomass in the soils amended with untreated
and anaerobically digested sludge, and the control soil
were insignificant. However, in the soil amended with
composted sludge, the biomass was found significantly
(P < 0.05) greater by a factor from 1.9 to 4.4 than
that determined in the control soil throughout the mea-
suring period of 90 days. This effect can be related
to the organic C matter contained both in this type of
sludge and bulking agent used in composted sludge.
The availability of wood shaving and sawdust organic
matter, used in composting as bulking agent, for soil
microflora is not as high so that the decrease of mi-
crobial biomass is slower than that found for the ap-
plication of sludge alone. The observed stimulation
by sewage sludge is in a good agreement with other
studies (Fließbach et al., 1994; Hue, 1995; Dar, 1997;
Moreno et al., 1999). These investigators attributed
the positive effects of sludge on heterotrophic soil mi-
croorganisms to the greater availability of carbon and
nutrients in sludged soil.
Although the sewage sludge contained additional
metal amounts, no decrease in the soil microbial
biomass was found as would be expected in ac-
cordance with the results of Chander and Brookes
(1991), McGrath (1994), McGrath et al. (1995) and
Valsecchi et al. (1995). This difference in the results
can be caused by a number of factors. The minimum
metal concentrations giving rise to the decrease of
Control soil
Composted sludge
4970 ± 950 a 2450 ± 540 b
3910 ± 2500 b 890 ± 240 a
2150 ± 410 c 924 ± 92 a
1980 ± 260 b 910 ± 46 a
2240 ± 310 b 812 ± 154 a
1640 ± 160 c 854 ± 247 a
1920 ± 330 b 824 ± 107 a
microbial biomass cited by McGrath et al. (1995) are
higher than metal contents determined for this study
if the soils with similar organic matter content and
pH (Woburn, UK) are compared. Ni content only
was higher in amended samples of grey forest soil
of Tatarstan than in the reference soil from Woburn,
UK. However, the sensitivity of the soil microbial
community toward this metal is the least among the
metals investigated. It should be also mentioned that
the duration of investigations cited above was longer
than that in this study.
3.3. Soil microbial activity
Soil respiration is considered a useful indicator of
soil microbial activity (Anderson and Domsch, 1990).
The application of all the sludges investigated affected
the microbial communities of the grey forest soil. This
effect and its duration depended on the type of sludge
applied. As shown in Table 5, the basal respiration
of the amended soil was significantly (P < 0.05)
greater than that of the control soil only in case of
composted sludge application. Corresponding values
for the soils treated with anaerobically digested and
untreated sludge did not differ from those measured
for the control soil. The average relative shift of the
basal respiration was found to be equal to 3.9, 1.3 and
1.8 for the soils amended with composted, anaerobi-
cally digested and untreated sludge, respectively. Basal
respiration decreased with time, especially in soils
amended with untreated sludges. A significant de-
crease in microbial respiration observed can be related
to the microbial consumption of easily biodegradable
 S.Yu. Selivanovskaya et al. / Agriculture, Ecosystems and Environment 86 (2001) 145–153 151

Table 5
Changes in basal respiration (mg CO2 –C kg−1 h−1 ) in samples of unamended grey forest soil (control) and in samples after application of
untreated, anaerobically digested and composted sludge
Time, day after Soils amended with Control soil
sludge application Untreated sludge Anaerobically digested sludge Composted sludge

1 21.8 ± 5.9 aa 14.1 ± 2.9 b 22.8 ± 5.0 a 7.6 ± 2.0 c

3 17.4 ± 2.1 a 6.7 ± 0.3 b 38.9 ± 5.8 c 6.2 ± 2.9 b
7 6.8 ± 0.5 a 9.1 ± 3.9 ac 30.6 ± 1.8 b 13.5 ± 3.9 c
15 10.9 ± 0.9 a 9.1 ± 2.9 a 27.4 ± 5.5 b 7.5 ± 1.5 a
30 18.4 ± 4.0 a 9.3 ± 1.9 b 30.5 ± 7.0 c 6.5 ± 2.0 b
60 5.7 ± 2.6 a 6.9 ± 2.5 a 13.6 ± 4.1 b 5.8 ± 3.2 a
90 9.5 ± 2.1 a 7.7 ± 1.9 a 21.5 ± 3.0 b 4.4 ± 1.1 c
a Mean ± standard error. Values followed by the same letter within each row are not significantly different at 5% level by Fisher’s

range test.

compounds during sludge treatment (Moreno et al.,
1999).
A relationship between soil respiration and micro-
bial biomass can be expressed using the parameter of
the metabolic quotients qCO2 . It can be considered
an indicator of the environmental disturbance of the
microbial population. Organic amendment of the soil
resulted in a significant (P < 0.05) increase in the
qCO2 from the initial value 3 typical for control soil
samples (Table 6). The untreated sludge contained
relatively high amounts of easily biodegradable or-
ganics, so that the appropriate value of the metabolic
quotient was greater (up to 21.5 ␮g CO2 –C mg−1 h−1 )
in comparison with the same parameter obtained for
the application of anaerobically digested and com-
posted sludges. The lesser qCO2 value in comparison
with that for untreated sludge was found for the soil
amended with composted sludge. Nevertheless, the
value 90 days after application remained higher than
that determined in control measurement. At the end
of the experiment, the qCO2 value approached that
of the control due to the protective and buffering ca-
pacity of the soil, except for that of the soil amended
with composted sludge. In the latter, the stabiliza-
tion probably requires more than the 90 days used
in the study. As stressed previously, the application
rates of the sludges were calculated in such a man-
ner that the amounts of metals in the soils treated
was about the same for all the samples (see Table 2),
although the soil samples treated differed in the
amount and quality, i.e., stability of organic matter.
In this respect, the observed changes in qCO2 can
be related to the main influence of organics, but not
metals.

Table 6
Changes in metabolic quotient (qCO2 ) (␮g CO2 –C mg−1 h−1 ) in samples of unamended grey forest soil (control) and in samples after
application of untreated, anaerobically digested and composted sludge
Time, day after Soils amended with Control soil
sludge application
Untreated sludge Anaerobically digested sludge Composted sludge

1 5.4 ± 1.9 aa 3.6 ± 1.6 b 4.6 ± 1.2 a 3.1 ± 1.1 b

3 15.5 ± 2.2 a 5.5 ± 0.3 b 9.9 ± 4.2 a 6.9 ± 4.5 b
7 6.9 ± 0.9 a 15.6 ± 8.2 b 14.2 ± 1.1 b 14.6 ± 4.7 b
15 12.6 ± 1.3 a 12.6 ± 4.9 a 13.8 ± 3.2 a 8.3 ± 1.7 b
30 21.5 ± 5.2 a 10.3 ± 3.0 bc 13.6 ± 3.6 c 7.9 ± 3.1 b
60 8.0 ± 4.6 a 16.6 ± 8.1 b 8.3 ± 2.8 a 6.8 ± 5.2 a
90 8.0 ± 2.5 ab 4.8 ± 1.5 b 11.2 ± 1.9 a 5.3 ± 1.5 b
a Mean ± standard error. Values followed by the same letter within each row are not significantly different at 5% level by Fisher’s

range test.
152 S.Yu. Selivanovskaya et al. / Agriculture, Ecosystems and Environment 86 (2001) 145–153
Table 7
Changes in N2 -fixation activity (mg N kg−1 for 24 h) in samples of unamended grey forest soil (control) and in samples after application
of untreated, anaerobically digested and composted sludge
Time, day after Soils amended with
sludge application Untreated sludge Anaerobically digested sludge
1 0.007 ± 0.0014 aa 0.02 ± 0.006 b
3 0.18 ± 0.02 a 0.11 ± 0.05 a
7 0.06 ± 0.007 a 0.69 ± 0.08 b
15 0.09 ± 0.016 a 0.24 ± 0.08 b
30 0.05 ± 0.02 a 0.84 ± 0.10 b
60 0.41 ± 0.03 a 1.0 ± 0.07 b
90 0.41 ± 0.09 ab 0.51 ± 0.15 b
a Mean ± standard error. Values followed by the same letter within each row are not significantly different at 5% level by Fisher’s
range test.
Soil microbial processes, like mineralization of or-
ganic matter or soil respiration, can be relatively little
affected by moderate levels of heavy metals, while the
processes carried out by few specialized organisms,
i.e., nitrogen fixation, are more sensitive (McGrath,
1994). Table 7 shows that the heterotrophic N2 -fixing
activity for soils amended with untreated sludge over
time was lower than or similar to that for the control
soils. This may be related to the availability of the
metals, which were probably greater in the untreated
biological sludge than in the treated ones, since the
metal concentration in all the soil samples was close
to each other (Table 2). This observation was similar
to that reported by Garcia et al. (1995) and Voutsa
et al. (1996). The level of N2 -fixation activity of the
soil treated with anaerobically digested and composted
sludge was significantly (P < 0.05) higher (by a fac-
tor from 1.1 to 27) than that for the control soil. Since
the N2 -fixation process is an energy consuming pro-
cess, the addition of the available organics present in
sewage sludge may enhance the process. The effect
was in a good agreement with the study of McGrath
(1994), which confirmed that the N2 -fixation was more
sensitive than soil respiration to soil metal content.
4. Conclusions
The investigation of the various parameters used as
indicators of the response of microbial community as
well as plants to the soil showed that the preliminary
sludge treatment can be of key importance for the ef-
fect observed. The estimation of specific parameters
Control soil
Composted sludge
0.55 ± 0.005 c 0.18 ± 0.03 d
0.81 ± 0.06 b 0.12 ± 0.05 a
0.69 ± 0.08 b 0.02 ± 0.014 c
0.37 ± 0.07 b 0.10 ± 0.015 a
0.36 ± 0.11 c 0.17 ± 0.06 d
0.85 ± 0.08 b 0.32 ± 0.01 a
0.84 ± 0.20 c 0.31 ± 0.04 a
of microbial community, i.e., biomass, respiratory and
N2 -fixing activity is more informative than the tradi-
tional methods of heavy metal analysis. The changes
in microflora after the sludge amendment are not
exhausted with the influence of heavy metal contam-
ination. As was shown, the choice of soil amendment
and sludge treatment call for the account of both
organic and inorganic materials. Thus, the absence
of positive effect on microbial biomass and activity
observed for the soil amended with the untreated and
anaerobically digested sludges can be caused by rela-
tively low level of organics. The maximum beneficial
effects were found for composted sludge. Its amend-
ment provides for higher amount of organic matter in
the soil as referred to the same amount of metals.
References
Anderson, J.P.E., Domsch, K.H., 1978. A physiological method
for the quantitative measurement of microbial biomass in soil.
Soil Biol. Biochem. 10, 215–221.
Anderson, T.H., Domsch, K.H., 1990. Application of eco-
physiological quotients (qCO2 and qD) on microbial biomasses
from soils of different cropping histories. Soil Biol. Biochem.
22, 251–255.
Anderson, T.H., Domsch, K.H., 1993. The metabolic quotient for
CO2 (qCO2 ) as a specific activity parameter to assess the effects
of environmental conditions, such as pH, on the microbial
biomass of forest soils. Soil Biol. Biochem. 25, 393–395.
Brendecke, J.W., Alexson, R.D., Pepper, I.L., 1993. Soil microbial
activity as an indicator of soil fertility: long-term effects of
municipal sewage sludge on an arid soil. Soil Biol. Biochem.
25, 751–758.
Brookes, P.C., 1995. The use of microbial parameters in monitoring
soil pollution by heavy metals. Biol. Fert. Soils 19, 269–279.
 S.Yu. Selivanovskaya et al. / Agriculture, Ecosystems and Environment 86 (2001) 145–153
Chander, K., Brookes, P.C., 1991. Effects of heavy metals from
past application of sewage sludge on microbial biomass and
organic matter accumulation in a sandy loam and silty loam
UK soil. Soil Biol. Biochem. 23, 927–932.
Chander, K., Brookes, P.C., 1993. Residual effects of zinc, copper
and nickel in sewage sludge on microbial biomass in a sandy
loam. Soil Biol. Biochem. 25, 1231–1239.
Dar, G.H., 1997. Impact of lead and sewage sludge on soil
microbial biomass and carbon and nitrogen mineralization. Bull.
Environ. Contam. Toxicol. 58, 234–240.
Fließbach, A., Martens, R., Reber, H.H., 1994. Soil microbial bio-
mass and microbial activity in soils treated with heavy metal co-
ntaminated sewage sludge. Soil Biol. Biochem. 26, 1201–1205.
Garcia, C., Moreno, J.L., Hernandes, T., Costa, F., Polo, A., 1995.
Effect of composting on sewage sludges contaminated with
heavy metals. Bioresour. Technol. 53, 13–19.
Gardiner, D.T., Miller, R.W., Badamchian, B., Azzari, A.S., Sisson,
D.R., 1995. Effects of repeated sewage sludge applications on
plant accumulation of heavy metals. Agric. Ecosyst. Environ.
55, 1–6.
Giller, K.E., Witter, E., McGrath, S.P., 1998. Toxicity of heavy
metals to microorganisms and microbial processes in agri-
cultural soils: a review. Soil Biol. Biochem. 10/11, 1389–1414.
GOST No. 26204-84, 1984. Metod opredeleniya podviznich form
phosphora i kaliya metodom Chirikova. Izdatelstvo Standartov,
Moskwa, SSSR (in Russian).
GOST No. 26713-85, 1985. Metod opredeleniya vlagi i suchogo
ostatka. Izdatelstvo Standartov, Moskwa, SSSR (in Russian).
GOST No. 26715-85, 1985. Metod opredeleniya obshego azota.
Izdatelstvo Standartov, Moskwa, SSSR (in Russian).
GOST No. 26717-85, 1985. Metod opredeleniya obshego phos-
phora. Izdatelstvo Standartov, Moskwa, SSSR (in Russian).
GOST No. 26213-91, 1992. Metod opredeleniya organicheskogo
veshestva. Izdatelstvo Standartov, Moskwa, Rossiya (in
Russian).
Hani, H., Siegenthaler, A., Candinas, T., 1996. Soil effect due
to sewage sludge application in agriculture. In: Rodriguez-
Barrueco, C. (Ed.), Fertilizer and Environment. Kluwer
Academic Publishers, Dordrecht, pp. 267–274.
Hue, N.V., 1995. Sewage sludge. In: Rechcigl, J.E. (Ed.), Soil
Amendments and Environmental Quality. Lewis Publishers,
Boca Raton, FL, pp. 199–247.
ISO 14240-2, 1997. Soil quality — Determination of soil microbial
biomass. Part 2: Fumigation–extraction method. International
standard, 12 pp.
Kasatikov, V.A., Ovcharenko, M.M., Kasatikova, S.M., Shabardina,
N.N., 1997. Vliyanie mineralnych udobreniy i osadkov
153
stochnych vod na uroven koncentracii v pochve ryada mikroele-
mentov. Agrochimiya 2, 81–85 (in Russian).
Leita, L., De Nobili, M., Muhlbachova, G., Mondini, C., Marchiol,
I., Zerbi, G., 1995. Bioavailability and effects of heavy
metals on soil microbial biomass survival during laboratory
incubation. Biol. Fert. Soils 19, 103–108.
McGrath, S.P., 1994. Effects of heavy metals from sewage sludge
on soil microbes in agricultural ecosystems. In: Ross, S.M.
(Ed.), Toxic Metals in Soil–Plant Systems. Wiley, New York,
pp. 247–274.
McGrath, S.P., Chaudri, A.M., Giller, K.E., 1995. Long-term
effects of metals in sewage sludge on soils, microorganisms
and plants. J. Ind. Microbiol. 14, 94–104.
Merzlaya, G.E., Zyabcina, G.A., Nesterovich, I.A., Fomcina, T.P.,
1995. Agroecologicheskaya ocenka ispolsovaniya osagkov
stochnych vod. Agrochimiya 5, 92–96 (in Russian).
Moreno, J.L., Hernandez, T., Garcia, C., 1999. Effects of
cadmium-contaminated sewage sludge compost on dynamics
of organic matter and microbial activity in an arid soil.. Biol.
Fert. Soils 28, 230–237.
SanPiN No. 2.1.7.573-96, 1997. Gigienicheskie trebovaniya k
ispolzovaniyu stichnich vod i ich osadkov dlya orosheniya
i udobreniya M. Informacionno-izdatelskii centr Minzdrava
Rossii, Moskva, p. 36 (in Russian).
Selivanovskaya, S.Yu., Latypova, V.Z., 1999. Nekotorye aspecty
normirovaniya kachestva i utulizazii osadkov stochnich vod.
Ecologicheskaya Chimiya 8 (2), 119–129 (in Russian).
Selivanovskaya, S.Yu., Latypova, V.Z., Naumova, R.P., Ravzieva,
G.M., 1997. On the possibility of involvement of microelements
of sewage sludge into biogeochemical circulation. Environ.
Radioecol. Appl. Ecol. 1, 13–19.
Tsadilas, C.D., Matsi, T., Barbayiannis, N., Dimoyiannis, D., 1995.
Influence of sewage sludge application on soil properties and
on the distribution and availability of heavy metal fractions.
Commun. Soil Sci. Anal. 26, 2603–2619.
Valsecchi, G., Giliotti, C., Farini, A., 1995. Microbial biomass,
activity, and organic matter accumulation in soils contaminated
with heavy metals. Biol. Fert. Soils 20, 253–259.
Van den Berg, J.J., 1993. Effects of sewage sludge disposal. Land
Degrad. Rehabil. 4, 407–413.
Voutsa, D., Zachariadis, G., Samara, C., Kouimtzis, Th., 1996.
Evaluation of the toxic content of sludges produced during the
biological treatment of municipal and industrial waste waters.
J. Environ. Sci. Health A 31 (3), 657–671.
Zvagincev, D.G., 1991. Metody Pochvennoy Microbiologii i
Biochimii. Publishing House MGU, Moscow, 304 pp. (in
Russian).