Open Journal of Soil Science
2011. Vol.1, No.1, 8-15
Copyright © 2011 SciRes. DOI:10.4236/ojss.2011.11002
Soil Biological and Biochemical Response to Cd Exposure
Reginald Ebhin Masto*, Rajkumar Ahirwar, Joshy George, Lal Chand Ram,
Vetrivel Angu Selvi
Central Institute of Mining and Fuel Research, Digwadih Campus, Dhanbad, India.
Received June 2nd, 2011; revised June 20th, 2011; accepted June 25th, 2011.
Though heavy metals can stimulate the activity of soil enzymes in smaller amounts, yet act as inhibitors, if present in high concen-
trations. Natural and anthropogenic heavy metal contamination and its disturbances on soils can be evaluated by using enzymatic
activities as sensors. To study the effects of Cd, soil added with known Cd concentrations (0, 10, 20, 50,100 and 200 mg/kg soil)
were incubated for a period of 30 days at 28°C. At intervals of 0, 5, 10, 20 and 30 days samples were withdrawn for enzyme assays
like dehydrogenase (DHA), catalase (CAT), phenol oxidase (PHE), and peroxidise (PER). In a separate experiment the effect of Cd
on active microbial biomass carbon (AMBC), basal soil respiration (BSR), and metabolic quotient were studied. AMBC showed a
reduction trend with increase in Cd concentration, and a maximum reduction of 47% was observed at 30th day for 200 mg/kg
treatment. BSR also showed the same trend, with a maximum decrease of 42% at the 30th day. With the rate of Cd amendments and
treatment period, DHA showed an inhibition trend; whereas maximum decrease was observed for 200 mg/kg treatment at 30th day.
CAT, PER, and PHE were found to be increased with Cd addition and remained at higher levels than in the control soil. These
changes can be attributed to the effect of Cd on microbial activities. Based on cluster analysis, AMBC appears to be the sensitive
indicators for the soil exposed to Cd contamination.
Keywords: Cadmium, Microbial Biomass, Basal Soil Respiration, Dehydrogenase, Catalase, Peroxidase, Phenol Oxidase,
Intensified urbanisation and associated anthropogenic activi-
ties cause extensive changes on soil and soil-related natural
resources. The study of enzymatic activities in soil is a useful
tool for assessing the functional diversity of soil microbial
communities or soil organic mass turnover (Kandeler et al.
1999). Soil biological activities and biochemical properties get
altered with addition of fertilisers, agricultural activities and
also with the contamination of chemicals like heavy metals.
Soil microbes get influenced by pollutants introduced into the
soil, which is manifested by changes in enzyme activities. In
this group, heavy metals are of special importance, which can
stimulate the activity of soil enzymes in smaller amounts, but
can also act as inhibitors if present in high concentrations
(Christensen et al., 1982; Frankenbereger et al., 1983; Wysz-
kowska et al., 2001). In general, an increase of metal concen-
tration influences soil microbial properties (e.g. respiration rate,
and enzyme activity), which appear very useful as indicators of
soil pollution (Brookes, 1995; Szili-Kovács et al., 1999). In
sewage sludge and phosphate fertilisers, Cd is one of the most
toxic and has been recognised as an environmental contaminant
of considerable interest in various human and animal diseases
(Bramley, 1990; Loganathan et al., 1996). Moreover Cd repre-
sents a group of heavy metals causing the most severe changes
in the biological properties of soils (Milosevic et al., 1997;
Landi et al., 2000; Lebedeva et al., 1995; Welp, 1999; Zheng et
The purpose of the study was to determine the influence of
cadmium contamination on soil microbial activities. Soil en-
zyme activities are the driving force behind all the biochemical
transformations occurring in soil. Several soil quality monitor-
ing programs employed microbial biomass, basal respiration,
and microbial community structure as indicators of soil envi-
ronmental quality (Doran and Parkin, 1994; Sparling, 1997,
Yao et al., 2000). Soil microbial biomass, which plays an im-
portant role in nutrient cycling and ecosystem sustainability,
has been found to be sensitive to increased heavy metal con-
centrations in soils (Giller et al., 1998; Huang and Khan, 1998).
Basal respiration is also commonly measured and indicates the
total carbon turnover. The metabolic quotient, i.e., the ratio of
basal respiration to microbial biomass, is inversely related to
the efficiency with which the microbial biomass uses the in-
digenous substrates (Anderson and Domsch, 1990) and can be a
sensitive indicator for revealing heavy metal toxicity under
natural conditions (Wardle and Ghani, 1995). Though there are
few studies on the effect of Cd on soil microbial activities, most
of them are carried out in temperate soils, where the microbial
activities are quiet higher as compared to tropical soils. Such
studies on tropical soils are limited. In view of above, this study
was undertaken to determine the effect of cadmium exposure
on the soil biological activities in a red soil from tropical region
Materials and Methods
Soil samples collected from Central Institute of Mining and
Fuel Research (CIMFR), Digwadih campus were selected for
the study, and analysed for their physico-chemical properties by
standard methods and data are shown in Table 1.The samples
were passed through 2 mm sieve, and added with required
quantities of cadmium chloride in solution form to attain dif-
ferent Cd concentrations of 0, 10, 25, 50, 100, 200 mg/kg soil.
The samples were then incubated at 28°C, while maintaining
R. E. MASTO ET AL. 9
the moisture content of the soil at field capacity level by adding
required amounts of water. Samples were taken out at 0, 5, 10,
15, 20, and 30 days of incubation, and analyzed for dehydro-
genase, peroxidase, phenol oxidase, and catalase activities. A
separate set of soil was incubated for 10 days to measure the
soil respiration rate and active microbial biomass.
Methods of Analys es
Dehydrogenase activity (DHA) was determined by adding 0.2 ml
of 3% sterile triphenyltetrazolium chloride (TTC) solution and
0.5 ml of 1% sterile glucose into a culture tube containing 1 g of
soil sample. After an incubation period of 24 hr at 28℃, 10 ml of
methanol was added and re-incubated at 28℃ for 8 hr. The
extracted triphenyl formazan (TPF) was measured by absorb-
ance at 485 nm using a spectrophotometer (Klein et al., 1971).
Catalase activity (CAT) was determined as the amount of H2O2
consumed by the soil as described by Xu and Zheng (1986).
Twenty five ml of 3% H2O2 was added to 5 g soil sample. After
incubation at 4℃ for 30 minutes, 25 ml of 1M H2SO4 was added
to it. The contents were filtered; 20 ml of 0.5M H2SO4 was then
added to the 5 ml filtrate. The resulting solution was titrated
against 0.005 M KMnO4 to measure the un-reacted H2O2.
Phenol oxidase (PHE) and peroxidise (PER) activities were
measured with L-DOPA (L-3, 4-dihydroxyphenylalanine) as
substrate in acetate buffer (Robertson et al., 1999). Phenol oxidase
activity was determined by adding 5 ml of 50 mM sodium acetate
buffer and 5ml of 5mM L-DOPA to 0.5 g soil sample. After in-
cubation, the solution was centrifuged and the supernatant was
measured by absorbance at 460 nm. Control was kept for each
sample by adding 5 ml of acetate buffer instead of L-DOPA. For
determination of peroxidase activity, H2O2 was added in addition
to L-DOPA, the increment in the absorbance at 460 nm due to
H2O2 was expressed as the peroxidase activity.
For determination of active microbial biomass carbon
(AMBC), 20 g soil at 60% WHC was placed in each of two
conical flasks. The soil in one flask was amended with nutrients
(120 mg glucose, 30 mg yeast extracts, 45 mg NH4Cl, 12 mg
MgSO4.7H2O, and 10 mg KH2PO4), the other flask was kept as
control, without nutrient amendment. A vial containing 5.0 ml of
0.5 M NaOH was placed in each of the flasks to trap evolved
CO2. The flaks were sealed and incubated in the dark for 24 h at
20°C. The trapped CO2 was measured by back titration with 0.5
M H2SO4.The AMBC was measured as follows:
AMBC = (CO2-C24amend – CO2-C24unamend) × AC (1)
where CO2-C24amend – CO2-C24unamend are the amount of CO2
evolved from the glucose-nutrient-amended and unamended
soils during 24 h incubation, respectively, and AC is the coeffi-
cient (0.283) to convert CO2-C into AMBC (Islam and Weil,
Basal soil respiration (BSR) was measured as the CO2 evolu-
tion from the un-amended moist soil adjusted to 60% WHC for
an incubation period of 10 days at 25 ± 1°C in the dark (Islam
and Weil, 2000). The BSR was calculated as follows:
BSR = (CO2-Csoil – CO2 -Cair)/10 (2)
where CO2-Csoil - is the amount of CO2 evolved from soil and
CO2-Cair is the atmospheric CO2 absorbed by 0.5 M NaOH in a
Metabolic quotient (qCO2) was calculated as BSR per unit of
The data were analyzed using a statistical software SYSTAT-
12. One-way analysis of variance was carried out to compare
the means of different treatments and least significant differ-
ences at P < 0.05 were obtained using Duncan’s multiple range
test (DMRT). The data were also subjected to Pearson correla-
tion analysis, and cluster analysis, to identify the relationship
between the variables and to find out the key soil parameters
that are sensitive to Cd exposure.
Results and Discussion
DHA activity showed a significant decline (P < 0.05) with
increase in Cd concentration (Figure 1). And the mean activity
decrease was found to be 15, 18, 35, 40, and 56% in case of 10,
20, 50, 100 and 200 mg/kg Cd treatments, respectively. The
reduction in AMBC (Figure 5) must have contributed to the
decrease in DHA as this is a group of intracellular enzyme pre-
sent in active microorganism in the soil (Nannipieri et al., 1990).
The increased sensitivity of DHA to metal contamination can
be explained by the fact that the dehydrogenase is active only
within living cells, intact, unlike other enzymes that act outside
the cell. DHA was found most sensitive to pollution with Cd
(Violeta, 2011). Other reason may be the interaction of heavy
metals with the enzyme substrate complex, denaturation of the
enzyme protein or interaction of Cd with protein-active group
(Nannipieri et al., 1994). Karaca et al (2002) and others confirm
the same pattern in their studies also. Similarly, the inhibition
extent was also obvious between different incubation periods,
and varied as the incubation proceeded, the highest inhibition
rate was detected in at 30th day. Sardar et al (2007) showed that
in the case of Cd treatments, DHA activity was significantly
inhibited, after 2 weeks of incubation. This highest inhibitory
effect of heavy metals on soil enzyme activities in the first two
weeks may be due to the sudden exposure of the microbes to
heavy metals. Later on the microbes may have adapted to the
polluted environment, and the enzyme activity tended to re-
cover. Similar results were obtained by Maliszews
ka-Kordybach and Smreczak, (2003) and Zhang et al. (2007).
Some initial physicochemical characteristics of the soil sample.
1 Clay (%) 22.0
2 Silt (%) 9.5
3 Sand (%) 68.5
4 Bulk density (Mg/m3) 1.44
5 Maximum water holding capacity (%) 32.54
6 pH 4.65
7 EC (dS/m) 0.054
8 LOI (%) 4.62
9 Organic carbon (%) 1.69
10 CEC 21.3
11 Nitrogen (mg/kg) 19.1
R. E. MASTO ET AL.
(ROS) such as hydroxyl radical (HO.), superoxide radical (O2
or hydrogen peroxide (H2O2). Catalase enzyme production and
extracellular release may be induced by exposure of the cells to
elevated levels of hydrogen peroxide (Ercal et al., 2001). Perei-
ra et al (2002) opined that the reactive oxygen species (ROS)
induced by Cd are metabolised by CAT in the peroxisomes.
Stimulation of CAT activity can be associated with effective
antioxidant defense system acting against oxidative stress
and/or compensating for the decrease in other antioxidant
Contrary to DHA, other enzymes like catalase, phenol oxi-
dase, and perioxidase increased significantly (P < 0.05) with
increase in Cd concentration (Figure 2). The mean CAT activity
has increased by 6, 11, 22, 29, and 41% in case of 10, 20, 50,
100 and 200 mg/kg Cd treatments respectively. Similarly, the
corresponding increase in PHE activity was 12, 25, 31, 38,
51%; and increase in PER was 4, 13, 20, 25, 32% respectively
(Figure 3 & 4). The increase in CAT, PER, and PHE may be
due to the increase in production of reactive oxygen species
Changes in dehydrogenase activity of soil added with Cd during the incubation period (Within each
incubation period, bars with the same alphabets are not significantly different at P < 0.05 level us-
Changes in catalase activity of soil added with Cd during the incubation period (Within each incu-
bation period bars with the same alphabets are not significantly different at P<0.05 level using
R. E. MASTO ET AL. 11
to play a prominent
rowth and the active-
l of Cd (Figure 5). The mean AMBC at 0, 10, 20, 50,
nan, 2009). Antioxidants are well known
role in the defense against free radicals in
under Cd stress. The effects of Cd on the g
ties of the antioxidant enzymes, catalase, s
plants. Catalase scavanges H2O2 by breaking it down directly to
form water and oxygen while peroxidise decomposes H2O2 by
oxidation of phenolic compounds. The increased activities of
catalase and peroxidase suggest that soil biological systems
depend on these antioxidative enzymes for elimination of H2O2
under Cd stress. Effect of Cd on the above soil enzymes is less
found in literature; however, there are reports on such enzymes
in plant system. Cui and Wang, (2006) reported an increase in
leaf peroxidase activity with Cd treatments while leaf catalase
activity decreased significantly. Similar declines in catalase
activity were reported under Cd stress in rice, cabbage, bean,
carrot, radish, and pea (Chaoui et al. 1997; Sandalio et al. 2001;
Shah et al. 2001; Pandey and Sharma 2002). However, in-
creased catalase activity was also observed in sunflower coty-
ledons and barley (Patra and Panda 1998; Gallego et al. 1999)
and glutathione reductase have been investigated in Crotalaria
juncea seedlings, where the CAT activity did not exhibit any
major variation in the roots following CdCl2 treatment, how-
ever, 2 mM CdCl2 induced a 6-fold increase in activity in the
leaves when compared to the untreated control (Pereira et al.,
Microbial Biomass, Respiration and Metabolic
The AMBC values significantly (p < 0.05) decreased with in-
d 200 mg/kg Cd treatments were 580, 471, 396, 384, 362,
and 310 mg/kg, respectively, which corresponds to 19, 32, 34, 38,
and 47% AMBC reduction. The reduction in AMBC
Changes in phenol oxidase activity of soil added with Cd during the incubation period (Within
each incubation period bars with the same alphabets are not significantly different at P<0.05 level
Changes in peroxidase activity of soil added with Cd during the incubation period (Within each in-
cubation period bars with the same alphabets are not significantly different at P<0.05 level using
R. E. MASTO ET AL.
Effect of Cd on (a) Active microbial biomass carbon, (b) Basal soil respiration, (c) Metabolic quotient (bars
with the same alphabets are not significantly different at P < 0.05 level using LSD.)
is probably due
iability or com-
ing to Zhang et al. (2008), MBC de-
/kg/day) BSR (mg CO2
to the decreased conversion of substrate into
iomass in the Cd contaminated soils, i.e re-
changes in population sizes due to changes in v
petitive ability. Accordnew m
duced primary production and resultant lower input of energy
(Chander & Brookes, 1991). Giller et al. (1997) expressed that
micro organisms differ in their sensitivity to metal toxicity and
sufficient metal exposure will result in immediate death of cells
due to disruption of essential functions, and to more gradual
creased with increasing Cd concentration in soil. These results
are in agreement with the findings of previous studies (Bhat-
tacharyya et al. 2008; Khan et al., 2010). But results of Fritze et
al., (2000) and Landi et al., (2000) showed that even at high Cd
contamination up to 1000 mg Cd/ kg microbial biomass C was
R. E. MASTO ET AL. 13
not found to be negatively affected. Results from another study
conducted in a laboratory on an agricultural sandy loam showed
a significantly lower biomass C in a Cd polluted soil at the very
low contamination level of 0.001799 mg Cd/kg (Griffiths et al.,
1997). Giller et al. (1998) reported that the microbial biomass C
in agricultural soils under long- term metal stress is reduced in
comparison to an unpolluted site. This shows that the compari-
son of these results is very difficult because the different soil
types, time frames, and metal concentrations, lead to different
bioavailable fractions of the metals. Moreover Cd is not an
essential element and so cannot have a direct positive influence
on the soil microbes. There may be an indirect effect of Cd on
the availability of other essential micronutrients. The different
soil types and locations contain different microbial communi-
ties which may not have the same sensitivity to Cd toxicity.
Microbial biomass alone does not provide information on
microbial activity. Some measure of microbial biomass turn-
over, such as BSR, is required for this assessment (Anderson
and Domsch, 1986; Sparling & Ross, 1993). The basal respira-
tion decreased by 18, 30, 32, 34, and 43% under 10, 20, 50, 100
and 200 mg/kg Cd treatments respectively (Figure 5). The re-
A and PHE/ CAT (Table 2), however, positive rela-
HA and AMBC/BSR.
AT, and nega-
ction in BSR may be due to the adverse effects of Cd on soil
microflora, which appeared to increase the accumulation of
organic matter as the heavy metal content increased, probably
because the biomass was less effective in mineralizing soil
organic matter under these conditions. Soil respiration studies
on forest soils showed a decreasing trend with Cd contamina-
tion level. (Landi et al., 2000). The microbial metabolic quo-
tient (respiration-to-biomass ratio) or qCO2 is increasingly be-
ing used as an index of ecosystem development (during which
it supposedly declines) and disturbance (due to which it sup-
posedly increases) (Wardle & Ghani, 1995). The mean qCO2 at
0, 10, 20, 50, 100, and 200 mg/kg Cd treatments were 0.109,
0.111, 0.112, 0.113, 0.115, and 0.117 mg/kg/day respectively.
The metabolic quotient increased by 2, 2.7, 3.4, 5.8 and 7.4%
under 10, 20, 50, 100, and 200 mg/kg Cd treatments respec-
tively (Figure 5). This may be due to the fact that under stress,
the soil micro organisms need to expend more energy to survive.
The greater demand for energy by microorganisms in order to
cope with the toxicity of Cd was also confirmed by the increase
in metabolic quotient (qCO2). Chander and Brookes (1991) and
Bardgett and Saggar (1994) reported a doubling of qCO2 upon
heavy metal contamination. An increased qCO2 indicates shift-
ing of energy from growth to maintenance in an ecosystem.
Biomass synthesis is less efficient under heavy metal stress and
biomass reduction in heavy metal contaminated soils is mainly
due to inefficient biomass synthesis. The shift towards catabolic
processes is often better reflected by increased metabolic quo-
tients (qCO2), i.e. the ratio of CO2 production rate to microbial
Correlations and Cluster Analysis
Significant negative correlation (P < 0.05) was observed be-
tion (P < 0.01) was observed between D
PHE had significant positive correlation with C
tive relation with AMBC/BSR. Negative correlation was ob-
served between CAT and AMBC/BSR, and positive correlation
with qCO2. AMBC had a significant positive correlation with
BSR. The responses to Cd amendment by different soil pa-
uster Combine Rescaled Distance Cl
0 5 10 15 20 25
PHE ─┘ ├───────────────────────────┐
DHA ────┬───┐ │ │
BSR ────┘ ├───────────┘ │
CAT ────────┘ │
Hierarchical dendrogram fo r soil parameters obtained by Ward’s hierarchical cluster i ng method.
Table 2. ns between the parameters of soil contaminated with Cd. Correlatio
DHA PHE PER CAT AMBC BSR qCO
DHA 1.00 –0.966** –0.704 –0.992** 0.933** 0.923** –0.799
PHE 1.00 0.662 0.961
** –0.974** –0.969** 0.761
P ER 1.00 0.724 –0.549 –0.539 0.632
CAT 1.00 –0.901* –0.889*0.854*
AMBC 1.00 0.999** –0.660
BSR 1.00 –0.637
*Correlation is significant at the 0.05 level (2-tailed); **Correlation is significant at the 0.01 level (2-tailed).
R. E. MASTO ET AL.
rameters were further clasy clusteanalysisRelative
homogeneous groups of vas were idntified bierarch
cal cluster analysis, using angorithm that startsach
variable in a separate clusbines cluster This w
one by Ward’s metho
osure of Cd as evidenced by the reduction
in the AMBC, BSR, and DHA activity; and increase in the
activities of CAT, PHE,robial quotient. AMBC
appears to be sensitive soil indicator for the effects that are
lication. This work was financially supported under the Net
Work Project (NWPuncil for Scientific
and Industrial Research (CSIR), Ministry of Science and Tech-
qD) on microbial biomasses from
soils of different cropping histories. Soil Biology and Biochemistry,
22, 251-255. doi:10.1016 094-G
sified br . ly
ter and com
dd, with Euclidean distances as the crite-
rion to form the clusters. For Cd amended soils, Figure 6 shows
three clusters: 1) PER-PHE-qCO2 2) DHA-BSR-CAT, 3)
AMBC. However, the clusters 1 & 2 could be joined together at
a relatively high level. The group of AMBC was remarkably
different from the other parameters in terms of Euclidian dis-
tances in cluster analysis. Judging from these results, the
AMBC appears to be the sensitive indicator for the effects of
Cd on the soil quality. Microbial biomass is well established as
an early indicator of gross changes in C input caused by pollu-
tion (Brookes and McGrath, 1984). In ecotoxicological studies,
the microbial biomass has been proposed as a sensitive indica-
tor to define the impact of contaminants such as metals on soil
biological functioning (Brookes, 1995; Dahlin et al., 1997;
Giller et al., 1998).
The soil biological and biochemical activities significantly
altered with the exp
PER and mic
sulting from Cd contamination. Further studies involving
different types of soils along the natural Cd contamination gra-
dient are needed to clarify the trends detected in this study.
We express our thanks to Director, Central Institute of Min-
ing and Fuel Research, Dhanbad, India for supporting this p
-017, XI FYP) of the Co
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