Directory UMM :Data Elmu:jurnal:S:Soil Biology And Chemistry:Vol32.Issue11-12.oct2000:
Soil Biology & Biochemistry 32 (2000) 1697±1705
www.elsevier.com/locate/soilbio
Factors in¯uencing the degradation of soil-applied endosulfan
isomers
Niranjan Awasthi, Rajiv Ahuja, Ashwani Kumar*
Environmental Biotechnology Section, Industrial Toxicology Research Centre, Post Box No. 80, Mahatma Gandhi Marg, Lucknow 226 001, India
Accepted 21 April 2000
Abstract
The addition of isolated bacterial cells to contaminated soils causes an enhanced degradation of endosulfan isomers. Various
factors, including the additional presence of carbon sources, pH, moisture content, concentration of endosulfan, and size of
inoculum, in¯uenced the degradation of endosulfan isomers. The degradation was faster in wet soils, as compared with the
¯ooded soils, and was inhibited by the presence of additional carbon sources such as sodium acetate and sodium succinate. The
degradation of endosulfan was not detectable at acidic pH and increased gradually to reach an optimal activity at pH 8.5. It
chemically converts into endosulfan diol at higher pH values. The rate of biodegradation progressed with the increase in
endosulfan concentration up to 5.0 mg gÿ1 soil, followed by an inhibitory eect at higher concentrations, reaching a total loss of
biodegradative activity at 10 mg gÿ1 soil. The addition of 2 106 bacterial cells gÿ1 soil was optimal for endosulfan degradation
and any further increase in inoculum size was of no additional advantage. Initial optimization of these factors is, therefore,
essential for successful bioremediation. 7 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Endosulfan; Chlorinated pesticide; Biodegradation; Contaminated sites; Bioremediation
1. Introduction
Endosulfan (6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9ahexahydro-6,9-methano-2,3,4-benzo(e )dioxathiepin-3oxide, CAS No. 115-29-7) is a chlorinated pesticide of
the cyclodiene group. Its technical preparation consists
of a- and b-isomers (70:30). It is used extensively,
throughout the world, as a broad spectrum insecticide,
on cotton crops, ®eld crops such as paddy, sorghum,
oil seeds and pulses, as well as vegetables and fruit
crops (Goebel et al., 1982). Endosulfan contamination
has been detected in soils, water, air and food products
because of its abundant usage and potential for environmental transport. It is extremely toxic to ®shes
and aquatic invertebrates (Verschueren, 1983; Sunderam et al., 1992) and is classi®ed as a `priority pollu-
* Corresponding author. Fax: +91-522-228227.
E-mail address: [email protected] (A. Kumar).
tant' by international environmental agencies (Keith
and Telliard, 1979). The persistence of endosulfan in
agricultural soils has been studied in many laboratories. Its life in soils has been estimated to be from
100 to 120 days (Rao and Murty, 1980; Kathpal et al.,
1997) to several months (Stewart and Cairns, 1974)
and the relative rates of dissipation for a- and b-endosulfan have also been shown to be dierent. This dissipation depends on a multitude of factors such as its
volatilization, alkaline hydrolysis and photodecomposition, besides the presence of fertilizer, crop pattern, atmospheric temperature, rain and biotic conversions,
among others (Goebel et al., 1982).
Bioremediation, which involves degradation of target chemicals by indigenous or added microbial cells,
is used to clean up sites contaminated by pollutants
such as pentachlorophenol (Miethling and Karlson,
1996; Barbeau et al., 1997), diesel oil (Margesin and
Schinner, 1997), herbicides (Kilbane et al., 1983;
Kaake et al., 1992), polyaromatic hydrocarbons (Kast-
0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 0 8 7 - 0
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N. Awasthi et al. / Soil Biology & Biochemistry 32 (2000) 1697±1705
ner et al., 1994; Mueller et al., 1996), petroleum products (Balba et al., 1991) and munition compounds
(Funk et al., 1993). In many other cases, addition of
microorganisms has failed to enhance degradation of
polluting chemicals, probably due to any or combination of factors like non-optimal temperature, pH,
moisture content, insucient number of added microorganisms or incompatible soil texture. Besides, the
concentration of polluting chemical might either be
too high to be toxic to the added microbes or too low
to induce the degradative activity (Goldstein et al.,
1985; Block et al., 1993; Morra, 1996).
Bacteria and fungi, which degrade endosulfan isomers in liquid culture have been isolated and characterized in many laboratories (El Zorgani and Omer,
1974; Martens, 1976; Miles and Moy, 1979; Mukherjee
and Gopal, 1994; Kullman and Matsumura, 1996;
Awasthi et al., 1997). Endosulfan diol, endosulfan sulfate, endosulfan aldehyde, endosulfan ether and endosulfan lactone have been demonstrated as the major
metabolites formed during its degradation. The environmental fate of endosulfan in dierent types of
contaminated soils has also been studied (Rao and
Murty, 1980; Antonious and Byers, 1997; Kathpal et
al., 1997; Kaur et al., 1998; Parkpian et al. 1998), but
the in¯uence of exogenous bacterial cells on endosulfan degradation has not been evaluated.
In order to develop a suitable technology package
for bioremediation of soils contaminated with endosulfan, we have studied the ecacy of an isolated bacterial culture (Awasthi et al., 1997) on the
biodegradation of soil-applied endosulfan isomers. The
in¯uence of factors, such as moisture content, pH, presence of additional carbon sources, size of inoculum
and initial pesticide load in soils, on endosulfan degradation were also evaluated.
were found to be more eective. For this study, each
of the isolated bacteria were grown separately in a rich
medium (Peptone 1.0 g; Yeast Extract 0.5 g; NaCl 0.5
g; dissolved in distilled water, pH adjusted to 7.5 and
made up to 100 ml) overnight, centrifuged at 10,000
rpm for 10 min, washed and resuspended in a mineral
medium (KH2PO4, 170 mg; Na2HPO4, 980 mg;
(NH4)2SO4, 100 mg; MgSO4, 4.87 mg; MgO, 100 mg;
FeSO4, 50 mg; CaCO3, 20 mg; ZnSO4, 80 mg;
CuSO45H2O, 16 mg; CoSO4. 15 mg; H3BO3, 6 mg; dissolved in 100 ml of distilled water, the pH of the medium was 7.0) to the density of 2 108 colony forming
units (CFU) mlÿ1. Equal volume of both the cultures
were mixed to make a stock co-culture. For degradation experiments, bacteria from stock co-culture
were grown in rich medium overnight, centrifuged,
washed twice and suspended in mineral medium to 2
108 CFU mlÿ1 before use.
2.3. Soils studied
Two soils, one (Soil A) from the Banthara ®eld
station of the National Botanical Research Institute,
Lucknow, containing, clay 60%, silt 30%, sand 10%,
organic carbon 0.38% at pH 8.7, and the other (Soil
B) from the Gheru Campus of Industrial Toxicology
Research Centre, Lucknow, containing clay 39%, silt
12%, sand 49%, organic carbon 0.59%, at pH 9.37,
were used. Collected soils were air dried and sieved (2
mm). Four hundred milliliters of distilled water containing 6.0 g of commercial endosulfan (35% EC) were
added to 1000 g of each soil and mixed thoroughly.
The concentration of the active ingredient was estimated to be 2.35 mg gÿ1 soil, i.e. 1.64 mg of a-endosulfan and 0.71 mg of b-endosulfan. After air drying
for 24±48 h, the soils were pulverized and used for
degradation studies.
2. Materials and methods
2.1. Chemicals
Commercial endosulfan (Parrysulfan, 35% EC, by
EID Parry, Chennai, India) was purchased from a
local market. Pure a- and b-endosulfan were obtained
from Farbwerke-Hoechst AG, Frankfurt, Germany. 2Phenoxy ethanol was purchased from Sigma, St Louis,
MO, USA. All other reagents were of analytical grade.
2.2. Bacterial cells
A bacterial co-culture consisting of two dierent
strains of Bacillus sp., isolated earlier from a contaminated site by selective enrichment, was used (Awasthi
et al., 1997). Either of the isolated bacteria can mediate the degradation of endosulfan but together they
2.4. Degradation of soil applied endosulfan and in¯uence
of external factors
Degradation of soil-applied endosulfan in uninoculated or inoculated conditions, was studied in parallel
sets of three containers each, for each variable and
time point of the study.
To study the degradation in soils, two sets of beakers (100 ml) were maintained for each soil. Ten
grams of endosulfan spiked soil was added to each
beaker. To one set (uninoculated), 3.0 ml of mineral
medium, and to another (inoculated) 3.0 ml of mineral
medium containing 2 108 CFU of bacterial cells
were added and mixed. Containers were covered with
perforated aluminium foil and incubated at 288C for
14 weeks. Distilled water was added weekly, to compensate for evaporational loss of water. Soil samples
N. Awasthi et al. / Soil Biology & Biochemistry 32 (2000) 1697±1705
were taken after 0, 1, 3, 5, 8, 12 and 14 weeks, and the
residual endosulfan concentrations were quanti®ed.
For the experiments, where eect of pH was to be
evaluated, portions of unspiked soil A (75 g each)
were adjusted to pH values of 3.0, 5.0, 7.5, 8.5, 10.0
and 12.0 by addition of either 2 N HCl or 1 N NaOH.
Adjustments to desired pH were made at least three
times over 10 days, till the pH values of the soils were
stabilized. Excess liquid from each soil was drained
after pH adjustment and commercial endosulfan was
added to yield the concentration of 2.35 mg gÿ1 active
ingredient. For each pH adjusted soil, two sets were
made containing 10 g soil in each beaker. One set
(uninoculated) received 3.0 ml of mineral medium, and
other (inoculated) received 3.0 ml of mineral medium
containing 2 108 CFU of bacterial cells. Soils were
incubated at 288C for 6 weeks and 1 g samples were
taken after 0, 3 and 6 weeks to quantify the residual
endosulfan present in each sample.
To study the eect of additional carbon sources,
four beakers, each containing 75 g of spiked soil A
were used. A 2% aqueous solution of glucose, sodium
succinate and sodium acetate were added to beakers 1,
2 and 3, respectively, to yield the ®nal concentration
of 10 mg gÿ1of additional carbon source. The fourth
beaker received no additional carbon source. Each soil
was then apportioned (10 g) into two sets of beakers.
One set received 3 ml of mineral medium and served
as uninoculated soils. The other set received 3 ml of
medium containing 2 108 CFU of bacterial cells. All
containers were incubated at 288C for 8 weeks. One
gram soil samples were taken after 0, 3, 5 and 7
weeks, and residual endosulfan concentrations were
quanti®ed.
To evaluate the eect of inoculum size, six sets of
beakers containing 10 g of spiked soil B were used.
Three milliliters of mineral medium containing 2 106 ,
2 107 , 2 108 , 4 108 and 109 CFU of bacterial
cells was added to sets 1±5, respectively. Three milliliters of mineral medium without any bacterial cells was
added to containers of set 6, which served as uninoculated controls. All the containers were incubated for 7
weeks at 288C. One gram soil samples were taken after
0, 1, 3, 5 and 7 weeks, and the residual endosulfan
concentrations were quanti®ed.
In the experiments where eect of wet and ¯ooded
conditions was to be investigated, 12 sets of beakers
containing 10 g of spiked soil A were used. Three
milliliters of mineral medium containing 2 108 CFU
of bacterial cells was added to a group of six sets
(inoculated). Three milliliters of mineral medium without bacterial cells was added to the other group of six
sets (uninoculated). Three sets, from both inoculated
and uninoculated groups, received an additional 20 ml
of mineral medium to represent ¯ooded samples. All
the beakers were incubated at 288C for 6 weeks. One
1699
¯ooded and one wet set from each group were harvested after 0, 3 and 6 weeks, and residual endosulfan
concentrations were quanti®ed.
When the eect of initial endosulfan concentration
on its own biodegradation was studied, commercial
endosulfan was added to 75 g portions of unspiked
soil to arrive at a concentration of 0.05, 0.1, 0.4, 2.0,
5.0 and 10.0 mg gÿ1. Two sets of three replicate beakers containing 10 g soil per beaker were made. One
set (uninoculated) received 3.0 ml of mineral medium
and other set (inoculated) received 3.0 ml of mineral
medium containing 2 108 CFU of bacterial cells.
Soils were incubated at 288C for 7 weeks. One gram
soil samples were taken after 0, 1, 2, 3, 5 and 7 weeks,
and residual endosulfan concentrations were quanti®ed.
2.5. Extraction and analysis of residual endosulfan from
the soils
Approximately 1.5 g of wet soil was removed from
the incubating soil samples at the sampling times mentioned earlier for dierent treatments, and air dried.
One gram of dried soil was transferred to a test tube
and extracted with 3 ml of ethyl acetate by vortexing.
The ethyl acetate layer was decanted after 5 min. This
extraction was repeated two more times. The ethyl
acetate fractions were pooled, passed through anhydrous sodium sulfate and evaporated at room temperature. The eciency of extraction was 8522%: In the
experiment with wet and ¯ooded soils, the volumes in
all the containers were raised to 25 ml with mineral
medium before harvesting. An equal volume of ethyl
acetate was added and samples were shaken on orbital
shaker at 220 rpm for 30 min. Contents were transferred to separatory funnel and the organic layer was
collected. The aqueous layer was extracted two more
times with 20 ml of ethyl acetate. Ethyl acetate fractions were pooled, passed through anhydrous sodium
sulfate and evaporated at room temperature.
Pesticide residue was dissolved in acetone and an aliquot containing 5±10 mg of endosulfan was spotted on
a thin layer chromatography sheet (Silica gel 60, 2 mm
thick). Chromatograms were developed in hexane:chloroform:acetone (9:3:1) and the separated spots
were visualized by spraying the chromogenic reagent
(AgNO3 in 2-phenoxy ethanol), followed by UV irradiation (Kovacs, 1965). The Rf for a-endosulfan, bendosulfan, endosulfan sulfate and endosulfan diol
were 0.67, 0.4, 0.3 and 0.1, respectively. For gas chromatography, the residual pesticide was dissolved in 1
ml of acetone, diluted 106 times with n-hexane, and
analyzed using Shimadzu GC-14B gas chromatograph,
®tted with 63Ni ECD detector and a stainless steel column 150 3 mm) ®lled with 80±100 mesh gas coated
with 1.5% OV-17 and 1.95% OV-210 as matrix. The
1700
N. Awasthi et al. / Soil Biology & Biochemistry 32 (2000) 1697±1705
temperature for column injector, and detector were
maintained at 220, 250, and 2808C, respectively, and
nitrogen (IOLAR grade-1) was used as carrier gas.
Retention times for a-endosulfan, b-endosulfan and
endosulfan sulfate were 11.6, 20.2 and 23.6 min, respectively.
2.6. Statistical analysis
The values of the residual endosulfan, obtained
from triplicate samples, were analyzed using analysis
of variance (ANOVA), and Fischer's LSD was used to
compare means. P values less than 0.05 were considered signi®cant.
3. Results
3.1. Degradation of soil bound endosulfan isomers
The degradation of endosulfan in uninoculated soil
A and soil B, measured by the disappearance of the
parent chemical after 14 weeks of incubation, was 20
and 38%, respectively (Fig. 1). Addition of isolated
bacterial cells enhanced this degradation to 90 and
78%, respectively. Degradation rates for a- and b-iso-
mers of endosulfan were comparable in uninoculated
or inoculated conditions in both the soils (Fig. 1). In
the inoculated soil samples, besides enhanced degradation of endosulfan, degradation of formed endosulfan diol was also observed (Fig. 2, lane 8.5 I).
Accumulation of endosulfan sulfate, a metabolite
known to be formed primarily by fungal activity (Martens, 1976; Kullman and Matsumura, 1996) and also
in the contaminated soils (Kathpal et al., 1997; Rao
and Murty, 1980) was not observed at any time during
the degradation of endosulfan.
3.2. Eect of soil pH on endosulfan degradation
In uninoculated soils, after 6 weeks of incubation,
no signi®cant degradation of endosulfan isomers at pH
3.0, slight degradation at pH 5.0 and a signi®cant
degradation at pH 7.5 and 8.5, was observed (Table 1).
In proportion to the degraded endosulfan, small
amounts of endosulfan diol were formed at pH 7.5
and 8.5, whereas at higher pH values, i.e. 10.0 and
12.0, almost all the added endosulfan was rapidly converted to endosulfan diol (Fig. 2). Inoculated soils presented no enhancement in degradation at pH 3.0,
slight at pH 5.0, and good to optimal (>40±50%)
enhancement at pH 7.5 and 8.5, respectively (Table 1).
At pH 10.0 and 12.0, since all the added endosulfan
was converted to endosulfan diol in uninoculated soils,
no further change in its degradation due to added cells
could be found. Formed endosulfan diol, however,
underwent degradation at pH 8.5, 10.0 and 12.0
(Fig. 2), suggesting that the bacterial activity towards
the degradation of endosulfan and its metabolites was
operating in these alkaline conditions also.
3.3. Eect of additional carbon sources
In uninoculated soils, during 7 weeks of incubation,
no signi®cant change in the degradation of endosulfan
isomers was observed in the presence of glucose,
sodium acetate or sodium succinate (Fig. 3). In inoculated soils, where no auxiliary carbon source was
added, 75% of the added endosulfan was degraded
after 7 weeks of incubation. A signi®cant inhibition
was observed in presence of sodium acetate and
sodium succinate, while the inhibition in presence of
glucose was statistically not signi®cant. The inhibition
was highest (100%) in the soils that received sodium
succinate, as the amounts of residual endosulfan in
uninoculated and inoculated soils, after 7 weeks of incubation, were identical.
Fig. 1. Degradation of endosulfan isomers in two dierent soils: aendosulfan (-w-w-), b-endosulfan (-q-q-) in uninoculated, and aendosulfan (-r-r-), b-endosulfan (-t-t-) in inoculated conditions.
Amounts of endosulfan recovered at 0 time were taken as 100%.
Vertical bars represent 2standard deviation of the mean of three
replicates.
3.4. Eect of inoculum size
Addition of 2 105 CFU gÿ1 soil had no signi®cant
eect on endosulfan degradation, when compared to
1701
N. Awasthi et al. / Soil Biology & Biochemistry 32 (2000) 1697±1705
Fig. 2. Thin layer chromatogram depicting the degradation of endosulfan isomers at dierent pH values, after 6 weeks of incubation: aES Ð aendosulfan, bES Ð b-endosulfan, ED Ð endosulfan diol, UI Ð uninoculated and I Ð inoculated.
enhanced the degradation of both isomers of endosulfan to 68±69% in non-¯ooded and 43±48% in ¯ooded
soils, after the same period of incubation (Table 2).
the uninoculated soil samples (Fig. 4). Addition of 2
106 cells stimulated a rapid degradation, and after 7
weeks of incubation 60% of the added endosulfan was
degraded. Addition of higher CFU, i.e. 2 107 , 4
107 and 108, resulted in an initial increase in endosulfan degradation P < 0:05 which attenuated with time
to show no signi®cant increase in endosulfan degradation over a period of 7 weeks (Fig. 4).
3.6. Eect of initial concentration of endosulfan on its
degradation
At low initial concentration of endosulfan i.e. 50 mg
gÿ1 soil, there was a rapid degradation of endosulfan
isomers in uninoculated soils. Here, addition of bacterial cells, had no appreciable in¯uence on the degradation rates (Fig. 5). The dierence in endosulfan
degradation rates, between uninoculated and inoculated soils, increased progressively up to 0.4 mg gÿ1
soil. At higher concentrations, endosulfan began to
3.5. Degradation in ¯ooded and non-¯ooded soils
In uninoculated soils, after an incubation of 6
weeks, the degradation of endosulfan isomers was better in the non-¯ooded condition (26±29%) than in the
¯ooded condition (15%). Addition of bacterial cells
Table 1
Eect of soil-pH on the degradation of endosulfan (ES) isomersa
Incubation (week)
Residual a-ES
(mg gÿ1 soil)
0
3
6
Residual b-ES
(mg gÿ1 soil)
0
3
6
a
pH 3.0
pH 5.0
pH 7.5
pH 8.5
UI
I
UI
I
UI
I
UI
I
1.4020.04
(100)
1.3820.05
(98)
1.3820.06
(98)
0.6020.03
(100)
0.5820.04
(97)
0.5820.03
(97)
1.4120.04
(100)
1.3720.04
(98)
1.3820.06
(98)
0.6020.04
(100)
0.5720.04
(95)
0.5720.04
(95)
1.4020.04
(100)
1.3720.04
(98)
1.3020.05
(93)
0.6120.04
(100)
0.5620.04
(93)
0.5320.04
(88)
1.3920.04
(100)
1.3520.05
(96)
1.2720.07
(91)
0.6020.03
(100)
0.5520.05
(92)
0.5120.04
(85)
1.4220.04
(100)
1.2520.04
(89)
1.2020.06
(86)
0.5920.04
(100)
0.5020.04
(83)
0.4620.05
(77)
1.4120.04
(100)
0.9320.05
(66)
0.5320.07
(38)
0.6020.04
(100)
0.3920.05
(65)
0.2220.07
(37)
1.4020.04
(100)
1.2420.05
(88)
1.1820.04
(84)
0.6120.03
(100)
0.5220.04
(87)
0.4820.05
(80)
1.3920.04
(100)
0.7220.06
(51)
0.3620.08
(26)
0.5920.03
(100)
0.3020.06
(50)
0.1620.06
(27)
Values given are average of three experiments 2 standard deviation. Numbers in parantheses represent percentage of endosulfan recovered,
taking the amounts at 0 time as 100%. UI Ð uninoculated, I Ð inoculated.
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N. Awasthi et al. / Soil Biology & Biochemistry 32 (2000) 1697±1705
Fig. 3. Eect of additional carbon sources on the degradation of
endosulfan isomers: unamended (-w-w-), and amended with glucose
(-q-q-), sodium acetate (-r-r-) or sodium succinate (-t-t-), UI
Ð uninoculated and I Ð inoculated. Amounts of endosulfan recovered at 0 time were taken as 100%. Vertical bars represent 2standard deviation of the mean of three replicates and where no bar is
shown it is less than the size of the symbol.
show an inhibitory eect on the degradative activity
and at the concentration of 10 mg gÿ1 soil, in both
uninoculated and inoculated soils, nearly no degradation of endosulfan was observed, up to 7 weeks of
incubation (Fig. 5).
4. Discussion
The environmental fate of organic pollutants in soils
is in¯uenced signi®cantly by the pH and texture of the
soil, and also by the presence of organic matter and
Fig. 4. Eect of inoculum size on the degradation of endosulfan isomer: uninoculated (-w-w-), and inoculated with 2 105 (-q-q-),
2 106 (-r-r-), 2 107 (-t-t-), 4 107 (-r-r-) and 108 (-D-D-)
CFU gÿ1 soil. Amounts of endosulfan recovered at 0 time were
taken as 100%. Vertical bars represent 2standard deviation of the
mean of three replicates and where no bar is shown it is less than the
size of the symbol.
copollutants. Accordingly, biodegradation of endosulfan isomers was studied in two dierent soils that have
contrasting properties in terms of their texture, pH, organic content, etc. When inoculated the degradation of
endosulfan was faster in soil A than in soil B, probably because of their dierent physico-chemical properties. During the degradation, the formation of
endosulfan sulfate, a metabolite known to accumulate
Table 2
Degradation of endosulfan isomers in non-¯ooded and ¯ooded soilsa
Time
(week)
Non-¯ooded
Flooded
a-Endosulfan
0
3
6
a
b-Endosulfan
a-Endosulfan
b-Endosulfan
UI
I
UI
I
UI
I
UI
I
1.4020.04
(100)
1.1220.04
(80)
1.0020.05
(71)
1.4120.04
(100)
0.6320.05
(45)
0.4520.06
(32)
0.6120.05
(100)
0.4820.04
(80)
0.4520.06
(74)
0.6020.04
(100)
0.2720.06
(46)
0.1820.07
(31)
1.4020.05
(100)
1.2220.05
(87)
1.1920.06
(85)
1.3920.04
(100)
0.8420.05
(60)
0.7220.05
(52)
0.6020.04
(100)
0.5520.04
(92)
0.5120.05
(85)
0.5920.05
(100)
0.4120.06
(68)
0.3420.06
(57)
Values given (mg gÿ1 soil) are average of three experiments2standard deviation. Numbers in parentheses represent percentage of endosulfan
recovered, taking the amounts at 0 time as 100%. UI Ð uninoculated, I Ð inoculated.
N. Awasthi et al. / Soil Biology & Biochemistry 32 (2000) 1697±1705
during incubation with certain fungi (El Zorgani and
Omer, 1974; Martens, 1976; Kullman and Matsumura,
1996) and also in contaminated soils (Rao and Murty,
1980; Kathpal et al., 1997; Lee et al., 1997; Kaur et
al., 1998) was not observed at any time. This could be
due to lack or non-functioning of fungi in the soils studied here. These results are in accordance with our
earlier studies, where, in culture conditions, endosulfan
sulfate was not formed (Awasthi et al., 1997).
Under uninoculated conditions, increased degradation of endosulfan isomers was observed at higher
pH values and is primarily due to its alkaline hydrolysis into endosulfan diol. This probably is the reason
for the increased degradation of endosulfan isomers in
soil B (pH 9.37) as against soil A (pH 8.5) at the earlier time points (Fig. 1). Under inoculated conditions,
however, the degradation of endosulfan isomers was
Fig. 5. Eect of initial endosulfan concentration on its degradation
in soil: a-endosulfan (-w-w-), b-endosulfan (-q-q-) in uninoculated,
and a-endosulfan (-r-r-), b-endosulfan (-t-t-) in inoculated conditions. Panels A±F represent experiments where initial concentrations of endosulfan were 0.05, 0.1, 0.4, 2.0, 5.0 and 10.0 mg gÿ1
soil, respectively. Amounts of endosulfan recovered at 0 time were
taken as 100%. Vertical bars represent 2standard deviation of the
mean of three replicates.
1703
optimal at pH 8.5. This could be due to better bioavailability of endosulfan, and optimal biotic activity
of cells at this pH value. Better degradation of contaminating diesel oil (Margesin and Schinner, 1997) as
well as herbicide atrazine (Hance, 1979) has also been
reported, under alkaline conditions.
Carbon sources, other than the target chemical, are
present in natural soils and may in¯uence degradation
rates. Accordingly, the eect of some of the common
carbon sources was evaluated on the biodegradation of
endosulfan isomers in soil. In inoculated soils, the presence of sodium acetate or sodium succinate inhibited
the degradation of endosulfan to dierent extents. The
inhibition of degradation in presence of additional carbon sources can also be among the reasons for less
degradation of endosulfan isomers in soil B (organic
content 0.59%) than in soil A (organic content
0.38%). In many other studies also, the presence of
more favorable carbon sources have been shown to
impede the degradation of less favorable chemicals,
e.g. xenobiotics (Hartline and Gunsalus, 1971; Sahu et
al., 1993). This could be due either to the mechanism
of catabolite repression (Hartline and Gunsalus, 1971;
Botsford and Harman, 1992) or decrease in the rates
of transcription either due to supercoiling of promoter
DNA (Assinder and Williams, 1990) or by decreased
binding of transcription factors (Holtel et al., 1994).
Exogenous microorganisms, when added to the
soils, run the risk of getting out-competed by native
microbial communities and also of predation by protozoans, etc. To arrive at the optimal number of bacterial cells for eective degradation of endosulfan, the
in¯uence of dierent inoculum sizes ranging from 2
105 to 108 CFU gÿ1 soil was studied. While the addition of 2 105 CFU caused no enhancement in the
degradation rates, addition of 2 106 or more cells
caused a substantial enhancement in the degradation
of endosulfan. The enhancement in the degradation
over 7 weeks was comparable to the inoculation with
2 106 , 2 107 , 4 107 or 108 CFU gÿ1 soil. It is
possible that during incubation in the soils, when the
added bacterial cells were 2 105 or below they were
out-competed or preyed upon by the indigenous micro¯ora. At higher inoculum densities i.e. 2 106 ,
2 107 , 4 107 , 108 CFU gÿ1, the bacterial population might have equilibrated to a common eective
level. In the experiments, where in¯uence of dierent
water regimes on endosulfan biodegradation was evaluated, the higher degradation of endosulfan in non¯ooded conditions, in uninoculated and inoculated
soils, was probably due to better oxygen availability,
as against ¯ooded conditions.
Concentrations of target pollutant can vary considerably in dierent contaminated soils and exert a
signi®cant in¯uence on the degradative activity of
microorganisms. While very high amounts of the pol-
1704
N. Awasthi et al. / Soil Biology & Biochemistry 32 (2000) 1697±1705
lutant can be toxic to the microorganisms, their low
concentrations might fail to induce degradative activity. At low initial concentrations of endosulfan, i.e.
50 and 100 mg gÿ1 soil, the degradation was very rapid
in both uninoculated and inoculated soils. At higher
concentrations, however, the degradation rates were
slower, leading to a total inhibition of the degradative
activity at the initial concentration of 10 mg gÿ1 soil.
The inhibition of degradation was probably due to the
cytotoxicity of endosulfan at this concentration.
In this study, we have used bacterial cells that had
been isolated and characterized for the degradation of
endosulfan isomers in liquid-culture conditions
(Awasthi et al., 1997), and have presented evidence for
their viability as well as capability to enhance the
degradation of the chlorinated pesticide, endosulfan, in
contaminated soils. The enhancement is in¯uenced
greatly by factors such as soil-pH, presence of carbonaceous material, water content, size of bacterial inoculum and concentration of the pesticide present in the
soils. Initial optimisation of these factors is therefore
essential before undertaking any bioremediation activity. Further, in view of the in¯uence of external factors on the degradation activity, half-lives of chemicals
in the soils reported in literature should be considered
with caution.
Acknowledgements
We thank Dr P.K. Seth, Director, Industrial Toxicology Research Centre, Lucknow for his constant
support to this work, Mr Neeraj Mathur of this institute for statistical analysis, Dr S. K.Tiwari of National
Botanical Research Institute, Lucknow, for analysis of
the soils and Dr Rakesh Jain of Institute of Microbial
Technology, Chandigarh, for identi®cation of the bacterial strains. Generous gift of endosulfan isomers and
metabolites by Hoechst, AgrEvo, Germany, and ®nancial assistance from Department of Biotechnology,
New Delhi is gratefully acknowledged.
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www.elsevier.com/locate/soilbio
Factors in¯uencing the degradation of soil-applied endosulfan
isomers
Niranjan Awasthi, Rajiv Ahuja, Ashwani Kumar*
Environmental Biotechnology Section, Industrial Toxicology Research Centre, Post Box No. 80, Mahatma Gandhi Marg, Lucknow 226 001, India
Accepted 21 April 2000
Abstract
The addition of isolated bacterial cells to contaminated soils causes an enhanced degradation of endosulfan isomers. Various
factors, including the additional presence of carbon sources, pH, moisture content, concentration of endosulfan, and size of
inoculum, in¯uenced the degradation of endosulfan isomers. The degradation was faster in wet soils, as compared with the
¯ooded soils, and was inhibited by the presence of additional carbon sources such as sodium acetate and sodium succinate. The
degradation of endosulfan was not detectable at acidic pH and increased gradually to reach an optimal activity at pH 8.5. It
chemically converts into endosulfan diol at higher pH values. The rate of biodegradation progressed with the increase in
endosulfan concentration up to 5.0 mg gÿ1 soil, followed by an inhibitory eect at higher concentrations, reaching a total loss of
biodegradative activity at 10 mg gÿ1 soil. The addition of 2 106 bacterial cells gÿ1 soil was optimal for endosulfan degradation
and any further increase in inoculum size was of no additional advantage. Initial optimization of these factors is, therefore,
essential for successful bioremediation. 7 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Endosulfan; Chlorinated pesticide; Biodegradation; Contaminated sites; Bioremediation
1. Introduction
Endosulfan (6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9ahexahydro-6,9-methano-2,3,4-benzo(e )dioxathiepin-3oxide, CAS No. 115-29-7) is a chlorinated pesticide of
the cyclodiene group. Its technical preparation consists
of a- and b-isomers (70:30). It is used extensively,
throughout the world, as a broad spectrum insecticide,
on cotton crops, ®eld crops such as paddy, sorghum,
oil seeds and pulses, as well as vegetables and fruit
crops (Goebel et al., 1982). Endosulfan contamination
has been detected in soils, water, air and food products
because of its abundant usage and potential for environmental transport. It is extremely toxic to ®shes
and aquatic invertebrates (Verschueren, 1983; Sunderam et al., 1992) and is classi®ed as a `priority pollu-
* Corresponding author. Fax: +91-522-228227.
E-mail address: [email protected] (A. Kumar).
tant' by international environmental agencies (Keith
and Telliard, 1979). The persistence of endosulfan in
agricultural soils has been studied in many laboratories. Its life in soils has been estimated to be from
100 to 120 days (Rao and Murty, 1980; Kathpal et al.,
1997) to several months (Stewart and Cairns, 1974)
and the relative rates of dissipation for a- and b-endosulfan have also been shown to be dierent. This dissipation depends on a multitude of factors such as its
volatilization, alkaline hydrolysis and photodecomposition, besides the presence of fertilizer, crop pattern, atmospheric temperature, rain and biotic conversions,
among others (Goebel et al., 1982).
Bioremediation, which involves degradation of target chemicals by indigenous or added microbial cells,
is used to clean up sites contaminated by pollutants
such as pentachlorophenol (Miethling and Karlson,
1996; Barbeau et al., 1997), diesel oil (Margesin and
Schinner, 1997), herbicides (Kilbane et al., 1983;
Kaake et al., 1992), polyaromatic hydrocarbons (Kast-
0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 0 8 7 - 0
1698
N. Awasthi et al. / Soil Biology & Biochemistry 32 (2000) 1697±1705
ner et al., 1994; Mueller et al., 1996), petroleum products (Balba et al., 1991) and munition compounds
(Funk et al., 1993). In many other cases, addition of
microorganisms has failed to enhance degradation of
polluting chemicals, probably due to any or combination of factors like non-optimal temperature, pH,
moisture content, insucient number of added microorganisms or incompatible soil texture. Besides, the
concentration of polluting chemical might either be
too high to be toxic to the added microbes or too low
to induce the degradative activity (Goldstein et al.,
1985; Block et al., 1993; Morra, 1996).
Bacteria and fungi, which degrade endosulfan isomers in liquid culture have been isolated and characterized in many laboratories (El Zorgani and Omer,
1974; Martens, 1976; Miles and Moy, 1979; Mukherjee
and Gopal, 1994; Kullman and Matsumura, 1996;
Awasthi et al., 1997). Endosulfan diol, endosulfan sulfate, endosulfan aldehyde, endosulfan ether and endosulfan lactone have been demonstrated as the major
metabolites formed during its degradation. The environmental fate of endosulfan in dierent types of
contaminated soils has also been studied (Rao and
Murty, 1980; Antonious and Byers, 1997; Kathpal et
al., 1997; Kaur et al., 1998; Parkpian et al. 1998), but
the in¯uence of exogenous bacterial cells on endosulfan degradation has not been evaluated.
In order to develop a suitable technology package
for bioremediation of soils contaminated with endosulfan, we have studied the ecacy of an isolated bacterial culture (Awasthi et al., 1997) on the
biodegradation of soil-applied endosulfan isomers. The
in¯uence of factors, such as moisture content, pH, presence of additional carbon sources, size of inoculum
and initial pesticide load in soils, on endosulfan degradation were also evaluated.
were found to be more eective. For this study, each
of the isolated bacteria were grown separately in a rich
medium (Peptone 1.0 g; Yeast Extract 0.5 g; NaCl 0.5
g; dissolved in distilled water, pH adjusted to 7.5 and
made up to 100 ml) overnight, centrifuged at 10,000
rpm for 10 min, washed and resuspended in a mineral
medium (KH2PO4, 170 mg; Na2HPO4, 980 mg;
(NH4)2SO4, 100 mg; MgSO4, 4.87 mg; MgO, 100 mg;
FeSO4, 50 mg; CaCO3, 20 mg; ZnSO4, 80 mg;
CuSO45H2O, 16 mg; CoSO4. 15 mg; H3BO3, 6 mg; dissolved in 100 ml of distilled water, the pH of the medium was 7.0) to the density of 2 108 colony forming
units (CFU) mlÿ1. Equal volume of both the cultures
were mixed to make a stock co-culture. For degradation experiments, bacteria from stock co-culture
were grown in rich medium overnight, centrifuged,
washed twice and suspended in mineral medium to 2
108 CFU mlÿ1 before use.
2.3. Soils studied
Two soils, one (Soil A) from the Banthara ®eld
station of the National Botanical Research Institute,
Lucknow, containing, clay 60%, silt 30%, sand 10%,
organic carbon 0.38% at pH 8.7, and the other (Soil
B) from the Gheru Campus of Industrial Toxicology
Research Centre, Lucknow, containing clay 39%, silt
12%, sand 49%, organic carbon 0.59%, at pH 9.37,
were used. Collected soils were air dried and sieved (2
mm). Four hundred milliliters of distilled water containing 6.0 g of commercial endosulfan (35% EC) were
added to 1000 g of each soil and mixed thoroughly.
The concentration of the active ingredient was estimated to be 2.35 mg gÿ1 soil, i.e. 1.64 mg of a-endosulfan and 0.71 mg of b-endosulfan. After air drying
for 24±48 h, the soils were pulverized and used for
degradation studies.
2. Materials and methods
2.1. Chemicals
Commercial endosulfan (Parrysulfan, 35% EC, by
EID Parry, Chennai, India) was purchased from a
local market. Pure a- and b-endosulfan were obtained
from Farbwerke-Hoechst AG, Frankfurt, Germany. 2Phenoxy ethanol was purchased from Sigma, St Louis,
MO, USA. All other reagents were of analytical grade.
2.2. Bacterial cells
A bacterial co-culture consisting of two dierent
strains of Bacillus sp., isolated earlier from a contaminated site by selective enrichment, was used (Awasthi
et al., 1997). Either of the isolated bacteria can mediate the degradation of endosulfan but together they
2.4. Degradation of soil applied endosulfan and in¯uence
of external factors
Degradation of soil-applied endosulfan in uninoculated or inoculated conditions, was studied in parallel
sets of three containers each, for each variable and
time point of the study.
To study the degradation in soils, two sets of beakers (100 ml) were maintained for each soil. Ten
grams of endosulfan spiked soil was added to each
beaker. To one set (uninoculated), 3.0 ml of mineral
medium, and to another (inoculated) 3.0 ml of mineral
medium containing 2 108 CFU of bacterial cells
were added and mixed. Containers were covered with
perforated aluminium foil and incubated at 288C for
14 weeks. Distilled water was added weekly, to compensate for evaporational loss of water. Soil samples
N. Awasthi et al. / Soil Biology & Biochemistry 32 (2000) 1697±1705
were taken after 0, 1, 3, 5, 8, 12 and 14 weeks, and the
residual endosulfan concentrations were quanti®ed.
For the experiments, where eect of pH was to be
evaluated, portions of unspiked soil A (75 g each)
were adjusted to pH values of 3.0, 5.0, 7.5, 8.5, 10.0
and 12.0 by addition of either 2 N HCl or 1 N NaOH.
Adjustments to desired pH were made at least three
times over 10 days, till the pH values of the soils were
stabilized. Excess liquid from each soil was drained
after pH adjustment and commercial endosulfan was
added to yield the concentration of 2.35 mg gÿ1 active
ingredient. For each pH adjusted soil, two sets were
made containing 10 g soil in each beaker. One set
(uninoculated) received 3.0 ml of mineral medium, and
other (inoculated) received 3.0 ml of mineral medium
containing 2 108 CFU of bacterial cells. Soils were
incubated at 288C for 6 weeks and 1 g samples were
taken after 0, 3 and 6 weeks to quantify the residual
endosulfan present in each sample.
To study the eect of additional carbon sources,
four beakers, each containing 75 g of spiked soil A
were used. A 2% aqueous solution of glucose, sodium
succinate and sodium acetate were added to beakers 1,
2 and 3, respectively, to yield the ®nal concentration
of 10 mg gÿ1of additional carbon source. The fourth
beaker received no additional carbon source. Each soil
was then apportioned (10 g) into two sets of beakers.
One set received 3 ml of mineral medium and served
as uninoculated soils. The other set received 3 ml of
medium containing 2 108 CFU of bacterial cells. All
containers were incubated at 288C for 8 weeks. One
gram soil samples were taken after 0, 3, 5 and 7
weeks, and residual endosulfan concentrations were
quanti®ed.
To evaluate the eect of inoculum size, six sets of
beakers containing 10 g of spiked soil B were used.
Three milliliters of mineral medium containing 2 106 ,
2 107 , 2 108 , 4 108 and 109 CFU of bacterial
cells was added to sets 1±5, respectively. Three milliliters of mineral medium without any bacterial cells was
added to containers of set 6, which served as uninoculated controls. All the containers were incubated for 7
weeks at 288C. One gram soil samples were taken after
0, 1, 3, 5 and 7 weeks, and the residual endosulfan
concentrations were quanti®ed.
In the experiments where eect of wet and ¯ooded
conditions was to be investigated, 12 sets of beakers
containing 10 g of spiked soil A were used. Three
milliliters of mineral medium containing 2 108 CFU
of bacterial cells was added to a group of six sets
(inoculated). Three milliliters of mineral medium without bacterial cells was added to the other group of six
sets (uninoculated). Three sets, from both inoculated
and uninoculated groups, received an additional 20 ml
of mineral medium to represent ¯ooded samples. All
the beakers were incubated at 288C for 6 weeks. One
1699
¯ooded and one wet set from each group were harvested after 0, 3 and 6 weeks, and residual endosulfan
concentrations were quanti®ed.
When the eect of initial endosulfan concentration
on its own biodegradation was studied, commercial
endosulfan was added to 75 g portions of unspiked
soil to arrive at a concentration of 0.05, 0.1, 0.4, 2.0,
5.0 and 10.0 mg gÿ1. Two sets of three replicate beakers containing 10 g soil per beaker were made. One
set (uninoculated) received 3.0 ml of mineral medium
and other set (inoculated) received 3.0 ml of mineral
medium containing 2 108 CFU of bacterial cells.
Soils were incubated at 288C for 7 weeks. One gram
soil samples were taken after 0, 1, 2, 3, 5 and 7 weeks,
and residual endosulfan concentrations were quanti®ed.
2.5. Extraction and analysis of residual endosulfan from
the soils
Approximately 1.5 g of wet soil was removed from
the incubating soil samples at the sampling times mentioned earlier for dierent treatments, and air dried.
One gram of dried soil was transferred to a test tube
and extracted with 3 ml of ethyl acetate by vortexing.
The ethyl acetate layer was decanted after 5 min. This
extraction was repeated two more times. The ethyl
acetate fractions were pooled, passed through anhydrous sodium sulfate and evaporated at room temperature. The eciency of extraction was 8522%: In the
experiment with wet and ¯ooded soils, the volumes in
all the containers were raised to 25 ml with mineral
medium before harvesting. An equal volume of ethyl
acetate was added and samples were shaken on orbital
shaker at 220 rpm for 30 min. Contents were transferred to separatory funnel and the organic layer was
collected. The aqueous layer was extracted two more
times with 20 ml of ethyl acetate. Ethyl acetate fractions were pooled, passed through anhydrous sodium
sulfate and evaporated at room temperature.
Pesticide residue was dissolved in acetone and an aliquot containing 5±10 mg of endosulfan was spotted on
a thin layer chromatography sheet (Silica gel 60, 2 mm
thick). Chromatograms were developed in hexane:chloroform:acetone (9:3:1) and the separated spots
were visualized by spraying the chromogenic reagent
(AgNO3 in 2-phenoxy ethanol), followed by UV irradiation (Kovacs, 1965). The Rf for a-endosulfan, bendosulfan, endosulfan sulfate and endosulfan diol
were 0.67, 0.4, 0.3 and 0.1, respectively. For gas chromatography, the residual pesticide was dissolved in 1
ml of acetone, diluted 106 times with n-hexane, and
analyzed using Shimadzu GC-14B gas chromatograph,
®tted with 63Ni ECD detector and a stainless steel column 150 3 mm) ®lled with 80±100 mesh gas coated
with 1.5% OV-17 and 1.95% OV-210 as matrix. The
1700
N. Awasthi et al. / Soil Biology & Biochemistry 32 (2000) 1697±1705
temperature for column injector, and detector were
maintained at 220, 250, and 2808C, respectively, and
nitrogen (IOLAR grade-1) was used as carrier gas.
Retention times for a-endosulfan, b-endosulfan and
endosulfan sulfate were 11.6, 20.2 and 23.6 min, respectively.
2.6. Statistical analysis
The values of the residual endosulfan, obtained
from triplicate samples, were analyzed using analysis
of variance (ANOVA), and Fischer's LSD was used to
compare means. P values less than 0.05 were considered signi®cant.
3. Results
3.1. Degradation of soil bound endosulfan isomers
The degradation of endosulfan in uninoculated soil
A and soil B, measured by the disappearance of the
parent chemical after 14 weeks of incubation, was 20
and 38%, respectively (Fig. 1). Addition of isolated
bacterial cells enhanced this degradation to 90 and
78%, respectively. Degradation rates for a- and b-iso-
mers of endosulfan were comparable in uninoculated
or inoculated conditions in both the soils (Fig. 1). In
the inoculated soil samples, besides enhanced degradation of endosulfan, degradation of formed endosulfan diol was also observed (Fig. 2, lane 8.5 I).
Accumulation of endosulfan sulfate, a metabolite
known to be formed primarily by fungal activity (Martens, 1976; Kullman and Matsumura, 1996) and also
in the contaminated soils (Kathpal et al., 1997; Rao
and Murty, 1980) was not observed at any time during
the degradation of endosulfan.
3.2. Eect of soil pH on endosulfan degradation
In uninoculated soils, after 6 weeks of incubation,
no signi®cant degradation of endosulfan isomers at pH
3.0, slight degradation at pH 5.0 and a signi®cant
degradation at pH 7.5 and 8.5, was observed (Table 1).
In proportion to the degraded endosulfan, small
amounts of endosulfan diol were formed at pH 7.5
and 8.5, whereas at higher pH values, i.e. 10.0 and
12.0, almost all the added endosulfan was rapidly converted to endosulfan diol (Fig. 2). Inoculated soils presented no enhancement in degradation at pH 3.0,
slight at pH 5.0, and good to optimal (>40±50%)
enhancement at pH 7.5 and 8.5, respectively (Table 1).
At pH 10.0 and 12.0, since all the added endosulfan
was converted to endosulfan diol in uninoculated soils,
no further change in its degradation due to added cells
could be found. Formed endosulfan diol, however,
underwent degradation at pH 8.5, 10.0 and 12.0
(Fig. 2), suggesting that the bacterial activity towards
the degradation of endosulfan and its metabolites was
operating in these alkaline conditions also.
3.3. Eect of additional carbon sources
In uninoculated soils, during 7 weeks of incubation,
no signi®cant change in the degradation of endosulfan
isomers was observed in the presence of glucose,
sodium acetate or sodium succinate (Fig. 3). In inoculated soils, where no auxiliary carbon source was
added, 75% of the added endosulfan was degraded
after 7 weeks of incubation. A signi®cant inhibition
was observed in presence of sodium acetate and
sodium succinate, while the inhibition in presence of
glucose was statistically not signi®cant. The inhibition
was highest (100%) in the soils that received sodium
succinate, as the amounts of residual endosulfan in
uninoculated and inoculated soils, after 7 weeks of incubation, were identical.
Fig. 1. Degradation of endosulfan isomers in two dierent soils: aendosulfan (-w-w-), b-endosulfan (-q-q-) in uninoculated, and aendosulfan (-r-r-), b-endosulfan (-t-t-) in inoculated conditions.
Amounts of endosulfan recovered at 0 time were taken as 100%.
Vertical bars represent 2standard deviation of the mean of three
replicates.
3.4. Eect of inoculum size
Addition of 2 105 CFU gÿ1 soil had no signi®cant
eect on endosulfan degradation, when compared to
1701
N. Awasthi et al. / Soil Biology & Biochemistry 32 (2000) 1697±1705
Fig. 2. Thin layer chromatogram depicting the degradation of endosulfan isomers at dierent pH values, after 6 weeks of incubation: aES Ð aendosulfan, bES Ð b-endosulfan, ED Ð endosulfan diol, UI Ð uninoculated and I Ð inoculated.
enhanced the degradation of both isomers of endosulfan to 68±69% in non-¯ooded and 43±48% in ¯ooded
soils, after the same period of incubation (Table 2).
the uninoculated soil samples (Fig. 4). Addition of 2
106 cells stimulated a rapid degradation, and after 7
weeks of incubation 60% of the added endosulfan was
degraded. Addition of higher CFU, i.e. 2 107 , 4
107 and 108, resulted in an initial increase in endosulfan degradation P < 0:05 which attenuated with time
to show no signi®cant increase in endosulfan degradation over a period of 7 weeks (Fig. 4).
3.6. Eect of initial concentration of endosulfan on its
degradation
At low initial concentration of endosulfan i.e. 50 mg
gÿ1 soil, there was a rapid degradation of endosulfan
isomers in uninoculated soils. Here, addition of bacterial cells, had no appreciable in¯uence on the degradation rates (Fig. 5). The dierence in endosulfan
degradation rates, between uninoculated and inoculated soils, increased progressively up to 0.4 mg gÿ1
soil. At higher concentrations, endosulfan began to
3.5. Degradation in ¯ooded and non-¯ooded soils
In uninoculated soils, after an incubation of 6
weeks, the degradation of endosulfan isomers was better in the non-¯ooded condition (26±29%) than in the
¯ooded condition (15%). Addition of bacterial cells
Table 1
Eect of soil-pH on the degradation of endosulfan (ES) isomersa
Incubation (week)
Residual a-ES
(mg gÿ1 soil)
0
3
6
Residual b-ES
(mg gÿ1 soil)
0
3
6
a
pH 3.0
pH 5.0
pH 7.5
pH 8.5
UI
I
UI
I
UI
I
UI
I
1.4020.04
(100)
1.3820.05
(98)
1.3820.06
(98)
0.6020.03
(100)
0.5820.04
(97)
0.5820.03
(97)
1.4120.04
(100)
1.3720.04
(98)
1.3820.06
(98)
0.6020.04
(100)
0.5720.04
(95)
0.5720.04
(95)
1.4020.04
(100)
1.3720.04
(98)
1.3020.05
(93)
0.6120.04
(100)
0.5620.04
(93)
0.5320.04
(88)
1.3920.04
(100)
1.3520.05
(96)
1.2720.07
(91)
0.6020.03
(100)
0.5520.05
(92)
0.5120.04
(85)
1.4220.04
(100)
1.2520.04
(89)
1.2020.06
(86)
0.5920.04
(100)
0.5020.04
(83)
0.4620.05
(77)
1.4120.04
(100)
0.9320.05
(66)
0.5320.07
(38)
0.6020.04
(100)
0.3920.05
(65)
0.2220.07
(37)
1.4020.04
(100)
1.2420.05
(88)
1.1820.04
(84)
0.6120.03
(100)
0.5220.04
(87)
0.4820.05
(80)
1.3920.04
(100)
0.7220.06
(51)
0.3620.08
(26)
0.5920.03
(100)
0.3020.06
(50)
0.1620.06
(27)
Values given are average of three experiments 2 standard deviation. Numbers in parantheses represent percentage of endosulfan recovered,
taking the amounts at 0 time as 100%. UI Ð uninoculated, I Ð inoculated.
1702
N. Awasthi et al. / Soil Biology & Biochemistry 32 (2000) 1697±1705
Fig. 3. Eect of additional carbon sources on the degradation of
endosulfan isomers: unamended (-w-w-), and amended with glucose
(-q-q-), sodium acetate (-r-r-) or sodium succinate (-t-t-), UI
Ð uninoculated and I Ð inoculated. Amounts of endosulfan recovered at 0 time were taken as 100%. Vertical bars represent 2standard deviation of the mean of three replicates and where no bar is
shown it is less than the size of the symbol.
show an inhibitory eect on the degradative activity
and at the concentration of 10 mg gÿ1 soil, in both
uninoculated and inoculated soils, nearly no degradation of endosulfan was observed, up to 7 weeks of
incubation (Fig. 5).
4. Discussion
The environmental fate of organic pollutants in soils
is in¯uenced signi®cantly by the pH and texture of the
soil, and also by the presence of organic matter and
Fig. 4. Eect of inoculum size on the degradation of endosulfan isomer: uninoculated (-w-w-), and inoculated with 2 105 (-q-q-),
2 106 (-r-r-), 2 107 (-t-t-), 4 107 (-r-r-) and 108 (-D-D-)
CFU gÿ1 soil. Amounts of endosulfan recovered at 0 time were
taken as 100%. Vertical bars represent 2standard deviation of the
mean of three replicates and where no bar is shown it is less than the
size of the symbol.
copollutants. Accordingly, biodegradation of endosulfan isomers was studied in two dierent soils that have
contrasting properties in terms of their texture, pH, organic content, etc. When inoculated the degradation of
endosulfan was faster in soil A than in soil B, probably because of their dierent physico-chemical properties. During the degradation, the formation of
endosulfan sulfate, a metabolite known to accumulate
Table 2
Degradation of endosulfan isomers in non-¯ooded and ¯ooded soilsa
Time
(week)
Non-¯ooded
Flooded
a-Endosulfan
0
3
6
a
b-Endosulfan
a-Endosulfan
b-Endosulfan
UI
I
UI
I
UI
I
UI
I
1.4020.04
(100)
1.1220.04
(80)
1.0020.05
(71)
1.4120.04
(100)
0.6320.05
(45)
0.4520.06
(32)
0.6120.05
(100)
0.4820.04
(80)
0.4520.06
(74)
0.6020.04
(100)
0.2720.06
(46)
0.1820.07
(31)
1.4020.05
(100)
1.2220.05
(87)
1.1920.06
(85)
1.3920.04
(100)
0.8420.05
(60)
0.7220.05
(52)
0.6020.04
(100)
0.5520.04
(92)
0.5120.05
(85)
0.5920.05
(100)
0.4120.06
(68)
0.3420.06
(57)
Values given (mg gÿ1 soil) are average of three experiments2standard deviation. Numbers in parentheses represent percentage of endosulfan
recovered, taking the amounts at 0 time as 100%. UI Ð uninoculated, I Ð inoculated.
N. Awasthi et al. / Soil Biology & Biochemistry 32 (2000) 1697±1705
during incubation with certain fungi (El Zorgani and
Omer, 1974; Martens, 1976; Kullman and Matsumura,
1996) and also in contaminated soils (Rao and Murty,
1980; Kathpal et al., 1997; Lee et al., 1997; Kaur et
al., 1998) was not observed at any time. This could be
due to lack or non-functioning of fungi in the soils studied here. These results are in accordance with our
earlier studies, where, in culture conditions, endosulfan
sulfate was not formed (Awasthi et al., 1997).
Under uninoculated conditions, increased degradation of endosulfan isomers was observed at higher
pH values and is primarily due to its alkaline hydrolysis into endosulfan diol. This probably is the reason
for the increased degradation of endosulfan isomers in
soil B (pH 9.37) as against soil A (pH 8.5) at the earlier time points (Fig. 1). Under inoculated conditions,
however, the degradation of endosulfan isomers was
Fig. 5. Eect of initial endosulfan concentration on its degradation
in soil: a-endosulfan (-w-w-), b-endosulfan (-q-q-) in uninoculated,
and a-endosulfan (-r-r-), b-endosulfan (-t-t-) in inoculated conditions. Panels A±F represent experiments where initial concentrations of endosulfan were 0.05, 0.1, 0.4, 2.0, 5.0 and 10.0 mg gÿ1
soil, respectively. Amounts of endosulfan recovered at 0 time were
taken as 100%. Vertical bars represent 2standard deviation of the
mean of three replicates.
1703
optimal at pH 8.5. This could be due to better bioavailability of endosulfan, and optimal biotic activity
of cells at this pH value. Better degradation of contaminating diesel oil (Margesin and Schinner, 1997) as
well as herbicide atrazine (Hance, 1979) has also been
reported, under alkaline conditions.
Carbon sources, other than the target chemical, are
present in natural soils and may in¯uence degradation
rates. Accordingly, the eect of some of the common
carbon sources was evaluated on the biodegradation of
endosulfan isomers in soil. In inoculated soils, the presence of sodium acetate or sodium succinate inhibited
the degradation of endosulfan to dierent extents. The
inhibition of degradation in presence of additional carbon sources can also be among the reasons for less
degradation of endosulfan isomers in soil B (organic
content 0.59%) than in soil A (organic content
0.38%). In many other studies also, the presence of
more favorable carbon sources have been shown to
impede the degradation of less favorable chemicals,
e.g. xenobiotics (Hartline and Gunsalus, 1971; Sahu et
al., 1993). This could be due either to the mechanism
of catabolite repression (Hartline and Gunsalus, 1971;
Botsford and Harman, 1992) or decrease in the rates
of transcription either due to supercoiling of promoter
DNA (Assinder and Williams, 1990) or by decreased
binding of transcription factors (Holtel et al., 1994).
Exogenous microorganisms, when added to the
soils, run the risk of getting out-competed by native
microbial communities and also of predation by protozoans, etc. To arrive at the optimal number of bacterial cells for eective degradation of endosulfan, the
in¯uence of dierent inoculum sizes ranging from 2
105 to 108 CFU gÿ1 soil was studied. While the addition of 2 105 CFU caused no enhancement in the
degradation rates, addition of 2 106 or more cells
caused a substantial enhancement in the degradation
of endosulfan. The enhancement in the degradation
over 7 weeks was comparable to the inoculation with
2 106 , 2 107 , 4 107 or 108 CFU gÿ1 soil. It is
possible that during incubation in the soils, when the
added bacterial cells were 2 105 or below they were
out-competed or preyed upon by the indigenous micro¯ora. At higher inoculum densities i.e. 2 106 ,
2 107 , 4 107 , 108 CFU gÿ1, the bacterial population might have equilibrated to a common eective
level. In the experiments, where in¯uence of dierent
water regimes on endosulfan biodegradation was evaluated, the higher degradation of endosulfan in non¯ooded conditions, in uninoculated and inoculated
soils, was probably due to better oxygen availability,
as against ¯ooded conditions.
Concentrations of target pollutant can vary considerably in dierent contaminated soils and exert a
signi®cant in¯uence on the degradative activity of
microorganisms. While very high amounts of the pol-
1704
N. Awasthi et al. / Soil Biology & Biochemistry 32 (2000) 1697±1705
lutant can be toxic to the microorganisms, their low
concentrations might fail to induce degradative activity. At low initial concentrations of endosulfan, i.e.
50 and 100 mg gÿ1 soil, the degradation was very rapid
in both uninoculated and inoculated soils. At higher
concentrations, however, the degradation rates were
slower, leading to a total inhibition of the degradative
activity at the initial concentration of 10 mg gÿ1 soil.
The inhibition of degradation was probably due to the
cytotoxicity of endosulfan at this concentration.
In this study, we have used bacterial cells that had
been isolated and characterized for the degradation of
endosulfan isomers in liquid-culture conditions
(Awasthi et al., 1997), and have presented evidence for
their viability as well as capability to enhance the
degradation of the chlorinated pesticide, endosulfan, in
contaminated soils. The enhancement is in¯uenced
greatly by factors such as soil-pH, presence of carbonaceous material, water content, size of bacterial inoculum and concentration of the pesticide present in the
soils. Initial optimisation of these factors is therefore
essential before undertaking any bioremediation activity. Further, in view of the in¯uence of external factors on the degradation activity, half-lives of chemicals
in the soils reported in literature should be considered
with caution.
Acknowledgements
We thank Dr P.K. Seth, Director, Industrial Toxicology Research Centre, Lucknow for his constant
support to this work, Mr Neeraj Mathur of this institute for statistical analysis, Dr S. K.Tiwari of National
Botanical Research Institute, Lucknow, for analysis of
the soils and Dr Rakesh Jain of Institute of Microbial
Technology, Chandigarh, for identi®cation of the bacterial strains. Generous gift of endosulfan isomers and
metabolites by Hoechst, AgrEvo, Germany, and ®nancial assistance from Department of Biotechnology,
New Delhi is gratefully acknowledged.
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