Journal of Environmental Management 152 (2015) 36e42

Contents lists available at ScienceDirect

Journal of Environmental Management

journal homepage: www.elsevier.com/locate/jenvman

Degradation of chlorpyrifos in tropical rice soils

Subhasis Das 1, Tapan K. Adhya*
Laboratory of Soil Microbiology, Division of Crop Production, Central Rice Research Institute, Cuttack 753006, India

a r t i c l e i n f o a b s t r a c t

Article history: Chlorpyrifos [O,O-diethyl O-(3,5,6-trichloro-2-pyridinol) phosphorothioate] is used worldwide as an

Received 24 June 2014 agricultural insecticide against a broad spectrum of insect pests of economically important crops
Received in revised form including rice, and soil application to control termites. The insecticide mostly undergoes hydrolysis to
14 January 2015
diethyl thiophosphoric acid (DETP) and 3,5,6-trichloro-2-pyridinol (TCP), and negligible amounts of
Accepted 16 January 2015
other intermediate products. In a laboratory-cum-greenhouse study, chlorpyrifos, applied at a rate of
Available online 21 January 2015
10 mg kg
1 soil to five tropical rice soils of wide physico-chemical variability, degraded with a half-life
ranging from 27.07 to 3.82 days. TCP was the major metabolite under both non-flooded and flooded
Keywords:
Chlorpyrifos
conditions. Chlorpyrifos degradation had significant negative relationship with electrical conductivity
Persistence (EC), cation exchange capacity (CEC), clay and sand contents of the soils under non-flooded conditions.
Degradation Results indicate that degradation of chlorpyrifos was accelerated with increase in its application fre-
Rice soils quency, across the representative rice soils. Management regimes including moisture content and
Soil properties presence or absence of rice plants also influenced the process. Biotic factors also play an important role in
Planted soil the degradation of chlorpyrifos as demonstrated by its convincing degradation in mineral salts medium
inoculated with non-sterile soil suspension.
© 2015 Elsevier Ltd. All rights reserved.

1. Introduction (Cnaphalocrocis medinalis) and leafhopper (Nephotettix virescens)

Organophosphorus insecticides are being increasingly used in
agriculture as a substitute for organochlorine and carbamate in-
secticides because of their high efficiency and lower persistence in
the environment (Eler
sek and Filipi
c, 2011). Chlorpyrifos [O,O-
diethyl-O-(3,5,6-trichloro-2-pyridyl) phosphorothioate], a phos-
phorothioate insecticide, has been commercially used since the
1960s. Globally, chlorpyrifos ranks first among the conventional
pesticide active ingredients in the agricultural sector with the
production of 3.64e4.99 million kg during 2007 (Grube et al.,
2011). In India, chlorpyrifos was the second most used agricul-
tural insecticide in 2013e14 at 9540 tons of formulation (Ministry
of Chemicals and Fertilizers, Govt. of India (2014)). Used particu-
larly for the control of broad-spectrum insect pests of economically
important crops (Cho et al., 2002), chlorpyrifos is intensively used
for effective control of gall midge (Orseolia oryzae), leaffolder
* Corresponding author. School of Biotechnology, KIIT University, Bhubaneswar
751024, Odisha, India.
E-mail address: adhyas@yahoo.com (T.K. Adhya).
1
Present address: The Energy and Resources Institute, Environmental and In-
dustrial Biotechnology Divn., Darbari Seth Block, IHC Complex, Lodhi Road, New
Delhi 110 003, India.
(Mallick et al., 1999) in rice, and soil applications to control termites
(Reticulitermes spp., Coptotermes spp., Heterotermes spp.)
(Sundaram et al., 1999). Like many other phosphorothioate in-
secticides with a PeOeC linkage, when applied to plants or soil,
chlorpyrifos is mostly hydrolysed with TCP [3,5,6-trichloro-2-
pyridinol] as the major transformation product (Getzin, 1981;
Racke, 1993).
Pesticide degradation in soil can be influenced by both biotic
and abiotic factors, which act in tandem and complement each
other in the soil environment (Alexander, 1994). Microbial activity
has been deemed to be the most influential and significant cause of
degradation of organophosphorus pesticides. However, several
physico-chemical factors such as pH, temperature, moisture con-
tent, organic carbon content, and pesticide formulation (Getzin,
1981) might also influence degradation of a pesticide molecule.
Chlorpyrifos bound to soil may be broken down by abiotic factors
like UV light, chemical hydrolysis and dechlorination
(Gebremariam, 2011; Roberts and Hutson, 1999). Chlorpyrifos un-
dergoes hydrolysis and produces several intermediate by-products
like diethyl thiophosphoric acid (DETP) and 3,5,6-trichloro-2-
pyridinol (TCP), and negligible amounts of chlorpyrifos oxon,
desethyl chlorpyrifos, desethyl chlorpyrifos oxon, and 3,5,6-
trichloro-2 methoxypyrimidine (Fig. 1; Racke, 1993). Initially, the

http://dx.doi.org/10.1016/j.jenvman.2015.01.025
0301-4797/© 2015 Elsevier Ltd. All rights reserved.
 S. Das, T.K. Adhya / Journal of Environmental Management 152 (2015) 36e42
Fig. 1. Transformation of chlorpyrifos in soil.
high rate of chlorpyrifos degradation in soils with alkaline pH was
attributed to chemical hydrolysis. However, the relationship be-
tween high soil pH and chemical hydrolysis was found to be weak,
since there was little degradation in several sterile high-pH soils
(Racke et al., 1996).
The dissipation data from temperate zones (Kollig and Kitchens,
1990) cannot be used to predict the behaviour of a pesticide
molecule in the tropics as most pesticides have much shorter half-
lives under tropical conditions (Racke et al., 1997; Laabs et al., 2002;
Menon et al., 2004). Information on the fate of chlorpyrifos under
the specific climatic conditions and representative soil environ-
ment of the tropics (Arbeli and Fuentes, 2007; Chai et al., 2013;
Racke et al., 1997) is rather limited, particularly in rice soils (Liang
et al., 2011; Zhang et al., 2012). Understanding of the fate and
behaviour of chlorpyrifos under tropical conditions is therefore
important both in agronomical and environmental terms. Rice
being one of the most important cereals grown almost exclusively
in tropical regions of Asia and Africa, and tropical rice being an
important agro-ecosystem with its unique feature of flooded soil,
we investigated the persistence and biodegradation of this phos-
phorothioate insecticide in five representative tropical rice soils
under both flooded and non-flooded conditions. Further, for an
effective pest control under field conditions, pesticides are often
applied repeatedly which may lead to its faster degradation and
undermine their efficacy (Arbeli and Fuentes, 2007). Hence, we also
investigated the possibility of accelerated degradation of chlor-
pyrifos in rice soils upon its repeated application.
2. Materials and methods
2.1. Soils
Five soils from rice-growing areas of India, widely varying in
their physicochemical characteristics were used in the study. The
soils were air-dried and ground to pass through a <2-mm sieve
before use. Physico-chemical characteristics of the soils were
determined according to Sparks et al. (1996). The data are reported
in Table 1.
37
2.2. Insecticide and metabolites
For persistence studies, a commercial formulation of 94%
chlorpyrifos 20 EC (Force, Nagarjuna Chemicals, Hyderabad, India)
was used. For degradation studies in culture medium and analytical
purposes, certified standards of chlorpyrifos (Dursban, 99.99% pu-
rity) and 3,5,6-trichloro-2-pyridinol (TCP, 99.99% purity) were ob-
tained from AccuStandard Inc, New Haven, CT, USA.
2.3. Soil incubation studies
Portions (20 g) of each soil were placed in separate sterile test
tubes (220  25 mm) and moistened with sterile distilled water to
maintain them at 60% moisture holding capacity (for the non-
flooded system) or flooded with 25 ml sterile distilled water (for
the flooded system) to have a standing water column of 5 cm, a
situation which simulates conditions observed in a rice field (Das
and Adhya, 2012). After incubation for 10 days at room tempera-
ture (28 ± 4  C), an aqueous suspension of the commercial
formulation of chlorpyrifos was added to the flooded or non-
flooded soils to provide a final concentration of 10 mg active
ingredient (a.i.) g
1 soil. Following addition of the insecticide, the
tubes were incubated at room temperature in the dark. Loss of
moisture during incubation, especially in the non-flooded tubes,
was compensated by adding the required quantity of sterile
distilled water at weekly intervals. At periodic intervals, residues of
chlorpyrifos in the soils were analysed by gas liquid chromatog-
raphy (GLC) following extraction and clean-up while amount of TCP
formed was quantified by high performance liquid chromatography
(HPLC).
2.4. Accelerated degradation of chlorpyrifos in retreated soils under
greenhouse conditions
Accelerated degradation of chlorpyrifos was studied under
greenhouse conditions as described by Bharati et al. (1998). An
aqueous suspension of chlorpyrifos was applied at 10 mg a.i. g
1 soil
to 5 kg of the respective soils contained in individual earthenware
38 S. Das, T.K. Adhya / Journal of Environmental Management 152 (2015) 36e42

Table 1
Physico-chemical characteristics of the soils used in the study.

Location Soil type pH EC Organic carbon Total nitrogen SO4 Fe content CEC (cmol kg
1 Soil separates
(ds m
1) (%) (%) (mg kg
1) (mg g
1) soil)
Clay Silt Sand
(%) (%) (%)

CRRI, Orissa Alluvial 6.21 0.51 0.86 0.09 21.53 8.62 15.0 25.9 21.6 52.5
Ersama, Orissa Coastal saline 6.70 8.8 0.82 0.23 32.84 11.94 32.4 35.3 50.8 13.9
Canning, West Saline silty 7.71 7.4 1.48 1.30 162.4 10.22 31.8 40.6 49.6 9.8
Bengal clay
Ranital, Orissa Alluvial 5.76 1.2 0.83 0.06 38.66 7.48 18.6 25.6 12.6 61.8
Hazaribag, Red 6.43 0.9 0.78 0.04 187.14 8.66 9.0 18.6 21.5 59.9
Jharkhand

pots (25.5  9.5 cm). Two series of pots (unplanted and planted to
rice) were maintained under four treatments viz. (1) pots retreated
with chlorpyrifos and maintained under non-flooded condition, (2)
pots retreated with chlorpyrifos and maintained under flooded
condition, (3) untreated control pots maintained under non-
flooded conditions and (4) untreated control pots maintained un-
der flooded conditions. Twenty-one-day-old rice seedlings (Oryza
sativa L, cv Naveen) planted in pots represented the planted series.
All the pots in three replicates each representing different treat-
ments were maintained in the greenhouse under appropriate water
regime with 60% moisture-holding capacity for non-flooded pots
and with standing water of 5 (±1) cm above the soil for flooded
pots. The moisture level of non-flooded and flooded pots was
maintained by weekly additions of the required quantity of sterile
distilled water. The first application of chlorpyrifos was made to the
soil in the pots at 10 days after flooding (unplanted pots) or
transplantation (planted pots). A second application was made 15
days later (25 days after flooding) and subsequent applications
were made at 30 days (40 days after flooding) and 45 days (55 days
after flooding).
At regular intervals, soil samples from pots treated as above
were collected as described by Panda et al. (1988) and tested for
chlorpyrifos degradation. In brief, soil samples from the surface
layer (10 ± 2 cm) of pots from both sets (unplanted and planted)
were collected separately by using a tube auger with a mark at
2 cm. Portions (1 g) of the soils thus removed were used to prepare
suspensions in sterile distilled water (10 ml) in pre-sterilized test
tubes (200  25 mm). Portions (10 ml) of sterile mineral salts
medium [(NH4)2HPO4$0.5; MgSO4, 7H2O$0.2, FeSO4, 7H2O$0.01,
K2HPO4$0.1, Ca(NO3)2 0.01 g L
1 in distilled water, pH 7.0] con-
tained in 100-ml Erlenmeyer flasks and supplemented with
10 mg ml
1 of chlorpyrifos, were separately inoculated with 1 ml of
soil suspension from both planted and unplanted pots. The flasks
were incubated under aerobic conditions in a shaker at room
temperature (28 ± 2  C). Uninoculated medium served as control.
At periodic intervals, contents of the whole flask of the inoculated
and uninoculated media were sacrificed from each duplicate set
and the amount of chlorpyrifos remaining in the medium was
determined by GLC after extraction with solvents. TCP [3,5,6-
trichloro-2-pyridinol], the degradation product of chlorpyrifos
was detected in mineral salts medium by HPLC but was not
quantified.
2.5. Extraction and analysis of residues
In the experiment on the persistence of chlorpyrifos in different
soils, residues of the parent insecticide and its metabolites were
extracted from the soil. For extraction of chlorpyrifos from soil, soil
plus water in each of the duplicate tubes were quantitatively
transferred to a 250-ml Erlenmeyer flask with distilled water and
acetone. The residues were subsequently transferred to hexane and
dried over sodium sulphate (Adhya et al., 1987). The hexane layer
after appropriate dilution with the same solvent was analysed for
chlorpyrifos by GLC. TCP extraction from soils was performed ac-
cording to the method reported by Baskaran et al. (2003) and
quantified by HPLC. Sample preparation and extraction of residues
were done in the laboratory in diffuse light. Details of GLC and HPLC
analysis are given in Supplementary information.
2.6. Measurement of Eh and pH
Soil samples (40 g) placed in 100-ml beakers were flooded with
50 ml sterile distilled water and incubated at room temperature.
After 10 days of flooding, 1 ml aqueous suspension of chlorpyrifos
was added to each sample to provide final concentration of 10 mg
a.i. g
1 soil. At periodic intervals, the redox potential of duplicate
soil samples was measured by inserting a combined platinum-
calomel electrode (Barnant Co IL, USA) into the soil and
measuring the potential difference in mV (Pal et al., 1979). Imme-
diately after the measurement of the redox potential, the pH of the
soil was measured with a portable pH meter (Philips model PW
9424, Philips Analytical, Cambridge, UK).
2.7. Statistical analyses
Individual data sets on residues of chlorpyrifos and other soil
parameters were statistically analysed using statistical package
CropStat Ver. 7.2 (International Rice Research Institute, Philippines).
Simple and multiple correlations between different half-lives of
chlorpyrifos and soil parameters were analysed by SYSTAT (SPSS
Inc., USA).
3. Results and discussion
3.1. Persistence of chlorpyrifos under non-flooded and flooded
conditions
The persistence of the commercial formulation of chlorpyrifos
was studied in five tropical rice soils under both non-flooded and
flooded conditions (Table 2). The amount of chlorpyrifos remaining
in the soil was plotted on a log scale against the time of incubation.
Degradation of chlorpyrifos in all the five soils followed a first-order
reaction as the plots yielded straight lines based on the equation,
C ¼ C0e
kt where C is the concentration of the insecticide remaining
in the soil after time t, C0 is the initial concentration and k is the
first-order kinetic constant. The half-life (t1/2) values (Table 2) ob-
tained by the function t1/2 ¼ ln2/k from these plots indicate that
chlorpyrifos is less persistent in all the five soils under flooded than
under non-flooded condition. Statistical validation of the data in-
dicates that among the five soils used in the study, degradation of
chlorpyrifos under non-flooded condition was the least in Hazar-
ibag, Ranital and CRRI soils in that order and the most in Ersama soil
 S. Das, T.K. Adhya / Journal of Environmental Management 152 (2015) 36e42 39

Table 2
Persistence of Chlorpyrifos in five tropical soils of varying physico-chemical characteristics under non-flooded and flooded conditions.

Incubation (days) Chlorpyrifosa recovered (mg g
1 soil)b

CRRI Ersama Canning Ranital Hazaribag

Non-flooded Flooded Non-flooded Flooded Non-flooded Flooded Non-flooded Flooded Non-flooded Flooded

0 10.03a (BDL)c 9.44a (BDL) 9.92a (BDL) 9.92a (BDL) 8.84a (tr) 9.33a (BDL) 10.03a (BDL) 9.74a (BDL) 9.93a (BDL) 9.64a (BDL)
2 8.60b (tr) 6.89b (tr) 9.48a (tr) 8.25b (0.26) 8.75a (tr) 8.02b (0.84) 8.06b (0.31) 6.09b (1.22) 9.66a (BDL) 8.99a (BDL)
4 6.67c (1.01) 5.73c (1.12) 9.09b (tr) 5.44c (1.87) 8.36a (0.6) 7.99b (0.62) 7.27c (0.64) 5.23c (1.76) 9.27a (tr) 8.73b (0.61)
6 6.33c (1.07) 5.81c (1.14) 7.00c (1.31) 2.84d (2.88) 8.14a (0.6) 7.94b (0.6) 6.03d (1.28) 4.81c (2.32) 8.53b (0.5) 7.91c (0.64)
10 6.76c (1.12) 5.37c (1.22) 4.87d (2.21) 1.84d (4.02) 7.87b (1.02) 7.68b (1.11) 5.76d (2.01) 4.17d (3.21) 8.35b (0.51) 7.37c (1.16)
15 5.76d (2.46) 4.64d (3.02) 4.17e (2.86) 0.59e (4.68) 5.87c (2.24) 5.16c (2.64) 5.46d (2.12) 3.34e (3.32) 7.36c (1.01) 5.94d (1.86)
20 4.48e (2.79) 2.79e (3.822) 3.10f (3.01) 0.37e (4.16) 3.47d (3.28) 3.21d (3.24) 4.48e (2.86) 2.29f (4.24) 6.48d (1.92) 4.79e (2.88)
30 3.46f (2.52) 1.67f (2.60) 2.99f (2.85) 0.32e (2.23) 3.04d (3.12) 1.90e (3.22) 3.46f (2.88) 1.27g (3.86) 4.46e (2.27) 2.97f (3.36)
k 0.0316 0.0518 0.046 0.1816 0.0404 0.0534 0.0314 0.0598 0.0256 0.0386
r2 0.9152 0.9506 0.8956 0.9626 0.9141 0.9448 0.9227 0.9615 0.9713 0.9854
t1/2 (d) 21.93 13.38 15.07 3.82 17.15 12.98 22.07 11.59 27.07 17.95
a
Chlorpyrifos was added to the flooded or non-flooded soil at 10 mg g
1 soil under laboratory conditions.
b
Mean of three replicate observations. In a column, means followed by a common letter are not significantly different (P < 0.05) by Duncan's multiple range test.
c
Values in parenthesis indicate the amount of TCP recovered; average of three replicate observations (BDL ¼ Below Detectable Limit; tr ¼ trace).

(Table 2). Under flooded conditions, chlorpyrifos was most persis-
tent in Hazaribag soil and the least in Ersama soil. Chlorpyrifos
degrades slowly in soils with low pH (5.5e6.5), and is stable under
acidic conditions (Singh et al., 2003). Initially the high rate of
chlorpyrifos degradation in soils with alkaline pH was attributed to
chemical hydrolysis. However, Racke et al. (1996) concluded that
the relationship between high soil pH and chemical hydrolysis was
weak, since there was little degradation in several high-pH soils
under sterile conditions. Under flooded conditions, the pH of the
soils increased to near neutrality within a few days of flooding
(Table 3). All the soils used in this study were microbially active, as
indicated by a sharp drop in the redox potential and simultaneous
increase in pH, following flooding (Liesack et al., 2004). Thus, the
near-neutral pH and high microbial activity could be the reasons for
faster degradation of chlorpyrifos under flooded conditions albeit
without any strong relationship with soil pH (Table 4). Detection of
TCP in almost equimolar amount from the soils studied, indicated
that hydrolysis is the major pathway of degradation of the insec-
ticide (Mallick et al., 1999; Racke, 1993). Concentration of TCP,
however, declined with time suggesting its further degradation.
A simple linear correlation analysis between t1/2 and different
soil properties (Table 4) under non-flooded and flooded conditions
indicate a significant negative relationship with EC, CEC, clay and
sand contents of the soils under non-flooded condition. Several
pesticides have been reported to be increasingly persistent under
saline conditions (Adhya et al., 1987). Increasing salinity is inhibi-
tory to growth and activity of micro-organisms (Zahran, 1997).
However, surface catalysed hydrolysis of several phosphorothioate
insecticides like chlorpyrifos is known to be accelerated at pH
beyond 7.0 (Adamson and Inch, 1973). Clay content in association
with oxides of Fe is known to increase the surface area and induce
surface catalysed hydrolysis (Felsot and Dahm, 1979). Thus, larger
contents of Fe oxides and clay probably induced hydrolysis of
chlorpyrifos in Canning and Ersama soils under non-flooded con-
ditions despite high salinity and near neutral to alkaline pH.
Conversely, acidic pH, lower Fe oxide and clay contents in CRRI,
Ranital and Hazaribag soils contributed to longer persistence of
chlorpyrifos under non-flooded conditions.
3.2. Enhanced degradation of chlorpyrifos in tropical rice soils
following repeated application
The degradation of chlorpyrifos after its repeated application to
CRRI soil is shown in Table 5 and follows almost similar pattern in
other soils (Supplementary tables S-1 to S-4). The half-life of
chlorpyrifos (Table 6) calculated by first order function in almost all
the five soils reduced exponentially in the order of first, second and
third application. The results suggest that repeated application of
chlorpyrifos to different soils significantly (p  0.05) enhanced its
degradation. During the same period, only marginal loss of chlor-
pyrifos was recorded from the un-inoculated control (Table 5).
Initial degradation of chlorpyrifos in the soils was little slow and the
rate of degradation gradually increased with its repeated applica-
tions. What was interesting in the present study is the role of rice
plants especially under flooded conditions, in influencing the
enhanced degradation of chlorpyrifos at least in Ersama, Canning
and Ranital soils. Thus in mineral salts (MS) medium inoculated
with CRRI soil, from pots with three repeat applications of chlor-
pyrifos and maintained under both non-flooded and flooded con-
ditions, the amount of chlorpyrifos recovered came down to very
low levels (Table 5). Flooded soil unplanted or planted to rice is
physically, chemically and microbiologically more active than its
non-flooded counterpart (Liesack et al., 2004). Rice plants are also
known to exert a positive influence on soil microbial activity
through the transport of oxygen to the root region as well as by the
release of root exudates (Watanabe and Furusaka, 1980).

Table 3
Changesa in the redox potential (Eh) and pH of the five tropical soils under flooded condition.

Incubation (days) Soil types

CRRI Ersama Canning Ranital Hazaribag

Eh (mV) pH Eh (mV) pH Eh (mV) pH Eh (mV) pH Eh (mV) pH

0 þ260 6.20 þ200 6.70 þ220 7.71 þ240 5.75 þ274 6.42
10
162 6.51
106 6.82
134 7.79
103 6.02
86 6.51
20
184 6.70
163 6.91
165 7.65
122 6.68
110 6.73
30
206 6.88
179 7.04
193 7.43
138 6.76
126 6.82
a
Mean of three replicate observations.
40 S. Das, T.K. Adhya / Journal of Environmental Management 152 (2015) 36e42

Table 4 Enrichment of chlorpyrifos-degrading micro-organisms has been

Correlations (r values) between half-life values and various soil parameters under considered to be the cause of enhanced degradation of the insec-
non-flooded and flooded conditions.
~ o et al., 2012; Singh et al., 2006). Thus, high microbial
ticide (Bricen
Soil parameters Moisture status activity in a flooded soil unplanted or planted to rice could be the
Non-flooded Flooded reason for enhanced degradation of chlorpyrifos following its
repeated application.
pH
0.528
0.063
Organic carbon
0.452 0.082 Accelerated biodegradation of soil-applied pesticides upon their
Total Nitrogen
0.532
0.016 repeated application is the result of soil-pesticide-microbe inter-
Fe content
0.785
0.695 action and can undermine the efficacy of the pesticides under
SO4 content 0.354 0.639
consideration (Racke, 1990). Unlike in the case of other organo-
Ec
0.892*
0.708
CEC
0.971**
0.756 phosphates, there has been no report of enhanced degradation of
Clay
0.925*
0.587 chlorpyrifos since its first use in 1965. It has been suggested that the
Silt
0.826
0.566 accumulation of TCP, which has antimicrobial properties, prevents
Sand 0.886* 0.591 the proliferation of chlorpyrifos-degrading microorganisms in soil
*Significant at P ¼ 0.05. (Racke et al., 1990). However, only recently accelerated degradation
**Significant at P ¼ 0.01. of chlorpyrifos is being reported from soil previously treated with
chlorpyrifos (Fang et al., 2008; Robertson et al., 1998), although
results had also been reported that the half-life of chlorpyrifos was

Table 5
Degradation of Chlorpyrifos in a mineral salts medium inoculated with suspension from unplanted and planted non-flooded or flooded CRRI soil untreated and treated with
commercial formulation of Chlorpyrifos.

Application Days Chlorpyrifosa recovered (mg ml
1 medium)b

Un-inoculated Non-flooded Flooded

Unplanted Plantedc Unplanted Planted

Untreated Pre-treated Untreated Pre-treated Untreated Pre-treated Untreated Pre-treated

1st 0 9.82a 9.52a 10.06a 9.41a 10.00a 9.82a 9.15a 8.92a 9.74a
5 9.55a 8.21b 8.98b 8.18b 8.89b 9.78a 8.29b 8.36a 5.67b
10 9.69a 6.93c 8.88b 6.41c 4.29c 7.98b 7.17c 5.69b 5.12b
15 9.54a 5.86d 2.97c 4.00d 2.21d 3.69c 1.85d 2.65c 2.62c
2nd 0 9.82a 10.03a 9.48a 10.09a 9.91a 9.89a 9.92a 9.84a 9.77a
5 9.63a 6.95b 8.77b 6.07b 5.83b 8.18b 4.04b 8.20b 5.84b
10 9.67a 5.39c 4.07c 6.30c 2.91c 4.41c 2.71c 5.05c 4.54c
15 9.61a 3.36d 2.48d 2.76d 2.22d 2.76d 1.38d 2.15d 1.85d
3rd 0 9.90a 9.57a 9.62a 10.08a 9.80a 9.72a 9.57a 9.86a 9.91a
5 9.69a 6.59b 7.71b 6.85b 5.17b 7.94b 3.82b 6.51b 2.38b
10 9.71a 2.72c 0.31c 1.84c 1.64c 4.17c 0.23c 4.01c 2.04b
15 8.99a 1.46d 0.29c 0.79d 0.25d 1.90d 0.00d 1.08d 0.00c
a
Chlorpyrifos was added to the mineral salts medium at 10 mg ml
1.
b
Mean of three replicate observations. In a column, means followed by a common letter are not significantly different (P < 0.05) by Duncan's multiple range test.
c
All the planted pots were planted with rice plants cv. Naveen.

Table 6
Persistence (DT50) of Chlorpyrifos in a mineral salts (MS) medium independently inoculated with suspension from five tropical soils unplanted and planted to rice under non-
flooded or flooded conditions and untreated or retreated with commercial formulation of Chlorpyrifos.

Soil Application frequency DT50 (days)a

Non-flooded Flooded

Unplanted Plantedb Unplanted Planted

Untreated Pre-treated Untreated Pre-treated Untreated Pre-treated Untreated Pre-treated

CRRI 1st 21.30a 9.46a 12.36a 6.60a 11.05a 7.05a 8.62a 8.56a
2nd 9.86b 7.24b 9.04b 6.69a 7.76b 5.88a 6.87a 6.62a
3rd 5.31b 3.46c 4.38c 3.89b 6.26b 3.01b 7.24a 4.39b
Ersama 1st 10.10a 6.20a 9.48a 8.12a 7.22a 5.48a 6.37a 6.05a
2nd 6.68b 4.99a 6.61b 1.66b 7.16a 3.51b 6.81a 3.21b
3rd 6.11b 5.60a 4.65c 1.42b 5.50b 3.04b 4.65b 2.83b
Canning 1st 9.51a 6.43a 13.03a 7.36a 16.30a 8.88a 10.02a 4.31a
2nd 7.78b 4.18b 12.75a 1.75b 7.96b 5.29b 10.05a 3.02b
3rd 6.74c 2.94c 6.92b 1.44b 7.82b 4.40c 1.44b 1.24c
Ranital 1st 7.59b 6.31a 8.06b 6.24a 6.26b 5.79a 5.90a 3.70a
2nd 6.64b 1.82b 6.32b 2.49b 11.16a 4.59a 6.53a 3.66a
3rd 15.61a 1.54b 21.24a 1.79b 8.46b 5.98a 5.68a 3.31a
Hazaribag 1st 18.88a 11.50a 10.45bc 6.98a 18.62a 14.68a 11.53a 8.73a
2nd 19.98a 11.84a 10.10ab 5.91b 14.10b 7.98b 9.55a 5.27b
3rd 16.01b 9.69b 7.98b 4.51b 10.60c 5.52c 7.32b 3.16b
a
Mean of three replicate observations; in a column, means followed by a common letter are not significantly different (P < 0.05) by Duncan's multiple range test.
b
All the planted pots were planted with rice plants cv. Naveen.
 S. Das, T.K. Adhya / Journal of Environmental Management 152 (2015) 36e42 41

Table 7
Persistence of Chlorpyrifos in a mineral salts medium inoculated with sterile and non-sterile suspension of three tropical rice soils retreated with Chlorpyrifos.

Incubation Chlorpyrifosa recovered (mg ml
1 medium)b

(days)
Uninoculated CRRI Ersama Canning

Inoculated with Inoculated with non- Inoculated with Inoculated with non- Inoculated with Inoculated with non-
sterile soil sterile soil suspension sterile soil sterile soil suspension sterile soil sterile soil suspension
suspension suspension suspension

0 9.74a ± 0.07 9.77a ± 0.09 9.75a ± 0.11 9.73a ± 0.13 9.99a ± 0.03 9.80a ± 0.12 9.85a ± 0.32
3 9.65a ± 0.12 9.56a ± 0.33 9.00b ± 0.25 9.53ab ± 0.34 9.02b ± 0.15 9.61ab ± 0.32 9.45a ± 0.28
5 9.51a ± 0.23 9.46a ± 0.14 8.21b ± 0.21 9.23abc ± 0.12 8.32c ± 0.54 9.43ab ± 0.05 9.33a ± 0.25
7 9.38a ± 0.33 8.80b ± 0.07 7.34d ± 0.27 8.99bc ± 0.23 6.22d ± 0.13 9.12b ± 0.21 6.87b ± 0.12
10 9.12a ± 0.34 8.63b ± 0.02 6.95d ± 0.20 8.65c ± 0.22 4.23e ± 0.12 9.01b ± 0.11 6.08c ± 0.32
a
Chlorpyrifos was added to the flooded or non flooded soil at 10 mg g
1 soil at 15 days interval to the pots under greenhouse conditions.
b
Average of three replicate observations ± mean deviation; in a column, means followed by a common letter are not significantly different (P < 0.05) by Duncan's multiple
range test.

extended with the number of applications (Singh et al., 2002). In
the present study, accelerated degradation of chlorpyrifos in five
tropical rice soils, widely different in their physic-chemical char-
acteristics, has been demonstrated under both non-flooded and
flooded conditions especially from planted pots.
3.3. Persistence of chlorpyrifos in sterile and non sterile suspension
of soil retreated with chlorpyrifos
The role of micro-organisms in the degradation of chlorpyrifos
was investigated in sterile vs non-sterile soil suspensions of three
flooded soils showing enhanced degradation of chlorpyrifos.
Chlorpyrifos disappeared faster in mineral salts medium inoculated
with non-sterile soil suspension than in medium inoculated with
soil suspensions sterilized by autoclaving (Table 7). In studies on
microbial conversion of chlorpyrifos, the insecticide was shown to
be hydrolysed producing metabolites including TCP. Enhanced
degradation of chlorpyrifos was reported following repeated
application of the parent compound (Fang et al., 2008; Robertson
et al., 1998) or its metabolite (Yang et al., 2005). In the present
study also TCP was detected as a metabolite in the degradation of
chlorpyrifos. This study demonstrates enhanced degradation of
chlorpyrifos, a chlorine-containing phosphorothioate insecticide,
after two or three additions of the parent compound to five tropical
rice soils.
4. Conclusions
The present study indicates that chlorpyrifos (10 mg g
1 soil)
undergoes degradation in tropical rice soils with widely divergent
physico-chemical properties. Hydrolysis was the major pathway of
degradation leading to the production of TCP as the major hydro-
lysis product in both non-flooded (aerobic) and flooded (anaerobic)
soils. Although, chlorpyrifos was not known earlier to undergo
enhanced degradation in retreated soils, current study provides
convincing evidence of enhanced degradation in flooded soil
planted to rice following repeated addition of the insecticide,
possibly due to enrichment of degrading microbial population. In
spite of the fact that chlorpyrifos is frequently used in rice culti-
vation and possibly in larger amounts, moisture status and pres-
ence of rice plants and associated microbial activity would
stimulate degradation and mineralization of the insecticide.
Acknowledgements
This work forms part of the Ph.D. dissertation submitted by the
senior author to the Utkal University, Bhubaneswar, Odisha, India.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.jenvman.2015.01.025.
References
Adamson, J., Inch, T.D., 1973. Possible relationships between structure and mecha-
nism of degradation of organophosphorus insecticides in the soil environment.
In: Proc 7th British International Fungicide Conference, pp. 65e72.
Adhya, T.K., Wahid, P.A., Sethunathan, N., 1987. Persistence and biodegradation of
selected organophosphorus insecticides in flooded versus non-flooded soils.
Biol. Fertil. Soils 4, 36e40.
Alexander, M., 1994. Biodegradation and Bioremediation. Academic Press Inc, San
Diego.
Arbeli, Z., Fuentes, C.L., 2007. Accelerated biodegradation of pesticides: an overview
of the phenomenon, its basis and possible solution, and a discussion on the
tropical dimension. Crop Prot. 26, 1733e1746.
Baskaran, S., Kookana, R.S., Naidu, R., 2003. Contrasting behaviour of chlorpyrifos
and its primary metabolite, TCP (3, 5, 6-trichloro-2-pyridinol), with depth in
soil profiles. Aust. J. Soil Res. 41, 749e760.
Bharati, K., Padhi, S., Adhya, T.K., 1998. Accelerated biodegradation of g-hexa-
chloroclohexane (g-HCH) in a flooded alluvial soil pretreated with g-HCH or its
metabolite 1,2,4-trichlorobenzene (TCB). Bull. Environ. Contam. Toxicol. 60,
858e863.
~ o, G., Fuentes, M.S., Palma, G., Jorquera, M.A., Amoroso, M.J., Diez, M.C., 2012.
Bricen
Chlorpyrifos biodegradation and 3,5,6-trichloro-2-pyridinol production by
actinobacteria isolated from soil. Int. Biodeterior. Biodegradation 73, 1e7.
Chai, L.-K., Wong, M.-H., Bruun Hansen, H.C., 2013. Degradation of chlorpyrifos in
humid tropical soils. J. Environ. Manag. 125, 28e32.
Cho, C.M.-H., Mulchandani, A., Chen, W., 2002. Bacterial cell surface display of
organophosphorus hydrolase for selective screening of improved hydrolysis of
organophosphate nerve agent. Appl. Environ. Microbiol. 68, 2026e2030.
Das, S., Adhya, T.K., 2012. Dynamics of methanogenesis and methanotrophy in
tropical paddy soils as influenced by elevated CO2 and temperature interaction.
Soil Biol. Biochem. 47, 36e45.
Eler
sek, T., Filipi

c, M., 2011. Organophosphorus pesticides e mechanisms of their
toxicity. In: Stoycheva, M. (Ed.), Pesticides e the Impacts of Pesticides Exposure.
Intech Publishers, pp. 243e260. www.intechopen.com.
Fang, H., Yu, Y.L., Wang, X.G., Chu, X.Q., Pan, X.D., Yang, X.E., 2008. Effects of
repeated applications of chlorpyrifos on its persistence and soil microbial
functional diversity and development of its degradation capability. Bull. Envi-
ron. Contam. Toxicol. 81, 397e400.
Felsot, A., Dahm, P.A., 1979. Sorption of organophosphorus and carbamate in-
secticides by soil. J. Agric. Food Chem. 27, 557e563.
Gebremariam, S.Y., 2011. Mineralization, Sorption and Desorption of Chlorpyrifos in
Aquatic Sediments and Soils. Ph.D. Thesis. Department of Civil and Environ-
mental Engineering, Washington State University, p. 198.
Getzin, L.W., 1981. Degradation of chlorpyrifos in soil: influence of autoclaving, soil
moisture, and temperature. J. Econ. Entomol. 74, 158e162.
Grube, A., Donaldson, D., Kiely, T., Wu, L., 2011. Pesticides Industry Sales and Usage:
2006 and 2007 Market Estimates. Biological and Economic Analysis Division,
Office of Pesticide Programs, U.S. Environmental Protection Agency, Washing-
ton, DC 20460, pp. 1e33.
Kollig, M.P., Kitchens, B.E., 1990. Problems associated with published environmental
fate data. Environ. Toxicol. Chem. 28, 95e103.
Laabs, V., Amelung, W., Pinto, A., Zech, W., 2002. Fate of pesticides in tropical soils of
Brazil under field conditions. J. Environ. Qual. 31, 256e268.
Liang, B., Yang, C., Gong, M., Zhao, Y., Zhang, J., Zhu, C., Jiang, J., Li, S., 2011.
Adsorption and degradation of triazophos, chlorpyrifos and their main hydro-
lytic metabolites in paddy soil from Chaohu Lake, China. J. Environ. Manage. 92,
42 S. Das, T.K. Adhya / Journal of Environmental Management 152 (2015) 36e42
2229e2234.
Liesack, W., Schnell, S., Revsbech, N.P., 2004. Microbiology of flooded rice paddies.
FEMS Microbiol. Rev. 24, 625e645.
Mallick, B.K., Banerji, A., Shakil, N.A., Sethunathan, N., 1999. Bacterial degradation of
chlorpyrifos in pure culture and in soil. Bull. Environ. Contam. Toxicol. 62,
48e55.
Menon, P., Gopal, M., Prasad, Rajender, 2004. Dissipation of chlorpyrifos in two soil
environments of Semi-Arid India. J. Environ. Sci. Health B39, 517e531.
Ministry of Chemicals and Fertilizers, Govt of India, 2014. Chemicals and Petro-
chemical Statistics at a Glance: 2014. Ministry of Chemicals and Fertilizers,
Govt. of India, New Delhi. http://chemicals.nic.in/MLCPCSTAT14.pdf.
Pal, S.S., Barik, S., Sethunathan, N., 1979. Effects of benomyl on iron and manganese
reduction and redox potential in flooded soil. J. Soil Sci. 30, 155e159.
Panda, S., Sharmila, M., Ramanand, K., Panda, D., Sethunathan, N., 1988. Persistence
of hexachlorocyclohexane isomers and carbofuran applied to surface and sub-
surface layers of a flooded soil. Pesticide Sci. 23, 199e207.
Racke, K.D., 1990. Implications of enhanced biodegradation for the use and study of
pesticides in the soil environment. In: Racke, K.D., Coats, J.R. (Eds.), Enhanced
Biodegradation of Pesticides in the Environment, ACS Symposium Series, vol.
426. American Chemical Society, Washington DC, pp. 269e282.
Racke, K.D., 1993. Environmental fate of chlorpyrifos. Rev. Environ. Contam. Toxicol.
131, 1e154.
Racke, K.D., Laskowski, D.A., Schultz, M.R., 1990. Resistance of chlorpyrifos to
enhanced biodegradation in soil. J. Agric. Food Chem. 38, 1430e1436.
Racke, K.D., Skidmore, M.W., Hamiltonm, D.J., Unsworthm, J.B., Miyamoto, J.,
Cohen, S.Z., 1997. Pesticide fate in tropical soils. Pure Appl. Chem. 69,
1349e1371.
Racke, K.D., Steele, K.P., Yoder, R.N., Dick, W.A., Avidov, E., 1996. Factors effecting the
hydrolytic degradation of chlorpyrifos in soil. J. Agric. Food Chem. 44,
1582e1592.
Roberts, T.R., Hutson, D.H., 1999. Metabolic Pathways of Agrochemicals e Part 2:
Insecticides and Fungicides. The Royal Society of Chemistry, Cambridge, UK,
pp. 235e242.
Robertson, L.N., Chandler, K.J., Stickley, B.D.A., Cocco, R.F., Ahmetagic, M., 1998.
Enhanced microbial degradation implicated in rapid loss of chlorpyrifos from
the controlled-release formulation suSConBlue in soil. Crop Prot. 17, 29e33.
Singh, B.K., Walker, A., Morgan, J.A.W., Wright, D.J., 2003. Effects of soil pH on the
biodegradation of chlorpyrifos and isolation of a chlorpyrifos-degrading bac-
terium. Appl. Environ. Microbiol. 69, 5198e5206.
Singh, B.K., Walker, A., Wright, D.J., 2002. Persistence of chlorpyrifos, fenamiphos,
chlorothalonil, and pendimethalin in soil and their effects on soil microbial
characteristics. Bull. Environ. Contam. Toxicol. 69, 181e188.
Singh, B.K., Walker, A., Wright, D.J., 2006. Bioremediation potential of fenamiphos
and chlorpyrifos degrading isolates: influence of different environmental con-
ditions. Soil Biol. Biochem. 38, 2682e2693.
Sparks, D.L., Page, A.L., Summer, M.E., Tabatabai, M.A., Helmke, P.A., 1996. Methods
of Soil Analysis, Part 3, Chemical Methods. American Society of Agronomy, USA,
p. 1358.
Sundaram, B., Rai, S.K., Ravendra, N., 1999. Degradation of bifenthrin, chlorpyrifos
and imidacloprid in soil and bedding materials at termiticidal application rates.
Pesticide Sci. 55, 1222e1228.
Watanabe, I., Furusaka, C., 1980. Microbial ecology of flooded soils. In: Alexander, M.
(Ed.), Advances in Microbial Ecology, vol. 4. Plenum Press, New York,
pp. 125e168.
Yang, L., Zhao, Y.-H., Zhang, B.-X., Yang, C.-H., Zhang, X., 2005. Isolation and char-
acterization of a chlorpyrifos and 3,5,6-trichloro-2-pyridinol degrading bacte-
rium. FEMS Microbiol. Lett. 251, 67e73.
Zahran, H.H., 1997. Diversity, adaptation and activity of the bacterial flora in saline
environments. Biol. Fertil. Soils 25, 211e223.
Zhang, X., Shen, Y., Yu, X.-Y., Liu, X.-J., 2012. Dissipation of chlorpyrifos and residue
analysis in rice, soil and water under paddy field conditions. Ecotoxicol. Envi-
ron. Saf. 78, 276e280.