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Bulletin of EnvironmentalContamination and Toxicology ISSN 0007-4861Volume 94Number 3 Bull Environ Contam Toxicol (2015)94:387-392DOI 10.1007/s00128-015-1465-0
Chlorpyrifos Degradation in Soils withDifferent Treatment Regimes Within NzoiaRiver Drainage Basin, Kenya
Gershom Kyalo Mutua, AnastasiahNjoki Ngigi & Zachary Moranga Getenga
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Chlorpyrifos Degradation in Soils with Different TreatmentRegimes Within Nzoia River Drainage Basin, Kenya
Gershom Kyalo Mutua • Anastasiah Njoki Ngigi •
Zachary Moranga Getenga
Received: 27 June 2014 / Accepted: 16 January 2015 / Published online: 24 January 2015
� Springer Science+Business Media New York 2015
Abstract Two organic amendments, filter mud compost
and Tithonia diversifolia leaves generated within a sugar-
cane growing area were used to enhance the degradation of
chlorpyrifos in soil. Filter mud compost and T. diversifolia
leaves significantly enhanced degradation of chlorpyrifos
in soils (p \ 0.05) with DT50 values of 21 and 24 days,
respectively. Furthermore, field degradation of chlorpyrifos
in soil with prior exposure to chlorpyrifos was significantly
enhanced (p = 0.034) with DT50 of 21 days compared to
30 days in soil with no previous exposure. Degradation of
chlorpyrifos in sterile and non-sterile soils were signifi-
cantly different (p = 0.023) with DT50 values of 161 and
27 days, respectively. Results show enhanced degradation
of chlorpyrifos in organically amended soils and soils with
prior exposure to the pesticide. These amendments show
promise in a continuing effort to reduce chlorpyrifos con-
centrations in soils.
Keywords Pesticide � Organic amendments � Application
history
Chlorpyrifos (CPF) [O, O-diethyl-O-(3,5,6-trichloro-2-
pyridyl) phosphorothionate] is a broad-spectrum non-sys-
temic organophosphate insecticide used for soil treatment
during planting (Tortella et al. 2010). It is used all over the
world due to its high effectiveness against target
organism(s). CPF is highly toxic to both aquatic and ter-
restrial organisms with a potential to bio-concentrate
(Racke 1993). It has a high mammalian toxicity and
exposure to unborn children causes delayed motor and
mental development (Rauh et al. 2006). It is moderately
persistent in soil with field half-lives ranging between 7
and 120 days (Kamrin 1997). In soil, it is degraded to
3,5,6-trichloro-2-pyridinol (TCP) and 2-methoxy-3,5,6-tri-
chloropyridine with TCP being the major transformation
product (Menon et al. 2004). CPF has been detected in sea
water and marine sediments (Delgado-Moreno et al. 2011),
drinking water wells (Bortoluzzi et al. 2007), river water
(Bhattacharjee et al. 2012) and soil (Wright et al. 1994).
Due to the environmental concerns associated with CPF
presence and accumulation in different environmental
matrices, there is a need to devise suitable, safe and eco-
nomically viable methods to degrade the chemical. Bio-
degradation plays an important role in the degradation of
CPF (Tortella et al. 2010). Studies within the Nzoia River
Drainage Basin (NRDB) have reported enhanced degra-
dation of pesticides as a result of biostimulation using
organic materials (Lalah et al. 2009; Jemutai-Kimosop
et al. 2012). However, this strategy has not been tested on
CPF, despite its extensive use in commercial sugarcane
farming in Kenya where its application rate is 0.94 kg of
active ingredient (a.i.) per hectare. In this study, field
experiments were conducted to evaluate effects of filter
mud compost (FMC) and Tithonia diversifolia leaves
(TDL) on degradation of CPF. Both FMC and TDL are
readily available in the NRDB. About 24,000 tons of FMC
are produced within the basin per year, while about
10 t ha-1 (tons per hectare) of TDL are harvestable per
year (Jemutai-Kimosop et al. 2012). In addition, there is no
documented information on the effect of repeated exposure
of CPF to soil on its degradation. This effect was
G. K. Mutua � Z. M. Getenga (&)
Department of Pure and Applied Chemistry, Masinde Muliro
University of Science and Technology,
P.O Box 190, Kakamega 50100, Kenya
e-mail: zgetenga@yahoo.com
A. N. Ngigi
Department of Physical Sciences, Multimedia University
of Kenya, P.O Box 30305, Nairobi 00100, Kenya
123
Bull Environ Contam Toxicol (2015) 94:387–392
DOI 10.1007/s00128-015-1465-0
Author's personal copy
investigated through both field and laboratory experiments
in the current study.
Materials and Methods
An analytical standard of CPF (98.5 %) (CAS RN
2921-88-2) was purchased from Dr. Ehrenstorfer GmbH
(Augsburg, Germany). CPF has a water solubility of
1.39 mg L-1 (25�C), log Kow of 4.7–5.3, Koc of 8,500 and
a vapour pressure of 2.0 9 10-5 mm Hg (25�C) (Racke
1993). A stock solution of 100 lg mL-1 was prepared in
acetonitrile and working solutions were prepared by
appropriate dilution. Formulated CPF (Chlorpyrifos S480
of 48 % w/v), polyvinyl chloride pipes (PVC), chemicals
and other materials were purchased from local suppliers.
FMC and fresh TDL were obtained from the Nzoia Sugar
Company estate which is located within the NRDB. These
organic materials were then characterized and prepared for
application. Elemental composition of FMC was 11.7 % C,
2.6 % N, 0.05 % P and 0.11 % K, while TDL had 24.2 %
C, 2.9 % N, 0.08 % P and 0.09 % K.
Field studies were carried out in the Nzoia Sugar
Company estate where CPF is extensively used. Soil from
the study area is sandy loam with C and N contents of
2.8 % and 0.14 %, respectively. Experiments were con-
ducted using PVC pipes of 45 cm long and a diameter of
6 cm (Lalah et al. 2009). Before setting up the experiment,
the amount of dry soil that the PVC pipe would accom-
modate was established to be 844.6 ± 72.5 g (n = 3). A
cultivated field without prior application history of CPF
was chosen for the study with organic amendments. The
field (well prepared with all weeds and stones carefully
removed) was subdivided into three sections, one for study
on FMC, another for the study on TDL and the third for the
control experiment. FMC was applied at the field appli-
cation rate of 30 t ha-1 giving an equivalent of 8.48 g per
pipe (10.04 mg g-1 of soil). Likewise, TDL was applied at
the field application rate of 5 t ha-1 which translated to
1.44 g per pipe (1.71 mg g-1 of soil). Pipes (one metre
apart) were then driven into the prepared soil with about
4 cm left protruding above the ground to avoid loss of the
applied CPF through surface runoff. Chlorpyrifos S480
(48 % a.i.) (24 lg g-1), corresponding to a field applica-
tion rate of 936 g of CPF a.i. per hectare, was surface-
applied to the soil in the PVC pipes. The pipes were then
filled with soil. Another cultivated field with a prior CPF
application history of 36 months, whose residual CPF and
TCP concentrations were below detection limit, was used
for the study on the effect of repeated application. CPF was
applied as described above. A control experiment for both
treatments was carried out on unamended non-history soil.
A total of 84 pipes were installed and used in the study.
Over the study period of 102 days, weather conditions
were monitored. Mean (±SD) relative humidity and evap-
oration rates were 49.5 % ± 14.6 % and 6.1 ± 1.4 mm
day-1, respectively. A mean (±SD) daily sunshine of
7.2 ± 1.4 h day-1 was recorded and the mean (±SD) wind
run was 119.2 ± 28.9 km day-1. The mean (±SD) air
temperature and soil temperature to a depth of 30 cm were
21.2 ± 1.0 and 26.6 ± 1.1�C, respectively. In the first
2 months of study, the amounts of rainfall were 250.7 and
63.8 mm, respectively, while 2.6 mm were recorded in the
last month.
A laboratory degradation experiment was carried out
using soil with prior CPF application history of 36 months
using 250 mL conical flasks. Moisture content of the soil
was determined gravimetrically. CPF (in acetonitrile) was
added drop-wise at the field application rate of 24 lg g-1
to aliquots of 3.5 g of oven-dried ground soil in flasks.
The flasks were then placed in a fume hood for 24 h for
the solvent to evaporate followed by restoration of the
moisture content to 60 % (gravimetric water content of
soil at 100 % of water holding capacity) by addition of
deionised water. Aliquots of 46.5 ± 0.1 g of dry soil
equivalent were then added to each flask, homogenized
and compacted to a volume of 38.46 mL at a soil density
of 1.3 g cm-3 (Getenga et al. 2009). Flasks were then
loosely covered with perforated aluminium foil. Soil
moisture content was maintained at 60 % during the entire
experimental period. Control set ups consisted of sterile
soils in which sterilization was achieved by addition of
1 % (w/v) sodium azide. Three replicates and a control
were sampled on day 0, 7, 21, 63 and 102 for both
treatments. Field and laboratory experiments were moni-
tored for 102 days.
During sampling, soil in the whole pipe was taken, air
dried and homogenized. After pre-treatment, soil sub-
samples of 50 ± 0.1 g in triplicate were extracted by the
Soxhlet method using 150 mL of methanol for 24 h (Wu
et al. 2011). Extracts were concentrated with a rotary
evaporator to about 2 mL prior to clean-up by solid phase
extraction (SPE). SPE cartridges (Strata C-18 E, 55 lm,
70 A, and 1000 mg/6 mL) mounted on a vacuum operated
phenomenex 24-position manifold set were first pre-con-
ditioned with 10 mL methanol and 10 mL distilled water.
Without allowing the cartridges to dry, extracts dissolved in
250 mL of water were passed through the cartridges and
analytes were eluted with 4 mL methanol. Analysis of CPF
residues was done by Shimadzu prominence LC-20AT high
performance liquid chromatograph equipped with a SPD-
20A prominence UV detector and a phenomenex C18
(250 9 4.60 mm, 5 micron, Luna 5u) column. The mobile
phase comprised 75 % acetonitrile and 25 % of 1 mM
H3PO4 (Mauldin et al. 2006) at a flow rate of
1.5 mL min-1. Aliquots of 10 lL were injected and
388 Bull Environ Contam Toxicol (2015) 94:387–392
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detected at 240 nm wavelength. The limit of detection and
quantification were based on noise to signal ratio of 3 and
10 and gave values of 0.013 ± 0.001 and 0.024 ±
0.017 lg mL-1, respectively. Recovery studies were car-
ried out on soil samples free of the analytes to check the
efficiency of the extraction method adopted. The mean
recovery of CPF was 85.2 % ± 4.5 %. Pesticide degrada-
tion rate and half-life were determined by first-order rate
equation, C = Co-kt. Statistical analysis of data was done
using SPSS 17.0 version software.
Results and Discussion
From the field study on organic amendments, there was a
rapid degradation of CPF up to the 35th day in the three
treatments (FMC-, TDL-amended and unamended soils),
followed by a slower phase thereafter (Fig. 1).
The plateau observed from the 63rd day indicates lack of
extractability or bioavailability of CPF residues as a result of
it being strongly bound to soil particles (Getenga et al. 2004).
It may also be due to reduced microbial activities as a result of
the accumulation of TCP (Racke et al. 1990). At the close of
the experiment, residue levels were 3.4 % ± 2.2 % in the
FMC-amended soil, 5.2 % ± 2.5 % in the TDL-amended
soil and 10.8 % ± 2.1 % (of applied amount) in the control
(unamended) soil. The first order kinetic model gave CPF
half-life values of 21, 24 and 30 days in the FMC- and TDL-
amended soils, and unamended soil, respectively. Degrada-
tion rates (lg g-1 day-1) were 0.033 (R2 = 0.92), 0.029
(R2 = 0.92) and 0.023 (R2 = 0.94) for FMC-, TDL-amen-
ded and unamended soil, respectively.
Degradation of CPF in the three treatments was com-
pared. There was a significant difference (p = 0.035)
between degradation in the FMC-amended and unamended
soil. Likewise, a significant difference (p = 0.040) was
observed between degradation of CPF in TDL-amended
and the unamended soil. Amending soil with FMC and
TDL increases soil organic matter, available N, soil res-
piration, soil aggregate stability, and reduces soil com-
paction leading to increased microbial activities (Cox et al.
2001). This resulted in the observed enhanced degradation
of CPF. Also FMC- and TDL-amended soils may have
upset the C–N ratio which led to utilisation of CPF to
regain the optimum nutrient balance (Lalah et al. 2009).
Soil in the study area had enough C content
(2.8 % ± 0.3 %) to ensure the presence of an active
autochthonous microbial population that could degrade
pesticides (Chowdhury et al. 2008) but insufficient N
(0.14 % ± 0.03 %). Optimal conditions in the study area,
like high soil temperature, neutral pH, relative high amount
of rainfall and well aerated soils favoured microbial growth
and activities as optimal microbial degradation of CPF
occurs between pH values of 6 and 8 and a temperature of
between 25 and 35�C (Liu et al. 2012). Bacterial strains
such as Klebsiella sp. (Ghanem et al. 2007), Bacillus
licheniformis (Zhu et al. 2010) and Pseudomonas putida
(Vijayalakshmi and Usha 2012) utilize CPF as carbon,
phosphorus and energy sources. TDL has a high C–N ratio
(8.2:1) compared to FMC (4.6:1), thus TDL-amended soil
supported less microbial growth compared to FMC-amen-
ded soil due to limited availability of N to microbes
(Tortella et al. 2010). As a result, FMC supported more
microbial population, hence the relatively faster degrada-
tion of CPF in the FMC-amended soil compared to the
TDL-amended soil. Elsewhere, enhanced degradation of
CPF has been reported in soil amended with other organic
materials like coconut husks, peanut shells (Romyen et al.
2007), farm yard manure, mushroom spent compost and
vermicompost (Kadian et al. 2012). Within the NRDB,
enhanced degradation of diuron was reported in soil
amended with FMC and TDL (Jemutai-Kimosop et al.
2012). The shorter half-lives of CPF obtained in amended
soils are consistent with those obtained by Romyen et al.
(2007), where the half-life was 56.8 days in unamended
soil, 46.5 days in soil amended with peanut shells, and
53.3 days in soil amended with rice husks.
Fig. 1 Degradation of CPF
under field conditions in the
three variants at an initial
application rate of 24 lg g-1
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From the field study on the effect of repeated applica-
tion, soils with and without prior CPF exposure showed an
initial fast degradation rate up to the 35th day followed by
a slower rate thereafter. By the 35th day, 94.9 % of the
applied CPF in soil with prior application history had
degraded compared to 75.1 % in the non-history soil. The
slower rate thereafter may have been due to accumulation
of the metabolite TCP, which has antimicrobial effects,
hence slowing down proliferation of CPF-degraders (Racke
et al. 1990). Additionally, CPF is strongly bound to soil
particles. On the 102nd day, CPF residues were
3.3 % ± 1.5 % and 10.8 % ± 2.1 % in soil with and
without application, respectively (Fig. 2).
Degradation of CPF in the two soils was significantly
different (p = 0.034). Half-life values in soil with and
without previous exposure to CPF were 21 and 30 days,
respectively. The degradation rate was 0.034 lg g-1 day-1
in soil with previous exposure compared to 0.023 lg g-1
day-1 in the non-history soil. Enhanced degradation of CPF
in soil with prior application history could be attributed to the
presence of adapted CPF-degraders developed as a result of
long exposure of soil to CPF (Fang et al. 2008). This adap-
tation gives them a competitive advantage over other
microbes as a result of reduced inhibitory effects of CPF on
microbial communities. These opportunistic microbes uti-
lise CPF as carbon and energy source helping them to pro-
liferate substantially (Singh and Walker 2006). Studies have
reported enhanced degradation of CPF due to previous
application of CPF or its analogues (Fang et al. 2008).
Enhanced degradation of CPF in both surface (0–15 cm) and
subsurface (40–50 cm) soils with repeated application of
CPF has also been reported (Sirisha 2006).
The contribution of microbes to the degradation of CPF
in soil was established through a laboratory experiment.
Degradation of CPF in sterile and non-sterile soils is shown
Fig. 3.
Degradation of CPF in the two sets was significantly
different (p = 0.021). By the 21st day, percent residues were
43.8 % ± 1.8 % and 73.8 % ± 2.8 % in non-sterile and
sterile soils, respectively. At the close of the experiment,
residue levels were 5.5 % ± 1.1 % and 64.3 % ± 2.3 % in
the non-sterile and sterile sets, respectively. Half-life values
were 27 days (k = 0.025 lg g-1 day-1) and 161 days
(k = 0.0043 lg g-1 day-1) in non-sterile and sterile soils,
respectively. The DT50 value obtained in the non-sterile soil
is slightly higher than that obtained by Fang et al. (2009)
Fig. 2 Degradation of CPF
under field conditions in soils
with and without prior
application history
Fig. 3 Degradation of CPF
under laboratory conditions in
sterile and non-sterile soil
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which ranged between 14 and 18 days. TCP formation in the
non-sterile set reached a maximum value on the 21st day
(29 % of the applied CPF) and reduced to 20 % by the end of
the experiment. In the sterile set, levels of TCP were 8.6 %
and 10.3 % on the 7th day and 102nd day, respectively.
Studies have reported enhanced degradation of CPF in non-
sterile soils showing that microbes play a significant role in
its degradation (Zhu et al. 2010). Degradation of CPF in
sterile soil is attributable to chemical degradation and some
of the residual microbes that may have resisted sterilization
by sodium azide.
Several CPF-degrading bacterial strains including
Enterobacter strain B-14 (Singh et al. 2004), Stenotroph-
omonas sp. strain YC-1 (Yang et al. 2006), Paracoccus sp.
strain TRP (Xu et al. 2008), Sphingomonas sp. strain Dsp-2
(Li et al. 2007), cyanobacterium, Synechocystis sp. strain
PUPCCC 64 (Singh et al. 2011) have been isolated from
diverse sources. Bacterial strains such as Flavobacterium
sp., Micrococcus sp. (Singh and Walker 2006) and Esch-
erichia coli (Ghanem et al. 2007) degrade CPF through
cometabolism without utilising it as a carbon source. Some
fungal strains such Aspergillus sp., Phanerochaete chry-
sosporium, Pencillium brevicompactum and Trichoderma
harzianum degrade CPF through catabolism (Singh and
Walker 2006). CPF can also be degraded by a bacterial
consortium as reported by Sasikala et al. (2012). Results
showed that organic amendments and repeated exposure of
CPF to soil significantly enhanced its degradation
(p \ 0.05). Therefore, FMC and TDL can be used effec-
tively to mitigate environments contaminated with CPF.
Acknowledgments We acknowledge the financial grant towards
this research by the National Commission of Science, Technology and
Innovation, Kenya (NCST/5/003/3rd CALL MSc/026). We also
acknowledge the contributions of Ms. Selly Kimosop towards the
completion of this study.
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