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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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