The Dissipation of Carbofuran in Two Soils with DifferentPesticide Application Histories within Nzoia River DrainageBasin, Kenya

Selly Jemutai-Kimosop • Francis O. Orata •

Isaac O. K’Owino • Zachary M. Getenga

Received: 27 September 2013 / Accepted: 11 February 2014

� Springer Science+Business Media New York 2014

Abstract The dissipation of carbofuran from soils within

the Nzoia River Drainage Basin in Kenya was studied

under real field conditions for 112 days. Results showed

significantly enhanced dissipation of carbofuran with half

life (DT50) values of 8 days (p = 0.038) in soils with prior

exposure to carbofuran compared to 19 days in soils with

no application history. At the end of the experiment, resi-

dues of 2.57 % and 9.36 % of the initial carbofuran applied

were recorded in the two types of soil, respectively. Car-

bofuran metabolites identified in the study were 3-keto

carbofuran and carbofuran phenol with 5.84 % and 15.0 %

remaining in soils with prior exposure, respectively. Soils

with no application history recorded 16.05 % and 12.82 %

of 3-keto carbofuran and carbofuran phenol metabolites,

respectively.

Keywords Pesticides � Persistence � Tropical region �Metabolites

Carbofuran (2,3-dihydro-2,2-dimethylbenzofuran-7-yl

methylcarbamate) is a broad spectrum carbamate insecti-

cide and nematicide that has been used worldwide for

control of pests in sugarcane, sugar beet, rice and coffee

farms (Trabue et al. 2001). In spite of its efficacy in pest

control, carbofuran is acutely toxic and has been linked to

wildlife mortalities in many countries (Gupta 1994; Otieno

et al. 2010). As a result, carbofuran use has been banned in

several countries (Khuntong et al. 2010). However, this

chemical is still imported into Kenya for restrictive use to

control foliar feeding insects in rice paddy fields including

the Bunyala rice irrigation scheme (Mohanty et al. 2009).

Consequently, carbofuran is among the leading surface and

ground water contaminants in Kenya (Otieno et al. 2010).

Its detection in various matrices has raised a lot of envi-

ronmental concerns based on its persistence in the envi-

ronment (Bermudez-Couso et al. 2011).

Dissipation of carbofuran in the environment occurs

through both abiotic and biotic processes (Kim et al. 2004).

Biomineralization of carbofuran results in complete

detoxification of a contaminated environment (Chung

2000). However, pesticides that are newly introduced into

soils are usually resistant to biodegradation by indigenous

soil microflora (Krishna and Philip 2008). Nevertheless,

studies have shown that repeated applications of the same

pesticide or its analogues may result in selective build up of

microbial populations capable of utilizing the compound as

a source of carbon, nitrogen and/or energy (Getenga et al.

2009). For example Trabue et al. (2001) reported acceler-

ated degradation of carbofuran in soils with prior exposure

to the same pesticide. Rozo et al. (2013) recorded increased

microbial population of carbofuran-degrading bacteria in

soils with 8 years of carbofuran application compared to

soils having three or 1 year of carbofuran exposure. A

similar trend was observed by Plangklang and Reungsang

(2013). Most of these studies have been conducted in

temperate regions; however, there are limited studies

detailing the degradation of carbofuran in soils with prior

exposure in tropical regions, particularly the Nzoia River

Drainage Basin (NRDB) where it is frequently used.

These soils have a good potential for enhanced degra-

dation of carbofuran arising from the long exposure to the

chemical (Mohanty et al. 2009). This phenomenon reduces

the level of persistence of carbofuran in the soil and its

S. Jemutai-Kimosop (&) � F. O. Orata �I. O. K’Owino � Z. M. Getenga

Department of Pure and Applied Chemistry, Masinde Muliro

University of Science and Technology,

P.O Box 190-50100, Kakamega, Kenya

e-mail: s_kimosop@yahoo.com

123

Bull Environ Contam Toxicol

DOI 10.1007/s00128-014-1234-5

potential to contaminate water bodies. Therefore, the study

aimed at comparing the dissipation of carbofuran in soils

with different pesticide application histories. This will

provide a baseline data for future studies in bioremediation

of carbofuran insecticide within the study area.

Materials and Methods

High purity ([99 %) pesticide standards; carbofuran and

and 3-keto carbofuran were purchased from Sigma Aldrich

(USA). Carbofuran phenol ([99 % purity) and surrogate

standard, 4-Bromo-3,5-dimethylphenyl-N-methylcarba-

mate (BDMC) of above 99.5 % were purchased from Dr.

Erhenstorfer (Augsburg, Germany). Methanol (high-per-

formance liquid chromatography and analytical grades)

was purchased from Kobian Kenya Limited. All other

chemicals were analytical grade and purchased from BDH

(UK). A LC-20AT Shimadzu liquid chromatograph

equipped with a SPD-10A VP Shimadzu UV–VIS detector

and a 250 9 4.6 mm Shim Pack ODS (VP-2) 5 lm column

were used for HPLC analyses.

This study was conducted in Bunyala rice irrigation

scheme nuclear estates in NRDB within the Lake Victoria

catchment area in Kenya (longitudes 3385603000 to

3481003000E and latitudes 0�3000S to 0�1103000N). Soils in

this area are clay loam with high cation exchange capacity

(37 me %) and acidic (pH = 4.6). They have low nutrient

content with organic carbon of 1.1 % and nitrogen levels of

0.08 %. Some of the chemical parameters of the soils are

presented in Table 1. Dissipation of carbofuran was con-

ducted in two rice fields with different pesticide application

records between the months of January and April, 2011

which was a relatively dry season. Weather conditions

during the experimental period are shown on Table 2. Prior

to field experimental set-up, reconnaissance studies were

done where information concerning the land use was

obtained. In addition, residue analysis was done for both

fields and results showed no quantifiable amounts of car-

bofuran in the fields without carbofuran use. One experi-

ment was set in a field without any history of carbofuran

use and another in a field that had been exposed to car-

bofuran for 4 years with application done once a year. The

two fields were 2 km apart. Dissipation studies were con-

ducted using poly vinyl chloride (PVC) tubes of 6 cm

diameter and 45 cm length (Lalah et al. 2009). The amount

of dry soil that the PVC pipe would accommodate was

established to determine the concentration of the pesticide

to be applied in the soil contained in the pipes. The dry

weight of soil sample per pipe (n = 3) was

1,000.59 ± 72.50 g. A total of 42 pipes were installed in

both fields leaving about 4 cm above the surface. All pipes

were spiked with carbofuran at a concentration of

12.82 lg/g in soil which was calculated from actual field

application rates for pest control. The pipe was then filled

with soil to avoid advection transport processes like loss

through surface run off (Lalah et al. 2009). Samples were

collected immediately after application and thereafter at

the 1st, 7th, 21st, 49th, 81st and 112th days. During each

sampling, the whole pipe was harvested, and samples were

immediately taken to the laboratory, air dried under shade,

homogenised and sieved through a 2 mm sieve.

After pre-treatment, soil sub-samples of 50.0 ± 0.01 g

in triplicate were extracted using three portions of 150 mL

methanol for 24 h on an orbital shaker at 200 revolutions

per minute. Extracts were then concentrated under reduced

pressure at 30�C until dryness. Re-dissolution was done

with 5 mL of methanol and diluted to 250 mL with double

distilled water.

The solution was then percolated through a C18 solid

phase extraction cartridge that had already been condi-

tioned by passing 10 mL methanol and 10 mL double

distilled water successively. The column cartridge was

dried with a vacuum manifold and analytes were eluted

with HPLC grade methanol. Clean extracts were dried over

anhydrous Na2SO4 in a florisil column that had been con-

ditioned with 10 mL of HPLC grade methanol to obtain a

Table 1 Chemical parameters of the two soil-types used in the experiments

Study site OC (%) N (%) P (lg/g) K (Cmol/Kg) Ca (Cmol/kg) Mg (lg/g) Fe (lg/g) Cu (lg/g) Zn (lg/g)

Soil with prior history 0.9 1.00 12.55 0.25 3.00 3.72 38.02 1.02 1.33

Soils with no history 1.2 0.06 13.20 0.45 2.90 3.75 38.98 0.86 1.32

Average 1.1 0.08 12.90 0.35 2.95 3.74 38.50 0.94 1.31

Table 2 Summary of weather data at the experimental site between

January and April, 2011

Parameter Jan Feb Mar Apr Avg.

Mean daily soil temp,

0–20 cm (�C)

27.05 28.15 26.98 26.52 27.18

Mean wind speed

(Km/h)

131.7 131.7 116.6 112.4 123.1

Evaporation rate (mm/

day)

7.52 7.78 5.63 4.90 6.46

Relative Humidity (%) 42.5 35 50.5 53 45.3

Sunshine (h/day) 8.5 7.5 6.8 7.0 7.5

Bull Environ Contam Toxicol

123

final volume of 10 mL which was filtered through a

0.45 lm nylon membrane prior to HPLC analysis. An

aliquot of 10 lL of sample was injected into the HPLC.

Carbofuran and its metabolites were detected at 280 nm

wavelength. Isocratic elution was performed with a mobile

phase consisting of 4:1 v/v HPLC grade acetonitrile and

double distilled water at a flow rate of 1 mL/min (Kim

et al. 2004).

Method performance was checked using samples spiked

with 0.01–2.5 lg/mL of 4-Bromo-3,5-dimethylphenyl-N-

methylcarbamate (BDMC) as a surrogate standard. Ana-

lytical curves and the related linear regression data indi-

cated a linear response of the UV detector for all standards

analysed with correlation coefficients (r2) above 0.99.

Selectivity of the method was verified by comparison of the

chromatograms obtained from samples spiked with known

concentration of standards and those of samples free of

carbofuran pesticide standards. Blank samples did not

present peaks at retention time of the standards. Moreover,

the chromatograms of the standards presented satisfactory

chromatographic resolution. Pesticides eluted at 3.8, 6.3,

8.4 and 9.4 min for carbofuran, carbofuran phenol, 3-keto

carbofuran and BDMC, respectively. Recovery experi-

ments were performed by spiking pristine soil samples with

concentration of 0.5, 1 and 2.5 lg/mL of the target com-

pounds and then run through the analytical procedure as a

check for matrix effects. Good recoveries were obtained

ranging between 83.4 ± 0.1 % and 95.6 ± 0.1 %. Exter-

nal calibration standards were prepared in methanol at

concentration ranges of up to 2.5 lg/mL. A midpoint cal-

ibration standard was injected into the instrument after

every ten samples, throughout the analysis, to check for

instrument response and drift. Repeatability of results was

evaluated weekly, where a sample of known concentration

was run through the whole analytical procedure. Sequential

extraction experiments were performed in order to evaluate

whether the method’s extraction procedure was capable of

completely removing the analytes from the environmental

matrices. The limit of detection (LoD) was taken as the

injected amount that resulted in a peak with a height at

least three times as high as the baseline noise level. The

limit of quantification (LoQ) of target chemicals was

evaluated for each sample based on the average blank

concentrations plus five times its standard deviation of ten

blanks. This value was then verified by the concentration in

a standard that yielded a standard to noise (S/N) ratio of

10:1. Pesticide dissipation half-life values were determined

by regression using first-order rate equation (Krutz et al.

2007). Data was analyzed using ANOVA to evaluate the

difference between various treatments. Separation of

means was done by paired t test at 95 % confidence limit

(Bermudez-Couso et al. 2011).

Results and Discussion

There was rapid dissipation within the first day of appli-

cation in both fields with 35.27 % and 38.62 % loss in soils

with prior exposure to carbofuran and soils without history

of carbofuran use, respectively (Fig. 1). At this time of

application, the chemical is loosely held to the soil and loss

through hydrolysis is possible. This is accelerated by the

low soil pH (4.6) and high tropical temperatures. Rapid

dissipation of carbofuran in rice field soil could also be due

to the low air–water partition coefficient, Kaw of

1.23 9 10-7 which indicates that carbofuran preferably

dissolves in aqueous phase (de Melo Plese et al. 2005).

However, volatilization of carbofuran was considered

minimal due to the low vapour pressure of 4.8 9 10-6

mmHg (Bermudez-Couso et al. 2011). Seven days after

application of carbofuran, 65.45 % loss was observed in

soils with prior carbofuran exposure compared to 48.01 %

Fig. 1 The dissipation of

carbofuran from two soils

differing in application history

in Bunyala rice paddy fields

Bull Environ Contam Toxicol

123

loss in soils with no application history. A slower rate of

carbofuran dissipation was observed after 21 days, reach-

ing a plateau 65 days after application. This may be

attributed to strong adsorption of carbofuran to soil organic

matter (Koc of 209) and bonding with ionic sites of silicate

clay (Krishna and Philip 2008; Bermudez-Couso et al.

2011). Formation of bound residues is also possible,

making the pesticide non-available to degrading microbes

resulting in slower dissipation rates (Trabue et al. 2001).

The low substrate concentration may also lead to reduction

of dissipation rates. The characteristic bi-phasic carbofuran

dissipation pattern is consistent with other findings (Lalah

et al. 2009). At the end of the experiment, 97.43 % of the

initial carbofuran applied had been lost in the field with

prior exposure to carbofuran, whereas 90.64 % of the

chemical was lost in soils with no history of carbofuran use

as shown in Fig. 1.

Dissipation of carbofuran was significantly enhanced

(p = 0.038) in soils with prior carbofuran history with

half-life (DT50) value of 8 days compared to 19 days in

soils with no application history. Repeated application of

carbofuran to the field may have resulted in an increase in

microbial communities capable of degrading the chemical

(Trabue et al. 2001). Adaptation of microbes to metabolize

carbofuran is also possible in soil continually exposed to

the pesticide probably due to horizontal gene transfer

(Khuntong et al. 2010). These results are in accordance

with other findings reporting enhanced carbofuran in soils

with prior history (Mohanta et al. 2012; Mujeeb et al.

2012). A study by Khuntong et al. (2010) reported carbo-

furan dissipation half-life of 8.9 days in a rice field, while

Campbell et al. (2004) reported half-life value of 40 days

in sand soils. Moderate persistence of carbofuran has been

reported in tropical soils with no prior exposure to the

chemical with dissipation half-life values ranging between

66 and 115.5 days (Lalah et al. 2001).

Several carbofuran-degrading microorganisms have

been isolated in soils with prior exposure to the chemical.

A study by Rozo et al. (2013) reported 48 isolates capable

of degrading carbofuran in soil with 8 years of carbofuran

application in contrast to 21 and 12 isolates from soils with

3 and 1 year of exposure of the insecticide, respectively.

Rozo et al. (2013) reported an increase in population of

bacterial communities and diversity of the isolates arising

from adaptation of the microorganisms to mineralize or

catabolize carbofuran after repeated application. Chung

(2000) reported accelerated degradation and alteration of

carbofuran degradative pathway in soils with prior history

of use. Some of the carbofuran-degrading bacteria isolated

from soils with prior history belong to the genera Pseu-

domonas, Verinia, Flavobacterium, Flexibacterium,

Enterobacter (Chaudhry and Ali 1988; Mohanta et al.

2012; Plangklang and Reungsang 2013). Slaoui et al.

(2007) isolated a fungus capable of degrading carbofuran

which belonged to the genus Gliocladium. Another study

by Omolo et al. (2012) reported carbofuran-degrading

bacteria belonging to Pseudomonas and Alcaligenes spe-

cies in soils exposed to carbofuran in other parts of Kenya.

Therefore, the soil in this study may have carbofuran-

adapted microbes which were not isolated.

Two carbofuran metabolites were identified in this

study. Their proportion in relation to the applied carbofuran

is presented in Fig. 1. The appearance of 10.75 % of 3-keto

carbofuran in soils with prior history within the first day of

application and decreased after 35 days indicates the pos-

sibility of carbofuran degradation by oxidative pathway

(Chung 2000). Accumulation of carbofuran phenol during

the entire experimental period indicates the possible deg-

radation by hydrolytic pathway (Chung 2000). Therefore, it

appears that the degradation of carbofuran in soils from

Bunyala study site occurs through both microbial oxidation

of the furan ring to form 3-keto carbofuran and hydrolytic

cleavage to form carbofuran phenol (Kim et al. 2004). In a

study by Mujeeb et al. (2012), 3-keto carbofuran was found

as the major metabolite and other metabolites such as

carbofuran phenol and 3-hydroxycarbofuran were also

reported. Chung (2000) reported carbofuran degradation

through both hydrolysis and oxidation to form 3-hydrox-

ycarbofuran and carbofuran phenol, respectively. Oxida-

tion of the latter gives 3-ketocarbofuran which is further

hydrolyzed to 3-hydroxycarbofuran phenol and 3-keto-

carbofuran phenol (Chung 2000). Similarly, Kim et al.

(2004) reported carbofuran phenol metabolite in soils

treated with carbofuran.

Results showed significantly enhanced dissipation

(p = 0.038) of carbofuran in soils within Bunyala study site

in soils with prior history of the pesticide use with DT50 value

of 8 days compared to 19 days in soils with no application

history. These results suggest that repeated application of

carbofuran may have stimulated the selection of microbial

communities with the capacity to efficiently degrade the

pesticide at a fast rate, significantly reducing the deleterious

effect by the chemical. Adapted microbes can therefore be

isolated from these soils as an alternative to control the

impact of carbofuran in the environment.

Acknowledgments We appreciate the research grant received from

the National Council for Science and Technology of Kenya (NCST/5/

003/CALL 2/137 and NCST/5/003/2nd CALL M.Sc/19), the schol-

arship by the German Academic Exchange Service (DAAD) and

Agnes Muyale for assistance in analysis.

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