ral
ssBioMed Cent
Malaria Journal
Open Acce
Research
Screening of pesticide residues in soil and water samples from
agricultural settings
Martin C Akogbéto*
1
, Rousseau F Djouaka
1
and Dorothée A Kindé-Gazard
2
Address:
1
Centre de Recherche Entomologique de Cotonou, 06 BP 2604, Bénin and
2
Faculté des Sciences de la Santé, Université d'Abomey-Calavi,
Bénin
Email: Martin C Akogbéto* - akogbeto@leland.bj; Rousseau F Djouaka - rousseaudj@yahoo.com; Dorothée A Kindé-
Gazard - kindegazard@yahoo.fr
* Corresponding author
Abstract
Background: The role of agricultural practices in the selection of insecticide resistance in malaria
vectors has so far been hypothesized without clear evidence. Many mosquito species, Anopheles
gambiae in particular, lay their eggs in breeding sites located around agricultural settings. There is
a probability that, as a result of farming activities, insecticide residues may be found in soil and
water, where they exercise a selection pressure on the larval stage of various populations of
mosquitoes. To confirm this hypothesis, a study was conducted in the Republic of Benin to assess
the environmental hazards which can be generated from massive use of pesticides in agricultural
settings.
Methods: Lacking an HPLC machine for direct quantification of insecticide residues in samples,
this investigation was performed using indirect bioassays focussed on the study of factors inhibiting
the normal growth of mosquito larvae in breeding sites. The speed of development was monitored
as well as the yield of rearing An. gambiae larvae in breeding sites reconstituted with water and soil
samples collected in agricultural areas known to be under pesticide pressure. Two strains of An.
gambiae were used in this indirect bioassay: the pyrethroid-susceptible Kisumu strain and the
resistant Ladji strain. The key approach in this methodology is based on comparison of the growth
of larvae in test and in control breeding sites, the test samples having been collected from two
vegetable farms.
Results: Results obtained clearly show the presence of inhibiting factors on test samples. A normal
growth of larvae was observed in control samples. In breeding sites simulated by using a few grams
of soil samples from the two vegetable farms under constant insecticide treatments (test samples),
a poor hatching rate of Anopheles eggs coupled with a retarded growth of larvae and a low yield of
adult mosquitoes from hatched eggs, was noticed.
Conclusion: Toxic factors inhibiting the hatching of anopheles eggs and the growth of larvae are
probably pesticide residues from agricultural practices. Samples used during this indirect assay have
been stored in the laboratory and will be analysed with HPLC techniques to confirm hypothesis of
this study and to identify the various end products found in soil and water samples from agricultural
settings under pesticide pressure.
Published: 24 March 2006
Malaria Journal 2006, 5:22 doi:10.1186/1475-2875-5-22
Received: 09 August 2005
Accepted: 24 March 2006
This article is available from: http://www.malariajournal.com/content/5/1/22
© 2006 Akogbéto et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 9
(page number not for citation purposes)

Malaria Journal 2006, 5:22 http://www.malariajournal.com/content/5/1/22
Background
The resistance of vectors to insecticides is a real handicap
to the use of insecticide-treated materials. The recurrent
presence on the agenda of most entomological research in
Africa of vector resistance, more specifically of Anopheles
gambiae resistance to insecticides, is due to the fact that
insecticide-treated materials remain the principal tool of
National Programmes of Malaria Control (NPMC) in the
fight against vectors. The first cases of resistance were
mentioned in the 1950s and 1960s with An. gambiae to
organochlorine. Then, the phenomenon was limited to
dieldrin and hexachlorocyclohexane (HCH) [1]. In Africa,
the first cases of dieldrin resistance in An. gambiae were
recorded in Burkina Faso in 1960 [2]. Ten years later, the
identification of dieldrin resistance and cases of DDT
resistance were reported in Togo, Senegal, Nigeria, Cam-
eroon and Guinea [3]. There is no relation between DDT
resistance and dieldrin resistance.
In the case of pyrethroids, resistance is mentioned rela-
tively late, in the 1990s. The first cases of pyrethroid resist-
ance were recorded in Côte d'Ivoire [4] and many other
cases have been described in Kenya [5], Benin [6,7],
Burkina Faso [7,8], Côte d'Ivoire [7] and Mali [9]. In cen-
tral Africa, cases of pyrethroid resistance have been
described in Cameroon and in the Central African Repub-
lic [10].
Despite the lack of concrete evidence, the use of insecti-
cides in households and of pesticides in agricultural set-
tings has been highlighted as a key factor contributing to
the emergence of vector resistance. Some believe that
resistance probably arose from the use of insecticide aero-
sols in households and some plants used for fumigation
over a long period in rural and urban areas. In Benin
Republic, Akogbeto and Yakoubou [6] suspect the emer-
gence of DDT resistance, recorded in An. gambiae from
meridian regions, to be related to two phenomena: (i) the
massive use of DDT and dieldrin for house-spraying
applications in southern villages from 1953 to 1960 dur-
ing WHO programmes of malaria eradication [11] and
(ii) the massive use of organochlorine in agricultural set-
tings during the 1950s [3]. However, the absence of con-
clusive data regarding the implication of house-spraying
in the selection of resistance seems to corroborate the
observation of Mouchet [12], that no case of resistance
has been recorded following the DDT house-spraying pro-
grammes performed over a period of 10 years in Madagas-
car, Thailand and South America.
Others believe that the emergence of resistance results
from massive use of insecticides against pests in agricul-
tural plantations. Recent studies conducted by Diabate et
constantly subjected to insecticide treatments, as com-
pared to the low frequency of kdr recorded in rural areas
where farmers are restricted to food crops for local con-
sumption with no pesticides. In Côte d'Ivoire, the kdr
mutation identified in resistant strains of An. gambiae was
probably selected as a result of the massive use of DDT
and pyrethroids against pests in cotton fields [7,8]. The
hypothesis of a relation between some agricultural prac-
tices and the emergence of resistance should not be
neglected. In Benin, insecticide treatments against pests in
cotton plantations are done twice each month, for an
average of three months (between July and October) each
year. These treatment periods coincide with the rainy sea-
sons and correspond to the period of high mosquito den-
sities and increased development of Anopheles larvae. In
vegetable farms, treatments are more regular and are done
throughout the year. Pesticide treatments release active
components into the environment of which some get
directly into the breeding sites of mosquitoes. There is a
high probability that insecticide residues can be found on
soil in agricultural areas and could exercise a selection
pressure on the larval stage on some populations of mos-
quitoes. To confirm this hypothesis, a study was carried
out to assess environmental hazards related to the use of
pesticides in agricultural settings. The study is a biological
evaluation to screen residual insecticides on soil and
water samples from vegetable farms subjected to pesticide
treatments.
Materials and methods
Study area
This study was conducted in Benin Republic, West Africa.
Two "test sites" and a "control site" were investigated. The
two test sites were vegetable farms (Houeyiho and Para-
kou). The vegetable area of Houeyiho is a big farm of 14
hectares in the town of Cotonou. In Houeyiho, more than
300 farmers are involved in the cultivation of a large vari-
ety of vegetables: cabbages, carrots, lettuces, amaranth,
cucumber etc. Farming at Houeyiho is associated with the
use of insecticides to fight pests. The Parakou vegetable
farm is located in the town of Parakou. This farm has sim-
ilar characteristics to that in Houeyiho, but it is smaller in
size and is less well-maintained. The control site selected
for this study was the backyard of the Centre for Research
in Entomology, Cotonou (CREC) located at Akpakpa, a
peripheral locality of Cotonou. This site has a watering
pool and its soil texture resembles that of the test sites. The
main difference between the control site and the two test
sites is the absence of insecticide pressure at the CREC
premises. A recent study conducted in the test sites (Akog-
béto et al, in press) confirmed the reality of the use of
insecticides in these agricultural settings. Pyrethroids, and
more specifically deltamethrin and cyfluthrin, are fre-Page 2 of 9
(page number not for citation purposes)
al [13] highlight the elevated levels of resistance genes,
kdr, in An. gambiae collected in cotton-growing areas and
quently used in the vegetable farms of Parakou and
Houeyiho.

Malaria Journal 2006, 5:22 http://www.malariajournal.com/content/5/1/22
Determination of levels of resistance of Anopheles
populations from the 2 study sites
Prior to the evaluation of insecticide residues, the suscep-
tibility to insecticides of An. gambiae samples from the two
test sites was determined. The test was based on WHO
standard protocols with two pyrethroids (permethrin 0,
75% and deltamethrin 0, 05%) and one organochlorine
(DDT 4%). The use of pyrethroids and organochlorine in
this test aimed to verify the presence of cross-resistance
between the two families of insecticides. Mosquitoes
exposed to insecticide papers in this test were three- to
five-day old females of Anopheles, having emerged from
larvae collected on the premises and in water spots found
in the vegetable plantations of Houeyiho and Parakou.
After exposure, dead and live Anopheles were separately
kept on silica gel for further analysis of molecular forms
and the identification of resistance genes (kdr). Total DNA
extraction was conducted using modified techniques of
Collins et al [14]. Amplification of DNA fragments were
based on PCR techniques using appropriate primers, as
described by Scott et al [15], for the identification of spe-
cies of the An. gambiae complex, that of Favia et al [16] for
molecular M/S forms, and that of Martinez Torres et al
[17] for kdr mutations. Amplified fragments were
migrated in electrophoresis tanks and bands were visual-
ized under UV lights
Protocol for biological evaluation of the presence of
insecticide residues in soil and water samples from selected
sites
Not having an HPLC machine for direct quantification of
insecticide residues in collected samples, the protocol pre-
sented does not enable an identification and a quantifica-
tion of insecticide residues in analysed samples. An
indirect bioassay focussed on the study of factors capable
of inhibiting the normal growth of mosquito larvae in
breeding sites. Developmental speed and the yield of rear-
ing An. gambiae larvae in breeding sites reconstituted with
water and soil samples collected in agricultural areas
under pesticide pressure, were monitored. Two strains of
An. gambiae were used in this indirect bioassay: the pyre-
throid-susceptible Kisumu strain and the resistant Ladji
strain. An. gambiae Ladji was selected from the locality of
Ladji at 5 km from Cotonou. The Anopheles population
was purely "M" form, the level of susceptibility to per-
methrin and DDT was respectively 65% and 50%. It was
in homozygous form, with an allelic frequency for the kdr
mutations of one [6,7].
The protocol used in this evaluation is mainly based on
comparison of the growth of larvae in test breeding sites
(water and soil samples from agricultural settings under
pesticide pressure) and in control breeding sites (water
field were taken to the insectary where they were used for
various simulations. Samples from Houyeiho and Para-
kou underwent series of simulations. The first set of breed-
ing sites were reconstituted with top soil from vegetable
farms mixed with water from control sites or mixed with
watering water collected in the farm. The second set of
breeding sites were made with soil collected in watering
pools found in both farms (Houeyiho and Parakou) and
used for the irrigation of vegetables. The control site
selected for this study is the backyard of the Centre for
Research in Entomology, Cotonou (CREC). This site has a
watering pool and soil and water samples collected from
CREC were closely similar to collections from study sites,
the only difference being the absence of insecticide pres-
sure at the CREC. Water from CREC was designated as
CREC-water, whereas soil samples were designated as
CREC-soil. The volume of water used in the reconstitution
of breeding sites was 1,000 ml. For breeding sites made
with a mixture of soil and water samples, 100 g of soil is
well mixed in 1,000 ml of water.
An average of 200 eggs of the susceptible Kisumu strain
was inoculated in each artificial breeding site. A similar
inoculation was repeated with the resistant Ladji strain
and for the different types of artificial breeding sites sim-
ulated. More than 4,000 eggs were inoculated and moni-
tored during this biological evaluation. Monitoring of the
growth of inoculated eggs led to assessing variations in
hatching rates of eggs, speeds of development of larvae
and yields of rearing larvae to adult mosquitoes with each
strain of mosquito and each type of artificial breeding site.
Data from breeding sites suspected to be contaminated
with insecticide residues were compared with those from
control sites. This comparison led to the confirmation of
the potential presence of factors inhibiting the develop-
ment of larvae. During this follow-up experiment, larvae
in all artificial breeding sites were fed with similar quan-
tity and type of food (well-ground cat biscuits mixed with
yeast powder).
Results
Level of susceptibility of Anopheles populations from the
three selected sites
A total of 775 females of An. gambiae were exposed to var-
ious types of impregnated papers for their levels of suscep-
tibility prior to biological evaluations of pesticides.
Results obtained were in line with past data published by
Akogbeto and Yakoubou [6] and Akogbeto et al [18] in the
same localities. Data recorded highlight elevated resist-
ance to permethrin at 0.75% and DDT at 4% in the local-
ity of Houéyiho (Table 1). In the vegetable farm of
Parakou, Anopheles species collected were susceptible to
pyrethroids despite the use of insecticides in the localityPage 3 of 9
(page number not for citation purposes)
and soil samples from similar areas but not under pesti-
cide pressure). Soil and water samples collected in the
against pests. These results could be explained by abun-
dance of populations of An. arabiensis (87.5%) in the area.

Malaria Journal 2006, 5:22 http://www.malariajournal.com/content/5/1/22
However, a reduced susceptibility to DDT was recorded at
4% (mortality rate = 90%) with An. gambiae from Para-
kou. In general, susceptibility tests conducted in this study
were confirmed with PCR analysis performed in the two
sites. High frequencies of kdr mutations were recorded at
Houéyiho (90%) and low frequencies at Parakou
10%)(Table 1).
Impact of agricultural treatments with insecticides on
hatching rates of An. gambiae eggs
The CREC-water taken alone offers favourable conditions
for hatching of An. gambiae eggs. When few grams of soil
samples from agricultural areas under insecticide treat-
ments are added into it, a decrease in hatching rates is
observed. The mean hatching rate recorded in control
breeding sites is 74.5% (n = 200) for the susceptible Kis-
umu strain and 86% (n = 150) for the resistant Ladji
strain. With breeding sites reconstituted with soil and
water samples from the vegetable farm of Houeyiho, a sig-
nificant drop in hatching rates was recorded with both
strains: 33.4% (n = 400) and 36.7% (n = 350), for Kisumu
and Ladji respectively (P-Kisumu = 0.00, X
2
= 89.9, df = 1:
P-Ladji = 0.00, X
2
= 102.69, df = 1). A similar observation
was made with breeding sites from the Parakou vegetable
farm. Here, the hatching rate of the Kisumu strain
dropped from 68.7% (n = 200) in the corresponding con-
trol sample to 45.9% (n = 200) in the test sample (P =
0.00005, X
2
= 20.6, df = 1). However, with the resistant
Ladji strain, similar hatching rates were recorded with
control samples (62.2%: n = 200) and samples suspected
to contain insecticide residues (63.4%: n = 200), (P =
0.75, X
2
= 0.1, df = 1) (Figure 1).
In other simulations, the decrease in hatching rate was
spectacular. In Houeyiho, for example, artificial breeding
sites formulated with top soil and water from watering
pools gave hatching rates as low as 13.2% with the Kis-
umu strain and 8.5% with the Ladji strain compared to
Impact of agricultural treatments with insecticides on
development of An. gambiae larvae
The development of larvae was estimated by quantifying
the proportion of first instars larvae reaching the pupae
stage. This is the best indicator to monitor when evaluat-
ing factors inhibiting larval growth. Contrary to the first
indicator, which is based on fast hatching process of eggs,
this second indicator takes into consideration the devel-
opmental cycle of larvae from first instars to pupae. This
cycle is relatively long and involves many interactions
between larvae and constituents of breeding sites which
allows, therefore, a good follow-up of expressions of
inhibiting factors in simulated breeding sites. In control
breeding sites, 84.7 to 99.3% of first instars larvae of the
Kisumu strain were able to reach pupae stage, giving a
mean of 97.2%. Similar figures were also recorded with
the resistant Ladji strain in controls (mean of 92.3%)(Fig-
ure 2). In breeding sites constituted of samples from agri-
cultural settings under insecticide pressure, breeding of
larvae gave very low rates of first instars larvae reaching
the pupal stage: 13.2% (n = 53) was recorded with Kis-
umu strains in breeding sites made from combinations of
top soil and water from watering pools of the Houeyiho
vegetable farm. With the same strain, 42.1% of larvae
reaching the pupal stage were recorded in breeding sites
from mixtures of top soil from Houeyiho and CREC-
water. With the Ladji strain, although the results obtained
in test samples were low compared to controls (92.3%), a
higher rate of Ladji larvae reaching pupal stage was
recorded with the two simulations: 53.3% (n = 30) and
60.8% (n = 125), respectively (Figure 2a). Cumulative
analysis of results recorded with the development of both
strains of An. gambiae on artificial breeding sites from agri-
cultural settings showed that Ladji larvae were less suscep-
tible to presumed toxic breeding sites (75% reaching the
pupae stage) compared to 53.9% of Kisumu larvae (P =
0.0064 X
2
= 7.41, df = 1). Similar observations were made
with samples from vegetable areas of Parakou. Simula-
Table 1: Susceptibility tests and PCR analysis on Anopheles populations from vegetable areas of Houeyiho and Parakou
Laboratory processing Houeyiho Parakou
Perm.0.75% Delta 0.05 DDT 4% Perm.0.75% Delta 0.05 DDT 4%
Mosquitoes exposed to insecticides 120 100 130 80 80 80
% Mortality 70 94 70 96 100 90
Mosquitoes tested for PCR 3 --80--
An. gambiae s.s. 0 1
An. arabiensis --70--
Freq. M 100 - - 40 - -
S 6
Freq. Kdr 90--10--Page 4 of 9
(page number not for citation purposes)
74.5% and 86% in control breeding sites, with both
strains respectively (Figure 1a).
tions from Parakou produced a relatively low develop-
ment of larvae in presumed toxic breeding sites (55.3%

Malaria Journal 2006, 5:22 http://www.malariajournal.com/content/5/1/22
Page 5 of 9
(page number not for citation purposes)
Hatching rates of eggs of 2 strains of An. gambiae in simulated breeding sites using samples of soil substrates and water from vegetable ar as of Houeyiho and ParakouFi ure 1
Hatching rates of eggs of 2 strains of An. gambiae in simulated breeding sites using samples of soil substrates
and water from vegetable areas of Houeyiho and Parakou. 1. Control: CREC-water. 2. Superficial soil from ridges in
vegetable areas + CREC- water. 3. Superficial soil in between ridges in vegetable areas + CREC- water. 4. Soil from watering
pools + Water from watering pools. 5. Soil from watering pools + CREC-water. 6. Water from watering pools alone. 7. Big
water pool found exclusively at Parakou farm. n : number of eggs inoculated in each simultation.
Fig.1a (Houeyiho)
0
10
20
30
40
50
60
70
80
90
100
12345
Simulations
%
H
a
t
c
h
e
d
e
g
g
s
An.gambiae Kisumu
An.gambiae Ladji
n=200
n=150
n=400
n=350
n=400
n=350
n=400
n=350
n=400
n=350
Fig. 1b (Parakou)
0
10
20
30
40
50
60
70
80
90
100
1234567
Simulations
%
H
a
t
c
h
e
d
e
g
g
s
An.gambiae Kisumu
An.gambiae Ladji
n=200
n=200
n=200
n=200
n=200
n=200
n=200
n=200
n=200
n=200
n=200
n=200
n=200
n=200

Malaria Journal 2006, 5:22 http://www.malariajournal.com/content/5/1/22
Page 6 of 9
(page number not for citation purposes)
Rate of 1st instars larvae getting to pupae stages in simulated breeding sites using samples of soil substrates and water from veg table areas of Houeyiho a d ParakouFi ure 2
Rate of 1st instars larvae getting to pupae stages in simulated breeding sites using samples of soil substrates and water from
vegetable areas of Houeyiho and Parakou.
Fig. 2a (Houeyiho)
0
10
20
30
40
50
60
70
80
90
100
12345
Similar Simulations as in Fig1
%
1
s
t
I
n
s
t
a
r
s
l
a
r
v
a
e
g
e
t
t
i
n
g
t
o
p
u
p
a
e
An.gambi aeKi sumu
An.gambi ae Ladj i
n=143
n=19
n=53
n=223
n=240
n=91
n=125
n=30
n=249
n=110
Fig.2b (Parakou)
0
10
20
30
40
50
60
70
80
90
100
1234567
Similar simulations as in Fig1
%
1
s
t
i
n
s
t
a
r
s
l
a
r
v
a
e
g
e
t
t
i
n
g
t
o
p
u
p
a
e
An.gambiaeKisumu
An.gambi ae Ladj i
n=131
n=125
n=107
n=65
n=109
n=71
n=74
n=117
n=142
n=149
n=71
n=119
n=118
n=162

Malaria Journal 2006, 5:22 http://www.malariajournal.com/content/5/1/22
for Kisumu strain: n = 92 and 87.2% for Ladji strain: n =
117) compared to controls (75.5%: n = 138 for Kisumu
and 83% n = 125 for Ladji) (P-kisumu = 0.0015, X
2
= 9.9,
df = 1: P-ladji = 0.38, X
2
= 0.76, df = 1)(Figure 2).
Impact of agricultural treatments with insecticide on the
yield of rearing An. gambiae
This yield expresses proportions of An. gambiae eggs reach-
ing adult stage. This is also a good indicator to assess tox-
icity of breeding sites in which eggs were inoculated.
Results globally obtained with this indicator were not
spectacular, with low figures obtained even with controls:
45% with the Kisumu strain and 48.5% with the Ladji
strain. When eggs were inoculated into artificial breeding
sites from agricultural areas, the yield of adult mosquitoes
was reduced by half. Comparisons made with both strains
throughout the experiment showed that the Kisumu strain
faced more difficulties in simulated breeding sites than
the Ladji strain. The growth of the Ladji strain was less
affected by simulations than the Kisumu strain (Figure 3).
When eggs of both strains were grown in artificial breed-
ing sites reconstituted with soil and water samples from
the two agricultural areas selected in this study, the yield
of resistant strains was always high compared to suscepti-
ble strains: 31.8% (n = 350) versus 24% (n = 400) in
Houeyiho (P = 0.01, X
2
= 5.56, df = 1)(Figure 3a) and
62.6% (n = 200) versus 24.1% (n = 200) in Parakou (P =
0.00000, X
2
= 69.39, df = 1)(Figure 3b).
Discussion and conclusion
The results of this study suggest that samples from agricul-
tural settings under pesticide pressure contain inhibitory
factors responsible for the reduced growth rate in larvae of
Anopheles Kisumu, with a lesser inhibitory effect on the
development of the resistant Ladji strain.
Parameters targeted during this study have been analysed
and interpreted separately according to their respective
values. Because of the short duration of hatching process
with Anopheles eggs, the hatching rate should be consid-
ered as a simple signal of toxicity of samples from agricul-
tural settings. The rapid hatching of eggs when introduced
in simulated breeding sites does not give room for expres-
sion of inhibitory factors on embryos. This hypothesis
probably explains similarities in data recorded in Para-
kou, where hatching rates of the resistant Ladji strain did
not appear to be influenced by artificial breeding sites
generated with water from watering pools of Parakou and
those from top soil mixed with CREC-water (63.4% and
62.2% respectively for the two sets of artificial sites).
Anopheles
strain had probably developed over time some capacity to
withstand low levels of toxicity. This assumption could
explain the low inhibitory impact of breeding sites on
hatching of Anopheles Ladji eggs recorded in this study.
As observed with hatching rates, larval development also
varies with respect to strains of Anopheles inoculated and
types of artificial breeding sites simulated. In the vegetable
farm of Houeyiho, breeding sites simulated with top soil
collected around vegetables seemed to inhibit larval
growth more than simulations with watering water and
soil from watering pools. In these two simulations, inhib-
itory effects are less spectacular. In breeding sites gener-
ated with water mixed with soil from watering pools,
84.7% of Anopheles Kisumu eggs were able to reach the
pupal stage, whereas, in simulations with water mixed
with soil collected around vegetables, only 13.2% of lar-
vae were able to reach the pupal stage. This consistent dif-
ference in results suggests an unequal distribution of
insecticide residues after treatment in vegetable farms of
Houeyiho. A similar trend was recorded with data from
vegetable areas of Parakou. However, in Parakou, simula-
tions using irrigation water collected from cultivated areas
seem more toxic compared to other simulations. A recent
study conducted by Akogbeto et al [18] reveals that several
chemicals are used in the vegetable farms of Houeyiho
and Parakou against field pests. These chemicals are
mainly pyrethroids, organophosphates and carbamates.
These compounds are used as single formulations or as
combinations of two or three insecticides of different fam-
ilies, the final aim being to generate a synergy of insecti-
cides for a better pest management. After pesticide
treatments in agricultural settings, residues of insecticides
get into mosquito breeding sites. These residues have
lethal effects on larvae of some populations of mosquito
whereas they exert a selective pressure on other popula-
tions, leading to a gradual tolerance of insecticide concen-
trations and to the emergence of resistant populations.
Insecticides used in public health against disease vectors
are similar to those used for years in agriculture. In Benin
Republic, pyrethroids were introduced in agriculture in
the 1970s and, after 30 years of continuous use, cases of
resistance may be found in some populations of insects.
With regard to the origins of vector resistance identified in
rural and urban areas, various diverging hypothesis are
put forward. Some authors incriminate pesticides used in
cotton farms and rice fields as the main source of selection
of resistance in several species of mosquito in rural envi-
ronments (Georghiou [19] in Central America, Asia and
Africa; Chandre et al. [7] and N'guessan et al. [20] in Côte
d'Ivoire; Diabate et al. [15] in Burkina Faso). The impor-
tant movement of young people from villages to towns, asPage 7 of 9
(page number not for citation purposes)
Ladji is a strain selected from a relatively polluted locality,
the locality of Ladji in peripheral region of Cotonou. This
a result of unemployement, has led to the development of
agricultural spaces within urban areas, where vegetables

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Yield of rearing to adults of An. gambiae eggs in simulated breeding sites using samples of soil substrates and water from vege-tab e areas of Houeyiho and ParakouF gure 3
Yield of rearing to adults of An. gambiae eggs in simulated breeding sites using samples of soil substrates and water from vege-
table areas of Houeyiho and Parakou.
Fig.3b (Parakou)
0
10
20
30
40
50
60
70
1234567
Similar Simulations as in Fig1
%
o
f
e
m
e
r
g
i
n
g
a
d
u
l
t
s
f
r
o
m
e
g
g
s
An. gambi ae Ki sumu
An. gambi ae Ladj i
n= 200
n= 200
n= 200
n= 200
n= 200
n= 117
n= 200
n= 200
n= 142
n= 149
n= 71
n= 119
n= 118
n= 162
Fig.3a (Houeyiho)
0
10
20
30
40
50
60
70
80
90
100
12345
Similar Simulations as in Fig1
%
o
f
e
m
e
r
g
i
n
g
a
d
u
l
t
s
f
r
o
m
e
g
g
s
An.gambiaeKi sumu
An. gambi ae Ladj i
n= 400
n= 400
n= 400
n= 400
n= 350
n= 350
n= 350
n= 350

Malaria Journal 2006, 5:22 http://www.malariajournal.com/content/5/1/22
are intensely cultivated. These vegetable farms found in
urban settings are active throughout the year because of
constant and high demands of urban populations. To
keep their productivity high and avoid shortages of vege-
tables in urban areas, farmers are forced to treat their
farms at relatively high frequencies. This condition
explains the increased use of insecticides in urban centres
as opposed rural areas. Pyrethroids in contact with water
undergo a rapid colloidal transformation, followed by
sedimentation. After pesticide treatment, insecticide resi-
dues are washed by rainfall and sediment in watering
pools located below cultivated areas. This is probably
what happens in Parakou, in view of the sloping terrain
where the vegetable farm is located. The terrain allows a
fast sweeping of pesticide residues down the slope and
could explain the high levels of toxicities recorded in
watering pools located below cultivated sections com-
pared to top soil collected on cultivated sections. In addi-
tion to this washing process, insecticide residues
generated by agricultural practices are also degraded by
strong sunrays recorded in this northern part of Benin
Republic. At Houeyiho, the washing phenomenon also
exists but is relatively low compared to the frequent and
massive use of insecticides. This insecticide pressure keeps
the level of insecticide residues in the soil high and may
explain the elevated toxicity observed with top soil. Data
from this study indicate that factors inhibiting the hatch-
ing of An. gambiae eggs and the development of their lar-
vae are insecticide residues from agricultural practices.
Some samples of soil and water from the three study sites
were stored in the laboratory and will be analysed by
HPLC to verify the hypothesis and to identify the chemical
compounds present in water and soil samples.
Authors' contributions
MCA conceived the study and participated in data inter-
pretation and manuscript preparation. RFD carried out
the study design, sample processing and data analysis.
DAKG participated in the design of the study and substan-
tially helped draft the manuscript.
Acknowledgements
This study was supported by funds from the WHO/MIM/TDR. We
acknowledge the MIM Task Force who recommended the proposal and Dr.
Olumide A.T. Ogundahunsi, current manager of the MIM Task Force. We
also acknowledge the Director of the Special Programme for Research and
Training in Tropical Diseases who approved the application.
References
1. Coz J, Hamon J: Importance pratique de la résistance aux
insecticides en Afrique au sud du Sahara pour l'éradication
du paludisme dans ce continent. Cahier ORSTOM, Sér Ent Méd
1963, 33:77-83.
2. Coz J, Davidson G, Chauvet G, Hamon J: La résistance des ano-
phèles aux insecticides en Afrique Tropicale et à Madagas-
car. Cahier ORSTOM, Sér Ent Méd Parasito 1968, 34:314.
Comité OMS d'experts des Insecticides. Cahiers Techniques
AFRO/WHO 1976, 585:30-31.
4. Elissa N, Mouchet J, Rivière F, Meunier JY, Yao K: Resistance of
Anopheles gambiae s.s. to pyrethroïds in Côte d'Ivoire. Ann Soc
belge, Méd Trop 1993, 73:291-294.
5. Vulule JM, Beach RF, Atieli FK, Roberts JM, Mount DL, Mwangi RW:
Reduced susceptibility of Anopheles gambiae to permethrin
associated with the use of permethrin-impregnated bednets
and curtains in Kenya. Med Vet Entomol 1994, 8:71-75.
6. Akogbéto M, Yakoubou S: Résistance des vecteurs du paludisme
vis-à-vis des pyréthrinoïdes utilisés pour l'imprégnation des
moustiquaires au Bénin, Afrique de l'Ouest. Bull Soc Path Exot
1999, 92:123-130.
7. Chandre F, Darriet F, Manga L, Akogbéto M, Faye O, Mouchet J, Guil-
let P: Status of pyrethroid resistance in Anopheles gambiae s.l.
Bull World Health Organ 1999, 77:230-234.
8. Diabaté A: Evaluation de la résistance des vecteurs du palud-
isme vis-à-vis des pyréthrinoïdes au Burkina Faso. In Thèse de
3ème cycle Université de Ouagadougou, Faculté de Sciences; 1999.
9. Fanello C, Petrarca V, Della Torre A, Santolamazza , Alloueche A,
Dolo G, Coulibaly M, Curtis CF, Touré YT, Coluzzi M: The pyre-
throid knock-down resistance gene in the Anopheles gambiae
complex in Mali and further evidence of reproductive isola-
tion within An. gambiae s.s. Insect Mol Biol 2003, 12:241-245.
10. Etang J, Manga L, Chandre F, Guillet P, Fondjo E, Mimpfoundi R, Toto
JC, Fontenille D: Insecticide susceptibility status of Anopheles
gambiae s.l. (Diptera: Culicidae) in the Republic of Cam-
eroon. J Med Entomol 2003, 40:491-497.
11. Joncour G: Lutte anti-palustre au Dahomey. Rapport no 13: Ministère de
la Santé Publique 1959.
12. Mouchet J: Mini-review: agriculture and vector resistance.
Insect Science and its Applications 1988, 9:297-302.
13. Diabaté A, Baldet T, Chandre F, Guiguemde TR, Guillet P, Hemingway
J, Hougard JM: First report of the kdr mutation in Anopheles
gambiae M form from Burkina Faso, West Africa. Parassitolo-
gia 2002, 44:157-158.
14. Collins FH, Finnerty V, Petrarca V: Ribosomal DNA probes differ-
entiate five cryptic species in the Anopheles gambiae com-
plex. Parasitology 1988, 30:231-240.
15. Scott JA, Brogdon WG, Collins FH: Identification of single speci-
men of the Anopheles gambiae complex by the polymerase
chain reaction. Am J Trop Med Hyg 1993, 49:520-529.
16. Favia G, Della Torre A, Bagayoko M, Lanfrancotti , Sagnon NF, Toure
Y, Coluzzi M: Molecular identification of sympatric chromo-
somal forms of Anopheles gambiae and further evidence of
their reproductive isolation. Insect Mol Biol 1997, 6:377-383.
17. Martnez-Torres DF, Chandre F, Williamson MS, Darriet F, Bergé JB,
Devonshire AL, Guillet P, Pasteur N, Pauron D: Molecular charac-
terization of pyrethroid knockdown resistance (kdr) in the
major malaria vector Anopheles gambiae s.s. Insect Mol Biol
1998, 7:179-184.
18. Akogbéto M, Chandre F, Baldet T, Diabate A, Sagnon NF, Traoré S,
Etang J, Boakye D: Evolution of pyrethroid resistance in Anoph-
eles gambiae in West Africa. In Final report, WHO/MIM/TDR PI
Meeting Addis Abbaba. 14–16 March 2005
19. Georghiou GP, Lagunes-Tejada A: Cases of resistance in Insecta.
The Occurrence of resistance to pesticides in arthropods.
Food and Agriculture Organisation of the United Nations: Rome 1991.
20. N'guessan R, Darriet F, Guillet P, Carnevale P, Traoré-Lamizana M,
Corbel V, Koffi AA, Chandre F: Resistance to carbosulfan in field
populations of Anopheles gambiae from Côte d'Ivoire based
on reduced sensitivity of acetylcholinesterase. Med Vet Ento-
mol 2003, 17:19-25.Page 9 of 9
(page number not for citation purposes)
3. WHO: Résistance des vecteurs et des réservoirs de maladies
aux pesticides. Vingt-deuxième rapport technique du