Environment International 32 (2006) 594 – 599

www.elsevier.com/locate/envint

Contamination of fish by organochlorine pesticide residues in the Ouémé

River catchment in the Republic of Bénin
Elisabeth Yehouenou A. Pazou a , Philippe Lalèyè b , Michel Boko c , Cornelis A.M. van Gestel d,⁎,
Hyacinthe Ahissou a , Simon Akpona a , Bert van Hattum e , Kees Swart e , Nico M. van Straalen d
a
Laboratoire de Biochimie et de Biologie Cellulaire, FAST/UAC, BP 526, Cotonou, Bénin
b
Laboratoire d'Hydrobiologie et d'Aquaculture, FSA/UAC, BP 526, Cotonou, Bénin
c
Laboratoire de Climatologie, FLASH/UAC, BP 526, Cotonou, Bénin
d
Institute of Ecological Science, Vrije Universiteit, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands
e
Institute of Environmental Studies, Vrije Universiteit, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands
Received 10 January 2006; accepted 11 January 2006
Available online 21 February 2006

Abstract

In the Republic of Bénin, aquatic ecosystems are subject to poisoning risks due to the inappropriate use of pesticides, such as washing of empty
bottles in rivers and using pesticides to catch fish. In some areas, cotton fields are located near riverbanks, increasing the probability of pesticide
emission to the river. To assess contamination levels in the Ouémé River catchment area, different fish species were collected from different
geographical areas along the river. DDT, its metabolites and isomers were the most frequently identified pesticides in fish flesh, α-endosulfan, β-
endosulfan, dieldrin, telodrin, lindane and octachlorostyrene were also detected. Concentrations of pesticide residues in fish ranged from 0 to
1364ng/g lipid. A preliminary risk assessment indicated that the daily intake of chlorinated pesticides by people consuming fish from the Ouémé
River still is rather low and does not present an immediate risk.
© 2006 Elsevier Ltd. All rights reserved.

Keywords: Organochlorine pesticide residues; Aquatic ecosystems; Pesticides; Ouémé River; Fish

1. Introduction used for human consumption. Agricultural lands cover a large

The Ouémé basin, with an area of 50,000 km2, is the largest
river catchment area in the Republic of Bénin. It drains part of
the northern, the central and the southern regions of the country
(Anthony et al., 2002). The Ouémé River provides drinking
water, water for domestic use and irrigation water. It also is an
important means of transportation for people of the area. It is
used for commercial transport and business between the
neighbouring country Nigeria and the Republic of Bénin.
Biodiversity of the Ouémé River is remarkably high, with a
large diversity of fish, molluscs, shellfishes, insects, amphi-
bians, reptiles and birds. The Ouémé River contains more than
120 fish species (Lalèyè et al., 2004), of which approx. 90% are
⁎ Corresponding author.
E-mail addresses: yehouenoue@yahoo.fr (E. Yehouenou A. Pazou),
kees.van.gestel@ecology.falw.vu.nl (C.A.M. van Gestel).
0160-4120/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envint.2006.01.003
part of the river basin with intensive agricultural practice
including a high use of chemicals like fertilizers and pesticides
for cotton and maize farming and the growing of vegetables. In
some areas, cotton fields are located very close to the river,
increasing the probability of pesticide emission to the river.
Moreover, inappropriate use of pesticides, e.g. to catch fish, and
washing of empty bottles in the river contribute to pesticide
emission (Osibanjo et al., 1994). As a consequence, pesticides
may distribute among the river ecosystem components, such as
water and sediment, and accumulate in biota.
The only study on pesticide residue levels in Bénin fish was
done by Okoumassoun et al. (2002), who identified some
organochlorine pesticides and measured vitellogenin levels in
the tilapia species Sarotherodon melanotheron sampled at five
sites in the Ouémé River. An investigation of the distribution of
organochlorine and organophosphorus pesticide residues in
different fish species from the river could provide more
 E. Yehouenou A. Pazou et al. / Environment International 32 (2006) 594–599 595

information on contaminant concentrations in this aquatic identified and quantified residues of pesticides in fourteen fish
ecosystem, and on the possible risk for the local population species from different trophic levels and from different
consuming fish from the river. The present study, therefore, geographical areas throughout the Ouémé River basin.

Fig. 1. Map of the Ouémé River catchment and its position in the Republic of Bénin and the African continent. The locations of the sampling areas are indicated (see
also Table 1).
596 E. Yehouenou A. Pazou et al. / Environment International 32 (2006) 594–599

2. Materials and methods
2.1. Study area and fish samples
The Ouémé River is the most important river in Republic of Bénin, its length
is 510 km and its catchment area measures approximately 50 000km2 (Fig. 1). It
springs out of the Tanéka hills (about 9°51′NS) in the Atacora Mountains and
flows into the Atlantic Ocean by the Cotonou channel. Its two important
tributaries are the Zou River (150km) and the Okpara River (200km). In 2003,
fish species were obtained, as a random distribution of fish caught, from
fishermen in Kpassa, Atchakpa-Béri, Toué, Bonou, Lowé and Houédo areas.
The local populations commonly consume all of the sampled fish species. The
fish species were identified using the taxonomic key of Lévêque et al. (1992).
2.2. Lipid content in fish
Fish flesh (muscle tissue) was dissected, freeze-dried and ground to a fine
powder with a mixer. The samples were transported to the Netherlands and
prepared for GC analysis. About two grams of fish powder were collected for
extraction. The extraction was performed in an accelerated solvent extractor
(ASE 200, Dionex, Sunnyvale, CA, USA) with 20ml of an n-hexane–acetone
mixture (9 : 1 v/v; Merck, Darmstadt, Germany). About 1ml of extract was
introduced into a small tube and concentrated under a gentle stream of nitrogen
gas. The small tubes were dried at 50°C overnight in a stove. The lipid content
of the fish muscle tissue was obtained before and after weighing and drying 1ml
of extract, and was expressed as percentage of dry weight.
Chlorinated pesticides were analysed in fish extracts by gas chromatography
in combination with electron capture detection (ECD) according to methods
similar as applied in previous studies (Leonards et al., 1997; Ten Hulscher et al.,
2003). Pesticide concentrations were expressed on a lipid content basis.
2.3. Clean-up
The quantity of extract to be used for clean up varied with each sampled
species, depending on its lipid content. To each extract, 2ml of isooctane
(Merck) were added and concentrated to approximately 1ml under a gentle
stream of nitrogen gas using a water bath (30 °C). Concentrated extracts were
introduced on to a multi-layered column containing a small amount of glass
wool at the bottom, 3 g of aluminium oxide (type Woelm, ICN, Fisher Scientific,
Landsmeer, The Netherlands) 15% deactivated with water. A thin layer of
anhydrous sodium sulphate (Promochem, Wesel, Germany) was placed on the
top (1cm). The column was conditioned with 4ml of petroleum ether–
diethylether (95 : 5v/v, Merck) before adding the concentrated extract. A total
volume of 17ml of petroleum ether–diethylether was used to elute the pesticides
from the column. The extracts were concentrated to about 0.5 ml under a gentle
stream of nitrogen gas (30°C). One ml of isooctane was added to the extract,
which was again concentrated to 0.5ml. To correct for variability due to
injection conditions, 100μl of an internal standard PCB 198 was added to each
extract. The extracts were transferred into vials for automated GC–ECD
analysis. The ECD response was calibrated using a standard mixture containing
32 compounds including 24 organochlorine pesticides and 8 polychlorinated
biphenyls.
2.4. GC–ECD analysis
Analysis of chlorinated pesticides in the fish species was carried out with a
gas chromatograph (Varian 3800, Walnut Creek, CA, USA) in combination with
an auto sampler (Varian 8200), and an electron capture detector (Varian EFC
13). The ECD temperature was 300 °C. Helium was used as the carrier gas with a
flow rate of 1ml/min and nitrogen make-up gas at a flow rate of 30 ml/min. The
injection volume was 3μl with a splitless injection mode. The following
injection temperature program was applied: 90°C (hold 3min), 30°C/min to
200°C (hold 15min), 5°C/min to 265°C (hold 5min), and 3°C/min to 275°C
(hold 15min). The column was an ultra performance capillary column (length of
50m, internal diameter of 0.20mm and film thickness of 0.33μm; Ultra2,
Agilent Technologies, Palo Alto, CA, USA). The organochlorine residue
components were identified by comparing their retention times with those of the
standard mixture of PCBs and organochlorine pesticides. Quantification was
based on comparison with calibration curves in the concentration range of 0 to
250ng/ml. The calibration line of the standard was slightly quadratic for almost
all compounds. Peak heights of analytes were all in the range of the calibration
line. Samples with higher peaks were diluted. The limit of detection (LOD) for
all pesticides including ∑DDT (pp′-DDE, op′-DDD, pp′-DDD′ op′-DDT, pp′-
DDT) was 0.1 ng/g lipid, and the limit of quantification (LOQ) was 0.3 ng/g.
Extracts were analyzed in onefold. Quality assurance measures applied in the
laboratory included rigorous contamination control procedures (strict washing
and cleaning procedures), monitoring of blank levels of solvents, equipment and
other materials, analysis of procedural blanks, recovery of spiked standards,
monitoring of detector response and linearity, and analysis of a reference
material. Recoveries of chlorinated pesticides in the reference material were
between 80% and 110% of certified concentrations.

Table 1
Fish species collected from different geographical areas along the Ouémé River in Bénin, their average body weight and lipid content
Areas (major crops) Family of Species name No. of Average fresh Lipid
species fish pooled bodyweight ± S.D. (g) content (%)
Kpassa (maize/vegetables) Cichlidae Oreochromis niloticus 2 104 ± 1.3 2.2
Cichlidae Sarotherodon galileus 1 163 2.5
Bétérou (maize) Clariidae Clarias gariepinus 1 265 6.4
Atchakpa-Béri (maize/cotton) Cichlidae Tilapia guineensis 1 98 4.9
Clariidae Clarias gariepinus 1 153 1.4
Claroteidae Chrysichthys nigrodigitatus 1 188 4.3
Toué (maize/orange trees) Cichlidae Tilapia zilli 8 64 ± 33 3.4
Polypteridae Polypterus e. endlicheri 1 81 2.0
Clariidae Clarias ebriensis 5 53 ± 17 6.3
Bonou (maize/cotton/vegetables) Clariidae Clarias gariepinus 2 254 ± 7 4.4
Lowé (maize/vegetables) Protopteridae Protopterus annectens 1 170 2.5
Clariidae Clarias gariepinus 6 63.3 ± 19 4.2
Clariidae Clarias ebriensis 5 32 ± 12 2.9
Mochokidae Synodontis schall 4 34 ± 17 1.4
Mochokidae Synodontis nigrita 6 44 ± 5 4.7
Cichlidae Schilbe intermedius 1 57 17.7
Claroteidae Chrysichthys auratus 3 135 ± 83 3.7
Mormyridae Hyperopisus bebe 3 38 ± 11 2.6
Houédo (vegetables) Clariidae Clarias gariepinus 1 249 4.6
Also indicated are the main crop types grown in the different areas along the river.
3. Results and discussion

Fish species collected from different geographical areas along the

Ouémé River, the number of fish used for pesticide analysis, their
average fresh bodyweight and lipid content are shown in Table 1.
There was a large variation in lipid contents between the fish species
sampled. But also within a species, lipid contents showed large
differences between areas. An extremely high lipid content of 17.7%
was found in Schilbe intermedius from Lowé. High lipid contents
were also found in Clarias gariepinus from Bétérou (6.4%) and C.
ebriensis (6.3%) from Toué. C. gariepinus from Atchakpa-Béri and
Synodontis schall from Lowé did contain the lowest lipid content
(1.4%). These differences in lipid contents may be due to differences
in species, age and size of the fish, but also to differences in feeding
habits and life strategies.
The levels of pesticide residues in fish are presented in Table 2.
Octachlorostyrene was detected in only one fish sample (Oreochromis
niloticus) from Kpassa, at a concentration of 9 ng/g lipid. Lindane (γ-
HCH) was found in only two fish samples from Toué (Tilapia zilli and
C. ebriensis) in concentrations of 3–65 ng/g lipid. Telodrin was found
in T. guineensis from Atchakpa-Béri and in C. ebriensis from Lowé in
concentrations of 4.1 and 6.9 ng/g lipid, respectively.
All fish samples contained DDT, its metabolites and isomers. The
pp′-DDT concentrations ranged from 13 to 1364ng/g lipid (Table 2),
with the lowest concentration in Protopterus annectens from Lowé and
the highest in C. gariepinus from Bonou. pp′-DDE and pp′-DDD
residues were also detected in all fish samples, op′-DDD in 58% and op
′-DDT in 84% of the samples. β-Endosulfan contaminated 68% of fish
samples with the highest concentration in S. intermedius (Table 2)
caught in the Lowé area. A high level of α-endosulfan (57 ng/g lipid)
598 E. Yehouenou A. Pazou et al. / Environment International 32 (2006) 594–599

concentrations were lower in the pelagic species (T. guineensis, S.
galileus, and Oreochromis niloticus) than in benthic species. Since
sediments from all areas did contain pesticides (Yehouenou A. Pazou
et al., 2006), uptake from sediments probably plays a role in benthic
species. Pesticide residues found in fish were compared to those
detected in sediments from the different areas (Yehouenou A. Pazou
et al., 2006). ∑DDT (pp′-DDE, op′-DDT, pp′-DDT, op′-DDD, pp′-
DDD) concentrations in sediments ranged between b0.1 and 809ng/
g OM, while ∑endosulfan levels varied between b0.1 and 164ng/g
OM. The wide ranges in sediment concentrations are illustrative of
spatial heterogeneity, which may partly be due to the fact that
different crops were grown along the river at the different sites
sampled for pesticide analysis (Table 1; Yehouenou A. Pazou et al.,
2006). No correlation was found between ∑DDT or ∑endosulfan
concentrations in fish and the corresponding levels in sediments.
When focusing on a single fish species occurring in different areas
(C. gariepinus), pesticide levels showed a negative correlation with
those in sediments.
From the comparison of fish concentrations with average
sediment concentrations at the different locations in the Ouémé
River reported by Yehouenou A. Pazou et al. (2006), bio-
accumulation factors (BAF) were calculated using pesticide con-
centrations in fish expressed on a lipid basis and in sediment on an
organic matter (OM) basis. BAF were highest for pp′-DDT (5.7–37 g
OM/g lipid), followed by op′-DDD (0.9–22 g OM/g lipid) and pp′-
DDE (1.0–26 g OM/g lipid), ∑endosulfan (0.6–11 g OM/g lipid),
and op′-DDT (0.13–4.2g OM/g lipid). In some cases, pesticides
were detected in fish while sediment concentrations were below the
detection limit. This suggests that fish did not only accumulate the
pesticides from sediment at the site of sampling but also from water
and maybe from sediment at other sites in the river. For more insight
in the factors explaining the pesticide concentrations in fish from the
Ouémé River, a more detailed study is needed of spatial and
temporal variations in fish, water and sediment concentrations.
To determine the potential risk of pesticide residues in fish for the
human population living along the Ouémé River, intake of four
major groups of pesticides (DDT, ∑endosulfan, lindane and dieldrin)
from fish consumption was estimated: Table 3. Fish consumption
was assumed to amount two fish per person per day. From Table 3, it
can be concluded that total pesticide intake by fish consumption is
rather low. To compare pesticide intake with Acceptable Daily
Intakes (ADI) mentioned by IPCS (2005), daily intakes of the
different pesticides are summed to calculate a toxic unit, which is the
sum of the intake per pesticide divided by its corresponding ADI.
From Table 3, it appears that the resulting toxic units are low, and,
depending on the fish species and the area, are a factor of 100–
10,000 below the summed ADI. It may therefore be concluded that
the human population consuming fish from the Ouémé River is not
at risk due to pesticide pollution of the fish. It should, however, be
kept in mind that we took a low fish consumption of only two
(small) fish per day as a starting point for our calculation. In
addition, no data is available on other sources of pesticide intake,
such as drinking water or eating crops containing pesticide residues.
Furthermore, this assessment just focused on a limited number of
organochlorinated pesticides, while nowadays many other types of
pesticides are in use in Bénin. Further research on the potential risk
of pesticides to the human population living along the Ouémé River
therefore is needed.

Table 3
Estimation of the potential risk of fish consumption of the local population along the Ouémé River
Areas Kpassa Bétérou Atchakpa-Béri Toué Toué Bonou Lowé Houédo
Species name O. niloticus C. gariepinus C. nigrodigitatus T. zilli P. endlicheri C. gariepinus S. intermedius C. gariepinus
Fish body (g w. w.) 104 265 188 64 81 254 57 249
Fish meat (g w. w.) 35 60 30 21 27 57 20 51
Fish meat (g d. w.) 11 27 19 6 8 26 6 23
Fat content (%) 2.2 6.4 4.3 3.4 2 4.4 17.7 4.6
Fat in meat (g) 0.24 1.73 0.82 0.20 0.16 1.14 1.06 1.06

Pesticide concentration in fish (ng/g lipid)

∑DDT 129 535 196 134 473 1642 1191 789
∑endosulfan 16 23 0 5.3 0 32 215 54
Lindane 0 0 0 6.5 0 0 0 0
Dieldrin 0 0 0 0 0 0 9

Pesticide intake per two fish (ng)

∑DDT 62.4 1849 320 54.7 151 3757 2530 1670
∑endosulfan 7.74 79.5 0 2.16 0 73.2 457 114
Lindane 0 0 0 2.65 0 0 0 0
Dieldrin 0 0 0 0 0 0 19.1 0

Daily intake in ng/kg body weight for adult person (60 kg)
DI ∑DDT 1.04 30.8 5.34 0.91 2.52 62.6 42.2 27.8
DI ∑endosulfan 0.13 1.32 0.00 0.04 0.00 1.22 7.61 1.90
DI lindane 0.00 0.00 0.00 0.04 0.00 0.00 0.00 0.00
DI dieldrin 0.00 0.00 0.00 0.00 0.00 0.00 0.32 0.00
Toxic unit 0.00013 0.0033 0.00053 0.00011 0.00025 0.0065 0.0087 0.0031
Based on fish weight (g. w. w. = gram wet weight), meat (g. d. w. = gram dry weight) and lipid contents, pesticide concentrations on a lipid basis and the assumption that
people consume two fish per day, daily intake (DI) is estimated. By comparison with Acceptable Daily Intakes (ADI) for different pesticides, a summed risk is
calculated, expressed as a toxic unit. ADI values for ∑DDT, ∑endosulfan, lindane and dieldrin are 10,000, 6000, 5000 and 100ng/kg bodyweight/day, respectively
(IPCS, 2005).
 E. Yehouenou A. Pazou et al. / Environment International 32 (2006) 594–599
4. Conclusion
Organochlorine pesticides contaminated all fish collected in
the Ouémé River basin with a concentration ranging from the
detection limit to 1364 ng/g lipid. Pesticide intake by people
consuming fish from the river, however, is still low and no
immediate risk is present.
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