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Trente ans de lutte contre l’onchocercose en Afrique de l’Ouest. Traitements larvicides et protection de l’environnement

Laurent Yaméogo
Christian Levêque
Jean-Marc Hougard

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Effects of Aerial Spraying of Chlorphoxim on the Brain Acetylcholinesterase Activity of Fish from Three Rivers in the Ivory Coast, West Africa

L. A. K. Antwi


The effect of aerial treatment of rivers with chlorphoxim, in connection with the ongoing Onchocerciasis Control Programme (OCP) in the Volta River Basin, on the brain acetylcholinesterase (AChE) activity of three fish species Tilapia galilaea, Tilapia zilli and Alestes nurse has been studied in the Ivory Coast. A cage experiment with Tilapia zilli, placed just below the breeding site of Simulium damnosum s.l. in the river Marahoué and aerially treated with chlorphoxim at a concentration of 0.05 mg litre-1 per 10 min of river discharge, showed a 16% reduction in the brain acetylcholinesterase activity of the caged Tilapia zilli after 2 h and about 20 % after 5 h of the river treatment. However, there was no reduction in the brain AChE activity of some Tilapia galilaea, Tilapia zilli and Alestes nurse randomly taken with a cast net from the rivers Bandama and N'zi, which had been similarly treated with chlorphoxim for about 10 months. It appears that during river treatment the fish avoid the impact of the larvicide by swimming downstream.

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1Since 1974, rivers in the Volta River Basin of West Africa have been treated with temephos (O, O, O', O'-tetramethyl-O, O'-thiodiphenylene phosphorothioate) to kill larvae of the Simulium damnosum complex, the vectors of river blindness (onchocerciasis), in connection with the ongoing Onchocerciasis Control Programme (OCP). However, from May 1980, it was detected that two forest species of the S. damnosum complex (S. sanctipauli and S. soubrense) had developed resistance to temephos on the lower Bandama river in the Ivory Coast (OCP, 1981). Later in the year, temephos resistance was observed on the rivers Marahoué and N’zi. These unexpected developments necessitated replacing temephos treatment of the three rivers with a series of weekly chlorphoxim (O, O-diethyl-2-chloro-α-cyanobenzylidene amino-oxyphosphonothioate) treatments, which began in October 1980.

2Toxicity of chlorphoxim to African fish species has not been studied extensively, but Galleta (1968) had reported the 24 h LC50 of chlorphoxim to Gambusia affinis to be 2 mg litre-1 while the 24h laboratory tests with 0.10 mg litre-1 and 1.0 mg litre-1 chlorphoxim on black bullhead gave mortalities of 10% and 80%, respectively (Anon., 1967). These studies did indicate that chlorphoxim might be more toxic to fish than temephos. Again, at the beginning of the OCP operations in 1974, some concern was expressed over the possible harmful effect such large-scale application of insecticide would have on the riverine fishery (Asibey, 1975, 1977). There was therefore a need to monitor closely the effect of the chlorphoxim treatment on the fish species in the treated rivers. This paper reports one such study on fish from the rivers Marahoué, N’zi and Bandama in the Ivory Coast, which have all been treated with chlorphoxim.


Description of the study area

3The Marahoué and N’zi rivers are tributaries of the river Bandama and all flow from north to south. Details of the hydrology and the physicochemical characteristics of the rivers have been given by Iltis & Lévêque (1982). At Danangoro on the river Marahoué, where the cage experiment was carried out (see below), the breeding site of the blackfly Simulium damnosum s.l. is an expanded section of the river containing huge rocks (Fig. 1). At the time of the river treatment, the water level was high and the river flow fast, forming small rapids on the rocks.

Fish collection

4Three commercial fish species, Tilapia galilaea, Tilapia zilli and Alestes nurse, were randomly caught daily for one week with a cast net from the treated rivers—the Marahoué at Danangoro, the N’zi at Dabakala and the Bandama at Marabadiasa—and from the untreated river Kadioni, which was used as the control.

Fig. 1. Expanded section of river Marahoué at Danangoro.

Cage experiment with Tilapia zilli in the river Marahoué

5To study the immediate impact of aerial applications of chlorphoxim to rivers on the fish brain acetylcholinesterase activity, a cage experiment was carried out using T. zilli in the river Marahoué during one of its weekly aerial sprayings with chlorphoxim.

6On the day of the river treatment, T. zilli were obtained with a cast net from an untreated barrage at Loka near Bouaké. The fish were transported live in plastic bags filled with aerated water to the riverside, where they were divided into groups according to size and distributed among two 52 x 52 x 52cm cages, each holding about 10 fish ranging from 8 to 12 cm in length. A set of 10 fish was taken for the control measurement. The cages were next placed in the river at a point below the breeding/treatment site (Fig. 1) 2 h before treatment. At about 1200 h the larvicide was applied by the OCP team as part of their routine treatment of the river from a helicopter at a point about 200 m upstream of the S. damnosum s.l. breeding site. According to an OCP estimate, the concentration of the larvicide at the breeding site of the blackfly was 0 05 mg litre–1 for 10 min of river discharge (Davies et al., 1978).

7The first cage was removed from the river 2 h after spraying and the second cage was removed after 5 h. The fish in each cage were placed in a polythene bag, labelled and placed in an ice-box for transportation to the laboratory for enzyme analysis.

Acetylcholinesterase activity measurement

8The colorimetric method of Ellman et al. (1961) was used to measure the acetylcholinesterase activity.

9Each fish was decapitated and the operculum removed. The fish head alone was weighed and homogenised using a Potter–Elvehjin homogeniser in a 0 1 M phosphate buffer solution (pH 7 0) so as to make a 10% solution. The fish homogenate solution was next diluted with the phosphate buffer to make a 0 1% solution, 4 0 ml of which was measured into a photometric cuvette, and 0 1 ml dithiobisnitrobenzoic acid (DTNB) solution added and thoroughly mixed. The spectrophotometric zero was set with this solution, after which 0 1 ml acetylthiocholine (ASCh) solution (0 2 M) was added. Immediately after mixing, the rate of the yellow colour production was followed by measuring the absorbance (A) at 412nm every 30s for 120s using a Coleman 295 spectrophotometer. Duplicate measurements were made on each fish homogenate. At the end of each measurement, 0 004 ml anticholinesterase solution was added to check for any non-enzymatic hydrolysis. No increase in the yellow colour was recorded after the addition of the anticholinesterase solution, indicating that there was no other hydrolysis than that due to the acetylcholinesterase.

10Change of absorbance per minute (ΔA min–1) was calculated and the rates converted to absolute units using the formula:

11μmol ASCh min–1 g–1 fish head wt

12where 1.36 x104 = extinction coefficient of DTNB,103 = conversion of mol litre–1 to mol ml–1, and 106 = conversion of mol ASCh min–1 g–1 head weight to μmol ASCh min-1 g1 head weight. The measurements were made at a room temperature of 25–30°C.


13The results of the AChE activity measurements are summarised in Tables 1 and 2. The enzyme activities are expressed as μmol acetylthiocholine (ASCh) hydrolysed per minute per gram fish head weight. Student’s t-test (p = 0 5) was used to compare the enzyme activity of.the fish from the treated rivers with their respective Controls.

14Table 1 shows that the brain AChE activity of the caged T. zilli was signifïcantly reduced by 16.32% after 2 h (p <0.05) of the river treatment and by 19.06% after 5 h (p <0.05). These results indicate that during the river treatment with chlorphoxim the larvicide induces a significant reduction in the brain acetylcholinesterase activity of fish present in the river water just below the S. damnosum s.l. breeding site. Near the point of the chlorphoxim release, the level of enzyme inhibition would be greater than the observed 20% because of the higher larvicide concentration.

TABLE 1. Acetylcholinesterase (AChE) Activity of Caged Tilapia zilli Kept in the River Marahoué During Chlorphoxim Treatment

TABLE 1. Acetylcholinesterase (AChE) Activity of Caged Tilapia zilli Kept in the River Marahoué During Chlorphoxim Treatment

15The increase in the level of the enzyme inhibition from 16% after 2 h to about 20% after 5 h of river treatment might have been caused by either the residual chlorphoxim left in the river after the bulk of the larvicide had passed downstream or the appearance of some metabolites of chlorphoxim more toxic than the parent compound. Metabolites of malathion were found to continue inhibiting AChE activity for several weeks after exposure was discontinued and the parent compound had disappeared from the water (Weiss, 1961). Verma et al. (1979) have also reported that cholinesterase of vertebrates remained inhibited for several weeks after exposure caused by oxygen analogue metabolites of thiophosphates.

16A similar cage experiment performed in the Black Volta river treated with temephos did not reveal any significant inhibitory effect on the fish brain AChE activity (Scheringa et al., 1981), providing further evidence that chlorphoxim is more toxic to fish than temephos.

17In contrast to the observed toxic effect of chlorphoxim treatment on the brain AChE activity of the caged T. zilli in the river Marahoué, the enzyme activity levels of A. nurse, T. galilaea and T. zilli randomly sampled with a cast net from the rivers Bandama and N’zi (Table 2) were not significantly different from their respective Controls (p> 0.05).

18Both rivers had had weekly treatments with chlorphoxim for about 10 months. It appears then that during treatment the majority of the fish in the treated rivers avoid the impact of the Chemical by swimming downstream. Avoidance reactions of fish to Chemicals in water have beeri observed by many workers including Hansen (1969), Scherer (1975) and Abban & Samman (1980). The large volume of flowing river water, especially during the rainy season when the chlorphoxim treatment of the rivers in the Ivory Coast is undertaken, ensures a rapid dilution of the larvicide from the dosing point, so that downstream below the breeding site the chlorphoxim is barely detectable. The fish near the treatment site can swim safely into the waters downstream of the breeding site.

TABLE 2. Acetylcholinesterase (AChE) Activity of Three Fish Species Randomly Taken from Two Rivers which had been Treated with Chlorphoxim


Fish species

No. of fish analysed

Fish head weight (g) (mean ± SD)

Total AChE activity in µmol ASCh min -1 g -1 head weight (mean ± SD)

Bandama (chlorphoxim-treated)

Tilapia zilli


3·25 ± 0·73

20·41 ± 3·73

Barrage de Loka (control)

Tilapia zilli


3·05 ± 1·69

20·15 ± 6·15

Bandama (chlorphoxim-treated)

Tilapia galilaea


2·66 ± 0·79

18·20 ± 5·96

N’zi (chlorphoxim-treated)

Tilapia galilaea


2·73 ± 0·77

17·70 ± 4.21

Kadioni (control)

Tilapia galilaea


2·46 ± 0·82

17·65 ± 5·08

Bandama (chlorphoxim-treated)

Alestes nurse


2·12 ± 0·50

13·11 ± 4·63

N’zi (chlorphoxim-treated)

Alestes nurse


2·52 ± 1·35

11·81 ± 3·11

Kadioni (control)

Alestes nurse


1·91 ± 0·56

11·11 ± 4·68

19Another factor which has contributed to the survival of the fish population in the treated rivers from the toxic effect of the larvicide is its low persistence in the environment (OCP, 1973). This property of chlorphoxim ensures that it does not accumulate to lethal levels in the treated rivers even after a series of prolonged treatments.

20No fish kill was observed during the chlorphoxim treatment of the river. However, the recorded 20% reduction in the caged fish brain AChE activity might have cause! the fish to exhibit some toxic symptoms such as a reduction both in their ability to tolerate reduced oxygen tension (Eaton, 1970) and in their feeding activities (Verma et al., 1979).

21In the laboratory, fish exposed to sublethal concentrations of organophosphorus compounds recover their brain AChE activities to the normal levels usually within one month (Weiss, 1959; Benke & Murphy, 1974). However, in the fish’s natural environment such recoveries could be much faster. It is therefore possible that in the treated rivers, fish that are exposed to chlorphoxim could recover their original acetylcholinesterase activity before the next river treatment.


22I wish to thank Professor J. H. Koeman, Dr J. J. T. W. A. Strik and Ms E. Scheringa, all of the Agricultural University, Wageningen, The Netherlands, for training experience in the measurement of AChE activity. The cage experiment was undertaken at the suggestion of Professor Koeman. Special mention must be made of Mr T. Sineyogo, at the OCP Headquarters, Ouagadougou, Burkina Faso, who collected the fish samples from the rivers. Finally, I thank Dr M. A. Odei of the Institute of Aquatic Biology, Achimota, Ghana, for making it possible for me to undertake this study, Funds for this study were provided by the OCP.



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Table des illustrations

Légende Fig. 1. Expanded section of river Marahoué at Danangoro.
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Titre TABLE 1. Acetylcholinesterase (AChE) Activity of Caged Tilapia zilli Kept in the River Marahoué During Chlorphoxim Treatment
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