Diflubenzuron (DFB, trade name dimilin 25 WP) is a chitin synthesis inhibitor widely used against forest insect pests in Algeria. Prior to implementation of these products as chemical agents for mosquito control, knowledge on their potential effects on non-target organisms and its behaviour in water are needed. Therefore, the present study was focused on DFB and aimed to use an HPLC procedure developed previously in order to obtain information on its degradation in freshwater and its bioconcentration in adult females of the fish Gambusia affinis (Cyprinodondiformes, Poeciliidae), which is one of the best candidates for biological control programs against mosquitoes. The adult females were exposed to dimilin (initial concentration 312 ng a.i./ml) for 28 days and residues analysis determined at different exposure times (0, 7, 14, 21 and 28 days). The concentration of DFB in freshwater decreased with exposure time while the amount of residues detected at the surface of the fish body increased progressively to reach a maximum at day 14 (162.7 ± 14.0 ng/fish) and declined thereafter during the exposure period. In addition, DFB incorporation into body increased with decreasing DFB concentration in water at each exposure time. The following average distribution was noted at the end of experiment (28 days): about 33% of the applied concentration was detected on the surface of fish body and was recovered by simple rinsing, and about 67% was found inside the fish body. A degradation in water and surface of fish occurred starting day 14 during the experimental period. Thus, about 40% of the initial concentration was degraded in freshwater after 28 days. The results are discussed to develop a better understanding of the degradation of dimilin in water and their potential effect on non-target organisms for its application for controlling mosquito.
Mosquitoes are the most important vectors of pathogens causing human and animal diseases [
Thereafter, this procedure was routinely applied for evaluation of DFB residues in different organs and tissues from several insect pests [23-25], and also to study DFB degradation in seawater [
Gambusia affinis (Baired and Girard, 1845) were collected from Kherraza River (4˚04'N, 04˚02'E) as previously described [
Dimilin® (wettable powder 25% active ingredient, a.i.), the trade name of diflubenzuron, and technical grade diflubenzuron (97.9% purity used as standard for quantification of residues) were kindly provided by Pr. G. Smagghe (Ghent University, Belgium). Dimilin was added to the rearing water at a final concentration of 312 ng a.i./mL. This concentration was chosen according to concentrations tested on several fish species [28-31]. Fish starved for 2 days were exposed to the insecticide for 28 days. Untreated fishes were also used as controls (For each series, three aquaria were used, each containing 30 fishes).
DFB residues in freshwater and in fishes (surface and tissues) were performed using high performance liquid chromatography (HPLC) procedure as described previously [
Each extract was dissolved in 200 µL of acetonitrilewater (50 - 50) and small aliquots (10 - 20 µL) were twice injected into a Waters HPLC (600 E) equipped with two pumps (M6000 A), a detector (Waters 996 operated at 254 nm), an automatic injector (WISP 710 B) and a M 740 system Controller Data module. A 125 mm × 4 mm i.d. Merck RP18 column (7 µm particles) was used with acetonitrile-water (50 - 50 by volume) as the mobile phase at a flow rate of 1 mL/min. Chromatography was performed at room temperature (about 25˚C). A calibretion curve was established with technical grade diflubenzuron (99.7% purity) using a Nec Power Mate 433 computer equipped with Waters Millenium Software.
Bioconcentration was estimated by using the bioconcentration factor (BCF) defined according to [
The normality of data was verified using the Kolmogorov-Smirnov test, and the homogeneity of variances was checked by Levene’s test. Data have been expressed by the mean ± standard deviation (m ± SD). Data were subjected to analysis of variance (ANOVA) followed by Tukey’s test. All statistical analyses were performed using MINITAB Software (Version 14, Penn State College, PA, USA) and p < 0.05 was considered to be a statistically significant difference. The number of fishes and repeats used are given with the results.
According to previous data, the response of the UV detector was linear from 2 ng to 1000 ng of DFB [
The amount of DFB residues detected on surface of the fish body increase progressively to reach a maximum within 14 day exposure (162.7 ± 14.0 ng/animal) and declined thereafter during the exposure period (
This increase was rather low, and it is only after 21 days of exposure that this amount became higher than that found on the fish skin.
At the end of experiment (28 days), the following average distribution was noted: about 33% of the applied concentration was detected on the surface of fish body and was recovered by simple rinsing, and about 67% was found inside the fish body. In our study, there was a progressive incorporation into fish body and a degradation in water and surface of fish occurred starting day 14 during the experimental period. Thus only about 40 % of the initial dose was degraded in water after 28 days. In addition, under the laboratory conditions the half-life of DFB in freshwater determined from the regression curve showing the degradation as function the time (Y = −2.72 + 1.52X; R2 = 0.979) was 34.71 days. The bioconcentration factor (BCF) (
G. affinis remains one of the best candidates for biological control programs against mosquitoes [13,14]. This fish has been the subject of several studies including be-
havioural ecology, histopathology, reproductive biology, endocrinology and toxicology [33-35].
The normal development of the ovaries has already been described [27,36] and the obtained data provide an experimental basis to study the activity of IGRs on females.
Several workers have already reported on the interactions of DFB with various components of the environ ment. Laboratory and field studies show that DFB can have adverse impacts on aquatic organism [29,37-39]. Both the tested IGR [
Most methods use liquid chromatography with UV detection for the analysis of benzylphenylurea compounds such as DFB [43,44]. HPLC was found to be one of the best methods due to its sensitivity [
In the present study, the rapid extraction procedure and the analysis by a sensitive HPLC method [
The amounts of DFB detected inside G. affinis indicate that there was a progressive accumulation into the fish body. The rate of DFB uptake increased with the DFB concentration in the freshwater. In the present study, the bioconcentration factor (BCF) ranged from 0.462 ± 0.089 (minimum value at day 7) to 3.115 ± 0.710 (maximum value at day 28), indicating that DFB has a potential to bioconcentrate in this fish starting day 21 of exposure period. Similarly, analysis of DFB in Oreochromis niloticus exposed to 100 µg/mL indicated a bioconcentration ratio of 29.8 fold after 48 h [
In conclusion, an HPLC method for determination of dimilin commercial formulation in freshwater and fish body was used. At the end of the exposure period (28 days), 60% of the compound was still detected in rearing water. According to our previous study, the compound appears more stable in freshwater than in seawater. Thus, dimilin can be used for controlling of mosquitoes in an integrated manner according to its relative stability in freshwater and its slight toxicity against this non-target fish species as reported previously. In order to complete the present finding, a further study is needed to determine the major metabolites of DFB resulting from its degradation under our experimental conditions.
The authors wish to thank Pr. G. Smagghe (Ghent University) for donating insecticides. This study was financed by the Algerian Fund for Scientific Research and by the Ministry of High Education and Scientific Research of Algeria (CNEPRU and PNR projects to Pr. N. Soltani).