Environ. Sci. Technol. 2006, 40, 4151-4157
Phototransformation of the Insecticide Fipronil: Identification of Novel Photoproducts and Evidence for an Alternative Pathway of Photodegradation M U R I E L R A V E T O N , * ,†,‡ A S M A E A A J O U D , †,‡ JOHN C. WILLISON,§ HEDDIA AOUADI,† MICHEL TISSUT,† AND PATRICK RAVANEL† Equipe Pertubations Environnementales et Xe´nobiotiques, Laboratoire d’Ecologie Alpine, UMR 5553 UJF/CNRS/UdS, Universite´ Joseph Fourier, BP 53X, 38041 Grenoble Cedex 9, France, and Laboratoire de Biochimie et Biophysique des Syste`mes Inte´gre´s, UMR 5092 UJF/CEA/CNRS, De´partement de Re´ponse et Dynamique Cellulaires, CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France
Fipronil is a recently discovered insecticide of the phenylpyrazole series. It has a highly selective biochemical mode of action, which has led to its use in a large number of important agronomical, household, and veterinary applications. Previous studies have shown that, during exposure to light, fipronil is converted into a desulfurated derivative (desulfinyl-fipronil), which has slightly reduced insecticidal activity. In this study, the photodegradation of fipronil was studied in solution at low light intensities (sunlight or UV lamp). In addition to desulfinyl-fipronil, a large number of minor photoproducts were observed, including diversely substituted phenylpyrazole derivatives and aniline derivatives that had lost the pyrazole ring. Desulfinylfipronil itself was shown to be relatively stable under both UV light and sunlight, with only limited changes occurring in the substitution of the aromatic ring. Since this compound accumulated to levels corresponding to only 30-55% of the amount of fipronil degraded, it was concluded that one or more alternative pathways of photodegradation must be operating. On the basis of the structurally identified photoproducts, it is proposed that fipronil photodegradation occurs via at least two distinct pathways, one of which involves desulfuration at the 4-position of the pyrazole ring giving the desulfinyl derivative and the other of which involves a different modification of the 4-substituent, leading to cleavage of the pyrazole ring and the formation of aniline derivatives. The latter compounds do not accumulate to high levels and may, therefore, be degraded further. The ecological significance of these results is discussed, particularly with regard to the insecticidal activity of the photoproducts.
Introduction Two recently commercialized phenylpyrazole compounds, fipronil (1) and ethiprole (2), are widely used on account of * Corresponding author phone: +33 476 51 46 80; fax: +33 476 51 44 63; e-mail:
[email protected]. † Universite ´ Joseph Fourier. ‡ These authors contributed equally to this work. § CEA-Grenoble. 10.1021/es0523946 CCC: $33.50 Published on Web 05/28/2006
2006 American Chemical Society
FIGURE 1. Chemical structures of fipronil and desulfinyl-fipronil. their effectiveness against a wide range of insects (3). In agriculture, fipronil (5-amino-3-cyano-1-[2,6-dichloro4-(trifluoromethyl)]phenyl-4-((trifluoromethyl)sulfinyl)pyrazole) (Figure 1) is mainly used against soil insects, such as wireworms (4), aquatic insects, such as the water rice weevil (5), and in locust control (6). The biochemical target of these insecticides is the γ-aminobutyric acid (GABA) gated chloride channel (7), resulting in a much more potent activity in insects than in mammals (8). Fipronil is currently active against insects resistant or tolerant to other insecticide families, such as organophosphates, pyrethroids, carbamates, and most of the cyclodienes (9). It is nontoxic to earthworms (10) and to certain aquatic species, such as Daphnia (11). After about 15 years of use, insect resistance or cross-resistance to fipronil has yet to appear, possibly as a result of its selective mode of action (9). The photosensitivity of fipronil has previously been demonstrated (12-14), with a desulfinyl derivative being formed as the major product (Figure 1). Some additional photoproducts have also been detected (13, 15). However, these studies were not carried out under natural conditions of illumination. The aim of the present study was to investigate the different pathways by which fipronil, in solution, might be degraded under sunlight or under equivalent conditions produced by a low intensity UV (290350 nm) lamp. Previously identified and some novel photoproducts were identified and quantified, and the time courses of photoproduct formation and fipronil degradation were measured. The insecticidal activity of the reaction mixture was assessed at various stages of the photodegradation process and compared with the values obtained with some purified photoproducts and metabolic derivatives. Finally, the ecological significance of these data is discussed.
Materials and Methods Chemicals. Fipronil (99.3% purity), desulfinyl-fipronil (97.8% purity), sulfide-fipronil (98.8% purity), amide-fipronil (99.8% purity), sulfone-fipronil (99.7% purity), amide-sulfonefipronil (92.3% purity), and acid-fipronil (97.9% purity) were provided by Aventis CropScience SA (CRLD, Lyon, France). 2,4,6-Trichloroaniline standard (97% purity) was purchased from Sigma. Photodegradation Experiments. Fipronil and desulfinylfipronil were solubilized in a water/ethanol mixture (50/50, v/v), pH 6.8, at an initial concentration of 700 µM. The solution (200 mL) was placed in a 500-mL crystallizer dish (diameter: 10 cm) and covered with a transparent film which transmitted UV light above 250 nm. In preliminary experiments under sunlight, the solution was stirred continuously and exposed to a photoperiod of 16 h of light and 8 h of darkness. Subsequent experiments in the laboratory were carried out under controlled conditions of temperature and light intensity. The solution was illuminated from above with a 8 W, F8T5/BLB (Sylvania) lamp (290-350 nm) at a distance of 10 cm, and the temperature was maintained at 20 ( 2 °C. All experiments were carried out in duplicate. At regular time VOL. 40, NO. 13, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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intervals, a volume of 20 mL was collected and extracted three times with a mixture of petroleum ether (bp 40-60 °C)/ethyl acetate (50/50, v/v). The lipophilic fraction was concentrated and resolubilized in 4 mL of ethanol prior to GC-MS analysis. The remaining aqueous fraction was analyzed by spectrophotometry (Uvikon 933, Kontron Instruments) to confirm the absence of phenylpyrazole products. Preliminary experiments comparing the effect of natural sunlight with that of the UV lamp showed the formation of a similar number of derivatives. For quantitative experiments, the UV lamp was chosen to allow for standardized and repeatable conditions to be used. To obtain sufficient quantities of unknown photoderivatives for GC-MS analysis, it was important to start the experiment with a high concentration of fipronil but without any risk of precipitation. After we tested increasing amounts of ethanol added to water and showed that the photodegradation mixture remained the same, the presence of 50% ethanol in the reaction mixture was chosen. Finally, control experiments performed in the dark showed that fipronil was chemically stable and was not detectably degraded under these conditions in the absence of light. Silylation. To determine the structure of novel photoproducts, samples were dried under vacuum and solubilized in 50 µL of acetonitrile and 100 µL of BSTFA-TCMS reagent (bis(trimethylsilyl)trifluoroacetamide/trimethylchlorosilane (99/1); Supelco). The reaction was carried out at 70 °C for 20 min, followed by incubation at room temperature for at least 2 h. After centrifugation (10 min, 14 000g), the samples were ready for GC-MS analysis. Silylation experiments indicated the number of exchangeable sites as -NH2, -OH, and -CO2H groups. Analyses. GC-MS analysis was carried out on a HP6840/ HP5973 apparatus (Agilent Technologies, Les Ulis, France) equipped with an MDN-12 fused silica capillary column (30 m, 0.25 mm internal diameter, 0.25 µm film; Supelco). The injector was used in the split mode, with a split ratio of 50:1 and an injection volume of 2.5 µL. The oven temperature was held at 70 °C for 4.5 min and then increased to 240 °C at a rate of 50 °C/min and held for a further 20 min. To detect fipronil, its standard metabolites, and new photoproducts, samples were analyzed in the full SCAN mode (50-550 mass range). The following retention times (minutes) were determined for standard chemicals: 2,4,6-trichloroaniline, 8.33; desulfinyl-fipronil, 10.16; sulfide-fipronil, 11.17; fipronil, 11.29; sulfone-fipronil, 13.11. Calibration curves were established for fipronil, its main pure derivatives, and aniline derivatives. For traces of unknown phenylpyrazoles and aniline derivatives, the calibration curves of fipronil and 2,4,6-trichloroaniline were used, respectively. Bioassays. The fourth-instar larvae of Aedes aegypti BoraBora strain were used for bioassays. The bioassays were performed in water, as described in Aajoud et al. (16), without feeding (Chaton et al. (11) demonstrated that feeding increased the toxicity of fipronil on A. aegypti by a factor of 3). The LC50 values were estimated by log-probit analysis. log Kow Estimation. The partition coefficient values were estimated using a reversal phase HPTLC-R18 F254 (Merck): solvent, methanol; references, trifluraline, rotenone, atrazine, carbofuran, and imidacloprid. The RM values were defined by the equation RM ) log(1/Rf - 1) (17). The equation of correlation between log Kow and RM values is
y ) -1.7403x2 + 3.9292x + 3.2438
R2 ) 0.9948
Results and Discussion Photodegradation of Fipronil. The exposure of fipronil to constant UV light for a period of 170 h led to the formation 4152
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FIGURE 2. Time course of fipronil photodegradation and photoproduct formation The results (n ) 2, SE does not exceed 5%) are expressed in equivalents of fipronil percents. (A) Fipronil exposed to UV light. The photoproduct group entitled “others” is constituted of about 6 new, minor unidentified derivatives. Key: b, fipronil; 9, desulfinyl derivatives; /, 4-unsubstituted derivatives; 2, sulfide derivatives; [, sulfone derivatives; 0, aniline derivatives; O, other derivatives. (B) Fipronil exposed to sunlight. Key: b, fipronil; 9, desulfinyl derivatives; 4, other derivatives. of a wide range of photoproducts, ranging from highly lipophilic to more hydrophilic compounds. The comparison of assays carried out under solar or UV lamp radiation gave the following results: (a) The evolution of the absorption spectrum of the solution between 200 and 400 nm was similar in both cases. (b) TLC chromatograms of the solutions exposed to sunlight showed the presence of numerous compounds having the same Rf values as those which were found after UV lamp irradiation. (c) GC-MS analysis of the extracts confirmed this situation through the identification of numerous common derivatives formed during the two experiments. (d) The time courses of fipronil degradation and the accumulation of desulfinyl-fipronil and other photoproducts were similar under both conditions (Figure 2). Taken together, these results suggest that the irradiation provided by the UV lamp mimics fairly closely the effect of sunlight. In the photodegradation experiments, six main photoproduct groups could be identified, based on the similarities in their chemical structures. Four of these groups are shown in Table 1 and include previously unidentified 4-unsubstituted, sulfide, and aniline derivatives. The other groups comprise the desulfinyl derivatives described below (Figure 3) and a group of compounds whose structure was not determined. This complexity disagrees with other studies which suggest the existence of a single phototransformation pathway with the desulfinyl derivative as the only product (14). GC-MS analysis was used to suggest plausible structures for several new photoproducts (Table 1). A possible 4-unsubstituted-fipronil derivative was identified, with a mass of 354, which was unable to be silylated. The fragmentation pattern was m/z 353 [M - 1]+, 325 [C6H2(Cl2)(CF3)(N2C3H)(NO2)]+, and 279 [C6H2(Cl2)(CF3)(N2C3H)]+, consistent with loss of H from the molecular ion, loss of an aldehyde group, and loss of NO2, respectively. The hypothetical structure
TABLE 1. Chemical Structures of Previously Identified and Novel Fipronil Photoproducts (Desulfinyl-fipronil and Derivatives Shown in Figure 3)a,b
a New derivative structures were established using mass spectra [MS, m/z (amu) (% relative abundance)] analysis and silylation experiments: (a) tR 10.9; nonsilylated, MS 353 [M - 1]+ (93), 325 [C6H2(Cl2)(CF3)(N2C3H)(NO2)]+ (31), 279 [C6H2(Cl2)(CF3)(N2C3H)]+ (100), 241 [C6H2(Cl2)(CF3)N2]+ (56), 213 [C6H2CL2(CF3)]+ (98), 69 [CF3]+ (18), other ions 229 (99), 166 (89), 81 (42); (b) tR 11.37, monosilylated, MS 386 [M]+ (16), 333 [M - F2(NH2)]+ (100), 317 [C6H3(CF3)ClC3N2(NH2)(CN)S]+ (31), 179 [C6H3(CF3)Cl]+ (78), 144 [C6H3(CF3)]+ (11), 69 [CF3] (48), other ions 351 (26), 221 (34), 207 (11); (c) tR 14.29, disilylated, MS 352 [M]+ (100), 255 [M - C2N(SH)(CN)]+ (52), 213 [C6H2Cl2(CF3)]+ (25), 143 [C6H2(CF3)]+ (13), other ions 290 (27), 179 (9), 69 (7); (d) tR 8.63, monosilylated, MS 229 [M]+ (100), 210 [M - F]+ (32), 194 [C6H2Cl(CF3)(NH2)]+ (11), 179 [C6H2Cl(CF3)]+ (22), other ions 158 (9); (e) tR 8.33, monosilylated, MS 195 [M]+ (100), 159 [M - Cl]+ (12), 124 [M - Cl2]+ (30), 88 [M - Cl3]+ (12), 73 [M - Cl3(NH2)]+ (8), other ions 97 (17), 62 (17); (f) tR 5.63, nonsilylated, MS 214 [M]+ (100), 195 [M - F]+ (25), 179 [C6H3(CF3)Cl]+ (79), 143 [C6H3(CF3)]+ (27), 74 [C6H3]+ (28), other ions 164 (23), 125 (19). b The asterisks in the table represent the following: (/) derivatives published by Hainzl and Casida (12); (//) derivatives published by Bobe´ et al. (13); (///) derivatives published by Ngim et al. (15).
FIGURE 3. Photodegradation products of desulfinyl-fipronil. proposed for this photoproduct was therefore 3-CHO,4-H,5NO2-fipronil. It could be formed from the 4-unsubstitutedfipronil derivative by transformation of the cyano group at position 3 and oxidation of the 5-amino group. Two novel sulfide derivatives were also detected. A minor product that could be monosilylated and showed a fragmentation pattern similar to that of desulfinyl-fipronil corresponded to the sulfide-fipronil with a monochlorinated phenyl group. In contrast, a photoproduct that accumulated to a higher level was detected, which presented a mass of 353 and which was able to be disilylated. The structure proposed on the basis of MS analysis (m/z 352, 255, 213) was
4-thiol-fipronil. In the sulfone group, the photoproduct corresponding to the sulfonic acid derivative previously described by Bobe´ et al. (13) and Ngim et al. (15) was detected at a trace levels, under UV and sunlight experiments. Finally, among the studied derivatives in the reaction mixture, the presence of compounds lacking the pyrazole ring was detected (Table 1). These corresponded to aniline derivatives with various chlorine substitutions on the benzene ring, thus testifying to the high degree of fipronil degradation under UV irradiation. The amide-fipronil derivative was not detected under our conditions. This was presumably due to the neutral pH of the reaction medium, since Ngim and Crosby (18) detected its presence under alkaline conditions and Bobe´ et al. (13) demonstrated its production at basic pH in the absence of light. Figure 2 shows the quantitative evolution of these six groups, during the 170-h UV exposure. A high rate of fipronil degradation was observed during the first 25-30 h, by which time the accumulation of desulfinyl-fipronil had declined to a very slow rate (Figure 2A), suggesting either the existence of another photodegradation pathway or the occurrence of VOL. 40, NO. 13, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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a steady state in the formation and degradation of the desulfinyl derivative. The rate of desulfinyl-fipronil accumulation also decreased with time in the sunlight experiment (Figure 2B) but did not reach a steady state after 70 h exposure. All fractions collected at 0, 24, 72, and 170 h were analyzed by GC-MS. Fipronil was rapidly photodegraded with approximately 50% removal after 60 h (Figure 2) and 75% after 170 h exposure. Degradation products such as sulfone (0.5%), 4-unsubstituted (0.5%), desulfinyl (0.3%), and sulfide (0.1%) were detected after only 1 h of exposure to UV light. At the end of the experiment, desulfinyl, sulfide, and 4-unsubstituted photoproduct groups represented 66% of the total mixture. Desulfinyl derivatives constituted the major group of photoproducts (31%), the principal molecule accumulating as a function of time being the desulfinylfipronil. The sulfide group represented 18% of the composition, with 9.5% 4-thiol-fipronil and 9% sulfide-fipronil. The 4-unsubstituted derivatives accumulated to a similar level. Sulfone derivatives accumulated to low levels, accounting for 1.5% at 24 h and 0.8% after 170 h, suggesting that they may be further degraded. Aniline derivatives were also minor components of the photoproduct mixture, reaching 1% of the total composition after 170 h. Photodegradation of Desulfinyl-fipronil. Exposure of a hydro-alcoholic solution of fipronil to UV light or sunlight led to the formation of desulfinyl-fipronil as the main product, as reported previously (12-15, 19). When pure desulfinylfipronil was exposed to UV light, the formation of two other derivatives (DFa, DFb) was observed, resulting from rearrangements on the aromatic ring (Figure 3). The DFb product had lost a chlorine substituent from the aromatic ring, whereas, in the DFa product, the chlorine atom at aromatic position 6 had been substituted by a CF3 group. No cleavage of the pyrazole ring was observed under the reaction conditions used. The time course of desulfinyl-fipronil photodegradation is presented in Figure 4. The desulfinyl-fipronil concentration decreased by only about 1.4%, i.e., from 700 to 690 µM in 96 h. In agreement with this, the two metabolites accumulated to only a very low level, with 1% of DFb and 0.2% of DFa being formed after 96 h of irradiation. These results show that desulfinyl-fipronil is chemically and photochemically fairly stable under UV light, in comparison to fipronil irradiated under the same conditions (Figure 4A). Existence of Two Photodegradation Pathways. The results presented above suggest the existence of two distinct pathways of fipronil photodegradation. The evidence for this is as follows: (1) Since the desulfinyl-fipronil produced by photodegradation is stable, then the kinetics of formation can be directly deduced from the accumulation curve. (2) As the rate of desulfinyl-fipronil production decreases sharply after 30 h, the phototransformation of fipronil, which continues at a relatively high rate, must occur via another pathway. (3) The accumulation kinetics of the other photoproducts identified suggest that the second pathway proceeds via cleavage of the pyrazole ring, yielding aniline derivatives, which are then further degraded. A hypothetical scheme of fipronil photodegradation based on these arguments is shown in Figure 5. Insecticidal Properties of Photodegradation Products. The insecticidal activity of the reaction mixture was tested at different times on Aedes aegypti using a standard assay. This test was chosen for its sensitivity and reproducibility and for the easy bioavailability of all molecules for the insect organism. Surprisingly, the toxicity of the reaction mixture did not decrease significantly during the photodegradation experiment, suggesting that the photoproducts themselves are insecticidal (Figure 4B). Such a result required to be understood, leading therefore to compare the insecticidal 4154
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FIGURE 4. (A) Comparative kinetics of fipronil and desulfinyl-fipronil photodegradation. The results (n ) 2, SE does not exceed 5%) are expressed in equivalents of fipronil percents. Key: b, fipronil; 9, desulfinyl-fipronil. (B) Variation in insecticidal activity during photodegradation. Key: light stippling, fipronil solution exposed to UV light; heavy stippling, desulfinyl-fipronil solution exposed to UV light. activity and lipophilicity (log Kow) of the main products accumulated in the studied reaction to that of other phenylpyrazole derivatives previously studied in our laboratory (Table 2). As reported previously (16), the LC50 values of sulfonefipronil and sulfide-fipronil were 2-fold lower than for fipronil itself. Since the lipophilicity of these derivatives was similar to that of fipronil (suggesting a similar concentration inside the membrane surrounding of the chloride channel), their increased toxicity might be explained by an increase in their specific activity on this chloride channel, at least in mosquitoes. Furthermore, a recent study has demonstrated that both fipronil and its sulfone derivative are able to block the GABA- and glutamate-chloride channels (20). Desulfinylfipronil also showed a log Kow similar to that of fipronil but was about 3 times less toxic, a decrease which can be attributed to the loss of the sulfur atom. A similar loss of toxicity was observed in Procambarus clarkia (Crustacea) (5, 21). Although no conclusions can be drawn about the toxicity of the 4-unsubstituted derivatives shown in Table 1, it is
FIGURE 5. Hypothetical photodegradation pathways of fipronil under aerobic conditions in water. The asterisk indicates the hypothetical pathway proposed by Bobe´ et al. (13).
TABLE 2. Comparative Toxicity of Fipronil and Various Photoproducts and Hydrophilic Metabolites in an Aedes aegypti Bioassaya product type photoproducts
hydrophilic metabolites
product names fipronil desulfinyl-fipronil sulfone-fipronil sulfide-fipronil 2,4,6-trichloroaniline amide-fipronil amide-sulfone-fipronil acid-fipronil
lipophilicity: log Kow 2.74b 2.42 2.81 2.42 4.06 1.10 0.70 -0.1
toxicity: LC50 (24 h), nM 19.9c 62.8c 8.8c 8.8c 268000 121.6c 1065 92260
a The concentration of ethanol (used to dissolve the pure compound) in the bioassay did not exceed 1% (v/v) and did not cause mortality. Assays were carried out in triplicate, and the average LC50(24 h) values were calculated. b This value was confirmed by a 14C-fipronil partition experiment between n-octanol/water, pH 6, 20 °C, triplicate. c Results published by Aajoud et al. (16).
likely that the other phenylpyrazole photoproducts identified, all of which retain the sulfur atom, are also lipophilic and, at least as toxic as fipronil. As a whole, all the results shown in Table 2 suggest that all the derivatives in which the phenylpyrazole ring is present possess high insecticidal properties as far as they are lipophilic. On the other hand, aniline derivatives, represented by 2,4,6-trichloroaniline, apparently have little insecticidal activity. Their mode of action is obviously not due to a specific effect on the chloride channel nor can it be explained by an uncoupling effect on oxidative phosphorylation, since these compounds are unable to
exchange protons at biological pH (4-8). The weak toxicity of these compounds may be due to their potential chaotropic activity (17). During the transformation of fipronil under natural conditions, another group of compounds is formed through hydrolysis of the 3-CN substituent (unpublished observations). The hydrophilicity of these metabolites is much greater than that of the principal photodegradation products, and their insecticidal activity is 6-5000 times lower than that of fipronil (Table 2). Thus, the photodegradation of fipronil leads to the initial accumulation of a mixture of products that retain VOL. 40, NO. 13, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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a high level of insecticidal activity, but the further degradation of these products, either by photolysis to aniline derivatives or by conversion to hydrophilic metabolites, should lead to a decrease in the overall toxicity. Ecological Impact. To deduce the ecological impact of fipronil photodegradation from experiments carried out under artificial conditions (UV light, high insecticide concentration, in the presence of 50% ethanol) requires caution. However, previous studies in our laboratory (16) and preliminary tests comparing the effects of UV lamp irradiation to solar irradiation, as well as the influence of increasing concentrations of ethanol and the effect of initial fipronil concentration (using 14C-fipronil to obtain the appropriate sensitivity at low concentration (16)), allowed us to conclude that the in vitro conditions chosen were qualitatively representative of what may occur in natura. Several studies have previously demonstrated that desulfinyl-fipronil is the major photodegradation product of fipronil (12-16, 19), and this point was clearly confirmed here. Indeed, in a recent study in which irradiation was carried out at high light intensities for short periods of time, at low fipronil concentrations, and in the presence of dissolved organic matter (DOM), desulfinyl-fipronil was the only photoproduct detected (14). The very different conditions used in the present study (low light intensities, long incubation time, high fipronil concentration, absence of DOM) allowed the detection and identification of a large number of additional photoproducts. As discussed above, the variety of derivatives observed could not be attributed to the presence of ethanol, since qualitatively similar results were obtained in the absence of ethanol and at low fipronil concentrations. Conversely, the use of a high initial concentration of fipronil (700 µM) in 50% ethanol allowed the formation of sufficient quantities of minor photoproducts for structural identification and prevented microbial development during the long-term incubation. Measurement of the kinetics of photoproduct formation and the identification of novel photoproducts led us to suggest that photodegradation of fipronil occurs via two distinct pathways (Figure 5). The first one leads to accumulation of a lipophilic, insecticidal derivative, desulfinyl-fipronil, the formation of which results from a complex, probably multistep, rearrangement of the 4-CF3-SO chain. The second pathway involves the formation of several phenylpyrazole derivatives, some of which possess a 4-sulfurated chain and others of which do not. Among this group of derivatives, one or more can be degraded further by opening of the heterocycle ring, leading to aniline compounds. However, these compounds never accumulate to high levels, suggesting either that their formation is relatively slow or that they are rapidly mineralized. The chemical or photochemical degradation of these compounds is poorly documented. However, in natura, the biological degradation of such compounds has been clearly demonstrated (22, 23). The contribution of this second pathway to fipronil degradation under natural conditions remains to be investigated. Situations in which it might be expected to work efficiently in natura are on the leaves of plants, after foliar treatment (acridian control; 3, 6), or on the hair of animals treated against fleas (24) and ticks (25). The degree of interaction between the two degradative pathways requires further study, (1) to identify clearly the starting point of each distinct pathway and (2) to look for possibilities of displacing the equilibrium between the two, to decrease the formation of desulfinyl-fipronil. The second pathway presents analogies with the biological pathway of metabolic degradation, which involves the sulfone- and sulfide-fipronil as intermediates and possibly also the thioland 4-unsubstituted-fipronil, and may lead to opening of the heterocycle ring (16). The fact that the 3-cyano group remained essentially unchanged during photodegradation (except for the 4-un4156
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substituted metabolite) is noteworthy. As the series of 3-cyano-substituted phenylpyrazoles identified in this work retain a high insecticidal activity, it would be interesting to determine whether the stably accumulated desulfinyl-fipronil could be biologically transformed by 3-amido or 3-carboxy substitutions, thus significantly reducing the insecticidal activity. Such a reaction is certainly possible, in the soil, in plants, and in water, as a result of biological hydrolysis. The overall ecological impact of fipronil photodegradation has therefore to be considered as a combination of reactions partly induced by light and partly due to living organisms. In this regard, we have observed that the desulfinyl-fipronil slowly disappears in the soil, in seed coated corn or sunflower cultures (unpublished results). This phenomenon might be due to the action of soil microorganisms and might therefore reduce the potential danger of desulfinyl-fipronil accumulation along food chains. It should also be pointed out that although some uses of fipronil, such as acridian control, involve exposure to light and might therefore lead to desulfinyl-fipronil formation, this is not the case for other applications, such as treatment within the soil, for instance in the control of wireworms, or inside plants, in which the insecticide is protected from light irradiation.
Abbreviations desulfinylfipronil
5-NH2-4-CF3-3-CN-1-[2,6-dichloro-4(trifluoromethyl)phenyl]pyrazole
sulfide-fipronil
5-NH2-4-CF3S-3-CN-1-[2,6-dichloro-4(trifluoromethyl)phenyl]pyrazole
amide-fipronil
5-NH2-4-CF3SO-3-CONH2-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]pyrazole
sulfone-fipronil
5-NH2-4-CF3SO2-3-CN-1-[2,6-dichloro4-(trifluoromethyl)phenyl]pyrazole
amide-sulfone- 5-NH 2 -4-CF 3 SO 2 -3-CONH 2 -1-[2,6fipronil dichloro-4-(trifluoromethyl) phenyl]pyrazole acid-fipronil
5-NH2-4-CF3SO-3-COOH-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]pyrazole
DFa
5-NH2-3-CN-1-[2-chloro-4,6-bis(trifluoromethyl)phenyl]-4-(trifluoromethyl)pyrazole
DFb
5-NH2-3-CN-1-[2-chloro-4-(trifluoromethyl)phenyl]-4-(trifluoromethyl)pyrazole
GABA
γ-amino butyric acid
Acknowledgments We are grateful for Dalila Azrou-Isghi and Joe¨lle Patouraux for technical assistance and to the Ministe`re del’ecologie et du de´veloppement durable of France for financial support.
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Received for review November 29, 2005. Revised manuscript received April 11, 2006. Accepted April 24, 2006. ES0523946
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