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a Dipartimento di Produzione Vegetale, Università della Basilicata, Via N. Sauro 85, I 85100 Potenza, Italy
b Dipartimento di Chimica, Università della Basilicata, Via N. Sauro 85, I 85100 Potenza, Italy
c L.A.C.E., Université Claude Bernard Lyon I, 43 Bd du 11 novembre 1918, F 69622 Villeurbanne Cedex, France
d GSF-National Research Centre for Environment and Health, Institute of Ecological Chemistry, Ingolstädter Landstrasse 1, D 85764 Neuherberg-München, Germany
* Corresponding author (bufo{at}unibas.it)
Received for publication February 9, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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,* transition. The role of free radicals in the photodegradation of acifluorfen was determined and a radical mechanism was proposed. Toxicity tests against Daphnia magna Strauss showed that acifluorfen was not toxic at a concentration of 0.1 mM; however, photoproducts formed after 36 h of UV exposure of the herbicide induced a remarkable toxicity to the test organism. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Diphenyl-ethers are highly active herbicides used for the selective control of broadleaf weeds in large-seeded legume crops. Acifluorfen 1, as illustrated in Scheme 1 (Fig. 1) , is a nitro-diphenyl ether herbicide (Johnson et al., 1978), acting as protoporphyrinogen oxidase inhibitor, and is used to control monocotyledonous and broadleaved weeds at rates in the range of 0.38 to 0.6 kg of active ingredient (a.i.) ha-1. Solubility in water is 120 mg L-1. It has acute oral toxicological effects on mammals with oral LD50 values between 1370 and 2050 mg kg-1 in rats, an acute percutaneus LD50 value of 3680 mg kg-1 for rabbit, and moderate fish toxicity between 17 and 61 mg L-1 (Worthing and Hance, 1994).
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Photochemical reactions of substituted diphenyl ethers in liquid phases have received attention (Nakagawa and Crosby, 1974; Ruzo et al., 1980), but no information is available on the photochemical behavior of acifluorfen and its photoinduced toxicity. It is only known that in aqueous solutions acifluorfen degraded following first-order kinetics, with loss of the carboxylic acid group (Pusino and Gessa, 1991). Diphenyl ether, 1,4-diphenoxybenzene, and some substituted diaryl ethers (methyl and/or methoxy derivatives) have been irradiated using unfiltered UV light from a high-pressure mercury lamp at 25°C. The reactions observed were cleavage of the ether bond(s), followed by H-abstraction from the solvent yielding phenols and benzene derivatives, and a photo-Claisen type rearrangement yielding 2- and/or 4-hydroxybiphenyl derivatives (Hageman et al., 1970). Another experiment showed that diphenyl ether was converted into o-phenylphenol, p-phenylphenol, and a small amount of phenol, by UV light in various solvents. p,p'-Ditolyl ether was similarly converted into 2-(p-tolyl)-4-methylphenol and p-cresol, indicating that the photochemical rearrangement proceeds via C–O bond cleavage and recombination of the radical (quinoid) fragments. These reactions were reported as intramolecular and occurred via an excited singlet state or via a short-lived triplet (Ogata et al., 1970).
Sunlight photolysis of the herbicide nitrofen (2,4-dichlorophenyl p-nitrophenyl ether) in aqueous methanol was represented as a photonucleophilic displacement of nitrophenate by the hydroxide ion of water, giving rise to 2,4-dichlorophenol and p-nitrophenol. Oxygen did not influence the formation of derivative photoproducts (Nakagawa and Crosby, 1974).
Other substituted diphenyl ethers were studied under 300 nm irradiation in water, cyclohexane, and methanol solutions. The major reaction pathways involved reductive dehalogenation, decarboxymethylation, reduction of nitro substituents, and cleavage of the ether linkage to yield phenols. Nucleophilic substitution by the solvent was also observed (Ruzo et al., 1980).
Photodegradation of 3-phenoxybenzoic acid was also investigated under xenon lamp irradiation (
> 290 nm) in aqueous phosphate buffer at pH 7. In these conditions hydroxylation of both phenyl rings and ether cleavage were the main degradation pathways, and ether cleavage proceeded via reaction with a hydroxyl radical excluding the photonucleophilic substitution by water molecule (Katagi, 1992).
The photochemistry of diphenyl ethers is relevant to the dependence of their activity on light absorption. Our objectives were to determine the photochemical behavior of acifluorfen in various solvents and the photoinduced toxicity arising under UV irradiation. Data obtained in this study will be useful in understanding the environmental behavior of this herbicide.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Ultraviolet spectra were recorded using a Uvikon 930 spectrophotometer (Kontron Instruments, Milan, Italy). Fluorescence and phosphorescence spectra were obtained with a Jobin Yvon (Opera-Milan, Italy) 3D spectrofluorimeter. The fluorescence spectrum was recorded in anhydrous acetonitrile using diphenyl-anthracene (DPA) as the actinometer. The phosphorescence spectrum was recorded, under liquid nitrogen, in the mixture of ethylether, 2-methylbutane, and ethanol (5:5:2) (EPA), using acetophenone as the actinometer.
The photodegradation of acifluorfen was studied for 140 h in a solar simulator (Suntest CPS+; Heraeus, Hanau, Germany) furnished with a 1.1 kW xenon arc (protected with a quartz plate; total passing wavelength: 300 nm < < 800 nm). The irradiation chamber was maintained at 25°C using both the circulating water from a thermostatic bath and a conditioned airflow. Another experiment was carried out for 50 h by use of a Pyrex reactor (250 mL) (Steroglass, Perugia, Italy) equipped with a high pressure mercury arc (Philips HPK 125 W; total passing wavelength: 240 nm < < 580 nm; Philips Lighting, Monza-MI, Italy) protected with a quartz thimble and surrounded by a water jacket to maintain temperature at 25°C.
Solutions (0.1 mM) of the herbicide in water, acetonitrile, methanol, and n-hexane were used in the degradation tests. Aqueous solutions were prepared using pH 7 phosphate buffers with an ionic strength of 0.05 M. All glassware was sterilized by autoclaving for 60 min at 121°C before use. All solvents and solutions were filtered using sterilized ultrafilters (0.2 µm). Aseptic handling materials and laboratory facilities were used throughout the study to maintain sterility. All working solutions were divided into two parts. One of these portions was used for the irradiation experiment, the other as a control, which was kept in the dark. All reactions were performed in triplicate in the atmospheric environment. In the experiments carried out under xenon lamp irradiation, quantum yields (Guittonneau et al., 1995) were also determined using uranyl oxalate as the actinometer (Murov et al., 1993).
In order to understand the mechanism of the photochemical degradation of acifluorfen the following experiments were performed with the solar simulator system (Suntest):
(i) Photodegradation of Compound 1 (0.10 mM in acetonitrile) either in the presence of naphthalene (1 mM), as a singlet quencher, or in the presence of biphenyl (0.25, 0.5, and 1 mM), as a triplet quencher;
(ii) Photodegradation of Compound 1 (0.10 mM in acetonitrile) either in the presence of 2,2'-azobisisobutyronitrile (AIBN, 1 mM), as a radical initiator, or in the presence of benzoquinone (1 mM), as a radical inhibitor;
(iii) Photodegradation of Compound 1 (0.10 mM in acetonitrile) without the presence of oxygen, by using argon to remove air from the reaction vessel.
A separate experiment was carried out under mercury arc irradiation for the identification of photoproducts. To avoid the formation of low quantities of many by-products, which could be a hindrance for understanding the degradation pathway, the reaction was stopped after 2 h of irradiation, when 20% of starting compound was converted, and after 36 h, when the reaction could be considered at completion (98% acifluorfen degradation).
Analytical Procedures
The depletion with time of Compound 1 was monitored via high performance liquid chromatography (HPLC) using a HP 1090 instrument (Hewlett–Packard, Palo Alto, CA) equipped with a diode array detector. A C18 PerkinElmer (Wellesley, MA) HS-5HCODS column (250 mm long, 4.6 mm i.d.), 5-µm particle size, was used at a 0.6 mL min-1 flow rate. An acetonitrile and water (7:3 [v/v]; H3PO4, pH = 3) mixture was used as the mobile phase. The retention time of acifluorfen was 5.84 min.
For the identification of photoproducts, a HP 5971 mass selective detector on a HP 5890 gas chromatograph was used (OV-1 capillary column between 70 and 250°C [12°C min-1]). Further analyses were achieved on a Finnigan SSQ 7000 mass spectrometer (ThermoFinnigan, Brenen, Germany), coupled with another HP 5890 chromatograph (DB 5-MS capillary column between 90 and 300°C [10°C min-1]).
The injection of pure Compound 1 in the gas chromatography–mass spectrometer (GC–MS) gave Compound 2, as illustrated in Scheme 1 (Fig. 1), as the unique detectable product because of the thermal degradation of acifluorfen in both the gas chromatographic systems. For this reason, flow injection technique (FIA) coupled with electrospray ionization (ESI) on quadrupole ion trap (QIT) mass spectrometer (Finnigan LCQ) was also used to characterize the reaction mixture obtained either at 2 or 36 h of irradiation. Standard or sample solutions (1 µL) were injected at a flow rate of 200 µL min-1 using a solvent system of acetonitrile and water (1:1 [v/v]; 5 mM NH4OAc, 1% formic acid). The heated inlet capillary was maintained at 130°C and nitrogen was employed as both the sheath and auxiliary gas (10 L min-1). Electrospray ionization was performed in the negative and positive ion modes and a typical ESI voltage was 1.3 kV. Under these conditions no thermal decomposition of the pure standard acifluorfen was observed.
The reaction mixture was extracted with ethyl acetate, concentrated under a mild vacuum in a rotary evaporator, and nitrogen fluxed. Separation of photoproducts was achieved by thin layer chromatography (TLC) on 60F254 plates (Merck Eurolab, Milan, Italy) using a solvent system of acetonitrile, chloroform, and methanol (30:5:65 [v/v/v]). Recovered products were analyzed by [1H] NMR spectroscopy. A Brucker (Rheinstetten, Germany) 300 AM instrument was used and the solvent was CDCl3.
Daphnia magna Test
Toxicity tests on D. magna were performed in triplicate following OECD Guideline 202 for testing of chemicals (Organisation for Economic Co-operation and Development, 1993). A D. magna Strauss monoclonal culture was used. The vitality of the test population used in each test was determined by a reference toxicant test with K2Cr2O7. The endpoint for effect determination was immobility after 24 h, which was used for calculation of the 24-h EC50 (effective concentration 50%, i.e., the concentration at which 50% of examined Daphnia were immobilized). An organism was defined to be immobile if it was not able to swim within 15 sec after gentle stirring. Probit analysis was used for the calculation of EC50 and 95% confidence limits.
The pure active ingredient of the herbicide (50 mg L-1) was dissolved in a buffer solution (pH 7). The test was performed in 20 mL of water saturated with oxygen. In each of the 20-mL flasks, 20 individuals not older that 24 h were used. The same experiment was carried out on the herbicide solutions after 2 h (20% acifluorfen degraded) and 36 h (98% of acifluorfen degraded) of irradiation, respectively. The test was carried out in the dark at 20°C. The water fleas were not fed during the test. The test compounds were by visual examination completely dissolved at the different test concentrations. A pre-test was necessary to chose the dilution range in the main test.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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* transitions. Table 1 summarizes the absorption maxima and the corresponding extinction coefficients evaluated by the Lambert–Beer law. Acifluorfen shows a low emission band in acetonitrile at 300 nm (exc = 288 nm) in the fluorescence spectrum (Fig. 3)
, and an emission band at 410 nm in the phosphorescence spectrum with 
ph = 1.5 x 10-3 (Fig. 4)
. These data allow the calculation of the energy of the first excited singlet state at 402.9 kJ mol-1 
and the energy of the lowest excited triplet state at 291.8 kJ mol-1. These values are compatible with the excited electron transfer from the higher level to the lower one (the direct transfer from the ground state to the triplet state is forbidden).
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Identification of Photoproducts
When the reaction was stopped after 2 h of irradiation (Sample A) the gas chromatography–mass spectrometry (GC–MS) spectra allowed the identification of two derivatives (Scheme 1 [Fig. 1]). The first, 2-chloro-1-(4-nitrophenoxy)-4-trifluoromethylbenzene (nitrofluorfen), is referred to as Compound 2 [MS, m/z (relative abundance): 319 (33%), 317 (M+, 100), 298 (13), 289 (15), 287 (40), 236 (67), 217 (14), 196 (13), 195 (12), 179 (11), 139 (13), 132 (10), 92 (14), 75 (19), 63 (18)]. The second, 5-[2-chloro-4-(trifluoromethyl) phenoxy]-2-nitrophenol (hydroxy-nitrofluorfen), is referred to as Compound 3 [335 (18%), 333 (M+, 52), 280 (17), 252 (100), 223 (11), 179 (8), 132 (8), 69 (16), 63 (19)].
When the photochemical reaction was allowed to run its course, after 36 h irradiation (Sample B), in addition to Compounds 2 and 3, other substances were detected in the reaction mixture (Fig. 7) . These included 2-chloro-4-(trifluoromethyl)phenol, referred to as Compound 4 [198 (33%), 196 (M+, 100), 179 (15), 177 (35), 161 (15), 146 (22), 132 (25), 113 (13), 63 (19)]; 5-trifluoromethyl-5'-nitrodibenzofuran, referred to as Compound 5 [281 (M+, 100%), 262 (10), 251 (25), 235 (23), 222 (20), 207 (68), 157 (11)]; and 2-[2-chloro-4-(trifluoromethyl)phenoxy]-cyclopentadien-1-ol, referred to as Compound 6 [278 (12%), 276 (M+, 28), 242 (14), 241 (100), 207 (9), 69 (16)]. In the last case (Sample B), many signals due to a host of small molecules ranging from 53 to 99 m/z were also recorded.
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The [1H] NMR spectra showed the formation of two photodegradation products in Samples A and B (chemical shifts are reported in 
): Compound 2, 7.70 (s, 1 H) and 7.30 to 7.00 ppm (m, 6 H); and Compound 3, 7.72 (s, 1 H), 7.51 (s, 1 H), 7.2 to 7.0 (m, 4 H), and 3.5 ppm (bs, 1 H). Compound 5 was also recognized in Sample B: 7.81 (s, 1 H), 7.75 (s, 1 H), and 7.2 to 6.9 ppm (m, 4 H).
Mechanism of Photodegradation
In order to elucidate the mechanism of the photochemical degradation of acifluorfen we wished to clarify whether the photochemical reaction occurred through the first excited singlet state or through the first excited triplet state.
Kinetic data of the reactions performed in the presence of either a singlet (naphthalene 
) or a triplet quencher (biphenyl 
) are shown in Table 3. The presence of a singlet quencher seems to inhibit the reaction while the presence of triplet quencher does not; on the contrary, we observed an increase of the degradation rate. This behavior is probably due to the capability of biphenyl to sensitize the reaction by energy transfer; in fact, the energy of the first excited singlet state of biphenyl is 418 kJ mol-1 (Murov et al., 1993), a value very close to acifluorfen (ES, 402.9 kJ mol-1). These results are in agreement with a mechanism involving the first excited singlet state. Furthermore, the degradation of Compound 1 was accelerated in the presence of a radical initiator (2,2'-azobisisobutyronitrile, AIBN). The presence of a radical inhibitor (benzoquinone) decreased the reaction rate (Table 3). These data also support the presence of radicals in the reaction.
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On the basis of all previous results we can formulate our opinion as to the initial mechanism of acifluorfen photodegradation (Scheme 1 [Fig. 1]): the first excited singlet state may undergo a homolytic photolysis of the carboxyl bond to give Compound 2 after abstraction of a hydrogen atom from the solvent. On the other hand, the radical intermediate can react with oxygen, giving Compound 3. The derivative Compound 2 is due to the decarboxylation of Compound 1, according to Pusino and Gessa (1991). Defoin et al. (1986) found the same behavior for phenylglyoxylic acid and ascribed a * transition to the CO2 loss under UV irradiation. In Compound 3, a hydroxyl group replaces the carboxylic function, as also found by Katagi (1992) on 3-phenoxybenzoic acid.
The products detected after 36 h of irradiation are depicted in Fig. 7. Compound 4 can be a residue due to the cleavage of the ether linkage as reported in the literature (Hageman et al., 1970). Product 5 could be derived from the cleavage of the ether linkage in Compound 1 followed by the rearrangement of the molecule (Scheme 2 [Fig. 8] ) in agreement with Ogata et al. (1970). In our case the dibenzofurane intermediate was not successively converted to the diphenyl derivative.
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Toxicity Evaluation
Daphnia magna has been used for many years as a standard aquatic test species. In fact, chronic and acute tests with this microcrustacean are among the most frequently performed in aquatic toxicology (Behechti et al., 1998; Mark and Solbé, 1998). The choice of D. magna for use as a standard test species was strongly influenced by the following factors:
(i) It can be cultured in the laboratory, although culture on defined synthetic media is rather expensive;
(ii) It represents the zooplankton community, a major element of the freshwater food chain;
(iii) As a species of worldwide occurrence, the ecological relevance of the test results is recognized.
Acifluorfen in the concentration range used for the test (5–50 mg L-1) did not show acute toxicity to the model organism. The 24-h EC values for the D. magna immobilization in the reaction mixtures were calculated referring to the initial content of the pure active ingredient. Organism swimming was not significantly reduced during the test performed in the dilution range of the mixture after a 2-h irradiation (20% acifluorfen transformed, giving 13% Compound 2 and 7% Compound 3). Otherwise, the products obtained after 36 h of irradiation were toxic since the 24-h EC50 was 35.1 mg L-1 (95% confidence limit, range 29.7–41.4 mg L-1) and 24-h EC10 was 6.7 mg L-1 (95% confidence limit, range 5.9–9.7 mg L-1). In the 36-h mixture, acifluorfen was 98% transformed, giving 24% Compound 2, 38% Compound 3, and many other by-products that were not identified with exception of substances shown in Fig. 7 (not quantified). Unfortunately, owing to poor recovery of each by-product, it was not possible to perform Daphnia tests for individual photoproducts to establish if one substance in particular is responsible for the toxicity found in the 36-h mixture.
To better understand such a result, octanol–water partition coefficients (Kow) were calculated from structures of acifluorfen and discovered metabolites by using the Clog P 4.0 program from BioByte (Claremont, CA) (Leo, 1993). The log Kow of acifluorfen was -4.73. All values for metabolites were positive, ranging from 3.35 to 5.35. The Kow coefficient has been assumed as a valid index for the evaluation of the capability of chemicals to interact with the organic and/or biological environment (Scheunert, 1992). We could not find a correlation between log Kow and the toxic effects of the metabolite mixture (after a 36-h irradiation) on zooplankton organisms. Our opinion is that the immobilization of D. magna may be due to a possible synergistic effect of several acifluorfen by-products in the aqueous environment.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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, halflife; k, kinetic constant; 
