Cyclodextrin-enhanced fluorescence and photochemically-induced fluorescence determination of five aromatic pesticides in water

Analytica Chimica Acta 360 (1998) 129±141

Cyclodextrin-enhanced ¯uorescence and photochemically-induced ¯uorescence determination of ®ve aromatic pesticides in water Atanasse Coly1, Jean-Jacques Aaron* Institut de Topologie et de Dynamique des SysteÁmes de l'Universite Denis DIDEROT, Paris 7, Laboratoire Associe au CNRS, URA 34, 1, rue Guy de la Brosse, 75005 Paris, France Received 14 July 1997; received in revised form 24 November 1997; accepted 3 December 1997

Abstract The effect of b-cyclodextrin (b-CD) and hydroxypropyl-b-cyclodextrin (HP-b-CD) aqueous solutions upon the ¯uorescence and photochemically-induced ¯uorescence (PIF) properties of ®ve pesticides, including coumatetralyl, pirimiphos-methyl, chlorpyriphos, deltamethrin and fenvalerate was investigated. A 1:1 stoichiometry was found for the b-CD and HP-b-CD complexes formed with all compounds. Binding constant values, ranging between about 90 and 830 Mÿ1 were calculated using the iterative nonlinear least-squares regression approach. Cyclodextrin-enhanced ¯uorescence and PIF methods were developed for the determination of these pesticides with linear dynamic ranges over two orders of magnitude, and limits of detection (LOD) between 0.2 and 54 ng mlÿ1 according to the compound. Application to the analysis of tap water and river water samples yielded satisfactory recoveries (88±116%). The method seems to be suitable for environmental water analysis. # 1998 Elsevier Science B.V. Keywords: Pesticides; Cyclodextrins; Fluorescence; Photochemically-induced ¯uorescence; Water analysis

1. Introduction Since their discovery in 1891 by Villiers [1], cyclodextrins (CDs) have been the object of a number of applications as well as of basic research works [2±8]. In the last twenty years, their interesting properties have allowed to improve analytical methodologies and develop new concepts in quantitative [8±14] as well as qualitative [8,15±18] analytical chemistry. Among the *Corresponding author. Fax: (+33) 01 44 27 68 14. 1 On leave from the Faculte des Sciences et Techniques DeÂpartement de Chimie, Universite Cheikh Anta DIOP, Dakar, SeÂneÂgal. 0003-2670/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved. PII S0003-2670(97)00721-6

various analytical methods investigated for CD uses [19], (e.g., UV±Vis spectrophotometry, NMR, electrochemical analysis, chromatography, electrophoresis, etc.), luminescence spectrometry remains one of the most interesting ones for the analysis and characterization of CD inclusion complexes, because of the signi®cant analyte signal increment that is frequently induced. Indeed, larger is the signal increase provoked by the organized medium, more sensitive and accurate is the analytical measurement. However, relatively few works have been devoted to the spectro¯uorimetric study of CD complexes with pesticides [20±23]. The ¯uorescence signals of pesticides such as warfarin, an anticoagulant rodenticide

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[20,21], bromadiolone [21] and coumatetralyl [22], known to be very intense in organic media, but quenched in aqueous solutions, have been restored in the presence of CD. The formation of a 1:1 inclusion complex of warfarin with b-cyclodextrin (b-CD) has been reported by Marquez et al. [20], who applied this ®nding to the ¯uorimetric determination of this rodenticide in irrigation water. The absorption and ¯uorescence spectral properties of the complexes of two plant growth regulators (2-naphthyloxyacetic acid and 1-naphthylacetic acid) with b-CD have been investigated in order to characterize the inclusion processes involved [23]. Recently, synchronous ¯uorescence with variable angle scanning has been proposed for the resolution of an arti®cial ternary mixture of aminocarb, carbendazim and coumatetralyl in 10ÿ2 M b-CD solutions [22]. More generally, ¯uorescence enhancements have been reported for a number of other organic compounds in the presence of b-CD [24±35] or chemically-modi®ed b-CD [13,36,37]. In contrast, the effect of CD on the photochemically-induced ¯uorescence (PIF) properties has been explored in the case of only two photoreactive phenothiazines [38,39]. In the present work, we investigated spectro¯uorimetrically the inclusion properties of b-CD and 2hydroxypropyl-b-CD (HP-b-CD) with ®ve aromatic pesticides, including coumatetralyl, pirimiphosmethyl, chlorpyriphos, deltamethrin and fenvalerate. The ®rst two compounds are naturally ¯uorescent [40,41], while the three others exhibit strong PIF signals in organic solvents [41±44] or in organized media [43]. Therefore, in this study, we evaluated the analytical usefulness of b-CD and HP-b-CD for the ¯uorescence and PIF determination of these pesticides. CD-enhanced ¯uorescence and PIF methods were applied to the analysis of spiked water samples. 2. Experimental 2.1. Reagents Coumatetralyl (99% m/m), pirimiphos-methyl (98% m/m), chlorpyriphos (99% m/m), deltamethrin (99% m/m) and fenvalerate (99% m/m) (HPLC analytical reagent-grade) were purchased from Riedel-de Haen (Hannover, Germany) and used as received. b-

CD and HP-b-CD with average molecular substitution of 0.8 (Aldrich, Milwaukee, WI, USA) were used without further puri®cation. Spectroscopic-grade solvents including methanol (Merck, Darmstadt, Germany) dimethyl sulfoxide (DMSO) and acetonitrile (Aldrich) were utilized. Distilled water was used for preparing aqueous solution of cyclodextrins. Buffer solutions of different pH (1±12) were purchased from: Acros Organics (Geel, Belgium), Aldrich, Fluka (Buchs, Switzerland) and Merck. Sodium dodecyl sulfate (SDS) analytical reagent grade was obtained from Acros Organics and used as received. 2.2. Apparatus All ¯uorescence and PIF measurements were performed on a Kontron SFM-25 spectro¯uorimeter controlled by a Geocom microcomputer Model CPD 1420E and equipped with a thermostated cell compartment. Uncorrected ¯uorescence spectra were recorded and memorized using a Kontron SFM-25 data control and acquisition program. An Osram 200W HBO high-pressure mercury lamp with an Oriel Model 8500 power supply was utilized for photolysis reactions. The photochemical set-up included a lightbox, consisting of a fan, the mercury lamp and a quartz lens. A standard Hellma 1 cm pathlength quartz ¯uorescence cuvette was placed on an optical bench at 30 cm from the mercury lamp. For photolysis the solutions were stirred magnetically during the irradiation. All measurements were performed at 2018C, using the thermostated cell holder and a Lauda Model K4R thermostatic bath. 2.3. Procedure 2.3.1. Solution preparation Stock standard solutions of the pesticides (10ÿ3 M) were freshly prepared by dissolving the compound either in methanol (for deltamethrin) or in acetonitrile (for the other pesticides). Serial dilutions were performed to obtain working standard solutions. All solutions were protected against light with aluminium foil and stored in a refrigerator. Stock solutions of b-CD (0.016 M) and HP-b-CD (0.02 M) were freshly prepared with distilled water, and serial dilutions were made from these stock solutions.

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The CD: pesticide inclusion complexes were prepared by transferring 5±50 ml aliquots of the pesticide working standard solution (prepared in organic solvent) into a 5 ml volumetric ¯ask and adjusting to marker with the required CD solution volume, 1 ml of the optimal pH buffer solution and distilled water. The solutions were then shaken vigorously before irradiation and/or analytical measurements. 2.3.2. Analytical measurements and photolysis reaction For the ¯uorimetric method, the ¯uorescence intensity was monitored at the ®xed analytical excitation and emission wavelengths of the complexes. For the PIF method, an aliquot of the CD: pesticide complex solution was placed in a quartz cuvette and irradiated at room temperature with UV light for a ®xed time. Curves of ¯uorescence intensity (IF) vs UV irradiation time (tirr) were constructed at the analytical excitation (ex) and emission (em) wavelengths of the CD: pesticide photoproduct complex using 3 min time intervals. Linear calibration curves were obtained at these ex and em values by measuring the PIF signal corresponding to the optimum tirr. All ¯uorescence and PIF intensity measurements were corrected for the solvent signal with the appropriate blank. Fluorescence measurements were carried out in triplicate while PIF intensities were measured on at least two pesticide concentrations. In all cases, the results were expressed as mean values. Microcal Origin, version 4.00, application software was used for the statistical treatment of the data and the iterative nonlinear least-squares curves ®tting procedure based on the Marquardt algorithm. 2.3.3. Chlorpyriphos extraction in river water 5 ml of spiked river water containing 3520 ng mlÿ1 of chlorpyriphos was introduced in a 25 ml volumetric ¯ask in which 10 ml of dichloromethane was added. The solution was then stirred ultrasonically during 10 min before being placed in a separating funnel. The organic phase was isolated while the aqueous layer was used for a second extraction. After a third extraction the organic phase was collected and gently evaporated to dryness in a bath at 408C. The residue was then dissolved in 5 ml of distilled water and used for the analysis.

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2.3.4. Water sample analysis Three aliquots (0.25, 0.5 and 0.75 ml) of the spiked stock standard solution of water samples were introduced in 5 ml volumetric ¯asks in which the CD solution convenient volume 1 ml of the optimal pH buffer solution, and the required amount of distilled water were added before irradiation and/or analytical measurements. In these conditions, the maximum concentration of tap water or river water samples introduced in the solution to be analyzed did not exceed 15% (v/v). 2.3.5. Determination of the cyclodextrin: pesticide complex binding constants Stoichiometry and binding constants of the CD complexes were determined by measuring the ¯uorescence of PIF intensity of the pesticide (®xed concentration and optimal irradiation time) at its wavelength of maximum emission as a function of different CD concentrations (ranged from 0.00 to 0.013 M in the case of b-CD or 0.00 to 0.02 M in the case of HP-b-CD) and analyzing the data by means of the modi®ed Benesi-Hildebrand approach (or double reciprocal plot method) for 1:1 or 2:1 inclusion complexes [12,23,38,39]. For better estimation of binding constants, the non linear regression analysis (NLR) method was used on the basis of the direct ®tting of the observed ¯uorescence intensity, F versus the initial CD concentration, [CD]0 [45]. The following equation was used in the case of a 1:1 inclusion complex: Fˆ

F0 ‡ F1 K1 ‰CDŠ0 1 ‡ K1 ‰CDŠ0

(1)

where F0 is the fluorescence intensity of the Eq. (1) analyte essentially in the absence of analyte, F1 is the fluorescence intensity when all guest molecules are complexed with CD, F is the measured fluorescence intensity at each CD concentration and K1 is the binding constant. NLR analysis requires a preliminary evaluation of the complex parameters from the linear plots. The curve fitting procedure based on the Marquardt algorithm is based on Eq. (1), allowing one to calculate F at each CD concentration through iteration, i.e., by varying the values of the initial parameters F0, F1 , and K1. The goodness of the fit between the calculated and

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experimentally measured values of F was judged by means of the value of 2. The K1 values that gave the best fit (i.e., smallest 2) between the calculated and experimental values of F were chosen as the experimentally-determined constants. 3. Results and discussion 3.1. Effect of pH The in¯uence of the pH on the ¯uorescence and PIF intensity of the complexes under study was investigated (Fig. 1). For the ¯uorimetric method, the ¯uorescence signal increased with increasing pH and reached a plateau for pH values between 6±12 for coumatetralyl and 9±12 in the case of pirimiphos-methyl. Consequently, the optimal pH value chosen was 10 for pirimiphosmethyl, while a non-buffered aqueous solution was preferred for coumatetralyl since the ¯uorescence intensity in this medium showed no difference relative to that obtained in the buffer solutions of pH 6±12.

Fig. 1. Effect of pH on the fluorescence and PIF intensity of cyclodextrin (10ÿ2 M):pesticide complexes, (A) coumatetralyl (10ÿ7 M):b-CD, exˆ314 nm, emˆ380 nm; (B) pirimiphosmethyl (210ÿ6 M):b-CD, exˆ305 nm, emˆ366 nm; (C) chlorpyriphos (10ÿ5 M):b-CD, exˆ378 nm, emˆ428 nm, tirrˆ20 min; (D) deltamethrin (410 ÿ6 M):HP-b-CD, e x ˆ291 nm, emˆ314 nm, tirrˆ15 min; (E) fenvalerate (410ÿ6 M):b-CD, exˆ293 nm, emˆ333 nm, tirrˆ10 min.

For the PIF method, the variation of PIF intensity of the complex as a function of the pH was characterized by a well-de®ned maximum obtained at pH values of 4, 10, 11, respectively for chlorpyriphos, fenvalerate and deltamethrin. For all compounds, the pH was maintained at the optimal value by adding 20% (v/v) of the corresponding buffer solution. 3.2. Fluorescence and PIF spectral properties in CDs Because of the ability of CD to form complexes, the ¯uorescence signal of the analyte is generally enhanced; indeed the ¯uorophore introduced into the CD internal cavity is isolated from the surrounding water molecules and its excited state is shielded from quenching processes. We investigated the effect of CDs on the ¯uorescence and PIF spectral properties of the pesticides under study. The ¯uorescence and PIF spectra were recorded in water and at various CD concentrations (Figs. 2 and 3). Except for chlorpyriphos and deltamethrin, the addition of increasing CD amounts to the pesticide aqueous solutions resulted in small shifts of the excitation and emission maximum wavelengths. For instance, in the case of pirimiphosmethyl, the increase of HP-b-CD concentration produced a progressive blue-shift of the emission peak and an enhancement of the ¯uorescence intensity (Fig. 2). In all cases, the increase of b-CD or HP-bCD concentration resulted in an enhanced ¯uorescence or PIF signal (Figs. 2 and 3); the relative magnitude of this enhancement was found to depend on the type of cyclodextrin employed. Except for fenvalerate, HP-b-CD gave a better ¯uorescence increment than b-CD (Table 1), probably because of the different complexing abilities, in agreement with literature results [13,37]. The CD enhancement factors varied between 1.5 and 7.3 according to the compound. These CD-induced ¯uorescence or PIF signal enhancements can be attributed to several factors [37], including increase in the radiative rate constants, decrease in the degrees of freedom and in molecular motion, prevention of collisional deactivation, increase of the favorable microenvironment polarity or viscosity, and shielding of the excited singlet states from water molecules and molecular oxygen or other species present in the bulk aqueous solution.

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Fig. 2. Fluorescence emission spectra of pirimiphos-methyl (410ÿ6 M) in (A) water; (B) 810ÿ4 M; (C) 210ÿ3 M; (D) 610ÿ3 M; (E) 10ÿ2 M of HP-b-CD; exˆ304 nm; pHˆ10.

Fig. 3. PIF emission spectra of chlorpyriphos (610ÿ6 M) in (A) water; (B) 810ÿ4 M; (C) 3.210ÿ3 M; (D) 6.410ÿ3 M; (E) 9.610ÿ3 M; (F) 1.2810ÿ2 M of b-CD; exˆ378 nm; pHˆ4; tirrˆ20 min.

3.3. Effect of media As shown in Table 1, we compared the ¯uorescence and PIF intensity of the pesticides in CD media,

sodium dodecyl sulfate (SDS) micellar solutions and several organic solvents. The organic solvent used for each compound was the one giving the largest ¯uorescence or PIF signal [40,42]. While SDS gave

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Table 1 Effect of various media on the fluorescence and PIF intensity of aromatic pesticides Compound

Solvent a

Buffer pH

ex/em (nm)

IF b

topt (min) c

Coumatetralyl 10ÿ7 M

H2 O b-CD HP-b-CD SDS DMSO H2 O b-CD HP-b-CD SDS DMSO H 2O H2 O b-CD HP-b-CD SDS SDS DMSO H2O H2 O b-CD HP-b-CD SDS SDS MeOH H2O H2 O b-CD HP-b-CD SDS SDS MeCN

None f None f None f None f ± 10 10 10 10 ± None f 4 4 4 None f 4 ± None f 11 11 11 None f 11 ± None f 10 10 10 None f 10 ±

312/390 314/380 316/385 308/398 331/404 304/385 305/366 305/366 301/366 300/361 378/428 378/428 378/428 378/428 378/428 378/428 371/436 291/313 ± 291/313 291/312 291/318 291/317 291/317 293/333 293/333 320/362 320/362 293/340 293/340 293/333

1.7 11.3 12.4 1.0 29.0 1.0 1.9 2.9 3.1 4.7 1.0 1.2 3.7 3.2 2.0 2.0 7.2 1.8 ± 2.7 4.1 3.7 2.3 1.0 1.4 1.0 3.5 2.6 10.2 4.2 4.2

± ± ± ± ± ± ± ± ± ± ND d ND d 20 20 30 30 30 ND d NF e 22 22 22 16 12 08 08 08 ND d 08 08 08

Pirimiphos-methyl 210ÿ6 M

Chlorpyriphos 610ÿ6 M

Deltamethrin 410ÿ6 M

Fenvalerate 410ÿ6 M

a

Cyclodextrin concentration: 10ÿ2 M; Sodium dodecyl sulfate (SDS) concentration: 510ÿ2 M. Relative fluorescence or PIF intensity, corrected for the solvent signal and normalized to the lowest fluorescence or PIF intensity for each compound. c Optimum irradiation time, corresponding to the maximum PIF intensity (IF). d ND means that topt is not defined since the curve of IF vs irradiation time (tirr) has no maximum and is characterized by a continuous increase of the signal: IF was measured for tirrˆ25 min. e NF means non fluorescent. f Measurements were done in distilled water. b

¯uorescence and PIF signal values comparable to those obtained in CDs, DMSO produced generally higher pesticide signals than CDs. Although DMSO gives the greatest ¯uorescence intensity, this does not necessarily mean that it is the recommended medium for ¯uorimetric assays of the pesticides. Indeed, DMSO has generally a high blank signal; moreover, the use of cyclodextrins presents a distinct advantage over DMSO, since no extraction procedure is needed for determining real samples of pesticides in water. As

a consequence, analysis of pesticides should be more rapid in CD media. 3.4. Effect of organic solvent on the inclusion complex formation The in¯uence of the amount of organic solvent in the predominantly aqueous cyclodextrin solutions was checked in the case of the coumatetralyl:b-CD complex formation. For 10ÿ2 M b-CD solution, the

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Fig. 4. Effect of acetonitrile on the fluorescence intensity of the coumatetralyl (210ÿ7 M):b-CD (10ÿ2 M) complex, exˆ314 nm, emˆ380 nm.

¯uorescence signal of the complex was unaffected for acetonitrile/water mixture in the 0±5% (v/v) range. The ¯uorescence intensity decreased with increasing acetonitrile percentage until a value of 30% (v/v), and then increased for larger amounts of acetonitrile (Fig. 4). Consequently, the allowed maximum percentage of any organic solvent was chosen to be 1% (v/v) for all measurements in the presence of b-CD or HP-bCD. The decrease of ¯uorescence intensity observed in the 5±30% (v/v) acetonitrile range is probably due to the competition of acetonitrile molecules with pesticides for binding sites inside the protective CD cavity. This is in agreement with the decrease of cyclodextrin-analyte binding abilities noted when the concentration of urea [46] or several organic solvents [24,28,37,47] increases. 3.5. Effect of CD concentration The effect of increasing CD concentration was to enhance progressively the ¯uorescence and PIF signals of all pesticides under study. This corresponds to the behaviour generally observed for other organic compounds [12,20,23,37±39]. Our results show a signi®cant ¯uorescence and PIF intensity enhance-

Fig. 5. Influence of (
) b-CD and (*) HP-b-CD concentrations on the PIF intensity of (a) chlorpyriphos (610 ÿ6 M), exˆ378 nm, emˆ428 nm, pHˆ4, tirrˆ20 min and (b) deltamethrin (410 ÿ6 M), ex ˆ291 nm, em ˆ312 nm, pHˆ11, tirrˆ22 min. Markers type: (solid) experimentally determined points, (open) calculated points by the nonlinear fitting approach.

ments with increasing CD concentration (Fig. 5). However, in the case of these pesticides, we found the existence of two different types of evolution of the ¯uorescence signal vs CD concentration. The ®rst type

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of curve obtained for chlorpyriphos and fenvalerate was characterized by a continuous increase of signal without any well-de®ned maximum (Fig. 5(a)). In the second one, the emission intensity increased with increasing cyclodextrin concentration and levelled off at b-CD and HP-b-CD concentrations of about 10ÿ2 M, reaching a plateau value (Fig. 5(b)). This second type of behaviour was observed for coumatetralyl, pirimiphos-methyl and deltamethrin in both cyclodextrins. In order to ensure complex formation, the optimal CD concentration selected for all pesticides was 10ÿ2 M. 3.6. Photolysis studies 3.6.1. Pesticides exhibiting native fluorescence To establish a model for the photodegradation pathways of the naturally-¯uorescent pesticides (coumatetralyl and pirimiphos-methyl) in environment, we investigated the in¯uence of UV irradiation on their ¯uorescence signal at analytical excitation and emission wavelengths in water as well as in b-CD and HPb-CD media. In all instances, a signi®cant decrease of ¯uorescence intensity occurred with UV irradiation time (tirr), indicating a relatively rapid photodecomposition of both pesticides (Fig. 6). A very interesting ®nding is that the addition of b-CD and HP-b-CD caused a drastic increase in the photolysis rate of both pesticides with a more marked effect in the case of coumatetralyl, this rate increase being more important for HP-b-CD than for b-CD. Indeed, within a 30 min irradiation time, it remained only about 0.4±0.5% of the coumatetralyl initial ¯uorescence intensity, in the presence of CDs. This shows that more than 99.5% of coumatetralyl is degraded photolytically. In contrast, for pirimiphos-methyl, the ¯uorescence signal levelled off at about 15.7 and 13.7% of the initial value after the same irradiation time, respectively for b-CD and HP-b-CD. Furthemore, this residual ¯uorescence signal continued to decrease slowly with increasing UV irradiation time. No formation of ¯uorescent photoproduct(s) was observed since no change in the emission spectra occurred upon irradiation. Satisfactory linear relationships were obtained for the photolysis kinetic curves of ln(I/I0) vs tirr for coumatetralyl indicating a ®rst-order behaviour. Pirimiphos-methyl obeyed an apparent ®rst-order kinetic law only for very short irradiation times

Fig. 6. Effect of UV irradiation time on the fluorescence intensity of (a) coumatetralyl (410ÿ7 M) and (b) pirimiphos-methyl (410ÿ6 M) in (*) water, (
) b-CD 10ÿ2 M, and (‡) HP-b-CD 10ÿ2 M.

(1 min). The estimated half-lives depended on the solvent system used and were about 17, 3 and 1 min in the case of coumatetralyl and 8, 8 and 0.5 min for pirimiphos-methyl, respectively, in water, b-CD and

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HP-b-CD. The identity of the photoproduct(s) is currently under investigation in our laboratory. These experiments allow us to point out an interesting result from an analytical standpoint, i.e., these aromatic pesticides should not be illuminated by intense UV light, during ¯uorimetric measurements, since irradiation reduces signi®cantly the ¯uorescence signal and therefore, would decrease the sensitivity of the method. On the other hand, ¯uorimetry appears useful for monitoring the degree of photodegradation of both pesticides in the environment. 3.6.2. Pesticides exhibiting PIF signal To evaluate the kinetics of the ¯uorophore formation, we studied the evolution of PIF intensity with UV irradiation time, both for free and CD-complexed pesticides In the case of deltamethrin and fenvalerate (pyrethroid insecticides), the ¯uorophore formation kinetics have been described previously in organic solvent as well as in micellar SDS solution [42,43]. No change of the photodegradation behaviour was observed in the presence of CD. For chlorpyriphos, although the photochemical pattern was like that of pyrethroid insecticides in organic and aqueous micellar media, a signi®cant difference was noted in the presence of b-CD. Indeed, the curves of PIF intensity vs tirr were found previously to obey two different types of evolution, including (i) an increase of PIF intensity followed by a continuous decrease, and (ii) a continuous increase of signal with no well-de®ned maximum value [42,43]. A third type of behaviour arises for the photolysis of chlorpyriphos in b-CD: the ®rst part of the kinetic plot, characterized by a PIF signal increase is followed by a plateau region (Fig. 7). This shows that a dynamic equilibrium is established between the chlorpyriphos photoproduct (identi®ed by GC-MS as a dechlorinated chlorpyriphos) and its degradation product(s). The general structure of the three pesticides supports the possibility of the formation of an inclusion complex with the ¯uorophore ring before irradiation, assuming that the photodegradation process only takes place in the CD cavity. The rather large PIF enhancement factors (within 1.5±7.3) obtained are in agreement with the latter hypothesis. Another feature is that most of the various photoproducts obtained for fenvalerate (6), deltamethrin (4) and chlorpyriphos (1) have potentially different complexing abilities and competition

Fig. 7. Evolution of PIF intensity with irradiation time at different chlorpyriphos concentrations, (*) 210ÿ6 M; (
) 610ÿ6 M; (*) 10ÿ5 M in b-CD 810ÿ3 M; exˆ378 nm, emˆ428 nm, pHˆ4.

rates for binding sites with CD which would rather decrease signi®cantly the PIF signal. The photoreactivity of the three pesticides depends on the nature of CD. Deltamethrin behaves similarly in b-CD and HP-b-CD following the ®rst type of mechanism whereas fenvalerate obeys the ®rst type of mechanism in b-CD and the second one in HP-bCD. In the case of chlorpyriphos, the third mechanism is involved in b-CD (Fig. 7) and the ®rst one in HP-bCD. 3.7. Determination of cyclodextrin:pesticide complex stoichiometry and association constants The stoichiometry of inclusion complexes formed with b-CD and HP-b-CD and the magnitude of the corresponding association constants constitute analytically useful data. A typical double reciprocal plot for HP-b-CD:coumatetralyl complex is shown in Fig. 8. For all pesticides, a linear relationship is obtained (for the entire range of CD concentrations tested) when 1/(FÿF0) is plotted against 1/[CD]0 , with correlation coef®cients within 0.987±0.998. This indicates the

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A. Coly, J.-J. Aaron / Analytica Chimica Acta 360 (1998) 129±141 Table 2 Binding constants of aromatic pesticides with cyclodextrins Compound Fluorimetric method Coumatetralyl Pirimiphos-methyl PIF method Chlorpyriphos Deltamethrin Fenvalerate

Cyclodextrin

K (Mÿ1)

b-CD HP-b-CD b-CD HP-b-CD

56542 61539 17134 73659

b-CD HP-b-CD b-CD HP-b-CD b-CD HP-b-CD

9028 11619 627104 828124 31661 15046

from synchronous ¯uorescence measurements (669 Mÿ1) [22]. For the other pesticides, there are no data in literature. Fig. 8. Double reciprocal plot for HP-b-CD:coumatetralyl complex. A linear relationship (*) was obtained when the data are plotted assuming a 1:1 reaction scheme, and a downward concave curve (
) when the data are plotted assuming a 2:1 stoichiometry.

validity of the 1:1 stoichiometry model. In contrast, a downward concave curve is obtained when these data are ®tted for a 2:1 complex. This suggests clearly that the stoichiometry of the complex is not 2:1. The estimated values of K1 and F1 were then used for the nonlinear curve-®tting procedure. The theoretical F values calculated from Eq. (1) using estimated values of K1 and F1 are shown in Fig. 5 (open markers). The convergence between the calculated and observed F values is excellent, (2 values between 0.0025±0.0801). Table 2 summarizes the binding constants obtained by ¯uorimetric and PIF methods. Except in the case of fenvalerate, HP-b-CD presents a slightly better complexing ability relative to b-CD. Chlorpyriphos exhibits low K1 values relative to the other pesticides, probably because of the steric hindrance due to the presence of three chlorine atoms in the molecule, which may inhibit complexation. No correlation is found between the binding constant values and the hydrophobicity of the pesticides, which indicates that steric effects are probably predominantly involved in complex formation. Our value of the b-CD:coumatetralyl binding constant complex is in satisfactory agreement with that recently reported

3.8. Analytical figures of merit To evaluate the analytical interest of the CD approach, we have established the calibration graphs under optimal conditions for the complexes formed in both CD media. Linear calibration plots were obtained, and the analytical ®gures of merit were determined. All data are given in Table 3 together with the correlation coef®cients (r), limits of detection (LOD), linear dynamic ranges (LDR), and relative standard deviations (RSD). For each compound, ¯uorescence and PIF measurements were performed at least for ten different concentrations. The LODs were particularly low, ranging from 0.2 to 54 ng mlÿ1. The LODs obtained in CD are much lower than those reported for deltamethrin by a PIF method in methanol (33 ng mlÿ1) [42] and SDS (11 ng mlÿ1) [43], chlorpyriphos by multivariate spectral analysis and derivative spectrophotometry (100 ng mlÿ1) [48] and coumatetralyl by synchronous ¯uorimetry in b-CD (17 ng mlÿ1) [22]. However, our LODs values are larger than those found for coumatetralyl by ¯uorimetry in ethanol (0.07 ng mlÿ1) [40], pirimiphosmethyl by ¯uorimetry in methanol (0.3 ng mlÿ1) [40] and fenvalerate by PIF in acetonitrile (9 ng mlÿ1) [42] and SDS (7 ng mlÿ1) [43]. An obvious advantage of our method is the ability to directly determine the pesticides in aqueous media without preliminary extraction and clean-up steps.

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Table 3 Analytical figures of merit for the fluorimetric and PIF determination of aromatic pesticides Compound Fluorimetric method Coumatetralyl Pirimiphos-Me PIF method Chlorpyriphos Deltamethrin Fenvalerate

Solvent a

ex/em (nm)

Concentration range (ng mlÿ1)

Slope b

rc

b-CD HP-b-CD b-CD HP-b-CD

314/380 316/385 305/366 304/366

2±60 1±60 24±1830 18±1830

0.95 0.96 0.84 0.97

0.999 0.999 0.998 0.999

b-CD HP-b-CD b-CD HP-b-CD b-CD HP-b-CD

378/428 378/428 291/312 291/312 320/362 320/362

106±4220 106±4220 50±2030 40±2540 84±2940 105±4200

0.93 1.03 0.82 1.06 0.92 0.92

0.998 0.998 0.997 0.997 0.993 0.997

topt e (min)

RSD f (%)

0.2 0.2 4.8 3.9

± ± ± ±

1.2 1.0 2.8 1.3

44 54 6.9 6.8 22 32

20 20 22 22 08 16

4.3 3.2 2.4 4.4 2.6 1.8

LOD d (ng mlÿ1)

a

Cyclodextrin concentration is 10ÿ2 M. Slope of the log±log calibration curves. c Correlation coefficient. d Limit of detection (LOD) was defined as the amount of analyte giving a signal-to-noise ratio of 3. e Optimum irradiation time, corresponding to the maximum PIF intensity. f Relative standard deviation, (nˆ3). b

3.9. Analytical applications To show the analytical applicability of the proposed method to authentic samples, recovery experiments of the pesticides under study were performed on the spiked tap water and the Seine river water samples, using the direct measurement procedure. Tap water and river water were spiked with 117, 3055, 3520, 2032, 4200 ng mlÿ1 of coumatetralyl, pirimiphos-methyl, chlorpyriphos, deltamethrin and fenvalerate, respectively, and the solutions were then stirred ultrasonically for 15 min before being kept in the dark and used as stock standard solutions. Analyses were carried out as described in the experimental section. 3.9.1. Tap water analysis Table 4 summarizes the results of three replicate analyses. The recoveries obtained ranged from 88 to 111% for the ¯uorimetric method and 94 to 108% for the PIF method. 3.9.2. River water analysis The Seine river water samples, freshly collected at Chatou, near Paris, were ®ltered with a Whatman N8 1 ®lter paper to eliminate the suspended organic matter. A pH value of 8.3 was found for these samples.

Tap water samples were free of any ¯uorescent dissolved species, while river water samples contained a ¯uorescent organic species with maximum wavelength values (exˆ361 nm, emˆ433 nm) relatively close to those of chlorpyriphos excitation (378 nm) and emission (428 nm) maxima, but far from those of the other pesticides. Consequently, the extraction procedure (described in the experimental section) was performed only for chlorpyriphos in order to avoid any interference from band overlapping. Preliminary tests showed that the ¯uorescent matter present in the river water was light resistant and no signi®cant change of the signal occurred in a 10ÿ2 M HP-b-CD and/or a pHˆ4 buffer solution. This is rationalized by assuming that no inclusion complex could be formed between this organic matter and HPb-CD. Although dichloromethane was not able to remove completely the organic matter, an interesting photochemical behaviour was observed after extraction. Indeed, before extraction, the percentages of remaining signal were 83 and 67% after, respectively, 8 and 25 min irradiation times, whereas, after extraction, the corresponding values were 40 and 29%. As a consequence of this signal difference, it was decided to study the recovery of chlorpyriphos in the latter conditions. The analytical data are shown in Table 4. It can be seen that satisfactory recoveries were

140

A. Coly, J.-J. Aaron / Analytica Chimica Acta 360 (1998) 129±141

Table 4 Determination of the aromatic pesticides in tap water and river water samples Compound Tap water analysis Coumatetralyl (HP-b-CD) Pirimiphos-methyl (b-CD) Chlorpyriphos (b-CD) Deltamethrin (HP-b-CD) Fenvalerate (b-CD) River water analysis Coumatetralyl (b-CD) Pirimiphos-methyl (HP-b-CD) Chlorpyriphos (HP-b-CD) Deltamethrin (b-CD) Fenvalerate (HP-b-CD)

a

Concentration (ng mlÿ1) Taken

Found a

5.9 11.7 17.5 152.7 305.5 458.2 176 352 528 101.6 203.2 304.8 210 420 630

6.00.6 13.01.5 19.00.9 142.45.2 306.313.2 402.311.9 1694 3297 49712 99.93.3 219.96.6 328.912.1 21013 42228 60533

103.1 111.3 108.4 93.2 100.3 87.8 95.9 93.5 94.1 98.4 108.3 107.9 100 100.5 96.1

5.9 11.7 17.5 152.7 305.5 458.2 176 352 528 101.6 203.2 304.8 210 420 630

6.80.1 13.20.4 20.10.2 159.43.6 320.05.1 476.74.6 1755 35812 57624 96.93.2 194.74.2 283.43.5 2087 41217 60314

115.6 112.6 114.6 104.3 104.8 104.0 99.1 101.7 109.1 95.4 95.8 92.9 98.8 98.0 95.7

Recovery (%)

Meanstandard deviation of three replicates.

obtained for all the pesticides with values ranging from 104 to 116% for the ¯uorimetric method and 96 to 109% for the PIF method. It is probable that a signi®cant portion of the dissolved substances in natural water affecting the action of photolysis through attenuation of UV light was eliminated after extraction, which reduced the matrix effect. In order to improve the recovery percentage by minimizing the in¯uence of the ¯uorescent matter or any other dissolved substance, we allowed a maximum percentage of 15% (v/v) of real water sample in the ®nal volume (5 ml) of the solution to be analyzed (see experimental section). Among both methods, PIF shows better recovery results than conventional ¯uorimetry method for real samples. This makes PIF a valuable tool for

water analysis. Moreover, we feel that it is possible to improve considerably the selectivity of our method in other complex environmental matrices by choosing convenient UV irradiation times, or by applying speci®c procedures such as derivative and synchronous spectroscopy and/or partial least-squares multivariate calibration. 4. Conclusion We have shown the usefulness of the effect of cyclodextrins for improving the sensitivity, selectivity and simplicity of the ¯uorimetric and PIF methods for the determination of ®ve widely-used pesticides in

A. Coly, J.-J. Aaron / Analytica Chimica Acta 360 (1998) 129±141

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