Degradation Mechanisms of Phoxim in River Water - Journal of

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J. Agric. Food Chem. 2011, 59, 312–321 DOI:10.1021/jf1029459

Degradation Mechanisms of Phoxim in River Water BIXIA LIN, YING YU,* XIAOGANG HU, DAYI DENG, LICAI ZHU, AND WEIJIE WANG School of Chemistry and Environment, South China Normal University, Guangzhou 510006, People’s Republic of China

Degradation of phoxim in river water was fully explored in this paper. Effects of pH, temperature, and photoirradiation on the degradation were investigated in detail. The results indicated that the degradation was characterized by a first-order process; UV irradiation and the increase of pH and temperature substantially accelerated the degradation. To fully characterize the degradation mechanism, HPLC-MS/MS was utilized to identify the degradation intermediates. Five intermediates were identified as phoxom, phoxom dimer, O,O,O0 ,O0 -tetraethyldithiopyrophosphate, O,O,O0 -triethyl-O0 -2hydroxyethyldisulfinylpyrophosphate, and O,O,O0 -triethyl-O0 -2-hydroxyethyldithiopyrophosphate. On the basis of the results of the intermediate analysis, the degradation pathways of phoxim under the present experimental conditions were proposed. Through conversion of a thiophosphoryl into a phosphoryl group, some phoxim was converted to phoxom, most of which further formed dimer. Another portion of phoxim transformed to O,O,O0 ,O0 -tetraethyldithiopyrophosphate via nucleophilic substitution and photolysis. Thereafter, O,O,O0 ,O0 -tetraethyldithiopyrophosphate underwent hydroxylation to form O,O,O0 -triethyl-O0 -2-hydroxyethyldithiopyrophosphate or sulfur oxidation first and then hydroxylation to produce O,O,O0 -triethyl-O0 -2-hydroxyethyldisulfinylpyrophosphate. The understanding of phoxim’s degradation mechanism in this study will be critical to its safety assessment and increase the understanding of the fate of phoxim in environment water. KEYWORDS: Phoxim; degradation; mechanisms; river water; HPLC-MS/MS

INTRODUCTION

Organophosphorus pesticides (Ops) are of great environmental concern due to their widespread use in the past several decades and their potential toxic effects, primarily on the nervous systems of organisms including humans. Moreover, some metabolites of Ops in the environment also have the same toxic effects as Ops do (1-3). Therefore, to fully understand Ops’ impact on the environment, we need to investigate both the parent pesticides’ and their corresponding metabolites’ impact. Thus, the degradation study of Ops has become increasingly important in recent years. Phoxim, an organophosphorus pesticide with high efficiency and low toxicity (4), is frequently employed in both agriculture and fisheries to control a variety of ectoparasites via dipping, spraying, or pour-on applications (5). As a result of its applications, it is one of the frequently detected pesticides in agricultural effluent and river water (6), causing concerns about its impact on the environment and humans and arousing great interest in investigating the metabolites of phoxim and the degradation mechanism. The investigation of phoxim’s metabolism in crops began 30 years ago. Hans-Ulrich et al. (7) have reported the metabolic mechanism and metabolite identification of phoxim in plants and the cell suspension cultures of soybean; four metabolites were characterized as oxime, primary amine, N-malonate, and N-malonic *Author for correspondence (phone þ8620-39310382; fax þ862039310382; [email protected]).

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acid conjugate of phenylacetonitrileamine. In another case, Walter et al. (8) have determined the degradation kinetics of phoxim contained in stored wheat and found two extractable products, the oxygen analogue and the S-ethyl isomer. The photodegradation mechanisms of phoxim have been studied by a number of groups, and some intermediates during the process have been characterized. When irradiated with natural sunlight, phoxim was photodegraded to O,O-diethylcyanobenzylideneaminothiophosphonate, which was recognized as a highly active acetylcholinesterase inhibitor (9). While irradiated by UV with m-TiO2 as the photocatalyst, phoxom, O,O-diethyl-O-methyl phosphorothioate and monoethyl phenylphosphonate were identified as the intermediates (10). To the best of our knowledge, the degradation mechanism of phoxim in environmental water has not been investigated so far and remains unclear. To understand the fate of phoxim after its release into environmental water, it is necessary to characterize the degradation kinetics, the potential degradation intermediates, and the degradation pathways in water. The understanding of phoxim’s degradation mechanism will be critical to its safety assessment. Therefore, we investigated the degradation kinetics and mechanism of phoxim in river water under different conditions. To thoroughly explore the degradation mechanism, great efforts have been made to identify potential intermediates during the degradation process by using HPLC-MS/MS. On the basis of the results from the HPLC-MS/MS study, five intermediates were identified. Two intermediates, phoxom and O,O,O0 ,O0 -tetraethyldithiopyrophosphate, have been previously identified (9, 10), and

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Table 1. Degradation Kinetic Parameters of Phoxim in River Water under Different Temperatures T/°C

kinetic eq

k/day-1

t0.5/days

5 15 25 35

ln[C]/[C0] = -0.0083t ln[C]/[C0] = -0.0138t ln[C]/[C0] = -0.0263t ln[C]/[C0] = -0.0486t

0.0083 0.0138 0.0263 0.0486

83.5 50.2 26.4 14.3

Figure 1. Chromatograms of (a) phoxim standard and degraded water samples extracted by (b) hexane, (c) dichloromethane, and (d) ethyl acetate.

Figure 3. Effect of pH values on phoxim degradation kinetics. Table 2. Degradation Kinetic Parameters of Phoxim in River Water under Different pH Values pH

kinetic eq

k/day-1

t0.5/days

2.00 5.00 7.00 9.00 12.00

ln[C]/[C0] = -0.0008t ln[C]/[C0] = -0.0020t ln[C]/[C0] = -0.0037t ln[C]/[C0] = -0.0200t ln[C]/[C0] = -0.0491t

0.0008 0.0020 0.0037 0.0200 0.0491

866.4 346.6 187.3 34.7 14.1

Figure 2. Effect of temperature on phoxim degradation kinetics.

three new intermediates, O,O,O0 -triethyl-O0 -2-hydroxyethyldithiopyrophosphate, O,O,O0 -triethyl-O0 -2-hydroxyethyldisulfinylpyrophosphate, and phoxom dimer, were identified and characterized for the first time. On the basis of the intermediates’ identification and kinetics results, degradation pathways were proposed, and a microorganism played a very important role in phoxim’s degradation in river water, which, however, has never been reported before. MATERIALS AND METHODS Materials. Phoxim standard (99.0% purity) was purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Methanol (HPLC-grade), hexane (AR), dichloromethane (AR), and ethyl acetate (AR) were all obtained from Kermel (Tianjin, China). All other reagents were of AR grade and from Chemical Reagent Factory (Guangzhou, China) unless otherwise noted. HPLC Analysis. HPLC was carried out on a HPLC-UV system (Shimadzu, Guangzhou, China) equipped with a photodiode array (PDA) detector, an LC 10AD pump, and a Platisil ODS column (250 mm 4.6 mm, 5 μm, Dikma). Unless specified otherwise, UV absorption was monitored at 254 nm and the injection volume was 20 μL. Solvent elution systems were as follows: System 1. Samples were eluted with a linear gradient of methanol (B) in water (A) at a flow rate of 0.8 mL/min. Solvent composition was changed from 70 to 100% B in 30 min and then kept at 100% B for 10 min. This solvent system was used to separate phoxim and its potential degradation intermediates when extraction was optimized. System 2. Samples were eluted with an isocratic elution of 85% methanol (B) in water (A) at a flow rate of 1 mL/min in 10 min. This system was used for Calibration Curve establishment and quantification of undegraded phoxim in river water for degradation kinetics study.

HPLC-MS/MS Analysis. LC-MS/MS was carried out on a Finnigan LCQ Deca XP Plus ion-trap mass spectrometer (Thermo Finnigan, Austin, TX) with a Platisil ODS column (250 mm 4.6 mm, 5 μm, Dikma). Electrospray ionization was achieved at a spray voltage of 5.0 kV and a capillary temperature of 350 °C. Collision-induced dissociation (CID) was achieved with Ar as a collision gas and at a collision energy of 20 eV. The mass spectrometer was operated in the positive ion mode, with nitrogen as the sheath gas (5 L/min), and the mass spectrometer parameters were optimized for stable fragment ions during the infusion of samples. Flow rate and UV absorbance were the same as for system 1 mentioned above; samples were eluted with an isocratic elution of 49% methanol (B) in water (A) for 22 min, and then the solvent was changed from 49 to 70% B in 13 min and held at 70% for 20 min. The injection volume was 10 μL. Calibration Curve. For the quantification of phoxim, a calibration curve was obtained by analyzing the peak areas of phoxim at concentrations of 0.5, 1.0, 3.0, 5.0, 7.0, and 10 mg/L. The curve has a linear correlation coefficient of >0.9991 with a regression equation of y = 10207x - 2204 (y = peak area; x = concentration, mg/L) in the range of 0.5-10 mg/L. Phoxim was eluted at 7.8 min under the chromatographic conditions used in system 2. Pretreatment Procedures. A 10 mL water sample was filtered through filter paper and extracted with the desired extractant (4 mL 3). The extracts were combined, dehydrated with anhydrous magnesium sulfate, and evaporated to dryness (rotary vacuum, 37 °C), then redissolved with 1 mL of methanol and eventually filtered through a syringe filter (Millipore, 0.45 μm pore size). Study of the Degradation Kinetics. Degradation kinetics experiments of phoxim were carried out in 200 mL quartz flasks with river water collected from Centre Lake of University Town (Guangzhou, China). The physical-chemical properties of the lake water were characterized as follows: conductivity, 184.7 S/m; mineralized degree, 159.6 mg/L; TPC (total plate count), 262.1 cfu/mL; and pH, 9.20.

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The initial concentration of phoxim in river water samples was set to 7 mg/L. Three different sets of phoxim solutions were set up to investigate the effects of temperature, initial pH, and photoirradiation on the degradation. Ten-milliliter samples were withdrawn from each reaction mixture at a time interval of 4 days using a syringe. The samples were pretreated according to the pretreatment procedures above, and phoxim in the reaction mixture was eluted with the solvent mentioned in system 2 and quantified from the calibration curve. All results were based on the average of triplicate experiments. Identification of Degradation Intermediates. In our experiments, the concentrations of degradation intermediates formed were very low compared with parent pesticide phoxim added. To obtain detectable signals of degradation intermediates on HPLC and LC-MS systems, phoxim was added to the river water samples, once fetched, to reach a fortified final concentration of 100 mg/L. The solutions were then laid airproof and stirred under natural solar irradiation beside laboratory windows. When most of the phoxim had degraded, the samples were treated according to the pretreatment procedure and then analyzed on the LC-MS system.

Figure 4. Effect of photoirradiation on phoxim degradation kinetics. For UV and solar irradiation experiment, the samples were irradiated from 7 a.m. to 7 p.m. and from sunrise to sunset in a day, respectively. For darkness experiments, the reaction solutions were kept in a black case for a whole day. Table 3. Degradation Kinetic Parameters of Phoxim in River Water in the Absence or Presence of Photoirradiation photoirradiation

kinetic eq

k/day-1

t0.5/days

darkness sunlight ultraviolet

ln[C]/[C0] = -0.0051t ln[C]/[C0] = -0.0265t ln[C]/[C0] = -0.1022t

0.0051 0.0265 0.1022

134.0 26.2 6.9

Figure 5. Total ion current chromatogram of phoxim degradation sample.

River water samples without phoxim were set as blank, whereas the phoxim reaction solutions at time zero were set as negative control. Both the blank and negative control solutions were treated and analyzed as the degradation samples were. The total ion current chromatogram and the full-scan mass spectra obtained from the reaction group were compared with those of the blank and the negative control to identify the potential degradation intermediates. These potential intermediates were then analyzed further by LC-MS/ MS. Their retention times, UV-vis absorption, MS2 spectra, and observed mass differences were compared with those of phoxim to elucidate their structures. RESULTS AND DISCUSSION

Extraction Optimization. Liquid-liquid extraction (LLE) is the classical pretreatment method for the separation and preconcentration of organophosphorus pesticide degradation intermediates and the original parent pesticides (11). Three extractants of different polarities, hexane, dichloromethane, and ethyl acetate, were tested in our experiments to identify a better extractant for the extraction of phoxim and its intermediates. Figure.1 reports the HPLC profiles corresponding to the phoxim degradation samples extracted by hexane, dichloromethane, and ethyl acetate, respectively. Samples extracted by ethyl acetate showed more signal peaks and much higher peak strength on HPLC than did samples obtained with the other two extractants. Samples extracted by hexane or dichloromethane showed only one apparent signal peak on HPLC, and the peak had a retention time of 18.7 min, the same as phoxim standard. Thus, this peak was identified as phoxim, and for the identification of phoxim’s degradation intermediates, ethyl acetate was used as the extractant. In addition, the recoveries of phoxim for samples extracted by hexane, dichloromethane, and ethyl acetate were compared to find the optimal extractant for phoxim enrichment. With dichloromethane, the recovery was 98.3-100.5% and RSD was 0.9-1.2%; with hexane, the recovery was 61.2-68.9% and the RSD, 1.4-2.3%; with ethyl acetate, the recovery was 87.1-89.0% and the RSD, 2.0-3.5%. Therefore, dichloromethane was identified as the best extractant for phoxim enrichment from water samples. Study of the Degradation Kinetics. Effects of temperature, pH values, and photoirradiation on the degradation kinetics were investigated. Effect of Temperature. Degradation reactions were carried out at four different temperatures, 5, 15, 25, and 35 °C, to investigate the effect of temperature on the degradation rate of phoxim and to obtain activation parameters. To maintain the desired temperatures, experiments were conducted in a constanttemperature incubator (GNP-9160, INCBTEL, Ningbo, China).

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Figure 6. Base peak chromatogram (a), MS (b), MS2 (c), and fragmentation pathways of M1 at m/z 323 (d).

All river water samples were used without pH adjustment, and the initial pH was 9.2, the same as that of the original river water. On the basis of the data (Figure 2), plots of ln[Ct]/[C0] versus time gave a linear profile (Table 1), indicating that the degradation process was a first-order reaction. The apparent degradation rate constants, k, were 0.0083 day-1 at 5 °C,

0.0138 day-1 at 15 °C, 0.0263 day-1 at 25 °C, and 0.0486 day-1 at 35 °C. Hydrolysis of most compounds is an endothermic reaction. This is confirmed by the fact that the increasing temperature tends to accelerate the hydrolysis process of compounds. According to van’t Hoff’s theory, the hydrolysis rate usually doubles with every

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10 °C increase in temperature (12). In our experiments, the degradation rate under different temperatures increased by a factor Q (Q = ktþ10/kt) between 1.7 and 1.9 with every 10 °C increase, which was tallied with van’t Hoff’s theory. A plot of lnk against 1/T gave a linear line in the temperature range 303-333 K and yielded the Arrhenius expression lnk = -5082.2 (1/T) þ 13.437, from which an activation energy of 97.18 kJ/mol was calculated. Activation energy is the energy difference between reactant molecules in the ground state and the transition state. It determines how rapidly the reaction occurs: large activation energy results in a slow reaction because few reacting molecules collide with enough energy to climb the high

activation energy barrier; on the contrary, small activation energy results in a rapid reaction because almost all reacting molecules are energetic enough to climb to the transition state (13). The activation energy obtained in this study was relatively small and similar to those of sulfonylurea herbicides (14). Effect of pH. pH values of 2.00, 5.00, 7.00, 9.00, and 12.00 were selected to investigate the effect of pH on the degradation kinetics. The experiments were carried out in 200 mL quartz flasks. The river water samples were adjusted to the desired pH by adding the appropriate phosphate buffer solution. Then the flasks containing 100 mL reaction solutions were capped with silicon stoppers and placed under natural sunlight for 20 days.

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The characteristics of degradation kinetics under five pH values are listed in Figure 3 and Table 2. The results indicated that phoxim was relatively stable in both acidic and neutral conditions with a slow degradation rate: after 20 days of reaction, the degradation ratio ([C0] - [C])/[C0] was 7. The degradation

half-lives of phoxim in river water were 866.4 days at pH 2.00, 346.6 days at pH 5.00, 187.3 days at pH 7.00, 34.7 days at pH 9.00, and 14.1 days at pH 12.00. In summary, phoxim was prone to decompose under alkaline condition, which was in agreement with the results of Zhou et al. (15). This is due to the fact that molecule’s phosphate ester bond is relatively unstable and prone to be

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hydrolyzed in alkaline conditions. In conclusion, pH was a major factor in determining the degradation rate of phoxim in river water. Effect of Photoirradiation. UV irradiation was conducted in a phytotron (DEZN-P-4, Xiamen, China) equipped with a UV lamp emitting at 254 nm. The river water samples containing

phoxim were irradiated from 7 a.m. to 7 p.m. every day with the UV lamp. In solar irradiation experiments, the samples were laid under natural sunlight and irradiated from sunrise to sunset every day. In control experiments under darkness, the samples were kept in a black case until analyzed.

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Figure 10. Base peak chromatogram (a), MS (b), and UV-vis absorption spectra of M5 at m/z 587 (c).

Data for the degradation kinetics of phoxim in the absence or presence of photoirradiation are presented in Figure 4 and Table 3. The degradation rates under ultraviolet or sunlight were 20 or 5 times faster than that in the darkness. Most pesticides show UV-vis absorption bands at relatively short UV wavelengths. UV irradiation will lead to the promotion of the pesticides to their excited singlet states, which may then intersystem cross to produce triplet states. Such excited states can then undergo other processes such as homolysis, heterolysis, photoionization, and the like (16). As a result, phoxim was susceptible to degradation by UV irradiation like many other pesticides. Because sunlight reaching the Earth’s surface contains a small amount of short-wavelength UV radiation, phoxim showed a slight lability to solar irradiation as well. Identification of Degradation Intermediates. With the method mentioned above, the tentatively assigned degradation intermediates of phoxim were characterized on a HPLC-MS/MS system. For river samples of phoxim after 3 months, five peaks were assigned to be potential degradation intermediates, plus one for phoxim, by comparison of the HPLC-MS/MS profiles of the reaction samples, blank, and negative control. These six peaks were characterized by their retention times (RT) and protonated molecular ions or sodium-adducted ions as follows: RT 15.64 min, m/z 323, labeled M1; RT 37.19 min, m/z 361, M2; RT 43.13 min, m/z 393, M3; RT 44.28 min, m/z 283, M4; RT 48.14 min, m/z 587, M5; RT 54.66 min, m/z 299, M0 (Figure 5). MS2 spectra of the degradation intermediates of phoxim were obtained by fragmentation of the protonated molecular ions or sodium-adducted ions and used for the elucidation of the molecular structures of the degradation intermediates. Of the six peaks, the retention time,

MS, and MS2 of M0 were the same as those of phoxim; thus, M0 was confirmed as phoxim. M1 has m/z of 323, and its formation was favored by the presence of photoirradiation, a conclusion from the comparison of its formation in the darkness and the photocatalyzed conditions. If the ion is the protonated ion, the MW may be 322. An intermediate of phoxim photocatalyzed degradation with MW 322 has been previously identified as O,O,O0 ,O0 -tetraethyldithiopyrophosphate (9). Therefore, M1 was tentatively assigned as O,O,O0 ,O0 -tetraethyldithiopyrophosphate. To further confirm the identity of M1, the fragmentation pattern of M1’s MS/MS was thoroughly studied. The characteristic ion at m/z 250 appearing in the MS2 spectrum of M1 was formed by loss of one -C2H5 and one -OC2H5. Another ion at m/z 194 was formed by the loss of three -C2H5 and one -OC2H5, an indication that M1 had four -OC2H5. A sodium-adducted fragment ion at m/z 177 was formed by the cleavage of an O-P bond, and a corresponding potassium-adducted fragment ion at m/z 208 represented such a cleavage. On the basis of the fragmentation analysis, M1 was confirmed as O,O,O0 ,O0 tetraethyldithiopyrophosphate. The base peak chromatogram, MS, MS2, and fragmentation pathways are listed in Figure 6. For M2, both the protonated molecular ion at m/z 339 and sodium-adducted ion at m/z 361 exist in the mass spectrum (Figure 7b); therefore, the MW is 338. The sodium-adducted ion at m/z 361 was selected for MS2 analysis due to its higher intensity; five fragment ions were observed at m/z 333, 305, 287, 259, and 225 (Figure 7c). The ion at m/z 333 was formed by losing one -C2H5 from its precursor ion at m/z 361; similarly, the ion at m/z 305 formed from the ion at m/z 333 and m/z 259 from m/z 287,

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Figure 11. Proposed transformation pathways of phoxim in river water.

both by losing one -C2H5 from the specific precursor. This indicates that M2 possesses at least three -OC2H5. Additionally, the molecular weight of M2 increases by 16 Da, compared with that of M1. Presumably, M2 was the hydroxylated product of M1, and the molecular structure is listed in Figure 7d. The ions at m/z 287 and 225 formed via losing H2O from m/z 305 or two -OH from m/z 259, respectively. For M3, both the protonated molecular ion at m/z 371 and the sodium-adducted ion at m/z 393 exist in the mass spectrum (Figure 8b). The sodium-adducted ion at m/z 393 was selected for MS2 analysis due to its higher intensity. Six fragment ions were observed at m/z 365, 337, 319, 301, 291, and 257 (Figure 8c). A difference of 32 Da was found between the ion at m/z 361 [M2 þ Na]þ and the ion at m/z 393 [M3 þ Na]þ, between the ion at m/z 339 [M2 þ H]þ and the ion at m/z 371 [M3 þ H]þ, between the ion at m/z 333 and the ion at m/z 365, between the ion at m/z 305 and the ion at m/z 337, between the ion at m/z 287 and the ion at m/z 319, between the ion at m/z 259 and the ion at m/z 291, and between the ion at m/z 225 and the ion at m/z 257 by comparison of M2 protonated/sodium-adducted molecular ions, and MS2 fragment ions with those of M3. This allowed us to infer that M3 was from the sulfur oxidation of M2. The molecular structure and fragmentation pathways are listed in Figure 8d. M4’s protonated ion was at m/z 283. Therefore, the MW of M4 was 282, which was 16 Da less than that of phoxim, indicating that M4 might be the desulfurization daughter product of phoxim, phoxom, which has been previously reported by Dai and co-workers (10) in their photolysis experiments. Therefore, M4 was tentatively assigned as phoxom. The fragment ions at m/z 255, 227, and 129 were observed in the MS2 spectrum (Figure 9c). Fragment ions at m/z 255 and 227 were formed by loss of one or two ethyl groups from the protonated molecular ion at m/z 283,

which suggested that M4 had two -OC2H5. A characteristic fragment ion at m/z 129 was present in M4 and phoxim (17), indicating both have the same fragment, -NC(CN)ph. All of the fragment ions above confirmed the suggestion that M4 was represented by phoxom. For M5, the ion at m/z 587 was chosen for MS2 analysis; however, no stable characteristic fragment ions were observed in MS2 analysis. Figure 10c shows the UV-vis absorption spectra of both M5 and phoxom. The UV-vis absorption spectrum of M5 was essentially the same as that of phoxom, both with two strong absorption peaks at 204 (203) and 280 nm. Considering the m/z difference and UV-vis absorption spectra, it was sensible to deduce that M5 might be a phoxom dimer and m/z 587 was the sodium-adducted molecular ion of M5. Degradation Pathways. On the basis of the results from the study on degradation kinetics and identification of the degradation intermediates, degradation pathways were proposed and presented in Figure 11. The degradation of phoxim in river water involved two main pathways. First, for the starting material phoxim, owing to the different polarities of the O-P bond, atom P became a reactive positive center(18), and nucleophilic attack (by H2O or OH-) at the tetrahedral phosphorus gave a bipyramidal intermediate A. Intermediate A was not stable, and it could react in a couple of potential ways: (1) A collapsed with expulsion of sulfide to produce phoxom (M4); (2) A collapsed with expulsion of alkoxide or iminoxide, and due to the steric hindrance of iminoxide group, its expulsion was favored. The expulsion of iminoxide would produce transient intermediates B, R-hydroxyiminophenylacetonitrile, and C, O,O-diethylthiophosphoric ester, which could condense under the photocatalysis to form M1, O,O,O0 ,O0 tetraethyldithiopyrophosphate by losing H2O.

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As for the degradation of phoxim, the nucleophilic attack of the tetrahedral phosphorus is normally the rate-determining step. OH- is a better nucleophile than H2O; therefore, alkaline solutions accelerate the degradation of phoxim. This was confirmed by the results from intimal solution pH effects on the degradation of phoxim in our study. Subsequently, sulfur oxidation happened to some portion of M1 (O,O,O0 ,O0 -tetraethyldithiopyrophosphate) to produce intermediate D, O,O,O0 ,O0 -tetraethyldisulfinylpyrophosphate. Then further hydroxylation happened to both M1 and intermediate D to produce M2, O,O,O0 -triethyl-O0 -2-hydroxyethyldithiopyrophosphate and M3, O,O,O0 -triethyl-O0 -2-hydroxyethyldisulfinylpyrophosphate. Microbial degradation is considered to be a major factor determining the fate of insecticides in the environment. Many authors indicated that the bacterial strains belonging to the different taxonomic groups have a great degradation potential of the organophosphorus insecticides and other pesticides (19-21). A great number of studies demonstrated hydroxylation is a common degradation process in microbial degradation. For example, DBH (4,40 -dichlorobenzhydrol) and DDOH (2,2-bis(4-chlorophenyl)ethanol)) are regarded as hydroxylated metabolites in the DDT (dichlorodiphenyltrichloroethane) biodegradation pathway (22). Diazinon can be also biodegraded to 2-isopropyl6-methyl-4-hydroxypyimidine via hydroxylation under aqua conditions with appropriate pH, temperature, and sunlight as well as the presence of microorganisms (23). In our study, the water samples fetched from the river, where TPC reached 262.1 cfu/mL, were used without sterilization, and the reaction system was laid under sunlight. The combined action of those factors might provide a biodegradation environment for phoxim degradation. Moreover, M2 and M3 were not formed in distilled water, where the TPC (total plate count) was 6.5 cfu/mL. Therefore, it was rational for us to consider the hydroxylated products M2 and M3 to be the microbial metabolites of phoxim. Whether these two hydroxylated metabolites are common in phoxim’s biodegradation needs further study. As for M4 (phoxom), most phoxom would further form a stable dimer (M5) in river water samples. Castillo et al. (24) suggested that oxidation forms of Ops have a higher stability than their precursors due to the higher electronegativity of PdO compared with PdS in aquatic environments. As the electron density of OdP is thicker than that of PdS, compounds combined with cholinesterase are more stable and inhibitory on the action of acetylcholinesterase, which indicates a higher toxicity of OdP than of SdP. Compared with the parent pesticide phoxim, its transformation intermediates such as phoxom and phoxom dimer in river water are more toxic and should be more closely monitored.

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Received for review July 30, 2010. Revised manuscript received November 20, 2010. Accepted November 21, 2010. Financial support was provided by the National Natural Science Fund of China (No. 20975041) and the Natural Science Fund of Guanddong Province in China (No. 10251063101000000).

Degradation Mechanisms of Phoxim in River Water - Journal of

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