Article pubs.acs.org/JAFC
Stereoselective Degradation and Transformation Products of a Novel Chiral Insecticide, Paichongding, in Flooded Paddy Soil Juying Li,†,§ Sufen Zhang,† Chengchen Wu,† Chao Li,# Haiyan Wang,† Wei Wang,† Zhong Li,# and Qingfu Ye*,† †
Institute of Nuclear Agricultural Sciences, Zhejiang University, Hangzhou 310029, China Shenzhen Key Laboratory of Environmental Chemistry and Ecological Remediation, College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen 518060, China # School of Pharmacy, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China §
S Supporting Information *
ABSTRACT: Paichongding is a chiral neonicotinoid insecticide currently marketed as racemate against sucking and biting insects. Under anaerobic condition, all paichongding stereoisomers underwent appreciable degradation in soil during 100 days of incubation, with estimated t1/2 values between 0.18 and 3.15 days. Diastereoselectivity in paichongding degradation was observed, with enantiomers (5S,7R)- and (5R,7S)-paichongding being more preferentially degraded in soils than enantiomers (5R,7R)- and (5S,7S)-paichongding. The half-lives of (5R,7R)- and (5S,7S)-paichongding were 3.05 and 3.15 days, respectively, as compared to 0.18 day for (5R,7S)- and (5S,7R)-paichongding. A total of nine intermediates were identified, of which depropylated paichongding was the predominant metabolite and appeared to be stable and recalcitrant to further degradation. Paichongding is degraded via denitration, depropylation, nitrosylation, demethylation, hydroxylation, and enol−keto tautomerism, producing chiral and biologically active products. These findings could have implications for environmental risk and food safety evaluations. KEYWORDS: paichongding, chiral pesticide, diastereoselective degradation, transformation products, degradation pathway
■
acetochlor, metalaxyl, metolachlor, and dimethenamid,16 ethofumesate,17 and epoxiconazole.18 Moreover, chiral intermediates with altered mobility and chemical/biological activity may be produced due to incomplete stereoselective degradation of chiral pesticides and can themselves be metabolized enantio-/stereoselectively, leading to unanticipated environmental effects. Therefore, it can be expected that such stereoselectivity will have important implications for us to understand the overall environmental risk of chiral contaminants.19 Paichongding has two asymmetrically substituted C atoms and consists of two diastereomeric pairs of enantiomers or four stereoisomers. Interestingly, the activities against alfalfa aphids of the four stereoisomers of paichongding differed significantly, with (5R,7S)- > (5S,7S)- ≥ (5S,7R)- > (5R,7R)paichongding.20 Li et al. reported that mineralization, extractable residues, and bound residue formation of paichongding were diastereoselective.21 Wang et al. found that after foliar application, no stereoselective absorption was observed among the enantiomers of paichongding, whereas significant stereoselective translocation in plants was observed between its epimers.11 Thus, information on the stereoselective degradation and environmental behavior of paichongding as well as its metabolites (e.g., the metabolic pathways) is crucial for assessing environmental and human health safety.
INTRODUCTION Neonicotinoids have become one of the most important classes of synthetic insecticides for controlling sucking insects on both crops and animals in the past decades.1 The popularity is largely due to their outstanding potency and strong systemic resistance against piercing−sucking pests and high efficacy for flea control on pets.2 However, they may affect pest insects as well as nontarget organisms such as pollinators.3 For instance, imidacloprid, one of the most widely used neonicotinoid insecticides, has been shown to cause a range of significant sublethal effects on honeybee and bumblebee colonies and thus has been banned from use in Europe.3 Paichongding is a novel nicotinic acetylcholine receptor (nAChR) agonist and shows excellent insecticidal activity. It is currently used for controlling a broad spectrum of sucking and biting insects, especially on imidacloprid-resistant insects.4−6 The application of 10% paichongding racemate suspension concentrate started from 2009 in China, and the production of paichongding was estimated at about 1000 tons annually with a treated area of approximately 3.3 million hectares.7 Chiral pesticides are of significant concern in recent years, as the biological activities of such compounds are generally stereoselective and the biologically mediated environmental processes usually exhibit significant isomer selectivity.8−13 Transformation usually affects the availability and hence the potential for uptake into plants or leaching to groundwater.14 Once in soil, one enantio-/stereoisomer of chiral pesticides may be degraded and transformed, whereas others are accumulated. Enantio-/stereoselective degradation in soils was observed for a variety of chiral pesticides such as benalaxyl,15 alachlor, © XXXX American Chemical Society
Received: June 21, 2016 Revised: September 5, 2016 Accepted: September 23, 2016
A
DOI: 10.1021/acs.jafc.6b02787 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
twice with 0.01 M CaCl2 (50 mL), 9:1 (v/v) acetonitrile/water (50 mL), methanol (50 mL), and then methylene chloride (50 mL) by shaking for 2 h each time and centrifugation for 10 min at 4000 rpm. Preliminary experiments showed that the sequential extraction efficiency of 14C activity ranged from 97.81 to 105.20% by this procedure. Identification of Degradation Intermediates. All supernatants were combined and concentrated on a rotary evaporator to near dryness and redissolved to 1 mL by methanol for further analyses. All analyses were performed in triplicate. The final sample extract was fractionated by high-performance liquid chromatography (HPLC). HPLC was performed on a Diamonsil C18 column (5 μm, 250 × 4.6 mm, Dikma Technologies, Lake Forest, CA, USA) at 30 ± 1 °C with a 2695 multisolvent delivery unit and a 2998 photodiode array (PDA) detector (Waters, Milford, MA, USA). A 20 μL aliquot of samples in methanol was injected. Mobile phase A was water acidified with 0.1% acetic acid, and mobile phase B was acetonitrile containing 0.1% acetic acid. The following gradient program was used (in reference to mobile phase B): 0−7 min, 15% B; 7−20 min, 15−35% B; 20−40 min, 35−100% B; 40−45 min, 100% B; and 45−50 min, re-equilibration with 15% B. The flow rate was 1.0 mL/min. The postcolumn eluent (1.0 mL) was collected at 1.0 min intervals and mixed with 10 mL of scintillation cocktail for 14C radioactivity measurement on LSC. The distribution of 14C as a function of postinjection run time was used to construct a chromatogram and quantify the fractions of parent compound and 14 C-containing metabolites. According to the constructed radioactive chromatograms, the final extracts from soils containing (5R,7R)-paichongding after 11 and 45 days of incubation, (5R,7S)-paichongding after 60 days of incubation, and (5S,7R)-paichongding after 60 and 100 days of incubation were selected for LC-MS/MS analysis. These extracts were also analyzed on a Waters 2695 HPLC system in tandem with a Bruker Esquire 3000plus ion trap mass spectrometer (Bruker Daltonik, Bremen, Germany), which was operated with electrospray ionization in positive ion mode. The chromatographic conditions of the HPLC analysis were applied in LC-MS/MS analysis. The ion-transfer capillary temperature was set to 250 °C. Pure nitrogen was used as sheath gas and auxiliary gas at a flow rate of 10 L/min and a pressure of 30 psi. All data were acquired and processed using the Esquire 5.0 software. Data Analysis. First-order degradation kinetic models were commonly used in degradation studies. The degradation of paichongding stereoisomers in soil not only follows simple firstorder kinetics but also shows a pattern where most stereoisomers decline at an initially rapid phase and in a second phase less rapidly. The following equations were used to calculate the degradation kinetics in this study:
The objective of this study was to elucidate the degradation pathways of paichongding in soil, with an emphasis on the identification of incomplete degradation products and stereoselective degradation, by using 14C labeling and LC-MS/MS. This information will be valuable for obtaining a more holistic assessment of environmental risks of paichongding and other neonicotinoid insecticides.
■
MATERIALS AND METHODS
Chemicals. The 14C-paichongding (1-((6-chloropyridin-3-yl) methyl)-7-methyl-[14C]8-nitro-5-propoxy-1,2,3,5,6,7hexahydroimidazo[1,2-a]pyridine) racemate with radiochemical and chemical purities >98% was synthesized as described by Li et al.4 The four stereoisomers of 14C-paichongding were separated as described by Li et al.21 The radiochemical and chemical purities of the four stereoisomers used here were >98%, and the specific radioactivities of 14 C-(5R,7R)-paichongding, 14C-(5S,7S)-paichongding, 14C-(5S,7R)paichongding, and 14C-(5R,7S)-paichongding were 1.89, 1.79, 1.69, and 1.90 mCi/mmol, respectively. Each 14C-isomer was individually tested, and the stock solution was prepared by dissolving the isomer in acetonitrile and diluted to 200 mg/L with distilled water. 2,5-Diphenyloxazole (7.0 g, Arcos Organics, Geel, Belgium) was mixed with 0.5 g of 1,4-bis(5-phenyloxazol-2-yl)-benzene (Arcos Organics) in a 35:65 (v/v) 2-methoxyethanol/dimethylbenzene mixture (1 L) to prepare the scintillation cocktail. All organic solvents and other chemicals used in this study were of analytical or HPLC grade. Soil Incubation. A fluvio-marine yellow loam soil was collected from the top 15 cm at a location in Hangzhou, Zhejiang, China. The soil had a pH of 7.0 and organic matter (OM) and N contents of 3.05 and 0.29%, respectively. Moist soil was air-dried at room temperature, passed through a 1 mm sieve, and stored at 4 °C in the dark before use. The percentages of sand (21%), silt (71%), and clay (8%), cationexchange capacity (10.83 cmol kg−1), and OM content (0.24%) were measured. The OM and N contents were determined by flash combustion of dried materials on an NA1500 nitrogen analyzer (Carlo Erba, Italy). Degradation experiments were performed by using individual 14Cstereoisomer under anoxic condition. Full details of the procedures used to extract and determine the radioactivity are described in our previous paper.21 Briefly, soils were pre-incubated for 7 days to revive microbial activity. A portion of 50 g of soil (dry weight equivalent) was spiked with 24.5 mL of each 14C-stereoisomer stock solution and then thoroughly mixed with 280 g (dry weight equivalent) of soil, yielding a final fortification level of 8.17 μg g−1 for individual stereoisomers. This application amount was 2−3 times higher than the recommended dose (450 g ai ha−1) to facilitate the quantification and identification of metabolites of paichongding by using LC-MS/MS. The specific radioactivities of 14C-(5R,7R)-paichongding, 14C-(5R,7S)-paichongding, 14C-(5S,7S)-paichongding, and 14C-(5S,7R)-paichongding were 1.55, 1.57, 1.48, and 1.40 kBq g−1 soil (dry weight), respectively. The uniformity of 14C distribution was verified by the radioactivity analysis of 1.0 g soil subsamples combusted on an OX-600 biological oxidizer (R. J. Harvey Instrument, USA). A portion of soils (10 g, dry weight equivalent) was weighed into a 40 mL glass vial and submerged by distilled water (10 mL). The vials were then placed in vacuum desiccators under a headspace of N2 and were incubated at 25 °C in the dark. The same amount of nitrogen-purged deionized water was added to compensate for evaporated loss of water. At 0, 5, 11, 20, 31, 45, 60, 76, and 100 days after the treatment, the flow-through vacuum desiccators were flushed for 30 min with a continuous nitrogen stream (99.99% pure). Soils without 14C-stereoisomers were incubated as controls. Extraction and Analysis. Three tubes were sacrificed at each sampling time for sequential extraction and analysis of the 14C radioactivity in different soil extracts. The soil−water mixture in its entirety was transferred to a 100 mL polypropylene centrifuge tube. The water layer was decanted after centrifugation at 4000 rpm for 10 min, and the soil pellet was resuspended by vortexing and extracted
first-order model
C t = C0 e −kt
(1)
biexponential model
Ct = C1 ek1t + C2 ek 2t
(2)
C0 is the radioactivity of chemical present at time 0, t is the incubation time (days), and k is the degradation rate (days−1) in eq 1. In eq 2, C1 and C2 are the initial concentrations of C at time 0 degraded through a first- and another first-order process, and k1 and k2 are the degradation rate constants 1 and 2 (days−1) (k1 > k2), respectively. Ct is the total amount of chemical present at time t. Significance was determined by one-way ANOVA at p = 0.05 using SPSS 19.0 (Armonk, NY, USA). Graphs were generated by using Sigma-plot 10.0 (Systat Software, San Jose, CA, USA).
■
RESULTS AND DISCUSSION Stereoselective Dissipation of Paichongding. The degradation profiles of 14 C-(5R,7R)-paichongding, 14 C(5S,7S)-paichongding, 14C-(5S,7R)-paichongding, and 14CB
DOI: 10.1021/acs.jafc.6b02787 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
fraction of 14C parent was consistently greater for 14C-(5R,7R)paichongding and 14C-(5S,7S)-paichongding than for 14C(5R,7S)-paichongding or 14C-(5S,7R)-paichongding (p < 0.05) (Figure 1), suggesting that 14C-(5S,7R)-paichongding and 14C-(5R,7S)-paichongding were more readily biodegradable than 14C-(5R,7R)-paichongding and 14C-(5S,7S)-paichongding. Results from our previous study conducted under the same conditions showed that the primary losses of paichongding were due to the formation of nonextractable or bound residue, which was diastereoselective, with enantiomers (5S,7R)-paichongding and (5R,7S)-paichongding being more preferentially bound to soils than enantiomers (5R,7R)paichongding and (5S,7S)-paichongding.21 Thus, the higher bound residue formation may contribute to the faster degradation of enantiomers (5R,7S)-paichongding and (5S,7R)-paichongding. In addition, results may also be rationalized on the level of the paichongding stereoisomerdegrading organisms (e.g., superiority of microbial populations or consortia preferentially degrading the RS- or SR-isomer over the RR- or SS-isomer) or on a molecular level (e.g., activation/ inhibition of RR- or SS-isomer-specific enzymes). ANOVA showed that degradation of enantiomers (i.e., (5R,7R)paichongding and (5S,7S)-paichongding; (5S,7R)-paichongding and (5R,7S)-paichongding) showed no significant difference (p > 0.05), suggesting that microbial communities responsible for degradation of paichongding enantiomers were not selective or that paichongding enantiomer-degrading microorganisms exhibited selectivity in both directions. Instead, the differences between epimers (e.g., (5R,7R)-paichongding and (5S,7R)paichongding, (5R,7R)-paichongding and (5R,7S)-paichongding, (5S,7S)-paichongding, and (5R,7S)-paichongding, (5S,7S)paichongding, and (5S,7R)-paichongding) were found to be significant at p < 0.05. The difference between the isomerspecific degradation in the soil (Figure 1) indicates that, unlike enantiomers, the diastereomers of paichongding may differ in both biological activities and physical−chemical properties. This would result in the selectivity between epimers. Our findings were in good agreement with the observation found by Buerge et al. that selectivity in the dissipation of cyproconazole was more statistically significant between epimers than between enantiomers.26 Formation of Intermediates. The metabolites of paichongding were separated and identified by HPLC-LSC combined with mass spectral confirmation. Figure S1 shows a radiochromatogram of (5R,7R)-paichongding along with its metabolites after 11 days of treatment. A total of nine additional radioactive bands were frequently detected in addition to the paichongding parent compound. These metabolites are labeled herein as M1−M9 with increasing retention times. The relative distribution of parent paichongding and the metabolites is shown in Figure 2. M1, with an HPLC relative retention time 3.7 min, was the first product formed and the most abundant metabolite in soil.
(5R,7S)-paichongding plotted against incubation time in soil are shown in Figure 1. All paichongding stereoisomers
Figure 1. Degradation kinetics of 14C-(5R,7R)-paichongding, 14C(5S,7S)-paichongding, 14C-(5S,7R)-paichongding, and 14C-(5R,7S)paichongding in soil under flooded conditions (n = 3).
underwent appreciable degradation in soil during the 100 days of incubation under flooded conditions, with the estimated t1/2 values between 0.18 and 3.15 days (Table 1). For instance, the fraction of 14C-(5S,7R)-paichongding decreased to 19.41 ± 1.46% only 5 days after treatment. At the end of incubation, it accounted for only 2.12 ± 1.01%, and 14C-(5R,7S)-paichongding could not even be detected, suggesting that 14C-(5R,7S)paichongding was completely transformed to other products or incorporated into the soil matrix as nonextractable or bound residue. The two-compartment model provided a better fit (r2 > 0.90, p < 0.0001) than the simple first-order equation for (5R,7S)paichongding and (5S,7R)-paichongding in soil (Table 1). After an initial rapid (5−20 days) degradation, the stereoisomers (5R,7S)-paichongding and (5S,7R)-paichongding further decreased at a slower rate. The faster degradation rate of (5R,7S)paichongding and (5S,7R)-paichongding in the first compartment might be due to the higher microbial biomass at the beginning or the fact that rapid degradation occurred within the soil−water phase, where (5R,7S)-paichongding or (5S,7R)paichongding-degrading microorganisms have easier access to the compound. Banerjee et al. demonstrated that the higher microbial biomass resulted in an initial faster degradation of trifloxystrobin, whereas the low microbial biomass led to a slow degradation at the end of the experiment.22 In the second compartment, (5R,7S)-paichongding or (5S,7R)-paichongding was likely adsorbed to soil particles, and the degradation rate may be governed by the slow desorption−diffusion processes, leading to a slower degradation. Similar good fits of biexponential model were found in the degradation experiments on sulfachloropyridazine,23 chlorpyrifos, λ-cyhalothrin, endosulfane α, and trifluraline,24 and estrone-3-sulfate.25 Significant differences were observed in 14C-paichongding parent residuals between different isomers. In general, the
Table 1. First-Order Rate Constants (k), Half-Lives (t1/2), and Coefficients of Determination (r2) Describing Dissipation of Paichongding in Soil under Anerobic Conditions isomer
C1
k1 (days−1)
(5R,7R)-paichongding (5S,7S)-paichongding (5R,7S)-paichongding (5S,7R)-paichongding
141.66 104.35 61.18 61.18
0.331 0.241 9.204 9.204
C2
38.82 38.82 C
k2 (days−1)
r2
DT50 (days)
p
0.0198 0.0198
0.9229 0.9545 0.9179 0.9179
3.15 3.05 0.18 0.18
