Chiral fungicide triadimefon and triadimenol: Stereoselective transformation in greenhouse crops and soil, and toxicity to Daphnia magna

Journal of Hazardous Materials 265 (2014) 115–123

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Chiral fungicide triadimefon and triadimenol: Stereoselective transformation in greenhouse crops and soil, and toxicity to Daphnia magna Yuanbo Li, Fengshou Dong, Xingang Liu, Jun Xu, Yongtao Han, Yongquan Zheng ∗ State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, 100193, China

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

• The stereoselective toxicity of triadimefon and triadimenol was first studied. • The enantioselective metabolism of triadimefon in vegetables was first investigated. • The enantioselective transformation was conducted under two different uptake-route.

a r t i c l e

i n f o

Article history: Received 9 July 2013 Received in revised form 19 November 2013 Accepted 24 November 2013 Available online 1 December 2013 Keywords: Triadimefon Triadimenol Stereoselectivity Degradation Aquatic toxicity

a b s t r a c t Various chiral pesticides are used in greenhouses to ensure high crop yields. However, detailed knowledge on the environmental behavior of such chiral contaminants with respect to enantioselectivity in the greenhouse has received little attention so far. Here, the widely used fungicide triadimefon was chosen as a “chiral probe” to investigate its enantioselective degradation and formation of triadimenol in greenhouse tomato, cucumber, and soil under different application modes. In addition, the stereoselectivity of individual isomers of triadimefon and triadimenol in aquatic toxicity were first studied. Significant differences in their acute toxicity to Daphnia magna were observed among the isomers. Under foliage application or soil irrigation application, S-(+)-triadimefon was preferentially degraded, resulting in relative enrichment of the more toxic R-(−)-enantiomer in tomato, cucumber, and soil. Further enantioselective analysis of converted triadimenol showed that the compositions of the four product stereoisomers were different and closely dependent on environmental conditions: the most toxic RS(+)-triadimenol was the most preferentially produced isomer in tomato under foliage treatment, while the RR-(+)-triadimenol was proved to be the highest amount of metabolite isomer in cucumber and soil under both treatment modes and in tomato under soil treatment. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Greenhouse crop systems that protect crops from adverse meteorological conditions and allow the production of high-value vegetables during the entire season are expending worldwide.

∗ Corresponding author. Tel.: +86 01 62815908; fax: +86 01 62815908. E-mail address: [email protected] (Y. Zheng). 0304-3894/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jhazmat.2013.11.055

The greenhouse production of vegetables is characterized by high planting density under high temperature and humidity conditions. These conditions cause the appearance of more pests and diseases in greenhouse crop systems than in open fields [1]. Thus, a large amount of pesticide is used in greenhouses to maintain high crop yield [2]. A major side effect of the use of pesticides is the potential risk they can cause to humans and the environment health. Concerning pesticides, it was estimated that chiral pesticides may account for >40% of currently used pesticides in China [3]. It is well

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known that enantiomers/stereoisomers may show different transformation in plants, humans, and the environment, and they may have different toxicity to humans and nontarget organisms [4–9]. Being primary producers in an ecosystem, plants are responsible for producing all the energy in the ecosystem [10]. Since large quantities of chiral pesticides applied in the greenhouse, the physiological changes due to chiral chemicals may affect the food chain and further ecosystems [11]. Clearly, understanding the specific environmental behavior of chiral pesticide enantiomers in greenhouse crops is essential to assess the risk of these chiral contaminants to ecological health and food safety. However, to date, studies on this subject are still limited and obviously lagging behind laboratory and open field studies [12]. Triadimefon (Fig. 1) is a broad spectrum systemic 1,2,4-triazole fungicide that has been extensively used to control powdery mildews and fungi in greenhouse crops by inhibiting steroid demethylation [13,14]. Triadimefon has a single chiral center and consists two enantiomers, S-(+) and R-(−) [15]. In plants, soils, and animals, triadimefon is known to undergo carbonyl reduction to a more fungi-active metabolite, triadimenol, which is also registered as an agricultural fungicide [16,17]. The metabolic transformation involves the reduction of the carbonyl group to an alcohol, resulting in the formation of a second chiral center in triadimenol (Fig. 1) [18]. Therefore, triadimenol has two diastereomers: A [enantiomers A1 (1R,2S) and A2 (1S,2R)] and B [enantiomers B1 (1R,2R) and B2 (1S,2S)], giving a total of four stereoisomers. Interestingly, the fungicidal activities of these stereoisomers differ greatly, and the 1S,2R isomer is up to 1000-fold more fungicidally active than the other three [19]. In addition, triadimenol diastereomer A is 10 times more acutely toxic to rats (oral LD50 ) than diastereomer B [16]. Since its first introduction as triazole fungicide, triadimefon has been used as a racemic mixture. An increasing number of studies have shown that many chiral pesticide residues occur non-racemically and can be metabolized enantioselectively, such as dichlorprop [20,21], fipronil [22,23], metalaxyl [24,25], diclofop [26], fenbuconazole [27], and beflubutamid [28]. Although environmental transformation of pesticides usually results in less harmful products, such products may be more harmful to the biological systems or the environment than parent pesticide; this has been shown, for example, with DDT [29]. However, so far chiral selectivity of their metabolites have been largely ignored. In view of the differences in fungicidal activity and toxicity among the stereoisomers, the possible stereoselective formation of triadimenol is an important issue for both human health and ecological risk assessment. Enantioselective transformation of triadimefon has been studied in laboratory soils and animals [13,16,17]. Results indicated that the direction and degree of the observed enantioselectivity often differ across various soils. Studies have also shown that different triadimenol stereoisomer compositions could be produced depending on soil type. However, little is known about the stereoselective nontarget toxicity of triadimefon and triadimenol, as well as the stereoselective biotransformation of triadimefon in plants, especially under greenhouse conditions, although an earlier study has shown that triadimefon was more persistence in greenhouse than in field conditions [14]. In the present study, the stereoselectivity of triadimefon and triadimenol isomers in acute toxicity to Daphnia magna were evaluated. The occurrence of stereoselective transformation of triadimefon was further investigated in two common greenhouse vegetables (tomato and cucumber) and soil. The investigation was conducted under two application modes (foliage treatment and soil treatment) to understand the differences in enantioselectivity under different uptake routes. Results of the research provide more comprehensive insights into the environmental and human risks posed by chiral pesticides.

2. Materials and methods 2.1. Chemicals and reagents Analytical standards of triadimefon (99.4% purity), triadimenol A isomer (racemate of RS enantiomer and SR enantiomer, 99.9% purity), and triadimenol B isomer (racemate of RR enantiomer and SS enantiomer, 99.9% purity) were kindly provided by the Bayer CropScience, Germany. The two triadimefon enantiomers and four triadimenol stereoisomers were prepared by normal chiral HPLC with a Chiralpak AD-H column (Daicel, Japan). Briefly, racemic triadimefon, triadimenol A, and triadimenol B of known quantities (1000 mg/L) were injected into the chiral HPLC system. The mobile phase fractions corresponding to the purer enantiomers were collected manually by observing their UV signals. The purity of each separated stereoisomer (enantiomer), checked with chiral HPLC using the same system, was >98%. HPLC-grade methanol, hexane, and 2-propanol were purchased from Sigma–Aldrich (Steinheim, Germany). Analytical-grade sodium chloride (NaCl), anhydrous magnesium sulfate (MgSO4 ), and acetonitrile (ACN) were purchased from Beihua Fine-Chemicals Co. (Beijing, China). Ultra-pure water was obtained from a Milli-Q system (Bedford, MA, USA). Primary secondary amine (PSA, 40 ␮m) and graphitized carbon black (GCB, 40 ␮m) sorbents were obtained from Agela Technologies Inc. (Tianjin, China). The mobile phase solvents were filtered through a 0.22 ␮m pore size filter membrane (Tengda, Tianjin, China) before use. 2.2. Aquatic toxicity bioassays Stereoselectivity in aquatic toxicity was evaluated by 48 h acute toxicity assays using D. magna as the test species. Stock organisms were obtained from the Chinese Academy of Protection and Medical Science (Beijing, China). Prior to testing, a sensitive test for Daphnia to potassium dichromate (K2 Cr2 O7 ) was performed as a positive control, and the LC50 (24 h) value was about 1.38, in the range of 0.6–1.7 mg/L [30]. Toxicity tests were performed as previously described [31]. Briefly, five neonates were transferred into glass breakers filled with 20 mL of blank or test solutions of various concentrations. The test solutions with the highest concentrations were prepared by adding a known concentration of the stereoisomer (or racemate) to the dilution water. Subsequent dilutions were prepared from solutions with the highest concentrations. The maximum content of acetone in the final test solutions was <0.1% (by volume), which had no effect on the survival of the test species. Seven concentrations [ranging from 2 mg/L to 22.8 mg/L and 3 mg/L to 34.17 mg/L for stereoisomers (or racemates) of triadimefon and triadimenol, respectively] and two controls (a test water control and a test acetone control) for each compound were tested. Four replicates were preformed for each treatment. The test animals were not fed and were incubated at 22 ± 1 ◦ C for 48 h. Mortality of the Daphnia was observed after incubation for 48 h. The concentration that caused 50% mortality of the test population (LC50 ) was determined from the survival data by a probit equation with SPASS 18.0. Tests were considered to be valid if control mortality was <10%. 2.3. Plant care and fungicide application in greenhouse The field experiments were conducted during March and April 2012. Tomato (Lycopersicon esculentum) seeds and cucumber (Cucumis sativus) seeds purchased from Beijing Baofeng Seeds Co. (Beijing, China) were cultivated to tomato and cucumber seedlings in greenhouse. Sixteen plots of working areas for tomato and cucumber were chosen at the experimental field, located at the experimental base of Institute of Plant Protection, Chinese Academy

Y. Li et al. / Journal of Hazardous Materials 265 (2014) 115–123

A

117

O

O

OH CH3

*

*

O

CH3

*

CH3 N

CH3

N

Carbonyl Reduction

N

Cl

CH3 CH3 N

Cl

N

N

Triadimefon

S

100

Diastereomer A 1R, 2S 1S, 2R %

Enantiomers R S

0

Diastereomer B 1R, 2R 1S, 2S

Triadimefon

10.00

20.00

30.00

40.00

SS

100

SR RS

Metabolism

B

Triadimenol

R

%

RR

0

Triadimenol

10.00

20.00

30.00

40.00

Time

Fig. 1. (A) Metabolic transformation of triadimefon to triadimenol (*chiral center). (B) Enantioselective LC–MS/MS (MRM) chromatogram of triadimefon and triadimenol mixed standards.

of Agricultural Sciences (LangFang, China), each with an area of 30 (10 × 3) m2 . A buffer zone was set up between plots. For each vegetable, three plots were used to avoid random error, and a fourth plot was used as control (without fungicides). The plots had not been treated with triadimefon or triadimenol for more than 3 years. The temperature inside greenhouse was 22 ± 10 ◦ C throughout the experiment. Rac-triadimefon (20% emulsifiable concentrate) was applied in two application modes (foliar spray and soil irrigation) at the same dosage of 250 g a.i. ha−1 (gram of active ingredient per hectare) at fruit setting stage of tomato and cucumber. Three representative fruit samples (approximately 1000 g each) from each plot were collected on day 0 (2 h after application) and at 1, 3, 5, 7, 10, 14, 21, and 28 d after treatment. Soil samples were the composite of 16–20 subsamples collected from a depth of 0–10 cm using a plastic coring tube (5 cm diameter) at increasing time intervals (0, 1, 3, 5, 7, 10, 14, 21, 28, 40, and 60 d). All tomato and cucumber samples were rinsed with distilled water to remove the exterior residues and dust, and then homogenized using a blender (Philips, China). Excess water was removed by pressing the samples with filter paper. Soil samples were air-dried at room temperature, homogenized, and then passed through a 2-mm sieve. The physicochemical characteristics of the soil was as follows: organic matter, 1.16%; pH (suspension of soil in 0.01 M CaCl2 , 1:2.5, w/w), 7.82; sand, 15.1%; silt, 45.1%; and clay 39.8%. The treated samples were stored at −20 ◦ C in the dark until analysis. 2.4. Extraction Samples were first thawed at room temperature. Extraction of triadimefon and triadimenol from cucumber, tomato, and soil was carried out by the QuEChERS method. In brief, 10 g of finely

homogenized sample (dry weight basis for soil) was weighed in a 50 mL Teflon centrifuge tube, then 5 mL of water (only for soil) and 10 mL of ACN were added. The mixtures were vigorously shaken for 30 min at 25 ◦ C in a water bath shaker (Dongming Medical Instrument, Harbin, China). Subsequently, 4 g of MgSO4 and 1 g of NaCl were added. The tubes were capped, immediately vortexed vigorously for 3 min, and then centrifuged for 5 min at a relative centrifugal force (RCF) of 2599 × g. Afterward, 1.5 mL of the ACN (upper) layer was transferred into a single-use 2 mL centrifuge tube containing 150 mg of anhydrous MgSO4 and an appropriate amount of sorbent (40 mg PSA and 10 mg GCB). The samples were vortexed again for 1 min and then centrifuged at 2077 × g RCF for 5 min. The resulting supernatant was then filtered using a 0.22 ␮m Nylon syringe filter for chromatographic injection. 2.5. Enantioselective LC–MS/MS analysis Waters ACQUITY UPLCTM system (Milford, MA, USA) equipped with a Chiralcel OD-RH column (Daicel Chemical Industries Ltd., Japan, 150 mm × 4.6 mm i.d., 5 ␮m particle size) packed with cellulose tris(3,5-dimethylphenylcarbamate) was used for the analysis of triadimefon and triadimenol. Simultaneous separation of the six enantiomers was carried out by gradient elution using solvent A (HPLC-grade methanol) and solvent B (5 mM ammonium acetate in ultrapure water) as mobile phase with the flow rate set to 0.3 mL/min. The gradient elution program was started at 65% A in 0–30 min. This composition was linearly increased to 80% in 30–32 min, and then maintained for 8 min (32–40 min) before returning to the initial conditions (65% A) in 2 min. The column was re-equilibrated at the initial mobile phase composition until the total run time of 45 min. The sample injection volume was 5 ␮L.

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Table 1 First-order rate constants (K), half-lives (T1/2 ), and correlation coefficients (R2 ) for the degradation of triadimefon enantiomers in tomato, cucumber, and soil. Experiment Tomato

Application mode Foliar spray Soil irrigation

Cucumber

Foliar spray Soil irrigation

Soil

Foliar spray Soil irrigation

a b

k (day−1 )

Enantiomer

T1/2 (days)a

R2

R-(−)-triadimefon S-(+)-triadimefon R-(−)-triadimefon S-(+)-triadimefon

0.1396 0.1841 0.0944 0.1108

5.06 3.77b 7.34b 6.26b

± ± ± ±

0.06 0.04 0.08 0.05

0.9469 0.9018 0.9178 0.9092

R-(−)-triadimefon S-(+)-triadimefon R-(−)-triadimefon S-(+)-triadimefon

0.2153 0.2194 0.1410 0.1420

3.22 3.16 4.92 4.88

± ± ± ±

0.03 0.02 0.06 0.05

0.9642 0.9610 0.9141 0.9029

R-(−)-triadimefon S-(+)-triadimefon R-(−)-triadimefon S-(+)-triadimefon

0.0525 0.0615 0.0461 0.0486

13.20b 11.27b 15.04b 14.26b

± ± ± ±

0.7 0.6 0.9 0.8

0.9450 0.9550 0.9473 0.9462

b

Values represent the means ± SDs (n = 3). Statistical significantly difference between R-(−)-triadimefon and S-(+)-triadimefon, P < 0.05 (Student’s paired t-test).

The temperature at the column and sample manager were kept at 25 and 4 ◦ C, respectively. Quantification was achieved using a triple quadrupole mass spectrometer (Waters Corp., Milford, MA, USA) equipped with an ESI source operating in the positive mode. MS analysis was performed in the multiple reaction monitoring mode. Typical conditions were as follows: for triadimefon, transitions m/z 294.3 → 69 and m/z 294.3 → 197 were used for quantification and confirmation when the collision energies were set at 22 and 16 V, respectively. For triadimenol, transitions m/z 296 → 70 and m/z 296 → 99 were used for quantification and confirmation when the collision energies were set at 16 and 15 V, respectively. The optimized cone voltages of triadimefon and triadimenol were 25 and 23 V, respectively. In this work, the elution orders of triadimefon and triadimenol were determined by measuring the optical rotation of each enantiomer using reversed-phase LC coupled with an online OR-2090 detector (Jasco, Japan) using the same mobile phase composition as the UV detection at 223 nm. Under the conditions described above, the two compounds give six separated peaks with the retention time of SR-(−)-triadimenol, RS-(+)-triadimenol, SS-(−)-triadimenol, RR-(+)-triadimenol, R-(−)-triadimefon, and S(+)-triadimefon were approximately 24.41, 25.98, 28.92, 31.81, 38.42, and 40.11 min, respectively, as shown in Figs. 1 and 4. A series of standard working solutions of racemic triadimefon and triadimenol for the linearity of the six stereoisomers was prepared from the stock solution by serial dilution in pure ACN. Corresponding matrix-matched standard solutions of the same concentrations were prepared by adding blank tomato, cucumber, and soil sample extracts to each serially diluted standard solution. Satisfactory linearities of triadimefon or triadimenol stereoisomers (n = 7) in the range of 0.0052–2 mg/L were obtained when the correlation coefficients (R2 ) were higher than 0.9992 in all cases. The matrix effect was evaluated for the enantiomers in tomato, cucumber, and soil samples, and the external matrix-matched standards were eventually utilized for quantification to obtain more realistic results. Recoveries of triadimefon and triadimenol were determined immediately after fortifications. Preliminary experiments showed that the recoveries of triadimefon and triadimenol were 83.8–103.4% in tomato, 81.4–97.6% in cucumber, and 78.1–92.5% in soil, respectively. The limits of quantification (signal-to-noise ratio of 10) for triadimefon and triadimenol were estimated to be 0.5–2.8 ␮g/kg in three matrices based on an acceptable RSD < 12.4%. 2.6. Kinetic analysis It was assumed that the degradation of the triadimefon enantiomers in two vegetables and soil accorded with pseudofirst-order kinetic. Also, the corresponding rate constant (k) were

calculated according to Eq. (1). The starting point of regressive functions was the maximum value of the enantiomers concentrations in vegetables and soils, and then decreased in following days. The half-life (T1/2 , day) was estimated from Eq. (2): C = C0 e−kt T1/2 =

0.693 ln 2 = k k

(1) (2)

The enantiomeric fraction (EF) [32] was used to express the enantioselectivity of a pair of enantiomers: EF =

(+) (+) + (−)

(3)

Here, (+) and (−) are peak areas of the (+) and (−) enantiomers of triadimefon, triadimenol-A, and triadimenol-B eluted from the Chiralcel OD-RH column, respectively (Fig. 4). The EF values ranged from 0 to 1, with EF = 0.5 representing the racemic mixture. 3. Results and discussion 3.1. Enantioselective degradation of triadimefon in tomato and cucumber after two different application modes Degradation of triadimefon enantiomers in tomato and cucumber fruits was investigated under the foliage and soil applications of (±)-triadimefon under greenhouse conditions. Estimated halflives of the two enantiomers ranged from 3.77 d to 7.34 d in tomato and 3.16 d to 4.92 d in cucumber (Table 1). The half-life (T1/2 ) of pesticides in plants is an important indicator of pesticide efficacy and pollution [38]. The persistence of triadimefon was consistently prolonged under soil application than under foliage application. For example, the T1/2 of R-(−)-triadimefon and S-(+)-triadimefon in tomato were 5.06 and 3.77 d after foliage application, respectively, which correspondingly increased to 7.34 and 6.26 d after soil application (Table 1). These observations were consistent with the high T1/2 values found in cucumber. As shown in Fig. 2, the increased persistence may be partly attributed to the fact that a more complex uptake and accumulation process exists in the soil irrigation treatment (i.e., reaching the maximum triadimefon enantiomer concentrations requires longer time). Enantioselectivity in triadimefon degradation was evaluated by monitoring changes in EF values at predetermined intervals. Fig. 3A and B shows the EF values in tomato and cucumber over time for both foliage and soil treatments. Under foliage applications, the changed EF values ranged from 0.510 to 0.805 and 0.489 to 0.532 for tomato and cucumber, respectively. Therefore, enantioselectivities was thus clearly different between the two plants. The results

Y. Li et al. / Journal of Hazardous Materials 265 (2014) 115–123

A

450.00

R-(-)-triadimefon

400.00

SR-(-)-triadimenol

200.00 200.00

RR-(+)-triadimenol

160.00 160.00

Tomato foliar spray

120.00 120.00

80.00 80.00

250.00

RR-(+)-triadimenol

Cucumber foliar spray

250.00 200.00 150.00 100.00

100.00 50.00

0.00 15

20

25

0.00

30

0

5

10

Time (d)

60.00

20

320.00

R-(-)-triadimefon

D

30

0

10

20

30

750.00

R-(-)-triadimefon

RS-(+)-triadimenol

Concentration (μg/kg)

Tomato soil irrigation

20.00

600.00

SS-(-)-triadimenol

240.00

RR-(+)-triadimenol

Cucumber soil irrigation

160.00

70

60

70

RR-(+)-triadimenol

RR-(+)-triadimenol

200.00

60

SR-(-)-triadimenol RS-(+)-triadimenol SS-(-)-triadimenol

SR-(-)-triadimenol

280.00

SS-(-)-triadimenol

50

R-(-)-triadimefon S-(+)-triadimefon

F

S-(+)-triadimefon

RS-(+)-triadimenol

40.00

40

Time (d)

E

S-(+)-triadimefon SR-(-)-triadimenol

30.00

25

Time (d)

50.00

Concentration (μg/kg)

15

Concentration (μg/kg)

10

Soil foliar spray

150.00

50.00

5

RR-(+)-triadimenol

200.00

40.00 40.00

0

RS-(+)-triadimeno l SS-(-)-triadimeno l

SS-(-)-triadimenol

300.00

0.00 0.00

S-(+)-triadimefon SR-(-)-triadimenol

300.00

RS-(+)-triadimenol

350.00 Concentration (μg/kg)

Concentration (μg/kg)

SR-(-)-triadimenol

R-(-)-triadimefon

C

S-(+)-triadimefon

RS-(+)-triadimenol SS-(-)-triadimenol

350.00

R-(-)-triadimefon

B

S-(+)-triadimefon

Concentration (μg/kg)

240.00 240.00

119

120.00 80.00

Soil soil irrigation

450.00

300.00

150.00

10.00 40.00 0.00

0.00

0.00 0

5

10

15

20

25

30

0

5

10

Time (d)

15

20

25

0

30

10

20

30

40

50

Time (d)

Time (d)

Fig. 2. Concentrations of triadimefon and triadimenol enantiomers/stereoisomers in tomato, cucumber, and greenhouse soil over time after two application modes.

indicate a high selectivity for the degradation of triadimefon in tomato and a lower enantioselectivity in cucumber. Compared with foliage application mode, a similar but weaker trend of the changes in EF (0.545–0.714) was observed with the soil application mode in tomato, whereas a stronger tendency (0.506–0537) was found in cucumber. The different EF patterns observed between foliage and

soil application modes was thought to be caused by various factors, such as differences in uptake routes, translocation, metabolism, and persistence behavior that were involved in the enantioselective process. Combining EFs in Fig. 3A and B, it can be concluded that the S-(+)-enantiomer was preferentially degraded over the R(−)-enantiomer in both vegetables. Preferential degradation of the

(C) Soil-TF

(B) Cucumber-TF

(A) Tomato-TF

0.700

0.550

0.850

foliar spray

0.800

soil irrigation

0.540

foliar spray 0.650

0.750

0.530 0.600

EF

0.520

EF

EF

0.700 0.650

0.510

0.550

0.600

soil irrigation

0.550

foliar spray

0.500

soil irrigation 0.500

0.490

0.500

0.480

0.450 0

5

10

15 Time (d)

20

25

0.450 0

30

5

20

25

30

0

10

20

foliar spray-TN A foliar spray-TN B

soil irrigation-TN B

60

70

soil irrigation-TN B 0.7 0.6

EF

EF

foliar spray-TN B

0.400

0.800

50

(F) Soil-TN

0.500

0.850

40

Time (d)

0.600

0.900

30

0.8

0.700

0.950

15 Time (d)

(E) Cucumber-TN

(D) Tomato-TN 1.000

EF

10

soil irrigation-TN A

0.300 0.750

0.5

foliar spray-TN B

soil irrigation-TN A

0.4 0.200

0.700

soil irrigation-TN A soil irrigation-TN B

0.600 0

5

10

15 Time(d)

0.3

0.100

0.650

20

25

0.000 30

foliar spray-TN A

foliar spray-TN A 0

5

10

15 Time (d)

20

0.2 25

30

0

10

20

30

40

50

60

Time (d)

Fig. 3. Enantiomer fractions (EF) of triadimefon triadimenol-A, and triadimenol-B in tomato, cucumber, and greenhouse soil after two application modes.

70

120

Y. Li et al. / Journal of Hazardous Materials 265 (2014) 115–123

100

A

R-(-)

100

R-(-)

B

C

100

R-(-)

S-(+)

10.00

20.00

30.00

0

40.00

100

soil, 40 days

cucumber, 14 days

S-(+)

10.00

20.00

30.00

0

40.00

10.00

SS-(-)

RS-(+)

RR-(+)

%

%

RS-(+)

%

20.00

40.00

Triadimenol

SR-(-)

RS-(+)

SR-(-)

10.00

30.00

SS-(-)

RR-(+)

Triadimenol SS-(-) RR-(+)

0

20.00

100

100

Triadimenol

S-(+)

%

%

%

tomato, 14days

0

Triadimefon

Triadimefon

Triadimefon

SR-(-)

30.00

40.00

Time

0

10.00

20.00

30.00

40.00

Time

0

10.00

20.00

30.00

40.00

Time

Fig. 4. Enantioselective LC–MS/MS (MRM) chromatograms showing the elution of triadimefon and triadimenol extracted from tomato, cucumber, and soil after foliage treatment of rac-triadimefon. Note the difference in the triadimenol stereoisomer pattern among the three matrices.

S-(+)-enantiomer resulted in relative enrichment of the aquatically active R-(−)-enantiomer (Table 2), as shown in Fig. 4A and B. Established data show that enantioselective degradation occurs frequently for chiral triazole fungicides in plants [38–41]. Interestingly, other studies have demonstrated that the direction of enantioselectivity for some triazole fungicides may vary depending on the plant species. For example, the R-(−)-enantiomer of tebuconazole was found to be enriched in cabbage. By contrast, a reverse enantioselectivity pattern was observed in cucumber, with the S-(+)-enantiomer being degraded more slowly than the R-(−)enantiomer [40]. Enzyme systems in plants may play an important role in the enantioselective transformation of chiral compounds, which may be responsible for the preferential biodegradation of one of the enantiomers. Results from this and earlier studies demonstrated that the enantioselective degradation patterns of triazole fungicides are plant specific and depend on the properties of the enzyme systems in each plant. 3.2. Enantioselective degradation of triadimefon in soil after two different application modes Triadimefon concentrations in soil measured after foliage application were consistently lower than those measured after soil application throughout the entire dissipation study. Fig. 2C and F shows changes in the concentrations of triadimefon enantiomers in the soil samples over time. As shown in Table 1, the halflives of both enantiomers from soil application were longer than those from foliage application. Specifically, the half-lives of R-(−)triadimefon and S-(+)-triadimefon in greenhouse soil were 13.20 d and 11.27 d under foliage treatment and 15.04 d and 14.26 d under soil treatment, respectively. After 60 d of treatment, the EF values increased gradually from 0.503 to 0.630 for foliage application and from 0.504 to 0.563 for soil application (Fig. 3C), suggesting a relatively higher enantioselectivity under foliage application. The increase in EF values was significant (P < 0.05, Student’s paired t-test), indicating that S-(+)-triadimefon was preferentially over R(−)-triadimefon. The typical LC–MS/MS (MRM) chromatogram is

shown in Fig. 4C. This enantioselectivity was in agreement with that in two vegetables. The different half-lives and rates of enantioselectivity between two application modes in soil may be attributed to differences in primary deposition concentrations after two treatments. Generally, higher concentrations of triadimefon under soil application had higher persistence in soil. Therefore, it is likely that conditions under soil treatment were more unfavorable for microbial growth, resulting in a relatively low microbial activity and thus a weak biodegradation of the triadimefon enantiomers. Enantioselective degradation of triadimefon in soils has been reported in two previous studies under laboratory conditions [13,17]. However, reversed enantioselectivity was observed in these investigations. Garrison et al. [13] showed that S-(+)triadimefon is preferentially lost in three soil types in the USA, which is consistent with the results of the present study. However, another study proved that R-(−)-triadimefon is degraded more rapidly than S-(+)-triadimefon in both alkaline and acidic soils in China [17]. These observations indicate that enantioselectivity frequently occurs in triadimefon degradation in soil under both greenhouse and laboratory conditions, but enantioselectivity may vary significantly with the environmental conditions. Enantioselectivity may be rationalized on the level of microbial populations or Table 2 LC50 values for enantiomers (stereoisomers) and racemates of triadimefon and triadimenol to D. magna. Compounds

48 h-LC50 (␮g/mL)a

95% confidence interval

R2

Rac-triadimefon R-(−)-triadimefon S-(+)-triadimefon Rac-triadimenol SR-(−)-triadimenol RS-(+)-triadimenol SS-(−)-triadimenol RR-(+)-triadimenol

7.097 4.603 9.723 15.924 11.624 8.894 28.204 17.047

5.304–9.622 3.485–5.793 7.664–12.993 12.295–22.467 9.181–15.012 6.762–11.480 18.184–77.760 13.477–23.237

0.972 0.997 0.977 0.938 0.981 0.949 0.972 0.954

R2 represents the correlation coefficient. a Significant differences (p < 0.05) were observed between enantiomers or stereoisomers and racemates.

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consortia or on the level of enzymes responsible for uptake or actual degradation, etc. [42]. Further studies are needed to characterize interactions of environmental factors, such as soil properties, redox conditions, and microbial structures, with enantioselectivity in the behavior of triadimefon. Moreover, chiral interconversion was also suggested to be critically important in understanding the enantioselectivity of chiral pesticides in the environment [17,28,42]. In fact, enantiomerization of triadimefon has been observed in soils [17]. However, further verification of the speculations whether enantiomerization occurs in the degradation of triadimefon cannot be confirmed in this work, because of the lack of large amounts of single enantiomer standards for field treatment. 3.3. Formation of triadimenol as the primary metabolite of triadimefon The typical chromatograms in Fig. 4 show the enantioselective degradation of triadimefon and concurrent formation of triadimenol stereoisomers. No other metabolites were analyzed in the present work. Fig. 2 indicates that the decrease in the amount of parent compound was accompanied by the increase in the concentration of the metabolite. After foliage application, triadimefon was determined up to approximately 27.8%, 68.8%, and 78.5% transformed to triadimenol in tomato, cucumber, and soil, respectively (Fig. 2A–C). The maximum formation of triadimenol, however, seems to be correlated with different environmental media. Triadimenol was further degraded in tomato and cucumber, whereas in soil, the concentration of triadimenol stereoisomers reached a plateau and no further removal was observed with the time of treatment. Potentially, one of reasons that triadimenol did not exhibit apparently disappearance in soil was the relatively short experimental time (60 d) in this work, longer incubation times could have eventually shown the loss of these stereoisomers. It is interesting that in tomato, the triadimenol stereoisomer concentration followed the order RS-(+)-triadimenol > RR-(+)triadimenol > SS-(−)-triadimenol > SR-(−)-triadimenol during most of the experimental period (Fig. 2A). It was apparent that the RS-(+)-triadimenol was the most preferentially produced isomer via the reduction reaction, and notably, which was also known as the most toxic isomer to D. magna (Table 2). Surprisingly, the stereoisomer concentration order observed in cucumber and soil was significantly different from that observed in tomato, which was determined to be RR-(+)-triadimenol > SS-(−)triadimenol > SR-(−)-triadimenol > RS-(+)-triadimenol during most of the experiment period in cucumber and any given time in soil (Fig. 2B and C). Dramatically, RR-(+)-triadimenol was documented to be the highest amount of metabolite isomer, whereas only a very small amount of RS-(+)-triadimenol was produced. In summary, when triadimefon was treated in different media and even in different species of plants, each produced its own characteristic pattern of stereoisomer compositions of the productions of triadimenol (Fig. 4A–C). Previous studies also showed that different triadimenol stereoisomer patterns produced from rac-triadimefon may closely depend on environmental conditions and may vary significantly from soil to soil and species to species. For instance, Li et al. [17] found that the triadimenol stereoisomer concentration converted from triadimefon followed the order RR-(+)-triadimenol > SS-(−)triadimenol > SR-(−)-triadimenol > RS-(+)-triadimenol in alkaline soil. These results are consistent with the present work in cucumber and soil. However, a different composition pattern was observed in acidic soil, i.e., RR-(+)-triadimenol > SR-(−)-triadimenol > SS-(−)triadimenol > RS-(+)-triadimenol. In another study, Garrison et al. [16] reported that after 480 min exposure of rainbow trout liver microsomes to racemic triadimefon results in the production of at least twice amounts of SS-(−)-triadimenol as any of the other three isomers, while the most fungi-toxic isomer SR-(−)-triadimenol is

121

barely detectable. In a subsequent publication, a similar pattern was also observed by the same research group in studies of the chiral transformation of triadimefon to triadimenol in three soil types under laboratory conditions [13]. The discrepancies in different compositions of triadimenol stereoisomers from reduction of triadimefon are possible because of there may be variations in the microbial populations or enzyme systems in different types of soils and different species involved in this isomer-specific transformation process. Besides, the degradation behavior of individual triadimenol stereoisomers may also be different [39,43]. Under soil treatment, the maximum formation of triadimenol were estimated to be 44.8%, 47.2%, and 65.5% in tomato, cucumber, and soil, respectively (Fig. 2D–F). The formation rates in cucumber and soil were consistently lower under soil application than under foliage application. Interestingly, the formation rate in tomato was higher compared with the foliage application. Furthermore, the concentration orders of the four triadimenol stereoisomers in cucumber and soil were in accordance with those under foliage treatment. However, a different tendency was observed in tomato, the data in Fig. 3D suggests that the general order was RR-(+)-triadimenol > RS-(+)-triadimenol > SS(−)-triadimenol > SR-(−)-triadimenol. It could be concluded that different application modes may significantly alter the transformation patterns. 3.4. Stereoselectivity in aquatic toxicity Since the use of triadimefon dissolved in water or adsorbed on soil particles can be easily transported into aquatic systems via surface runoff. Triadimenol, for example, has been detected in water samples from ditches and streams at concentrations of up to a few microgram per liter [17]. Thus, some non-target aquatic organisms like D. magna may be exposed to different toxic effects to triadimefon and triadimenol. The LC50 values of individual stereoisomers and racemates to D. magna were used to evaluate the stereoselectivity of the two compounds in the aquatic toxicity tests, which is a relatively insensitive index used to evaluate the toxicity of a compound, with a lower value indicating a more toxic potency. As shown in Table 2, triadimefon was approximately 2.3 times more toxic than its metabolite triadimenol. In contrast to these results, triadimefon has been reported to be nearly fourfold more toxic than triadimenol to black fly larvae [33]. Differences in xenobiotic metabolism pathways may partly explain the relative differences observed in the toxicities of triadimefon and triadimenol between species. The t test indicated a significant difference in LC50 among stereoisomers of the same compound. For triadimefon, the R(−)-enantiomer was approximately 2.1 times more toxic than the S-(+)-form. For triadimenol, the decreasing order of toxicity to D. magna was RS-(+)-triadimenol > SR-(−)-triadimenol > RR(+)-triadimenol > SS-(−)-triadimenol. Specifically, the differences among the stereoisomers ranged from 1.3 to 3.2 times. It is interesting to note that SR-(−)-triadimenol, the most active stereoisomer against the target fungi (up to 1000-fold more active than the other three), was less toxic to D. magna. For triadimefon, previous study indicated that it is no significant difference in fungical activity between the two enantiomers [15]. These inconsistencies in stereoselectivity between fungicidal and aquatic toxicity suggest that different modes of action may be shared between the target fungi and aquatic invertebrates. While the toxicity testing is done for the individual isomers of triadimefon and triadimenol, limited experiments were done to determine whether enantiomerization occurred in the toxicity assay, which may has a great influence on the stereoselective toxicity of two compounds. In most cases, only one of the two enantiomers contributes to the majority of the acute aquatic toxicity of the chiral pesticides. For example, the toxicity differences against D. magna for

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the enantiomers of leptophos [34], fonofos [5], isocarbophos [31], and lactofen [35] were approximately 20-, 15-, 50-, and 47-fold, respectively. In addition, a maximum of 13-fold difference in stereoselective toxicity was observed among the four stereoisomers of chloramidophos [36]. However, until recently, little effort was made to elucidate the stereoselective toxicity of chiral triazole fungicides. Huang et al. [37] previously demonstrated that the (−)hexaconazole was about five times more toxic to algae than the (+)-hexaconazole based on individual 96 h-EC50 value. Previous and present results suggest that the enantiomers of triazole fungicides often exhibit differential toxicity. Toxicological effects of the racemate cannot be predicted simply by adding the individual effects of the enantiomers. Thus, currently available data on the toxicity of racemic mixtures of triazole fungicides are not reliable. Understanding the enantioselective toxicity of this structurally related class of fungicides is critical for environmental risk assessment and merits extensive investigation. 4. Conclusion In this study, we demonstrated that enantioselectivity may be reflected not only in the degradation of the parent enantiomers of triadimefon but also in the formation kinetics of the triadimenol stereoisomers under greenhouse conditions. The different uptake routes may significantly influence the chiral preference. It is important to emphasize that the six stereoisomers of triadimefon and triadimenol are independent entities with respect to their biological properties. Significant stereoselectivity was first observed in the acute toxicity of triadimefon and triadimenol to aquatic nontarget organisms incorporated with the transformation may have some environmental implications. Monitoring for the racemate, as currently practiced, will provide inadequate basis for assessing the environmental risks of the parent compound triadimefon. Taking the transformation of triadimefon in tomato as an example, the more toxic R-(−)-triadimefon was enriched in tomato and the most preferentially produced isomer RS-(−)-triadimenol was also exhibited the highest toxicity among the stereoisomers. Therefore, data derived only from racemic triadimefon will underestimate the ecotoxicological effects of this chiral contaminant. To summarize, the stereoselectivity in the toxicity, degradation, and transformation is of vital importance and needs to be taken into account for a more comprehensive environmental risk assessment of triadimefon. Acknowledgments This work was financially supported by the foundation established by the National Natural Science Foundation of China (31272071, 31071706, and 31171879) and the National Basic Research Program of China (The 973 Program, Grant No. 2009CB119000).

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