Dissipation and Enantioselective Degradation of Plant Growth

Dec 19, 2012 - ... persistence of paclobutrazol in open field surface soil at mango tree ... differences of the two chemicals in open field and green ...
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Dissipation and Enantioselective Degradation of Plant Growth Retardants Paclobutrazol and Uniconazole in Open Field, Greenhouse, and Laboratory Soils Chengwang Wu,† Jianqiang Sun,† Anping Zhang,†,* and Weiping Liu‡ †

International Joint Research Centre for Persistent Toxic Substances (IJRC-PTS), College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032, China ‡ IJRC-PTS, MOE Key Laboratory of Environmental Remediation and Ecosystem Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China S Supporting Information *

ABSTRACT: Greenhouses are increasingly important in human food supply. Pesticides used in greenhouses play important roles in horticulture; however, little is known about their behavior in greenhouse environments. This work investigates the dissipation and enantioselctive degradation of plant growth retardants including paclobutrazol and uniconazole in soils under three conditions (i.e., open field, greenhouse, and laboratory). The dissipation and enantioselective degradation of paclobutrazol and uniconazole in greenhouse were different from those in open field; they were more persistent in greenhouse than in open field soil. Leaching produced by rainfall is responsible for the difference in dissipation. Thus, local environmental impacts may occur more easily inside greenhouses, while groundwater may be more contaminated in open field. Spike concentrations of 5, 10, and 20 times the concentrations of native residues were tested for the enantioselective dissipation of the two pesticides; the most potent enantioselective degradation of paclobutrazol and uniconazole occurred at the 10 times that of the native residues in the greenhouse environments and at 20 times native residues in open field environments. The higher soil activity in greenhouses than in open fields was thought to be responsible for such a difference. The environmental risk and regulation of paclobutrazol and uniconazole should be considered at the enantiomeric level.

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INTRODUCTION Triazole plant growth regulators are important chemicals used to improve agronomic productivity in various crops such as vegetable, wheat, rice, and trees.1,2 The chemicals include paclobutrazol [(2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2(1H-1,2,4-triazol-1-yl) pentan-3-ol] and uniconazole [(E)(RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-1(1H-1,2,4-triazol-1yl) pent-1-en-3-ol ] as important plant growth retardants. Both paclobutrazol and uniconazole are carbon chiral due to the presence of the asymmetrically substituted carbon atoms in the triazol alkyl moiety (Figure SI-1 in the Supporting Information (SI)). In theory, paclobutrazol consists of two pairs of enantiomers with (2R, 3R)- and (2S, 3S)-configurations, and (2S, 3R)- and (2R, 3S)-configurations because of two stereogenic centers. However, the production process for paclobutrazol gives only (2R, 3R)-(+) and (2S, 3S)-(−)enantiomers because of steric−hindrance effects. Uniconazole has an asymmetric carbon and thus exists as a single pair of enantiomers with (R)-(−)- and (S)-(+)-configuration. In both cases, plant growth retardation activity is mainly from (S)enantiomer. The application of only (S)-enantiomer in place of racemic products has been recommended in many previous © 2012 American Chemical Society

studies. The use of only the active enantiomer is beneficial in many ways, such as reduction of application rate and amount of chemicals and prevention of nontarget toxicity from potential side-effects of inactive enantiomer.1,2 However, both paclobutrazol and uniconazole have been applied as racemic forms due to limitations of the production techniques and have been discharged into the environment as racemic forms. Soil is a very important environmental matrix and often acts as ‘source’ and ‘sink’ for numerous pollutants. It is also a more suitable media for enantioselective degradation of chiral pesticides than water and atmosphere because of the greater abundance of microbes.3 Enantioselective degradation in soils was observed for a wide variety of chiral pesticides such as organochlorine pesticides,4−7 aryloxyphenoxypropionate herbicides (including mecoprop,8−12 dichlorprop,9,10 fenoxapropethyl,13 and fluazifop-butyl12,14), triazole fungicide (including fenbuconazole,15 epoxiconazole and cyproconazole 16), pyrethReceived: Revised: Accepted: Published: 843

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roid insecticides,17 organophosphorus pesticides (such as ruelene 9), and acetamide pesticides.18−21 Chiral triazole plant growth retardants may also undergo enantioselective degradation in soils. However, there is little knowledge about the enantioselective degradation of chiral triazole plant growth retardants in soil. Soil for crop production is generally used under two scenarios: open fields and greenhouses. Greenhouses have a long history, and the use of greenhouse for crop growth has increased in recent years because of the need for increased food production in marginal environments. It is expected that the use of greenhouse will continue to grow worldwide with the increase of human population.22 However, in contrast to the rapid growth of greenhouse use, studies on the environmental behavior of pesticides in greenhouses are very limited and obviously lagging behind field studies. Paclobutrazol and uniconazole have been successfully employed to improve growth performance of plant cultivated in open field and greenhouse soils.1,2 A complete understanding of the dissipation of these chiral plant growth retardants must, therefore, include some knowledge of their enantioselectivity under different soil conditions. In this study, we investigated the dissipation and enantioselective degradation of paclobutrazol and uniconazole in open field surface, greenhouse surface, and laboratory soil so as to probe the effect of greenhouse soil conditions on the environmental behavior of the enanitomers of these chiral plant growth retardants.

spiking solution was added into 500 mL of distilled water at each location and was then mixed with soil of 1 kg thoroughly for several minutes. Following spiking, a subsample (t = 0) was collected in triplicate, wrapped in cleaned aluminum foil, sealed in plastic bags, and stored at −20 °C until analysis. The rest of the spiked soil was placed back into the holes that were dug in the experimental area and leveled with the surrounding soil, to recreate undisturbed conditions. The soil was sampled in triplicate after 15, 60, and 140 days, respectively. Soils used for laboratory test were only sampled from the greenhouse and divided into two subsamples, one of which was sterile for 2 h at 121 °C and the other one was not. The sterile and nonsterile soils were transferred into two containers separately and were placed in an incubator at 25 °C. The soils were sampled for analysis from the containers after 15, 60, and 180 days and were kept in the freezer until analysis. Determination of Soil Properties. The soil physicochemical characteristics including soil pH values (pH(KCl)), total organic carbon (TOC), texture, and microbial biomass content (Cbio) were measured using the method described in previous studies.6,23,24 The procedures are included in text of the SI. Mobility and Leaching of Pesticides in Soil. Paclobutrazol (200 μg) and uniconazole (200 μg) were added into a glass column (diameter of 20 mm and length of 250 mm) packed with 10 g of soil and eluted with 50 mL of water, equivalent to the rainfall amount during the field experiment period in the studied area. The soil column was subsequently divided into four layers (0−5 cm, 5−10 cm, 10−15 cm, and 15−20 cm), and concentrations of paclobutrazol and uniconazole in each soil layer were determined to evaluate the mobility and leaching of pesticides in soil column. To evaluate the effect of rainfall on dissipation of paclobutrazol and uniconazole in open field surface soil and greenhouse surface soil, paclobutrazol (200 μg) and uniconazole (200 μg) were spiked in soil, and then, the spiked soils were wrapped by steel nets and placed in open field and greenhouse by the method that was used in incubation test. After a heavy rainfall, four layers of soil at vertical (0−5 cm, 5−10 cm, 10−20 cm, 20−30 cm, and 30−40 cm) and horizontal directions (5 −10 cm, 10− 15 cm, 15−30 cm, and 30−40 cm) were sampled, respectively, and then, the concentration of paclobutrazol and uniconazole in each soil layer was determined. The mass of pesticides in each layer was determined by the concentration of pesticides in each soil layer multiplied by soil weigh of corresponding layer. Extraction, Cleanup, and Analysis. An aliquot of a 10 g sample of soil was extracted with 150 mL of hexane/acetone (v/v = 1/1) using a Soxhlet apparatus for 20 h. The extract was evaporated under vacuum to 5 mL, and the volume was further reduced to 1−2 mL by high-purity nitrogen blowdown. Samples were cleaned using a glass column (diameter of 10 mm and length of 250 mm) packed with anhydrous sodium sulfate (1 g), florisil (6 g), and anhydrous sodium sulfate (1 g) from the bottom to top. The column was eluted with 70 mL of hexane containing 30% (v/v) acetone, the effluents containing the two pesticides were collected, and the solvent was exchanged into hexane by rotary evaporation and high pure nitrogen blowdown and adjusted to a volume of 1 mL for analysis. The quantification of the sample was performed using an Agilent gas chromatograph 6890 equipped with a Nickel 63 electron capture detector (μECD) equipped with a HP-5 column (30 m, 0.25 mm i.d., 0.25 μm film thickness; Agilent Technologies Inc.). The chromatographic conditions were as follows: carrier gas, nitrogen 1 mL min−1; temperature

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EXPERIMENTAL SECTION Chemicals. All racemic standards (paclobutrazol and uniconazole) were obtained from Jiangsu Sevencontinent Green Chemical Co., Ltd. (Zhangjiagang, China). The florisil (60−100 mesh) and anhydrous granular sodium sulfate of pesticide residue grade, were purchased from Merck China (Shanghai, China) and Hangzhou Huipu Co. Ltd. (Hangzhou, China), respectively, were extracted for 48 h in a Soxhlet apparatus, and then were baked in the oven at 450 °C for 12 h before use. All solvents were of high performance liquid chromatography (HPLC) grade. Incubation of Pesticides in Soil under Open Field, Greenhouse, and Laboratory Conditions. Incubation of pesticide in open field surface soil and greenhouse surface soil were conducted on a farm in Fuyang in southeastern China. The distance between the open field and the greenhouse was about 3 m. Soils (0−5 cm) were sampled from three locations 2 m apart in each of the open field and greenhouse and were then transferred to laboratory for native pesticides residues that were already present in the soils before our study and soils properties analysis before incubation. The native residues of paclobutrazol were 4.1 ± 0.11 ng g−1 and 3.8 ± 0.12 ng g−1 for open field and greenhouse surface soil, respectively, and of uniconazole, 6.2 ± 0.15 ng g−1 and 5.9 ± 0.12 ng g−1, respectively. No significant difference in residues and enantiomeric fractions of the two pesticides was observed between the open field surface soil samples and greenhouse surface soil samples. The same procedure was employed for the incubation in greenhouse and open field, as described in the following. Incubation soils were sampled and spiked at each location in situ. The spiking solution was prepared in acetone containing 40 mg L−1 paclobutrazol and 60 mg L−1 uniconazole. Volumes of 0.5, 1, and 2 mL were used to result in the spike concentrations (on a dry weight basis) about 5, 10, and 20 times higher than the concentrations of native residues of each sampling point. The 844

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Table 1. Physicochemical and Microbial Parameters of Soils in Open Field and Greenhouse open field greenhouse

pH

TOC (%)

clay (%) 0.05 mm

Cbio (mg C/kg·dm)

Cbio/ TOC

6.1 ± 0.15 6.3 ± 0.058

3.1 ± 0.25 2.9 ± 0.15

29 ± 0.58 31 ± 0.0

51 ± 0.58 48 ± 1.2

19 ± 0.58 21 ± 1.2

223 ± 11 289 ± 19

72 100

Table 2. Dissipation of Paclobutrazol and Uniconazole in Open Field Surface Soils and Greenhouse Surface Soilsa paclobutrazol I

uniconazole

II

III

t t t t

= = = =

0d 15 d 60 d 140 d

25 ± 0.11b (0%)c 22 ± 0.15 (15%) 14 ± 0.092 (46%) 5.0 ± 0.12 (80%)

47 ± 0.65 (0%) 27 ± 0.29 (43%) 17 ± 0.26 (64%) 9.0 ± 0.19 (81%)

t t t t

= = = =

0d 15 d 60 d 140 d

24 ± 0.21 (0%) 22 ± 0.32 (9%) 16 ± 0.45 (33%) 9.0 ± 0.19 (62%)

44 37 36 23

± ± ± ±

0.48 0.34 0.45 0.26

(0%) (16%) (18%) (47%)

94 71 52 28

± ± ± ±

85 80 65 44

± ± ± ±

Open Field 2.3 (0%) 1.7 (25%) 0.84 (45%) 0.29 (70%) Greenhouse 1.1 (0%) 0.76 (6.0%) 0.67 (23%) 0.45 (48%)

I

II

III

43 ± 1.6 (0%) 37 ± 0.12 (14%) 25 ± 0.11 (41%) 7.0 ± 0.17 (85%)

70 ± 0.38 (0%) 32 ± 1.2 (54%) 19 ± 0.89 (72%) 9.0 ± 0.42 (83%)

132 ± 3.2 (0%) 112 ± 3.6 (15%) 75 ± 1.1 (64%) 22 ± 0.97 (83%)

39 35 26 18

± ± ± ±

0.32 0.74 0.34 0.24

(0%) (11%) (33%) (54%)

69 53 43 33

± ± ± ±

1.2 (0%) 0.92 (23%) 0.36 (38%) 0.78 (52%)

125 ± 3.8 (0%) 102 ± 2.3 (17%) 86 ± 0.83 (34%) 60 ± 0.12(52%)

a

I, II, and III correspond to spike concentrations 5 times, 10 times, and 20 times higher than native concentration, respectively. bConcentrations, ng g dw−1. cValues in brackets are dissipation percentages. −1

program, 60 °C ramped at 20 °C min−1 to 220 °C and held for 15 min; inlet temperature, 250 °C; and detector temperature, 320 °C. Enantiomer analysis was done by the gas chromatograph with a chiral capillary column BGB-172 (20% tert-butyldimethylsilylated-β-cyclodextrin in OV-1701, 30 m, 0.25 mm i.d., 0.25 μm film thickness; BGB Analytik AG, Boeckten, Switzerland). Samples (2 μL) were injected splitless, and split was opened after 1.0 min. The initial oven-temperature was set at 90 °C. After a 1-min hold, oven-temperature programs were used as follows: 15 °C min−1 to 160 °C, 2 °C min−1 to 200 °C, hold 3 min, 15 °C min−1 to 230 °C, hold 56 min. Nitrogen was used as the carrier gas at a flow rate of 1 mL min−1. The injector and detector temperatures were set at 250 and 320 °C, respectively. Quality Control/Quality Assurance. Method blanks (solvent) were processed by extracting and analyzing 10 g of sodium sulfate using the same procedure as that used for samples. A method blank was analyzed with each batch of samples. The “limit of detection” (LOD) was defined as the ratio between the amount of target compound that had a chromatographic peak with signal/noise ratio of 10 and the dry weight of soil sample. The LOD values for paclobutrazol and uniconazole were 0.32 and 0.25 ng g−1, respectively. All the method blanks were less than 5% of the lowest residues values of the samples, and the pesticide concentrations reported were not corrected by method blank. Spike recovery experiments were conducted by spiking the two pesticides into 10 g of soils and subsequently analyzing in the same manner as was done for samples. A spike sample was run with every batch of samples. After correcting for the native amounts in the soil, the mean recovery was 90% for paclobutrazol and 86% for uniconazole. Enantiomers of paclobutrazol and uniconazole were prepared by the method described in previous studies.1,2 Briefly, a racemic standard was separated by a HPLC equipped with a Chiralpak-AD chiral columun and chiral circular dichlorism detector, giving a pair of enantiopure standards that can be manually collected at the outlet of HPLC according to a positive or negative peak on the chromatogram. The resolved enantiopure standards were injected into an Agilent 6890 GC

instrument equipped with ECD for assignment of the correspondence between enantiomers and peaks in the gas chromatography (GC) chromatogram. The elution order of enantiomers of the two pesticides on chiral BGB-172 column was (2S, 3S)-(−)-paclobutrazol, (2R, 3R)-(+)-paclobutrazol, (S)-(+)-uniconazole, and (R)-(−)-uniconazole. The enantioselective degradation of the two pesticides was evaluated using DEVrac, which is defined as deviation of the enantiomer fraction (EF) from racemic (DEVrac = absolute value of 0.500 − EF).4 Thr EF was calculated by the ratio of peak area of the (+)-enantiomer to sum peak area of the (+) and (−) enantiomer eluting from the chiral BGB-172 column. Racemic standards were injected every six samples, and the average DEVrac values of the standards were 0.001 ± 0.000004 for paclobutrazol and 0.002 ± 0.000006 for uniconazole, which were not significantly different from 0.000, indicating the reproducibility of the method was reliable in measuring DEVrac. All the comparisons were statistically evaluated by the student t-test.25

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RESULTS AND DISCUSSION Physicochemical and Micobial Soil Properties. Changes in the soil environment can alter physicochemical and microbial soil properties, such as pH, total organic carbon (TOC), soil texture, and biomass carbon (Cbio). Among these soil parameters, Cbio is the most rapidly changeable parameter; thus, it is thought to be sensitive indicator of changes in soil quality. An increase of the ratio of Cbio/TOC is indicative of organic matter losses.24 The measured soil pH, TOC, texture, and Cbio are listed in Table 1. The results of the student t-test revealed that there was no significant difference for most of the soil parameters between open field surface soil and greenhouse surface soil except Cbio, indicating that Cbio was affected by subtle changes of soil environment. Greenhouse surface soil had a higher Cbio than open field surface soil, leading to a higher ratio of Cbio/TOC in greenhouse surface soil than in open field surface soil, suggesting greenhouse cultivation would lead to the loss of soil organic matter and further produce adverse effects on producibility of soil. 845

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Dissipation of Paclobutrazol and Uniconazole in Open Field and Greenhouse Surface Soils. Paclobutrazol and uniconazole residues declined consistently with time in open field surface soil as well as in greenhouse surface soil (Table 2), suggesting the dissipation of paclobutrazol and uniconazole had no lag period or the lag period was less than 15 days. The results of the student t-test indicated that the dissipation of the two chemicals in the open field was statistically different from that in the greenhouse. A mean of 77 ± 6.1% (range 70% −81%) of the paclobutrazol was dissipated in open field surface soil after a 140 day long-term dissipation experiment. However, paclobutrazol was slowly dissipated in greenhouse surface soil throughout the 140 days experiment period, with mean value of 52 ± 8.4% (range 47− 62%) of the initial concentration still present after 140 days. With respect to uniconazole, the dissipation percentages vary from 83% to 85% (mean value, 84 ± 1.2%) for open field surface soil and from 52% to 54% (mean value, 53 ± 1.2%) for greenhouse surface soil. Generally, higher concentrations of paclobutrazol and uniconazole had higher persistence in open field surface soil. Similar results were observed for persistence of paclobutrazol in open field surface soil at mango tree basins.26,27 However, such a pattern did not appear in the dissipation of paclobutrazol and uniconazole in greenhouse surface soil, which indicates paclobutrazol and uniconazole are most persistent at medium concentrations (i.e., concentrations of 10 times native residues). The difference of concentration effects on dissipation patterns of paclobutrazol and uniconazole between open field surface soil and greenhouse surface soil might be due to different soil microbial community or activity between open field and greenhouse, which was indicated by higher Cbio values in greenhouse surface soil than in open field surface soil. The first-order kinetics was not used to calculate the half-life of paclobutrazol and uniconazole dissipation in this study, because, in most cases, dissipation of three-quarters of initial concentration, which is prerequisite of the first-order kinetics application, was not achieved at the end of the experiment. It may be argued that extending the experiment period may achieve the requirement. However, the greenhouse was only used for crop cultivation during the period of about 140 days in the studied area. Therefore, the last sampling time was set at the 140th day in this work. Although the exact half-life of dissipation cannot be calculated by the first-order kinetics, it can be roughly estimated by dissipation percentage of initial concentration at the last sampling time. The estimated dissipation half-lives of paclobutrazol were 70 days in open field surface soil and over 140 days in greenhouse surface soil, and for uniconazole, they were about 50 days and 140 days, respectively, in the two different soils. These results suggested that dissipation of paclobutrazol and uniconazole was much faster in open field surface soil than in greenhouse surface soil. This is surprising because degradation of paclobutrazol and uniconazole in greenhouse surface soil was expected to be faster than that in open field surface soil due to faster degradation at higher temperature in greenhouse, and degradation was generally thought to be the dominate pathway of dissipation of pesticides in soil.28 Although the environmental factors relating to volatilization, such as atmospheric mixing height and wind direction and speed,29 are very different between open field and greenhouse, they cannot produce obvious effect on the concentration differences of the two chemicals in open field and green house soils. This is because the volatilization is not

the important dissipation way for the two chemicals in the soils.30,31 Thus, it can be assumed that pathways other than degradation and volatilization, such as leaching and runoff, might also contribute to the dissipation of paclobutrazol and uniconazole in open field surface soil. Mobility and Leaching of Paclobutrazol and Uniconazole in Soil. The tests on leaching of paclobutrazol and uniconazole in soil columns under laboratory conditions were carried out to initially assess the leaching potential of paclobutrazol and uniconazole in soil. The results are tabulated in Table 3 and indicate that paclobutrazol of 53%, 31%, 6%, and Table 3. Leaching of Paclobutrazol and Uniconazole in Soil Columns

a

soil layers

paclobutrazol

0−5 cm 5−10 cm 10−15 cm 15−20 cm

106 ± 2.5a (53%)b 61 ± 1.2 (31%) 12 ± 0.54 (6.0%) 5.4 ± 0.22 (2.7%)

unconazole 91 43 28 15

± ± ± ±

2.1 (46%) 0.98 (22%) 0.87 (14%) 0.68 (8%)

Concentrations, ng g−1dw−1. bPercentage of loaded amount.

2.7% were distributed in soil layers of 0−5 cm, 5−10 cm, 10− 15 cm, and 15−20 cm, respectively. Paclobutrazol of 7.3% was eluted from the soil column. For uniconazole, the distribution percentages in soil column were 46% for 0−5 cm, 22% for 5− 10 cm, 14% for 10−15 cm, and 8% for 15−20 cm, and spiked amount of 12% was eluted out. The results indicate that paclobutrazol and uniconazole had potentials to penetrate into deep soil layers, and moreover, uniconazole was more potent than paclobutrazol in penetration and leaching. A further study was conducted to investigate the effect of rainfall on dissipation of the two pesticides by determining concentrations of the two pesticides in open field and greenhouse soils at different layers in vertical and horizontal directions after a heavy rainfall. It was found that paclobutrazol and uniconazole were distributed into each soil layer in vertical direction for open field condition, while most of the paclobutrazol and uniconazole remained in surface soil of 0−5 cm for greenhouse condition (Table 4), suggesting that penetration produced by rainfall gave a greater contribution to the dissipation of the two pesticides in open field surface soil than that in greenhouse surface soil. Another interesting finding was that mobility in vertical direction was much higher than that in horizontal direction for both paclobutrazol and uniconazole in open field soil. This was indicated by higher residues of paclobutrazol and uniconazole in soil in vertical direction than in horizontal direction (Table 4). The results implied that paclobutrazol and uniconazole had direct risks to groundwater due to their high persistence and mobility potential. Groundwater is often pumped to the surface for agricultural and industrial application, and eventually discharged into surface water. Moreover, both paclobutrazol and uniconazole exhibited strong stimulatory effect on growth of Anabaena sp. and Microcystis aeruginosa, which are dominant species in cyanobacteria blooms.1,2,32 Thus, great concern should be paid to contamination of surface water by application of groundwater containing paclobutrazol and uniconazole. Degradation of Paclobutrazol and Uniconazole in Sterile and Nonsterile Soil. Tests on degradation of paclobutrazol and uniconazole in sterile and nonsterile soils under laboratory conditions were conducted to evaluate the contribution of abiotic degradation and biodegradation to dissipation of paclobutrazol and uniconazole in soil. Similar to 846

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Table 4. Distribution of Paclobutrazol and Uniconazole in Vertical and Horizontal Soil Layers paclobutrazol

a

soil layers

open field

0−5 cm 5−10 cm 10−20 cm 20−30 cm 30−40 cm

73 ± 5.7a (37%)b 64 ± 1.2 (32%) 22 ± 8.9 (11%) 15 ± 6.8 (7.5%) 6.2 ± 1.5 (3.1%)

0−5 cm 5−15 cm 15−30 cm 30−40 cm

0.64 ± 0.066 BLD BLD BLD

uniconazole open field

greenhouse Vertical Distribution 189 ± 8.9 (95%) 7.0 ± 1.2 (3.5%) BLD BLD BLD Horizontal Distribution BLD BLD BLD BLD

42 37 28 21 24

± ± ± ± ±

3.5 4.5 1.5 1.2 1.3

(21%) (19%) (14%) (10%) (12%)

0.51 ± 0.064 0.26 ± 0.066 BLD BLD

greenhouse 190 ± 5.8 (95%) 5.0 ± 2.5 (2.5%) BLD BLD BLD BLD BLD BLD BLD

Mass, ng. bPercentage of loaded amount.

the dissipation of paclobutrazol and uniconazole in open field surface soil and greenhouse surface soil, residues of paclobutrazol and uniconazole also decreased with the increase of incubation time in sterile and nonsterile soils (Table 5),

greenhouse surface soil gave a more significant contribution to the degradation of easily degraded paclobutrazol than that of difficultly degraded uniconazole. The results suggest that the degradation was the dominant dissipation pathway for paclobutrazol and uniconazole in greenhouse surface soil. The degradation of paclobutrazol and uniconazole in nonsterile soil contained abiotic degradation and biodegradation, while only abiotic degradation occurred in sterile soil.28 On the basis of the above hypothesis, it can be deduced that the difference between degradation in nonsterile soil and degradation in sterile soil was contributed by biodegradation. Therefore, it can be estimated that paclobutrazol dissipation of 52% in greenhouse surface soil consisted of abiotic degradation of 34% and biodegradation of 18%, while abiotic degradation and biodegradation contributed 30% and 23% to 53% of uniconazole dissipation in greenhouse surface soil, respectively. Comparing the dissipation of the pesticides in open field surface soil and that in greenhouse surface, it can be estimated that dissipation produced by rainfall contributed at least 24% and 31% to general dissipation of paclobutrazol and uniconzaole, respectively, in open field surface soil. Enantioselective Degradation of Paclobutrazol and Uniconazole in Open Field Surface Soil, Greenhouse Surface Soil, and Laboratory Soil. The DEVracs data for paclobutrazol and uniconazole over time for open field surface soil and greenhouse surface soil are illustrated in Figure 1. Results for DEVracs of paclobutrazol and uniconazole under laboratory conditions are presented in Figure 2. The student ttest indicated DEVracs of open field, greenhouse, and laboratory experiments were statically different for paclobutrazol and uniconazole in the last sampling soils. Significant changes in the DEVracs for spiked samples were observed for paclobutrazol and uniconazole in open field surface soil and greenhouse surface soil over the duration of the experiment. The DEVracs increased from 0.000 at day 0 to 0.131 at day 140 for paclobutrazol in open field surface soil, and to 0.114 in greenhouse surface soil. The DEVracs increased from 0.000 to 0.142 for uniconazole in open field surface soil and to 0.121 in greenhouse surface soil. The different DEVracs patterns between open field and greenhouse were also thought to be caused by the differences in their soil microbial community or activity because only microbial degradation of chiral pollutants was thought to be enantioselective. As in the open field and greenhouse experiments, a consistent increase of DEVracs was observed for both paclobutrazol and uniconazole in unsterile samples, which

Table 5. Degradation of Native Paclobutrazol and Uniconazole Residues in Nonsterile and Sterile Soils paclobutrazol nonsterile t=0d t = 15 d t = 60 d t = 180 d

3.9 ± 0.20a (0%)b 3.3 ± 0.091 (15%) 2.1 ± 0.10 (46%) 1.4 ± 0.014 (64%)

sterile 4.5 ± 0.51 (0%) 3.8 ± 0.32 (16%) 3.4 ± 0.11 (24%) 2.6 ± 0.091 (42%)

unconazole nonsterile 5.9 ± 0.12 (0%) 5.2 ± 0.13 (12%) 4.5 ± 0.11 (24%) 2.7 ± 0.011 (54%)

sterile 6.1 ± 0.16 (0%) 5.2 ± 0.25 (15%) 5.1 ± 0.012 (16%) 4.2 ± 0.034 (31%)

a Concentrations, ng g−1 dw−1. bValues in brackets are dissipation percentages.

indicating the degradation of the two pesticides in sterile and nonsterile soil was a continuous process and did not have an obvious lag period. The results in Table 5 show that about 64% of paclobutrazol were degraded in nonsterile laboratory soil after 180 days incubation, while the degradation percentage of paclobutrazol was only 42% for sterile laboratory soil throughout the same incubation period. The degradation percentages of uniconazole were 54% for nonsterile soil and 31% for sterile soil. The results indicated that the degradation of paclobutrazol was faster than that of uniconazole for both sterile and nonsterile soil. The opposite tendency was observed for dissipation of paclobutrazol and uniconazole in open field surface soil, which showed uniconazole was more easily dissipated than paclobutrazol. This implies that penetration produced by rainfall might compensate for degradation of uniconazole and make a greater contribution to dissipation of uniconazole than to dissipation of paclobutrazol in open field surface soil. The comparison between dissipation in greenhouse surface soil and degradation in nonsterile soil indicates that the dissipation percentages in greenhouse surface soil were close to the degradation percentages in nonsterile soil for uniconazole. Different from uniconazole, the degradation percentages of paclobutrazol in nonsterile soil was higher than its dissipation percentages in greenhouse surface soil. This was not surprising because longer incubation time in nonsterile soil than in 847

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Environmental Science & Technology

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DEVracs change of the native paclobutrazol and uniconazole in the sterile soils after a long-term incubation might also be produced by the incomplete sterilization. Additionally, soil might contain some ingredients that can lead to the occurrence of the enantioselective degradation of the native paclobutrazol and uniconazole in the sterile soils by acting as chiral catalysts. Another possible reason is that the accessibility/extractability of enantiomers might be different due to enantiomers being trapped by the organic matter at different rates in longer terms in soils.33 The verification of the assumptions is out of the scope of the present study and will be investigated in future studies. Agricultural and Environmental Implications. Results of the dissipation data for paclobutrazol and uniconazole indicate that they were more persistent in greenhouse surface soil than in open field surface soil. This is contradictory to the expectation considering the possible higher temperature and microbial activity in greenhouse soil. Leaching into deep soil layers due to rainfall had a larger impact on the dissipation than on the degradation of the two pesticides and thus led to a rapid dissipation in open field surface soil. The persistence of the two pesticides in greenhouse surface soil increased the possibility of phytotoxicity to the next crops, whereas the leaching in open field surface soil would produce environmental risk to groundwater and also contaminate surface water by groundwater input. To reduce the environmental impact of the two pesticides, small dosage is recommended for application of the two pesticides in greenhouses, and application of the two pesticides should be avoided during intensive rainfall periods for the open field. A previous study has shown that (S)-(+)-enantiomer of uniconazole is more active than (R)-(−)-enantiomer.2 The present study indicates that (R)-(−)-uniconazole is more persistent than (S)-(+)-uniconazole. Thus, application of pure (S)-(+)-uniconazole instead of the racemate of uniconazole would greatly benefit environmental safety by reducing application dosage and persistence of uniconazole and unknown environmental risks posed by (R)-(−)-uniconazole. With respect to paclobutrazol, (2S, 3S)-(−)-enantiomer is more active and more persistent than (2R, 3R)-(+)-enantiomer in most cases,1,34 suggesting that the application of active enantiomer instead of the racemate is a good choice for agricultural purposes but not so good for environmental impacts. Overall, the enantioselectivity of paclobutrazol and uniconazole was observed for both activity and degradation. Therefore, enantioselectivity must be considered in future risk assessment and regulatory decisions for paclobutrazol and uniconazole.

Figure 1. Time trends for DEVracs in the spiked field and greenhouse soils: (A) paclobutrazol (open field); (B) paclobutrazol (greenhouse); (C) uniconazole(open field); (D) uniconazole(greenhouse). “5 times, 10 times, 20 times” represent spike concentrations 5 times, 10 times, and 20 times higher than native concentration, respectively.

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Figure 2. Time trends for DEVracs of native paclobutrazol (A) and uniconazole (B) residues in the laboratory incubation.

ASSOCIATED CONTENT

S Supporting Information *

Determination of soil properties (Text SI-1), molecular structures of paclobutrazol and uniconazole (Figure SI-1), time trends for EFs of paclobutrazol and uniconazole in the spiked field and greenhouse soils (Figure SI-2), and time trends for EFs of native paclobutrazol and uniconazole residues in the laboratory incubation (Figure SI-3). This information is available free of charge via the Internet at http://pubs.acs.org.

showed DEVracs increase from 0.051 and 0.036 at day 0 to 0.129 and 0.150 at day 180 for paclobutrazol and uniconazole, respectively. Combining EFs in Figures SI-2 and SI-3 (SI), it can be concluded that quite a rapid degradation of (+)-enantiomers for paclobutrazol and uniconazole occurred under the three soil conditions. Higher DEVracs of the native paclobutrazol and uniconazole in the sterile soils were found at day 180 as compared to day 0. Similar results were observed for degradation of native transchlordane and cis-chlordane residues in the sterile woodland soil, which was explained by incomplete sterilization of soil and/or the spatial variability of EFs in the pooled soil.5 The

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86 571 8832 0534. Fax: +86 571 8832 0884. E-mail: [email protected]. 848

dx.doi.org/10.1021/es3041972 | Environ. Sci. Technol. 2013, 47, 843−849

Environmental Science & Technology

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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

This work was funded by the National Natural Science Foundation of China (Grant Nos. 41073090, 20837002, and 40973077), and the National Basic Research Program of China (2009CB421603). Valuable comments from the anonymous reviewers are highly appreciated.

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dx.doi.org/10.1021/es3041972 | Environ. Sci. Technol. 2013, 47, 843−849