JES-00108; No of Pages 9 J O U RN A L OF E N V I RO N ME N TA L S CI EN CE S X X (2 0 1 4 ) XX X–XXX

Available online at www.sciencedirect.com

ScienceDirect www.journals.elsevier.com/journal-of-environmental-sciences

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Liezhong Chen1,2,⁎, Yanli Li2 , Ting Wang2 , Yali Jiang2 , Kai Li2 , Yunlong Yu1

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1. College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310029, China. E-mail: [email protected] 2. State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

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Microencapsulated chlorpyrifos: Degradation in soil and influence on soil microbial community structures

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Article history:

Degradation kinetics of microencapsulated chlorpyrifos (CPF-MC) in soil and its influence on 16

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Received 27 December 2013

soil microbial community structures were investigated by comparing with emulsifiable 17

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Revised 1 April 2014

concentration of chlorpyrifos (CPF-EC) in laboratory. The residual periods of CPF-MC with 18

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Accepted 9 June 2014

fortification levels of 5 and 20 mg/kg reached 120 days in soil, both of the degradation curves 19

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did not fit the first-order model, and out-capsule residues of chlorpyrifos in soil were 20 maintained at 1.76 (±0.33) and 5.92 (±1.20) mg/kg in the period between 15 and 60 days, 21 respectively. The degradation kinetics of CPF-EC fit the first-order model, and the residual 35 22

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Pesticide

periods of 5 and 20 mg/kg treatments were 60 days. Bacterial community structures in soil 23

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Controlled-release formulation

treated with two concentrations of CPF-MC showed similarity to those of the control during the 24

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test period, as seen in the band number and relative intensities of the individual band on DGGE 25

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Side effects

gels (p > 0.05). Fungal community structures were slightly affected in the 5 mg/kg treatments 26

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Soil microbiology

and returned to the control levels after 30 days, but initially differed significantly from control 27

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Keywords:

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in the 20 mg/kg treatments (p < 0.05) and did not recover to control levels until 90 days later. 28 The CPF-EC significantly altered microbial community structures (p < 0.05) and effects did not 29

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disappear until 240 days later. The results indicated that the microcapsule technology 30 prolonged the residue periods of chlorpyrifos in soil whereas it decreased its side-effects on 31

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soil microbes as compared with the emulsifiable concentration formulation.

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Introduction

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The use of macromolecular substances as semi-permeable or contact-breakable membranes to encapsulate pesticides by chemical, physical or physico-chemical mechanisms is one approach to achieve microcapsule (MC) formulations with controlled-release properties (Heller, 1980; Dailey and Dowler, 1998; Tsuji, 2001). MC formulation is an advanced formulation that has several advantages over traditional formulations, including increased stability in the environment, reduced leaching from soil, and improved activity (Mogul et al., 1996; Frederiksen and Hansen, 2002; Bagle et al., 2012; Hack et al., 2012; Alonso et al., 2013). Since the Pennwalt Company

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© 2014 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. 33 Published by Elsevier B.V. 34

first developed Parathion-methyl MC in 1974, over 200 agro-chemical companies have participated in the development and application of MCs. About 60 pesticides are commercially available in MC formulation today, including chlorpyrifos, avermectin and alachlor (Hua, 2010; Hack et al., 2012; L.L. Wang et al., 2013). Chlorpyrifos (O,O-diethyl-O-(3,5,6-trichloro-2-pyridinyl) phosphorothionate, CPF) is a broad-spectrum organophosphorous insecticide that is widely used to control agricultural pests. Banning of the use of carbofuran has promoted CPF as an important substitute for controlling the underground pests in China (Jiang, 2008). However, the extensive use of CPF has led to contamination of the environmental and food matrix in some regions (Álvarez et al.,

⁎ Corresponding author.

http://dx.doi.org/10.1016/j.jes.2014.09.017 1001-0742/© 2014 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.

Please cite this article as: Chen, L., et al., Microencapsulated chlorpyrifos: Degradation in soil and influence on soil microbial community structures, J. Environ. Sci. (2014), http://dx.doi.org/10.1016/j.jes.2014.09.017

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1.3. Soil treatment

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Soil samples were pre-incubated at 25°C in the dark for 6 days. Then, the samples were separately treated by a predetermined amount of CPF in MC and EC formulations following proper dilution with distilled water, to achieve a certain concentration of insecticide and obtain soil moisture of 60% of the soil water holding capacity. Five treatments, including a control, recommended dosages (MC and EC, 5 mg (active ingredient)/kg), and four times the recommended dosages (MC and EC, 20 mg (active ingredient)/kg), were used in this experiment. The control treatments received the same amount of sterilized distilled water without CPF. Each treatment was performed in triplicate. Dosed samples (8 kg per treatment) were mixed thoroughly and transferred to 30 cm × 20 cm × 15 cm polypropylene containers. Each container was covered with black fabric and incubated in a climate chamber at 25°C. Soil moisture was determined and maintained by regular addition of sterilized water every 2 days. At fixed intervals of 0 (2 hr), 7, 15, 30, 45, 60, 90, 120, and 240 days after treatment, aliquots of the soil sample (60 g) were collected to determine residues of CPF and soil microbial community structures.

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1.4. Determination of residual CPF in soil

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was sieved (2 mm) to remove stones and debris, analyzed with standard protocols (Institute of Soil Science, Academia Sinica, 1979) and classified as silt loam with the following characteristics: sand 20.8%, silt 74.3%, clay 8.0%, organic matter content 3.23%, water holding capacity 41.2%, cationic exchange capacity 9.6 cmol/kg, total nitrogen 0.18% and pH 6.8.

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2013; Romeh and Hendawi, 2013; Q. Wang et al., 2013). CPF MC was recently developed as an alternative to conventional formulations to control pests with reduced application amounts and environmental effects (Frederiksen and Hansen, 2002; Zhu et al., 2010). Over 50 MC products containing CPF as an active substance have been registered and used in China (The data cited from Ministry of Agriculture of the People's Republic of China). However, while many studies on the product development and efficacy evaluation of CPF MC have been reported (Frederiksen and Hansen, 2002; Montemurro et al., 2002; Lláce et al., 2010; Zhu et al., 2010; Guo et al., 2011), as yet the environmental safety has not been assessed. Microbes play important roles in soil fertility through their functions in nutrient cycling and organic matter decomposition (Wainwright, 1978). Most pesticides will eventually reach the soil following application, even foliar application, to affect the stability of the soil microbial community and ultimately influence soil fertility and plant productivity (Omar and Abdel-Sater, 2001; Singh and Singh, 2005). Intensive studies have been conducted to investigate CPF distribution, absorption, transfer and degradation in soil (Redondo et al., 1997; Li et al., 2005; Singh et al., 2006; Van-Emmerik et al., 2007; Gebremariam et al., 2012). The influences of CPF on soil microbes, including microbial biomass, microbial diversity, microbial populations, microbial respiration, and enzymatic activities, have also been studied frequently (Singh et al., 2002a,b; Menon et al., 2004, 2005; Adesodun et al., 2005; Shan et al., 2006; Fang et al., 2009; Dutta et al., 2010; Wang et al., 2010). While certainly informative, most of these studies, however, do not focus on formulation, which is an important factor influencing the side effects of CPF. Organic solvent or emulsifiable concentration (EC) formulations of CPF could be classified as disposable release formulations. After introduction into the soil, CPF exerts maximum stress on soil microbes. This stress gradually weakens with CPF degradation. As the MC form of the pesticide provides a membranelike obstruction, CPF is never in direct contact with the soils. In this case, soil microbes may be challenged by a progressive stress and then stabilize to certain levels for a longer period of time. One could hypothesize that the response of the soil microbes to the CPF MC may be distinct from their responses to non-controlled-release formulations. To the best of our knowledge, few studies have focused on evaluation of the effects of controlled-release CPF on the soil microbial communities. Therefore, the present study investigates the response of soil microbial communities to the stress from CPF MC and compares findings with CPF EC results, along with determination of the degradation kinetics of the two formulations. DGGE was used to analyze the PCR products of region 3(V3) of the bacterial 16S rDNA and region ITS1 of the fungal DNA, to elucidate the DGGE patterns of microbial community structures among the different treatments.

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1. Materials and methods

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1.1. Chemicals

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Commercial formulations of CPF, in 40% EC and 36% MC, were obtained from DOW Chemical Co., USA and Xten Chemical Co., Japan, respectively. CPF standard (99.5%) was purchased from Shanghai Pesticide Research Institute, China.

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1.5. Determination of out-capsule CPF in soil

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The soil sample used in this study was collected from a field (0–20 cm) in Zhejiang Academy of Agricultural Sciences, Hangzhou, China that was used for growing rice for cultivation of pesticide-susceptible strains of rice pests, and had not been treated with any pesticide for the previous 10 years. The soil

Five soil samples without CPF were spiked separately with EC and MC mixtures and the last concentration of CPF in soil was 20 mg/kg, and the percentage of CPF from EC in each sample was 0%, 5%, 25%, 50% and 100%, respectively. The soil samples (20 g) were then transferred to a 2.5 cm × 25 cm glass column,

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CPF residues in soil were determined by the method proposed by Chu et al. (2008), with the shaking time optimized to 4 hr. Soil samples were extracted by acetone–petroleum ether (1:1, V/V). The extracts were prepared for gas chromatography (GC) analysis after filtration, washing by 3% sodium sulfate, drying by anhydrous sodium sulfate, concentration on rotary evaporator, and dissolution in 10.0 mL of acetonitrile. CPF residues were determined by a Shimadzu GC-2010 (Shimadzu Corp., Japan) equipped with a Ni63 electron capture detector and a fused silica capillary column (HP-5, Supelco Corp., USA) (30 m length, 0.32 mm internal diameter, and 0.33 μm film thickness). The operating conditions were as follows: injector port, 280°C; detector, 300°C; column, 240°C; carrier gas (N2) flow rate, 50 mL/min; and injection volume, 2 μL. Three replicate analyses were carried out at four different spiking levels to test the validity of the aforementioned method for extracting CPF from the soil. Soil samples without CPF were spiked with MC and EC at concentrations of 0.1, 1.0, 5.0, and 20.0 mg/kg.

Please cite this article as: Chen, L., et al., Microencapsulated chlorpyrifos: Degradation in soil and influence on soil microbial community structures, J. Environ. Sci. (2014), http://dx.doi.org/10.1016/j.jes.2014.09.017

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1.6. Soil DNA and microbial DNA extraction

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Soil DNA extraction was performed in 0.25 g soil samples (wet weight) using a MOBIO soil DNA isolation kit; microbial DNA extraction was performed via an ultraclean microbial DNA isolation kit (MOBIO Laboratories, Solana Beach, USA) according to the manufacturer's specifications; here, 30 sec bead-beating was substituted for vortex mixing.

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1.7. PCR-DGGE

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1.8. Statistical analysis

Soil DNA extracted as described in Section 1.6 was initially amplified by PCR. All PCRs were performed in an automated 201 Q10 thermal cycler (Mastercycler, Eppendorf, Germany). Amplifi202 cations were carried out in a final volume of 50 μL containing 203 1 × PCR buffer, 5 mmol/L MgCl2, 2 mmol/L dNTPs, 10 μmol/L 204 each of primers, 2.5 U TransStart Taq DNA Polymerase from 205 Transgene, and 1 μL (~10 ng) of DNA template (chromosomal 206 DNA or diluted PCR product).

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The forward primers ITS1f 5′-CTT GGT CAT TTA GAG GAA GTA A-3′ and ITS1f-gc 5′-CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GCT TGG TCA TTT AGA GGA AGT AA-3′ and the reverse primers ITS2 5′-GCT GCG TTC TTC ATC GAT GC-3′ and ITS4 5′-TCC TCC GCT TAT TGA TAT GC-3′ were used. Nested PCR was used to amplify the ITS1 region of fungal rDNA. A fragment of approximately 700–900 bp comprising both ITS1 and ITS2 was amplified in the first PCR reaction using the primer pair ITS1f/ITS4 and soil DNA as the template. Amplification products from the first PCR reaction were diluted 1/10 and 1 μL of this dilution was used as the template in a second round of PCR for specific amplification of the ITS1 region using primer pair ITS1f-gc/ITS2. A GC clamp was added to stabilize the melting behavior of the PCR product for DGGE analysis.

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The 16S rDNA PCR amplification was carried out using soil DNA as the template and the bacterial-specific primers, 8F (5′-AGA GTT TGA TCC TGG CT-C AG-3′) and 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′). Nested-PCR for the V3 region was performed on the 8F/1492R PCR products using primers 341F-gc (5′-CGC CCG CCG-CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GCC TAC GGG AGG CAG CAG-3′) and 518R (5′-ATT ACC GCG GCT GCT GG-3′). Amplicons with the GC clamp were analyzed by agarose gel electrophoresis (0.8 (W/V) agarose, 120 V, 30 min) and ethidium bromide staining to determine their integrity and yield and then stored at − 20°C for subsequent DGGE analysis.

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1.7.3. Analysis of PCR products by DGGE

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A DCode universal mutation detection system (Bio-Rad, USA) was used for DGGE analysis. Approximately 20 μL of PCR

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2. Results

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CPF recoveries from soil fortified with EC and MC concentrations of 0.1, 1.0, 5.0, and 20.0 mg/kg ranged from 91.82% to 101.01% with a relative standard deviation (RSD) less than 5.68%, and 92.86% to 100.58% with an RSD less than 5.22%, respectively. These data suggested that the proposed extraction method was satisfactory for extraction of CPF from the test soil (Table 1). The elution method was used to determine the content of out-capsule CPF in MC-treated soil. Samples of CPF EC and MC with different mixing ratios were used to determine the optimal volume of eluent. When the percentage of CPF from EC was 100%, there was no controlled-release CPF in the soil, and the amount of eluted CPF reached 0.382 mg with 95.5% recovery when the eluent volume was 150 mL. The amount of eluted CPF remained stable even as the volume of eluent increased to 250 mL. When the percentage of CPF MC in the mixture was increased, the elution curves showed characteristics similar to the EC trial. The amounts of eluted CPF remained stable when the volume of eluent was 150 mL (Fig. 1). This suggested that CPF from EC (out-capsule) in the mixture could be mostly eluted by 150 mL of eluent. The second stage elution curves showed that the amount of CPF that could be seen as originating from inside capsules was very little. Thus, the optimal volume of the eluent was determined as 150 mL.

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2.2. CPF degradation in soil

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Fig. 2 shows the variation of CPF residues in soil with time. The kinetics of CPF residue in MC-treated soil was observed to be significantly different from that observed in EC-treated soil. In the EC-treated soil, CPF dissipated rapidly via a first-order kinetics model. The fitted equations for the dissipation of CPF (EC) were y = 5.118e−0.112x (R2 = 0.9951) and y = 19.921e−0.117x (R2 = 0.9872) with half-lives 6.2 and 5.9 days at fortification levels of 5 and 20 mg/kg, respectively. The dissipation of CPF in MC-treated soil was significantly slower than that in EC-treated soil. On day 60 after treatment,

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The banding patterns of DGGE were analyzed using Quantity One v4.62 software (Bio-Rad). Principal component analysis (PCA) of DGGE data was performed using Matlab V7.01 software (MathWorks Inc., USA).

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products per sample was loaded onto 8% (m/V) polyacrylamide gel (40% acrylamide/Bis, 37.5:1, m/m) containing a linear denaturing gradient from 40% to 55% for ITS1f-gc/ITS2 amplicons or 45% to 60% for 341F-gc/518R amplicons (l00% denaturant corresponds to 40% formamide + 7 mol/L urea). The gel was run for 90 min at 45 V followed by 6 hr at 120 V in 1× TAE buffer at a constant temperature of 60°C. Following electrophoresis, the gel was rinsed, stained for 20 min in an ethidium bromide solution (0.5 mg/L), and destained thrice for 1 min each time in water. DGGE profile images were digitally captured and recorded (Tanon 2500 Gel Image System, Tanon, China).

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covered with quartz sand, and eluted with different dosages of acetone–petroleum ether (1:1, V/V) within 1 min. Eluted CPF analysis was conducted as described in Section 1.4. The optimum volume of the eluent was determined by setting an eluent gradient at 30, 50, 100, 150, 200, and 250 mL. Each trial was replicated three times.

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Please cite this article as: Chen, L., et al., Microencapsulated chlorpyrifos: Degradation in soil and influence on soil microbial community structures, J. Environ. Sci. (2014), http://dx.doi.org/10.1016/j.jes.2014.09.017

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Table 1 – Average recoveries and relative standard deviation (RSD) of two formulations of chlorpyrifos fortified in soil.

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91.82 96.35 99.45 101.01 92.86 97.47 100.21 100.58

5.68 2.35 0.88 0.21 5.22 2.82 0.74 0.24

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PCR-DGGE analysis results of the ITS1 region of fungal DNA at different periods were used to detect fungal community structures (FCS) in soil. The results were also divided into three periods: days 0, 7, and 15, days 30, 45, and 60, and days 90, 120, and 240; all of the replicates showed similar results. As shown in Fig. 4a and b, the FCS in soil were not clearly altered by initial application of MC and EC of CPF. Seven days later, the stability of the soil FCS was affected by the CPF. The intensity of band b2 on DGGE gel was weakened significantly in these samples, and the distance from the points of treatment samples (6, 7, 8, and 9) to the control (10) on the PCA substantially increased. The FCS in MC-treated soil recovered to control level on day 15; however, EC-treated soils showed no such recovery. The FCS in soil treated with high concentrations of CPF, regardless of the formulation, were affected more noticeably than the FCS in soil treated with low concentrations of the pesticide after 30, 45 and 60 days. PCA indicated that points 2, 4, 7, 9, 12, and 14 were farther from the controls (points 5, 10,

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PCR-DGGE analysis of the V3 region of bacterial 16S rDNA sequence fragments was used to detect bacterial community structures (BCS) in soil. The experiment was divided into three periods: days 0, 7, and 15, days 30, 45, and 60, and days 90, 120, and 240; all of the replicates showed similar results. As can be clearly seen from Fig. 3a and b, noticeable changes in the DGGE pattern could be exhibited by the PCA of the DGGE data. Points 1–5 clustered together, which indicated that the BCS in the soil collected 2 hr after being treated with

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CPF was dissipated by only 34.8% and 49.5% at 5 and 20 mg/kg, respectively. CPF then appeared to dissipate rapidly and nearly completely disappeared on day 120. To understand the mechanism by which CPF in MC treatments dissipated slowly in soil, the variation pattern of the content of out-capsule CPF was monitored. The maximal level of out-capsule CPF was found on day 7 after treatment. CPF levels decreased slightly from day 7 to day 15, by 21.1% and 29.0% in 5 and 20 mg/kg-treated soil, respectively. Average concentrations of 1.76 (±0.33) and 5.92 (±1.20) mg CPF/kg were observed in soils treated with 5 and 20 mg CPF/kg from day 15 to day 60, respectively. Thereafter, the variation pattern was similar to that observed for the total residue of CPF in MC-treated soil.

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MC and EC were similar to that in the control soil. Although the BCS of the control changed within the first 7 days, all points that corresponded to day 7 and day 15 of MC treatment and control samples clustered together and could be regarded as one group. The long distances from points 6 and 7 to 10 and from 11 and 12 to 15 suggested that the BCS in EC-treated soil were greatly altered on day 7 and day 15 after treatment. Although most of the bands were found in each lane on the DGGE gel, different bands were found among the lanes, such as B13 and B14, which emerged only in lanes that corresponded to day 7 and day 15 of EC-treated samples. B4 and B5 were noticeably depressed in the EC-treated samples on day 7, and B5 recovered obviously on day 15. Bands B15 and B16 in MC-treated samples were stronger than those in EC-treated samples. The successions of the BCS in MC-treated and control soil were very slight and stable and remained similar from day 30 to day 60. By contrast, the microbial communities in EC-treated soil were still different from those of the control. All points corresponding to the MC-treated and control samples were basically aggregated in the same region (Fig. 3d). The positions of the points of two EC-treated samples shifted noticeably from day 30 to day 60, but were a significant distance away from the points of control and MC treatments. This result suggested that the BCS in the EC-treated soil were unstable during this period. DGGE gel showed that bands B4, B9, B10, and B11 were depressed obviously in EC-treated samples, whereas B17 was enhanced in MC-treated samples during this period (Fig. 3c). During later periods of the experiment, the microbial communities in the soil of different treatments slowly showed stability, and differences among treatments narrowed with time (Fig. 3e and f). Although differences between the EC and control (or MC) groups remained more noticeable than those between MC groups and the control on day 90 and day 120, all points on the PCA were very close to each other on day 240. Band B4 was still depressed in lanes 1, 2, 6, and 7 on the DGGE gel, but recovered to near-control levels during the last days of the experiment.

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Fig. 1 – Effects of eluent volume on elution efficiency of CPF in soil.

Please cite this article as: Chen, L., et al., Microencapsulated chlorpyrifos: Degradation in soil and influence on soil microbial community structures, J. Environ. Sci. (2014), http://dx.doi.org/10.1016/j.jes.2014.09.017

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Fig. 2 – Degradation kinetics of CPF at concentration of 5 (a) and 20 (b) mg/kg in soil.

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3. Discussion

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In lab experiments, the reported half-lives of CPF in soil ranged from 3 to 450 days depending on soil properties and experimental conditions (Singh et al., 2002b; Chu et al., 2008; Fang et al., 2009; Chai et al., 2013; Karpuzcu et al., 2013; Xiong et al., 2013). In our investigation, the half-lives of CPF (EC) in soil were about 6 days; these were close to the results of Fang et al. (2009) and Chu et al. (2008) where half-lives of 3 to 18 days were observed. However, the degradation of CPF in our experiment was still faster than those in most of previous studies (Singh et al., 2002b; Chai et al., 2013). Although there were diverse influential factors, such as pH value and composition of soil, microbial degradation may play a more important role in CPF degradation. The results of subsequent works by us showed that there were several strains capable of utilizing CPF as a sole source of carbon and energy in EC-treated soils (data not shown here). While the half-life of CPF in soil varies over a wide range, the degradation curves of CPF under conventional formulations basically conform to a first-order kinetics model (Yucel et al., 1999; Singh et al., 2002a,b; Chu et al., 2008; Fang et al., 2009; Dutta et al., 2010). However, CPF MC showed excellent controlledrelease properties in soil, and the degradation kinetics of CPF did not fit the first-order model. While degradation and diffusion occurred soon after CPF was released to the soil (Mogul et al., 1996), the CPF concentration out of the capsules was

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maintained at a stable level for a long period of time because of the continuous supply from the capsules, until the osmotic pressure between the inside and outside of the capsules was not enough to release any more CPF. This result showed that MC formulation of pesticides could afford longer periods of efficacy than conventional formulations at the same application dosage. Because of this, MC formulation provides an alternative solution to repeated, high-dosage pesticide application that reduces environmental pollution but controls pests, weeds or diseases effectively. The MC formulation may yield effects on the environment that differ from those presented by conventional formulations due to its unique release pattern (Hack et al., 2012). In this work, although CPF concentrations in MC-treated soils reached levels achieved by the corresponding EC treatments on day 7 and remained at higher levels than those of EC treatments in the following days, the BCS in MC-treated soil were still more similar to the control samples than the EC-treated ones. This meant that the original bacteria in the soil were affected seriously when stressed by CPF sharply, but exhibited more tolerance and adaptability under gradual increases in CPF stress. While fungi were more sensitive than bacteria to CPF and showed effects of CPF in succeeding days, FCS of MC treatments and control were closer than those of EC treatments and control, especially during the late period of the experiment. Moreover, the FCS in MCtreated soils recovered to the control level more quickly than those in EC-treated soils. The results indicated that CPF MC was more beneficial than CPF EC in protecting the stability of soil microbial communities, and this was very important to preserve the stability of soil ecosystems (Imfeld and Vuilleumier, 2012). Fang et al. (2009) and Shan et al. (2006) had proved that the functions of soil microbes were initially affected by CPF, but recovered to the control levels later on. In this experiment, functions of soil microbes recovered to control level on day 30 (data not provided in this paper), however, the composition of the microbial communities in control and EC-treated soils differed obviously before day 120. It was clear that soil microbial communities had to finish the functional reconstruction to resist the stress of CPF before recovering completely in composition. Microbes that were represented by enhanced bands on DGGE

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and 15) than points corresponding to low concentration treatments. On the DGGE gel, band b12 was found in the lanes 1, 3, 7, 8, 13, and 14, and b11 was enhanced in lanes 2 and 4 (Fig. 4c and d). The DGGE and PCA results showed that the band patterns of CPF-treated and control soil gradually became similar at later experimental periods. Although points 1, 2, and 6 were still dispersed, most of the points clustered together as determined by PCA. This result suggested that the effects of CPF EC on the soil fungal community were more persistent than those of MC (Fig. 4e and f).

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Fig. 3 – PCR-DGGE analysis of V3 Region of the soil bacterial 16S rDNA (a, c and e) and PCA based on the corresponding DGGE fingerprints (b, d and f). Lanes 1 to 5, 6 to 10, and 11 to 15 in DGGE fingerprints denote the treatments of 5 mg CPF/kg (EC), 20 mg CPF/kg (EC), 5 mg CPF/kg (MC), 20 mg CPF/kg (MC) and control, respectively. The numbers 1 to 15 on PCAs were corresponded to the lane numbers of the DGGE. M was the marker lane. Days 0, 7, 15, 30, 45, 60, 90, 120, and 240 represented the sampling day after inoculation.

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gel of EC-treated samples might remedy the functional damage that was caused by other microbes depressed by CPF. In MCtreated soils, however, the microbial community structures

and functions were more stable than those in EC-treated soils. 453 This result indicated that soil microbes appeared to undergo 454 two processes to adapt to the stress presented by EC and MC 455

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formulations of CPF and recover their function. Compared with the former, the latter process was more beneficial in preserving the stability of the soil environment and maintaining the limited energy available in the soil space. Microbial degradation is an important pathway of pesticide dissipation in soil. Continuous stress by CPF enhanced the ability of soil microbes to degrade CPF (Robertson et al., 1998; Fang et al., 2008). Fang et al. (2008) found that the influence of CPF on soil functional diversity was weakened with repeated applications of CPF. On day 21 after the third application, a strain that could degrade CPF was isolated from the soil. The soil microbes in MC-treated soil were also challenged by continuous stress from CPF over a long period. DGGE and PCA results showed no noticeable difference between the MC groups and the control, but there were several specific bands in MC-treated samples that were not found in control samples (for example: B15 and B17), and these bands might play key roles in the adaptation of soil microbes to CPF stress even if the main community structures were not altered significantly. Certainly, further studies are necessary to determine whether the microbes corresponding to these bands are associated with degradation functions or not. DGGE analysis accurately showed the changes in soil microbial community structure (Pascaud et al., 2012). As a PCR-based method, however, DGGE also presents several limitations. For example, a single band on DGGE gel may represent one or several microbes (Gao et al., 2012). Besides, only colonies that are present in a certain proportion can be reflected by the gel, and one DGGE image cannot fully map the true conditions of a soil microbial community (Gao et al., 2012). Experiments in this study clearly showed the kinetics of BCS and FCS treated with EC and MC

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Fig. 4 – PCR-DGGE analysis of ITS1 Region of the soil fungal DNA (a, c and e) and PCA based on the corresponding DGGE fingerprints (b, d and f). Lanes 1 to 5, 6 to 10, and 11 to 15 in DGGE fingerprints denote the treatments of 5 mg CPF/kg (EC), 20 mg CPF/kg (EC), 5 mg CPF/kg (MC), 20 mg CPF/kg (MC) and control, respectively. The numbers 1 to 15 on PCAs were corresponded to the lane numbers of the DGGE. M was the marker lane. Days 0, 7, 15, 30, 45, 60, 90, 120, and 240 represented the sampling day after inoculation. formulations of CPF, and differences in the effects of the two formulations on microbial community structures were especially emphasized. Development of microencapsulation is encouraged as a substitute for traditional formulations nowadays. There is limited information about the environmental effects of MCs because they became commercially available only recently. Most studies on CPF are devoted to evaluating the environmental influence of this pesticide, whereas few works are focused on its controlled-release formulations. Results from this work indicated that the microcapsule technology improved the utilization of CPF as well as decreased the sideeffects of CPF on soil microbes. However, further works should be conducted to assess the risks of this product to the environment, including ecological risks to birds, fish, and other non-target organisms, the pollution risks to water and agro-products, and so on.

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This work was supported by the National High Technology R&D Program of China (Nos. 2013AA102804D, 2012AA06A204), the National Natural Science Foundation of China (Nos. 21177111, 42171489), the Key Scientific and Technological Innovation Team Program of Zhejiang Province (No. 2010R50028), the Zhejiang Provincial Natural Science Foundation (No. LZ13D010001), and the Hangzhou Science and Technology Development Item (No. 20110232B11). The authors also thank Dr. H. Jiang, Dr. X. Wang, and Dr. P. J. Zhang for making constructive comments on our manuscript.

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Adesodun, J.K., Davidson, D.A., Hopkins, D.W., 2005. Micromorphological evidence for changes in soil faunal activity following application of sewage sludge and biocide. Appl. Soil Ecol. 29 (1), 39–45. Alonso, M.L., Laza, J.M., Alonso, R.M., Jiménez, R.M., Vilas, J.L., Fañanás, R., 2013. Pesticides microencapsulation. A safe and sustainable industrial process. J. Chem. Technol. Biotechnol. 89 (7), 1077–1285. http://dx.doi.org/10.1002/jctb.4204. Álvarez, M., Mortier, C., Fernández Cirelli, A., 2013. Behavior of insecticide chlorpyrifos on soils and sediments with different organic matter content from provincia de Buenos Aires, República Argentina. Water Air Soil Pollut. 224 (3), 1453. Bagle, A.V., Jadhav, R.S., Gite, V.V., Hundiwale, D.G., Mahulikar, P. P., 2012. Controlled release study of phenol formaldehyde microcapsules containing neem oil as an insecticide. Int. J. Polym. Mater. Polym. Biomater. 62 (8), 421–425. Chai, L.K., Wong, M.H., Hansen, H.C.B., 2013. Degradation of chlorpyrifos in humid tropical soils. J. Environ. Manag. 125, 28–32. Chu, X.Q., Fang, H., Pan, X.D., Wang, X., Shan, M., Feng, B., et al., 2008. Degradation of chlorpyrifos alone and in combination with chlorothalonil and their effects on soil microbial populations. J. Environ. Sci. 20 (4), 464–469. Dailey, O.D., Dowler, C.C., 1998. Polymeric microcapsules of cyanazine: preparation and evaluation of efficacy. J. Agric. Food Chem. 46 (9), 3823–3827. Dutta, M., Sardar, D., Pal, R., Kole, R.K., 2010. Effect of chlorpyrifos on microbial biomass and activities in tropical clay loam soil. Environ. Monit. Assess. 160 (1–4), 385–391. Fang, H., Yu, Y.L., Wang, X.G., Chu, X.Q., Pan, X.D., Yang, X.E., 2008. Effects of repeated applications of chlorpyrifos on its persistence and soil microbial functional diversity and development of its degradation capability. Bull. Environ. Contam. Toxicol. 81 (4), 397–400. Fang, H., Yu, Y.L., Chu, X.Q., Wang, X.G., Yang, X.E., Yu, J.Q., 2009. Degradation of chlorpyrifos in laboratory soil and its impact on soil microbial functional diversity. J. Environ. Sci. 21 (3), 380–386. Frederiksen, H.K., Hansen, H.C.B., 2002. Starch-encapsulated chlorpyrifos: release rate, insecticidal activity and degradation in soil. J. Microencapsul. 19 (3), 319–331. Gao, G.P., Yin, D.H., Chen, S.J., Xia, F., Yang, J., Li, Q., et al., 2012. Effect of biocontrol agent Pseudomonas fluorescens 2P24 on soil fungal community in cucumber rhizosphere using T-RFLP and DGGE. Plos One 7 (2), e31806. Gebremariam, S., Beutel, M., Yonge, D., Flury, M., Harsh, J., 2012. In: Whitacre, D.M. (Ed.), Reviews of Environmental Contamination and Toxicology 215. Springer, New York, pp. 123–175. Guo, R.F., Huang, B.B., Wei, Y.X., Wu, Z.J., Wu, G., 2011. Preparation and characteristics analysis of microspheres of chlorpyrifos and polylactic acid. Chin. J. Pestic. Sci. 13 (4), 409–414. Hack, B., Egger, H., Uhlemann, J., Henriet, M., Wirth, W., Vermeer, A. W.P., et al., 2012. Advanced agrochemical formulations through encapsulation strategies? Chem. Ing. Tech. 84 (3), 223–234. Heller, J., 1980. Controlled release of biologically active compounds from bioerodible polymers. Biomaterials 1 (1), 51–57. Hua, N.Z., 2010. Development and recent progress of pesticide microencapsulates (I). Modern Agrochem. 9 (9), 10–14. Imfeld, G., Vuilleumier, S., 2012. Measuring the effects of pesticides on bacterial communities in soil: a critical review. Eur. J. Soil Biol. 49, 22–30. Institute of Soil Science, Academia Sinica, 1979. Soil Physical and Chemical Analysis. Shanghai Science and Technology Press, Shanghai, China. Jiang, S.K., 2008. Present situation and prospect of chlorpyrifos industry. Agrochem. Res. Appl. 12 (3), 1–3.

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Karpuzcu, M.E., Sedlak, D.L., Stringfellow, W.T., 2013. Biotransformation of chlorpyrifos in riparian wetlands in agricultural watersheds: implications for wetland management. J. Hazard. Mater. 244–245, 111–120. Li, K., Xing, B., Torello, W.A., 2005. Effect of organic fertilizers derived dissolved organic matter on pesticide sorption and leaching. Environ. Pollut. 134 (2), 187–194. Lláce, E., Dembilio, O., Jacas, J.A., 2010. Evaluation of the efficacy of an insecticidal paint based on chlorpyrifos and pyriproxyfen in a microencapsulated formulation against Rhynchophorusferrugineus (Coleoptera: Curculionidae). J. Econ. Entomol. 103 (2), 402–408. Menon, P., Gopal, M., Prasad, R., 2004. Influence of two insecticides, chlorpyrifos and quinalphos, on arginine ammonification and mineralizable nitrogen in two tropical soil types. J. Agric. Food Chem. 52 (24), 7370–7376. Menon, P., Gopal, M., Parsad, R., 2005. Effects of chlorpyrifos and quinalphos on dehydrogenase activities and reduction of Fe3+ in the soils of two semi-arid fields of tropical India. Agric. Ecosyst. Environ. 108 (1), 73–83. Mogul, M.G., Akin, H., Hasirci, N., Trantolo, D.J., Gresser, D.J., Wise, D.L., 1996. Controlled release of biologically active agents for purposes of agricultural crop management. Resour. Conserv. Recycl. 16 (1–4), 289–320. Montemurro, N., Grieco, F., Lacertosa, G., Visconti, A., 2002. Chlorpyrifos decline curves and residue levels from different commercial formulations applied to oranges. J. Agric. Food Chem. 50 (21), 5975–5980. Omar, S.A., Abdel-Sater, M.A., 2001. Microbial populations and enzyme activities in soil treated with pesticides. Water Air Soil Pollut. 127 (1–4), 49–63. Pascaud, A., Soulas, M.L., Amellal, S., Soulas, G., 2012. An integrated analytical approach for assessing the biological status of the soil microbial community. Eur. J. Soil Biol. 49, 98–106. Redondo, M.J., Ruiz, M.J., Font, G., Boluda, R., 1997. Dissipation and distribution of atrazine, simazine, chlorpyrifos, and tetradifon residues in citrus orchard soil. Arch. Environ. Contam. Toxicol. 32 (4), 346–352. Robertson, L.N., Chandler, K.J., Stickley, B.D.A., Cocco, R.F., Ahmetagic, M., 1998. Enhanced microbial degradation implicated in rapid loss of chlorpyrifos from the controlled-release formulation suSCon® Blue in soil. Crop. Prot. 17 (1), 29–33. Romeh, A., Hendawi, M., 2013. Chlorpyrifos insecticide uptake by plantain from polluted water and soil. Environ. Chem. Lett. 11 (2), 163–170. Shan, M., Fang, H., Wang, X., Feng, B., Chu, X., Yu, Y., 2006. Effect of chlorpyrifos on soil microbial populations and enzyme activities. J. Environ. Sci. (China) 18 (1), 4–5. Singh, J., Singh, D.K., 2005. Bacterial, azotobacter, actinomycetes, and fungal population in soil after diazinon, imidacloprid, and lindane treatments in groundnut (Arachis hypogaea L.) Fields. J. Environ. Sci. Health B 40 (5), 785–800. Singh, B.K., Walker, A., Wright, D.J., 2002a. Persistence of chlorpyrifos, fenamiphos, chlorothalonil, and pendimethalin in soil and their effects on soil microbial characteristics. Bull. Environ. Contam. Toxicol. 69 (2), 181–188. Singh, B.K., Walker, A., Wright, D.J., 2002b. Degradation of chlorpyrifos, fenamiphos, and chlorothalonil alone and in combination and their effects on soil microbial activity. Environ. Toxicol. Chem. 21 (12), 2600–2605. Singh, B.K., Walker, A., Wright, D.J., 2006. Bioremedial potential of fenamiphos and chlorpyrifos degrading isolates: influence of different environmental conditions. Soil Biol. Biochem. 38 (9), 2682–2693. Tsuji, K., 2001. Microencapsulation of pesticides and their improved handling safety. J. Microencapsul. 18 (2), 137–147.

E

REFERENCES

U

N

C

O

R

R

E

C

T

51 6

Please cite this article as: Chen, L., et al., Microencapsulated chlorpyrifos: Degradation in soil and influence on soil microbial community structures, J. Environ. Sci. (2014), http://dx.doi.org/10.1016/j.jes.2014.09.017

584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652

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Van-Emmerik, T.J., Angove, M.J., Johnson, B.B., Wells, J.D., 2007. Sorption of chlorpyrifos to selected minerals and the effect of humic acid. J. Agric. Food Chem. 55 (18), 7527–7533. Wainwright, M., 1978. A review of the effects of pesticides on microbial activity in soil. J. Soil Sci. 29 (3), 287–298. Wang, F., Yao, J., Chen, H., Chen, K., Trebše, P., Zaray, G., 2010. Comparative toxicity of chlorpyrifos and its oxon derivatives to soil microbial activity by combined methods. Chemosphere 78 (3), 319–326. Wang, L.L., Wang, Z., Zhang, B.H., Xiao, K.F., 2013a. Study on preparation of abamectin microcapsule with interfacial polymerization. Adv. Mater. Res. 634–638, 1090–1094. Wang, Q., Yang, J., Li, C., Xiao, B., Que, X., 2013b. Influence of initial pesticide concentrations in water on chlorpyrifos toxicity and

removal by Iris pseudacorus. Water Sci. Technol. 67 (9), 1908–1915. Xiong, J.F., Tang, X.Y., Zhou, G.M., Guan, Z., Wu, L.M., 2013. Dispersive solid phase extraction coupled with HPLC-UV for simultaneous determination of chlorpyrifos and 3,5,6-trichloro-2-pyridinol in soil samples. Anal. Methods 5 (2), 536–540. Yucel, U., Ylim, B., Gozek, K., Helling, C.S., Sarykaya, Y., 1999. Chlorpyrifos degradation in Turkish soil. J. Environ. Sci. Health B 34 (1), 75–95. Zhu, L., Wang, Z.H., Zhang, S.T., Long, X.Y., 2010. Fast microencapsulation of chlorpyrifos and bioassay. J. Pestic. Sci. 35 (3), 339–343.

F

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N C O

R

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E

C

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Microencapsulated chlorpyrifos: degradation in soil and influence on soil microbial community structures.

Degradation kinetics of microencapsulated chlorpyrifos (CPF-MC) in soil and its influence on soil microbial community structures were investigated by ...
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