Residual Behaviors of Six Pesticides in Shiitake from Cultivation to

Article pubs.acs.org/JAFC

Residual Behaviors of Six Pesticides in Shiitake from Cultivation to Postharvest Drying Process and Risk Assessment Tan Liu,† Cunzheng Zhang,§,# Jing Peng,† Zhiyong Zhang,§,# Xing Sun,§,# Hui Xiao,† Ke Sun,† Leiqing Pan,† Xianjin Liu,§,# and Kang Tu*,† †

College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China Key Laboratory of Food Quality and Safety of Jiangsu Province, Jiangsu Academy of Agricultural Science, Nanjing 210014, People’s Republic of China # Key Laboratory of Control Technology and Standard for Agro-product Safety and Quality, Ministry of Agriculture, Nanjing 210014, People’s Republic of China §

ABSTRACT: The dissipation of six pesticides (carbendazim, thiabendazole, procymidone, bifenthrin, λ-cyhalothrin, and βcyfluthrin) in shiitakes from cultivation to postharvest drying process was investigated, and the dietary exposure risk was estimated thereafter. The field trial study indicates that the half-lives of carbendazim, thiabendazole, and procymidone were much shorter than those of bifenthrin, λ-cyhalothrin, and β-cyfluthrin. Furthermore, the effects of two drying processes on the residues and processing factors (PFs) were investigated. The results showed that hot-air drying resulted in higher residues than sunlight exposure drying. Both drying processes led to pesticide residue concentration (with PF > 1), except for thiabendazole upon sunlight exposure treatment. The estimated daily intakes (EDIs) ranged from 0.06% of the acceptable daily intake (ADI) for thiabendazole to 42.43% of the ADI for procymidone. The results show that the six pesticide residues in dried shiitakes are still within acceptable levels for human consumption on the basis of a dietary risk assessment. KEYWORDS: shiitakes, pesticide residues, dissipation behavior, drying process, processing factor, dietary risk assessment

1. INTRODUCTION

under cultivation conditions, and it is therefore necessary to profile the residual behaviors of these six pesticides. The postharvest drying process is one of the most effective ways to preserve shiitakes because fresh shiitakes are perishable and easily turn brown as a consequence of oxidation and microbial decomposition due to the high moisture content.13 Currently, sunlight exposure drying and hot-air drying are two widely used techniques for preserving shiitakes in industrial processing.14 Different drying methods result in different residual behaviors of pesticides. Carbras et al. found that sunlight-drying is more effective for phosalone and vinclozolin degradation, whereas oven-drying is more effective for the degradation of iprodione and procymidone in the processing of raisins.15 The drying process has been found to potentially increase the concentration of pesticides due to water loss, ultimately leading to consumer exposure risks.16 Some studies reported the presence of pesticide residues in certain dried fruits and vegetables (raisins, apricots, peppers, and spring onion).15−20 The Joint Meeting of Pesticide Residues (JMPR) in 2004 reported a processing factor (PF) value of 3.7 for prochloraz on dried mushroom.21 To the best of our knowledge, there are no published results of PF values for the pesticides tested in the current study on dried shiitakes. The risk assessment of pesticide residues in dried shiitakes was evaluated by comparing the estimated daily intake (EDI)

Shiitake (Lentinus edodes) is the second most cultivated edible mushroom in the world, accounting for about 25% of the worldwide production of mushrooms.1 China is the world’s largest producer and exporter of shiitakes, with an annual production output of around 5.02 million tons.2 As has already been established, shiitakes contain various bioactive compounds such as polysaccharides, β-glucose, RNA complexes, degraded nucleic acid, and triterpene compounds. As such, shiitakes exhibit antitumor, antiviral, and antioxidant activities.3,4 Shiitakes are susceptible to a variety of diseases and insect infestations due to the high temperature and humidity required for adequate fruiting body growth. Therefore, pesticides are usually used to control disease outbreak and insect infestations during cultivation. The pesticides are sprayed directly on the fruiting body or are premixed with the substrates, which may lead to the accumulation of pesticide residues, potentially harmful to humans and the environment.5,6 Thiabendazole and β-cyfluthrin are two registered and commonly used pesticides for shiitakes in China.7,8 Although not registered in China, four pesticides (carbendazim, procymidone, bifenthrin, and λcyhalothrin) are still used for shiitakes by some cultivators, causing exporting issues due to international restrictions.9 In the United States and Canada, thiabendazole is registered for mushrooms.10,11 Investigative studies of the uptake of thiabendazole in three edible fungi (shimeji, king oyster, and oyster) revealed that the pesticide concentration increased within the incubation days 3−7.12 However, little information is available on the behaviors of these six pesticides in shiitakes © XXXX American Chemical Society

Received: September 8, 2016 Revised: November 4, 2016 Accepted: November 7, 2016

A

DOI: 10.1021/acs.jafc.6b04027 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Table 1. Summary of the Physicochemical Properties, Maximum Residue Limits (MRLs) on Mushroom, and Acceptable Daily Intakes (ADIs) of the Six Pesticides

a Log Kow = logarithm of octanol−water partion coefficient. bMRLs and ADIs data from Pesticide European Union (EU).32 cMRL data from Agricultural Standards of China.34 dMRLs data from Chinese National Standards.33 eMRL not currently established in China on edible fungi.

cyhalothrin (purity ≥ 98%), and β-cyfluthrin (purity ≥ 94.9%) were purchased from Dr. Ehrenstorfer GmbH Trade Co., Ltd. (Augsburg, Germany). The corresponding physicochemical properties of the pesticides are presented in Table 1. Standard stock solutions of carbendazim and thiabendazole were prepared in methanol, whereas standard stock solutions of procymidone, bifenthrin, λ-cyhalothrin, and β-cyfluthrin were prepared in n-hexane. The substances obtained through commercial sources included carbendazim 50% wettable powder (WP) and bifenthrine 2.5% emulsifiable concentrate (EC) (Guanlong Agrochemical Inc., Hebei, China), thiabendazole 50% suspension concentrate (SC) (Syngenta Crop Protection Inc., Suzhou, China), procymidone 50% WP (Zhongtian Bangzheng Biotechnology Inc., Hebei, China), λ-cyhalothrin 2.5% microemulsion (ME) (Taifeng Chemical Inc., Zhejiang, China), and β-cyfluthrin 4.0% EC (Suke Agrochemical Inc., Jiangsu, China). Acetonitrile, methanol, and nhexane were of chromatographic grade and were purchased from Merck (Darmstadt, Germany). Primary−secondary amine (PSA) and octadecylsilane (ODS) were purchased from Agela Technologies (Tianjin, China). 2.3. Field Trials. Three separate field trials were conducted in an experimental greenhouse located in Nanjing and according to the guidelines on pesticide residue trials.23 Two trials for the field dissipation behavior study were carried out by different application treatments (premixing and spraying) during the cultivation of shiitakes, and one trial was conducted for the study of the drying process. The mycelia of shiitakes obtained from enterprises of edible fungus strains (Lingbaoyang, Heinan, China) were inoculated into sterilized substrates (seed hull 79%, wheat bran 20%, gesso powders 1%, and water content 55%) and were cultivated following the procedures as described in Cultivation and Processing of Edible Fungi.24 After 90 days of cultivation, the inoculated substrates were moved into the soils (with densities of approximately 20 substrates/m2), and fruiting began approximately 1 month thereafter. The average maximum and minimum temperatures during this period were 26.4 and 17.8 °C, respectively. The average relative humidity in the greenhouse was 75−90%.

with an acceptable daily intake (ADI) of the pesticide. The EDI of a pesticide can be based on trial data and processing factors.22 The ADI is measured for the chronic toxicity of a pesticide as a health safety limit. Therefore, the EDI value of a pesticide below its ADI value is generally considered to be safe. To the best of our knowledge, no report on the dietary risk assessment of the pesticides in dried shiitakes can be found in the literature. The aims of this study were (1) the investigation of the residue dissipation behaviors of six pesticides in shiitakes under open field conditions via two application treatments (premixing and spraying); (2) the effect comparison of two drying methods (sunlight exposure and hot air) on the residue dissipation behaviors of these six pesticides in shiitakes as well as the determination of the corresponding processing factors; and (3) the evaluation of the dietary exposure risk of these pesticides in dried shiitakes based on field trial data and processing factors.

2. MATERIALS AND METHODS 2.1. Instruments. High-performance liquid chromatography (HPLC) analyses were performed with an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with an ultraviolet detector. Chromatographic separation was carried out using an Agilent Zorbax SB-C18 column (250 mm × 4.6 mm, 5 μm). Gas chromatography (GC) was conducted with an Agilent GC 7890 (Agilent Technologies) equipped with a μ-electron-capture detector (μECD) and an HP-5MS Ultra Inert capillary column (30 m × 0.25 mm, 0.25 μm). Other instruments included a Tissue Lyser (CK1000; Thmorgan, Beijing, China), a hot-air oven (GZX-9240MBE; Boxun, Shanghai, China), and a nitrogen evaporator (N-EVAP 112; Organomation Associates, Inc., Berlin, MA, USA). 2.2. Chemicals and Reagents. The pesticide standards carbendazim (purity ≥ 99%), thiabendazole (purity ≥ 98%), procymidone (purity ≥ 99.6%), bifenthrin (purity ≥ 99.5%), λB


Article

Journal of Agricultural and Food Chemistry 2.3.1. Pesticides for Trials of Premixing Treatment. Each of the six pesticides was premixed with the substrates before inoculation of the shiitake mycelia. Two dosage levels were used: 1:1250 (w/w, single dosage) for the ratio of the pesticide to the substrate and 1:625 (w/w, double dosage). The substrates without addition of any pesticide were used as control. A total of 15 substrates were used for each treatment. Experiments were conducted in triplicates. The prepared substrates premixed with pesticides were diluted with 300 mL of water and mixed with other culture compost as mentioned previously, stirred until homogeneous, and sealed into polyethylene bags using an SL-ZKJ sealer. After sterilization (100 °C/8 h), the shiitake mycelia were inoculated to the substrates and cultivated conventionally (as described under section 2.3). The shiitake samples were collected 4, 16, 48, and 96 h after the fruiting bodies of the shiitakes grew to a height of 2 cm. The samples were stored at −20 °C until further analysis. 2.3.2. Pesticides for Trials of Spraying Treatment. The spraying treatment was designed as residue dynamic trials, consisting of six treatment plots and one control plot without application of the pesticide. Each plot consisted of an area of 6 m2. Multimixing different pesticides could result in low production of shiitakes.25 Therefore, the pesticides were divided into three groups: A (carbendazim and thiabendazole), B (procymidone and bifenthrin), and C (λ-cyhalothrin and β-cyfluthrin). The shiitakes were sprayed with pesticides after the shiitake fruiting bodies grew to an average height of 2 cm. Application dosages for six pesticides were performed at two dosage levels, single dosage (recommended dosage) and double dosage (2 times the recommended dosage) using a JACTO-HD sprayer (nozzle diameter of 0.6 mm, flow rate of 250 mL/min) (Table 2). Each group of

for 20 days until the overall moisture content of shiitakes dropped to 12.8%. This ensured the final products were according to quality standards (moisture content of shiitakes < 13%).1 Randomly dried shiitakes were collected at intervals of 0, 1, 3, 5, 7, 10, 13, 17, and 20 days. As shown in Figure 1, the average of maximum and minimum temperatures was 33.3 and 26.3 °C, respectively.

Figure 1. Temperatures during sunlight exposure drying.

2.4.2. Hot-Air Drying. The shiitake samples were subjected to hotair drying using an air oven. The conditions were as follows: the initial temperature was 30 °C, then increased to 50 °C at a rate of 5 °C/h, then increased to 55 °C at a rate of 1 °C/2 h. The temperature was then held for 4 h and then increased to 60 °C at a rate of 1 °C/12 min, and the temperature was held for another 1 h. The drying process lasted for 20 h until the moisture content of shiitakes dropped to 12.5%. This slow heating process is commonly used by a variety of shiitake-processing companies, ultimately improving the quality of the dried shiitakes in China. During the drying process, the shiitakes were randomly collected at intervals of 0, 1, 3, 5, 7, 10, 13, 17, and 20 h. 2.5. Extraction of Pesticide Residues. Ten grams of a thoroughly homogenized fresh sample or 2.0 g of a dried sample was weighed into a 50 mL Teflon-coated centrifuge tube. Then, 40 mL of acetonitrile was added, and the mixture was shaken using a Tissue Lyser for a total of 10 min at 1000 strokes/min; afterward, 2.0 g of sodium chloride was added, and the mixture was shaken for another 1 min. The mixture was then centrifuged at 10000g for 10 min. Then, 5 mL of the upper layer was transferred into a centrifuge tube containing 50 mg of PSA and 50 mg of ODS.27 The resulting mixture was shaken for 1 min and centrifuged at 10000g for 5 min. Four milliliters of the supernatant was transferred to a 10 mL glass test tube and was evaporated to dryness under a stream of nitrogen (40 °C). For carbendazim and thiabendazole, the residues were dissolved in 1 mL of methanol, whereas n-hexane was used for the other four pesticides. For carbendazim and thiabendazole, the final extract was analyzed by highperformance liquid chromatography (HPLC), and gas chromatography was used (GC) for the other compounds. 2.6. GC Analysis. The chromatographic conditions were as follows: detector temperature, 300 °C; inlet temperature, 250 °C; oven temperature program, initial temperature of 120 °C, held for 1 min, increased at 18.75 °C/min to 270 °C, held for 13.25 min; carrier gas, nitrogen, 1 mL/min; makeup gas, nitrogen, 20 mL/min; injection volume, 1 μL; splitless mode. 2.7. HPLC Analysis. An isocratic elution was performed with a mobile phase of methanol/water (70:30, v/v) at a flow rate of 0.4 mL/ min and an injection volume of 10 μL. The column was kept at room temperature, and the detector wavelength was set to 285 nm. 2.8. Dissipation Rate Kinetics Model. The dissipation trends of six pesticides in shiitakes were analyzed using MATLAB 2010b software (MathWorks Inc., Natick, MA, USA) according to the following equation:

Table 2. Application Dosage (g (aia)/m2) of the Field Trail for the Six Pesticides trial for spraying treatment

a

trials for drying treatment

pesticide

single dosage

double dosage

carbendazim thiabendazole procymidone bifenthrinc λ-cyhalothrin β-cyfluthrin

0.1500 0.7500 0.0450 0.0040 0.0013 0.0022

0.3000 1.5000 0.0900 0.0080 0.0026 0.0044

double or 4 times dosage 0.3000 1.5000 0.0900 0.0160 0.0052 0.0088

ai, active ingredient.

pesticide dosage level was diluted with 2 L of water and sprayed in each plot (6 m2). Four pesticides (carbendazim, procymidone, bifenthrin, and λ-cyhalothrin) are not registered in China for use on edible fungi. Therefore, the recommended dosages of these compounds referred to pesticides for use in other plants.8 The shiitake samples were randomly collected at 0, 4, 8, 16, 32, 48, 64, 80, and 96 h intervals after application of the pesticides for treatment. All samples were stored at −20 °C until further analysis. 2.3.3. Pesticides for Trials of Drying Treatment. To ensure that quantifiable target pesticide residues were deposited on the shiitakes for subsequent processing studies, some commercial pesticide formulations were applied individually at an exaggerated dosage (2 or 4 times the recommended dosage) based on OECD guidelines.26 The recommended dosages of four unregistered pesticides referred to pesticides for use in other plants (Table 2).8 The trial plot was 15 m2. The six pesticides were diluted with 5 L of water and applied by spraying the pesticide directly on the ripe shiitake fruiting bodies. Approximately 30 kg of shiitakes, exhibiting uniform maturity and uniform sizes, were randomly collected 8 h after application of the pesticides. All samples were placed in polyethylene bags for the subsequent drying processes. 2.4. Drying Processes. 2.4.1. Sunlight Exposure Drying. After harvesting, the shiitake samples were subjected to drying by sunlight

first‐order kinetics model: Ct = C0 e−kc C

(1)


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Table 3. Recovery (%) and RSD (%) for the Six Pesticides in Two Matrices at Three Spiking Levels (mg/kg) (n = 3) carbendazim

thiabendazole

procymidone

λ-cyhalothrin

bifenthrin

β-cyfluthrin

sample

spike level

recovery

RSD

recovery

RSD

recovery

RSD

recovery

RSD

recovery

RSD

recovery

RSD

fresh shiitakes

0.02 0.1 1

87.8 84.7 78.2

9.2 6.7 8.3

83.4 91.2 82.6

10.1 4.8 3.2

92.7 86.2 89.8

3.7 3.1 7.0

102.3 81.5 80.6

12.4 5.8 6.9

78.9 98.2 76.8

6.7 9.0 4.2

86.7 92.1 91.8

5.4 4.0 5.2

dried shiitakes

0. 1 0.5 5

88.4 97.6 95.1

5.4 3.7 8.9

87.9 98.2 97.6

8.4 7.9 5.7

87.2 104.6 98.5

6.4 5.4 9.7

104.5 107.2 86.2

8.5 4.5 7.8

75.8 98.4 97.2

4.1 8.4 9.6

81.5 96.7 98.2

9.1 5.8 9.9

Table 4. Residues (mg/kg) of the Six Pesticides in Shiitakes by Premixing Treatment (Mean ± SD, n = 3)a h

carbendazim

thiabendazole

procymidone

bifenthrin

single dosage

4 16 48 96

0.058 0.037 0.071 0.088

± ± ± ±

0.004 0.002 0.003 0.010

c d b a

0.079 0.049 0.068 0.103

± ± ± ±

0.003 0.002 0.005 0.011

b c b a

0.040 0.040 0.060 0.080

± ± ± ±

0.001 0.003 0.001 0.004

double dosage

4 16 48 96

0.100 0.069 0.124 0.154

± ± ± ±

0.003 0.003 0.006 0.018

c d b a

0.119 0.097 0.116 0.151

± ± ± ±

0.002 0.006 0.006 0.007

b c b a

0.081 0.056 0.098 0.142

± ± ± ±

0.002 c 0.005 d 0.002 b 0.012c a

c c b ac

λ-cyhalothrin

β-cyfluthrin

NDb ND ND ND

ND ND ND ND

ND ND ND ND

0.048 ± 0.005 a 0.026 ± 0.001 b ND ND

0.020 ± 0.002 ND ND ND

0.028 ± 0.002 a 0.022 ± 0.001 b ND ND

a

Values in the same column followed by different letters are significantly different (P < 0.05). bNot detected ( 0.9992). As shown in Table 3, three fortification levels were analyzed for each pesticide in two matrices, the average recovery rates ranged from 75.8 to 107.2% for six pesticides, and the RSD ranged from 3.1 to 12.4%. The recovery and precision for D


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Table 5. Residues (mg/kg), Kinetic Models, and Half-Lives (h) of Carbendazim, Thiabendazole, and Procymidone (Mean ± SD, n = 3) carbendazim h

a

single dosage ± ± ± ± ± ± ± ± ±

thiabendazole

double dosage

0 4 8 16 32 48 64 80 96

6.082 5.231 4.389 3.300 1.828 1.148 0.838 0.692 0.642

0.376 0.268 0.123 0.347 0.081 0.042 0.074 0.039 0.022

first eq R2 half-life

Ct = 5.925e−0.03375t 0.989 20.5

single dosage ± ± ± ± ± ± ± ± ±

procymidone

double dosage

11.960 ± 0.288 10.213 ± 0.209 9.125 ± 0.541 6.905 ± 0.186 4.540 ± 0.134 2.880 ± 0.109 1.905 ± 0.102 1.285 ± 0.026 0.905 ± 0.031

4.655 3.440 2.799 1.724 0.705 0.369 0.220 0.145 0.130

Ct = 11.64e−0.0294t 0.997 23.6

Ct = 4.537e−0.0595t 0.996 11.7

0.202 0.223 0.094 0.051 0.032 0.018 0.001 0.003 0.003

9.455 7.813 6.174 3.918 1.605 0.805 0.570 0.492 0.452

± ± ± ± ± ± ± ± ±

0.461 0.538 0.010 0.186 0.028 0.009 0.004 0.005 0.001

Ct = 9.481e−0.0529t 0.996 13.1

single dosage 2.871 2.556 2.120 1.591 0.941 0.669 0.495 0.415 0.379

± ± ± ± ± ± ± ± ±

double dosage

0.177 0.251 0.084 0.029 0.058 0.025 0.019 0.025 0.006a

6.169 5.314 4.520 3.823 2.550 1.627 1.116 0.727 0.472

Ct = 2.787e−0.0298t 0.982 23.2

± ± ± ± ± ± ± ± ±

0.288 0.154 0.067 0.094 0.091 0.022 0.075 0.014 0.026a

Ct = 5.950e−0.0271t 0.996 25.6

The values were higher than EU MRLs.

Table 6. Residues (mg/kg), Kinetic Models, and Half-Lives (h) of Bifenthrin, λ-Cyhalothrin, and β-Cyfluthrin (Mean ± SD, n = 3) λ-cyhalothrin

bifenthrin h

a

single dosage ± ± ± ± ± ± ± ± ±

0 4 8 16 32 48 64 80 96

0.134 0.121 0.115 0.102 0.081 0.064 0.046 0.041 0.039

0.012 0.004 0.001 0.003 0.002 0.004 0.004 0.001 0.001a

first eq R2 half-life

Ct = 0.130e−0.0146t 0.991 47.5

double dosage 0.442 0.412 0.384 0.336 0.274 0.215 0.171 0.104 0.098

± ± ± ± ± ± ± ± ±

0.012 0.020 0.012 0.012 0.015 0.007 0.008 0.004 0.006a

Ct = 0.439e−0.0157t 0.995 44.1

single dosage 0.056 0.054 0.052 0.051 0.045 0.035 0.028 0.024 0.022

± ± ± ± ± ± ± ± ±

0.004 0.003 0.004 0.004 0.002 0.001 0.002 0.002 0.002a

Ct = 0.057e−0.0102t 0.982 68.2

β-cyfluthrin

double dosage 0.123 0.114 0.102 0.098 0.084 0.076 0.062 0.050 0.038

± ± ± ± ± ± ± ± ±

0.003 0.006 0.007 0.004 0.010 0.002 0.005 0.004 0.001a

Ct = 0.119e−0.01073t 0.982 64.6

single dosage 0.079 0.076 0.075 0.072 0.058 0.048 0.033 0.027 0.024

± ± ± ± ± ± ± ± ±

0.009 0.005 0.005 0.007 0.002 0.004 0.002 0.001 0.003a

Ct = 0.082e−0.0126t 0.980 54.9

double dosage 0.203 0.184 0.170 0.155 0.128 0.112 0.088 0.057 0.042

± ± ± ± ± ± ± ± ±

0.009 0.004 0.005 0.010 0.011 0.004 0.004 0.002 0.002a

Ct = 0.198e−0.0140t 0.984 49.4

The values were higher than EU MRLs.

after treatment (Table 4). The reason for this finding is most notably due to the small amounts of bifenthrin, β-cyfluthrin, and λ-cyhalothrin being absorbed from the substrate, whereas they cannot translocate in the plants. This may be due to the fact that these pesticides have contact action (acting on the surface). The dilution effect due to the growth of the fruiting body caused a decrease in pesticide residues.18 It can also be concluded from further inspection of the results that the dissipation behavior of pesticides in the shiitakes is influenced by various factors, including the absorption capacity of shiitakes from the substrate (increasing effect) and whether the pesticides could translocate in the shiitakes (increasing effect) during fruiting body growth (decreasing effect).31 3.2.2. Spraying Treatment. The residues, kinetic results, and half-lives of six pesticides are shown in Tables 5 and 6. No residues of the six tested pesticide were detected in samples from the control plot. The dissipation of pesticide residues in shiitakes fits well within a first-order kinetic model (R2 > 0.980). The longest and shortest half-lives were found in λcyhalothrin with 68.2 h and thiabendazole with 11.7 h, respectively. The half-lives of the tested six pesticide residues follow a general trend: thiabendazole < carbendazim < procymidone < bifenthrin < β-cyfluthrin < λ-cyhalothrin. The

differences in half-lives of the six pesticides may result from the different physicochemical properties such as molecular weight and octanol−water partition coefficient (Kow), both of which have been shown to influence the ability of a chemical to pass through plant cuticles.31 At present, four pesticides (carbendazim, procymidone, bifenthrin, and β-cyfluthrin) are not registered in China for use on edible fungi. Thiabendazole is registered for use on mushrooms by premixing treatment with preharvest interval (PHI) for 56 days; λ-cyhalothrins is registered for use on edible fungi by spraying the fruiting body with a PHI of 7 days.8 However, the maturation time of the fruiting body of shiitakes is only 96 h, and these PHIs appear not suitable for the use on shiitakes. Thus, it is necessary to investigate whether the final residues at harvest (96 h) meet the MRL requirements set by the European Union (EU)32 and China33 (Table 1). As shown in Tables 4−6, the final residues of the six pesticides applied by both application treatments were found to be below the MRLs of edible fungi in China. For the premixing treatment, the final six pesticide residues in shiitakes were below the standards of EU MRLs on cultivated fungi, except for procymidone. For the spraying treatment, only carbendazim and thiabendazole were below the standard of EU MRLs. The six pesticides applied at E


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Figure 2. Dissipation trends of six pesticides in dried shiitakes during sunlight exposure drying (A, B) and hot-air drying (C, D) on a dry weight basis.

Table 7. Kinetic Models and Half-Lives of the Six Pesticides in Two Drying Processes sunlight exposure drying pesticide carbendazim thiabendazole procymidone bifenthrin λ-cyhalothrin β-cyfluthrin

kinetic model Ct Ct Ct Ct Ct Ct

= = = = = =

−0.0549t

27.58e 25.96e−0.0784t 19.74e−0.0615t 3.649e−0.0383t 2.234e−0.0237t 2.686e−0.0357t

R2

hot-air drying half-life (days)

0.908 0.917 0.943 0.946 0.946 0.907

12.6 8.8 11.3 18.1 29.3 19.4

kinetic model Ct Ct Ct Ct Ct Ct

= = = = = =

−0.0260t

26.43e 25.62e−0.0431t 19.86e−0.0373t 3.799e−0.0274t 2.251e−0.0153t 2.718e−0.0209t

R2

half-life (h)

0.907 0.925 0.911 0.963 0.933 0.938

26.6 16.1 18.6 25.3 45.4 33.2

dissipation behaviors of bifenthrin, β-cyfluthrin, and λcyhalothrin. For the hot-air drying process, the temperature of the oven was 30−53 °C in the beginning of the first 10 h. Then, the temperature increased to 53−60 °C in the last 10 h. Similar to sunlight exposure drying, the dissipation rates of carbendazim, thiabendazole, and procymidone increased as a result of rising temperatures. 3.3.2. Kinetic Models and Half-Lives of the Six Pesticides. The parameters of calculated kinetic models and half-lives for the six pesticides are provided in Table 7. It can be found that the half-lives of carbendazim, thiabendazole, and procymidone were shorter than those of the other three pesticides tested in the sunlight exposure drying. The reason might be due to their different liposolubility; carbendazim, thiabendazole, and procymidone have log Kow values ≤3.3, whereas the other three have log Kow values ≥6.0. The high liposolubility (log Kow values ≥ 6.0) of the pesticides can be strongly retained by the waxes of the shiitakes skin,30 making their reduction slowly by drying. Moreover, the pesticides may be degraded by certain enzymes in edible fungi.36,37 Because of the different chemical structures of the pesticide, the regulatory mechanisms of pesticide degradation by enzymes may be different, resulting in different dissipation behaviors in shiitakes. Moreover, bifenthrin, λ-cyhalothrin, and β-cyfluthrin were pyrethroid pesticides, which exhibit a similar chemical structure. λ-Cyhalothrin and β-cyfluthrin feature a common structural scaffold, namely, phenoxybenzoic acid (PBA).38,39 The common metabolite of

tested dosages (single and double dosage) by premixing treatment are safe and recommended in real production situation. Therefore, further studies are required to evaluate the risk of the residues from the spraying treatment for the six pesticides on shiitakes. 3.3. Effects of Two Drying Methods on the Residue Dissipation Behaviors. 3.3.1. Dissipation Trends of the Six Pesticides. Due to different water contents in the fruiting bodies of shiitakes during the drying process, the residue of pesticides was calculated on a dry weight basis. The residue (Cd) of pesticides can be defined as Cd (mg/kg) = (Cw/100 − moisture content) × 100%, where Cw (mg/kg) represents the residues of pesticide after drying, and the moisture content (%) of the shiitake samples was measured following the National Standard of China.20,35 The dissipation rates of carbendazim, thiabendazole, and procymidone dropped slowly first and then more rapidly during the two drying processes (Figure 2A,C). For bifenthrin, β-cyfluthrin, and λ-cyhalothrin, the changes of dissipation rates follow an opposite trend: a quick reduction could be observed in the beginning, however, steadily slowing later (Figure 2B,D). For the drying procedure using sunlight exposure, the temperature was 24.6−29.9 °C in the beginning of the first 8 days and 27.4−35.5 °C during the last 12 days (Figure 1). This implies that the carbendazim, thiabendazole, and procymidone may be sensitive to high temperature, with their dissipation rate increasing with rising temperature, whereas the temperature may exhibit little impact on the F


Article


drying, the shiitakes were dehydrated over a much shorter time period using hot-air drying. Therefore, hot-air drying did not lead to a complete removal of pesticides in the shiitakes. On the other hand, the pesticides exhibited different dissipation behaviors in shiitakes using different drying methods. Using sunlight exposure drying, the dissipation of pesticides is thought to be largely determined by the combined effects of light and heat. Light plays an important role in the reduction of the pesticide concentrations. In hot-air drying, light is absent and the removal of pesticides through sublimation or evaporation in this enclosed environment seems to be diminished. Cabras et al. reported that sunlight-drying was more effective for the degradation of phosalone and veinclozolin in raisins, whereas oven-drying is reported to be more effective for the removal of iprodione and procymidone.15 3.4. Determination of Processing Factors. PFs can be routinely incorporated into a dietary exposure assessment to render the results more reflective of actual exposure.22 PFs reflect the ratio between the pesticide residues in the processed commodity (dried shiitakes) to that in the raw agricultural commodity (fresh shiitakes). A PF < 1 indicates that there is a reduction of pesticide by the processing method (reduction factor), whereas a PF > 1 indicates no reduction in weight or volume (concentration factor).43 The processing factors describe the proportional amount by which residues change (on a wet weight basis) when the food is being processed.44 For this study, the residues of carbendazim, thiabendazole, and procymidone in shiitakes exhibited similar change trends by both drying methods, increasing at the beginning and declining thereafter (Figure 4A,C). The reason for this phenomenon can be explained by the finding that the water loss rate of shiitakes was greater than the dissipation rate of pesticides, resulting in a concentration effect of the pesticides in shiitakes at an early stage of the drying process. Upon continuation of the drying process, the water loss rate of shiitakes declined and became lower than the dissipation rate of the pesticides. Particularly true for thiabendazole, the concentration of the residues was

bifenthrin is 2-methyl-3-phenyl benzoate, which features a structure similar to that of PBA.40 Pyrethroids do not easily degrade due to the particularly stable PBA scaffold.41 It can be seen from inspection of the results that pesticides with similar physicochemical properties also exhibit similar dissipation behaviors.42 3.3.3. Comparison of Two Drying Methods on the Final Residues in Dried Shiitakes. The removal rate (RR) of pesticides can be defined as RR (%) = (Cb − Ca)/Cb, where Cb and Ca (mg/kg) represent the average pesticide residues before and after drying, respectively.20 As shown in Figure 3, the

Figure 3. Removal rates of six pesticides in dried shiitakes by two drying methods.

removal rate of pesticides by sunlight exposure drying (36.2− 94.6%) was apparently higher than that of hot-air drying (26.0− 68.1%). Sunlight exposure drying proved to be more efficient than hot-air drying for the removal of the six pesticides. Compared to the long time needed for sunlight exposure

Figure 4. Dissipation trends of six pesticides in dried shiitakes during sunlight exposure drying (A, B) and hot-air drying (C, D) on a wet weight basis. G


Article

Journal of Agricultural and Food Chemistry Table 8. Processing Factors (Mean ± SD, n = 3) and Hazard Quotients (HQ; %) of the Six Pesticides for Shiitakes final PFs pesticide carbendazim thiabendazole procymidone bifenthrin λ-cyhalothrin β-cyfluthrin

sunlight exposure drying 1.71 0.42 1.22 3.43 3.77 4.13

± ± ± ± ± ±

0.17 0.03 0.08 0.13 0.21 0.20

HQs of dried shiitakes (with PFs) hot-air drying

sunlight exposure drying

hot-air drying

± ± ± ± ± ±

6.04 0.06 18.16 1.08 4.77 3.63

14.48 0.35 42.43 1.30 5.55 4.43

4.10 2.47 2.85 4.38 4.56 5.03

0.37 0.16 0.26 0.28 0.19 0.21

3.53 0.14 14.89 0.29 0.97 0.88

Funding

lower than the initial residues after sunlight exposure drying. This finding may be due to thiabendazole easily degrading upon sunlight exposure.45,46 The residues of bifenthrin, λ-cyhalothrin, and β-cyfluthrin increased during the two drying processes (Figure 4B,D). This finding is also due to the greater water loss rate of shiitakes compared to the pesticide dissipation rate. This ultimately results in an increase in pesticide concentration. The highest levels of PF for carbendazim, thiabendazole, and procymidone were determined to be 3.24 on the 10th day, 2.43 on the 10th day, and 3.09 on the 13th day for the sunlight exposure drying process, respectively. In the hot-air drying process, the maximum PFs for carbendazim, thiabendazole, and procymidone were found to be 4.51, 3.55, and 3.82, observed at the 17th, 10th, and 10th hour, respectively. The final PFs for the six pesticides were >1 in the two drying processes, except for thiabendazole upon sunlight exposure drying (Table 8). Shabeer et al. reported that the residues of dimethomorph and cymoxanil first increased and then decreased, whereas the concentration of famoxadone residues increased during raisin processing, resulting in a PF value >1 for all pesticides in raisins.16 3.5. Dietary Risk Assessment for Dried Shiitakes. The hazard quotient indicates an acceptable risk if it is lower than 100%, and a higher value represents a higher risk. The HQs for the six pesticides are summarized in Table 8. The HQ values for all tested pesticides were much lower than the safety level (100%). The HQ value of procymidone was relatively high, especially in the hot-air drying process (42.43%). Thiabendazole exhibits a particularly low HQ value of 0.06−0.35%, resulting in a negligible exposure risk upon consumption of dried shiitakes. For a realistic estimation, the PF value should be considered.47 In this work, the HQ values for dried shiitakes (with PF) were significantly higher than the HQ values for fresh shiitakes (without PF). Therefore, dietary exposure risk from pesticides through consumption of dried shiitakes should be considered. Meanwhile, the HQ values for dried shiitakes using hot-air drying were higher than the HQ values using sunlight exposure drying. As a consequence, the dietary risk for the six pesticides in the hot-air-dried shiitakes was found to be higher. However, our studies of dried shiitakes showed that the residual concentration for each tested pesticide can be considered acceptable (HQ < 100%), with a low risk to consumers.

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HQs of fresh shiitakes (without PFs)

This work is supported by the National Natural Science Foundation of China (31471797), Special Fund for Agroscientific Research in the Public Interest (No. 201303088), and PADA. Notes

The authors declare no competing financial interest.

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Residual Behaviors of Six Pesticides in Shiitake from Cultivation to

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