Pesticide residues in yuza (Citrus junos) cultivated using ordinary and environmentally friendly cultures

Abstract

An analysis of seven pesticides in 160 yuza samples (100 by ordinary culture and 60 by environmentally friendly culture) was conducted. The detection rate of carbendazim was the highest and the levels were up to 5.15 mg/kg. The degradation order of the seven pesticides in yuza tea was as follows: acequinocyl>chlorpyrifos>spirodiclofen>carbendazim>deltamethrin>phosalone, prothiofos.

Introduction

Citrus junos, also called yuza, is one of the most popular fruits on the market in South Korea. Yuza fruits have been traditionally used to make yuza tea. Almost all parts of yuza including the peel and seed are processed to make yuza tea.¹⁾ Therefore, it is important to monitor pesticide residues in yuza and yuza tea. Yuza is cultivated by both ordinary and environmentally friendly cultures. Environmentally friendly cultures are conducted with very minimal or no chemical pesticides or chemical fertilizers.²⁾

Various pesticides are commonly applied as a chemical protection for yuza. Chlorpyrifos (O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate) is used as an organophosphate insecticide on oranges, cotton, corn, and vegetable crops. Acequinocyl (3-dodecyl-1,4-dihydro-1,4-dioxo-2-naphthyl acetate) is an acaricide with contact action that is used to control several species of mites on agriculture crops and ornamentals.^3,4) Spirodiclofen (3-(2,4-dichlorophenyl)-2-oxo-1-oxaspiro[4,5]dec-3-en-4-yl 2,2-dimethylbutyrate) belongs to the newly discovered acaricidal group of spirocyclic tetronic acid derivatives.⁵⁾ Carbendazim (methyl benzimidazol-2-ylcarbamate) is the principal degradation product of two parent compounds such as benomyl (methyl 1-(butylcarbamoyl)benzimidazol-2-ylcarbamate) and thiophanate-methyl (dimethyl 4,4′-(o-phenylene)bis(3-thioallophanate)). Therefore, the tolerances of benomyl and thiophanate-methyl are generally expressed as carbendazim.⁶⁾ Benomyl is a very unstable compound and is mostly converted into carbendazim both directly on fruits and during analysis.⁷⁾ In contrast, thiophanate-methyl is more stable than benomyl.⁸⁾ Thus, to elucidate the amount of benomyl and thiophanate-methyl, an analysis of carbendazim is carried out.

In this study, various analytical tools such as GC-nitrogen phosphorus detection (GC-NPD), GC-MS and HPLC-photodiode array detection (HPLC-PDA) were used to analyze pesticides in yuza. Chlorpyrifos, prothiofos, and phosalone were simultaneously analyzed by GC-NPD. Acequinocyl, spirodiclofen, and carbendazim were determined by HPLC-PDA. Deltamethrin was detected by GC-MS. In addition, the kinetics of the dissipation of the pesticides sprayed onto yuza was determined and their half-lives were calculated by the kinetic model. The study objectives were: (1) to analyze pesticide residue levels over time in yuza cultivated under ordinary or environmentally friendly conditions; (2) to elucidate the kinetic parameters for the degradation of pesticides.

Materials and Methods

1. Chemicals, apparatus, and sampling of yuza

Seven pesticide standards (chlorpyrifos, prothiofos, phosalone, deltamethrin, acequinocyl, spirodiclofen and carbendazim) were purchased from Sigma-Aldrich Inc. (Milwaukee, WI, USA). Acetone, acetonitrile, petroleum ether, and dichloromethane were purchased from Burdick & Jackson (Muskegon, MI, USA). Methanol, water, and hexane were purchased from the J. T. Baker Chemical Co. (Phillipsburg, NJ, USA). All solvents used were HPLC grade. The phosphoric acid (Daejung, Seoul, Korea) and acetic acid (Duksan, Seoul, Korea) were 99% pure. Anhydrous sodium sulfate and sodium chloride were obtained from Daejung (Seoul, Korea).

A blender (HR 2860 model, Philips, Eindhoven, the Netherlands) and homogenizer (model 17506, Omni International Inc., Waterbury, CT, USA) were used to mix samples. The funnel shaker used for the extraction was obtained from Jeio Tech (Reciprocal shaker, RS-1, Seoul, Korea). A rotary evaporator (Buchi Rotavapor R110, Brinkmann Instruments, Inc., Westbury, NY, USA) and analytical nitrogen evaporator (S-040, Tokyo Rikakikai Co., Ltd., Japan) were used to evaporate the solvent. A solid-phase extraction vacuum manifold (Supelco, Rome, Italy) was used to clean up the samples. Florisil cartridges (1 g/6 mL) and amino propyl cartridges (1 g/6 mL) were purchased from Phenomenex (Torrance, CA, USA). Silica cartridges (1 g/6 mL) were purchased from Waters Corporation (Milford, MA, USA).

A total of 160 samples (100 cultivated by ordinary culture and 60 cultivated by environmentally friendly culture from the same plot) were collected from Hansung Food Co., Ltd. (Goheung-gun, Jeollanam-do, Korea) and the Goheung area from 2010–2011. In the case of the environmentally friendly culture, no organic pesticides were allowed and one third of the recommended chemical fertilizer was used for cultivation. To avoid mutual contamination of yuza, fruits were collected and wrapped separately before being delivered to our laboratory. The samples were delivered within 1 day under cooling conditions.

2. Analytical conditions for pesticides

Analyses of chlorpyrifos, prothiofos and phosalone were performed on a 6890 series GC, from Agilent Technologies (Palo Alto, CA, USA) equipped with an NPD and an Agilent Technologies 7683 autosampler. Separations by GC-NPD were carried out using a DB-5 capillary column (30 m×0.25 mm i.d., 0.25 µm, Agilent Technologies). The temperature of the split injector was held constant at 260°C. The injection volume was 1 µL, and the flow rate of the carrier gas (helium) was 1.0 mL/min. The column temperature program was 120°C (held 2 min), increased to 220°C at 10°C/min, increased to 250°C at 7°C/min, and finally increased to 280°C at 7°C/min (held 15 min). The data were acquired and processed using ChemStation software (Agilent Technologies).

GC-MS analyses of deltamethrin were performed on an Agilent Technologies GC 6890N gas chromatograph equipped with a mass spectrometer (MS 5975). Separation was carried out using a DB-5ms capillary column (30 m×0.25 mm i.d., 0.25 µm, Agilent Technologies). The temperature of the splitless injector was held constant at 260°C. The injection volume was 2 µL, and the flow rate of the carrier gas (helium) was 1.0 mL/min. The column temperature program was 80°C (held 2 min), increased to 220°C at 25°C/min, and finally increased to 280°C at 2°C/min (held 5 min). The data were acquired and processed using ChemStation software.

HPLC analyses of acequinocyl were performed on Waters 1525 HPLC equipped with a Waters 717 plus autosampler and a Waters 2998 PDA. The column used was a Zorbax Extend-C18 (4.6 mm×250 mm i.d., 5 µm, Agilent Technologies). The mobile phase was filtered through a 0.45-µm-membrane filter (JHWP, Millipore, Cork, Ireland) and degassed before using. Isocratic elution was with aqueous 0.1% phosphoric acid : acetonitrile (12.5 : 87.5, v/v) for 30 min. The injection volume was 10 µL, the flow rate was 0.8 mL/min, and the HPLC column temperature was maintained at 40°C. Peaks were detected at 250 nm. Empower Software (Waters) was employed for data collection and processing.

The HPLC system and column for the analysis of carbendazim and spirodiclofen were the same as for the acequinocyl analysis. The A and B mobile phases were water and methanol. The optimal gradient elution program was as follows: 0–45 min, 10% A; 45–50 min, 50% A; and 50–60 min return to the initial conditions. The injection volume was 10 µL, the flow rate was 1.0 mL/min, and the HPLC column temperature was maintained at 40°C. UV absorbance was simultaneously detected at 254 nm (for spirodiclofen) and 279 nm (for carbendazim). Data were acquired and processed using Empower Software.

3. Preparation of standard solutions and sample preparations for the analysis of pesticides

Stock standard solutions of the pesticides were prepared by accurately weighing 100 mg of pesticide in a 100-mL volumetric flask and diluting to volume with acetone for chlorpyrifos, prothiofos, phosalone, and deltamethrin analysis and with methanol for acequinocyl, spirodiclofen, and carbendazim analysis. Stock standard solutions were stored at −20°C until analysis. Working standard solutions used in the validation studies were prepared as follows: A working standard solution of 100 µg/mL was prepared by shifting 1 mL of the stock standard solution to a 10-mL volumetric flask and diluting to volume with acetone for chlorpyrifos, prothiofos, phosalone and deltamethrin analysis and with methanol for acequinocyl, spirodiclofen and carbendazim analysis. A working standard solution of 10 µg/mL was prepared by diluting a working standard solution of 100 µg/mL tenfold in the same way: 0.5, 1, 3, 5, 8, 10, and 20 µg/mL.

The sample preparation procedure for the analysis of pesticides was from the method of the Korean Food Code and the previous reoprt.^2,9)

4. Method validation

Prior to applying real samples, the analytical methods were validated through limit of detection (LOD: signal-to-noise ratio of 3), limit of quantification (LOQ: signal-to-noise ratio of 10), linearity, precision, and recovery. An external standard method was used for calibration. LOD and LOQ were determined by diluting the standard solution.¹⁰⁾ The linearity of the standard curves was estimated at concentrations of 0.5, 1, 3, 5, 8, 10 and 20 µg/mL (n=3). The recovery assay of the pesticides was determined by spiking a standard pesticide aqueous solution in five replicates. Precision was calculated as relative standard deviation (RSD). The intraday precision and interday precision were based on analyses of the standard solution five times in 1 day and on five consecutive days, respectively.

5. Experimental design for the kinetic model

Yuza cultivated in the conventional way was sprayed with a mixture of chlorpyrifos, prothiofos, phosalone, deltamethrin, acequinocyl, spirodiclofen, and carbendazim at the recommended normal doses with a sprayer. The amounts of 7 pesticides dissolved in 30 mL aqueous solution of the mixture were from 0.003 to 0.03 g. Yuza sprayed with 30 mL of pesticide mixture was processed for yuza tea in the laboratory, because we usually consume yuza tea rather than yuza worldwide. The sprayed yuza was sliced and the seed was separated. Table sugar was added to the sliced yuza (1 : 1) and aged for 1 week in the shade. Sampling was conducted after 2 hr, and 1, 2, 3, 5, 7, and 10 days from the last pesticide spraying. The weight of yuza did not increase during the sampling period; thus, the dilution of pesticide residue was not affected by growth. Kinetic models employed to estimate the half-life of the pesticide residues in yuza tea after pesticide treatment such as first-order (FO), zero-order (ZO), and second-order (SO) models are empirical formulas for estimating the correlations between time (t) and concentration (µg/g) of pesticide residue.^11,12)

Results and Discussion

1. Method validation and pesticide residue levels in yuza

The calibration curve parameter, recovery rates, LOD, and LOQ were measured for the accurate and precise analysis of the pesticides. The standard curve showed satisfactory linearity in a range from 0.5–20 µg/mL with determination coefficients of 0.9874–0.9999. The LOQ of each pesticide was 0.06–0.33 µg/mL. The recovery rates of the seven pesticides ranged from 83.4 to 103.2%. The RSD of the intraday and interday precision and accuracy for the analysis were lower than 13.8% and 19.1%, respectively. The results showed that the precision of this method for analyzing pesticide residue in yuza and yuza tea was satisfactory.

The results of the pesticide analysis in yuza cultivated by ordinary and environmentally friendly cultures (2010–2011) and the maximum residue limits (MRLs) are summarized in Table 1. Prothiofos and deltamethrin were not detected in yuza cultivated by ordinary culture in 2010. Chlorpyrifos was found at up to 0.108 mg/kg in a single sample of yuza cultivated by ordinary culture. Spirodiclofen revealed a range of contamination of up to 1.889 mg/kg in 16 of 50 yuza plants cultivated by ordinary culture. The 37 yuza cultivated by ordinary culture contained the highest level of carbendazim at 5.148 mg/kg. In 2011, spirodiclofen and carbendazim were detected at lower than 0.334 and 3.840 mg/kg, respectively. The other pesticides were not detected or quantified. Chlorpyrifos, prothiofos, phosalone, deltamethrin and acequinocyl were not detected or quantified in yuza cultivated by environmentally friendly culture in 2010. Spirodiclofen and carbendazim were found in eight of 30 yuza samples at up to 0.801 mg/kg and in 13 of 30 yuza samples at up to 2.907 mg/kg, respectively. Carbendazim was detected in eight samples at concentrations ranging from ND to 0.340 mg/kg in 2011. The remaining pesticides were not detected or quantified. Carbendazim was detected in all samples. The pesticide residues in all yuza samples were lower than the MRLs established by Korean legislation. However, because of possible health effects and widespread use, continuous monitoring of carbendazim is necessary in the future. In addition, higher levels of the tested pesticides were present in yuza samples produced in 2010 than in 2011.

Table 1. Results of the pesticide residue analysis in yuza cultivated by ordinary and environmentally-friendly culture

Detected pesticide	Yuza cultivated by ordinary culture					Yuza cultivated by environmentally-friendly culture					Korean MRLsc) (mg/kg)
	No. of samples analyzed per year	Samples with residues		Detection range (mg/kg)		No. of samples analyzed per year	Samples with residues		Detection range (mg/kg)
		2010	2011	2010	2011		2010	2011	2010	2011
Chlorpyrifos	50	1 (2%)	0 (0%)	ta)	t	30	0 (0%)	0 (0%)	NDa)	ND	0.50
				−0.108
Prothiofos	50	0 (0%)	0 (0%)	NDb)	t	30	0 (0%)	0 (0%)	ND	ND	0.05
Phosalone	50	0 (0%)	0 (0%)	t	t	30	0 (0%)	0 (0%)	ND	t	2.00
Deltamethrin	50	0 (0%)	0 (0%)	ND	ND	30	0 (0%)	0 (0%)	ND	ND	0.50
Acequinocyl	50	0 (0%)	0 (0%)	t	t	30	0 (0%)	0 (0%)	tb)	t	1.00
Spirodiclofen	50	16 (32%)	5 (10%)	0.299	0.274	30	8 (27%)	0 (0%)	0.283	t	2.00
				−1.889	−0.334				−0.801
Carbendazim	50	37 (74%)	27 (54%)	0.113	0.088	30	13 (43%)	8 (27%)	0.029	0.111	7.00d)
				−5.148	−3.840				−2.907	−0.340

^a) t: LOD<values<LOQ. ^b) ND: values<LOD. ^c) MRLs: maximum residue limit of yuza tea. ^d) MRLs: maximum residue limit of other citrus fruits.

2. Kinetic parameters for pesticide degradation

Ten days after spraying, the degradation rates of chlorpyrifos and acequinocyl exceeded 90% and 100%, respectively. The degradation order of the seven pesticides was as follows: acequinocyl>chlorpyrifos>spirodiclofen>carbendazim>deltamethrin>phosalone, prothiofos (Table 2). Because the half-lives of prothiofos, phosalone, and deltamethrin were longer than those of the others, their application doses should be reduced. The doses of acequinocyl and chlorpyrifos could be increased to improve productivity as they were under the MRLs. Three kinetic models were employed to characterize the best-fit kinetic model. Among the theoretical models, FO and SO models were the best-fit models for the pesticides residues, judging from the significance of the coefficient of determination and the standard error (Table 3). Therefore it is recommended that the half-life of the pesticide be assessed from the best-fit model rather than from the FO kinetic model.^11,12)

Table 2. Concentrations and degradation rates of pesticides residues in yuza tea treated with various pesticides

Days after treatment (D)	Chlorpyrifos		Prothiofos		Phosalone		Deltamethrin		Acequinocyl		Spirodiclofen		Carbendazim
	R(µg/g)a)	D(%)b)	R	D	R	D	R	D	Ra)	Dbb)	R	D	R	D
0	0.157±0.026	0	0.158±0.013	0	0.087±0.006	0	0.134±0.033	0	0.081±0.032	0	0.082±0.036	0	0.553±0.136	0
1	0.093±0.005	41	0.154±0.013	3	0.078±0.004	10	0.130±0.028	3	0.041±0.048	49	0.070±0.009	15	0.161±0.037	26
2	0.089±0.011	43	0.149±0.011	6	0.074±0.001	15	0.129±0.042	4	0.028±0.005	65	0.056±0.013	32	0.092±0.047	39
3	0.072±0.003	54	0.134±0.004	15	0.073±0.006	16	0.118±0.028	12	0.028±0.050	65	0.040±0.008	51	0.084±0.005	44
5	0.047±0.005	70	0.124±0.026	22	0.069±0.001	21	0.101±0.011	24	0.003±0.022	96	0.027±0.011	67	0.073±0.042	52
7	0.028±0.005	82	0.119±0.007	25	0.068±0.001	22	0.091±0.016	33	N.D.	100	0.013±0.007	84	0.053±0.024	65
10	0.016±0.014	90	0.115±0.050	27	0.063±0.002	28	0.077±0.012	43	N.D.	100	0.009±0.003	89	0.036±0.009	76

^a) R: residue (mean±standard deviation of nine replicates). ^b) D: degradation rate (%).

Table 3. Statistical parameters to estimate models and half-lives of pesticides in the yuza tea

Pesticide	Models (ID)	Kinetic equation	ka)	r2	Half-life (t1/2, days)
Chloropyrifos	FOb)	y=0.1363e−0.217x	0.217	0.987	3.19
	ZOc)	y=−0.0122x+0.1206	0.012	0.830	4.94
	SOd)	y=5.3301x+1.7065	5.331	0.932	0.32
Prothiofos	FO	y=0.155e−0.034x	0.034	0.903	20.38
	ZO	y=−0.0046x+0.1546	0.004	0.886	16.80
	SO	y=0.2572x+6.4261	0.257	0.914	24.98
Phosalone	FO	y=0.0813e−0.027x	0.027	0.895	25.66
	ZO	y=−0.002x+0.0812	0.002	0.861	20.30
	SO	y=0.3786x+12.275	0.378	0.922	32.42
Deltamethrin	FO	y=0.1386e−0.059x	0.059	0.985	11.74
	ZO	y=−0.0062x+0.1361	0.006	0.980	10.97
	SO	y=0.5797x+7.0038	0.579	0.980	12.08
Acequinocyl	FO	y=0.0887e−0.587x	0.587	0.910	1.18
	ZO	y=−0.0069x+0.0534	0.006	0.713	3.86
	SO	y=53.284x−36.572	53.28	0.732	0.68
Spirodiclofen	FO	y=0.0841e−0.237x	0.237	0.979	2.92
	ZO	y=−0.0075x+0.0724	0.007	0.901	4.82
	SO	y=10.316x+1.0486	10.31	0.939	0.10
Cabendazim	FO	y=0.1326e−0.131x	0.131	0.973	5.29
	ZO	y=−0.01x+0.1259	0.01	0.872	6.29
	SO	y=1.9562x+6.2346	1.95	0.964	3.18

^a) Rate constant or regression coefficient. ^b) First Order Kinetics (Equations: ln[R]_t=ln[R]₀−k t, Half-life: ln 2/k, [R]_t and [R]₀: molar % of pesticide residues at time t and 0 days, respectively, k: rate constants or regression coefficient). ^c) Zero Order Kinetics (Equations: [R]=[R]₀−k t, Half-life: [R]₀/2k). ^d) Second Order Kinetics (Equations: 1/[R]_t=1/[R]₀+k t, Half-life: 1/k[R]₀).

Acknowledgment

This study was supported by the R&D Convergence Center Support Program, Ministry for Food, Agriculture, Forestry, and Fisheries, Republic of Korea.

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