Multi-Residue Determination of Pesticides in Vegetables on Dalian Market by Gas Chromatograph, 2009-10

This paper presents results from surveillance of pesticide multi-residues in vegetables carried out in 2009-10. 420 samples of 10 different types of fresh vegetables were analyzed for their pesticide multi-residue contents using gas chromatograph and NY/T 761-2008 pesticide multi-residue screen methods. The residues exceeded MRLs of forbidden pesticides found were: carbofuran 0.110 mg/kg (kidney bean) and methamidophos 0.037 mg/kg (celery) in January 2009, methamidophos 0.037 mg/kg (tomato) in May 2009, aldicarb 0.013 mg/kg (kidney bean) in September 2009, omethoate 2.200 mg/kg (celery) in November 2009, carbofuran 0.052 mg/kg (green pepper) in April 2010, parathion 0.056 mg/kg (celery) and carbofuran 0.030 mg/kg (celery) in July 2010. Also, chlorpyrifos used as unforbidden pesticide was most frequently found above MRL, rape (0.820 mg/ kg) and celery (0.365 mg/kg) in January 2009, celery (0.330 mg/kg) in May 2009, lettuce (0.298 mg/kg) in September 2009, rape (0.910 mg/kg) in April 2010 and lettuce (0.230 mg/kg) in July 2010. In addition, cypermethrin used as unforbidden pesticide was found above MRL only once in rape (1.270 mg/kg) in May 2009 and none of unforbidden pesticides above MRL was found in November 2009 and January 2010. Most of the samples (96%) were up to the national standard.


Introduction
Nowadays, a growing demand for safe and nutritional agricultural products requires more green vegetables grown without the usage of pesticides [1][2][3][4][5][6][7]. Over 800 pesticides belonging to over 100 different chemical classes are extensively used to protect crops before and after harvest from infestation by pests and plant diseases in agriculture all over the world [1,2]. Although pesticides play an important role in increasing production and ensuring quality in agricultural practice, residues will evaporate into the air, flow into the rivers, settle in the soil, pollute the productions and transfer to the human bodies, which will cause potential harm to the human being and pose a major threat to biodiversity [3]. Therefore, the issue of produce security, environmental pollution and human health that caused by the usage of pesticides are seriously concerned worldwide.
Since the 21st century, numerous researches on the effects of pesticide multi-residue to quality and security of agricultural products have been carried up successively [5]. The Southeastern Poland monitoring programme for 2004-05 covered 747 samples of 39 different types of fresh fruits and vegetables which were analyzed for pesticide multi-residue contents, and there were 27 samples (3.6%) residues exceeded national MRLs [8]. A total of 4404 samples of fruits and vegetables (34% of Danish origin and 66% from other countries) were analyzed for pesticide multiresidue contents , and approximately 89 pesticides were detected which were more frequently found in fruit (60%) than in vegetables (18%) [6]. The present study reports the results from Dalian market (2009-10) of an on-going vegetable monitoring programme conducted by Liaoning Province Agriculture Academic Sciences, is aimed at ensuring that consumers are not exposed to unacceptable pesticide multi-residue levels.

Extraction
The samples (25 g) were accurately weighed into a 100 ml polypropylene centrifuge tube and extracted with 50 ml acetonitrile, a high-speed homogenate machine was used to blend at 20000 rpm for 2 min. Then, the mixture was filtered through a piece of filter paper and the combined filtrate (about 40-50 ml) was collected into a 100 ml measuring cylinder containing 5-7 g NaCl. The measuring cylinder was sealed up with a plug and oscillated tempestuously for 1 min. After that, left it to stand for 30 min at room temperature to make acetonitrile phase and water phase stratification. A total of 10 ml acetonitrile were sucked up to a 150 ml beaker which was kept in a water bath at 80°C ventilating with nitrogen or air slowly for evaporating to nearly dryness.

Purification
The methods of purification are different among organophosphorus pesticides, organochlorine and pyrethroid pesticides and carbamate pesticides. The specific content and operation methods are brief introduced as follows.
Organophosphorus pesticides: Acetone (2 ml) was added into the above residue and the beaker was covered with aluminium foil for purifying. The ready solution was transfered to a 15 ml scale centrifugal tube absolutely and acetone (about 3 ml) was used to wash the beaker for three times. The total volume collected was adjusted to exactly 5 ml and blended with a whirlpool mixer. The samples were filtrated with filter membrane (0.22 μm) before determination.
Organochlorine and pyrethroid pesticides: An aliquot of 10 ml n-hexane was added and the beaker was covered with aluminium foil for subsequent use. The Florisil PR column was eluted beforehand and conditioning using 5 ml mixed solution of acetone and n-hexane (mass fraction 10:90) and 5 ml n-hexane successively. When the dissolvent reached to the surface of column adsorption layer, the above sample was immediately poured into Florisil PR column and recovered with a 15 ml scale centrifugal tube. The beaker was washed twice with 5 ml mixed solution of acetone and n-hexane (mass fraction 10:90), then the effluent solution went through Florisil PR column again. The scale centrifugal tube containing the whole leacheate was laid in a pressure blowing concentrator at 50°C for evaporating to less than 5 ml and scaled to 5 ml with n-hexane. After that the mixed solution was blended with a whirlpool mixer. The samples were filtrated with filter membrane (0.22 μm) before determination. amino column was eluted in advance and conditioning using 4 ml mixed solution of methyl alcohol and dichloromethane (mass fraction 1:99). The above sample was rapidly added into the amino column as the dissolvent reached to the surface of column adsorption layer. The eluent was gathered with a 15 ml centrifuge tube and 2 ml mixed solution of methyl alcohol and dichloromethane (mass fraction 1:99) was used to wash the beaker twice, then the eluent went through the amino column and combined. The total solution collected was evaporated to dryness in a pressure blowing concentrator at 50°C under a nitrogen stream. Afterwards, the residue was accurately scaled to 2.5 ml with methyl alcohol and mixed with a whirlpool mixer for detecting.

Analytical methods
By the method of NY/T 761-2008 pesticide multi-residue screen methods for determination of organophosphorus pesticides, organochlorine pesticides, pyrethroid pesticides and carbamate pesticedes in vegetables and fruits, the samples were evaluated with Agilent 6890N gas chromatograph (with detectors of FPD, ECD and NPD). The chromatographic columns and SPE were from Agilen and the reagents used in the tests were all analytical reagents completely. Each sample determined twice parallelly, taking the means as the report results.
Organophosphorus pesticides: A gas chromatograph system (Agilent 6890N, Palo Alto, USA) equipped with an autosampler was used to analyse the residue of organophosphorus pesticides. The gas chromatograph separation was performed using a 50% poly phenyl dimethyl siloxane column (DB-17, 30 m × 0.53 mm × 1.0 μm) (A) and a 100% poly dimethyl siloxane column (HP-1, 30 m × 0.53 mm × 1.5 μm) (B) with the injector temperature of 220°C and the detector temperature of 250°C. The temperature of the column was programmed as follows: 150°C (2 min); 8°C/min to 250°C (12 min). Helium (≥ 99.999%) was used as a carrier gas at a flow of 10 ml/min, hydrogen (≥ 99.999%) was used as a burning gas at a flow of 75 ml/min and the air was used as an aided gas at a flow of 100 ml/min. The sample was divided into two parts and injected by a double autosampler at the same time. The standard solution of 1 ml was injected for comparing. The peak area of sample from column A was compared with the peak area of standard solution for quantitative analysis. If the retention time of the sample from the double columns and standard solution differ with in ± 0.05 min, the unknown component could be concluded.
Organochlorine and pyrethroid pesticides:

Quality assurance procedures
A gas chromatograph method using an external standard was developed for quantitative analysis (each sample for twice) and double columns for qualitative analysis. The test results were reliable when the standardized recovery reached to 70%-130%, whereas if the results out of rang, it needed to retest.

Results and Discussion
Maximum residue limits (MRLs) are defined as the highest concentrations of pesticide multi-residues (mg/kg). However, MRLs are not safety limits, and exposure to residues in excess of an MRL does not automatically imply a hazard to health

Conclusions
The monitoring programme for 2009 and 2010 on Dalian market covered 420 samples of 10 different vegetables. The samples were analysed for 27 pesticides of which 7 (carbofuran, methamidophos, aldicarb, omethoate, carbofuran, chlorpyrifos and cypermethrin) were detected in the samples. Pesticides were easier to keep on celery, rape, lettuce and kidney bean than the other vegetables. Comparatively speaking, owing to more