Xingwei YAO,Hui LI,Lili LIU,Feng WU,Yu XIAO,Xiaozheng SHAN,Zhenghua WEN,Guobao NIU,Hanmin JIANG,Deling SUN*

1.Tianjin Kernel Vegetable Research Institute,Tianjin 300384,China;2.Tianjin Institute of Agricultural Quality Standard and Testing Technology,Tianjin 300381,China

Since China’s reform and opening up,vegetable production in China has developed stably.In 2011,vegetable planting area was 19.67 million hm2,and vegetable yield was up to 0.679 billion t in China.China has become the he largest vegetable production and consumption country in the world at present.However,with the constant increase of vegetable planting area and yield in China,farmland cropping index has improved year by year;due to changes in ecological conditions and wide planting of foreign vegetable varieties,plant diseases and insect pests have occurred frequently,and diseases’and pests’resistance to pesticide has increased gradually.Because of the big increase in pesticide usage,pesticide residue has become in creasingly serious,so that China’s ecological and production environment have been damaged greatly,and people’s foot safety and health have been threatened seriously.

Scientists have studied methods of reducing pesticide usage and pesticide residue and making crop production stable.Transgenic technology has been successfully applied in many crops such as cotton[1],corn and veg etable,and lots of new varieties with resistance to diseases and pests have been bred,so the technology had contributed greatly to reduction of pesticide usage and protection of ecologicalenvironment.For instance,Huanget al.[2]have proposed new ideas of using nonchemical methods(rice genetic diversity and establishment of ecological environment with rice and pond organisms)to control diseases and pests;Molinari[3]has sug-gested that plants’resistance to pests can be used to resist damage by parasitic nematode,thereby greatly reducing pesticide usage and residue;Xu Xia et al.[4]have proved that pesticide residue of bagged grapes decreases by 45.00% -84.85% compared with grapes without bags;using gas chromatography,Liu Fangfang et al.[5]have selected cucumber germplasm resources with low pesticide residue from 28 cucumber samples,which lays foundation for breeding of new varieties oflow-residue cucumber;Chen Zhende et al.[6]have demonstrated that there are obvious differences between various varieties of Chinese cabbage in terms of pesticide residue.

Cauliflower,one of main vegetables in China,is good for nutrition and has a delicious flavor;it is beautiful and has anti-cancer efficacy,so it is deeply liked by consumers.The edible organ of cauliflower is ball-flower,and the degree of ball covered by inner leaves of cauliflower can be grouped into unprotected,partially protected and well protected ball according to the agricultural standard NY/T 2430-2013[7].In order to study the difference between cauliflower varieties with ball wellprotected and unprotected in terms of pesticide residue and its causes,Jinpin 70,a new male sterile cauliflower variety with ball well protected,and Xuebao with ball unprotected were sprayed with high-toxic pesticide phorate and efficient biopesticide emamectin benzoate at a regular dose to investigate the impact of different cauliflower varieties on the pesticide residue,which has great significance for reducing current pesticide residue and environmental pollution,assure vegetable and ecological security,and promoting sustainable development of vegetable industry.

Materials and Methods

Pesticides

Emamectinbenzoateproduced by Shandong Dongtai Agrochemical Limited Company(the active ingredient content is 5%,and it is missible oil)is an efficient and low-toxicity pesticide that has been applied in cauliflower production most widely at present;phorate produced by Tianjin Gengyun Pesticide Plant(the active ingredient content is 55%,and it is missible oil)is a highly toxic pesticide that can be used in an experiment only[8-10].

Materials

Jinpin 70,a new male sterile cauliflower variety with ball well protected,was provided by Tianjin Kernel Vegetable Research Institute,and Xuebao with ball unprotected was provided by Japan Sakata Seed Company.

Experimental design

When the diameter of ball of Jinpin 70 was up to 10 cm,50 plants were chosen randomly and were divided into two groups.Phorate and emamectin benzoate were sprayed on the balls evenly,and their dosages are 750 and 300 g/hm2respectively.On the first(2 h after spraying),third,fifth,seventh and tenth day after spraying,five balls were collected from each treatment,and 1/4 of each ball was collected and mixed together to detect pesticide residue.Each treatment had three repetitions,and Xuebao and Jinpin 70 with ball unprotected (all inner leaves were removed before spraying)were compared with Jinpin 70 with ball well protected in terms of pesticide residue.

Extraction and purification

Firstly,10.00 g of the smashed samples was put in a centrifuge tube(50 ml).After 20 ml of acetonitrile and 10 ml of water were added to the centrifuge tube,the centrifuge tube was oscillated in tissue grinding mill for 2 min and for 1 min after a moderate amount of sodium chloride was added to it.After a little rest,the centrifuge tube was centrifuged for 5 min at a speed of 4 000 r/min,and 10 ml of the supernatant was concentrated and then was dissolved in 4 ml of acetonehexane solution(V∶V=50∶50).

PSA solid phase extraction column was sprayed with 6 ml of acetone and 6 ml of acetone-hexane solution(V ∶V=20 ∶80),and then the samples above were transferred to PSA solid phase extraction column;the sample bottles were washed by 5 ml of acetone-hexane solution(V∶V=20∶80)for two times,and they were transferred to PSA solid phase extraction column.Afterwards,the column was cleared by 45 ml of acetone-hexane solution(V ∶V=50 ∶50),and then all eluate was collected and concentrated at 40℃;it was dissolved by acetonitrile and filtered by 0.22 μm filter membrane.

Instrumental analysis

Determination of phorate contentConditions of FPD gas chromatography are shown as follows:Agilent 6890N gas chromatograph was adopted here,and temperature 250℃was at the inject port,while splitless injection was adopted;chromatographic column (DB-1701)is 30 m×0.25 mm × 0.25 μm in size,and its velocity of flow was 1.3 ml/min;furnace temperature was 120℃220℃(3.5 min);detector(P-FPD)temperature was 250℃,and H2(75 ml/min)and air(100 ml/min)were a supplement,while injection volume was 2 μl;carrier gas was N2.

Determination of emamectin benzoate contentConditions of ultrahigh performance liquid chromatography are shown as follows:Waters ultra-high performance liquid chromatograph was adopted here,and chromatographic column was Acquity UPLC BEH C18(2.1 mm×50 mm,1.7 μm);sample size of 10 μl,and column temperature was 30℃;mobile phase was acetonitrile -0.1% methanoic acid solution(V∶V=70∶30),and its velocity of flow was 0.2 ml/min.Conditions of mass spectrometry detection are shown as follows:electrospray ionization(ESI)adopted positive ion mode;capillary voltage was 3.0 kV;temperature of ion source was 110℃;desolvation gas temperature was 380℃;desolvation gas flow was 550 L/h;cone gas flow was 50 L/h;detection mode was multiple reaction monitoring scanning mode (MRM),and mass spectrum acquisition parameters are shown in Table 1.

Results and Analysis

Changes of phorate residue incauliflower

Two hours after cauliflower was sprayed with phorate, phorate residues in Xuebao with ball unprotected,Jinpin 70 with ball unprotected,and Jinpin 70 with ball well protected were 0.173,0.123 and 0 mg/kg respectively,and there were extremely significant differences between various treatments.On the third day after spraying,phorate residues in Xuebao and Jinpin 70 with ball unprotected de-creased to 0.081 and 0.055 mg/kg respectively;phorate residue in Jinpin 70 with ball well protected increased,but it was extremely significantly lower than that of Xuebao and Jinpin 70 with ball unprotected,reducing by 87.65% and 81.82% compared with them.On the fifth day after spraying,phorate residues in Xuebao and Jinpin 70 with ball unprotected continued to reduce,and phorate residue in Jinpin 70 with ball well protected decreased to 0.004 mg/kg,significantly lower than that of Xuebao.On the seventh day after spraying,except phorate residue in Xuebao was detected,there was no phorate residue in Jinpin 70 with ball unprotected,and Jinpin 70 with ball well protected,and there were no significant differences between various treatments.On the tenth day after spraying,there was no phorate detected in all treatments(Table 2).

Table 1 Mass spectrometry detection conditions of emamectin benzoate

Table 2 Variations of phorate residue in cauliflower

As shown in Fig.1,phorate residue in cauliflower tended to decrease gradually in all treatments after spraying;in comparison with Jinpin 70,phorate residue in Xubao was high and reduced to 0 on the 10thday.Within 10 days after spraying,phorate residue in Jinpin 70 with ball well protected was extremely low,not higher than 0.010 mg/kg,showing that inner leaves of cauliflower can prevent the ball from being polluted by phorate.

Variations of emamectin benzoate residue in cauliflower

Two hours after cauliflower was sprayed with emamectin benzoate,emamectin benzoate residues in Xuebao and Jinpin 70 with ball unprotected were 0.154 and 0.111 mg/kg respectively,and the latter was 27.92% lowerthan the former,while emamectin benzoate residue in Jinpin 70 with ball well protected was 0 mg/kg respectively,and there were extremely significant differences between various treatments.On the third day after spraying,emamectin benzoate residues in Xuebao and Jinpin 70 with ball unprotected and well protected increased to 0.391,0.372,and 0.075 mg/kg respectively,and emamectin benzoate residue in Jinpin 70 with ball well protected decreased by 80.82% and 79.84% respectively compared with Xuebao and Jinpin 70 with ball unprotected,while there were significant differences between various treatments.On the fifth dayafter spraying, emamectin benzoate residues in all treatments dropped greatly,and emamectin benzoate residue in Jinpin 70 with ball well protected was the lowest(0.009 mg/kg),reducingby67.86% and57.14% compared with other treatments.On the seventh day after spraying,emamectin benzoate residues in all treatments continued to decrease,and emamectin benzoate residues in Jinpin 70 with ball unprotected and well protected reduced to 0.001 mg/kg,while there were no significant differences between various treatments.On the tenth day after spraying,there was no emamectin benzoate detected in all treatments(Table 3).

Table 3 Changes of emamectin benzoate residue in cauliflower

According to Fig.2,the changing trends of emamectin benzoate residues in Xuebao and Jinpin 70 with ballunprotected were almostthe same,that is,it increased obviously on the 3rdday and then declined.In comparison to Xuebao and Jinpin 70 with ball unprotected,emamectin benzoate residue in Jinpin 70 with ball well protected was low,but its changing trend was similar to that of Xuebao and Jinpin 70 with ball unprotected.

Conclusions and Discussion

Jinpin 70,a variety of cauliflower with the largest cultivated area in China,has extremely long inner leaves to protect its ball well[11].The experiment show that phorate and emamectin benzoate residues in Jinpin 70 with ball well protected were low,that is,they reduced by 76.47% -87.65% and 67.86% -80.82% respectively compared with Xubao from the 3rdto the 5thday after spraying,so Jinpin 70 has obvious advantages of edible safety;they decreased by 9.23% -81.82% and 57.14% -79.84% respectively compared with Jinpin 70 with ball unprotected,indicating that inner leaves of Jinpin 70 can prevent the ball from being polluted by phorate and emamectin benzoate,thereby improving its edible safety.It was found that the changing trends of phorate and emamectin benzoate residues in Xuebao and Jinpin 70 with ball unprotected were almost the same,showing that there was no obvious difference between various varieties of cauliflower in phorate and emamectin benzoate residues.

Phorate,a high-toxic,efficient and broad-spectrum pesticide,can kill pests with piercing-sucking or chewing mouthparts.According to the national standard,phorate residue in vegetables should not be detected[12].In the experiment,there was no phorate residue in Jinpin 70 with ball well protected on the 7thday after spraying,and phorate residue in Jinpin 70 with ball well protected was always lower compared with Xuebao and Jinpin 70 with ball unprotected.The the national standard stipulates that the maximum residue of emamectin benzoate in tomato and cucumber is 0.02 mg/kg[13].In the experiment,emamectin benzoate residue in Jinpin 70 with ball well protected on the 5thday after spraying was 0.009 mg/kg,obviously lower than the national limit,showing that it is very safe to eat Jinpin 70 with ball well protected.In a word,inner leaves of cauliflower with ball well protected can reduce pesticide residue in the cauliflowerand improve its edible safety obviously.

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