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Determination of Chlorpyrifos in Apple from the Resen Region

2017

The aim of this research is to determine the presence of chlorpyrifos in two varieties of apples: Golden Delicious and Idaret on two different locations (Evla and Krveni) from Resen region in the country. Chlorpyrifos is organophosphate pesticide (insecticide) used to protect apples from insects which can cause significant damage in apple production. The apples are analyzed in four development phases and in each phase the presence of chlorpyrifos is determined. Chlorpiryfos analysis of the apples is performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by an extraction/separation cleansing with acetonitrile and the dispersive SPE - QuEChERS - method. The results are compared with the maximum residue levels (MRL) prescribed by the legislation of the Republic of Macedonia. The conclusion is that chlorpyrifos is variously represented in most phases, regardless of the phase and location where apple is grown. At certain phase the concentration exceedes MRL, howe...

Introduction

The apple is the most important deciduous fruit consumed during a year. Its fruits can be frozen or used for various types of products (juice concentrate, apple acid and etc.) [1]. In Republic of Macedonia, the most common fruits are: apples with about 62%, 13% plums, cherries 7%, peaches 7%, table grapes 7% and other types of fruit (pears, apricots, almonds, walnuts, etc.) with about 4% [2]. Domestic consumption of apples is 12 kg per citizen [3]. It is estimated that about 25% of the total production of apples is delivered to domestic market while the remaining 75% is processed or sold on foreign markets [2]. Fruit production occupies about 2, 75% of the arable agricultural land, about 14 000 ha with 9 000 000 trees concentrated in regions at an altitude of 300-800 m (including the Resen region). In Macedonia, the total number of trees apple is 4 082 328, fruitful trees 3 811 548 with total production of 136 931 t or 36 kg per tree. In Resen, the number of fruit trees is 2 458 300, out of which fruitful trees are 2 256 800 while apple production is 90 450 tons of apples or 40 kg of apples per tree [3]. This shows us that apple is mostly cultivated in Resen. It is said that apple is a measure for the development of fruit growing in every country, since increasing the number of apple trees it increases the intensity of fruit growing production, globally and conversely [4]. In Resen are grown the following varieties of apples: Idaret, Golden Delicious, Mutcu, Red Delicious, Jonagold, Granny Smith, Fuji, Gala and Rubistar [5]. Idaret is a variety of apple grown in the Moscow, Idaho, by Lejf Verner. Diploid and secondary lush variety. Blooms early. The fruit is coarse to very coarse, with a round shape. The epidermis is firm, elastic and smooth. The main color is green to red. The inner part of the fruit is white, mixed with chippings of green color, firmly, crispy, with good structure, juicy and sweet. value is less than the p -critical value. If the obtained p-value is greater than or equal to the limit p-value, it says that the samples don't differ significantly. In the first case, rejecting the null hypothesis, while the second is accepted [21,22]. We made Student t-test for hypothesis: does the presence of chlorpyrifos in both varieties of apples taken from the Kriveni and Evla are significantly different? The aim of our research is to analyze the pesticide in two varieties of apples from the region which has the most apples, at two different locations.

Matherials and methods

For the analysis were taken two varieties of apples: Golden Delicious and Idaret. Samples were collected from two different locations (Evla and Kriveni) in four developmental phases of apples: I -phase, apple at the size of hazelnuts; II -phase apple at the size of a walnut; III -phase, early ripening of apple; IV -phase during the apple harvest. They were analyzed fresh apples with peel and mesocapr. Date of sampling for analysis is as follows 31. 05. 2016, 09. 07. 2016, 21. 08. 2016, 25. 09. 2016 for each phase. LC-MS/MS has become the most frequently used analytical method for quantification of polar pesticides in food [13][14][15][16][17]. Chlorpyrifos analyze was performed with modern methods LC-MS/MS following acetonitrile extraction/partitioning and clean-up by dispersive SPE -QuEChERS-method with MKS EN 15662 : 2011 method. This method is accredited in a flexible range of the Institute for Accreditation of the Republic of Macedonia (IRAM) for MKS EN ISO/IEC 17025 : 2006 under number LT -036 [18]. The QuEChERS method uses acetonitrile, with the application of adequate combination of salts, dinatrijum hidrogen citrata seskvihidrata, water-free magnesium sulfate and sodium chloride with the purification procedure by primary-secondary amine (PSA) with the addition of water-free magnesion sulfate which results in a better separation of phases without dilution [19,20]. The homogenized sample of apple with weight of 10 g is transferred at 50 ml centrifuge tube. Add 10 ml of acetonitrile and e.g. 100 μl of the internal standard solution. Shake vigorously for 1 min. This is the first extraction step. After extraction on a vortex mixer for 1 minute, 4 g of magnesium sulfate anhydrous (MgSO4), 1 g of sodium chloride (NaCl), 1 g of trisodium citrate dehydrate and 0.5 g of disodium hydrogencitrate sesquihydrate were added and the mixture was shaken for 1 min and centrifuged for 5 minutes at 3000 rpm. This is the second extraction with phase separation. After centrifugation, 1 ml of the extracts was transferred into a clean-up tube containing 900 mg of MgSO4 and 150 mg of primary-secondary amine (PSA). Shake for 30 sec. Centrifuge for 5 minutes at 4500 rpm. The cleaned and acidified extracts are transferred into autosampler vials to be used for the multiresidue determination. Analyses were carried out with a liquid chromatograph (LC) (Agilent 6420) with triple quadrupole mass spectrometry.

Results and discussion

The results are presented with graphs and diagrams. In order to see if the insecticide chlorpyrifos in analyzed fruit is within the limits, the obtained values are compared with the Maximum Residue Levels (MRL) according the legislation of the Republic of Macedonia [9]. In this phase, its concentration is 0.52 mg / kg and it is slightly above the allowed values. In the other phases chlorpyrifos is not present or is below the limit of detection. At the location Kriveni the chlorpyrifos is more present than Evla and it is present in all three phases at a concentration that is acceptable according to the legislation of the Republic Macedonia. It is seen that during harvest time, apples does not contain residues of chlorpyrifos.

The presence of chlorpyrifos in Golden Delicious in two different locations is presented in Figure 2. In Golden Delicious of Evla the chlorpyrifos is present in all four phases with the highest concentration of 0.19 mg / kg in the first phase and the lowest concentration of 0.03 mg / kg in the fourth phase, which is approximately 17 times less than the maximum allowed. At the location Kriveni it is present in the first two phases. The concentration of chlorpyrifos is reduced from 0.28 mg / kg in the first phase and finds with 0.05 mg / kg in the second phase which is 10 times smaller than the maximum residue levels.

Figure 2

Fig. 2. The presence of chlorpyrifos in Golden Delicious from Evla and Kriveni and compared with the MRL

In the third and fourth phase the chlorpyrifos is not present in the analyzed samples.

We made a comparison of the presence of chlorpyrifos in both varieties of apples at both locations (Figure 3 and Figure 4).

Figure 3

Fig. 3. The presence of chlorpyrifos in Idaret andGolden Delicious from location Evla

Figure 4

From figure 3 it may be noted that in the location Evla this pesticide is present in Golden Delicious in all phases and in Idaret only in the first phase. The obtained statistical values are presented in Table 1. In Table 1 is presented the mean of value (x) on the concentration of chlorpyrifos shown in mg / kg at the two varieties of apples from both locations. The value of t-test for Idaret at both locations is 0.534169 and it is lower than the critical t-value 3.182446. The value of t-test for Golden Delicious from both locations is 0.385922 and is less than the critical value of t-test. That means that null hypotheses can be accepted and that there is a significant difference in the presence of chlorpyrifos in apples from both locations. In both varieties of apples p-values are greater than the critical p-value. That means that null hypothesis can be accepted but the difference between the samples is not statistically significant.

Table 1

Conclusion

Analyzed are the two varieties of apples: Golden Delicious and Idaret from two different locations Kriveni and Evla in Resen region. Chlorpyrifos is detected by LC -MS / MS and QuEChERS extraction is applied with standard method MKS EN 15662: 2011. Analyses show that at location Evla chlorpyrifos is represented in Idaret only in the first phase while in other phases is not. In Golden Delicious at location Evla the chlorpyrifos is represented in all four phases but with approximately 17 times lower concentration than the maximum allowed in the fourth phase. At the location Kriveni chlorpyrifos in Golden Delicious is present in both phases whit allowed concentration, while in Idaret is present in all three phases. Although chlorpyrifos is available in two varieties of apples they are safe for consumption. Statistical analysis of data about apple from Kriveni and Evla showed that the use of chlorpyrifos depends on the variety of apple but not of the location where it is grown. These are the data obtained for analyzed apples. The investigation will be continue and further and other varieties of apples as well as in other locations.

Fig. 3 .Fig. 4 .

Vezirka JANKULOSKA, Ilija KAROV, Gorica PAVLOVSKA, Ilija BUZLEVSKI, Determination of chlorpyrifos in apple from Resen region, Food and Environment Safety, Volume XVI, Issue 1 -2017, pag. 34 -39









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