T.C.
TARIM ve KÖYİŞLERİ BAKANLIĞI
Koruma ve Kontrol Genel Müdürlüğü
Communiqué on Preservatives Used at the Surfaces of Citrus Fruits and Qualitative and Quantitative Analysis Methods thereof
Authorization Law: Turkish Food Codex
The Official Gazette: 05.03.2002-24686
Communication No : 2002/19
Objective
Article 1- The objective of this Communiqué; is to determine certain preservatives for the surface treatment of citrus fruits intended for human consumption and qualitative and quantitative analysis methods thereof for residue levels in and on the products.
Scope
Article 2- This Communiqué covers the preservatives for the surface treatment of citrus fruits and qualitative and quantitative analysis methods thereof for residue in and on the products.
Legal basis
Article 3- This Communiqué has been prepared in accordance with the “Turkish Food Codex Regulation” published in Official Gazette dated 16/11/1997, reiterated No. 23172.
Analysis Methods
Article 4- Analysis Methods mentioned in this Communiqué are applied as follows:
a)Sampling methods for control of certain preservatives used at the surfaces of citrus fruits are applied according to Annex-1.
b) The list of preservatives used at the surfaces of citrus fruits is given in Annex-2.
c) Quantitative analysis of biphenyl residuals in citrus fruits are performed according to the method in Annex-3.
d) Quantitative analysis of OPP and SOPP residues in citrus fruits are performed according to the method in Annex-4.
e) Qualitative analysis of biphenyl, orthophenylphenol (OPP) and sodium orthophenylphenate residues at the peels of citrus fruits are performed according to the method in Annex-5.
Registration and Inspection
Article 5- The inspected establishments are subject to legal procedure according to the Decree Law No 560, dated 24/6/1995, on Production, Consumption and Inspection of Foodstuffs, in case the results of the above mentioned analysis methods are found inappropriate.
Inspection
Article 6- The provisions of this Communiqué are inspected by Ministry of Health, and the Ministry of Agriculture and Rural Affairs, according to the provisions of Decree Law No: 560.
Compliance with European Union
Article 7- This Communiqué has been prepared within the framework of compliance with European Union having regard to the Commission Directive No.67/427/EEC .This Communiqué does not embrass the laboratories from using their own legislations and scientifically valid methods.
p>EnforcementArticle 8- This Communiqué enters into force on the date of publication.
Execution
Article 9- The provisions of this Communiqué are executed by the Minister of Agriculture and Rural Affairs and the Minister of Health
ANNEX-1
Sampling Procedure of Citrus Fruits
To Control Preservatives
A. Taking of Samples
The samples should be taken using scientific methods which ensure that the samples are representative of the lot to be analyzed.
I. The samples must satisfy at least the following requirements:
1. Packaged products (crates, paperboard boxes or similar containers)
| Number of package in each lot | up to 20 | 21-500 | 501-1000 | 1000 or more |
| Number of package to be sampled (min) | 1 | 2 | 3 | 4 |
| Quantity of fruit sampled in each pack (kg) | 2 | 2 | 2 | 2 |
2. The products in bulk,
| Bulk (kg) | up to 20 | 21-500 | 500 or more |
| Quantity to be sampled from bulk (kg) | 2 | 4 |
II. Lot is the group of products which have the same characteristics such as variety, degree of ripeness, type of packaging etc.
B. Packaging and delivery of the samples to the required destination:
1. Samples shall be transported in air-tight containers.
2. Containers shall be sealed.
3. Packaged samples shall be delivered as quickly as possible to the laboratories.
ANNEX-2
Preservatives
| Numbering | Naming | Using Conditions |
| a) Except surface treatment of tropical plants, b) During trade of citrus fruits: 1. Residue amounts in whole citrus fruits should not exceed below: |
||
| E 230 231 |
Biphenyl (Diphenyl) Orthophenylphenol |
For biphenyl 70mg/kg, for orthophenylphenol and orthofenylfenate E di sodium; Isoelement or together in kind of 12mg/kg 2) Following procedures should be applied: |
| E 232 | Ortophenylphenate disodiujm | - In wholesale trading the expression of “Canned by .........” should be indicated on the outer face of package and on the bill. - On retail sales, safe information to prevent misunderstanding of the consumer should apparently be indicated. |
ANNEX-3
Qualitative Analysis Of Biphenyl, Orthophenylphenol And Sodium Orthophenylphenate Residues in the Peels of Citrus Fruits
1. Objective
Method of analysis explains how to determine the presence of biphenyl, orthophenylphenol (OPP) and sodium orthophenylphenate (SOPP) residues in the peels of citrus fruits. Accuracy limit of this method is about 5 mg for biphenyl, 1 mg for OPP or SOPP. 5 mg (5 ppm) biphenyl and 1 mg (1 ppm) OPP is equivalent to the amount in the peels of citrus fruits.
When the citrus fruits are treated with these preservatives, residues are present in high amount in the fruit peels. In case these preservatives are determined in the fruit peels, it is necessary to perform quantitative analysis of residues in whole fruit.
2. Principle
Extract is prepared from the peel by using dichloromethane in acidic medium. Extract is concentrated and separated by thin layer chromatography using silica gel. Presence of biphenyl, OPP and SOPP is determined by means of fluorescence and color tests.
3. Reagents
3.1. Cyclohexane (in analytical purity)
3.2. Dichloromethane (in analytical purity)
3.3. HCl solution; 25% (weight/volume)
3.4. Silica gel; GF 254 Merck or equivalent
3.5. 0.5% solution of 2,4,7-trinitroflorenon (TNF) solution; is prepared in acetone (Fluca, B.D.H or equivalent).
3.6. 0.1% solution of 2,6- dibromo-benzocinon-chloroimid solution; prepared in ethanol. If the solution is stored in the refrigerator, it stays stable for about a week.
3.7. Concentrated ammonia solution; concentrated, specific gravity:0.9
3.8. Standard 1%.solution of biphenyl solution,prepared with syclohexane
3.9. Standard 1%solution of orthophenylphenol solution, prepared with syclohexane.
4. Required Apparatus
4.1. Mixer
4.2. Flask, 250 mL, inlet section as trimmed
4.3. Cooled reflux condenser
4.4. Rotary evaporator
4.5. Micropipettes
4.6. Thin layer Chromatographic apparatus with 20X20 cm plates
4.7. UV lamp (254 nm); should have enough intensity to make 5 mg biphenyl spots visible.
4.8. Pulverising equipment
4.9. Oven
5. Method
5.1. Preparation of sample and extraction
All the fruits in the sample are divided half. Half of each fruit is kept for quantitative analysis. About 80 g of peels are taken from the other half of the fruits. After chopping these peels, they are broken up in the mixer and put in a flask of 250 mL. To this 1 mL of 25% HCl and 100 mL of dichloromethane are added. Mixture is heated under reflux cooler for 10 minutes. Following the cooling process and rinsing the reflux cooler with 5mL dichloromethane, the mixture is filtered through a fluted filter. After putting some porous material in the flask of the evaporator filtrate is added to this and fitted on the evaporator. It is evaporated till there remains 10 mL filtrate under low pressure at 60 °C. Rotary evaporator should stay in fixed position in order not to cause any loss by forming biphenilyn film on the walls of the flask.
5.2. Chromatography
30g silica gel and 60 mL water are put in the mixer and mixed for 1 minute. The mixture is divided into 5 plates of chromatography and spread to form a film with a thickness of about 0.250 mm. These covered plates are subjected to stream of hot air for about 15 minutes and kept for 30 minutes in the oven of 110 °C.
Once the plates are cooled down, each plate is divided into parallel strips of 2 cm wide. 50 mL extract to be analyzed is dropped at a minimum distance of 1.5 cm from the edge of the strips, as close to each other. Minimum one strips is reserved for control samples containing 1 mL (10 mg) biphenyl and OPP standard solution residues.
Chromatography plates are developed with Cyclohexane: dichloromethane (25:95) mixture in the process tank which is covered with a filter paper beforehand.
5.3. Detection and Identification
Presence of biphenyl and OPP is determined by appearance of the spots in UV light of 254 nm. SOPP has changed into OPP during the extraction in acid medium, its presence can not be distinguished from OPP. The products are identified as follows:
(a) When TNF solution is sprayed on to the plates, biphenyl is identified as yellow spots under the day light.
(b) After spraying 2,6- dibromo-benzokinon-4-chloroimid on to the plates, when subjected to stream of hot air and then exposure to an ammonia-saturated atmosphere; OPP is identified as blue spots.
ANNEX-4
Quantitative Analysis of Biphenyl Residues in Citrus Fruits
1. Objective
Analysis method explains how to identify biphenyl residues quantitatively in whole fruit. Accuracy of the method is ±10% for the fruits where biphenyl is more than 10 mg (10 ppm) per kg fruit.
2. Principle
After distillation in acid medium and extraction by Cyclohexane, extract is separated by means of thin layer chromatography using silica gel. Chromatogram is developed and biphenyl is rinsed out from the medium and determined spectrophotometrically at 248 nm.
3. Reagents
3.1. Concentrated sulfuric acid
3.2. Silicone- based antifoaming emulsion
3.3. Cyclohexane (in Analytical Purity)
3.4. Hexane (in Analytical Purity)
3.5. Ethanol (in Analytical Purity)
3.6. anhydrous sodium sulphate
3.7. Silica gel GF 254 Merck or equivalent
3.8. standard 1% solution of pure biphenyl, prepared with Cyclohexane. By diluting with Cyclohexane, solutions of 0.6 mg/mL, 1 mg/mL and 1.4 mg/mL are prepared.
4. Required Apparatus
4.1. Mixer (1 liter capacity)
4.2. Distillation flask with modified Clevenger type separator, 2 L (Figure 1)
4.3. Cooled reflux condenser
4.4. Graduated flask, 10 mL
4.5. Micropipettes, 50 mL
4.6. Thin layer chromatographic apparatus with 20X20 cm plates
4.7.Oven
4.8. Centrifuge
4.9. Conical centrifuge tube, 15 mL
4.10. UV Spectrophotometer
5. Method
5.1. Sample preparation and extraction
All the fruit in the sample are divided into two. Half of each fruit is kept for qualitative analysis of biphenyl, OPP or SOPP residues. The other halves are put all together and shreded in a mill or crushed until a homogeneous mixture is obtained. Two samples of minimum 200 g are collected from this homogeneous mixture. Each sample is placed in the mixer together with 100 mL water and mixed at a slow speed for a few seconds. Water is added to fill up at a ratio of ¾ of the mixer capacity and mixed at maximum speed for 5 minutes. The resulting puree obtained is removed into the distillation flask of 2 liter. Mixer is rinsed with water and this water is added to the flask. Total volume of water including the rinsing water of the mixer should be 1 L. A few porous granule together with 1 mL of sulfuric acid and 1 mL of anti foaming emulsion is added to the mixture. Separator and reflux condenser are fitted on to the flask. Distilled water and 7 mL of Cyclohexane are poured into the separator until the level is well past the lower arm of the lateral return tube. This is distilled for about 2 hours. Distillate collected in the separator is collected in a graduated flask of size 10 mL. The separator is rinsed with 1.5 mL Cyclohexane which is then added into the graduated flask and filled up to its volume with Cyclohexane. Finally a little anhydrous sodium sulphate is added and the mixture is shaken.
5.2. Chromatography
30 g silica gel and 60 mL water are placed in the mixer and mixed for 1 minute. The mixture is divided into 5 chromatographic plates and spread as to form a layer approximately 0.250 mm thick. These covered plates are subjected for 15 minutes to stream of hot air and waited for 30 minutes in the oven of 110 °C.
Once the plates are cooled down, each plate is divided into 4 parallel strips of 4,5 cm wide. 50 mL extract to be analyzed is dropped at a minimum distance of 1.5 cm from the edge of the strips, as close to each other. Standard solutions in 3 different concentrations (0.6 mg/mL, 1 mg/mL and 1.4 mg/mL solutions) (corresponding respectively to 30, 50 and 70 mg levels of biphenyl) are dropped on the strips in the same way and amount.
If the analysis is to be performed in series, standard solutions are not necessarily be placed on each plate. Standard curve can be drawn by calculating the mean values obtained from at least 5 plates with the same standard quantities.
5.3. Development of Chromatogram and elution
Chromatograms are developed in the process tank which is previously covered with filter paper in 17 cm depth containing hexane. Plates are air dried. The areas in which the biphenyl is localised are identified under the 254 nm UV light and equal areas are marked with rectangles.
Marked areas on the plate are immediately scraped by a clean spatula. In the mean time, 10 mL ethanol is added to remove biphenyl from the scraped plate and shaked for 10 minutes. Later, the mixture transferred in the centrifuge tubes are centrifuged at 2500 rpm for 5 minutes.
A sample control area of the same size is also subjected to the same process. If the analysis is being performed in series, this control area is taken from the unused strip of the plate.
If the analysis is being performed individually, it is taken from one of the strips containing standard solution located below the area containing the biphenyl.
5.4. Spectrophotometric determination
The supernatant liquid provided after centrifuging is determined against control solution extracted from the area that does not contain biphenyl at 248 nm.
6. Calculation of results
Standard curve is drawn with the absorbents determined against 30, 50 and 70 mg biphenyl values. A linear curve passing through the center should be formed. Amount of biphenyl in the sample is found in the curve in ppm.
ANNEX-5
Quantitative Analysis of Orthophenylphenol (OPP) and Sodium Orthophenylfenate (SOPP) Residues in Citrus Fruits
1. Objective
Method of analysis explains how to determine OPP and SOPP residues in whole fruit quantitatively. The method gives out low results of about 10-20 % in average for about 12 ppm of OPP and SOPP Scope.
2. Principle
Extract is purified right after distillation in acidic medium and extraction with di-isopentyl ether and treated with 4-amino-2,3-dimethyl-1-phenyl-3-pyrazolin-5-one (=4-aminoantipyrine) solution. A red color is formed at a intensitivity measurable at 510 nm in spectrophotometer.
3. Reagents
3.1. Orthophosphoric acid, of 70 %
3.2. Silicone based antifoaming emulsion
3.3. Di-isopentyl ether (in Analytical Purity)
3.4. Purified Cyclohexane: shaked three times with NaOH of 4% and washed three times with distilled water.
3.5. NaOH solution, of 4%
3.6. Buffer solution, pH=10.4 : 6.64 g boric acid, 8.00 g KCl and 93.1 mL 1 N NaOH solution are put in graduated flask of 2 liter capacity. Shaked and bring up to calibration mark with distilled water.
3.7. Reagent I: 1.0 g 4-amino-2,3-dimethyl-1-phenyl-3-pyrazolin-5-one (4-aminotipyrine) is dissolved in 100 mL of distilled water.
3.8. Reagent II: 2 g potasium ferrocyanide is dissolved in 100 mL of distilled water. Reagent I and II are stable in dark bottles for nearly 14 days.
3.9. Silica gel
3.10. Standard solution: 10 mg of pure OPP is dissolved in 1 mL of 0.1 N NaOH. It is diluted to 100 mL with 0.2 mL of Sodium borate (1 mL=100mg).For the standard solution, it is diluted in the ratio of 1:10 with the buffer solution.
4. Required Apparatus
4.1. Shredding or crushing mill
4.2. Mixer
4.3. Distillation flask with modified Clevenger type separator, 1 L (Figure 1)
4.4. Cooled reflux condenser
4.5. Infra-red bath
4.6. Separating funnel, 200 mL
4.7. Graduated cylinders, 25 and 100 mL
4.8. Graduated flasks, 25 and 100 mL
4.9. Pipettes, from 1 mL to 10 mL
4.10. Pipette, 0.5 mL graduated
4.11. Spectrophotometer (with 5 cm cells)
5. Method
All the fruit in the sample are divided into two. Half of each fruit is kept for qualitative analysis of biphenyl, OPP or SOPP residues. The other halves are put all together and shreded in a mill or crushed until a homogeneous mixture is obtained. Two samples of minimum 250 g are taken from this homogeneous mixture.
Each sample is placed in the mixer together with 500 mL of water and mixed until a homogeneous mixture is obtained where the oily cells are no longer perceptible. Depending on the estimated amount of OPP, 150-300g puree is taken and put in distillation flask of 1 L. Water is added to raise the weight of mixture up to 500 g. After adding 10 mL of 70% orthophosphoric acid, a few porous granule and 0.5 mL antifoaming emulsion are also added. It is then fixed to the cooled reflux condenser and separator. 10 mL of di-isopentyl ether is put in the separator and the flask is heated gently in infrared bath , without allowing the puree to boil or foam. After distillation for about 6 hours, contents of separator are poured in separation funnel of 200 mL. Separator and cooler is rinsed with first 60 mL Cyclohexane and later 60 mL of water. Rinsing solutions are added in to the separation funnel. The mixture is shaken vigorously and right after the separation takes place, aqueous phase is discarded.
In order to extract OPP, organic phase is shaken vigorously with 10 mL of 4% NaOH five times, each time for 3 minutes. Alkaline soltions are combined, neutralised to pH 9-10 with orthophosphoric acid in the presence of phenolphtalein and diluted to 100 mL with water. A pinch of silica gel is added in order to clarify the turbid solution. Solution is shaken , filtered through dry, fine-grain filter. Since the colouring is developed with the maximum of accuracy and precision using quantities of OPP of between 10 and 70 mg , the sample of between 0.5 and 10 ml of solution is taken, taking into account the quantities of OPP which might be expected to be found. Sample is placed in a 25 mL graduated flask and to this 0.5 mL reagent I, 10 mL buffer solution and 0.5 mL reagent II are added. It is shaken and filled up to its volume with buffer solution. 5 minutes later intensity of red color at 510 nm is measured in comprasion with a control sample containing no extract. The color does not loose its intensity within 30 minutes. Standard curve is prepared under the same conditions by using OPP standard solution.
6. Observations
It is suggested to make parallel spectrophotometric determination with different volume of neutralised alkaline extracts for each analysis.
For the fruits which are not treated with these preservatives, the reading in comprasion with blank, give values of up to 0.5 ppm for oranges and 0.8 ppm for lemons.