1. INTRODUCTION
⌅Most of the edible oils on the market shelves are made ready for consumption by refining. With the help of the refining process, sensory characteristics such as taste, smell, appearance, and shelf stability are provided (Pudel et al., 2011Pudel F, Benecke P, Fehling P, Freudenstein A, Matthäus B, Schwaf A. 2011. On the necessity of edible oil refining and possible sources of 3-MCPD and glycidyl esters. Eur. J. Lipid Sci. Technol. 113, 368-373.). Due to the application of high temperatures, process contaminants may be formed as a result of some reactions (hydrolysis, oxidation) (Shahidi, 2005Shahidi F. 2005. Bailey’s Industrial Oil and Fat Products, Edible Oil and Fat Products: Processing Technologies. https://www.google.com/books?hl=tr&lr=&id=4AbyDwAAQBAJ&oi=fnd&pg ). 3-monochloropropane-1,2-diol (3-MCPD) and glycidyl esters (GE) are formed during several processes including frying, cooking, distillation, and refining (Zhou et al., 2014Zhou H, Jin Q, Wang X, Xu X. 2014. Effects of temperature and water content on the formation of 3-chloropropane-1, 2-diol fatty acid esters in palm oil under conditions simulating deep fat frying. Eur. Food Res. Technol. 238, 495-501.). 3-MCPD and GE could be formed during the refining of crude oils, especially in the deodorization stage where high temperatures are applied (230-250 °C) (Özdikicierler et al., 2016Özdikicierler O, Yemişçioğlu F, Saygın Gümüşkesen A. 2016. Effects of process parameters on 3-MCPD and glycidyl ester formation during steam distillation of olive oil and olive pomace oil. Eur. Food Res. Technol. 242, 805-813. https://doi.org/10.1007/s00217-015-2587-7 ). These researchers stated that the formation of contaminants accelerated with the increase in monoglyceride, diglyceride, and chloride ion concentrations in the environment. Cyclic acyloxonium ions and glycidyl esters are formed when mono and diglycerides are formed as a result of triglyceride hydrolysis when edible fats and oils are exposed to high temperatures. These compounds are converted into 3-MCPD with the presence of chloride ions (Hamlet et al., 2011Hamlet CG, Asuncion L, Velısek J, Dolezal M, Zelinkova Z. 2011. Formation and occurrence of esters of 3-chloropropane- 1 , 2-diol ( 3-CPD ) in foods : What we know and what we assume. Eur. J. Lipid Sci. Technol. 113 (3) 279-303. https://doi.org/10.1002/ejlt.201000480 ). In the presence of water and at high temperatures, triglyceride is hydrolyzed to diglyceride and monoglyceride. In glycerol and glycerides, 3-MCPD ester formation increases with increasing salt concentration and reaches its maximum level with approximately 15% water content (EFSA, 2016EFSA. 2016. Risks for human health related to the presence of 3-and 2-monochloropropanediol (MCPD), and their fatty acid esters, and glycidyl fatty acid esters in food (Vol. 14), Wiley Online Library.). 3-MCPD are formed as a result of the replacement of the acyl or hydroxyl groups in the acylglycerol molecule with the chloride ion (Svejkovska et al., 2006Svejkovska B, Dolezal M, Velisek J. 2006. Formation and decomposition of 3-chloropropane-1, 2-diol esters in models simulating processed foods. Czech J. Food Sci. 24 (4), 172.). In the absence of water in the environment, pre-hydrolysis of the acyl groups cannot occur, but the hydroxyl group of the glycerol molecule and the chlorine are directly replaced (EFSA, 2016EFSA. 2016. Risks for human health related to the presence of 3-and 2-monochloropropanediol (MCPD), and their fatty acid esters, and glycidyl fatty acid esters in food (Vol. 14), Wiley Online Library.). It has been stated that precursors such as monoglycerides, diglycerides, and chloride ions which cause 3-MCPD formation, as well as parameters such as processing temperature and duration, play a significant role in 3-MCPD formation (Sevindirici et al., 2018Sevindirici G, Özdikicierler O, Yemişçioğlu F. 2018. 3-Mcpd and Ge Risk in Refined Vegetable Oils: Structure, Formation Mechanism, Legal Regulations and Mitigation Techniques. Gida / J. Food 43, 886-895. https://doi.org/10.15237/gida.GD18053 ). In addition, it was observed by Shimizu et al. (2012)Shimizu M, Vosmann K, Matthäus B. 2012. Generation of 3-monochloro-1, 2-propanediol and related materials from tri-, di-, and monoolein at deodorization temperature. Eur. J. Lipid Sci. Technol. 114 (11), 1268-1273. that glycidol was formed from di- and monoacyl glycerols in the absence of chlorine ions at temperatures of 240 °C and above. However, Destaillats et al. (2012)Destaillats F, Craft BD, Sandoz L, Nagy K. 2012. Formation mechanisms of monochloropropanediol (MCPD) fatty acid diesters in refined palm (Elaeis guineensis) oil and related fractions. Food Addit. Contam. Part A Chem. Anal. Control Exp. Risk Assess, 29 (1), 29-37. https://doi.org/10.1080/19440049.2011.633493 determined that the majority of MCPD diesters are formed above 200 °C by the reaction of organochlorines with triacylglycerols. Additional experiments by these researchers confirmed that this reaction could be initiated during palm oil deodorization with hydrogen chloride (HCl) gas released through the thermal degradation of the organochlorines present in the oil. Since toxicological studies have shown that the free form of 3-MCPD is carcinogenic (Cho et al., 2008Cho W-S, Han BS, Nam KT, Park K, Choi M, Kim SH, Jeong J, Jang DD. 2008. Carcinogenicity study of 3-monochloropropane-1, 2-diol in Sprague-Dawley rats. Food Chem. Toxicol. 46, 3172-3177.), 3-MCPD has possible health risks. Similar to 3-MCPD, glycidol is also highly risky for health because of its epoxy ring structure (IARC, 2013IARC, 2013. Working Group on the Evaluation of Carcinogenic Risks to Humans. Some chemicals present in industrial and consumer products, food and drinking-water. IARC monographs on the evaluation of carcinogenic risks to humans, 101, 9.). The International Agency for Research on Cancer has classified 3-MCPD as a “potential carcinogen to humans” in group 2B. Glycidyl, on the other hand, was stated to have mutagenic and carcinogenic properties and classified in group 2A, which means that it was “probably carcinogenic to humans” (IARC 2013IARC, 2013. Working Group on the Evaluation of Carcinogenic Risks to Humans. Some chemicals present in industrial and consumer products, food and drinking-water. IARC monographs on the evaluation of carcinogenic risks to humans, 101, 9.). According to the report published in 2018, the tolerable daily intake of 3-MCPD was recommended as 2 µg·kg-1 body weight (EFSA 2018EFSA. 2018. Update of the risk assessment on 3-monochloropropane diol and its fatty acid esters (Vol. 16). https://doi.org/10.2903/j.efsa.2018.5083 ).
Regulation EU 2020/1322, amending Regulation EC No. 1881/2006, applies as of January 1st 2021 with regard to foods in the EU. This specifies maximum quantities for free 3-MCPD in hydrolyzed plant protein and soya sauce as well as maximum quantities for glycidyl fatty acid esters, expressed as glycidol, in vegetable oils and fats, fish oils and other marine oils which are placed on the market for consumers or for use as an ingredient in foods (maximum quantity 1 mg·kg-1) (EU, 2020EU. 2020. COMMISSION REGULATION (EU) 2020/1322 of 23 September 2020 amending Regulation (EC) No 1881/2006 as regards maximum levels of 3‐monochloropropanediol (3-MCPD), 3-MCPD fatty acid esters and glycidyl fatty acid esters in certain foods.). In addition, EU regulation set the maximum level for GE at 1 mg·kg-1 in vegetable oils and fats which are aimed for the final consumer or as an ingredient in food. In addition, the level of GE for vegetable oils and fats destined for baby food and processed cereal-based food is set at 0.5 mg·kg-1 (EU, 2020EU. 2020. COMMISSION REGULATION (EU) 2020/1322 of 23 September 2020 amending Regulation (EC) No 1881/2006 as regards maximum levels of 3‐monochloropropanediol (3-MCPD), 3-MCPD fatty acid esters and glycidyl fatty acid esters in certain foods.). With the same regulation, maximum quantities for the sum of free and fatty acid bound 3-MCPD (analyte group) are set for the same foods for which maximum levels for GE are established. Due to the low 3-MCPD formation potentials, a stricter maximum level (1.25 mg·kg-1) for the sum of 3-MCPD and 3-MCPD fatty acid esters, expressed as 3-MCPD is set for oils and fats from coconut, maize, rapeseed, sunflower, soybean, palm kernel and olive oils and mixtures of oils and fats with oils and fats only from this category. A higher maximum level of 2.5 mg·kg-1 applies to other vegetable oils (including olive pomace oils), fish oils and oils from other marine organisms and mixtures of oils and fats with oils and fats only from this category. For oil mixtures from both categories with quantitatively known ingredients, the category-related maximum levels apply to single ingredients. In the case of oil and fat mixtures of unknown composition, the higher maximum level of 2.5 mg·kg-1 applies. For vegetable oils and fats, fish oils and other marine oils which are destined for the production of baby food and processed cereal-based food for infants and young children the maximum level for the sum of 3-MCPD and 3-MCPD fatty acid esters, expressed as 3-MCPD is set to 0.75 mg·kg-1. Finally, for infant formula, follow-on formula and foods for special medicinal purposes for infants and young children as powders the maximum level is addressed as 0.125 mg·kg-1 for powders and as 15 µg·kg-1 for liquids. These limits set by the EU are valid for European countries.
Although there are many studies in the literature on the detection of 3-MCPD and glycidol in different food products, few studies have been conducted in Türkiye on the determination of 3-MCPD and glycidol in potato chips (Önal et al., 2016Önal B, Özdikicierler O, Yemişçioğlu F. 2016. Türkiye piyasasında satışa sunulan patates cipslerinde 3-MCPD esterleri ve glisidil esterleri miktarları. Akad. Gıda 17, 267-274.), in steam distillation of olive oils (Özdikicierler et al., 2016Özdikicierler O, Yemişçioğlu F, Saygın Gümüşkesen A. 2016. Effects of process parameters on 3-MCPD and glycidyl ester formation during steam distillation of olive oil and olive pomace oil. Eur. Food Res. Technol. 242, 805-813. https://doi.org/10.1007/s00217-015-2587-7 ), in baking in biscuit making (Mogol 2014Mogol BA. 2014. Mitigation of Thermal Process Contaminants by Alternative Technologies. Institute of Sciences of Hacettepe University.), or in frying stages (Deniz Şirinyıldız et al., 2019Deniz Şirinyıldız D, Aydın E, Öztürk Y, Avcı T, Yıldırım A, Yorulmaz A. 2019. Türk piyasasindan toplanan bitkisel yağlar ve margarinlerde 3-MCPD yağ asidi esterlerinin düzeyi. Gida / J. Food 44, 491-497. https://doi.org/10.15237/gida.GD19039 ). These processes applied to the oils mentioned above may differ from region to region. In this respect, we think that it is important to examine the edible fats and oils offered for sale in the country’s markets in terms of these process contaminants. Considering the results of this study, the daily intake limits determined by the relevant authorities for consumers may not be exceeded. In addition, manufacturers can make efforts to limit these compounds. Within the scope of this study, sunflower oil (7 brands), hazelnut oil (4 brands), corn oil (6 brands), natural extra virgin olive oil (7 brands), riviera olive oil (7 brands), margarine and shortening (9 brands), peanut oil (2 brands), and fish capsules (4 brands) which are sold and consumed widely in the markets of Türkiye were investigated. In total, 3-MCPD and glycidol levels were detected in 46 samples.
2. MATERIALS AND METHODS
⌅2.1. Materials
⌅The different cooking oils, fish oils, margarines and shortenings used in the study were obtained from local markets in Türkiye in 2019. Their names and number of brands are shown in Table 1. All of these oils, except for virgin olive oil and fish oils, are refined oils. Virgin olive oil is obtained by cold pressing., that is, it is not refined. Riviera oil consists of 20% virgin olive oil and 80% refined olive oil. 3-MCPD and 3-chloro-1,2-propane-1,1,2,3,3-d5-diol (3-MCPD-d5), glycidyl stearate, diethyl ether, methanol, sodium hydroxide, sodium bromide, ethyl acetate, phenylboronic acid (PBA), acetone and toluene were obtained from Sigma-Aldrich (Steinheim, Germany). The purity of the chemicals was ensured.
| Sample names | Number of brands |
|---|---|
| Sunflower oil (Refined) | 7 |
| Hazelnut oil (Refined) | 4 |
| Peanut oil (Refined) | 2 |
| Corn oil (Refined) | 6 |
| Virgin Olive Oil (Cold-pressed) | 7 |
| Riviera olive oil (Blend from refined and cold pressed) | 7 |
| Pastry oil (Shortening) (Refined) | 3 |
| Margarine (Refined) | 6 |
| Fish oil (capsule) (Native-Refined) | 4 |
| Total number of samples | 46 |
2.2. Methods
⌅2.2.1. Preparation of samples
⌅The lipid fractions of margarine and fats were obtained by centrifugation of the molten product, then filtered through anhydrous sodium sulfate by removing the upper oil phase. In margarines and shortenings, analyses were performed only for the lipid phase. Encapsulated fish oils were removed from the capsules with a sterile needle. Bottled edible oils were taken with a straw directly from the bottle in which they were kept in the market. All oil samples were stored at 4 °C in a dark environment until the experiments.
2.2.2. Analysis of 3-MCPD and glycidol
⌅3-MCPD and glycidol levels were determined based on the standard method of DGF C VI 18 (10) (DGF, 2011DGF, 2011. German Society for Fat Science Standard Method C-VI 18 (10) Fatty-Acid-Bound 3-Chloropropane-1, 2-Diol (3-MCPD) and 2, 3-Epoxipropane-1-ol (Glycidol): Determination in Oils and Fats by GC/MS (Differential Measurement), German Standard Methods for the Investigation of Fats, Fat Products, Surfactants and Related Substances, Frankfurt, Germany) with some modifications. In part A of this method, 3-MCPD, corresponding to the sum of bound 3-MCPD and bound glycidol was determined, as in the DGF C VI 17 (10) method. We modified the DGF C VI 18 (10) method by applying the DGF C VI 17 (10) method in part A. We modified the DGF C VI 18 (10) method part B so that only the amount of ester-bound 3-MCPD was determined. We did not intend to determine 2-MCPD in this part B. Therefore, the relevant standard was not used. Both methods which form the basis of our method, namely C-VI 17 (10) and C-VI 18 (10), have been developed and approved only for the analysis of edible oils and fats. The flow chart of processing steps is shown in Figure 1. According to this method, isotope-labeled 3-MCPD-d5 was used as an internal standard. All oil samples were derivatized after esterification and neutralization processes and injected into the GC/MS (Shimadzu GC-2010 Gas Chromatography-QP-2010 Ultra Mass Spectrometry System, Shimadzu Corporation, Kyoto, Japan) device. The operating conditions of the GC/MS device are provided in Table 2.
| Device: | Shimadzu GC-2010 |
| Detector: | EI+, SIM Mode |
| Internal standard Mass: | m/z = 201 or 150 (3-MCPD- d5) m/z = 196 or 147 (3-MCPD) |
| Column: | TRB - 5MS, 30m × 0.25mm × 0.25µm |
| Gases: | Helium 2.1 mL/min. |
| Temperature: | Oven Program: 60 °C (1 min), 6 °C·min-1 to 190 °C 190 °C - 280 °C to 20 °C /min (10-30 min) |
| Split: | 20 mL·min-1. |
| Splitless time: | 1.5 min. |
Solutions used in esterification, neutralization, and derivatization in analysis. A sodium methylate solution with methanol (NaOCH3): 0.5 molar methanol solution of sodium methylate (27 grams of sodium methylate tart) was dissolved in 1 liter of methanol.
Solvent mixture A: 8/2 (v/v) mixture of tertbutyl methyl ether and ethyl acetate
Sodium chloride (NaCl) solution: 20% (m/v) aqueous solution of sodium chloride
Sodium bromide (NaBr) solution: 60% (m/v) aqueous solution of sodium bromide
Solvent mixture B: A mixture of 30 mL of sodium chloride solution and 1 mL of acetic acid (v/v) (prepared daily)
Solvent mixture C: A mixture of 30 mL of sodium bromide solution and 1 mL of acetic acid (v/v) (prepared daily)
Derivatization solution: 2.5 grams of phenylboronic acid dissolved in a mixture of 19 mL of acetone and 1 mL (m/v) distilled water
Internal standard stock solution: 2000 mg·L-1 (m/v) of 3-MCPD-d5 dissolved in ethanol
Internal standard solution: Prepared from the internal standard stock solution by dissolving in tertbutyl methyl ether (20 mg·L-1).
The first step in the determination of 3-MCPD and glycidol based on the indirect DGF C-VI 18 (10) method was to evaluate the efficiency of the conversion from glycidol to 3-MCPD following the method used for Assay A. Figure 2 shows the amount of 3-MCPD formed as a function of the amount of glycidol (in the form of glycidyl stearate) in a spiked blank oil (olive oil) at seven different levels (0.05; 0.1; 0.25; 0.5; 1; 2.5 and 5 mg·kg-1). A linear regression of the type y = mx + b was performed, the reciprocal slope (1/m) provided the conversion factor (t) (Lucas et al., 2017Lucas D, Hoffmann A, Gil C. 2017. Fully Automated Determination of 3-MCPD and Glycidol in Edible Oils by GC / MS Based on the Commonly Used Methods ISO 18363-1, AOCS Cd 29c-13, and DGF C-VI 18 (10). GERSTEL Application Note, 18 (191), 1-6.).
Two-stage analysis was performed. In the first step (A), the total amount of 3-MCPD and glycidol in the sample, expressed as 3-MCPD, was calculated according to Eq.1. Here, 3-MCPD and glycidol were not separated. Therefore, the solvent was prepared with sodium bromide instead of sodium chloride in the second step (B) of the analysis in order to prevent the conversion of glycidol to 3-MCPD (Karl et al., 2016Karl H, Merkle S, Kuhlmann J, Fritsche J. 2016. Development of analytical methods for the determination of free and ester bound 2-, 3-MCPD, and esterified glycidol in fishery products. Eur. J. Lipid Sci. Technol. 118, 406-417. https://doi.org/10.1002/ejlt.201400573 ). In the second step, pure or bound 3-MCPD was determined according to the Eq.2. The glycidol levels were calculated by subtracting the result from the second stage from the result from the first stage and multiplying the conversion rate of glycidol to 3-MCPD (Eq.3).
A3-MCPD (1)= Mass fraction of 3-MCPD (mg·kg-1) detected in the first step
Q (147)= Peak area of 3-MCPD determined in the first step
Q (150)= Peak area of 3-MCPD - d5 determined in the first step
Cd5 - 3 - MCPD (1)= Concentration of internal standard (mg·kg-1) used in the first step
B3-MCPD (2)= Mass fraction of 3-MCPD (mg·kg-1) detected in the second step
Q (147)= Peak area of 3-MCPD determined in the second step
Q (150)= Peak area of 3-MCPD-d5 determined in the second step
Cd5 - 3 - MCPD (2)= Concentration of internal standard (mg·kg-1) used in the second step
WGlycidol= Mass fraction of glycidol in samples (mg·kg-1)
t= In the equation in the created calibration graph (Figure 2), the ratio of 1/m (y=1.2051x+0.0024, R2=0.9984).
A3-MCPD (1)= Mass fraction calculated in the first step
B3-MCPD (2)= Mass fraction calculated in the second step
2.3. Statistical analysis
⌅All data were statistically analyzed using SPSS (version 20.0 for Windows, SPSS Inc., Chicago, Illinois) package program by conducting one-way analysis of variance (ANOVA), and defining a significant difference at P < 0.05 by Duncan’s test. All measurements were performed with triplicate fresh samples, and values were expressed as means ± SD of triplicates from each independent experiment.
3. RESULTS AND DISCUSSION
⌅3.1. The levels of 3-MCPD and glycidol in sunflower oils
⌅The levels of 3-MCPD and glycidol determined in the sunflower oil samples are presented in Figure 3A. The amounts of 3-MCPD in the sunflower oil samples belonging to 7 different brands were determined in the range of 0.02-0.44 mg·kg-1. The highest 3-MCPD were determined in SO6, the lowest 3-MCPD were obtained from the SO2 sample. On the other hand, while the lowest glycidol was found in the SO5 sample, the highest glycidol was detected in the SO4 sample. It is noteworthy that the amount of glycidol was higher compared to 3-MCPD (Figure 3A). The levels of 3-MCPD in the sunflower oil samples were found to be SO6 > SO7 > SO3 > SO1 > SO4 > SO5 >SO2. On the other hand, the levels of glycidol were found to be SO4 > SO1 > SO7 > SO6 > SO2 > SO3 < SO5. Kuhlmann (2011)Kuhlmann J. 2011. Determination of bound 2,3-epoxy-1-propanol (glycidol) and bound monochloropropanediol (MCPD) in refined oils. Eur. J. Lipid Sci. Technol. 113, 335-344. https://doi.org/10.1002/ejlt.201000313 found 3-MCPD values in sunflower oils in the range of 0.1-2.1 mg·kg-1 and glycidol values in the range of 0.1-0.4 mg·kg-1. The same author found 3-MCPD levels in the range of 0.08-0.96 mg·kg-1 and glycidol levels in the range of 0.02-0.90 mg·kg-1 in sunflower oil in another study (Kuhlmann 2016Kuhlmann J. 2016. Analysis and occurrence of dichloropropanol fatty acid esters and related process-induced contaminants in edible oils and fats. Eur. J. Lipid Sci. Technol. 118, 382-395. https://doi.org/10.1002/ejlt.201400518 ). Zelinkova et al. (2006)Zelinkova Z, Svejkovska B, Velisek J, Dolezal M. 2006. Fatty acid esters of 3-chloropropane-1,2-diol in edible oils. Food Addit. Contam. 23, 1290-1298. https://doi.org/10.1080/02652030600887628 determined 3-MCPD values of less than 0.1 and 0.3 mg·kg-1 in two types of sunflower oil. Weißhaar and Perz (2010)Weißhaar R, Perz R. 2010. Fatty acid esters of glycidol in refined fats and oils. Eur. J. Lipid Sci. Technol. 112, 158-165. found 3-MCPD values for sunflower oils of less than 1.0 mg·kg-1 and glycidol values of less than 0.4 mg·kg-1. The results of the current study partially overlap with these literature findings. The majority of our glycidol values were higher than those. The possible reason for this variation in glycidol levels may be the difference in parameters such as temperature and time applied in refining. The glycidol in SO1 and SO4 samples were determined as 1.02 and 2.42 mg·kg-1, respectively. They were determined in the range of 0.06-0.83 mg·kg-1 in other samples. Similarly, Kalkan et al. (2021)Kalkan O, Topkafa M, Kara H. 2021. Determination of effect of some parameters on formation of 2-monochloropropanediol, 3-monochloropropanediol and glycidyl esters in the frying process with sunflower oil, by using central composite design. J. Food Compos. Anal. 96, 103681. https://doi.org/10.1016/j.jfca.2020.103681 determined glycidol in the range of 0.06-0.72 mg·kg-1 in sunflower oil during the frying of french fries in their research.
3.2. The levels of 3-MCPD and glycidol in margarines and shortenings
⌅The levels of 3-MCPD and glycidol determined in different kinds of margarine and shortenings are presented in Figure 3B. The 3-MCPD concentration was found in the range of 0.14-1.17 mg·kg-1, and the glycidol concentration in the range of 0.41-3.83 mg·kg-1 for 6 different kinds of margarine. The results obtained for margarine and shortening are valid only for the oil phase. The concentrations of 3-MCPD and glycidol in three shortenings were in the range of 0.12-0.68 mg·kg-1 and 1.98-6.46 mg·kg-1, respectively. It was observed that the glycidol level was higher than 3-MCPD in all kinds of margarine except for M1 and M3 samples (Figure 3B). The glycidol level was found significantly higher in M6 samples compared to the other samples (p < 0.05). No significant changes were observed among the M4, M5, and M6 samples in terms of 3-MCPD values. Similarly, the 3-MCPD values in M2 and M3 samples were not statistically different. In terms of glycidol, there was no significant difference between M1 and M3, or M4 and M5. By looking at the shortenings, the amounts of glycidol were found significantly higher than the 3-MCPD (p < 0.05). The amount of glycidol in S2 shortening samples was approximately 2.5-3 times higher than the other samples. Custodio-Mendoza et al. (2019)Custodio-Mendoza JA, Carro AM, Lage-Yusty MA, Herrero A, Valente IM, Rodrigues JA, Lorenzo RA. 2019. Occurrence and exposure of 3-monochloropropanediol diesters in edible oils and oil-based foodstuffs from the Spanish market. Food Chem. 270, 214-222. https://doi.org/10.1016/j.foodchem.2018.07.100 determined that the total bound 3-MCPD concentrations in the lipid fractions of margarines were in the range of 0.11-2.61 mg·kg-1. The 3-MCPD values were found in the range of 0.79-1.60 mg·kg-1 in five samples (Li et al., 2015Li C, Nie SP, Zhou Y qiang, Xie MY. 2015. Exposure assessment of 3-monochloropropane-1, 2-diol esters from edible oils and fats in China. Food Chem. Toxicol. 75, 8-13. https://doi.org/10.1016/j.fct.2014.10.003 ), 0.4-4.5 mg·kg-1 in 37 samples (Weißhaar 2011Weißhaar R. 2011. Fatty acid esters of 3-MCPD: Overview of occurrence and exposure estimates. Eur. J. Lipid Sci. Technol. 113, 304-308. https://doi.org/10.1002/ejlt.201000312 ), and 0.09-0.43 mg·kg-1 in four kinds of margarine (fat portion) and 0.50 mg·kg-1 in a vegetable shortening (Becalski et al., 2015Becalski A, Feng S, Lau BPY, Zhao T. 2015. A pilot survey of 2- and 3-monochloropropanediol and glycidol fatty acid esters in foods on the Canadian market 2011-2013. J. Food Compos. Anal. 37, 58-66. https://doi.org/10.1016/j.jfca.2014.09.002 ). In a report published in the Netherlands (Boon and te Biesebeek 2016Boon PE, te Biesebeek JD. 2016. Preliminary assessment of dietary exposure to 3-MCPD in the Netherlands. RIVM Letter report 2015-0199 pp 1-43. https://www.rivm.nl/bibliotheek/rapporten/2015-0199.pdf ), 3-MCPD concentrations were reported as 0.16-1.8 mg·kg-1 in seven margarines and shortenings. These findings are in agreement with the current results. The glycidol concentrations (0.15-5.5 mg·kg-1) determined by Weißhaar (2011)Weißhaar R. 2011. Fatty acid esters of 3-MCPD: Overview of occurrence and exposure estimates. Eur. J. Lipid Sci. Technol. 113, 304-308. https://doi.org/10.1002/ejlt.201000312 in 22 margarines (fat portion) were similar to our findings. Deniz Şirinyıldız et al. (2019)Deniz Şirinyıldız D, Aydın E, Öztürk Y, Avcı T, Yıldırım A, Yorulmaz A. 2019. Türk piyasasindan toplanan bitkisel yağlar ve margarinlerde 3-MCPD yağ asidi esterlerinin düzeyi. Gida / J. Food 44, 491-497. https://doi.org/10.15237/gida.GD19039 found 3-MCPD concentrations in the range of 0.57-4.54 mg·kg-1 in 14 margarines obtained from the market in Türkiye. The possible reason why these values were higher in margarines compared to many other oils may be salt and water contents. As known, palm oil is generally used to produce margarine. Palm oil is a fruit oil and contains more water than seed oils. Therefore, it is more sensitive to hydrolysis reactions. The high levels of monoglyceride and diglyceride formed as a result of these reactions cause the formation of 3-MCPD and glycidol (Shahidi and Zhong, 2005Shahidi F, Zhong Y. 2005. Lipid oxidation: measurement methods. Bailey’s industrial oil and fat products. Bailey’s industrial oil and fat products, Ed.: F Shahidi, John Wiley & Sons Inc. (Eds.), Hoboken, NJ, 357-385.). On the other hand, the presence of organochlorine compounds in palm oil has been implicated as a potential source of chlorine for the formation of 3-MCPD (Nagy et al., 2011Nagy K, Sandoz L, Craft BD, Destaillats F. 2011. Mass-defect filtering of isotope signatures to reveal the source of chlorinated palm oil contaminants. Food Addit. Contam: Part A, 28 (11), 1492-1500.). While the palm tree is growing, it absorbs chloride ions from the soil and water, which paves the way for the formation of 3-MCPD (Anonymous, 2018Anonymous, 2018. Proposed draft code of practice for the reduction of 3-monochloropropane-1,2-diol esters (3-MCPDE) and glycidyl esters (GE) in refined oils and products made with refined oils, especially infant formula. Joint FAO/WHO Food Standards Programme, 12-16 March. Codex Alimentarius Commission Committee on Food Additives and Contaminants.). In addition, the formation of 3-MCPD in palm oil is directly related to the oil’s exposure to high temperatures during refining (Franke et al., 2009Franke K, Strijowski U, Fleck G, Pudel F. 2009. Influence of chemical refining process and oil type on bound 3-chloro-1,2-propanediol contents in palm oil and rapeseed oil. LWT-Food Sci. Technol, 42 (10), 1751-1754. https://doi.org/10.1016/j.lwt.2009.05.021 ; Weißhaar, 2008Weißhaar R. 2008. 3-MCPD-esters in edible fats and oils-a new and worldwide problem. Eur. J. Lipid Sci. Technol. 110 (8), 671-672.).
3.3. The levels of 3-MCPD and glycidol in corn oils
⌅The levels of 3-MCPD and glycidol determined in corn oils are presented in Figure 3C. The 3-MCPD and glycidol values were determined to be in the range of 0.05-0.37 mg·kg-1, and 0.06-0.95 mg·kg-1, respectively in six corn oils. The glycidol levels were found higher than 3-MCPD in corn oil samples except for CO1 samples. While the mean value of 3-MCPD in corn oils was 0.22 mg·kg-1, the mean glycidol value was 0.56 mg·kg-1. The difference between the values of the samples was found statistically significant (p < 0.05). Among the samples, the CO1 sample appears to be more stable in terms of these process contaminants. Kuhlmann (2011)Kuhlmann J. 2011. Determination of bound 2,3-epoxy-1-propanol (glycidol) and bound monochloropropanediol (MCPD) in refined oils. Eur. J. Lipid Sci. Technol. 113, 335-344. https://doi.org/10.1002/ejlt.201000313 found that the 3-MCPD value in corn oil was 0.2 mg·kg-1 and the glycidol value was 0.7 mg·kg-1. The result of the current study was similar to these findings. In another study, the amounts of 3-MCPD and glycidol levels in corn oils were found to be < 1.7 and < 0.6 mg·kg-1, respectively (Weißhaar 2011Weißhaar R. 2011. Fatty acid esters of 3-MCPD: Overview of occurrence and exposure estimates. Eur. J. Lipid Sci. Technol. 113, 304-308. https://doi.org/10.1002/ejlt.201000312 ). Zelinkova et al. (2006)Zelinkova Z, Svejkovska B, Velisek J, Dolezal M. 2006. Fatty acid esters of 3-chloropropane-1,2-diol in edible oils. Food Addit. Contam. 23, 1290-1298. https://doi.org/10.1080/02652030600887628 reported 3-MCPD values of less than 0.3 and 0.372 mg·kg-1 in crude and refined corn oils, respectively. These results are also in good agreement with our findings. Deniz Şirinyıldız et al. (2019)Deniz Şirinyıldız D, Aydın E, Öztürk Y, Avcı T, Yıldırım A, Yorulmaz A. 2019. Türk piyasasindan toplanan bitkisel yağlar ve margarinlerde 3-MCPD yağ asidi esterlerinin düzeyi. Gida / J. Food 44, 491-497. https://doi.org/10.15237/gida.GD19039 found that the 3-MCPD concentration was 0.51-2.49 mg·kg-1 in five corn oil samples. The values determined in the current study were lower than those findings.
3.4. The levels of 3-MCPD and glycidol in hazelnut and peanut oils
⌅The amount of 3-MCPD and glycidol levels in hazelnut and peanut oils are exhibited in Figure 4A. While the 3-MCPD concentration was found as 0.06 mg·kg-1 in HO4 samples, it was found as 1.13-2.12 mg·kg-1 in the other three hazelnut oils (HO1, HO2, and HO3). Deniz Şirinyıldız et al. (2019)Deniz Şirinyıldız D, Aydın E, Öztürk Y, Avcı T, Yıldırım A, Yorulmaz A. 2019. Türk piyasasindan toplanan bitkisel yağlar ve margarinlerde 3-MCPD yağ asidi esterlerinin düzeyi. Gida / J. Food 44, 491-497. https://doi.org/10.15237/gida.GD19039 found that the 3-MCPD concentration in three hazelnut oils was in the range of 0.24-0.45 mg·kg-1. Our results (except HO4) were higher than these values. The possible reason for this variation may be the difference in parameters such as temperature and time applied in refining. The amounts of glycidol in hazelnut oil samples were determined to be in the range of 0.54-2.63 mg·kg-1. While there was no significant difference between the 3-MCPD values of HO2 and HO3 samples, the difference between 3-MCPD amounts in these samples and others was found statistically significant (p < 0.05). The same trend was followed for the glycidol amounts in the samples. The order of 3-MCPD and glycidol levels determined in hazelnut oil samples were found to be HO2 > HO3 > HO1 > HO4. Kuhlmann (2011)Kuhlmann J. 2011. Determination of bound 2,3-epoxy-1-propanol (glycidol) and bound monochloropropanediol (MCPD) in refined oils. Eur. J. Lipid Sci. Technol. 113, 335-344. https://doi.org/10.1002/ejlt.201000313 found the 3-MCPD value at 19 mg·kg-1 and the glycidol value at 0.5 mg·kg-1 for hazelnut oil. Zelinkova et al. (2006)Zelinkova Z, Svejkovska B, Velisek J, Dolezal M. 2006. Fatty acid esters of 3-chloropropane-1,2-diol in edible oils. Food Addit. Contam. 23, 1290-1298. https://doi.org/10.1080/02652030600887628 reported that the amount of 3-MCPD in unrefined crude hazelnut oil was less than 0.1 mg·kg-1. According to the results obtained in the current study, it was important to indicate that these compounds were formed in higher concentrations in hazelnut oils compared to the other oil samples analyzed in the study. It is well known that fruit oils such as hazelnut oil are more prone to hydrolysis because of their high water content compared to seeds. It has been determined that the risk of 3-MCPD formation during the refining of these oils is quite high because of the formation of mono and diglycerides as a result of the hydrolysis reaction and the chlorine ion in the structure of the fruits which pass into the oil (Xu et al., 2016Xu G, Liu D, Zhao G, Chen S, Wang J, Ye X. 2016. Effect of Eleven Antioxidants in Inhibiting Thermal Oxidation of Cholesterol. J. Am. Oil Chem. Soc. 93, 215-225.).
In two peanut oils, 3-MCPD concentrations were found to be 0.90 and 0.11 mg·kg-1, and glycidol concentrations were found at 1.47 and 0.45 mg·kg-1 (Figure 4A). Li et al. (2016)Li C, Li L, Jia H, Wang Y, Shen M, Nie S, Xie M. 2016. Formation and reduction of 3-monochloropropane-1,2-diol esters in peanut oil during physical refining. Food Chem. 199, 605-611. https://doi.org/10.1016/j.foodchem.2015.12.015 found that the 3-MCPD contents in 3 different peanut oils after deodorization to be 0.43-0.62 mg·kg-1. Kuhlmann (2011)Kuhlmann J. 2011. Determination of bound 2,3-epoxy-1-propanol (glycidol) and bound monochloropropanediol (MCPD) in refined oils. Eur. J. Lipid Sci. Technol. 113, 335-344. https://doi.org/10.1002/ejlt.201000313 determined that the 3-MCPD content was 0.1-0.9 mg·kg-1 and glycidol content was 0.4-1.1 mg·kg-1 in peanut oils. Similar results were found for the 3-MCPD level by Zelinkova et al. (2006)Zelinkova Z, Svejkovska B, Velisek J, Dolezal M. 2006. Fatty acid esters of 3-chloropropane-1,2-diol in edible oils. Food Addit. Contam. 23, 1290-1298. https://doi.org/10.1080/02652030600887628 at < 0.1 mg·kg-1 and Li et al. (2015)Li C, Nie SP, Zhou Y qiang, Xie MY. 2015. Exposure assessment of 3-monochloropropane-1, 2-diol esters from edible oils and fats in China. Food Chem. Toxicol. 75, 8-13. https://doi.org/10.1016/j.fct.2014.10.003 at 0.45-1.18 mg·kg-1. The results of the studies given above are similar to our observations. The amounts of 3-MCPD and glycidol levels determined in these peanut oils we used in our study showed a significant difference (p < 0.05). The amount of 3-MCPD in PO1 oil was 8 times higher than that of PO2, and the amount of glycidol was approximately 3 times higher.
3.5. The levels of 3-MCPD and glycidol in extra virgin olive oils
⌅The 3-MCPD and glycidol levels were not detected in 7 different extra virgin olive oils used in the study. It was expected that extra virgin olive oil does not contain any traces of 3-MCPD or glycidol since it is mechanically extracted without any heat treatments. The 3-MCPD in edible oils were studied by Jedrkiewicz et al. (2016)Jedrkiewicz R, Kupska M, Glowacz A, Gromadzka J, Namiesnik J. 2016. 3-MCPD: A Worldwide Problem of Food Chemistry. Crit. Rev. Food Sci. Nutr. 56, 2268-2277. https://doi.org/10.1080/10408398.2013.829414 and according to the obtained results from that study, MCPD were not detected in cold-pressed or unrefined edible oils. Similarly, the 3-MCPD and glycidol in extra virgin olive oils were not determined in the study by Custodio-Mendoza et al. (2019)Custodio-Mendoza JA, Carro AM, Lage-Yusty MA, Herrero A, Valente IM, Rodrigues JA, Lorenzo RA. 2019. Occurrence and exposure of 3-monochloropropanediol diesters in edible oils and oil-based foodstuffs from the Spanish market. Food Chem. 270, 214-222. https://doi.org/10.1016/j.foodchem.2018.07.100 . On the other hand, Zelinkova et al. (2006)Zelinkova Z, Svejkovska B, Velisek J, Dolezal M. 2006. Fatty acid esters of 3-chloropropane-1,2-diol in edible oils. Food Addit. Contam. 23, 1290-1298. https://doi.org/10.1080/02652030600887628 determined the amount of 3-MCPD to be less than 0.1 mg·kg-1 in virgin olive oils and in the range of 0.3-2.4 mg·kg-1 in refined olive oils.
3.6. The levels of 3-MCPD and glycidol in riviera olive oils
⌅According to the International Olive Council classification, riviera olive oil is an oil consisting of a mixture of refined olive oil and extra virgin olive oil, and is suitable for consumption. It has a free acidity, expressed as oleic acid, of not more than 1.00 gram per 100 grams and its other physico-chemical and organoleptic characteristics correspond to those fixed for this category in this standard (IOOC, 2021IOOC, 2021. Trade standard applying to olive oils and olive pomace oils. COI/T.15/NC No 3/Rev. 17. International Olive Oil Council, 15 (3), 1-17.). Riviera olive oil is the oil whose properties are improved by mixing natural olive oil in different proportions ranging from 5 to 20% with refined olive oil (Türkoğlu et al., 2012Türkoğlu H, Kanık Z, Yakut A, Güneri A, Akın M. 2012. Some Properties of Olive Oils Sold in Nizip and Surroundings. J.Agric. Fac. HR.U. 16, 1-8.). The amounts of 3-MCPD and glycidol levels determined in riviera olive oil samples are shown in Figure 4B. The 3-MCPD values varied between 0.16-1.69 mg·kg-1 (p < 0.05). The highest level was determined in ROO3 oil, the lowest level was determined for ROO7 oil. The amounts of glycidol were found in the range of 0.19-3.53 mg·kg-1 (p < 0.05). It is noteworthy that the variation in glycidol concentration was higher than the variation in 3-MCPD concentration. Kuhlmann (2011)Kuhlmann J. 2011. Determination of bound 2,3-epoxy-1-propanol (glycidol) and bound monochloropropanediol (MCPD) in refined oils. Eur. J. Lipid Sci. Technol. 113, 335-344. https://doi.org/10.1002/ejlt.201000313 found 3-MCPD amounts in the range of 0.3-1.2 mg·kg-1 and glycidol amounts in the range of 0.1-0.4 mg·kg-1 in riviera olive oils. While the amounts of 3-MCPD in this study were similar to our findings, the amounts of glycidol only partially matched our results. Weißhaar and Perz (2010)Weißhaar R, Perz R. 2010. Fatty acid esters of glycidol in refined fats and oils. Eur. J. Lipid Sci. Technol. 112, 158-165. determined that the average 3-MCPD value was 1.2 mg·kg-1 and glycidol was 0.3 mg·kg-1 in six riviera olive oil samples.
3.7. The levels of 3-MCPD and glycidol in fish oils
⌅The 3-MCPD and glycidol contents detected in four different brands of fish oil are shown in Figure 4C. The 3-MCPD and glycidol values were not determined in the FO3 sample. The highest amount of 3-MCPD was obtained for the FO4 sample at 0.11 mg·kg-1. While the glycidol could not be detected in the FO3 sample, the highest amount was found in the FO4 sample at 0.69 mg·kg-1. Kuhlmann (2011)Kuhlmann J. 2011. Determination of bound 2,3-epoxy-1-propanol (glycidol) and bound monochloropropanediol (MCPD) in refined oils. Eur. J. Lipid Sci. Technol. 113, 335-344. https://doi.org/10.1002/ejlt.201000313 reported that the amounts of 3-MCPD and glycidol levels in different fish oils were less than 0.05 and 0.025 mg·kg-1, respectively. Jedrkiewicz et al. (2016)Jedrkiewicz R, Kupska M, Glowacz A, Gromadzka J, Namiesnik J. 2016. 3-MCPD: A Worldwide Problem of Food Chemistry. Crit. Rev. Food Sci. Nutr. 56, 2268-2277. https://doi.org/10.1080/10408398.2013.829414 determined that the 3-MCPD content in refined fish oils was in the range of 1.5-5.5 mg·kg-1. The results of the present study were lower than these findings. The reason for this might be that the refining processes changed according to the fish type.
4. CONCLUSIONS
⌅This study surveyed the contamination levels of 3-MCPD and glycidol in edible oils collected in 2019 in Türkiye. The results for different oils and fats showed that 3-MCPD and glycidol levels did not occur in crude or natural oils and fats. 3-MCPD and glycidol were commonly detected in different amounts in refined oils and fats. The highest 3-MCPD amounts were found in some hazelnut and riviera olive oils. Glycidol was found above the limit values in most margarine, shortening, riviera and hazelnut oils. It is known that fruit oils such as hazelnut oil, olive oil and palm oil are more prone to hydrolysis because of their highwater content compared to seeds. It is thought that 3-MCPD and glycidol formation may be higher during the refining of these oils because of the hydrolysis reaction of triglycerides to form mono and diglycerides and the chlorine ion in the structure of the fruits, which pass into the oil. It was observed that the amounts of 3-MCPD did not exceed a certain level while the glycidol presented significantly higher maximum amounts. As expected, 3-MCPD and glycidol levels were not found in extra virgin olive oils as they were not subjected to refining processes. While these compounds were not detected in one brand in fish oils, 3-MCPD and glycidol were determined at 0.12 mg·kg-1 and less than 0.69 mg·kg-1, respectively in other fish oil samples. 3-MCPD and glycidol limit values in Türkiye are generally compatible with European regulations. When evaluated in this respect, the majority of margarine, shortening, hazelnut and riviera oils are above the glycidol limit value. 3-MCPD was above the limit in most of the hazelnut oils. The formation of 3-MCPD and glycidol can be limited by reducing their precursors such as monoglycerides and diglycerides in hazelnut, olive and palm oils before high temperature processing, as well as by reducing the palm oil content in margarine and shortening. A dietary risk assessment for 3-MCPD and glycidol was not performed in this study. In future studies, in the light of these data, the risk assessments of edible oils and fat-containing foods should be made.