DETECTION OF MICROPLASTIC RESIDUES-DEVELOPING A METHOD FOR PHTHALATES IN HONEY

In this pilot study, a method for the determination of phthalates in honey was developed. Th e following phthalates are included: dimethyl phthalate, diethyl phthalate, di-isobutyl phthalate, di-n-butyl phthalate, bis(2-ethylhexyl) phthalate, and di-(n-octyl) phthalate. For the preparation of the samples, the method of liquid-liquid extraction with hexane with an ultrasonic bath was used. Th e analysis of the prepared samples was performed using gas chromatography and a mass detector. Th e method is reliable, sensitive, and reproducible with a detection limit of 0.28 1.38 μg/kg. Th is paper presents the results of testing samples of honey stored in glass and plastic packaging for three years in order to determine the migration of phthalates. Dimethyl phthalate was not found in the tested samples stored in plastic and glass packaging. Diethyl phthalate was not found in samples stored in glass packaging while the concentration of diethyl phthalate in samples from plastic packaging was 3.34 μg/kg. Th e concentrations of diisobutyl phthalate, di-n-butyl phthalate and bis(2-ethylhexyl) phthalate, determined in samples from glass packaging were 5.32, 1.32 and 4.45 μg/ kg, and in honey samples from plastic packaging 15.84, 16.01 and 14.44 μg/kg. Concentrations of di-(n-octyl) phthalate were less than the LOQ in both types of samples.


INTRODUCTION
Nowadays, plastic is increasingly used for food packaging due to the low cost of materials, its potential for thermal sealing, optical properties, and it is also suitable for making diff erent shapes and sizes. Due to these properties, plastic products for food packaging and beverages have surpassed the use of materials such as glass or tinplate. Plastic packaging has many advantages -it is light, resistant, and easy to shape, i.e. it can be formed in diff erent shapes and sizes and thus adapted to diff erent types of food, from solids to liquids. Plastic packaging provides good protection against damage. However, from the chemical point of view, when it comes to biodegradability, its harmful eff ects are the subject of a great number of research papers today. EU Regulation 1935 dating from 2004 is based on the fact that all materials or objects that come into direct or indirect contact with food must be inert enough to prevent the transfer of things to food in the quantities that are large enough to endanger human health or cause unacceptable changes in food composition or deterioration of its organoleptic properties. Plastic can be decomposed into compounds harmful to human health. Th ese are divided into organic and inorganic compounds. Th e fi rst group includes amines, phenols, and phthalates. In order to obtain soft er and more fl exible plastic products, so-called soft eners are added during their production, and phthalates are the subject of this pilot study.
Diesters of 1,2-benzenedicarboxylic acid, better known as phthalates, are a group of man-made chemicals widely used in industry (Meeker et al, 2009). Th ey are present primarily in plastic products, toys, medical instruments, industrial materials, food, and clothing. Th ese compounds have the ability to disrupt the function of the endocrine system. Th e eff ects depend on the dose, duration of action, and developmental stage of the organism. Th e fetus, newborn, and children in puberty are the most vulnerable categories. Exposure to phthalates begins in the intrauterine period, as they freely pass through the placental barrier. It is believed that the side eff ects of phthalates can be manifested through neurocognitive disorders, allergies, asthma, testicular cancer, liver and kidney damage, insulin resistance and obesity, thyroid dysfunction, and respiratory system irritation. Phthalates in females can lead to anovulation, premature puberty, and changes in the duration of pregnancy (Butala et al, 2004;Yen et al, 2011;Bajkin et al, 2014). Bis(2-ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP), benzyl butyl phthalate (BBP) and di-isobutyl phthalate (DIBP) ('the four phthalates') are listed in Annex XIV to Regulation (EC) No 1907/2006 as substances that are toxic for reproduction, category 1B. Th erefore, phthalate toxicity poses a signifi cant risk to human health.
According to the "Rulebook on the Restrictions and Ban of Production, Placing on the Market and Use of Chemicals ("Offi cial Gazette of the Republic of Serbia RS", 90/2013, 25/2015, 2/2016, 44/2017, 36/2018 and 9/2020) DBP, DEHP, BBP are prohibited for use in toys and objects intended for child care in concentrations higher than 0.1% of plasticized materials, while di-"isononyl" phthalate (DINP), di-"iso decyl" phthalate (DIDP), di-n-octyl phthalate (DNOP) are prohibited for use in toys and items intended for the care of children that children can put in their mouths in concentrations higher than 0.1% of plasticized materials. Given the harmful eff ects of phthalates on hu-man health and the fact that they are increasingly found in plastic food packaging, there is a justifi ed need to place clearly defi ned restrictions on these compounds in food packaging, and food as well. Th e honey whose production process does not enable contamination with phthalates, except in the case of packaging and storage in plastic packaging, is the sample whose analysis could prove the migration of phthalates in the product. Also, certain types of honey crystallize during storage-some faster and some slower. Although crystallization is a natural property of honey, a large number of consumers do not like crystallized honey, so in order to decrystallize it, they heat it, which increases the migration of phthalates. In order to prove this, it is necessary to analyse the phthalate content in honey samples. Th e method of liquid-liquid extraction with hexane and the analysis of the stored samples using gas chromatographymass spectrometry (GC-MS) were chosen. Th e goal of this study is to develop and validate a method for the determination of phthalates in honey.

Honey samples
For the purpose of determining the presence of phthalates in honey, ten samples of diff erent origin were collected. Honey samples were randomly collected from two sources: honey samples that were brought to the Scientifi c Veterinary Institute "Novi Sad" (NIV-NS) in plastic jars and honey samples that were collected from NIV-NS's bee yard in glass jars. All the samples were in their original packaging and were transferred to the laboratory, properly labeled and stored in a dark place at room temperature for 3 years.

Reagents and materials
Standards of phthalate acid esters (PAE)" were investigated in this study, namely dimethyl phthalate (DMP; C10H10O4), diethyl phthalate (DEP; C12H14O4), diisobutyl phthalate (DiBP; C16H22O4), dibutyl phthalate (DBP; C16H22O4), bis(2-ethylhexyl) phthalate (DEHP; C24H38O4), di-n-octylphthalate (DnOP); C24H34O4)) they were purchased from dr. Ehrenstorfer GmbH (Germany). In Table 1, analytical data include Abbreviation, Retention time and Qualitative and Quantitative ion monitoring. N-Hexane was HPLC grade (Carlo Erba, Milan, Italy). Th e solutions of each phthalate were prepared at concentrations of 1 mg/mL. Phthalates solutions at diff erent concentrations (0.005, 0.01, 0.1, 0.05, 0.5 μg/mL) were prepared by dilution in n-hexane. Th e solutions were stored in vials at −20 °C. In order to avoid cross-contamination due to reagents, materials, and laboratory equipment, a thorough cleaning procedure was performed: the glassware was soaked and washed in acetone, dried at 140 °C for at least 4 h. All the solvents used in the analysis were tested in order to check the potential presence of PAE contamination using GC-MS analysis. Ultrapure water was produced by a Milli-Q system (Millipore, Bedford, USA).

Sample pretreatment
Th e amount of 5 g of honey and 10 mL of ultra-pure water was put into a 100 mL screw-cap glass centrifuge tube with a conical bottom and vigorously vortexed to for at least 1 min in order to form a homogeneous solution. Aft er that, the solution was mixed with 10 mL of hexane, and submitted to extraction by shaking in a mechanical shaker for 40 min. Th en, the organic phase was separated by centrifugation at 3000 rpm for 10 min and collected. Th e sample was once again extracted with 10 mL of hexane and the abovedescribed procedure was repeated. Th e two portions of supernatant were collected and transferred to a clear conical fl ask and evaporated to dryness at 40 °C with a rotary evaporator. Th e residue was dissolved in 1.0 mL of hexane and the fi nal solution was used for GC−MS analysis (Zhou et al, 2014). Th e method precision was evaluated, as described by Zhou et al. (2014). Th e retention times of the peaks and target ions, obtained from the standard solution of phthalates served as a base point for the phthalate's determination in samples.

GC-MS Analysis and Instrumentation
Th e identifi cation of phthalates was based on a comparison of retention times of the peaks and target ions with those obtained from a standard mixture of phthalates (standards supplied by instrument manufacturer). Th e quantification was based on external calibration curves prepared from the standard solution of each of the examined phthalates.
Th e GC operating conditions are shown in Table 2. Th e verifi cation of the peaks was carried out based on the retention times and target ions were compared to those of external phthalates. A solvent blank was also analyzed, phthalates were detected at the concentrations lower than LOQ.

Method Validation
Method validation and quality control were conducted following the European Commission SANTE /11813/2017 Regulation (European Commission, 2017). Th e method was validated in terms of the optimal linearity (r 2 > 0.99). Precision was evaluated by repeatability in triplicate (50.0 μg/kg, n = 10) and it ranged from 0.79 -5.72%. Recovery ranged from 88.51% to 112.23%. Th e obtained results are shown in Table 3.

Concentration of phthalates in honey
Th e pilot study results are presented in Table 4.

DISCUSSION
Various analytical methods, liquid and gas chromatographic techniques with diff erent detectors have been used to determine phthalates over the years. In this study, performance development methods included verifi cation of linearity, limit of detection (LOD) and limit of quantifi cation (LOQ), precision, recovery using a Guardiennes for pesticides residues (SANTE/2019). Th e calibration curves were obtained by measuring standard solutions injected in fi ve level of concentration (0.005; 0.01; 0.05; 0.1; 0.5 μg/mL). Figure 1. shows chromatograms of solvent blank (water and hexane) and standard solution, which means that the applied parameters of the method will enable phthalates separation. Th e linearity of the method was determined by calibration in fi ve calibration levels and good linearity characterized by a coeffi cient of linearity was obtained for all phthalates of interest > 0.999 (r 2 > 0.999). LOQ and LOD were determined by injecting fi ve consecutive samples of the fi rst calibration point (0.005 ug / mL) with an acceptable accuracy of ± 20%. Th e accuracy was assessed on the basis of the data obtained by injecting standard solutions in fi ve replicates in two calibration levels. It ranged between 0.78 -5.72. Recovery was determined by spiking a sample of honey into two concentration levels and two mean values obtained are shown as a result. Recovery values range between 88.51 -112.23% which is in line with the Sante guidelines. Th e result of the recovery, which corresponds to the recommendation for residual determinations, also shows us that the applied phthalate extraction method is satisfactory. During the development of the method, no calibration method was used through the matrix, but a blank sample was taken that underwent the same preparation procedure as the honey sample and contained water and hexane. Th e obtained results on the presence of phthalates in the blank are shown in Table 4. Namely, the values of phthalates in the blank are quantifi ed, but below the LOQ values with a low level of confi dence and therefore are not shown. However, it is important to point out that the solvent blank must work due to the wide distribution of phthalates. Notardonato et al (2020) got linearity > 0.999, but recovery was in the range from 69.3 to 98.8%. Zhou et al (2014) also got good linearity and recovery was between 82.9 -110.9%. while the start temperature was not exactly the same, but it was similar (between 90 and 100 °C). However, the response for each compound was linear with r 2 greater than 0.999 for all phthalates compounds and for all authors.
In this study we analyzed and found phthalates in honey. Some other authors also reported phthalates contamination of honey (Notardonato et al, 2020;Zhou et al, 2014;Lo Turco et al, 2016). Th e concentration that we registered was much lower than that found by other authors who analyzed more samples (Notardonato et al., 2020;Lo Turco et al, 2016).
DMP and DnOP were not detected in these ten honey samples, while other analyzed phthalates were present. Th e maximum contents of PAEs were within 16 As some plastic can migrate from food through diff erent materials, the EU Commission has defi ned the presence and the levels of small amounts of additives in food. Particularly, according to the EU Regulation No. 10/2011 and 2005/2018, the safety limit defi ned by each specifi c migration limits (SMLs) in food, for DMP, DEP, DiBP, and DnOP is 60 mg/kg, and 0.3, 0.05, and 1.5 mg/ kg for DBP, BP-A, and DEHP, respectively. Th e limit of 60 mg/kg requires consideration: this high value means that the additive is permitted to be used in the polymer production for food packaging and there are no restrictions. Th e LOQs that we had were lower than the SML set by the EU Commission: this means that the method investigated is sensitive enough to analyze the threshold limits of the diff erent compounds in the collected honey samples.

CONCLUSION
In this study, a fast and reliable method for the determination of six phthalates from honey was developed. Using liquid-liquid extraction, satisfactory recovery values were obtained and the analysis was performed on GC-MS. Th is paper can be considered as a pilot study for the determination of phthalates in honey and honey products. During the analysis, phthalates were found even in blank solvent, in concentrations lower than LOQ, and their presence even in the blank indicates the need to expand the study. During the test, phthalates were found in all tested honey samples. Given the fi ndings, in the future, we will focus on potential sources of phthalate contamination in the process of production of honey and honey products and will continue more extensive testing of these contaminants not only in honey but also in other foods.

Author's Contribution
B.K. draft ing the manuscript and have made substantial contributions to basic idea; B.K. and J.P. carried out the GC-MS analysis and have been involved in draft ing the manuscript; J.V. carried out sample collection and sample preparation and performed the statistical analysis; J.V. and R.R. have been involved in draft ing the manuscript; B.Đ. have been involved in data collection; J.V. and R.R. revised the manuscript critically.