Introduction
In recent years, plastic packaging materials have become increasingly prevalent in human daily life due to industrialization and urbanization. Among these materials, over thirty different types of plastic are utilized by the Food industry, including polyethylene terephthalate (PET) and high-density polyethylene (HDPE) which are commonly used in food and drinking packaging. polyethylene terephthalate (PET) in particular has gained popularity due to its stability, low cost, transparent and portability making it a popular choice for producing plastic bottles for water and beverages. However, there is growing concern over the potential release of harmful chemicals from plastic materials into drinking water, beverages under certain conditions [1-4]. Moreover, the possibility of phthalate esters being present in bottled beverages increases due to various factors, such as storage durations, temperature change, sunlight exposure; and pH. Secondly, the migration process of phthalates can increase due to ageing and the breakdown of plastic packaging. Photolysis can cause abiotic degradation of phthalates in plastic water bottles and beverages [5-8].
Plastic products often contained various additives that make them flexible, durable and transparent. phthalate esters, also known as plasticizers are one such additives [9-11]. These common plasticizers are di-esters of 1,2-benzenedicarboxylic acid (phthalic acid), and are widely used in plastic industry to improve flexibility, durability and softness of plastic materials [12-16]. However, due to their nature, phthalates can easily migrate from plastic materials into drinking water, or beverages during production and storage [17-19]. The migration of PAEs’ depends on their concentrations in the packaging polymer, the acidity of content in contact with packages, and the lipid content [20-23]. Dimethyl phthalate (DMP), Diethyl phthalate (DEP), Dibutyl phthalate (DBP), Benzyl butyl phthalate (BBP), Di-(2-ethylhexyl) phthalate (DEHP), Di-n-octyl phthalate (DnOP), Di-iso-nonyl phthalate (DiNP), and Di-iso-decyl phthalate (DiDP) are most commonly used in industry as plasticizers [24, 25].
Phthalate esters are considered as ubiquitous environmental pollutants due to extensive usage. They are known to have adverse effects on human health and considered as endocrine disruptor. The United State Environmental Protection Agency (USEPA) recognizes phthalates as endocrine disruptor and has classified as priority pollutants due to their potential risks to human health and environment. In response to these risk, the use of phthalates (DBP, DEHP, DMP, DEP and DnOP) has been banned in various consumers products by USA, EU, Australia and responsible agencies in Canada [26- 30]. Phthalates have been known to caused functional impairment in development and reproduction in both animals and human, as revealed by epidemiological studies. In particular, dibutyl phthalate (DBP), di-2 (ethyl hexyl) phthalate (DEHP), butyl benzyl phthalate (BBP) and certain phthalate metabolites have been found Teratogenic in animals. In rodents, phthalates have been identified as anti-androgenic and as a cause of reduced testosterone level, as well as prolonged estrous cycles and lower estradiol levels [31]. Phthalates are a type of endocrine disruptor that can have various adverse effects on human health. They have been linked to breast cancer and prostrate cancer, infertility, obesity, asthma, liver damage, disorder of androgenic attention deficit and function, mutagenicity, teratogenicity. Additionaly, exposure to phthalate has been associated with development abnormalility and social impairments in children, including deficit hyperactivity disorder (ADHD) [32-34].
Food contamination with phthalate esters has become a matter of public concern in recent year. Beverages can be easily contaminated by phthalate esters during packaging, transportation, and storage processes. Due to complexity of the sample matrix and the low levels of phthalates in food and beverages, determining phthalates in beverage samples is a challenging task [17, 35, 36]. Temperature and content of a PET bottles play a vital role in determine the rate and extent of leaching of chemicals from the bottle matrix into the water and beverages. The duration of storage, concentration of compound, its types, nature, and solubility in foodstuffs are the main factors that influence the leaching of chemicals in water or beverage contents. Storing plastic products under ideal conditions is essential. Different manufacturers advocate for optimal storage conditions such as indoor storage, keep in cool, dry place, and away from direct sunlight. The technical testing of all plastic containers focuses on two specific attributes: first ensuring minimum leaching of compounds from plastic matrix to drinking contents and the second confirming that any compounds that might leach from plastic to the liquid medium do not pose risk to the human health. The food and drug authority (FDA) has stated that exposing the bottle to higher temperature may lead to a higher degree of leaching of compounds from plastic to water or beverages. However, it is common for people to expose plastic bottled beverages to intense sunlight during transportation from production to selling points. These beverage bottles are some time left in sunlight at markets or in cars for longer periods before consumption. For that reason it is necessary to determine whether exposure to these storage factors can impact on the leaching of compounds from plastic matrix into the beverages [53-55]. Therefore, we conducted this study to access the phthalate esters in beverage bottles and milk purchased from markets of Lahore in year 2021 stored under different storage condition to evaluate the effect of these conditions on phthalate leaching through HPLC.
Materials and Methods
Chemicals
Dibutyl phthalate (DBP), Dimethyl phthalate (DMP), Di-n-octyl phthalate (DNOP), Diethyl phthalate (DEP), Acetonitrile, Chloroform, Carbon tetrachloride, Trichloroacetic acid, Lead acetate, Methanol and Sodium chloride used in research work were purchased from Sigma merk. Stock solution of phthalate esters (~99.00%) were prepared in mixture of acetonitrile: water (v/v). and stored at 4oC until analysis.
Sample Extraction
Beverage Samples Extraction
Phthalate esters were extracted from beverage bottles by ultrasound and vortex-assisted dispersive liquid-liquid micro extraction with some modification as describe below [37]. For this purpose, 2.5 ml of beverage samples and 0.5g of sodium chloride were added in 10 ml of conical centrifuge tube. 2 ml of methanol and 500 µl of chloroform were added in the next step. The tube was placed in ultrasonic bath for 30 sec then vortexed for 4 min and centrifuged for 3 min at 4000 rpm and chloroform phase was separated into the glass test tube and evaporated under nitrogen flow. The residue was dissolved with the mobile phase and then filter through 0.45 µm syringe filter before injected into HPLC-DAD under the following conditions given in Table 1.
Milk Samples Extraction
Phthalate esters were extracted from milk samples by dispersive liquid-liquid micro extraction according to Yan et al. [38] with some modifications. Before extraction of phthalates, lipid layer needs to be removed from milk. For this purpose, first milk samples were mixed with trichloroacetic acid and centrifuge at 10,000 rpm for 5 min. The fatty layer was removed and added lead acetate solution into supernatant, centrifuge at 10,000 rpm for 5 min. The supernatant was separated into 10 ml centrifuge tube and mixed with sodium chloride. In next step, 800 µl methanol and 40 µl carbon tetrachloride were injected rapidly into milk samples. The mixture was manually shaken for several seconds and further mixed by ultrasonic bath for 2.0 min. Then finally centrifuge at 4000 rpm for 5 min. the solvent evaporated under the nitrogen flow, residue dissolved into mobile phase and filter through 0.45 µm syringe filter injection into HPLC- DAD conditions given in Table 1.
Results and Discussions
In this study, we examined the retention time of four phthalate esters: DMP (1.819), DEP (5.596), DBP (8.683), and DnOP (14.250), as shown in Fig. 1. The HPLC method was validated in term of linearity, LOD, precision and accuracy. The linearity was obtained in the range of 0.25-100µg/ml for beverages with coefficient (R2) ranging from 0.9965-0.9992, and for milk samples linearity range was 0.25-100 µg/ml with coefficient ranging from 0.9935 to 0.9989, indicating good linearity and LOQ was 0.5 µg/ml for all the four phthalate esters, summarized in Table 2. The 1st and 2nd day of precision and accuracy were evaluated in six replicates of beverages and milk samples at three different concentration, coefficient of varience (CV) presented as precision. For beverages, 1st and 2nd day precision was 0.34-5.93 and 0.53-5.89 respectively and the accuracy of the method was within 94-98% as shown in table 3. For milk samples 1st and 2nd day precision was 0.74-3.64 and 0.07-3.44 respectively (Table 3) and the accuracy of the method was within 91-99%. The result indicated that analytical method is accurate and valid. Among the above-mentioned phthalate esters, only DBP and DEP could be determined quantitatively in beverages and milk samples and their detection frequency are 72% and 61% in beverage samples and 45% in milk samples. None of the fresh beverage samples contained any tested phthalates. Previous research has also failed to identify any phthalates other than diethyl hexyl phthalate (DEHP), which was not included in the list of tested phthalates in the current study. Razali et al. [8] did not find any phthalate in fresh PET bottles of sparkling (carbonated), mineral and drinking (non-carbonated) water. Montuori et al. [39] detected DMP, DEP and DBP in PET and glass water bottles and their concentration was twelve times higher in PET than in glass bottled water. However, all packaged milk samples analyzed in other countries by various researchers contained phthalate athough at low concentrations. DBP and DEHP were the most common phthalates detected in these studies, with concentrations ranging from 3.08-5.03 ng/g for DBP and 0.41-4.00 ng/g for DEHP [40-42], of which DBP is consistent with our findings. The detection of phthalates in freshly packaged milk samples is due to the contribution of severals factors along the processing chain. Mechanical milking, phthalate containing cattle feeds, commercial feeds, packaging materials and water source are the main sources of phthalate contaminations at both farm and industrial level [43-45]. Studies conducted in China, Iran and India have revealed higher concentrations of phthalates in packaged milk samples compared to raw milk samples, suggesting that the production and packaging process contribute to this contamination [45-47].
Table 1.
Analytical conditions of phthalate esters by HPLC-DAD.
Table 2.
Analytical performance of the developed method.
The results of phthalates concentrations in milk samples are shown in Table 4. Diethyl phthalate and dibutyl phthalate were detected in most of milk samples tested for phthalate esters on purchase day (time 1) and after expiry day (time 2). Concentrations of both the phthalates increased with storage and indicated by higher values at time 2. The concentrations differ significantly among the milk brands ranging 1.146-4.45 µg/ml for brand A, nd-1.963 µg/ml for brand B, nd-1.599 µg/ml for brand C, nd-1.682 µg/ml, for brand D nd-0.932 µg/ml for brand E and 2.071-7.734 µg/ml for brand F. lowest DBP concentration was detected in brand C (0.77 µg/ml) and highest in brand F (2.70 µg/ml). Maximum concentration of DEP was detected in brand F (7.73 µg/ml) and minimum in brand F (0.93 µg/ml).
Table 3.
1st day and 2nd day precision and accuracy for analysis of phthalate esters in beverage and milk samples.
Table 4.
Concentrations (µg/ml) of phthalate esters in milk samples.
Table 5.
Concentrations (µg/ml) of phthalate esters in beverage brands.
Table 5 summarizes the concentrations of phthalates released in beverage brands, Diethyl phthalate (DEP) was detected in samples stored at 4ºC with concentration ranging ND-69 µg/ml. In at room temperature bottles, Diethyl phthalate (DEP) was detected with concentration ranging from ND-120 µg/ml. Outdoor storage showed DEP concentration lying in the range of ND-130 µg/ml. Similarly, dibutyl phthalate leached in beverage samples stored at 4ºC concentrations ranging from ND-75 µg/ml, but even at room temperature, range of 5.6-77 µg/ml, although outdoor storage showed its leaching in samples similar to that of DEP concentrations ND-97 µg/ml. The remaining two tested phthalates (DMP and DnOP) were not detected in any of the beverage samples stored at any of the storage conditions. The reason could be probably their limited use in industrial levels and in bottle industry. Irrespective of storage conditions, brands A, B and E give the lowest average concentrations of DBP with no detection and brand D gives the highest average concentrations of DBP equal to 97 µg/ml. DBP individual concentrations of the brands were ranged ND-9.8 µg/ml for brand B, 75-87 µg/ml for brand C, 41-97 µg/ml for brand D, ND-19 µg/ml for brand E, and 9.2-26 µg/ml for brand F. For DEP, brands B, C and D give no detection as the lowest values, while, brand E gives 130 µg/ml as the highest values. Individual ranges were 11-55 µg/ml for brand A, ND-62 µg/ml for brand C, ND-106 µg/ml for brand D, 69-130 µg/ml for brand E, 37-110 µg/ml for brand F. The detection of phthalates after 4 months of storage in beverage bottles, compared to no detection in the beverage bottles analyzed before storage, and the increase in phthalate concentration in milk samples after storage, compared to their levels detected in packaged milk samples analyzed upon purchase, are indicators of phthalate leaching from plastic matrix of the packaging materials. For both beverages and milk samples, the order of the concentration of the two detected phthalates was DEP>DBP. The increase in phthalate concentration after 45 days of storage, compared to the phthalate content measured before storage served as evidence of phthalate leaching as observed in bottle water storage under conditions by Jeddi et al. [18] and Rastkari et al. [2]. When measuring phthalate esters leaching in different types of consumable plastic products Ahmed et al. [50] also found DMP and DBP to have the highest leaching in juices and soft drinks comparable to five different phthalates. In another study, Ayofe et al. [51] observed phthalate quantity in plastic coke in decreasing mean concentration of phthalate esters in order of DEHP>DMP>DEP>DphP. Likewise in another study, Yin et al. [35] analyzed eleven phthalate esters from carbonated soft drinks, mineral water bottles, fruit juices and tea beverages through HPLC analysis. Phthalates were detected in range of 0.24-34.3 µg/L in all beverage samples.
Table 6.
Detection frequency of DBP and DEP in beverage and milk samples.
| Sample nature | % of positive samples | % of samples > MRL of US EPA (6-8 µg/L) |
|---|---|---|
| Milk (n= 12) | DEP = 75 | 8.3 |
| DBP = 58.3 | 0 | |
| Beverages (n = 18) | DEP = 72.22 | 72 |
| DBP = 66.66 | 61 |
The maximum contaminant level (MCL) for DEHP in drinking water bottles set by USEPA, EU and WHO is 6 µg/L and 8 µg/L, which have been used for comparison as standard in this study because no limits have been set as permissible or maximum contaminant levels for any of the other phthalates by any national or global authority [41, 52, 56]. In this study, the mean concentration of diethyl phthalate and dibutyl phthalate in all milk samples were below the standard value, while in beverages the mean concentration of DBP and DEP were higher than standard values. Table 6 summarizes a comparison of detection frequency of DBP and DEP in milk and beverage samples and the comparison of their concentration with MRL (6 and 8 µg/L) set by USEPA, EU and WHO. Diethyl phthalate exceed the MRL in 8.3% of the positive milk samples and in 72% of the positive beverage samples. While, dibutyl phthalate exceed MRL in none of positive milk samples although it surpass the MRL in 61% of the positive beverage samples. These values confirm with the values obtained by other researchers previously. For instance, Moradian et al. [41] detected DEP (nd-0.02 µg/l), and DBP (0.02-0.04 µg/L) in milk samples. Similarly, Ayofe et al. [51] detected DMP, DEP, and DEHP in coca cola at concentration range 1.32-10.22 µg/l, nd-2.86 µg/l, and 7.43-18.67 µg/l respectively. Likewise Yin et al. [53] found DBP in mineral water (8.98-11.5 µg/l), in carbonated soft drink (5.03-16.5 µg/l), in tea beverage (2.22-12.6 µg/l), and in fruit juice (nd-20.6 µg/l).
The average concentration of both detected phthalate was calculated separately for each of storage condition, regardless of the tested brands. These average were plotted in Fig. 2. Which illustrates that outdoor (sunlight exposure) storage is the most prone to phthalate leaching, on the other hand, refrigerated condition (4oC) is the safest in this respect, on average, as this offered the highest storage temperature. In other words, probability of both DBP and DEP leaching increases as the temperature increases. The trend of phthalate leaching in plastic bottles is affected by storage conditions, with the highest leaching occurring in bottles stored outdoor, followed by room temperature and 4ºC. This is consistent with previous studies, which have found that phthalate leaching occurs as a results of hydrolysis, with temperature acting as a catalyst. Bottles that are stored at higher temperatures are more likely to support leaching of phthalates and have higher concentrations of phthalates than bottles stored at lower temperatures. This also justifies the highest level of leaching in bottles stored outdoor. Schmid et al. [48] reported an increase in DEHP in PET-bottled water that was stored in direct sunlight upto 34ºC for 17 hours. In a study of phthalate leaching in juices during storage, Arfaeinie et al. [49] found a positive correlation between the storage temperature, time and sunlight exposure of juice containers, and the extraction of phthalate esters (DnBP and DEHP) from highly acidic juices. Jeddi et al. [18] also deduced that freezing temperature (4ºC) does not support phthalate leaching in acidic beverages, hydrolysis controls the production and leaching of phthalates whose rate increases with increase temperature imply that high temperature allows more leaching than the lower ones.
Conclusions
The present research was conducted to assess the impact of storage conditions on the potential migration and concentration of phthalate esters in beverages and milk, using high-performance liquid chromatography to determine the risk of these phthalates on consumer health. The study found that diethyl phthalate DEP had highest frequency of leaching, followed by dibutyl phthalate (DBP). Furthermore, DEP was detected in a comparative high concentration when compared to DBP. Among the storage conditions, outdoor storage was found to be the most damaging, as it supported the highest degree of phthalate leaching from plastic bottles into beverages. On the other hand, storing beverages and milk at 4ºC was found to be safest as it did not support a high degree of hydrolysis that causes phthalate leaching. As a result, it is recommended that beverage bottles and packaged milk should be stored under 4ºC conditions if they are required to be stored for a long duration.
