Introduction

Bisphenol A (BPA) is an industrial chemical produced in large quantities worldwide and is commonly used intentionally (intentionally added substances-IAS) as a monomer in the synthesis of polycarbonate (PC), a plasticizer in the production of epoxy resins, and an additive in the production of polyvinyl chloride (PVC)1. This substance is widely used in packaging materials, including plastic containers, kitchenware, and the inner lining of cans that have direct contact with food. The widespread use of BPA in packaging has raised significant concerns about its potential migration into food products and human exposure to this substance2, because this compound is not chemically bound to the polymer matrix and can easily migrate into the food it contacts throughout its life cycle, even under improper storage conditions3,4. From a health perspective, based on the results of a review study5; BPA is concerning due to its estrogen-like properties and its ability to act as an endocrine-disrupting chemical (EDC). This substance is associated with reproductive problems, premature puberty, hormone-dependent tumors (breast and prostate cancer), and abnormalities including menstrual cycle irregularities, fertility disorders, endometriosis, polycystic ovary syndrome (PCOS), and more. Furthermore, exposure to BPA has been linked to diabetes, obesity, epigenetic effects, impaired immune system function, increased risk of cardiovascular diseases, thyroid dysfunction, and kidney disease. The health risks of BPA do not end here and, through transgenerational epigenetic mechanisms, it can also affect future generations, meaning its effects can persist in the population for decades.

Among the various materials used in the production of beverage bottles, polyethylene terephthalate (PET), a semi-crystalline polymer with a high molecular weight, is widely used for packaging beverages because it possesses desirable physical properties, including strength combined with lightness, transparency, and durability3,6,7,8. Additionally, PET exhibits good barrier properties against gases and moisture, which helps preserve the quality and extend the shelf life of packaged beverages. Furthermore, PET is highly recyclable and contributes to sustainability efforts7,9. To enhance the strength, lifespan, and transparency of PET, phthalates (PAEs) or other inorganic species (e.g., Sb2O3 for synthesis) are commonly used as IAS in the production of PET bottles3,10.

However, in addition to IAS7,11, some non-intentionally added substances (NIAS) may also be found in plastic products, the source of which is uncertain1,12. Although PET is generally considered free of BPA due to its production process, various studies have shown that BPA is indeed present in bottled beverages stored in PET12,13,14, indicating potential contamination during production processes or from other sources6,7. This phenomenon is mainly attributed to several factors, including process contamination during the manufacturing process, water source, bottle cap, environmental pollutants, and recycling processes in the production of PET materials3,7,15. This is further supported by studies that have shown BPA contamination in bottled water in different geographical locations, indicating that this problem is not limited to specific regions and is a multifaceted contamination14,16,17,18. The role of recycling in the contamination of PET bottles cannot be ignored. Research has shown that recycled PET may contain residual BPA from previous uses, which can then leach into new products17,19. Furthermore, according to some studies, environmental factors such as exposure to high temperatures and longer storage times potentially lead to higher concentrations of migrated chemical compounds11,20. For example, the study by Baz et al.20 showed that BPA concentration levels in bottled water samples increased from 9.46 ng/L at room temperature to 16.13 ng/L and 14.7 ng/L after the samples were exposed to sunlight and a boiling water bath, respectively. Massahi et al.11 also reported increased migration of PAEs at higher temperatures (40 °C) compared to lower temperatures (4 °C) (p < 0.05). The implications of these findings are significant, as they indicate that consumers may unintentionally ingest BPA from PET bottles and experience its health effects, and this may even be influenced by storage conditions.

Doogh (yogurt drink), a traditional Iranian fermented dairy beverage prepared from diluted yogurt, constitutes an important part of daily beverage consumption in Iran. This acidic drink is usually flavored with dried mint, rose petals, or other spices and provides high-quality protein, vitamins, calcium, and minerals. Doogh is also considered a good source of probiotics, which may contribute to gut health and improve the immune system. This traditional drink is currently very popular and has gained recognition in other countries besides Iran21,22. Currently, PET bottles are widely used for packaging doogh due to their unique properties and are stored under various conditions. The idea of how serious the risk of BPA is in bottled doogh is now being reinforced. Previous studies have investigated the chemical migration of BPA into dairy products and yogurt-based drinks, and packaging, processing equipment, and storage tanks have been identified as potential sources of contamination23. However, so far, no study has been conducted regarding the investigation of BPA in bottled doogh under different storage conditions.

Therefore, although PET bottles are often marketed as BPA-free, the evidence mentioned above suggests that BPA can still be present in the composition of these bottles and the doogh itself due to various contamination routes. Considering the widespread consumption of doogh in Iran and the potential health risks associated with BPA exposure, along with evidence suggesting that storage conditions affect the release of compounds from bottle materials, there is an urgent need to investigate the migration of BPA into this traditional beverage. The aim of this pioneering study is to investigate the effects of various storage conditions, often encountered by consumers or sellers, on the migration of BPA from PET bottles into doogh for the first time. In addition, by conducting a human health risk assessment, this research aims to provide valuable insights for consumers, manufacturers, and regulatory agencies regarding the safety of consuming doogh from PET bottles.

Materials and methods

Study design and sample collection

The study should have been conducted in different phases with three main factors: temperature (= for 30 days), storage time (in room temperature ≈ 25 °C), and light conditions (for 30 days). Three popular small-sized doogh brands (≈ 250 ml) in Iran were selected based on a simple researcher-made questionnaire. All three brands of doogh had a mint flavor. To cover all the conditions under investigation, the following design was used:

  • 3 temperature levels (4, 25, and 45 °C) × 3 brands = 9 samples.

  • 4 storage time levels (7 days, 15 days, 1 month, and 2 months) × 3 brands = 12 samples.

  • 2 light condition levels (sunlight exposure and shade) × 3 brands = 6 samples.

Therefore, the total number of samples was 27 samples (9 samples from each brand). Samples of the desired doogh brands with specific production dates were randomly purchased from grocery stores (Kermanshah Province). Doogh with the most recent production date was purchased. Manipulation and damage to the sample packaging during collection and transfer were also avoided. The samples were immediately transferred to the laboratory in covered containers. Upon arrival at the laboratory, the samples were stored in a refrigerator at 4 °C.

Storage conditions

Different storage temperatures

A temperature of 4 °C was considered to simulate the storage conditions of doogh in the refrigerators of distribution centers and homes. The relevant samples were placed in a laboratory refrigerator with the temperature set at 4 °C. 25 °C was considered to simulate the storage conditions of doogh at ambient room temperature under normal warehousing or store conditions. The high temperature (45 °C) was considered to simulate improper storage conditions and the exposure of doogh to intense heat (such as hot weather conditions or storage in hot and unventilated places). The relevant samples were kept in an oven with the temperature set at 25 °C and 45 °C. At the end of the storage period (30 days), the relevant samples were removed from the refrigerator and oven.

Different storage durations

Four storage time periods (at room temperature ≈ 25 °C) were selected to investigate the effect of storage duration on the amount of BPA migration. 7 days: simulating short-term storage after production and initial distribution. 15 days: simulating medium-term storage in distribution centers or homes. 1 month: simulating longer-term storage in warehouses or homes. 2 months: simulating long-term storage until near the expiration date. For each storage time, the doogh samples were removed from the storage conditions at the end of specified time intervals.

Different light conditions

Two light exposure conditions (shade and sunlight) were investigated. Samples pertaining to shade conditions were kept inside a dark cabinet within a closed cardboard box and away from any direct light (sunlight or artificial light). Samples pertaining to sunlight exposure conditions were placed outdoors in direct sunlight. At the end, after 30 days, the respective samples were removed from the simulated light conditions.

Analysis method

The study determined BPA levels in doogh samples using solid-phase extraction (SPE) for cleanup and pre-concentration, followed by high-performance liquid chromatography with an ultraviolet detector (HPLC-UV) for quantification. Furthermore, considering the successful application of this method in the study by Hadjmohammadi and Saeidi24 and their recommendations for determining BPA levels in dairy matrices, the analytical method used in the present research was adapted with slight modifications from their study.

Reagents, standards, and materials

In this study, reagents with the highest purity grade and without the need for further processing were used. BPA with a purity of more than 99% was purchased from Sigma-Aldrich (St. Louis, MO USA). Methanol (MeOH), n-hexane, hydrochloric acid (HCl), sodium hydroxide (NaOH), sodium dihydrogen phosphate (NaH2PO4), and disodium hydrogen phosphate (Na2HPO4) were all obtained with analytical grade from Merck (Darmstadt, Germany). SPE cartridges of the C18 type (500 mg sorbent mass, 6 mL volume) were obtained from Waters (Sep-Pak). Syringe filters (PTFE, 0.45 μm pore size) and membrane filters (Nylon, 0.45 μm pore size) were also obtained from Merck Millipore (Burlington, MA, USA). Deionized (DI) water was prepared using a Milli-Q purification system (Millipore Corporation, MA, USA). High-purity nitrogen gas (≤ 99.99%) was used. A calibrated pH meter (Metrohm 827 pH Lab, Herisau, Switzerland) was used for pH adjustments. In addition, a vacuum manifold capable of processing several SPE cartridges simultaneously, coupled with a vacuum pump, was used.

Preparation of standard solutions and reagents

A BPA stock standard solution (500 mg/L) was prepared by accurately weighing 50.0 mg of BPA standard and dissolving it in MeOH in a 100 mL Class A volumetric flask; this solution was stored in an amber glass vial at 4 °C and was stable for approximately 3 months. Two BPA intermediate standard solutions with concentrations of 50 mg/L and 5 mg/L were prepared by diluting 10.0 and 1 mL, respectively, of the 500 mg/L stock standard solution in 100 mL class A volumetric flask with MeOH and stored at 4 °C. BPA working standard solutions with concentrations of 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, and 20.0 mg/L were prepared by appropriate serial dilutions of the intermediate standard solutions with the initial mobile phase composition (DI water/MeOH, 30:70 v/v).

Phosphate buffer (0.05 M, pH 6.5) was prepared by dissolving appropriate amounts of NaH2PO4 and Na2HPO4 in DI water to reach a total phosphate concentration of 0.05 M and carefully adjusting the pH to 6.5 using dilute NaOH or HCl solution. The mobile phase (DI water/MeOH, 30:70 v/v, pH 6.5) was prepared by mixing DI water and MeOH in a 30:70 (v/v) ratio and adding the required amount of phosphate buffer with pH 6.5 to reach a final pH of 6.5, then filtered through a 0.45 μm membrane filter and thoroughly degassed under vacuum before use. The SPE washing solution was prepared by mixing DI water and MeOH in an 80:20 (v/v) ratio, and the pH was adjusted to 7.0 if necessary, using dilute NaOH or HCl. The elution solvent for SPE was HPLC-grade MeOH.

Sample preparation and extraction

The bottled doogh sample was initially subjected to thorough agitation to ensure complete homogenization. Subsequently, a 5.0 mL aliquot of the homogenized doogh was transferred to a 50 mL beaker, and 20 mL of a DI water/MeOH mixture (17:3, v/v) was added. The resulting mixture was stirred with a magnetic stirrer at ambient temperature for 10 min. The pH of the mixture was adjusted to 3.0 using HCl during the stirring process to prevent the ionization of BPA. Subsequently, the mixture was passed through a 0.45 μm membrane filter to separate precipitated solids, and the clear supernatant was collected for SPE.

The SPE cartridge was conditioned by passing 5 mL of n-hexane, 5 mL of MeOH, and 10 mL of DI water under gentle vacuum (flow rate approximately 1–2 mL/min), ensuring that the cartridge bed did not dry out between conditioning steps or before sample loading. The filtered doogh extract was passed through the conditioned SPE cartridge at a controlled flow rate of approximately 2–3 mL/min. To remove interfering substances, the cartridge was washed with 20 mL of DI water and subsequently with 10 mL of washing solvent (DI water/MeOH, 80:20, v/v, pH 7.0). Following this, the cartridge bed was dried under gentle vacuum for 2–4 min to remove any residual washing solvent. The retained BPA was eluted by passing 4.0 mL of MeOH at a slow flow rate (approximately 1 mL/min) through the cartridge, and the eluate was collected in a clean collection tube; the solvent was allowed to soak the cartridge bed for 1 min prior to elution. The methanolic eluate was evaporated to dryness under a gentle stream of nitrogen gas at approximately 40–50 °C. The dried residue was precisely reconstituted in 1.0 mL of the mobile phase (DI water/MeOH, 30:70, v/v, pH 6.5) and thoroughly vortexed to ensure complete dissolution. Finally, the reconstituted solution was filtered through a 0.45 μm syringe filter (PTFE) into an HPLC vial prior to injection. The preparation, extraction, and injection procedures were performed in triplicate for each bottled doogh.

System and chromatographic conditions

An HPLC system (PerkinElmer, Norwalk, CT, USA) equipped with a manual injector, and a UV detector (model LC-95) was used. A C18 column (Waters, 250 mm length × 4.6 mm internal diameter) was used for separation. The separation of compounds was performed isocratically with a mobile phase consisting of DI water and MeOH (30:70, v/v) buffered at pH 6.5 (using a 0.05 M phosphate buffer). The mobile phase flow rate was set at 1.0 mL/min, and the column temperature was maintained at 25 °C throughout the analysis. The sample injection volume was 10 µL, and compound detection was carried out using a UV detector at a wavelength of 282 nm.

Calibration and method validation

In this study, by examining the chromatograms of the standard solutions, the expected RT was approximately 5.6 min. Quantification was performed using the external standard method. The calibration curve was created by injecting prepared working standard solutions (0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, and 20.0 mg/L) in triplicate, which showed a coefficient of determination (r2) of 0.9999 and a regression equation of y = 61.67x + 3.97.

The linearity range was confirmed for concentrations of 0.1 to 20.0 mg/L with a coefficient of determination (r2 = 0.9999). The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.09 mg/L and 0.28 mg/L, respectively. The accuracy of the method was assessed by analyzing spiked doogh samples at two levels (0.4 mg/L and 4 mg/L, three replicates each), and the mean recoveries ranged from 98.2 to 103.6%. Precision was evaluated through inter-day analysis (3 different days) of a standard solution by calculating the %RSD, which was found to be 8.7%. In addition, all glassware used was washed with MeOH and DI water before the experiment to prevent any contamination.

Exposure and health risk assessment

The estimated daily intake (EDI) of BPA for each individual, in milligrams per kilogram of body weight per day (mg/kg bw/day), through the consumption of doogh, was calculated for each individual using Eq. (1).

$$\:\text{E}\text{D}\text{I}\:(\text{m}\text{g}/\text{k}\text{g}\:\text{b}\text{w}/\text{d}\text{a}\text{y})\:=(\text{C}\:\times\:\text{I}\text{R})/\text{B}\text{W}$$
(1)

where, “C” is the concentration of BPA in doogh (mg/L) under different storage conditions, “IR” is the daily consumption rate of doogh in L/day, which is 0.1515 L/day for adults and 0.0873 L/day for children, obtained using a researcher-developed questionnaire, and “BW” is the body weight of consumers in kg, which was 70 kg for adults and 15 kg for children11.

After calculating the EDI, the Hazard Quotient (HQ) was calculated according to Eq. (2) to assess the risk associated with the consumption of doogh containing BPA. In this equation, the Reference Dose (RfD) of BPA determined by the United States Environmental Protection Agency (US EPA) is 0.05 mg/kg bw/day25,26.

$$\:\text{H}\text{Q}\:=\text{E}\text{D}\text{I}/\text{R}\text{f}\text{D}$$
(2)

Finally, if HQ < 1, the risk of exposure to BPA through doogh consumption is negligible, and there is no serious concern. However, if HQ ≥ 1, the risk of exposure to BPA through doogh consumption may be significant, and this issue requires further evaluation to ensure consumer health27.

Statistical analysis

GraphPad Prism software (version 10.4.1) was used for statistical analysis and the creation of relevant graphs. To assess the normal distribution of the data, the Kolmogorov-Smirnov test were employed. The significance level (α) was set at 0.05 for all statistical tests used. The comparison of mean BPA concentrations between three or more groups was performed using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons test for pairwise comparisons between groups. The independent t-test (also known as the unpaired t-test) was used to compare the mean BPA concentrations between two independent groups.

Results

Temperature effect

The results of storing doogh for 30 days at different temperatures showed that increasing the storage temperature significantly increased the BPA concentration in the doogh. As shown in Fig. 1, the average BPA concentration in all three doogh brands showed an increasing trend with rising temperature, and this increase was statistically significant (for all pairwise comparisons between two specific temperatures; p < 0.001 or better). Specifically, at 4 °C, which is known as the typical refrigeration temperature, the BPA level was the lowest for all three brands. For example, in doogh brand #1, the BPA concentration at 4 °C was 0.160 ± 0.002 mg/L. When the temperature increased to 25 °C, the BPA concentration in the same brand was 0.189 ± 0.003 mg/L, which indicates a significant increase compared to the 4 °C temperature (about 18% higher, p < 0.0001). At 45 °C, the BPA concentration increased to its highest level; for instance, in the brand #1, this amount was 0.318 ± 0.005 mg/L, which is almost double its value at 4 °C (p < 0.0001). A similar pattern was observed in the other brands as well, such that the highest BPA level was observed at 45 °C and the lowest level at 4 °C for all three brands.

Fig. 1
figure 1

Concentration (mean ± SD) and pairwise comparisons of the mean BPA concentration between different storage temperatures—for the three doogh brands (a: Brand #1, b: Brand #2, c: Brand #3). ***p < 0.001, ****p < 0.0001. Error bars represent SD.

Full size image

The findings indicate that the response of each doogh brand to temperature conditions varies. Figure 2 shows a comparison of the average BPA concentration among different brands at each storage temperature. At 4 °C, Brand #1 had the highest BPA concentration (0.160 ± 0.002 mg/L) and Brand #3 had the lowest concentration (0.139 ± 0.001 mg/L); the difference in average BPA concentration between brands at this temperature was significant (for all pairwise comparisons between two specific brands; p < 0.01 or better). At 25 °C, Brand #3 showed the highest concentration (0.197 ± 0.003 mg/L) and Brand #2 showed the lowest concentration (0.171 ± 0.003 mg/L). At this temperature, like 4 °C, all differences between brands were statistically significant (for all pairwise comparisons; p < 0.05 or better). At 45 °C, Brand #1 had the highest concentration (0.318 ± 0.005 mg/L) and Brand #2 had the lowest concentration (0.269 ± 0.004 mg/L). At this temperature as well, like the previous two temperatures, all differences between brands were statistically significant (for all pairwise comparisons; p < 0.01 or better).

Fig. 2
figure 2

Concentration (mean ± SD) and pairwise comparisons of the mean BPA concentration between different doogh brands - for three storage temperatures (a: 4 °C, b: 25 °C, c: 45 °C). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars represent SD.

Full size image

Storage duration effect

The storage time of doogh (at room temperature ≈ 25 °C) was also identified as an influential factor in the BPA level. Based on Fig. 3, as the storage time lengthened, the BPA concentration in the samples also increased. The data shows that for all three doogh brands, increasing the storage time (over a period of 1 week to 2 months) had a positive and significant effect on the BPA level (for all pairwise comparisons between two specific brands; p < 0.01 or better). In the early days of storage, the BPA level was relatively low. For example, in Brand #1, the BPA concentration in the first week was measured as 0.115 ± 0.004 mg/L. After 2 weeks (15 days), the BPA concentration in the samples related to the same brand slightly increased (0.129 ± 0.003 mg/L), and this increase and the difference between the average concentrations were statistically significant (p < 0.01). As the storage time of the doogh samples was extended to 30 days, the increase in BPA concentration was again significant; the BPA concentration in the samples related to Brand #1 was 0.187 ± 0.003 mg/L (at this stage, the difference in average concentration compared to the first week and 2 weeks was highly significant; p < 0.0001). At the end of 60 days of storage for Brand #1 samples, the BPA concentration was at its highest, such that for Brand #1, the BPA concentration was 0.228 ± 0.003 mg/L. This amount is about twice that of the first week, and its difference from the values at the beginning of the period was statistically highly significant (for example, the difference between 60 days and 7 days; p < 0.0001). The results for Brands #2 and Brands #3 also showed a similar increasing trend in BPA due to increased storage time; generally, Brand #2 had slightly lower values at each time point, and Brand #3 had slightly higher values. However, like Brand #1, Brands #2 and Brands #3 also showed a significant increase in BPA levels during the 2 months of storage, which was statistically significant (p < 0.01 or better).

Fig. 3
figure 3

Concentration (mean ± SD) and pairwise comparisons of the mean BPA concentration between different storage times—for the three doogh brands (a: Brand #1, b: Brand #2, c: Brand #3). **p < 0.01, ****p < 0.0001. Error bars represent SD.

Full size image

Figure 4 shows the comparison of BPA concentrations between brands at each specific storage time. Accordingly, the rate of changes in BPA concentration varies among different brands. At the end of one week of storage, although differences were observed between the average BPA concentrations for the brands (range 0.107 ± 0.003 mg/L for Brand #2 to 0.115 ± 0.004 mg/L for Brand #1), these differences were not significant (p > 0.05). At the end of 15 days, Brand #3 (0.136 ± 0.003 mg/L) had the highest average BPA level and was significantly higher than Brand #1 (0.129 ± 0.003 mg/L) (p < 0.05) and Brand #2 (0.126 ± 0.002 mg/L) (p < 0.01). Although the BPA concentration in Brand #1 was slightly higher than in Brand #2 at the end of 15 days, it was not significant (p > 0.05). At the end of 30 days, Brand #3 (0.194 ± 0.004 mg/L) showed the highest BPA level compared to Brand #1 (0.187 ± 0.003 mg/L, p > 0.05) and Brand #2 (0.175 ± 0.004 mg/L) (p < 0.001). The higher BPA concentration in Brand #1 compared to Brand #2 was also significant (p < 0.05). At the end of 60 days, like 30 days, Brand #3 (0.238 ± 0.004 mg/L) showed the highest BPA level compared to Brand #1 (0.228 ± 0.003 mg/L (p > 0.05) and Brand #2 (0.217 ± 0.005 mg/L) (p < 0.001). The difference in BPA concentration between Brand #1 and Brand #2 was also significant (p < 0.05).

Fig. 4
figure 4

Concentration (mean ± SD) and pairwise comparisons of mean BPA concentration between different doogh brands—for four storage times (a: 7 days, b: 15 days, c: 30 days, d: 60 days). ns: not significant, *p < 0.05, **p < 0.01. Error bars represent SD.

Full size image

Light conditions effect

According to Fig. 5, the BPA concentration in samples stored (for 30 days) under sunlight was significantly higher than in samples stored in darkness (shade). For all three tested doogh brands, the BPA concentration increased very significantly (p < 0.0001) under light conditions compared to shade conditions. More specifically, the average BPA concentration of Brand #1 stored in darkness was 0.181 ± 0.003 mg/L, while for the same brand, the concentration for samples exposed to sunlight was reported as 0.340 ± 0.005 mg/L. This almost twofold increase for Brand #1 is statistically very significant (p < 0.0001). For Brand #2, which had the lowest BPA levels, the BPA concentration under shade storage conditions was 0.174 ± 0.002 mg/L, but under sunlight exposure conditions for the same brand, the concentration was reported as 0.304 ± 0.007 mg/L, indicating an approximately 75% increase in BPA concentration (p < 0.0001). Similarly, for Brand #3, the concentration was 0.194 ± 0.003 mg/L under shade conditions and 0.320 ± 0.011 mg/L under sunlight exposure for the same brand, showing about a 65% increase in concentration (p < 0.0001).

Fig. 5
figure 5

Concentration (mean ± SD) and comparison of the mean BPA concentration between shade and sunlight conditions—for the three doogh brands (a: Brand #1, b: Brand #2, c: Brand #3). ****p < 0.0001. Error bars represent SD.

Full size image

In Fig. 6, slight but mostly significant differences are observed in BPA concentration between different brands under a specific light condition. Clearly, under shade conditions, Brand #3 (0.194 ± 0.003 mg/L) had a higher concentration than Brand #2 (0.174 ± 0.002 mg/L) (p < 0.001) and Brand #1 (0.181 ± 0.003 mg/L) (p < 0.01). The higher BPA concentration in Brand #1 compared to Brand #2 was also not significant (p > 0.5). Under sunlight conditions, the trend was different from the shade, such that here Brand #1 (0.340 ± 0.005 mg/L) showed a higher concentration than Brand #2 (0.304 ± 0.007 mg/L) (p < 0.01) and Brand #3 (0.320 ± 0.011 mg/L) (p < 0.05). Here, the higher BPA concentration in Brand #3 compared to Brand #2 was not significant (p > 0.5).

Fig. 6
figure 6

Concentration (mean ± SD) and pairwise comparisons of the mean BPA concentration between different doogh brands - for two light storage conditions (a: in the shade, b: under the sunlight). ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001. Error bars represent SD.

Full size image

BPA intake and its associated health risk

Effect of temperature

Based on the results presented in Table 1, with an increase in storage temperature (= 30 days of storage), the daily exposure and the consumer HQ also increase. For example, for the children’s age group, which typically has higher exposure per unit of consumption due to lower body weight, the EDI related to the consumption of doogh brand #1 was estimated to be approximately 9.3 × 10− 4 mg/kg bw/day when stored at 4 °C, which corresponds to an HQ of about 0.019. The same brand under storage conditions of 25 °C had an EDI of about 1.1 × 10− 3 mg/kg bw/day and an HQ of 0.022, and under conditions of 45 °C (the highest temperature), the EDI for children increased to about 1.9 × 10− 3 mg/kg bw/day, with a corresponding HQ of about 0.037. A similar trend was observed for other brands in the children’s group. Therefore, the exposure and potential risk of BPA exposure in children when consuming doogh stored in a very warm environment (45 °C) is approximately twice that of consuming the same doogh from a refrigerator (4 °C). The values for adults were generally lower than those for children, due to the higher body weight for adults. For example, for doogh brand #1 at 45 °C, the adult EDI was calculated to be 6.9 × 10− 4 mg/kg bw/day, resulting in a corresponding HQ of 0.014. This value is approximately half the HQ for children under the same temperature conditions. At 4 °C, the adult HQ for brand #1 was about 0.0069 (≈ 0.007), which is again less than half of this value for children (0.019). The pattern of changes for other brands was similar; that is, the adult HQ at 45 °C reached a maximum of 0.012–0.014, and at 4 °C it was about 0.006–0.007.

Table 1 Exposure and health risks resulting from BPA for children and adults due to consumption in different brands of Doogh stored at different temperatures (4, 25, and 45 °C).
Full size table

Effect of storage duration

The results of the BPA exposure assessment through doogh consumption for different storage times (≈ room temperature) are presented in Table 2. The findings of this phase of the study are also consistent with the BPA concentration results under these storage conditions and show that a longer storage time leads to an increase in the consumer’s potential exposure to BPA. For the children’s age group, the EDI was lowest when consuming fresh doogh (7-day storage) and increased with longer storage periods. For example, for doogh brand #1, the children’s EDI for a 7-day storage time was 6.7 × 10−4 mg/kg bw/day, with a corresponding HQ of 0.013. With the same brand’s product stored for 15 days, the EDI increased slightly, and the corresponding HQ increased to about 0.015. At 30 days of storage, the children’s EDI for brand #1 reached 1.1 × 10−2 mg/kg bw/day and the HQ reached 0.022, and finally at 60 days, the EDI reached 1.3 × 10−2 mg/kg bw/day and the HQ reached 0.027. Therefore, for this brand, the children’s HQ, which increased from 0.013 in approximately the first week of storage to 0.027 at the end of 2 months of storage, shows an almost two-fold increase in HQ. A similar trend was observed in other brands as well. In other words, during 2 months of doogh product storage, children’s daily exposure to BPA can be about twice as high compared to 1 week of storage. For doogh brand #1, the adult HQ increased from 0.0050 in the short-term storage period (7 days) to 0.0099 at the end of 60 days.

Table 2 Exposure and health risks resulting from BPA for children and adults due to the consumption of different brands of Doogh stored in different time intervals (7, 15, 30, and 60 days).
Full size table

Effect of light conditions

Based on the data presented in Table 3, light conditions (shade vs. direct sunlight) have a significant impact on the level of exposure and health risk associated with BPA in doogh. The results show that storing doogh (= 30 days) exposed to sunlight, compared to shade conditions, can dramatically increase consumers’ exposure to BPA. This increase is reflected in the EDI and HQ indices. For the children’s group, consuming doogh stored in the shade resulted in an HQ of approximately 0.020–0.023 (depending on the brand), while consuming doogh after storage in sunlight increased the HQ to approximately 0.035–0.040. For example, for doogh brand #1, children’s EDI under shade conditions was 1.1 × 10− 3 mg/kg bw/day, which showed a corresponding HQ of 0.021, but for this brand under sunlight conditions, the EDI increased to 2.0 × 10− 3 mg/kg bw/day, and the HQ reached 0.040. The situation was similar in the adult age group. The HQ for adult consumers for brand #1 increased from approximately 0.0078 in the shade to 0.015 in the sun.

Table 3 Exposure and health risks resulting from BPA for children and adults due to the consumption of different brands of Doogh stored under shade and sunlight conditions.
Full size table

The important point is that in all the storage scenarios examined, the HQ for both age groups was < 1. This means that based on the HQ criterion, BPA exposure through the consumption of these doogh samples is likely within the safe range and does not exceed the recommended limit.

Discussions

Effects of storage conditions

The results of this research showed that various storage conditions, including temperature, duration, and exposure to light, have a significant effect on the migration of BPA from the packaging into the doogh. In general, increasing the storage temperature led to a significant and substantial increase in the BPA concentration in the products of all three brands (Fig. 1). Based on the results, samples stored in the refrigerator (4 °C) showed the lowest levels of BPA, while at 25 °C (≈ room temperature), the BPA concentration was slightly higher, and under warm conditions (45 °C), this increase in BPA level was much greater (p < 0.001 or better). For example, in one of the brands, increasing the temperature from 4 °C to 45 °C almost doubled the BPA concentration in the same brand. This increase in concentration is consistent with the hypothesis that both the diffusion process and the mass transfer of some chemical substances present in the composition of polymeric materials increase significantly at high temperatures compared to low temperatures. In fact, at higher temperatures, the contaminant molecules (in this case, BPA) gain more energy, which allows them greater ability to overcome intermolecular forces and migrate from the polymer structure towards the food substance. Consequently, these observations may be due to structural changes and the potential degradation of polymeric materials at higher temperatures14,28,29,30,31. It has been reported that boiling water significantly increased the migration of BPA from epoxy bottles32.

with increasing storage time of the bottled doogh, the BPA concentration in the samples (all three brands) significantly increased compared to shorter storage times (p < 0.01 or better). For example, in one brand, during two months of sample storage, the BPA level almost doubled compared to shorter storage conditions (7 days) (p < 0.0001) (Fig. 3). Naturally, longer storage times lead to longer contact of the contents with the bottle wall, providing more opportunity for interactions between the chemical compounds of the polymer matrix and the bottle contents, and ultimately may lead to increased migration of compounds14,29,33,34.

The other variable investigated was light conditions; doogh samples that were exposed to direct sunlight had significantly more BPA than similar samples kept in the shade. This difference in average concentration was also significant (p < 0.0001; approximately a doubling of BPA concentration was observed under light) (Fig. 5). In fact, sunlight irradiation, in addition to increasing temperature, can accelerate the migration of chemical compounds by degrading packaging polymers and breaking chemical bonds31,35.

These findings emphasize that storing doogh at lower temperatures, away from light, and for shorter periods can minimize consumer exposure to BPA. These observed patterns in doogh align with the results of previous studies on BPA migration from packaging materials. In one study, no significant increase in BPA was observed in the initial weeks of storing water at 25 °C, but when the temperature was raised to 35 and 45 °C, the amount of BPA increased considerably, and the effect of temperature even surpassed the effect of storage time. Also, exposing plastic bottles to ultraviolet light for several hours caused a noticeable increase in BPA migration35. In another study, PET bottles containing water released up to approximately 38.9 ng/L of BPA at 70 °C - over 1 week, which shows an almost twofold increase compared to cool conditions (4 °C) with approximately 18.7 ng/L36. In a study conducted on PET bottles, a positive and significant correlation (p < 0.05) was observed between the storage duration and the increase in DEHP and DBP concentrations compared to the initial measured values (immediately after purchase). Also, after two months of storage, a positive and significant correlation was observed between temperature and the increase in DEHP concentration34. A research by Massahi et al.31 showed that the PAEs studied (including DEHP, DBP, and BBP) were identified in both shaded and direct sunlight storage conditions in new PET bottles filled with distilled water. Data analysis showed that the difference in the average concentration of all three phthalate compounds between the shaded and sunlight storage conditions was statistically significant (p < 0.001). Specifically, the average concentration of each of these compounds was significantly higher under sunlight exposure compared to shaded storage. A study by Jeddi et al.37 also showed that storing bottled water at higher temperatures and for longer times increases the transfer of PAEs from the bottle to the water, with the highest transfer rate occurring at 40 °C after 45 days. Another study showed that temperature plays the most significant role in the migration of antimony from plastic bottles to water, with the highest migration rate observed at 75 °C, pH = 7, and after 5 days of storage38. Another study showed that the migration of BPA from new PC baby bottles to the water they contained at temperatures of 40 °C and 95 °C was 0.03 and 0.13 ppb, respectively. Based on the results of that study, it was found that the rate of BPA migration from the bottle to water increases rapidly at temperatures above 80 °C39. These results, consistent with our study’s findings. However, one study reported a decrease in DEHP concentration in samples stored outdoors for 3 months40; this suggests that under specific conditions, environmental factors such as temperature and sunlight may play a role in the degradation of some packaging chemical compounds over time40. Furthermore, some studies did not report a significant change in PAEs levels after storing PET bottles at different temperatures for specific time periods41,42,43.

The average concentration of BPA in the bottled doogh samples of this study under conventional conditions was approximately 0.1 to 0.2 mg/L (100–200 µg/L) and increased to approximately 0.3 mg/L under the most extreme storage conditions. Table 4 presents a comparison of the results of the present study with the results of some other studies on dairy products. In most cases, the BPA concentration in this study is higher compared to the values in other studies. For example, the average BPA range in different milk samples in a study in Italy was 0.016 to 0.265 µg/L44, which is an order of magnitude lower than that of doogh in the present study. Of course, the type of packaging and product conditions vary in different cases. These differences suggest that the composition of doogh packaging, the nature of the doogh itself, and its processing conditions may influence BPA migration. Furthermore, in more realistic conditions, multiple factors may simultaneously affect the quality of storage conditions for a packaged product and cause more noticeable changes.

Table 4 Comparison of BPA concentration in the present study with the results of some studies.
Full size table

Regarding the inherent characteristics of different products, difference in BPA concentration between different brands (which were subjected to various conditions) was statistically significant in most cases (Figs. 2 and 4, and Fig. 6). These results indicate that the specific characteristics of each doogh brand, such as pH level, composition and type of additives, chemical composition and even the purity percentage of PET bottles, as well as initial production and storage conditions at the factory, can have a significant impact on the migration rate of these compounds. These factors can likely influence chemical reactions and affect the leaching process of chemical compounds into the doogh11,14,29,33,45. In this regard, a study showed that cooking oil, compared to mineral water, provides a more favorable environment for the migration of PAEs from packaging to food. Also, the migration of these compounds significantly increased at higher temperatures, longer contact times, and higher dynamic frequencies46. Additionally, it has been shown that the detectable percentage of BPA in canned food samples is much higher than in non-canned foods47. A study by Goodson et al.48 generally showed that BPA migration mainly occurs during the initial processing and that factors such as storage temperature, its duration, or damage to the can do not significantly affect the increase in migration of this substance. These results can indicate the significant impact of food production processes on the level of chemical contaminants. In addition, differences in packaging material, for example in terms of the purity percentage of PET in this study, may also explain some of the observed variations49,50. Complementing the mentioned findings, another study that investigated the migration of BPA from different bottles found that at room temperature, the transfer rate of this substance from PC bottles to water is between 0.2 and 0.3 mg/L. In contrast, BPA migration from aluminum bottles with an epoxy (resin-based) inner lining to water was in a wider range between 0.08 and 1.9 mg/L32. These results indicate that assuming a constant storage factor and even the contents of the bottle, the greater fluctuation in the rate of BPA migration to the contents inside the bottles can be due to differences in the material of the bottles and the percentage of their constituent chemical compounds.

BPA exposure and risk characteristics

The important question in this study is whether the BPA levels observed in doogh can pose a health risk to consumers. Based on a RfD of 50 µg/kg bw/day25,26, the study results (Tables 1, 2 and 3) showed that even consumption of doogh stored under the worst conditions (very high temperature, long storage time, and exposed to sunlight) does not cause BPA exposure to exceed the established limit; that is, the HQ corresponding to the maximum EDI was calculated to be < 1 for both children and adults. For children and adults, the maximum BPA intake through doogh consumption was approximately 2.0 × 10− 3 and 7.4 × 10− 4 mg/kg bw/day, respectively; which yielded corresponding HQ of 0.04 and 0.015. It is important to note that these results should not make us too comfortable, because although based on the findings of this study, doogh itself likely contributes a relatively small share to the total daily BPA intake of most individuals, this share may be significant in people with high consumption of this beverage or other packaged foods. Even BPA intake from various other sources is also a concern47,51,52. Furthermore, this issue can be of greater importance in vulnerable populations5. However, it should be noted that the increase in HQ when the product warms up (Table 1) indicates a significant relative increase in potential risk. Although this risk is still below the hazardous level, its increase should not be ignored. in addition, the increase in HQ with time (Table 2) indicates that drinking fresh dairy products can reduce exposure to migrating chemical compounds. For example, the children’s HQ (0.013 at 7 days vs. 0.027 at two months) suggests that consuming the same brand of doogh with a two-month delay can make a difference of about two times in BPA intake. furthermore, the increasing of exposure due to storage under sunlight (Table 3) is a noteworthy point for food safety. This means that improper storage conditions (such as leaving the bottle in the sun) can reduce the consumer’s safety margin.

A study conducted in Italy reported an EDI of 0.002 µg/kg bw/day due to the occurrence of BPA in raw buffalo milk53, which is much lower than our results. Another study in Italy reported an HQ < 1 for BPA in raw milk54. Studies in China55 and Greece56 also reported HQ < 1 in milk. Based on a study conducted by Jeddi et al.37, it was found that the EDI of various PAEs for children, under different storage temperature conditions and storage times of mineral water bottles, varied between 0.01 µg/kg bw/day (BBP) and 0.24 µg/kg bw/day (DEHP); although the compounds measured are different from the present study, the daily intake of PAEs in that study is much lower than the BPA intake in the present study. Also, the HQ attributed to the total PAEs present in bottled water, in the worst-case temperature and storage time scenario, was calculated to be 0.012, which is significantly lower than the acceptable limit. A study of bottled waters of different brands showed that the daily intake of DEHP, BPA, and NP through water consumption constitutes less than 0.1% of the permissible daily intake values of these compounds14. Another study also reported that the HQ resulting from exposure to three types of PAEs through the consumption of various bottled beverages and liquid foods for both children and adults was significantly lower than the acceptable limit and was generally in the range of 1.5 × 10− 6 to 2.8 × 10[− 311.

Limitations

This study provides important results regarding the migration of BPA into doogh under different storage conditions, but acknowledging its limitations can provide areas for future research.

  1. 1.

    The findings of this study may not be generalizable to all doogh products on the market or other beverages packaged in PET, as the impact of differences in bottle material composition, manufacturing processes, and product formulation on BPA migration rates requires further investigation.

  2. 2.

    Unlike this study, future research could analyze the raw PET materials, bottle caps, and the doogh product before packaging to determine the origin of the BPA and thus act more targetedly to reduce contamination.

  3. 3.

    In this study, the health risk assessment was focused solely on BPA. That is, it did not consider the potential migration of other NIAS or other plasticizers such as PAEs that might be present. This could be investigated in future studies.

  4. 4.

    The exposure assessment was based on consumption data from a researcher-developed questionnaire and standard body weights. The accuracy of the EDI could be increased in future work by using national standard data.

Conclusion

This research examined BPA levels in packaged doogh stored in PET bottles under various conditions and evaluated the associated health risks. The results showed that higher storage temperatures (from 4 °C to 25 °C and 45 °C), longer storage durations (from 7 to 60 days), and exposure to direct sunlight, compared to shade, significantly increased BPA levels. Although the impact of storage conditions followed a consistent trend across brands, significant variations in BPA concentrations were noted between brands, likely due to differences in packaging materials, doogh formulations, or production processes. In all storage scenarios, the HQ for children and adults remained < 1. However, unfavorable conditions nearly increased HQ compared to optimal storage (cool, short-term, shaded), emphasizing the need for proper storage management to minimize BPA exposure, even in the absence of serious health risks. In the beverage industry, safer, food-grade packaging materials free from BPA and other harmful substances should be prioritized. Additionally, controlling storage conditions such as refrigeration or cool, shaded, well-ventilated spaces and implementing a FEFO (First Expire First Out) system for long-term storage can further mitigate possible health risks.