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Doogh is a traditional Iranian yogurt-based drink that is served flavored or unflavored and carbonated or non-carbonated. Bisphenol A (BPA) is an endocrine disrupting analyte, which poses significant dangers to public health. The goal of our investigation was to assess the BPA content in doogh samples from Tehran along with risk assessment by using the Monte Carlo method. A nano-adsorbent of magnetized iron-based multi-walled carbon nanotubes (MWCNT-Fe3O4) was used with gas chromatography-mass spectrometry (GC/MS) to evaluate the mentioned contaminant. The average amount of BPA in doogh samples was 3.50 µg/L (ranged 0.63 to 6.75 µg/L). BPA concentrations in all doogh samples were within the standard limit. In addition, the health risks of BPA intake through doogh were assessed. The results of multivariate statistical evaluation highlighted the relationship between BPA concentrations and independent variables (volume, brand, packaging type, storage conditions, pH, fat, salt, and trans fatty acid content). According to the updated tolerable daily intake (TDI) established by the European Food Safety Authority (EFSA), the 50th percentile for the target hazard quotient of BPA in doogh samples was 2.22E + 0 for adults and 7.83E + 0 for children (THQ > 1). This evidence suggests chronic consumption of doogh from plastic or metal containers may endanger human health. The intake of BPA through doogh samples poses adverse health risks to Iranian consumers.
Introduction
Doogh is one of the traditional Iranian beverages that is commercially produced and widely consumed. It is commonly prepared by combining yogurt, water, salt, and aromatic vegetables, providing an especially taste1,2. Doogh is rich in protein, calcium, and B vitamins and is a good alternative to carbonated drinks3. It also contains beneficial probiotics such as Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, which supports gut health and improves the immune system3,4.
Today, contamination of food products by endocrine disruptors has attracted significant concern worldwide, and research on this critical issue is still in progress5,6. Previous research suggests that chemical contamination in dairy products can come from a variety of sources, including industrial activities, agricultural chemicals, veterinary drugs, processing materials, and packaging7. Milk and dairy products may be contaminated by endocrine disruptors present in fats due to their high fat content, and some endocrine disruptors have been reported in milk and dairy products, such as organochlorine pesticides, dioxins, and polychlorinated biphenyl’s2,8,9. Packaging protects food products from physical damage, chemical spoilage, and microbial contamination, thereby maintaining the quality of the packaged food product and extending its shelf life. Packaging materials are usually selected based on product characteristics, processing, storage, and intended use. However, the research studies reported that some chemical contaminants, such as phthalates, trace elements, and bisphenols, may be migrating from packaging materials to food2,8,10,11.
Bisphenol A (BPA) is widely used in food packaging materials to create polycarbonate plastics and epoxy resins12. BPA consists of two phenol groups and exhibits favorable physical and chemical characteristics13. Since BPA contains hydroxyl groups directly bound to its aromatic ring, it can be converted into ethers, salts and esters14. BPA can migrate to packaged food from polycarbonate containers via physical and chemical mechanisms1,5,6. BPA is considered a potential threat to human health because of its endocrine-disrupting properties, particularly its estrogenic effects. Some investigates propose that BPA exposure might disrupt brain growth and the natural immune systems. Additionally, BPA exposure is a factor contributing to stroke prevalence13. BPA also exhibits neurotoxic effects, causing oxidative stress and triggering apoptosis15. Ingesting BPA can impact children’s brain function and prostate glands, trigger immune responses, alter behavior, elevate blood pressure, and potentially raise the risk of diabetes and heart disorders16. In the case of BPA in food products, the commission of EU has set a migration limit of 600 µg/kg17. The Reference Dose (RfD) of BPA was established at 50 µg/kg bw/day by the U.S. EPA18 and Tolerable Daily Intake (TDI) at 0.0002 µg/kg/day by the European Food Safety Authority (EFSA) (2023)19, respectively. Moreover, the Canadian Health Organization suggested the TDI of BPA at 25 µg/kg bw/day20.
Magnetic nanoparticles (MNPs) have become a significant trend in various scientific and applied fields, including biotechnology, pharmaceuticals, electronics, and industrial processes. In addition, carbon nanotubes (CNTs) have attracted much attention in recent years. These cylindrical structures are considered potential alternatives for reinforcing composite materials due to their distinctive tubular configuration and strong adsorption capabilities. Depending on how many graphene layers rolled up to form CNTs, there are 2 types of CNTs: MWCNTs (multi-walled) and SWCNTs (single-walled)6. Magnetic multi-walled carbon nanotube composites result from combining magnetite (Fe3O4) with multi-walled carbon nanotubes (MWCNTs). These hybrids harness the distinctive characteristics of both MWCNTs and MNPs. The connection strength between MWCNTs and MNPs within the composite is robust enough to withstand mechanical energy. These merits prove that magnetic CNTs hold significant analytical capability as efficient adsorbents for determining certain substances6. Chromatographic techniques are commonly used to analyze bisphenols, including GC-MS, UPLC-MS/MS (ultra-performance liquid chromatography-mass spectrometry), and HPLC (high-performance liquid chromatography), which GC-MS is more accurate, cheaper and more practical6.
Doogh is a widely consumed dairy product in the Iranian food basket. BPA is an endocrine disruptor, so its presence in food products raises concerns worldwide. Prior studies have examined the chemical migration of BPA into dairy products and yogurt based drinks, and packaging, equipment of processing, and storage tanks have been identified as potential sources of pollution1,21. To our knowledge, there is currently no comprehensive study (case studies have been seen) on BPA levels in doogh in the world. In this study, we applied a practical and accurate method utilizing magnetic multi-walled carbon nanotubes decorated with Fe3O4 (MWCNTs-Fe3O4) for the extraction and analysis of BPA in Iranian doogh beverages through the MSPE-GC/MS technique. “Indeed, this new method not only increases the sensitivity and selectivity of BPA detection, but also addresses growing concerns about food safety and exposure to the chemical in everyday consumer products. By assessing the risk of BPA exposure in children and adults, our research provides valuable insights into public health implications and emphasizes the need for careful monitoring of food products to protect vulnerable populations from potential endocrine disruptors.” This innovative method paves the way for more effective environmental and health assessments in food safety investigation. Therefore, the aim of the present study was to investigate the BPA content in commercial doogh using magnetic MWCNTs and GC/MS. Also, the human health risks associated with BPA exposure in ingested doogh were investigated.
Materials and methods
Materials
Sulfuric acid (H2SO4), nitric acid (HNO3), sodium acetate (C2H3NaO2), ethylene glycol (C2H6O2), iron(III) chloride hexahydrate (FeCl3·6H2O), n-hexane (C6H14), methanol (CH3OH), sodium chloride (NaCl), and amyl alcohol (C5H11OH) were brought from Merck Co., (Darmstadt, Germany). BPA standard sourced from Sigma-Aldrich (West Chester, PA; USA). All other chemicals applied in our research were of analytical grade.
Sample collection
A total of 64 samples of doogh were collected from the supplying stores in Tehran, Iran. In fact, doogh samples were purchased in two forms: carbonated (32 samples) and non-carbonated (32 samples). Also, doogh samples were divided into 4 groups based on packaging (can, PET bottle, glass, and plastic bag). These 4 groups of samples were obtained from two storage conditions of 4 °C (refrigerator) and 25 °C (ambient temperature). Finally, the collected samples were transported to the laboratory and stored at refrigerator temperature (4 °C) until further analysis.
Preparation of the MWCNT-Fe₃O₄ composite
Functionalizing MWCNTs with groups of carboxyl and hydroxyl
Initially, 2 g of multi-walled carbon nanotubes (MWCNTs) were added to 20 mL of HNO3 and 60 mL of H2SO4 to form functional groups with negative charges. Next, the solution was ultrasonicated for 6 h to attach groups of carboxyl and hydroxyl to MWCNTs. Finally, deionized water (100 mL) was added to mixture, then filtered and dried for 12 h at 60 °C6.
Magnetization MWCNTs with Fe3O4
In this step, 6 g of FeCl3·6H2O, 1 g of the functionalized MWCNTs and 7 g of sodium acetate were added to 100 mL ethylene glycol and was shaken for 30 min. The prepared solution was refluxed for 16 h, and after cooling (at room temperature), 100 mL ethanol was added and then was shaken for 10 min. The solution was filtered and washed with deionized water. Finally, the synthesized magnetic MWCNTs (MWCNT-Fe3O4) was dried at 50 °C for 24 h22.
Scanning electron microscopy (SEM), X-ray diffraction (XRD) and fourier transform infrared (FT-IR)
Morphological analysis was performed utilizing scanning electron microscope (SEM, PHILIPS, and S360 Mv2300), phase identification of the prepared adsorbent was conducted by X-ray Diffraction (XRD, Philips, X’PertPro 2002) and Fourier Transform Infrared (FT-IR, Nicolet model 510)22.
Bisphenol a measurement
Sample Preparation
First, 10 mL of doogh samples and 100 µL of BPA-d14 (0.1% in ethanol) as internal standard were added to 0.1 g of MWCNT-Fe3O4 and 0.5 g of NaCl, vortexed for 20 min, then the supernatant was removed. To separate BPA from the adsorbent, n-hexane (2 mL) was added to MWCNT-Fe3O4, then vortexed for 10 min and simultaneously placed in an ultrasonic bath. Next, the absorbent was gathered on the side of the tube utilizing a magnet (external), and the supernatant was filtered using a 0.45 μm syringe filter and withdrawn to a vial. The solution was dried at 25 °C and stored at refrigerator temperature. To the sample extracted from the previous step, 50 µL of MSTFA (N-Methyl-N-trimethylsilyltrifluoroacetamide) derivatizer was added and incubated at 50 ᵒC for 1 h. The dried contents of the vial were solved in 0.1 mL of methanol, and 1 µL of the solution was injected into the GC-MS6.
GC-MS analysis
The analysis of samples was conducted employing GC (Varian-450) with a 300-MS triple quadrupole mass spectrometer (Varian Inc., USA) and equipped with a DB-1701 MS capillary column (Varian Inc., USA, film thickness: 0.25 μm; internal diameter: 0.25 mm; column length: 30 m). The flow rate of helium carrier gas (99.999%) was 1 mL/min. The sample injection volume was 1.0 µL in the split and splitless injection mode. Additionally, for the detector, the SIM mode (selected ion monitoring) was applied. The temperature of the ion source was 250 °C, and the quadrupole temperature was 250 °C with 70 eV. The temperature instructions for the oven were as follows: the temperature initial was 150 °C with 1 min hold, and ramp was 270 °C at 20 °C/min with 28 min hold. Figure S1 shows chromatogram of standard sample6.
Quantification method
To the research of linearity, 5 samples (in series) were ready by adding levels of standard (5–1000 µg/L) on different days (in triplicate) and then were injected into the GC-MS (in duplicate). Moreover, for each triplicate, 2 control negative samples were prepared, (1) without sample and (2) without matrix. To draw a BPA’s curve of calibration, the attained data were applied. By the estimation of the relative standard deviation (RSD), the precision was evaluated, also known as the coefficient of variation (CV), which was lower than 8%, presenting the efficiency of the technique for repeatability (intra-day) and for accuracy of intermediate (inter-day). By the pollution with 0.20 µg/L of BPA analyte under analysis, 5 samples (equally) were ready for repeatability. The mentioned samples were evaluated according to the recommended method, from the extraction stage (all on the same day) to the examination by GC-MS. To verify the accuracy of the study, the standard addition technique was used, which involved adding various known amounts of validated BPA standards to the matrix (before the preparation of sample)6.
pH, Salt, Fat, and trans fatty acid measurement
pH, salt, trans fatty acid and fat were determined according to Iran National Standards Organization (INSO) numbers 2852, 694, and 10,29223.
Human health risk assessment
To assess the health risk of BPA via doogh consumption by Iranian adults and children, the estimated daily intake (EDI) and target hazard quotient (HQ) were computed by the following formulas24:
1
2
Where C is the amount of BPA in doogh samples (mg/kg), IR is the doogh ingestion rate in Iran (0.009 Kg/day)25, BW is average body weight (for children, and adults is 15 and 70 Kg, respectively), and tolerable daily intake (TDI) as the non-carcinogen risk, which was acquired from the European Food Safety Authority (EFSA), for bisphenol A (BPA) of 0.0002 µg/kg/day26. A Monte Carlo simulation (Oracle@ Crystal Ball, Oracle Corporation, USA) assessed uncertainty. Simulation accuracy was determined by critical parameter configuration, and the implementation involved 10,000 iterative cycles (Table S1).
Statistical analysis
In this study, the obtained data were analyzed utilizing SPSS v 27. Kolmogorov-Smirnov was utilized to assess the normality of the research parameters. Additionally, the test of Kruskal–Wallis was utilized to examine significance among groups. We calculated Spearman correlation coefficients between the concentrations of BPA in the doogh samples with their corresponding salt, fat, trans fatty acid, gas presence, and pH values. Significance level considered p < 0.05. Principal component analysis (PCA) was applied for studying data structure and identifying similarities among samples. Because of the chaotic nature of the collected data, which may include errors and invalid entries, and because the format and scale vary, data processing is necessary after collection. The data was normalized using the mean normalization formula. Before processing the data with factor analysis, the suitability of data for factor analysis was checked by Kaiser–Meyer–Olkin (KMO) test and Bartlett’s sphericity test. Next, we performed linear dimensionality reduction using principal component analysis (PCA).
Results and discussion
SEM image
Figure 1 shows that the SEM images of MWCNT and MWCNT-Fe3O4. Figure 1 shows that the MWCNT tubes are randomly and irregularly intertwined. It can be clearly seen that Fe3O4 adheres to the MWCNT surfaces like knots and its distribution is almost uniform. The surface of NWCNTs remains intact after loading with iron nanoparticles, but becomes rougher. In general, the diameter of MWCNT-Fe3O4 particles ranges from 34 to 38 nm. The Fe3O4 nanoparticles do not completely surround the MWCNTs, so the adsorbent provides efficient adsorption capacity of the MWCNTs and suitable ferromagnetic and catalytic properties of Fe3O4. Therefore, MWCNT is available as a carrier for good dispersion of the Fe3O4 particles, which results in high adsorption capacity of the adsorbent.
Fig. 1 [Images not available. See PDF.]
SEM images of MWCNTs (a), and MWCNT-Fe3O4 (b).
XRD image
The XRD patterns of MWCNT and MWCNTs-Fe3O4 are displayed in Fig. 2a and b, respectively. X-ray diffraction pattern of MWCNTs shows typical diffraction peaks at 2θ = 26.31° and 2θ = 31.127°, corresponding to the (0 0 2) and (5) crystallographic planes of MWCNT, with reference codes 00–041-1487 and 00–046-0945, respectively, which is similar to the previous findings27. After the deposition of the iron nanocomposites, distinct diffraction peaks indicate good crystallinity in the Fe3O4 samples, with a notable peak at 2θ = 35.452° (reference code 00–003-0863). The strong peak at 2θ = 26.31° remains, corresponding to the (0 0 2) crystallographic plane of MWCNT. These XRD results confirm that Fe3O4 was successfully coated onto the MWCNTs using the co-precipitation method.
Fig. 2 [Images not available. See PDF.]
XRD images of MWCNTs (a), and MWCNT-Fe3O4(b).
FT-IR analysis
FT-IR spectra of MWCNT and MWCNT-Fe3O4 are revealed in Fig. 3. This broadening can be attributed to several factors, including particle size distribution, the presence of different chemical components (multi-walled carbon nanotubes, iron oxide, and silver), and potential interactions between the nanoparticles. The FT-IR spectrum of MWCNT-Fe3O4 displays a broad peak in the 3000–3500 cm⁻¹ range, corresponding to the stretching frequency of the O-H bond in hydroxyl groups. The peak around 1586 cm⁻¹ is associated with the Fe-O-Fe stretching vibration in Fe3O4, while the peak at 1610 cm⁻¹ is linked to the stretching mode of C = C double bonds and the bending vibrations of H-N single bonds. This assignment (1586 cm⁻¹) is further validated by the absence of conflicting peaks and alignment with material-specific characteristics (e.g., nanostructure, coordination environment)28. A significant peak in the 3000–3500 cm⁻¹ region, indicative of OH groups, narrows after BPA extraction, suggesting a reduction in the number of OH-containing groups produced by the extraction29. Additionally, the vibrational peaks near 1180–1460 cm⁻¹, attributed to Metal-OH, show a reduction, indicating a decrease of hydroxyl following BPA extraction. This suggests that surface hydroxyl groups (M-OH) were substituted by adsorbed BPA molecules, playing a crucial role in BPA extraction. Researchers propose several potential mechanisms: (1) BPA interacts hydrophobically with the adsorbent; (2) π-π electron donor-acceptor interactions, (3) hydrogen connection between the BPA and O-containing functional groups, and (4) substitution of OH on the adsorbent surface with BPA. According to the FT-IR analysis after BPA extraction, the substitution of OH between BPA and the MWCNT-Fe3O4 surface appears to be a primary mechanism in the adsorption process.
Fig. 3 [Images not available. See PDF.]
FT-IR spectra of MWCNT (a), and FT-IR spectra of MWCNT-Fe3O4 after extraction (b).
Method validation results
Under optimized conditions, the performance of this technique was examined. A series of standard solution containing 5–1000 µg/L of BPA were utilized for plotting the curve of calibration. The curves of calibration were constructed by plotting the peak areas versus the levels of the individual compound with triplicate measurements. The LODs (limits of detection) were calculated based on a signal-to-noise ratio (S/N) of 3 and the LOQs (limits of quantification) were calculated based on an S/N of 10. The LODs were obtained in the range of 0.01 µg/L. The percentage of recovery was higher than 86.9%, which serves to demonstrate the accuracy and precision of the technique (Table 1).
The results achieved in our research were analogous and confirmed researches with other procedures. In analogous research, Massahi et al. analyzed BPA in doogh samples with method of SPE/HPLC-UV and reported the LOD, LOQ, R2, average recovery range and RSD were 0.09 mg/L, 0.28 mg/L, 0.9999, 98.2 to 103.6% and 8.7%, respectively1. Besides, Jiao et al. applied MWCNT-MNP and GC-MS/MS for the BPA’s measurement and stated the recovery was 90.3–103.7% with RSD less than 10%. The R2 and LOD were 0.9988 and 0.001 µg/L, respectively30. In other reserach, Zang et al. prepared graphene grafted magnetic microspheres and utilized as the adsorbent in MSPE technique for the BPA’s measurement. They stated the LOD was 10.0 ng/L, the recovery was in the range from 93.5% to 99.5% (inter-day) and from 93.9% to 104.3% (intra-day), with the RSD varying from 3.1% to 5.7% (inter-day) and from 2.1% to 5.8% (intra-day)31. Additionally, Hazrati-Raziabad et al. investigated BPA level with method of derivatization by GC/MS and reported the R2, recovery, LOD, LOQ, and RSD were 0.991, 99.8%, 0.001 µg/L, 0.0035 µg/L, and 8.9%, respectively32.
Table 1. Outcomes of method validation.
Compound | LOD (µg/L) | LOQ (µg/L) | RSD (%) | R2 | Recovery (%) |
|---|---|---|---|---|---|
BPA | 0.001 | 0.0035 | 8 | 0.994 | 86.9 |
BPA concentrations and other parameters in Doogh samples
Figure S 1,2. shows examples of chromatograms of real samples. In our investigation, Table 2 shows the 6 parameters of physicochemical (salt, fat, trans fatty acid, volume, pH values, and concentrations of BPA) results of 32 samples of doogh. The mean of BAP in all samples was 3.5 µg/L (in the range of 0.63–6.75 µg/L).
Table 2. The concentrations of BPA, salt, fat, trans fatty acid, gas presence, and pH values in the Doogh samples.
Fat% | pH | Salt% | Trans fatty acid% | Sample_volume (mL) | BPA_concentration (µg/L) | |
|---|---|---|---|---|---|---|
Minimum | 0.10 | 3.20 | 0.61 | 0.00 | 240.00 | 0.63 |
Maximum | 1.50 | 4.60 | 0.85 | 0.65 | 1500.00 | 6.75 |
Mean | 0.94 | 3.92 | 0.78 | 0.09 | 541.07 | 3.50 |
Median | 0.85 | 4.06 | 0.80 | 0.02 | 320.00 | 3.41 |
Std. Deviation | 0.50 | 0.50 | 0.05 | 0.20 | 406.19 | 1.87 |
Salt, fat, Trans fatty acid and pH content ranged from 0.61 to 0.85%, from 0.10 to 1.50%, from 0.00 to 0.65%, and from 3.2 to 4.6, respectively.
BPA levels in different types of different Doogh packaging (PET, cup, can, and plastic bag)
Table shows the BPA levels in different doogh packaging separately. BPA concentrations were in the following decreasing order based on doogh packaging materials: cup (mean = 3.83 µg/L) > can (mean of 3.81 µg/L) > PET (mean = 3.25 µg/L) > plastic bag (mean = 3.14 µg/L). There was no statistically significant difference in the concentrations of BPA in different doogh packaging (p > 0.05).
The results of our study show a clear relationship between the type of yogurt packaging and the amount of bisphenol A (BPA) migration. We observed that the average BPA concentration in glass packaging (3.83 µg/L) was the highest, followed by cans (3.81 µg/L) with a small difference, while PET bottles had the lowest average concentration of 3.25 µg/L and plastic bags had the lowest concentration of 3.14 µg/L. This trend suggests that some materials, especially glasses and cans, are more susceptible to BPA migration compared to PET and plastic bags. The differences in BPA levels can be attributed to factors such as the chemical composition of the packaging materials, the interaction between the doogh and the packaging during storage, and the temperature conditions6. Our findings highlight the need for further investigation into the mechanisms of BPA migration across different packaging types and emphasize the importance of selecting safer packaging materials for food products to minimize consumer exposure to harmful chemicals.
In similar research, Massahi et al. analyzed BPA in doogh samples with method of SPE/HPLC-UV and reported higher temperatures (e.g., 0.139–0.160 mg/L at 4 °C vs. 0.269–0.318 mg/L at 45 °C), longer storage (e.g., 0.107–0.115 mg/L at 7 days vs. 0.217–0.238 mg/L at 60 days), and direct sunlight exposure (e.g., 0.174–0.194 mg/L in shade vs. 0.304–0.340 mg/L in sunlight) increased (significantly) migration of BPA1. Also, koti et al. analyzed BPA by MSPE-GC/MS method in soft drink samples and reported the BPA’s average level in all samples was 1.83 ± 1.16 µg/mL (ranging from 0.45 to 5.10 µg/L)6. Our findings was similar to the research by Kovačič et al., which found the maximum amount of BPA in canned beverages is 2 to 4 times more than in plastic containers33. In contrast, Moid ALAMMARI et al. reported that the elevated BPA migration in PET bottles was higher than in cans34. Kang et al. and Mercogliano et al. didn’t found BPA in non-canned foods, however, they reported that the BPA in canned foods and recommended that BPA may migrate from packaging materials into dairy products35. In the research by Kumar et al., BPA mean canned Soft drinks ranged 0.01 µg/L from 0.014 µg/L, respectively, which was less than the BPA concentration in this study36. In the research by Geens et al., BPA mean and range in Canned Beverages was 1 µg/L and 0–8 µg/L respectively which was less than BPA concentration in this study37.
Table 3. Comparisons of BPA levels in different Doogh packaging (PET, cup, can, and plastic bag).
Package | Fat% | pH | Salt% | Trans fatty acid% | Sample_volume (mL) | BPA_concentration (µg/L) | |
|---|---|---|---|---|---|---|---|
Can | Minimum | 0.51 | 3.20 | 0.80 | 0.00 | 320.00 | 1.43 |
Maximum | 1.50 | 4.60 | 0.82 | 0.02 | 320.00 | 5.16 | |
Mean | 0.95 | 3.82 | 0.81 | 0.01 | 320.00 | 3.81 | |
Median | 0.51 | 3.40 | 0.82 | 0.00 | 320.00 | 3.56 | |
Std. Deviation | 0.52 | 0.69 | 0.01 | 0.01 | 0.00 | 0.98 | |
Cup | Minimum | 0.79 | 3.20 | 0.75 | 0.00 | 240.00 | 1.72 |
Maximum | 1.50 | 4.40 | 0.85 | 0.25 | 250.00 | 6.48 | |
Mean | 1.07 | 4.03 | 0.79 | 0.06 | 244.00 | 3.83 | |
Median | 0.79 | 4.20 | 0.75 | 0.04 | 240.00 | 3.07 | |
Std. Deviation | 0.39 | 0.48 | 0.05 | 0.11 | 5.48 | 1.89 | |
Pet | Minimum | 0.10 | 3.20 | 0.61 | 0.00 | 250.00 | 0.63 |
Maximum | 1.50 | 4.40 | 0.85 | 0.13 | 1500.00 | 6.75 | |
Mean | 0.92 | 3.97 | 0.79 | 0.03 | 694.44 | 3.25 | |
Median | 1.50 | 4.20 | 0.80 | 0.02 | 250.00 | 2.23 | |
Std. Deviation | 0.70 | 0.41 | 0.07 | 0.04 | 555.90 | 2.34 | |
Plastic | Minimum | 0.79 | 3.50 | 0.75 | 0.00 | 900.00 | 0.87 |
Maximum | 0.90 | 4.30 | 0.80 | 0.65 | 1000.00 | 6.43 | |
Mean | 0.86 | 3.90 | 0.78 | 0.39 | 960.00 | 3.14 | |
Median | 0.90 | 4.05 | 0.80 | 0.65 | 1000.00 | 0.98 | |
Std. Deviation | 0.06 | 0.34 | 0.03 | 0.35 | 54.77 | 2.57 | |
P-value | 0.798 | 0.283 | 0.396 | 0.00 | 0.00 | 0.735 | |
BPA levels in different types of temperature (refrigerator, and room)
Table 4 shows the impact of temperature (refrigerator, and room) on BPA levels in doogh samples. According to this table, BPA levels were higher at room temperature (3.34 ± 1.77 µg/L) compared to refrigerator temperature (2.88 ± 2.07 µg/L), but no significant difference was found among the BPA levels stored at room temperature and in the refrigerator (p > 0.05).
Ucheana et al. indicated that BPA concentrations in plastics tend to be higher at room temperature compared to refrigerator temperatures38. While BPA concentrations remained statistically similar (p > 0.05) under the study’s storage conditions, BPA migration rates showed a significant difference (p < 0.05)38. Studies have shown that increased temperature, such as that found at room temperature, can accelerate the migration of BPA from plastic into stored liquids. Conversely, lower temperatures, like those in a refrigerator, tend to slow down this migration process38.
Table 4. Comparisons of BPA levels in different temperature (refrigerator, and room).
Temperature | Fat% | pH | Salt% | Trans fatty acid% | Sample volume (mL) | BPA_concentration (µg/L) | |
|---|---|---|---|---|---|---|---|
Refrigerator | Minimum | 0.10 | 3.10 | 0.75 | 0.00 | 240.00 | 0.63 |
Maximum | 1.50 | 4.28 | 0.85 | 0.65 | 1500.00 | 6.48 | |
Mean | 1.01 | 3.82 | 0.80 | 0.07 | 509.09 | 2.88 | |
Median | 0.90 | 4.05 | 0.80 | 0.02 | 320.00 | 2.23 | |
Std. Deviation | 0.51 | 0.49 | 0.03 | 0.19 | 427.07 | 2.07 | |
Room | Minimum | 0.10 | 3.20 | 0.20 | 0.00 | 240.00 | 0.87 |
Maximum | 1.50 | 4.28 | 0.85 | 0.65 | 1500.00 | 6.75 | |
Mean | 0.90 | 3.90 | 0.77 | 0.11 | 561.76 | 3.34 | |
Median | 0.79 | 4.06 | 0.80 | 0.02 | 320.00 | 3.23 | |
Std. Deviation | 0.51 | 0.44 | 0.15 | 0.21 | 404.06 | 1.77 | |
P-value | 0.439 | 0.533 | 0.233 | 0.514 | 0.365 | 0.63 | |
BPA levels in different brands of Doogh samples
Table 4 indications the amounts of BPA concentrations in different brands of doogh samples. Based on this table, brand B had a higher level of contamination than brand A. As is clear from Table 4, the average level (µg/L) of BPA concentrations in brand A doogh samples was 2.84 ± 1.63 (µg/L), and in brand B was 3.49 ± 2.09 (µg/L). There was no statistically significant difference in the concentrations of BPA in different brands (p > 0.05).
Table 4. Comparisons of BPA levels in different brand (A, and B).
Brand | Fat% | pH | Salt% | Trans fatty acid% | Sample_volume (mL) | BPA_concentration (µg/L) | |
|---|---|---|---|---|---|---|---|
A | Minimum | 0.90 | 4.03 | 0.80 | 0.02 | 250.00 | 0.71 |
Maximum | 1.50 | 4.60 | 0.85 | 0.65 | 1500.00 | 6.19 | |
Mean | 1.37 | 4.32 | 0.81 | 0.16 | 609.29 | 2.84 | |
Median | 1.50 | 4.30 | 0.80 | 0.03 | 320.00 | 2.84 | |
Std. Deviation | 0.26 | 0.19 | 0.02 | 0.26 | 482.37 | 1.63 | |
B | Minimum | 0.10 | 3.20 | 0.61 | 0.00 | 240.00 | 0.63 |
Maximum | 0.79 | 4.30 | 0.82 | 0.25 | 1000.00 | 6.75 | |
Mean | 0.52 | 3.52 | 0.78 | 0.03 | 472.86 | 3.49 | |
Median | 0.51 | 3.45 | 0.80 | 0.00 | 320.00 | 3.44 | |
Std. Deviation | 0.26 | 0.37 | 0.06 | 0.07 | 316.17 | 2.09 | |
P-value | 0.000 | 0.000 | 0.028 | 0.013 | 0.208 | 0.192 | |
Correlation based on all observations
Correlation based on all observations is crucial to a study because it provides a framework for organizing and interpreting data. Its purpose is to uncover patterns, relationships, and significant differences within BPA in doogh, enabling valid conclusions and informed decisions. Correlation coefficients and statistical significance were assessed using Spearman’s test. Table 6 presents the correlation coefficients of BPA concentrations in the doogh samples. These concentrations were not significantly associated with BPA levels when considering salt, fat, trans fatty acids, gas presence, sample volume, temperature, packaging, and pH values across all sample levels (p > 0.05, Kruskal-Wallis, spearman-test). This may suggest that packaging type is the dominant factor affecting BPA migration, while other variables may have less influence.
There was no statistically significant difference in the concentrations of BPA for any of the parameters, brand, packaging type, and temperature (Tables 2 and 3, and 4). The pH value in doogh varied from 3.20 to 4.60 (mean = 3.92). Based on research, raising the pH level can speed up the hydrolysis of beverage containers39. To put it differently, the stability of the packaging could be influenced by the pH level40. These results were not in accordance with study by Benhamada et al.41, which found a significant correlation between pH and BPA levels. The highest average amount of BPA in doogh samples was from brand B (3.49 µg/L), and the lowest average concentration of BPA in doogh was from brand A (2.84 µg/L). The maximum content of BPA was in the doogh with cup packaging inside the refrigerator in brand B (6.75 µg/L), and the lowest amount of BPA related to doogh in the PET packaging at room temperature in brand B (0.63 µg/L).
BPA can be transferred to cow’s milk and then to doogh through a variety of routes, from packaging, water and factory equipment. In packaging, BPA is often found in the lining of cans or plastic containers, which can be released into doogh due to factors such as heat, acidity and the presence of fat (fat or free fatty acids in food can help BPA particles migrate into food systems). Considering the lipophilic nature and chemical structure of BPA, it should have a high affinity for fat. However, existing research has not shown any significant relationship between fat content (FFA) and BPA and long-term storage in various packaging. BPA can enter milk indirectly through the animal’s system or during processing when water contaminated with BPA is used for drinking by cows or in cleaning processes. In addition, factory equipment made of or coated with materials containing BPA can release BPA into milk during production or packaging42,43. The combination of these factors could lead to the presence of BPA in dairy products, including doogh. Future research should focus on refining these findings by expanding sample sizes or examining different packaging conditions, such as long-term storage or exposure to extreme temperatures, to more accurately assess their impact on BPA transfer.
Table 6. The concentrations of BPA in the Doogh samples with their corresponding salt, fat, trans fatty acid, gas presence, and pH values.
Spearman’s rho (Correlation Coefficient) | fat | pH | Salt | Trans fatty acid | Sample volume | BPA concentration |
|---|---|---|---|---|---|---|
Fat | 1.00 | |||||
pH | 0.79 | 1.00 | ||||
Salt | 0.22 | 0.00 | 1.00 | |||
Trans fatty acid | 0.55 | 0.56 | 0.06 | 1.00 | ||
Sample volume | 0.00 | 0.02 | 0.17 | 0.26 | 1.00 | |
BPA concentration | −0.05 | −0.15 | 0.03 | −0.13 | 0.11 | 1.00 |
Multivariate analysis
Principal Component Analysis (PCA) is a flexible statistical procedure utilized to simplify complex datasets containing observations and variables by distilling them into their most essential components. These components are derived as optimized linear combinations of the original variables and represent directions of maximum variance within the data. In our investigation, PCA was employed to explore the relationship among several independent variables such as volume, brand, packaging type, storage conditions, pH, fat, salt, and trans fatty acid content and BPA levels. The analysis of BPA content in doogh, is illustrated in Fig. 4. The primary aim was to uncover general distribution patterns or similarities in BPA content across samples. As the reduction in Euclidean space progressed, the samples demonstrated increasing correlations. The outcomes revealed that the samples clustered into three main groups, reflecting the relationships among BPA levels in doogh. PCA identified two principal components that together explained 70.1% of the total variance, specifically, (31% for PC1, 23.1% for PC2 and 16% for PC3). In this study, the KMO value was 0.65 and the p value of the Bartlett test of sphericity was significant (p < 0.001). The analysis of variance explained by each component highlighted that those capturing more variance represented more significant information, which is crucial for reducing dimensionality.
In the parameter plot of doogh (see Fig. 4), the first principal component was primarily characterized by fat content positively and by brand negatively. The second component was positively associated with packaging type, trans fatty acids, and sample volume. The third component was negatively related to BPA content. BPA levels in doogh samples had a positive correlation with fat content. Overall, the PCA findings were highly effective in assessing similarities and differences within and between groups, demonstrating that BPA levels in doogh samples could be clearly distinguished based on these principal components.
Fig. 4 [Images not available. See PDF.]
Principal component analysis (PCA) of doogh (a yogurt-based Iranian drink).
Assessment of human health risk
Figure 5 shows the estimated daily intake (EDI) values of BPA exposure through consuming doogh for Iranian children, and adults. The results showed that the rank order of the EDI values for each type of packaging was cup > can > PET > plastic bag, as presented in Table S2. The 50th percentile EDI values of BPA in all doogh samples for children and adults were 1.57E-6 and 4.5E-7 mg/kg bw/day, respectively. According to EFSA guidelines, EDI values for children, and adults were higher than the TDI (2E− 6 mg/kg bw/day) of BPA44. If THQ values more than one, it is considered an expected potential health risk and vice versa45,46. The results showed that the rank order of THQ values for each type of packaging was cup > can > PET > plastic bag, as presented in Table S2. The 50th percentile THQ values in all doogh samples for adults and children were 2.22E + 0 and 7.83E + 0, respectively (Fig. 5). THQ values for examined groups were higher than 1, and consumption should be considered as a potential non-carcinogenic health risk. The updated tolerable daily intake (TDI) established by EFSA implies that consuming doogh may pose health hazards, particularly to young children, who are more vulnerable to chemical exposure.
In comparison with previous research, Bemrah et al. concluded that the range of ADI values of BPA for adults and children was 7.7E− 5 −87 E− 5 mg/kg bw/day and 119 E− 6 −141 E− 6mg/kg bw/day, respectively47. Value of ADI migrated from canned foods in Korea reported by Lim et al. was 15 E− 4 mg/kg bw/day, and hazard index value was 3E− 248. Kumar et al. stated that the amount of BPA in Plastic packaging for milk in EDI and HQ was 18 E− 4 mg/kg bw/day and 0.36, and for cold drinks were 63 E− 5 mg/kg bw/day and 1.316. A related investigation in Australia by Marchiandi et al. found that the daily intake of BPA from non-alcoholic drinks packaged in cans, plastics, and glass ranged from 8.9E− 5 to 4.23E− 04 mg/kg/bw/day for different age groups, values which are approximately 2000 times above the safety threshold49. Sources of BPA and considerations for cumulative exposure come from various origins, mainly through contamination of food and drinks from plastic containers, canned foods, and food processing tools. Strategies to reduce exposure include selecting BPA-free products and limiting canned food intake32.
Fig. 5 [Images not available. See PDF.]
EDI and THQ values of bisphenol A exposure through doogh (a yogurt-based Iranian drink) consumption by children, and adults.
Conclusion
The chief objective of this investigation was to determine levels of BPA in doogh samples using MWCNT-Fe3O4/GC-MS. The prepared adsorbent (MWCNT-Fe3O4) demonstrated high efficiency and sensitivity, as confirmed by GC/MS analysis. The successful synthesis of MWCNT-Fe3O4 was verified through XRD patterns, FT-IR spectra, and SEM images, which also confirmed its effectiveness and adsorption capacity. It is important to highlight that both the preparation and construction procedures of the adsorbent presented in current research are innovative, and the resulting material shows a strong ability to adsorb and desorb BPA, even in complex environments like food and beverages. According to the obtained results of method validation, including recovery (86.9%), RSD (12.3%), and LOD (0.001 µg/L), this method was suitable for measuring bisphenol A in food matrixes. The average amount of BPA in doogh samples was 3.50 µg/L. The average contamination in different packages was in the order of cups > cans > PETs > plastics. But, there was no statistically significant difference in the concentrations of BPA in different doogh packaging (p > 0.05). Health risk modeling through probabilistic assessment demonstrates that consuming doogh poses a significant health threat (THQ > 1). This study is limited by its limited sample size and cross-sectional design, which precludes causal interpretation. The doogh samples only represent recent exposure and may not reflect long-term accumulation. Sources of environmental exposure were not assessed. Further longitudinal research is needed to understand the ongoing health effects of BPA exposure and to find effective measures to control BPA -related risks. Implementing such measures would not only enhance BPA regulation but also establish a framework for managing structurally analogous compounds. Expanded monitoring should prioritize high-risk food categories, including dairy products and liquid consumables (particularly drinks), all requiring thorough safety evaluations.
Author contributions
Nabi Shariatifar: Conceptualization, Supervision, Design of study, Methodology, Writing- Reviewing and Editing, Majid Arabameri: Design of study, Methodology, Writing- Reviewing and Editing. Hassan Hamedi: Visualization, Investigation, Data curation, Validation. Zahra Rezapour: Methodology, Software, Writing- Original draft, Validation.
Data availability
Data is provided within the manuscript or supplementary information files.
Declarations
Competing interests
The authors declare no competing interests.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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