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Detection and Characterisation of Microplastics in Food Grade Salts in India
* Corresponding author: Dr. Gunimala Chakraborty, Department of Molecular Genetics and Cancer, Nitte University Centre for Science Education and Research, NITTE (Deemed to be University), Paneer Campus, Deralakatte, Mangaluru 575022, Karnataka, India. gunimala@nitte.edu.in
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Received: ,
Accepted: ,
How to cite this article: Vittal R, Xavier S, Mohan M, Chakraborty A, Chakraborty G. Detection and Characterisation of Microplastics in Food Grade Salts in India. J Health Allied Sci NU. doi: 10.25259/JHASNU_126_2025
Abstract
Objectives
Plastic waste accumulation is a growing menace affecting both aquatic and terrestrial environments. One of the primary concerns associated with plastic pollution is the accumulation of microplastics (MPs) in the marine ecosystem, which is a matter of grave concern because marine resources are one of the primary contributors to the human food supply. In addition, the marine environment possesses a plethora of bioactive compounds that are used in a wide variety of products, intended for human use. One of the easiest routes of MPs ingestion from the marine environment is through salt, an indispensable ingredient in cooking. This study aimed to analyse commercial brands of sea salt and rock salt for the presence of MPs.
Material and Methods
A total of thirty different brands of salts available in India were collected and analysed by Nile red fluorescent staining (NR) and characterizing the plastic polymers by Fourier transform infrared spectroscopy (FTIR).
Results
The results indicate a high prevalence of Microplastics (MPs) in sea salt samples, ranging from 13 to 27 particles/100g, and in rock salt, ranging from 8 to 29 particles/100g. Both plastic microfibers and microparticles were detected, ranging between 2-14 particles/100g and 2-27 particles/100g, respectively. Microplastic sizes varied from 19.45 μm to 512.91 μm in sea salt and 29.69 μm to 1432.85 μm in rock salt. Fourier transform infrared spectroscopy (FTIR) identified polyethylene terephthalate (PET) as the most prevalent polymer (37%) in salt samples, followed by polyvinyl chloride (PVC-25.9%), polypropylene (PP-22.2%), polyethylene (PE -11%), and polystyrene (PS -3.7%).
Conclusion
This study highlights yet another source of MPs ingestion by humans. Given the fact that salt is a preservative, a taste enhancer, and a source of an essential micronutrient, there is an imminent need for potential mitigation techniques to ensure MP-free salts for human consumption.
Keywords
Fourier transform Infrared spectroscopy
Marine pollution
Microplastic
Polyethylene terephthalate
Sea salt
INTRODUCTION
In recent years, the health impacts of plastic pollution have drawn closer attention on a global scale, particularly on the microscopic but alarming fragments known as microplastics (MPs). MPs, often defined as plastic particles smaller than 5 mm in size, are pervasive in our environment and can be encountered anywhere from the deepest ocean depths to the most isolated mountain ranges.[1,2] Marine plastic pollution has become a significant worldwide environmental issue, alongside ozone depletion, climate change, and ocean acidification.[3] Since its first commercial, favourable factors like stability, lightweight, and low production costs. However, over the last few decades, excessive production and use have led to environmental contamination in an unprecedented manner worldwide.[4] MPs can easily infiltrate the aquatic environment due to anthropogenic activities.[5]
Studies have shown that MPs enter the human body by consuming food and water from the ecosystems infiltrated with them.[6] Indeed, MPs have been detected in the digestive system, feces,[7-9] blood,[10] and lungs[11] of humans. Ingestion of MPs has been shown to cause detrimental effects in model system-based research, primarily through the polymers’ disruption of endocrine receptor functions.[12] MPs are now considered emerging food pollutants as they can move up the food chain.[13] Thus, understanding the number of MPs the human body is inadvertently taking is crucial in establishing consumption guidelines to minimise pollution effectively and analyse health hazards associated with MP exposure.[14] Salt is an indispensable component in every cuisine worldwide. It serves as a taste enhancer, a source of micronutrients, and a preservative. Therefore, daily salt consumption is inevitable, as is the case for water and air.[15,16] An estimated 300 million tonnes of salt were consumed globally in 2018, of which approximately 11.6% (including table salts and food processing) was for human consumption.[17] Table salt is classified into different types according to their origins, such as sea salt, lake salt, rock salt, and river or well salt. Sea salt and lake salt are obtained by evaporation, whereas rock salts are produced by mining a mineral rock called halite.[18] Hence, its quality is affected by anthropogenic activities. Although the daily intake of salt is considerably less than other food items obtained from ecosystems likely to be contaminated by MPs, it is still a matter of concern simply because salt is used in every form of cooking.[19] The Food and Agriculture Organization of the United States (FAO) and the World Health Organization (WHO) recommend consuming no more than 5 g of salt per day,[20] however, the average salt consumption in India is relatively high at 11 g/day.[21]
To detect MPs, many fluorescent dyes such as Nile red, rhodamine B, safranin T, and fluorescein iso-phosphate can label plastic polymers and produce a fluorescent colour. Among these fluorescent dyes, Nile red, a lipophilic dye (9-diethylamino-5H-benzo[α] phenoxazine-5-one), outperforms others because of its high fluorescence intensity, good affinity towards polymer, and shorter incubation time.[22,23] Nile red staining not only helps in qualitative detection but also helps in enumerating the number of MP fibres/particles.[24] The spectral analysis methods commonly used to characterise MPs include Fourier-transform infrared (FT-IR) spectroscopy and Raman spectroscopy, with FTIR being the preferred choice.[25,26] This study primarily focused on detecting the presence of MPs in different types of salts and their characterisation by FTIR.
MATERIAL AND METHODS
Collection of salt samples
Thirty commercial salts (18 brands of sea salt and 12 brands of rock salt) with one sample from each brand were purchased from various retail markets, supermarkets, and sales outlets in and around Mangaluru city, Karnataka, India. Packed, sealed, and FSSAI-certified salts were chosen only, and three packets were purchased for each salt brand.
Preparation of Nile red (NR) solution
The NR solution was prepared in acetone at 1 mg/mL. This was then diluted to a 1:10 ratio with ultra-pure deionised water to yield a working concentration of 10 μg/mL and stored at 4°C in an amber coloured glass bottle. NR stain adheres to the plastic surface and fluoresces at a specific wavelength of 532 nm.[27,28]
Optimisation of Nile red interaction with salt
To determine the accuracy of the absorbance value of the Nile red stain upon interaction with salt, various concentrations of salt solutions ranging from 0 mg/mL to 100 mg/mL were prepared by mixing salt with ultra-pure deionised water. Further, 0.1 mL of NR was added and incubated for 30 minutes in the dark at room temperature. Later, the absorbance was measured at 532 nm using a spectrophotometer. All the samples were prepared in glass bottles to avoid the interference of plastics.
Sample preparation
A stock solution of the salt samples was prepared by dissolving 100 mg of the salt in 1000 mL of deionised water in a borosil® glass bottle under the laminar hood. Then, 100 mL of the salt solution from the stock was taken into a sterile borosil® glass bottle and added to 1 mL of the NR solution stock. The bottle was recapped and incubated for 30 minutes in the dark at room temperature. Further, the solution was vacuum filtered through a glass fibre filter (Whatman grade 934-AH, 55 mm diameter, 1.5 μm pore). For each analysis, 100 mL of deionised water was injected with 1 mL of Nile red, filtered, and labelled as a blank sample, which served as a negative control for estimating background noise in fluorescence intensity or ruling out laboratory contamination. This procedure was applied to all the salt samples.
Microscopy identification
To visualise the MPs, 100 mL of the prepared stock salt solution of all 30 samples was filtered through a glass fibre filter (Whatman grade 934-AH, 55 mm diameter, 1.5 µm pore) and was visualised for fluorescence in a dark condition using a fluorescent microscope (Leica DM2500, Germany). The samples in the filter paper were placed in uncovered petri plates on the stage under the RFP (Red fluorescence protein) filter at 5x magnification, and the fluorescent particles were counted manually by dividing the microscopic field into four quadrants. The fluorescence particles were photographed, and the particle size was counted using the Leica Application (LasX, Leica, Germany).
Characterisation of polymers using FTIR spectroscopy
To identify the type of polymer from the representative salt samples,100 mL of the prepared stock salt solution was filtered through a glass fibre filter (Whatman grade 934-AH, 55 mm diameter, 1.5 µm pore). The filtrate was washed with 200 μL of deionised water, collected in a glass tube, and subjected to FTIR (BRUKER Alpha-2, Germany). After analysis, the result was enumerated through a graph of spectra.
Comparative analysis of heat-treated salt samples
To assess the impact of heating on MPs within the chosen samples, which were previously identified with confirmed MP presence, we mimicked the cooking process. 10 mg of salt was added to 100 mL of double-distilled water and boiled for 15 minutes. Similarly, 10 mg of salt was added to the 100 mL of pre-boiled water (85⁰C); later, the samples were subjected to FTIR analysis.
Statistical analyses
Data are presented as mean ± SD for microparticles and microfibers found in the salt samples.
RESULTS
Optimisation of Nile red interaction with salt
The optimisation of NR interaction with salt was carried out using salt solutions of different concentrations, ranging from 0 to 100 mg/mL, and measuring the absorbance at 532 nm. The OD values obtained for all the concentrations tested ranged from 0.25-0.26, which indicated minimal fluctuations across the spectrum of sodium chloride (NaCl) concentrations [Table 1].
| Tubes no. | Volume of NaCI (mL) | Volume of deionized water (mL) | Cone. Of NaCI (mg/mL) | Volume of NR | Incubation for 30mins in Dark | Absorbanceat 532nm |
|---|---|---|---|---|---|---|
| 1. | 0 | 10 | 0.0 | 0.1 | 0.257 | |
| 2. | 0.001 | 9.999 | 0.01 | 0.1 | 0.250 | |
| 3. | 0.01 | 9.99 | 0.1 | 0.1 | 0.250 | |
| 4. | 0.1 | 9.9 | 1.0 | 0.1 | 0.26 | |
| 5. | 1.0 | 9.0 | 10 | 0.1 | 0.251 | |
| 6. | 10 | 0 | 100 | 0.1 | 0.25 |
Detection and quantification of MPs from salts
Out of 30 different brands of salts tested, 28 showed MPs, except for two flour salts (processed salt: P1-P2). Based on the fluorescent images, the polymers were categorised as microparticles and microfibers [Figure 1]. The identified microfibers ranged from 2-14 particles/100 g, whereas the microparticles ranged from 2-27 particles/100 g of sample. The number of MPs ranged from 13-27 particles/100 g in sea salt and 8-29 particles/100 g in rock salt. Standard deviation for the mean of microparticles in salt samples was 13.38 ± 3.79 particles/100g sea salt, 13.00 ± 8.34 particles/100g in rock salt, and zero in processed salt and the microfibres. The values were 4.63 ± 5.11 particles/100g sea salts, 5.17 ± 4.17 particles/100g rock salts, and processed salt had zero particles. Further, the size of MPs analysed ranged from 19.45 μm-512.91 μm in sea salts and from 29.69 μm-1432.85 μm in rock salt [Table 2].
- Representative images of MPs detected in different salt samples. Images a, b, c, and d represent microplastic microfibres, and E and F represent microplastic particles. MPs: Microplastics.
| Samples | Microparticles/100 g | Microfibers | Total MPs (Microparticles + microfibers) | Size (µm) |
|---|---|---|---|---|
| S1 | 15 | - | 15 | 512.91 |
| S2 | 19 | 5 | 24 | 80.5 |
| S3 | 14 | - | 14 | 198.56 |
| S4 | 12 | 12 | 37.91 | |
| S5 | 22 | 2 | 24 | 33.72 |
| S6 | 15 | 3 | 18 | 90.64 |
| S7 | 13 | - | 13 | 155.72 |
| S8 | 12 | 9 | 21 | 325.7 |
| S9 | 11 | 7 | 18 | 237.74 |
| S10 | 13 | - | 13 | 19.45 |
| S11 | 14 | 13 | 27 | 238.41 |
| S12 | 15 | 4 | 19 | 492.13 |
| S13 | 12 | 4 | 18 | 176 |
| S14 | 13 | 13 | 84.31 | |
| S15 | 9 | 14 | 23 | 205.98 |
| S16 | 5 | 13 | 18 | 451.48 |
| R1 | 23 | 23 | 126.84 | |
| R2 | 26 | 26 | 412.18 | |
| R3 | 9 | 8 | 17 | 302.44 |
| R4 | 27 | 2 | 29 | 166.1 |
| R5 | 2 | 6 | 8 | 754.38 |
| R6 | 3 | 9 | 12 | 125.5 |
| R7 | 11 | 10 | 21 | 541.23 |
| R8 | 13 | 9 | 22 | 138.67 |
| R9 | 9 | 11 | 21 | 29.69 |
| R10 | 15 | . | 15 | 1432.85 |
| R11 | 7 | 3 | 10 | 787.13 |
| R12 | 11 | 4 | 15 | 968.39 |
| P1 | - | - | - | - |
| P2 | - | - | - | - |
MPs: Microplastics, S: Sea salt, R: Rock salt, P: Processed salt.
Characterisation of polymer type by FTIR spectroscopy
Confirmation of the Nile red staining by FTIR spectroscopy analysis identified diverse polymer particles such as polyvinyl chloride (PVC), polyethylene terephthalate (PET), PS, polypropylene (PP), and polyethylene (PE) in the analysed salt samples. The identity of the MP polymers was determined using unique absorption spectra based on the presence of distinct functional groups. The spectral peaks of the PVC showed at 618.51 cm-1, 632.51 cm-1, 1331.57 cm-1, and 2813.64 cm-1, PET at 1246.99 cm-1, 1411.20 cm-1, 1724.5 cm-1, 2360.7 cm-1 and 2913.8 cm-1, PS at 1039.55 cm-1, 1482.04 cm-1, and 2933.63 cm-1, PP at 1367.5 cm-1, 1724.5 cm-1 and 2913 cm-1, and PE at 710.51 cm-1, 1388.98 cm-1, 1466.06 cm-1, 1724.56 cm-1, and 2913.84 cm-1 [Figure 2]. The results identified PET as the most common polymer present in the samples after PVC, PP, PE, and PS.
- Representative FTIR spectra show the presence of (a) PS (brown) and PVC (red), (b) PET (purple), (c) PP (orange) and PE (blue). FTIR: Fourier transform infrared spectroscopy. PS: Polystyrene, PVC: Polyvinyl chloride, PET: Polyethylene terephthalate, PP: Polypropylene, PE: Polyethylene.
Characterisation of heat-induced salt
The confirmed MP sample (R4) was subjected to FTIR analysis to check the effect of the heating process on the polymers. The samples showed similar prominent peaks of PVC at 619.76 cm-1, 697.16 cm-1, and 1428.33 cm-1, PET at 1420.09 cm-1, 1453.73 cm-1, 1509.30 cm-1, 2352.58 cm-1, 3050.34 cm-1, and 3421.56 cm-1, PS at 697.16 cm-1, 756.47 cm-1, 1491.08 cm-1, and 3031.93 cm-1, PP at 852.40 cm-1, 2851.34 cm-1, 2914.44 cm-1, and 2957.46 cm-1, and PE at 716.62 cm-1, 2851.34 cm-1, and 2914.44 cm-1 [Figure 3a].
- Representative FTIR spectra, (a) salt was added during the heating process, (b) salt was added after heating, show the presence of PS (brown), PVC (red), PET (purple), PP (orange), and PE (blue). FTIR: Fourier transform infrared spectroscopy. PS: Polystyrene, PVC: Polyvinyl chloride, PET: Polyethylene terephthalate, PP: Polypropylene, PE: Polyethylene.
The MP sample (R4), when added after the heating process, still showed similar peaks of PVC at 607.10 cm-1, 634.17 cm-1, 682.40 cm-1, 1945.46 cm-1, and 2915.34 cm-1, PET at 1345.46 cm-1, 1405.19 cm-1, 2341.27 cm-1, and 2915.34 cm-1, PS at 693.52cm-1, 1026.99 cm-1, 1442.49 cm-1, and 2849.34 cm-1, PP at 1380.69 cm-1, 2835.13 cm-1, and 2915.34 cm-1, and PE at 722.14 cm-1, 1380.69 cm-1, 1466.07 cm-1, 2849.34 cm-1, and 2915.34 cm-1 [Figure 3b]. The R4 sample, which underwent different experimental methods, was subjected to FTIR and showed similar polymers present.
DISCUSSION
MP contamination has become a global problem due to its pervasive environmental presence and harmful effects on organisms.[29] MPs can absorb waterborne pollutants and/or release dangerous chemicals that can pose risks to human health. Their small size, variable shape, applied coatings, and large surface make detecting, identifying, and quantifying MPs and nanoplastics (NPs) challenging.[30] Several studies have shown the presence of MPs in marine resources in alarming proportions, including those not meant for human consumption, which led to the hypothesis that sea salt may contain MPs.[31] The World Health Organization states that recommendation of the recommended daily intake is less than 5 g/day. The detection of MPs in food-grade salts raises an important concern for human consumption. This study identified the MPs in rock and sea salt through Nile red staining. Maes et al., proposed the use of Nile red dye for the identification of MPs.[32] Although several other dyes bind, Nile red has high specificity, high fluorescent emission, and shorter incubation time, and, hence, it is the most widely accepted stain. And NR has become one of the most widely accepted methods for rapid, preliminary detection of MPs across environmental and food matrices. In our study, thirty different brands of salt (rock salt and sea salt) were analysed for the presence of MPs using the NR staining method, using a protocol described earlier.[33] Upon exposure to NR, MPs were visualised under fluorescence microscopy at an excitation wavelength of 532 nm, allowing differentiation from mineral or organic debris. The selection included brands manufactured in different regions of India to represent a broad consumer distribution. More than 93% of the samples showed the presence of MPs at variable concentrations and of different types. Two samples, P1 and P2, did not show any presence of MPs, which could result from the purification processes followed in these samples. Similarly, Karami et al., investigated 17 commercial salt brands from eight different countries and stated that one of the samples showed no MP count. The amount of MP in rock salt and sea salt varied in this study, with rock salt having fewer MP particles than sea salt.[34] A similar result was obtained by Yang et al., showing sea salts contain a higher quantity of MPs than rock salts, which is most likely due to the huge quantity of marine plastic litter that is present in marine ecosystems.[35] However, the size of MPs in sea salt was less than that in rock salt, which could be because of the fewer manufacturing steps compared to sea salt and a contributing factor to the disparity in MP particle counts.
FTIR spectroscopy is one of the most powerful tools for identifying the respective chemical polymers in MPs.[36,37] The salt samples analysed in this study were examined through FTIR spectroscopy at a wavelength of 500-4000 cm-1 to determine the kind of (distinct) polymer. The data were displayed as an FTIR spectra graph with peaks at specific wavenumber positions. The absorption peaks for C-H, C-H2, C-H3, C-O, C=O, C=C, C-Cl, and =C-H- with benzene group were detected between 600 – 780 cm-1, 1050- 1250 cm-1, 1330-1420 cm-1, 1600-1680 cm-1, 2840-2950 cm-1, and 3049 cm-1, respectively. Previous studies have validated the absorption spectra for the MPs obtained from the salt samples showing prominent peaks that were identical to the peaks identified for specific polymers such as PVC between 605-700cm-1, 1330-1430 cm-1, 1945 cm-1, and 2813-2916 cm-1,[38,39] PET between 1246-1421 cm-1, 1508-1725 cm-1, 2340-2361 cm-1, 2913-2916 cm-1, and 3050-3422 cm-1,[40,41] polystyrene (PS) 690-757 cm-1, 1026-1492 cm-1, and 2848-3032 cm-1,[42] polypropylene (PP) at 852 cm-1, 1367-1725 cm-1, and 2835-2958 cm-1[43,44] and polyethylene (PE) at 709-723 cm-1, 1380-1725 cm-1, and 2849- 2916 cm-1.[28,45] In our samples, PET was found to be the most abundant polymer (37%), followed by PVC (26%) PP (22%), and PE (11%). PS (1%) was the least abundant polymer, an observation like the results shown by Lee et al.[46] The results indicated an increase in the presence of PVC in salt compared to the study conducted to analyse the presence of MPs in edible sea salt, where they found only 1-3% PVC in salt.[47] This could be due to the excessive use of PVC, one of the most versatile plastics in urbanisation in developing countries like India.
MPs decompose in different nitrogen and oxygen environments at specific thermal ranges, leaving carbon residues.[48] The FTIR analysis of the simulated cooking process revealed no alteration in the MP polymers present, indicating that the MPs in the salt remain in the body even after cooking, posing a risk of accumulation through ingestion in the body. The heat and moisture during cooking may release MPs from the salt into the cooked food. The extent of leaching may depend on factors such as the type of plastic, cooking duration, and temperature. Ingesting MPs, even at low levels of exposure, has raised concerns about the impact on human health, their tendency to bioaccumulate, and their ability to adsorb and transport toxic chemicals.[48] Studies have suggested that MPs can accumulate in the body and may have adverse effects; however, the full extent of these effects is not yet fully understood.[49]
The salts represent only one of the many dietary sources of MPs, like seafood, drinking water, bottled beverages, sugar, etc.[49] Similarly, a study showed that humans may be ingesting tens of thousands of MP particles annually through various food sources, including salt.[49] Thus, the salt contamination adds to the MPs in the human diet. Particle size is critical; particles >150 µm are less likely to translocate across the gut barrier, whereas those <20 µm can cross epithelial linings and reach systemic circulation, suggesting systemic exposure and raising questions about long-term health consequences.[50] MPs may act as carriers of toxic chemicals or infections, which results in inflammation, oxidative stress, or affects the gut microbiota.[51,52]
Many research investigations showed concern about MPs’ ingestion via food, including salt. Whereas prolonged exposure to MPs on human health is still under study, there is rising evidence hinting at their negative health effects. A migration of MPs from the gastrointestinal tract to other organs has also been observed by current investigations using in vitro and in vivo models, which stimulate both localised and systemic effects.[53] Due to our findings and emerging research on MPs from salt samples, which is a universally consumed commodity which highlighting the importance of continued monitoring and risk assessment of MPs in dietary sources.
HIGHLIGHTS
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MPs were found to be more in sea salt than in rock salt
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MPs from salt samples were detected using Nile red staining
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FTIR confirms the presence of different polymers, with PET as the most prevalent
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The analysis showed the presence of microfibers and microparticles with varied sizes and numbers
CONCLUSION
The presence of MPs in table salt raises severe concerns about their potential impact on human health, as salt is commonly used as a flavour enhancer and food preservative. Random analysis of common available brands of sea and rock salts showed the presence of MPs in 93% of the samples tested. To date, few reports have examined the presence of MPs in commercial salts. Hence, the results of this study assume significance as further research on this topic, particularly on the impact of persistent ingestion of MPs on human health, is much warranted. Salt is an indispensable ingredient in all kinds of cuisines and is often used in uncooked form in salads and other ready-to-eat products. Thus, MP contamination of salt may be considered a public health concern, and focused attention on its removal during the purification processes is necessary to ensure a safe product for the public.
Acknowledgment
The authors would like to thank NITTE (Deemed to be University) for providing facilities and infrastructure for the research. The infrastructure support provided by NITTE-GOK COE | AQUAMARIN, Government of Karnataka, is gratefully acknowledged.
Ethical approval
Institutional Review Board approval is not required. The study does not involve any clinical samples or animal models; therefore, ethical approval is not applicable.
Declaration of patient consent
Patient’s consent not required as there are no patients in this study.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
Use of artificial intelligence (AI)-assisted technology for manuscript preparation
The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.
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