Detection and Characterization of Microplastics in Commercial Salts in India

doi:10.21203/rs.3.rs-3893146/v1

Plastic waste accumulation is an ever-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 ecosystem, particularly in the marine ecosystem. Microplastics pollution in marine environment is a matter of grave concern because marine resources are one of the primarily contributors to 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 marine environment is through salt, an indispensable ingredient in cooking. This study aimed at analysing commercial brands of sea salt and rock salt for the presence of MPs by Nile red fluorescent staining (NR) and characterizing the plastic polymers by Fourier Transform Infrared Spectroscopy (FTIR). A total of thirty different brands of salts available in India were collected and analysed. The results indicate that presence of MPs is highly prevalent in sea salts with variable number, particles size and polymer types. In sea salt samples, the number of MPs ranged between 13- 27 particles/100g whereas in rock salt, it ranged between 8- 29 particles/100g. Both plastic microfibers and MPs were detected in the categories of samples analysed, ranging between 2- 14 particles/100 g for microfibers and 2- 27 particles/100g for microparticles. The size of MPs ranged between 19.45μm - 512.91μm in sea salts and between 29.69μm– 1432.85μm in rock salt. FTIR Spectroscopy identified polyethylene terephthalate as the most prevalent polymer (37%) in the salt samples, followed by polyvinyl chloride (25.9%) polypropylene (22.2%), polyethylene (11%), and polystyrene (3.7%). 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.

Sea salt

Rock salt

Microplastic

Marine pollution

FTIR

Polyethylene terephthalate

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). Microplastics, often defined as plastic particles smaller than 5 millimetres in size, are pervasive in our environment and can be encountered anywhere from the deepest ocean depths to the most isolated mountain ranges (Lee et al. 2019; Peixoto et al. 2019). Marine plastic pollution has emerged as one of the main worldwide environmental issues, alongside ozone depletion, climate change, and ocean acidification (Galloway and Lewis 2016). 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 (Lofty et al. 2023). MPs can easily infiltrate the aquatic environment due to anthropogenic activities (Choudhury et al. 2023). Studies have shown that MPs make their way into the human body through the consumption of food and water from the ecosystems that are infiltrated with MPs (Carbery et al. 2018). Indeed, MPs have been detected in the digestive system (Schwabl et al. 2019; Amelia et al. 2021), blood (Leslie et al. 2022), and lungs (Jenner et al. 2022) of humans. Ingestion of MPs has been shown to cause detrimental effects in model system-based research, primarily through disruption of endocrine receptor functions by the polymers (Ullah et al. 2023). MPs are now considered emerging food pollutants as they can move up the food chain (Nakat et al. 2023). Thus, understanding the number of MPs that the human body is inadvertently taking in is crucial to establishing consumption guidelines to effectively minimize pollution and to analyse health hazards associated with MP exposure (Lee et al. 2021). Salt is an indispensable component in every cuisine around the world and it not only serves as a taste enhancer but also serves as a source of micronutrients and as a preservative. Therefore, daily consumption of salt is inevitable as it is for water and air (WHO 2012; Barboza et al. 2018). 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 (Ore et al. 2020). 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 the mining of a mineral rock called halite (GOI 2017) and hence its quality is affected by anthropogenic activities. Although the daily intake of salt is considerably less compared to other food items that are 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 (Zhang et al. 2020). The Food and Agriculture Organisation of the United States (FAO) and the World Health Organisation (WHO) recommend consuming no more than 5 g of salt per day (Mozaffarian et al. 2014), however, the average salt consumption in India is quite high at 11 g/day (Johnson et al. 2019).

Several fluorescent dyes such as Nile red, rhodamine B, safranin T, and fluorescein iso-phosphate can label plastic polymers and hence are used in detecting MPs. Among these fluorescent dyes, Nile red, a lipophilic dye (9-diethylamino-5H-benzo[α] phenoxazine5-one) outperforms others because of its high fluorescence intensity, good affinity towards polymer, and shorter incubation time (Sturm et al. 2023; Meyers et al. 2022). Nile red staining not only helps in qualitative detection but also helps in enumerating the number of MP fiber/particles (Shim et al. 2016). The spectral analysis methods that are commonly used in the characterization of MPs include Fourier-transform infrared (FT-IR) spectroscopy and Raman spectroscopy, with FTIR being the preferred choice (Thiele et al. 2023; Mazlan et al. 2023). This study primarily focused on detecting the presence of MPs in different types of salts and their characterization by FTIR.

Collection of salt samples

Thirty different brands of commercial salts (18 brands of sea salt and 12 brands of rock salt) were purchased from different retail markets, supermarkets, and sales outlets in and around Mangalore City, Karnataka, India. Salts that are packed and sealed were only chosen and three packets were purchased for each salt brand.

Preparation of Nile Red (NR) solution

Stock solution of Nile red (NR) solution was prepared in acetone at 1µg/mL. This was then diluted to a 1:10 ratio with ultra-pure deionized 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 (Cassola et al. 2017; Mohan et al. 2023).

Optimization of Nile Red Interaction with Salt

To determine the accuracy of the absorbance value of NR 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 deionized water. Further, 0.1mL of NR was added and incubated for 30 minutes in the dark at room temperature. Later, the absorbance was measured at 532nm using a spectrophotometer. All the samples were prepared in glass bottles to avoid the interference of plastics.

Sample preparation

100 mL of the salt solution from the stock was taken into a sterile glass bottle and injected with 1mL of NR solution stock and the bottle was recapped and incubated for 30 minutes in the dark at room temperature. Further, the solution was vacuum filtered through a glass fiber filter (Whatman grade 934-AH, 55 mm diameter, 1.5 µm pore). Filters were then examined under fluorescence microscopy. For each analysis, 100 mL of deionized water collected in the lab 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.

Comparative analysis of heat-induced salt

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. 10mg of salt was added to the 100mL of double distilled water and boiled for 15 minutes. Similarly, 10mg of salt was added to the 100mL of pre-boiled water (85⁰C), later the samples were subjected to FTIR analysis.

Microscopy Identification

The glass fiber filter papers were visualized for fluorescence in a dark condition using a fluorescent microscope (Leica DM2500, Germany). The samples 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 4 quadrants. The fluorescence particles were photographed and the particle size was counted using Leica Application (LasX, Leica, Germany).

Characterization of polymers using FTIR spectroscopy

To identify the type of polymer, 100 mL of the prepared stock salt solution was filtered through a glass fiber filter (Whatman grade 934-AH, 55 mm diameter, 1.5 um pore). The filtrate was then washed with 200µL of deionized 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.

Optimization of Nile Red Interaction with Salt

The optimization of NR interaction with salt was carried out using salt solutions of different concentrations ranging from 0 to 100mg/mL and measuring the absorbance at 532nm. The OD values obtained for all the concentrations tested ranged from 0.25- 0.26, which indicated minimal fluctuations across the spectrum of NaCl concentrations (Table I).

Detection and quantification of MPs from salts:

Out of 30 different brands of salts tested, 28 brands showed the presence of MPs except for two brands of flour salt (processed salt: P1-P2). Based on the fluorescent images, the polymers were categorized as microparticles and microfibers (Fig.1). The identified microfibers ranged from 2- 14 particles/100g whereas the microparticles ranged from 5- 26 particles/100g of sample. In total, the number of MPs ranged from 3- 27 particles/100g in sea salt and 8-29 particles/100g in rock salt. Further, the size of MPs analyzed ranged from 19.45μm - 512.91μm in sea salts and 29.69μm – 1432.85μm in rock salt (Table II).

Characterization of polymer type by FTIR spectroscopy

Confirmation of the Nile red staining by FTIR spectroscopy analysis identified diverse polymer particles such as PVC, PET, PS, PP, and PE in the salt samples analysed. The identity of the MP polymers was done on the 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 (Fig.2).

Characterization 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(Fig.3A).

The MP sample (R4), when added after the heating process still showed the 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(Fig.3B). The R4 sample, which underwent different experimental methods subjected to the FTIR showed similar polymers present.

Microplastic contamination has turned into a global problem due to its pervasive presence in the environment and harmful effects on organisms (Cole et al. 2011). 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 the detection, identification, and quantification of MPs and NPs a challenging attempt (Sturm et al. 2021). Several studies have shown the presence of MPs in marine resources in alarming proportions, including in those that are not meant for human consumption, which led to the hypothesis that sea salt may contain MPs (Sivahami et al. 2021). In this study, the MPs were identified in both rock salt and sea salt through Nile red staining. In 2017, Maes et al proposed the use of Nile red dye for the identification of MPs. Although several other dyes bind, Nile red has high specificity, high fluorescent emission, and shorter incubation time and hence is the most widely accepted stain. In our study, thirty different brands of salt (rock salt and sea salt) were analyzed for the presence of MPs using the NR staining method using a protocol that was described earlier (Makhdoumi et al. 2023). 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 be the result of efficient purification processes followed in these samples. In this study, the number of MP in sea salt and rock salt varied with higher MP particles found in sea salt compared to rock salt. A similar result was obtained by Yang et al. (2015), 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. However, the size of MPs in sea salt was lesser than that in rock salt which could be because of the fewer manufacturing steps compared to sea salt, and a contributing factor for the disparity in MP particle counts.

FTIR spectroscopy is one of the most powerful tools for identifying the respective chemical polymers in MPs (Ivleva et al. 2021; Moses et al. 2023). The salt samples analyzed in this study were examined through FTIR spectroscopy at a wavelength of 500- 4000cm^− 1 to determine the kind of (distinct) polymer the data were displayed as a graph of FTIR spectra that had peaks at specific wavenumber positions. The absorption peaks for C-H, C-H₂, C-H_3, C-O, C = O, C = C, C-Cl, =C-H- with benzene group were detected between 600–780cm^− 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 (Park et al. 2018; Park et al. 2020), PET between 1246–1421 cm^− 1, 1508–1725 cm^− 1, 2340–2361 cm^− 1, 2913–2916 cm^− 1 and 3050–3422 cm^− 1(Pereira et al. 2017; El-Saftawy et al. 2014), polystyrene (PS) 690–757 cm-1,1026–1492 cm-1 and 2848–3032 cm-1 (F et al. 2013), polypropylene (PP) at 852 cm^− 1,1367–1725 cm^− 1 and 2835–2958 cm^− 1 (Mazhar et al. 2014; Ibor et al. 2023) and polyethylene (PE) at 709–723 cm^− 1,1380–1725 cm^− 1and 2849–2916 cm^− 1(Mohan et al. 2023; Asgari et al. 2014). In our samples, PET was found to be the most abundant polymer (37%), followed by PVC (26%) PP (22%), and polyethylene (11%). Polystyrene (1%) was the least abundant polymer, an observation similar to the results shown by Lee et al 2019. The results indicated an increase in the presence of PVC in salt as compared to the study conducted to analyse the presence of MPs in edible sea salt where they found only 1–3% PVC in salt (Vidyasakar et al. 2021). This could be due to the excessive use of PVC which is one of the most versatile plastics in urbanization in developing countries like India.

MPs decompose at specific thermal ranges in different nitrogen and oxygen environments leaving carbon residues (Rozman et al. 2021). The FTIR analysis of the simulated cooking process revealed no alteration in the MP polymers present indicating that the MPs in the salt remains intake 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 in minimal amounts has raised concerns about the potential impact on human health. Studies have suggested that MPs can accumulate in the body and may have adverse effects, however, to date the full extent of these effects is not yet fully understood (CK Seth and A Shriwastav 2018).

The presence of MPs in table salt raises serious 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 as a public health concern and focused attention on its removal during the purification processes is a must to ensure a safe product to the public.

MPs: Microplastics

NR: Nile red

FTIR: Fourier Transform Infrared Spectroscopy

PET: Polyethylene terephthalate

PVC: Polyvinyl chloride

PE: Polyethylene

PP: Polypropylene

PS: Polystyrene

WHO: World Health Organization

GOI: Government of India

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript.

Author Contributions

RV: Involved in data analysis and prepared the original draft.

SX: Performed the experiments.

MM: Partly performed the experiments and involved in data analysis.

GC: Conceptualized the study, supervised the work and edited the drafted manuscript.

AC: Supervised the work, participated in the data analysis and revision of the manuscript

Acknowledgments

The authors would like to thank the Nitte University Centre for Science Education and Research (NUCSER) and Nitte (Deemed to be University) for providing facilities and infrastructure for the research.

Supplementary materials

NIL

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Table I. Optimization of the interaction of NR with salt at OD at 532nm

Tubes no.	Volume of NaCl (mL)	Volume of deionized water (mL)	Conc. Of NaCl (mg/mL)	Volume of NR	Incubation for 30mins in Dark	Absorbance at 532_nm
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

Table II. Composition of microparticles, the total number of MPs per 100gm of sample analyzed, and their sizes in all the thirty salt samples analyzed.

Samples	Microparticles	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	-	-	-	-

*S - Sea salt, R- Rock salt, P- Processed salt

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Graphical Abstract

Detection and Characterization of Microplastics in Commercial Salts in India

Status:

Version 1

Abstract

Figures

Introduction

Materials and Methods

Collection of salt samples

Preparation of Nile Red (NR) solution

Optimization of Nile Red Interaction with Salt

Sample preparation

Comparative analysis of heat-induced salt

Microscopy Identification

Characterization of polymers using FTIR spectroscopy

Results

Discussion

Conclusion

Abbreviations

Declarations

References

Tables

Supplementary Files

Status:

Version 1