Assessing the impact of heavy metal residues in food and drug packaging on the standard bacterial strains: a biofilm perspective

ABSTRACT

The presence and migration of heavy metals from food and drug packaging materials into consumables pose significant health concerns. This study explored the effects of vanadium, arsenic, cadmium, and mercury contained in the digest of packaging materials on biofilms formed by standard strains of Escherichia coli ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 70063, and Enterococcus faecalis ATCC 29212. Results showed that vanadium at 1.2 µg/ml promoted strong biofilm formation in all tested bacteria, while cadmium (1 µg/ml) and mercury (6.6 µg/ml) supressed biofilm formation. Arsenic at 0.6 µg/ml initially facilitated biofilm formation, but its effectiveness decreased with higher concentrations. This interference of heavy metals digests on biofilm formation in the gut microbiota is a concern, as leached heavy metals into food when consumed could disrupt the balance of human intestinal flora and homeostasis.

KEYWORDS:

• Heavy metals
• drug packaging
• Pseudomonas aeruginosa
• biofilm
• human intestinal flora

1. Introduction

Food packaging serves much more than merely keeping a product in place. It safeguards commodities and maintains the quality and freshness of food. Packaging may add contaminants to the food, which may have unknown health effects (Claudio, 2012). Food safety is improved by the reduction of bacterial contamination due to food packaging (Hanning et al., 2009). Even though food packaging is crucial for convenience and sanitation, research of this type highlights the need for a deeper comprehension of the extent and effects of chemical contamination of food via packaging. Migration of metal ions from packaging material is the most significant source in terms of quantities (Marsh & Bugusu, 2007; Reimer et al., 2010).

Study by Bitel et al. (2007) reported the leaching of chemicals and additives from packaging materials into food. The Food and Agriculture Organization (FAO) and Codex Alimentarius Commission (CAC), founded by the World Health Organization of the United Nations (WHO) in 1961, have set regulations to protect consumer health and validate ethical business practises in the food industry. Standards are established for assessing the safety of foods in addition to norms for individual food, their labelling, hygiene, additives, pesticide residues, and procedures. As per CAC, the permissible limit of vanadium is 1.8 µg/ml, cadmium 7 µg/ml, mercury 1.4 µg/ml, and arsenic 1.6 µg/ml (Codex and SDGs, 2020). These harmful metals are known to leach out of packing materials into the food item, and the rate of leaching changes with temperature and food simulant pH (Dong et al., 2014).

A biofilm is a collection of microbial cells that permanently attach to a surface and are typically contained within a polysaccharide matrix. Adherence to the surface offers several benefits, including resistance to antimicrobial agents, the acquisition of novel genetic characteristics, and the availability of nutrients and metabolic coherence (Kokare et al., 2009). Development of bacterial biofilm involves a complex mechanism, and quorum sensing is one of the main regulatory systems, which is critical for intestinal homeostasis in human gut. In the human gut, the microbes coexist with each other on the mucus layer of the host and work together, either in free-floating planktonic form or as an adhering biofilm on outer mucosal layer (Hussain et al., 2020). By forming biofilm in the gut, the microbes protect the human intestine from harmful germs. This biofilm act as mechanical, chemical, biological, and immune barrier of the host gut (Tian et al., 2023). Biofilm also helps the human gut to stay healthy by protecting it from heavy metals that may be ingested along with food and water. These biofilms have a variety of physiological and pathological activities, such as supporting metabolic processes, modulating immunological responses, and preserving gut health (Yousi et al., 2019). However, increased number of heavy metals might disrupt the biofilm and affect the normal function of human gut (Deng et al., 2020).

Packaging materials are contaminated through anthropogenic activities (Rehder, 2013). Heavy metals like arsenic, cadmium, vanadium, and mercury enter the environment through sources like phosphate fertilizers, water treatments, waste incineration, and industrial processes (Shawai et al., 2017). Thus, entry of heavy metals into food chain, air, water, and soil poses a risk to humans. Consumption of leached heavy metals along with food can lead to several diseases and ailments, including obesity, autism, neurological deterioration, cardiovascular disease, and respiratory illness in humans (Campanale et al., 2020). Clinical studies have highlighted the association between heavy metal exposure and neurological disorders, cardiovascular diseases, kidney dysfunction, and cancer, among the immunocompromised individuals (Bakulski et al., 2020; Hong et al., 2014; Jaishankar et al., 2014). Accumulation of these metals in human tissues over time exacerbates long-term health concerns. Understanding and addressing these sources of heavy metal exposure is crucial for effective health risk mitigation (Alissa & Ferns, 2011; Genchi et al., 2020).

Exposure of animals to heavy metals like mercury and cadmium can disrupt the homeostasis of gut microbiome, which has a substantial impact on gut health (Assefa & Köhler, 2020). Arsenic exposure also increases drug resistance in gut flora (Chi et al., 2017). Study by Mohapatra et al. (2020) has explained in depth that bacteria in polluted environments form biofilms, offering potential for bioremediation due to their complex structures and gene transfer capabilities.

Given the multifaceted sources and pervasive nature of heavy metals, it is crucial to recognize the health concerns they pose and adopt proactive measures to minimize exposure. This includes monitoring the quality of water, food, and air for heavy metal contamination, as well as seeking alternatives to packaging materials that may contain these hazardous elements. By understanding the profound impact of heavy metals on overall health and addressing their sources, we can take significant steps towards safeguarding human health and well-being.

Paucity of information on the risk of chronic intake of heavy metals from packaging material on human gut bacteria tempted us to conduct the present study. Though biofilm is formed by most of the gut bacteria, it is difficult to study the entire gut microbial flora. Hence, we studied the biofilm-forming capacity of standard strains of bacteria in the presence of heavy metal salts and digest of packaging materials (DPMs) containing heavy metals.

2. Materials and methods

2.1. Study setting

The Institutional Ethics Committee approval [Ref: IEC KMC MLR 04–2021/131] was obtained, and the research was carried out at the Department of Microbiology, Kasturba Medical College, Mangalore.

2.2. Heavy metal salts used and stock solution preparation

Laboratory-grade heavy metal salts of vanadium (vanadium pentoxide), arsenic (arsenic trioxide), cadmium (cadmium acetate) and mercury (mercuric chloride) with a purity range of 98.5–99.9% were procured from Intelligent Materials, Pvt. Ltd., Nanoshel group of Companies, Punjab, India. Each of these salts of heavy metals was dissolved in sterile distilled water to prepare 100 µg/ml stock solution. The stock solution was sterilized by autoclaving, and the volumes and concentrations are given in Table 1.

Table 1. Luria Bertani broth with different concentrations of digests of packaging material/heavy metal salt solution for the detection of biofilm production.

Name of heavy metal in the DPM/heavy metal salt solution	Amount of LB broth added to each well of microtiter plate (µl)	Amount of heavy metal salt solution/digests added to LB broth (µg/ml)	Final concentration of heavy metal in each well (µg/ml)
Vanadium/vanadium pentoxide	176.8	3.2	1.6
	176.4	3.6	1.8
	176	4	2.0
	175.6	4.4	2.2*
Arsenic/arsenic trioxide	178	2	1.0
	177.6	2.4	1.2
	177.2	2.8	1.4
	176.8	3.2	1.6*
Cadmium/cadmium acetate	166.8	13.2	6.6
	166.4	13.6	6.8
	166	14	7.0
	165.5	14.4	7.2*
Mercury/mercuric chloride	178	2	1
	177.6	2.4	1.2
	177.2	2.8	1.4*
	176.8	3.2	1.6

* Additional concentrations tested for some bacteria, as MIC value was different as stated in Table 2.

Table 2. Biofilm-forming ability of standard stains of bacterial isolates in the presence of standard heavy metal salts and heavy metal digests from packaging material.

Heavy metals tested (CAC permissible level)	ATCC strains tested (MIC values)	Concentration (µg/ml)	Type of heavy metal	Type of biofilm produced
Vanadium (V:1.8µg/ml)	E. coli (2 µg/ml)	1.6	Standard	Strong
			Digest	Moderate
		1.8	Standard	Moderate
			Digest	Moderate
		2.0	Standard	Weak*
			Digest	Weak
	P. aeruginosa (2 µg/ml)	1.6	Standard	Strong
			Digest	Strong
		1.8	Standard	No biofilm*
			Digest	No biofilm
		2.0	Standard	No biofilm
			Digest	No biofilm
	K. pneumoniae (2 µg/ml)	1.6	Standard	Moderate
			Digest	Moderate
		1.8	Standard	Moderate
			Digest	Moderate
		2.0	Standard	No biofilm
			Digest	No biofilm
	E. faecalis (2.2 µg/ml)	1.8	Standard	Strong
			Digest	Strong
		2.0	Standard	Weak
			Digest	Weak
		2.2	Standard	Weak
			Digest	Weak
Arsenic (As:1.6µg/ml)	E. coli (1.6 µg/ml)	1.2	Standard	No biofilm
			Digest	No biofilm
		1.4	Standard	No biofilm
			Digest	No biofilm
		1.6	Standard	No biofilm
			Digest	No biofilm
	P. aeruginosa (1.6 µg/ml)	1.2	Standard	No biofilm
			Digest	No biofilm
		1.4	Standard	No biofilm
			Digest	No biofilm
		1.6	Standard	No biofilm
			Digest	No biofilm
	K. pneumoniae (1.6 µg/ml)	1.2	Standard	No biofilm
			Digest	No biofilm
		1.4	Standard	No biofilm
			Digest	No biofilm
		1.6	Standard	No biofilm
			Digest	No biofilm
	E. faecalis (1.4 µg/ml)	1.0	Standard	Weak
			Digest	Weak
		1.2	Standard	No biofilm
			Digest	Weak
		1.4	Standard	No biofilm
			Digest	No biofilm
Cadmium (Cd:7.0µg/ml)	E. coli (7 µg/ml)	6.6	Standard	Moderate
			Digest	No biofilm
		6.8	Standard	Moderate
			Digest	No biofilm
		7.0	Standard	No biofilm
			Digest	No biofilm
	P. aeruginosa (7.2 µg/ml)	6.8	Standard	Moderate
			Digest	No biofilm
		7.0	Standard	No biofilm
			Digest	No biofilm
		7.2	Standard	No biofilm
			Digest	No biofilm
	K. pneumoniae (7 µg/ml)	6.6	Standard	Weak
			Digest	No biofilm
		6.8	Standard	Weak
			Digest	No biofilm
		7.0	Standard	Weak
			Digest	No biofilm
	E. faecalis (7 µg/ml)	6.6	Standard	Weak
			Digest	No biofilm
		6.8	Standard	Weak
			Digest	No biofilm
		7.0	Standard	Weak
			Digest	No biofilm
Mercury (Hg:1.4µg/ml)	E. coli (1.6 µg/ml)	1.2	Standard	Weak
			Digest	No biofilm
		1.4	Standard	Weak
			Digest	No biofilm
		1.6	Standard	No biofilm
			Digest	No biofilm
	P. aeruginosa (1.4 µg/ml)	1.0	Standard	No biofilm
			Digest	No biofilm
		1.2	Standard	No biofilm
			Digest	No biofilm
		1.4	Standard	No biofilm
			Digest	No biofilm
	K. pneumoniae (1.4 µg/ml)	1.0	Standard	No biofilm
			Digest	No biofilm
		1.2	Standard	No biofilm
			Digest	No biofilm
		1.4	Standard	No biofilm
			Digest	No biofilm
	E. faecalis (1.4 µg/ml)	1.0	Standard	Weak
			Digest	No biofilm
		1.2	Standard	Weak
			Digest	No biofilm
		1.4	Standard	No biofilm
			Digest	No biofilm

* Weak {O.Dc < O.D ≤ (2 · O.Dc)}, Moderate {(2 · O.Dc) < O.D ≤ (4 · O.Dc)}, Strong {(4 · O.Dc) < O.D}, and No biofilm producers {O.D ≤ O.Dc} (Walawalkar et al., 2016).

2.3. Preparation of DPM

Initially, 13 different types of commonly used food and drug packaging materials such as aluminium cans, leak-proof bags, cardboard, tetra packs, cellophane, tissues, sachets, aluminium bags & boxes, plastic bags & containers, medicinal blister packets and medicinal closures (one each weighing 10 grams) were subjected to microwave-assisted digestion (Mukhi et al., 2023). Qualitative and quantitative analysis by inductively coupled plasma optical emission spectroscopy (ICP-OES) showed that digests of cardboard, sachet and medicinal closure contained the highest quantity of heavy metals. Hence, we chose to analyse five samples each of cardboard, sachet and medicinal closure.

Digestion of 10 g each of cardboard, sachet and medicinal closure was done by Microwave-assisted digestion as per the USEPA 3051 guidelines (Eskilsson & Björklund, 2000) Briefly, the packaging materials were cut into small pieces and individually dissolved in concentrated nitric acid followed by concentrated hydrochloric acid in a laboratory microwave unit. The sample with the acids was placed in a quartz microwave vessel and sealed. Sealed vessels containing medicinal closures, cardboard and sachets were heated in the microwave for 120, 90 and 45 min respectively. Vessels were cooled; contents were filtered, centrifuged and diluted using distilled water. The pH of the digest was adjusted to 7.4. These extreme conditions did not harm the metal ions. Hence, the packaging materials were digested at these temperatures (Tchounwou et al., 2012). ICP-OES was used to quantify the amount of heavy metal present in each of the DPM. Analytical grade reagents, chemicals [Sigma Aldrich, Germany] and double distilled water were used for the preparation of solutions and dilutions.

2.4. Sterility of the DPM

Digests of cardboard, sachets and medicinal closures with heavy metals were sterilized by autoclaving at 121°C for 15 min. The concentration of respective heavy metals in the DPMs was determined by ICP-OES and dissolved in sterile distilled water to bring the concentration to 100 µg/ml. This was used as a stock solution to incorporate different concentrations of heavy metals into Luria Bertani (LB) broth (Table 1). Since the digest of cardboard had 2.7 µg/ml of vanadium, it was dried in hot air furnace at 80°C to evaporate the solvent and increase the concentration. The concentrated DPM of cardboard containing vanadium was dissolved in distilled water to obtain the stock solution with a concentration of 100 µg/ml. These stock solutions of heavy metal digests of cardboard, sachet and medicinal closures were used to study the biofilm forming capacity of the standard strains of bacteria that represents gut flora.

2.5. Bacterial cultures and media

Standard strains of E. coli ATCC 25923, P. aeruginosa ATCC 27853, K. pneumonia ATCC 70063, and E. faecalis ATCC 29212 available in the microbiology department stock culture collection were used in the study. LB broth (Media Pvt. Ltd. Mumbai, India) was incorporated with the different sub-inhibitory concentrations of sterile DPM and heavy metal salt solution as per Table 1.

2.6. Controls

Sterile LB broth incorporated with heavy metal salt and digest of heavy metals from the packaging was used as sterility control (negative control). LB broth without heavy metal inoculated with different strains of bacteria was used as growth control (positive control).

2.7. Biofilm production by microtiter plate method

Biofilm formation was performed in a sterile 96-well microtiter plate (Sri Durga laboratory supplies, Mangalore, Karnataka). Briefly, a colony of each of the ATCC strains, E. coli ATCC 25923, P. aeruginosa ATCC 27853, K. pneumonia ATCC 70063, and E. faecalis ATCC 29212, was grown for 24 hin peptone water at 37°C. The growth of each isolate was diluted in sterile saline (1:20) to match the turbidity of 0.5 McFarland’s standard. Varying sub inhibitory concentrations of heavy metal stock solution and LB broth as per Table 1 was added to different wells of microtiter plate. To each well, 20 µl of 1:20 diluted ATCC bacterial culture was added. Plates were incubated for 48 h for 37°C. To confirm the bacterial viability, we subcultured the content of the microtiter well on to blood agar plate, after which, the contents of the microtiter plate were removed, tapped, and washed thrice with phosphate buffer saline. It was air-dried for 30 min in an inverted position and was then stained with 0.1% crystal violet for 10 min. Excess stain was removed by gentle tapping and plates were washed three times with distilled water. The biofilm was fixed by adding 200 µl of ethanol (80%) for 10 min (Turki et al., 2012). The optical density (OD) was measured at 570 nm in a microtiter plate reader (Multiskan FC Filter-based Microplate Photometer; Thermo Fisher Scientific). Three standard deviations (SDs) above the mean of the negative control were considered as cut off (O.Dc). Based on the cut off values, biofilm producers were classified as weak {O.Dc < O.D ≤ (2·O.Dc)}, moderate {(2·O.Dc) < O.D ≤ (4·O.Dc)}, strong {(4·O.Dc) < O.D}, and non biofilm producers {O.D ≤ O.Dc} (Walawalkar et al., 2016).

2.8. Statistical analysis

All the experiments were performed in triplicates. The mean and SD of the triplicates was taken as a result. Microsoft Excel was used to calculate the mean and SD.

3. Results

3.1. Quantification of heavy metals in packaging materials

Qualitative and quantitative analyses of DPMs by ICP-OES showed that cardboard, sachet and medicinal closures had the highest quantity of heavy metals among the 13-packaging material analysed. The remnants of food/drugs if any sticking to the packaging material got degraded during heating (60− 80°C) for prolonged time and acid treatment used for their digestion process. Digest of cardboard had 2.7 µg/ml of vanadium, sachets had 155 µg/ml of cadmium and 213 µg/ml of mercury, and medicinal closure had 101.41 µg/ml of arsenic. These concentrations of heavy metals in the DPMs were higher than the permissible levels specified by CAC, which is a cause of concern. CAC acceptable values for vanadium are 1.8 µg/ml, cadmium 7 µg/ml, mercury 1.4 µg/ml, and arsenic 1.6 µg/ml (Codex & SDGs, 2020).

3.2. Biofilm production of heavy metal salts and digests of cardboard, sachet, and medicinal closures

We studied the Biofilm-forming capacity of standard strains of bacteria in the presence of sub-inhibitory concentration of heavy metal salts (standard) and digest of heavy metals from food and drug packaging materials using LB broth. At the end of the incubation period, there was no growth in LB broth with various concentrations of heavy metal without bacterial inoculum, which indicated the sterility of heavy metal digests and LB broth. LB broth without heavy metals inoculated with various ATCC bacterial strains showed good growth, which indicated that the batch of LB broth prepared supported the bacterial growth.

CAC has given permissible limits for vanadium, cadmium, mercury and arsenic in packaging material as shown in Table 2. In the presence of vanadium digest, E. coli formed a moderate biofilm at 1.6 µg/ml and a weak biofilm at 2 µg/ml. P. aeruginosa formed a strong biofilm at 1.6 µg/ml but could not form a biofilm at higher concentrations. K. pneumoniae formed a moderate biofilm at 1.6 µg/ml but failed to form a biofilm at higher concentrations of vanadium. E. faecalis also formed a strong biofilm at 1.8 µg/ml but not at 2.2 µg/ml of vanadium. The results of biofilm formation/inhibition at different concentration of Va, A, Cd, and Hg metal salts and DPM are depicted in Table 2. For mercury digests, all the tested organisms failed to form a biofilm even at lower concentrations. We subcultured the contents of microtiter well after 48 h onto blood agar plate to confirm the viability of bacterial inoculum in the microtiter plate. Subculture from microtiter wells showed good growth on blood agar. This confirms that lack of biofilm was not due to killing of bacteria by heavy metals but due to inhibition of biofilm formation by the heavy metals

4. Discussion

Papers and paperboards are widely used in food packaging all over the world. According to European standards and CAC, the packaging material should be free of plasticizers, heavy metals and other chemicals (Codex and the SDGs, 2020; Nordic guidance for authorities, industry and trade, 2022). In our study, digests of cardboards, sachets and medicinal closures had varying amounts of heavy metals, which were higher than the European and CAC standards (Table 1). Earlier Indian study has reported the presence of heavy metals like aluminium, arsenics, cobalt, chromium, nickel, lead and vanadium in more than the permissible concentration in food packaging material made of paper and paperboards (Sood & Sharma, 2019). However, they have not studied sachets and medicinal closures for heavy metal contamination. To the best of our knowledge, other Indian studies are not available to compare our data. Vanadium, mercury, arsenic, and cadmium are common contaminants in food packaging materials that seep out at low levels and if consumed along with food are known to cause diarrhoea, nausea, vomiting and have negative impact on gut microbiota (Enrique Conti, 2006; Ubeda et al., 2020).

Microbial biofilms in the human gastrointestinal (GI) tract helps the gut to survive extensive conditions, any attack from the foreign substances or pathogens, thus acting as mechanical, chemical, biological and immunological barrier (Donlan & Costerton, 2002; Tian et al., 2023). Biofilms play a role in bolstering host defences, exchange of nutrition between the microbiota and the host, and preventing the colonisation of pathogenic bacteria. Thus, biofilm acts as defence mechanism that enables the bacteria to endure and thrive in situations with high metal. Metals can also influence cell surface adhesion and/or cell-to-cell aggregation process, promoting biofilm formation and, consequently, its resistance (Macfarlane & Dillon, 2007). However, the public, conservationists, and environmental policymakers still have a limited understanding of heavy metals contaminants in food and its possible impact on gut microbiota and human health.

This study was intended to know the effect of heavy metals on E. coli ATCC 25923, P. aeruginosa ATCC 27853, K. pneumoniae ATCC 70063, and E. faecalis ATCC 29212 which are the representative of prominent gut microbiota. E. coli is frequently found in the human GI tract, as either a commensal, probiotic, or pathogen (Laughlin et al., 2000). P. aeruginosa, though a hospital pathogen, is also a part of normal gut flora (Min & Xuefeng, 2015). E. faecalis and K. pneumoniae are the flora of the mouth, and intestines, of healthy individuals (Vuotto et al., 2014; Yan & Bassler, 2019). In our study, these representative strains showed weak to no biofilm formation at the CAC permissible concentration of heavy metal digests and heavy metal salts (Table 2). Thus, our findings show CAC permissible concentration of V, Cd, As and Hg supress or inhibit the biofilm formation (Table 2). Furthermore, the concentrations chosen for the study included the minimum inhibitory concentration of the heavy metal in presence of the bacteria and two concentrations below the MIC as mentioned in Table 2.

In the present study, DPMs had higher concentration of heavy metals than the CAC permissible levels. The food packed in these materials when consumed may have serious impact on gut microbiota if they leach into food. Earlier studies report heavy metals to be detrimental to people and, at greater levels, can cause cancer, dysfunction of immune system, kidney, and nervous and many more organs (Muncke et al., 2020). Since bacterial strains were not able to form biofilms even at CAC permissible concentrations, these levels should be regulated and scrutinised well by the governing bodies because biofilms are critical for the intestinal homeostasis (Hussain et al., 2020). Since accumulation of heavy metals is a risk for human health, it is better to avoid them for further use in packaging materials. The United Nations sustainable development goals (SGD 3 & 12) encourages ensuring sustainable consumption and production of food for good health and wellbeing. Hence, it is better to use packaging materials free of heavy metals to reduce the risk to the health of humans and the environment.

The present study has used only four representative bacteria of the gut microbiome to know the effect of heavy metals in packaging material on biofilm. Further studies are necessary to quantify biofilm in order to show the dependency of biofilm biomass on the heavy metal concentration. As microbiomes are unique to an individual, studies involving transcriptomics and metagenomics are necessary to know the metal microbe and host gut interactions. Moreover, it is necessary to study the number and quantity of each heavy metal leached into food items packed and stored at specified storage conditions. It is also important to know the acute toxicity and risk of these heavy metals on food contact material/consumers, which is the limitations of the study.

5. Conclusions

The effect of heavy metal digests on biofilm formation by four standard bacterial strains which represents gut microbiota showed concentration dependent variation in biofilm suppression or inhibition. Hence, presence of heavy metals in food and drug packaging materials may have harmful effect on gut microbiota and human health. However, packaging materials containing heavy metals may be avoided while packing and storing food for a sustainable consumption and production for good health and wellbeing, as heavy metals have impact on human health and on the environment. The impact of these heavy metals on the aerobic and anaerobic gut flora isolated from healthy human faeces samples requires more research.

Author contributions

All authors contributed to the study conception, design, writing, and reviewing of the final manuscript. The details are summarised as follows:

Conceptualization: [S.M., D.B., R.M.S.].

Methodology: [S.M., D.B.].

Formal analysis and investigation: [S.M., D.B., R.M.S., P.M.].

Writing – original draft preparation [S.H., S.M.].

Editing and revising the manuscript: [D.B., S.M., R.M.S.].

Approval of the final version of the manuscript: [S.M., D.B., R.M.S., P.M., S.H.].

Acknowledgements

The authors would like to acknowledge Department of Microbiology, KMC, Mangalore, and Department of Chemical Engineering, National Institute of technology, Karnataka, for providing all the facilities required during the course of this research.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data can be made available upon request from the corresponding author.

Additional information

Funding

This work is supported by the Rotary club of Dombivili Suncity Part of Rotary District 3142 under Rotary International [Club I.D – 84860].