BIBECHANA Vol. 20, No. 1, April 2023, 21–26 ISSN 2091-0762 (Print), 2382-5340 (Online) Journal homepage: http://nepjol.info/index.php/BIBECHANA Publisher:Dept. of Phys., Mahendra Morang A. M. Campus (Tribhuvan University)Biratnagar Overall migration of microplastics in mineral water and non-alcoholic beverages Jagjit Kour1,∗, Pratima Bhatt1, Shobha Basnet2 1Department of Chemistry, Tri-Chandra Multiple Campus, T.U., Nepal, 2Zest Laboratories and Research Center (P) Ltd, Nepal ∗Corresponding author. Email: jagjitkour00@gmail.com Abstract In today’s world, packaging plays a crucial role in enhancing, enclosing, and shielding the materials that are used from procurement to handling and storage, processing, manufactur- ing and finally to the consumer. Packaging shields a product’s contents from contamination, spoiling and makes it simple to store and transfer anywhere. Man-made plastic breaks down into little particles as a result of numerous external pressures. The degradation of plastics produce nano or microplastic. Health issues arise when microplastics (MPs) leak from plastic food packaging and are consumed by people. Though plastic packaging is abundant, consumers from developing nation are not aware of the risk associated with plastic food packaging. The purpose of this study is to demonstrate occurrence of microplastics in plastic food packaging and highlight the risks involved in consumption of microplastics. In this research, the migra- tion of microplastics from different plastic containers were noted in accordance to the Bureau of Indian Standards IS - 9845-1998. The IS 9845: 1998 method was utilized in this investi- gation to determine the overall migration of various plastics used to package mineral water and beverages. Distilled water and 3 % acetic acid (w/v) were utilized as food simulators for the analysis. The migration of microplastics in mineral water were found to range from 0.38 ppm to 0.54 ppm while in case of non-alcoholic beverages overall migration ranged from 0.29 to 75.75 ppm. Among the 12 samples of non-alcoholic beverages, one sample exceeded the maximum limit of microplastics as specified by WHO standard. Keywords Microplastics, simulant, plastic, distilled water, migration . Article information Manuscript received: December 22, 2022; Accepted: March 28, 2023 DOI https://doi.org/10.3126/bibechana.v20i1.53457 This work is licensed under the Creative Commons CC BY-NC License. https://creativecommons. org/licenses/by-nc/4.0/ 1 Introduction Plastics are man made synthetic polymers with dif- ferent properties like low cost, lightweight, easy to excess, durability etc., due to which plastics are used in our daily life in every aspect from house- hold to large industrial scale [1]. Plastics are used in the pharmaceutical industry, agricultural indus- tries, automobiles, storage, clothing, transporta- tion, packaging, etc. This increases the produc- tion of plastics each day [2]. Around 60% plastic packaging is used for food and beverage and rest is 21 http://nepjol.info/index.php/BIBECHANA jagjitkour00@gmail.com https://doi.org/10.3126/bibechana.v20i1.53457 https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ Jagjit Kour et al./ BIBECHANA 20 (2023) 21-26 22 used for other purposes like cosmetics, medicines, etc. [3]. During storage and distribution packaging protects food against physical, chemical and bio- logical hazards. Plastics are composed of large or- ganic molecules so they are printable, moldable and heat sealable [4]. Usually plastics used for the pack- ing mineral water and non-alcoholic beverages are polyethylene terephthalate (PET), polypropylene (PP) etc., and belong to thermoplastic group and can be recycled [5]. Ethylene glycol and dimethyl terephthalate under goes polycondensation reaction to give polyethylene terephthalate. This type of plastic is used for packing food and beverages since they are stable towards temperature and moisture [6, 7]. Transferring of chemicals from plastic ma- terial to food is known as leaching or migration. It is a process that follows Fick’s law of diffusion. Leaching can occur by direct transfer and gas trans- fer phase [8]. As different layers films are applied in packaging, so the chemicals from the films leach to the drinks through diffusion as well as partition method. The method of leaching chemicals from outer layer of plastic to drink is known as set-off [9]. Plastic is one of the important and useful prod- uct in our life and it has made our lives easy in many ways. At the same time it also has negative impact in our life, affecting our health and envi- ronment in different ways. Plastic itself is chemi- cally inactive but different additives present in the plastics may cause chemical reactions which lead to migration. When drinks and foods are aggres- sive, then they react with plastic in which they are packed, in such condition chemicals from plas- tic leaches to drink [10]. Plastic degrades into small particles like micro and nano plastic by different ex- ternal forces caused by humans. Article published by Thompson in 2004 where he used the term mi- croplastic which attracts the people’s attention [11]. Microplastics once deposited in environment, are difficult to remove. Plastic materials are degraded into microplastics and nano plastics by the photo- oxidation process [12]. Microplastics are plastic particles of various shape, size and dimensions rang- ing from 1 µm to 5 mm and are not soluble in wa- ter [13]. There are two types of microplastics found in our environment, primary microplastic and sec- ondary microplastic. Micro plastics that are man- ufactured into small size are called primary MPs where as microplastics degraded from large plastic particles are called secondary MPs [14]. Primary microplastics can enter directly to the environment and are made for different purposes such as for cos- metics, industrial use, personal care, cleaning prod- ucts, beads, plastic pellets etc., and the secondary microplastics are obtained through fragmentation of plastic by aging [15–17]. Presence of microplas- tic in environment increases pollution and affect the health of living beings and destroys the productiv- ity of soil [18]. In developing nation like Nepal where plastic food packaging is very abundant due to various fac- tors such as lack of alternatives, lack of awareness and busy lifestyle. Due to that microplastic leach- ing has become an alarming concern for people. Studies and research carried out in advanced na- tions have properly demonstrated the qualitative and quantitative analysis of microplastic leaching and migration through use of sophisticated tools and apparatus. Those advanced llevel researches have also shown the negative effects caused by mi- croplastic leaching from plastic packaging on human health as well as the environment along with the mitigating measures and alternative approaches. However, in developing nations, these kind of es- earches have not been conducted thoroughly with adequate analysis so as to aware people and pro- vide alternatives. This research is conducted on a small scale to highlight the quantitative measure- ments of microplastic leaching as a result of plastic food packaging and also to relate different kind of impacts it has on environment and human health. This issue is brought forward to people’s attention and can be used for more detailed studies and re- search in coming days. This study is based on the quantitative deter- mination of microplastics leached from the plas- tic containers packed with mineral water and non- alcoholic beverages. Indian standard has specified various types of simulants and test conditions for various type of foods which is given in table 1 and 2. The choice of simulant depends upon the type of food products used, time-temperature condition and also depend upon the use of food products which is given in table 3. Bureau of Indian Stan- dard IS 9845:1998 has classified the drinks and food products into seven categories. These drinks and foods have different simulants and test conditions, which is based on the pH and fat content of the drinks and food products which is given in table 2. In the present study simulant A (distilled wa- ter) was used for mineral water and simulant B (3 % acetic acid) was used for non-alcoholic beverages. European Union (EU), Bureau of Indian Standard (BIS) set the limits for migration of microplastic for food and beverage products as 60 ppm or 60 mg/L. This is the upper limit for overall migration. If the migrating substance has color then that is not suitable for packaging drinks and foods even if the extractive value is below the limit [19]. Jagjit Kour et al./ BIBECHANA 20 (2023) 21-26 23 Table 1: List of food simulants. Simulant A Distilled water Simulant B Acetic acid 3 % Simulant C1 Ethanol 10% Simulant C2 Ethanol 50 % Simulant D n-heptane Simulant E Mixture of synthetic triglycerides or sunflower oil or rectified olive oil Table 2: Categories of drinks and foods with simulants. S.N. Type Discriptions Example Simulant 1 1 3Aqueous non-acidic foods (pH>5) without fat Honey, rasgulla, yeast paste and mineral water etc. A 2 2 Aqueous acidic foods with no fat (pH≤5) Fruit juices, carbonated beverages, lemonade, etc. B 3 3 Alcoholic beverages: a. Concentration of alcohol less than 10 % b. Concentration of al- cohol above 10 % Beers, pharmaceutical syrups Whisky, Wine, brandy, etc. C1 C2 4 4 Fats and processed dry foods with surface fat or volatile oil, oils Vegetables, ghee, biscuits, chocolates, tea and coffee powder D 5 5 Non-acidic foods (pH>5) with excess fat and excess moisture content Butter, bread, pastry, milk-based sweets, ice- cream, etc. A and D 6 6 Acidic foods (pH< 5) with excess fat and excess mois- ture content Different types of pickles, tomato ketchup, cheese, mayonnaise, etc. B and D 7 7 Dry processed foods hav- ing no fat Different types of cereals, pulses, dry fruits, vegeta- bles, salt, sugars, oats, etc. No end test Table 3: Simulants with time-temperature conditions. S.N. Conditions Types of food and drinks Time- temp of water Time- temp of 3% acetic acid Time- temp of 10 % alcohol Time- temp of 50 % alcohol Time-temp of n-heptane 1 High temper- ature heat sterilized 1, 2, 4 121 0C 2h 121 0C 2 h - - 66 0C for 2h 2 Hot filled or pasteurized above 660C, below 100 0C 1, 2, 4, 5 and 6 100 0C 2h 100 0C 2h - - 49 0C for half an hour 3 Pasteurized or hot filled below 66 0C 1 to 6 70 0C 2h 70 0C for 2h 70 0C for 2h 70 0C for 2h 38 0C for an hour 4 Filled and stored at room temp and also in refrigerated and frozen condition 1 to 6 40 0C for 10 days 40 0C for 10 days 40 0C for 10 days 40 0C for 10 days 38 0C for an hour Jagjit Kour et al./ BIBECHANA 20 (2023) 21-26 24 2 Methodology Test Standard: The overall migration of vari- ous plastic containers were determined with IS 9845:1998 method. 2.1 Determination of conjugative weight of Beakers It is very important for this research work to take the conjugative weight of the beakers into consid- eration. Hence, Fifteen beakers of 100 mL each were cleaned and were placed inside the oven with temperature 100 0C for 2h. Then the beakers were removed from the oven and placed in side the desic- cator. After cooling inside the desiccator, weight of the beakers were measured. The process of cooling and weighing were repeated till the constant weight was determined. This process of conjugative weight determination was carried out for all the beakers. 2.2 Preparation of sample solution Glacial acetic (3 mL) was placed in a volumatic flask and 97 mL purified water was added to form 100 mL 3 % acetic acid solution. 2.3 Sample selection The Samples were randomly selected from differ- ent areas inside Kathmandu. Sample selection was done on the basis of their storage condition, type of products mostly used by children and teenagers, plastic condition and local manufacturing products. Three samples of same batch number were taken. 12 Mineral water bottles of four different brands and 12 bottles of non-alcoholic beverages from four different brands were also taken for the testing (two different brands of mango drinks and two different brands of litchi drinks). 2.4 Procedure The plastic bottles used for the analysis were rinsed with distilled water for three times. The pH value of each sample was noted. After that, the plastic bot- tles were filled to their normal capacity with their respective simulant and the lids were closed. Son- ication was carried out to avoid formation of bub- bles. Consequently, the bottles were then placed inside the oven at specified temperature for re- quired time. Then after, the plastic bottles were removed from the oven and content of each bottle was poured into the beaker. The bottle was rinsed with fresh simulant and then again poured into the same beaker. In case of mineral water sample, three plastic bottles of four different brands were taken and their pH value was read then water from plastic bottle was removed. The plastic bottles were rinsed with distilled water three times. Then the bottles were filled with simulant A (distilled water) to their nor- mal capacity and lids were closed. Sonication was carried out to avoid formation of bubbles. Then those filled bottles were placed inside the oven at temperature 40 0C for duration of 10 days. Then the water bottles were removed and the content of each bottle was poured into the beaker whose con- stant weight was already measured. The bottle was rinsed with fresh simulant and then again poured into the same beaker. Similarly, in case of non-alcoholic beverages, same procedure was applied but simulant B (3 % acetic acid) was used as food simulant and the time and temperature condition were also same as in case of mineral water samples. Blank test was also car- ried out for all the samples. 2.5 Calculation of extractive amount Beaker content was evaporated to dryness and the beaker was kept inside the oven at 100 0C to obtain absolute dryness. Then the beaker was removed from the oven and kept in the desiccator for half an hour. After the beaker was cooled, it’s conjugative weight was noted as 0.1 mg. Amount of extract was calculated in ppm. Similarly, blank test was also done without the sample and this process was done for all other samples. Extractive Amount (Ex) = M/V * 1000 ppm or mg/L where, M = mass of extract in mg - value of blank test and V = total volume of simulant applied for each sample in mL 3 Results and Discussion Overall migration of microplastics in mineral wa- ter and non- acholic beverages were carried out us- ing food simulants A (distilled water) and B (3 % acetic). It was seen that every plastic packaging leaches the microplastic in different quantities de- pending on type of plastic container, storage tem- perature, storage duration, pH value of drink and type of drink packed in the container. 3.1 From water bottles Distilled water was used as food simulant in the overall migration test for mineral water bottles. Amount of microplastic found in plastic bottles of mineral water is different for different brands. Table 4. shows the amount of extractive for different min- eral water bottles. Microplastics abundance ranges from 0.38 ppm to 0.54 ppm. The migration of mi- croplastics in mineral water are within the limit pro- vided by EU. Jagjit Kour et al./ BIBECHANA 20 (2023) 21-26 25 Table 4: Overall migration in mineral water. Sample No. Simulant (mL) Amount of Extractive triplicate (ppm) 1 1000 0.4466 2 1000 0.0333 3 1000 0.0666 4 1000 0.7100 Table 5: Overall migration in non-alcoholic beverages. Sample No. Simulant (mL) Extractive ( ppm) 1 (Lichee drink) 150 75.0560 2 (Lichee drink) 170 5.6399 3 (Mango drink) 250 0.4266 4 (Mango drink) 250 0.2933 3.2 From water bottles 3 % Acetic acid was used as food simulant for the non-alcoholic beverages. 12 samples of four differ- ent brands along with blank were studied for over- all migration. Table 5. shows the amount of ex- tractive for different non-alcoholic beverage bottles. The results show that among 12 samples of mango and lichee drinks only one sample of lichee drink (sample no. 1) exceeded the limit of migration of microplastics given by EU. 3.3 From water bottles As the present study is based on the quantita- tive measurements only and depicts the preliminary analysis of microplastic migration based on plastic packaging of mineral water and non-alcoholic bever- age that resulted in above mentioned findings. Ac- cording to Oßmann et al., 2018, in blank samples, 384–468 microplastic particles per liter were dis- covered. Mineral water included anywhere between 2649 and 2857 microplastics per liter in single-use PET bottles, 4889 to 5432 microplastics per liter in multi-use PET bottles, and 6292 to 10,521 mi- croplastics per liter in glass bottles whereas accord- ing to Shruti et al. (2020), microplastics in a range of sizes (0.1-3 mm) and hues, including blue, black, and brown, were discovered in non-alcoholic bever- age such as soft drinks, energy drinks, and cold tea. Hence, end to end relative data is not available for comprehensive comparison. 4 Conclusion The overall migration limit is 60 mg/L or mg/kg or ppm for plastic container used for food packaging as given by the EU. Present study showed that the mi- gration in mineral water occurred in the range 0.38 ppm to 0.54 ppm. However, the migration limit was within the range specified by the EU. In case of non-alcoholic beverages, overall migration of mi- croplastics exceeded the limit provided by EU in one sample only. This might be due to the type of plastic container, storage conditions, duration of storage and quality of the drink. However, the oc- currence of microplastic leaching was evident which indicates that every plastic leaches which is an en- vironmental and health risk. This shows that long term use of these kind of drinks affect the human health gradually and especially for children the risk is very high. The microplastics are consumed along with the food and drinks and after consumption it might disrupt the metabolic activities inside the body that can cause different types of diseases in the long run. Therefore, consumption of these kinds of foods and drinks packed in the plastic containers should be discouraged. Alternative solutions such as use of bio-degradable plastic containers, use of paper packaging and awareness on plastic leaching to general public should be conducted. To facilitate this, relevant policies should be implemented from local bodies and government entities. Moreover, traditional plastic packaging practice should be pe- nalized and use of bio-degradable options should be promoted. Acknowledgement The authors would like to extend heartfelt grati- tude to the University Grant Commission (UGC), Bhaktapur, Nepal for funding the research work un- der the Small Research Development and Innova- tion Grant (SRDIG) Award no. 76/77-S and T-8 and Zest Laboratories and Research Center, Balkot, Bhaktapur, Nepal for carrying out all the experi- ments for this research work. Jagjit Kour et al./ BIBECHANA 20 (2023) 21-26 26 References [1] C. Wang, J. Zhao, B. Xing. Environmen- tal source, fate, and toxicity of microplastics. Journal of Hazardous Materials, 407:124357, 2020. 10.1016/j.jhazmat.2020.124357 [2] J. Chen, W. Wang, H. Liu, X. Xu, J. Xia. A review on the occurrence, distribution, charac- teristics, and analysis methods of microplastic pollution in ecosystems. 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