Assessment of Arsenic, Vanadium, Mercury, and Cadmium in Food and Drug Packaging

Background Food and drug packaging materials are an integral part of our everyday life. Noxious elements can inadvertently be included in packaging materials in various stages of their production. Adulterants, adhesives, colorants and heavy metal interference are the common sources of contamination in food packaging materials. Heavy metal toxicity has far-reaching ill effects on living organisms. The present study aimed at qualitatively and quantitatively analysing heavy metal content of various materials that are used for food and drug packaging in India. Methods The qualitative detection was done by rapid assay and heavy metals were quantified with the help of inductively coupled plasma-optical emission spectrometry (ICP-OES). A total of thirteen types of food and drug packaging materials were procured from local market and analysed for four heavy metals viz. arsenic (As), vanadium (V), mercury (Hg) and cadmium (Cd). The concentration of each heavy metal in the samples was compared with the permissible values published by the European Council. Results Heavy metals were qualitatively detected in ten out of thirteen samples. Among the ten samples mercury and arsenic were detected the most followed by cadmium and vanadium. Quantitative estimation by ICP-OES showed presence of vanadium and cadmium in ten samples and arsenic and mercury in all the thirteen samples above the permissible range. Conclusions The notable elevation in mercury concentration, followed by cadmium, arsenic and vanadium registering the least, presents a potential health hazard to consumers and compromises the food quality.


Results
Heavy metals were qualitatively detected in ten out of thirteen samples.Among the ten samples mercury and arsenic were detected the most followed by cadmium and vanadium.Quantitative estimation by ICP-OES showed presence of vanadium and cadmium in ten samples and arsenic and mercury in all the thirteen samples above the permissible range.

Conclusions
The notable elevation in mercury concentration, followed by cadmium, arsenic and vanadium registering the least, presents a potential health hazard to consumers and compromises the food quality.

Introduction
Packaging materials are defined as any substance or item that comes in contact with food and drug including containers, cans, bottles, cartons, boxes, cases and covering material such as foil, film, metal, paper, wax paper or cloth. 1 The main objective of packaging is to provide protection from foreign materials while in-transit and help in maintaining the shelf life of food products.Packaging is a field, combining science and technology used for protecting products during distribution, storage, sale and use.It can also be applied to the procedure of evaluating, designing and manufacturing a packed item.Storage and preservation instructions, directions for use, expiry or use by date, and design of packaging materials among others are crucial information which give cognoscibility to the brand and increase the visibility on the shelf. 2,35][6] Leaching can be explained as any process that allows transfer from surface to the core particles. 7With reference to food packaging, the term "leaching" can be defined as the movement of undesirable particles from packaging materials to the packaged particle.The European Council Standard requires that various contaminants like aromatic amines, benzophenones, polyaromatic hydrocarbons, plasticizers and heavy metals be controlled and analyzed in food packages. 8Heavy metals are a huge source of environmental pollution. 9he toxicity of heavy metals has harmful effects on biological systems as they do not undergo biodegradation.They accumulate in living organisms, causing several diseases and disorders even when present in very low concentrations. 10e toxic effects of vanadium are particularly seen in lung and stomach tissues, resulting in pneumonia, bronchitis and breach in the gastric mucosal lining. 11Increased concentration of arsenic in humans can induce epigenetic changes and genetic mutations causing cancer. 12Cadmium has been used in industries for a long time.Toxicity due to cadmium can affect kidneys, and the reproductive, skeletal, and respiratory systems, causing proteinuria, kidney stones, loss of bone density and mineralisation, destruction of mucous membranes, pneumonitis, testicular necrosis, and affects steroid hormone synthesis. 13Mercury pollution impacts human health leading to developmental flaws in children and Minamata disease 14 Metals like vanadium and mercury may be added as additives as a part of manufacturing process or may be an unintentional adulterant released from the moulds used specifically in the plastic industry. 15 this study locally procured food and drug packaging materials were used.The study started with digestion of packaging materials followed by preliminary qualitative analysis.All the samples were analysed for four heavy metals using spot test for vanadium, senSafe Boris's mercury detection strips, swab test for cadmium and aquasol arsenic detector kit.After analysing the presence or absence of heavy metals all the samples were quantified using ICP -OES (Inductively coupled plasma optical emission spectrometry).The use of ICP-OES is advantageous for sample preparation since it eliminates the need for several dilutions because it can identify multiple components from an analysis.When plasma energy is applied externally to a sample, the constituent elements are stimulated and excited atoms return to their low energy positions leading to emission of rays and the spectrometer captures these to calculate the photon wavelength.The position of the photon rays determines the element type and the strength of the rays establishes the component of each element. 16VISED Amendments from Version 2 In response to reviewer's suggestions, significant enhancements have been incorporated into the manuscript, with reference to arsenic, vanadium, mercury, and cadmium in food and drug packaging materials.One notable modification is in the title of the manuscript focusing four heavy metals considered in the study.The updated guidelines issued by the European Council for Food Packaging Association, specifically the Nordic guidance for authorities, have been included in the manuscript to explain the permissible limits of the select heavy metals found in food and drug packaging materials.These guidelines provide essential insights and regulations regarding the permissible levels of heavy metals in food contact materials, serving as a crucial reference point for evaluating the safety and compliance of packaging materials.Moreover, the inclusion of calibration graphs and detailed specifications on the detection limits for each heavy metal analysed enhances the methodological accuracy and rigor of the study.By presenting this information, the manuscript offers readers an inclusive understanding of the analytical techniques employed and the precision of the measurements obtained, reinforcing the credibility and reliability of the research outcomes.In addition, the discussion section has been elaborated to provide more inclusive consistent analysis of the findings, drawing upon relevant studies to emphasize the significance of heavy metal content in food and drug packaging materials.These corroborative evidences have led to the addition of five more references in the manuscript that elucidates the multifaceted implications of heavy metal exposure on public health and environmental sustainability, fostering a more comprehensive understanding of the issue among readers and stakeholders.The improvements made to the manuscript not only enhance its scholarly credibility and scientific understanding, but also stress upon the need for development of evidence-based policies and regulatory frameworks to assess the heavy metal content within the permissible limits in food and drug packaging materials.
The detection for arsenic (As), vanadium (V), mercury (Hg) and cadmium (Cd) was quantified in ppm (parts per million).The detection limits are stated in the standard curves (Figure 10) viz., V -0.000011 ppm, As -0.000156 ppm, Cd -0.000442 ppm, Hg -<0.001ppm. 17nsidering the wide applications of the packaging material, stringent regulation is necessary due to elevated concentration of heavy metals in them.The Environmental Defence Fund 18 states that these heavy metals can be toxic and the route of ingestion can be packaging materials. 19Sometimes, odorous compounds from the food packaging may get transferred to the food items and affect the foods' flavour.This results in considerable nutritional loss and consumer dissatisfaction.Although food and drug packaging are a multi-billion industry, studies on the presence of heavy metal in these packaging materials in India are lacking.The assessment of heavy metals vanadium, cadmium, arsenic and mercury in thirteen types of locally procured food and drug packaging materials was done by qualitative analysis showcasing presence or absence of heavy metals in them and then confirming their presence using quantitative technique of ICP-OES (Inductively coupled plasma optical emission spectrometry).

Methods
This study was conducted at the Department of Biochemistry, Kasturba Medical College, Mangalore, Karnataka and Department of Chemical Engineering, National Institute of Technology, Suratkal, Karnataka.The Institutional Ethics Committee (IEC) approval was obtained prior to the conduct of the study.
A total of eleven food packaging samples and two drug packaging materials were bought from the local market.The samples included aluminium cans, leak-proof bags, cardboard, tetrapaks, cellophane, tissues, sachets, aluminium bags and boxes, plastic bags and containers, as well as medicinal blister packets and medicinal closures.The packaging materials were collected, cleaned with distilled water and dried in a hot air oven.The samples were cut into small pieces using scissors and metal cutters, and 10 grams each of the sample were weighed using a calibrated electronic weighing scale.

Analysis of toxins in the samples
Packaging material was collected and cleaned as mentioned above.A total of 10 grams of weighed samples were digested using standard acid digestion technique as described by USEPA 305 (United States Environmental Protection Agency amendment no 3050(B). 20Operating conditions for microwave-assisted digestion were followed as per the USEPA 3051. 21The operating conditions were followed as per original protocol.Digestion of the complete sample takes place in the process leaving the heavy metals in solution form.Digested solutions were cooled at room temperature and filtered through 0.45-μm microfilters.Prior to inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis, the samples were again filtered using 0.25-μm filters ensuring a clear filtrate.Analytical grade reagents such as concentrated hydrochloric acid (36%), sulphuric acid (98%) and nitric acid (68%) procured from Sigma Aldrich Chemicals Private Ltd, Bangalore, India were used.Double distilled water was procured from the Chemical Engineering lab at the National Institute of Technology, Karnataka using Accumax Distillation Unit for the extraction of the probable toxins from the samples.

Qualitative analysis
Post digestion of the sample, preliminary tests were performed to qualitatively confirm the presence of heavy metals.The following tests were performed in triplicates.
Spot test for vanadium: The digested solution was treated with 0.1% sodium salicylate solution in a medium of 20 ml syrupy phosphoric acid.A turquoise blue colour obtained indicated a positive result. 22The blue colour is spotted when vanadium forms vanadium hydroxyamide naphthol ternary complex with the hydrogen-free radical. 22nSafe Boris's mercury detection strips were used for the quick and easy detection of low levels of mercury procured by Industrial Test Systems Ltd, U.S.A. Dithizone in the digested fluid acts as a sensitive reagent for the determination of mercury in acidic media.Dithizone forms coloured primary and secondary dithizonates complexes with mercury. 23The presence of a yellow to ochre colour indicates a positive result for presence of mercury.
Swab test for cadmium: a clean cotton swab was dipped in 70% alcohol and dried, after which it was immersed in the digest and air-dried.The indicator was prepared in a small cup containing 1,5-diphenycarbazone and alcohol (70%) in a concentration of 70:30.The cotton swab dipped in the indicator turns violet-blue colour confirming the presence of cadmium.The coloured complex is formed when cadmium reacts with diphenylcarbazone. 24quasol arsenic detector kit was used following the manufacturer's instructions.Briefly, (i) an arsenic silica reagent (ASR) test paper was placed on the black lid of the test bottle, using a forceps provided and making sure that the hole in the lid was covered by the test paper.(ii) The blue disc on the black lid was fixed gently.(iii) Precaution was taken not to touch the test zone.(iv) A 5 ml sample digest was taken in the test bottle with the syringe provided.(v) Three demitasse spoons of ASR-1 were added and gently swirled for a minute.(vi) Six demitasse spoons of ASR-2 were added to the above and immediately tightly screw-capped with the blue disc as prepared above.(vii) The test bottle was allowed to stand for 15 minutes, with intermittent swirling.(viii) The ASR test paper was removed from the lid and dipped in water for two seconds and excess water shaken off.The colour obtained on the test paper was compared with the colour comparison chart provided, at the end of five and eight minutes.A yellow tint indicated a positive test.

Quantitative analysis
All thirteen samples were digested as described above, labelled and subjected to ICP-OES to quantify the chosen heavy metals.Multi-element standard (REICPCAL29A) was used to standardize and calibrate the metal concentration. 25tandards of all four metals were run in triplicates using an inductively coupled optical plasma emission spectrometer (ICP-OES) from Agilent Technologies (U.S.A) with Expert software version of 7.100.6821.61355and firmware version of 2994.

Results
All tests were run in triplicates to ensure consistency in results.The average of the three readings was taken for computation and analyses.

Digestion
Microwave-assisted digestion was carried out using hydrochloric acid and sulphuric acid in a concentration of 80:20 26 used for samples like aluminium can and leak-proof bag.Dehydrator aid proof digestion used a combination of sulfuric acid and nitric acid in the ratio of 80:20 was used for medicinal blister packets, tetrapak, sachets, plastic containers and medicinal closures.Acid digestion with a combination of hydrochloric acid and nitric acid in ratio of 80:20 respectively was employed for the digestion of cellophane, tissue cardboard, aluminium bags, box and plastic container.The time taken for digestion ranged from 210 minutes to 34 minutes (Table 1).Figure 8. Aquasol arsenic detector test.

Quantitative analysis
Heavy metals were detected in the all the thirteen samples.The levels of heavy metals ranged from 0.29 -40.8 ppm for vanadium,1.7 -236.2 ppm for arsenic, 1.53 -546 ppm for mercury and 2.2 -337 ppm for cadmium.The maximum permitted quantity (ppm) as suggested by the European Council for Food Packaging Association (Nordic guidance for authorities) is; As -0.002 ppm, V -0.01 ppm, Hg -0.003 ppm, Cd -0.005 ppm. 27Vanadium was found within the permissible limits in three samples (tissue, sachet, cellophane).Arsenic, mercury and cadmium were above the permissible limit in all the studied samples.Arsenic and mercury were the most common heavy metal contaminants and cadmium and vanadium were the least (Table 3).

Discussion
In the current study, eleven commercially available food packaging materials and two drug packages were analysed for the presence of four heavy metals, namely vanadium, arsenic, mercury and cadmium.Packaging materials constitute the mainstay of the modern food industry.Hence, it is important to study their interference with food/drug present within the packaging material.As research has shown leaching of toxins from packaging materials, monitoring the heavy metal leaching is essential to prevent harmful effects to the human body. 6nadium is a heavy metal which is used as an additive in stainless steel as well as a catalyst for manufacturing sulfuric acid.It is also used in glass coating and lacquering in aluminum cans to give strength to the material.Vanadium is used as an amalgam in manufacturing cans with stainless steel to give it tensile strength.Vanadium toxicity is known to cause abdominal discomfort by interfering with mucosal lining leading to nausea, bloating, diarrhoea and vomiting in the initial stages.Prolonged exposure can result in renal and neural damage. 28Qualitative tests detected the presence of vanadium in only three samples.However, ICP-OES values showed presence of vanadium higher than permissible amounts in 10 samples.The study by Imtiaz et al. 29 stated that vanadium causes oxidative stress in human cells and alteration in human metabolism, reduces enzymatic activities and disturbs membrane integrity in humans. 29This oxidative stress leads to the formation of pentavalate vanadium which is its most toxic and mobile form.Arsenic, found in higher concentration in all the study materials (detected by six samples qualitatively), is a heavy metal that can act as a toxin due to its high presence in water which is used for material cleaning and lacquering processes. 30rsenic is also known to be present as one of the food packaging colorants used in the packaging industry. 31Arsenic could enter the food packaging material during the cleaning process or as an adulterant of iron source.Arsenic has been used as a common ingredient in many pesticides and herbicides in the past.It is known cause skin lesions and cancer in humans. 32The International Agency for Research on Cancer (IARC), a part of the World Health Organisation (WHO) has stated that exposure to arsenic is the leading cause of lung, bladder and skin cancer. 33The National Toxicology Programme (NTP) is composed of several different government agencies, including the National Institute of Health (NIH), the Center for Disease Control and Prevention (CDC), and the Food and Drug Administration (FDA).In its most recent Report on Carcinogens, the NTP classifies arsenic and inorganic arsenic compounds as known to be human carcinogens.The study by Park et al. stated that arsenic is present in paper in contact with food such as any kind of wrapping, and paper board, and presents a risk to consumer safety as increased ingestion in humans can cause skin lesions and gastric cancers. 34rcury dissolves in aluminum at room temperature and is added as an additive in aluminum cans and foils to enhance the shelf-life of the seafood packed inside. 35Ingestion of mercury is the leading cause of Minamata disease.Mercury was detected qualitatively in eight samples and quantitatively in amounts greater than those permissible in sachet and plastic bag only.The mercury absorbed in the body mainly concentrates in the kidneys and brain. 36The half-life of mercury in the body is about 70 days.Inorganic mercury is mainly absorbed through the respiratory tract, but is also absorbed through the skin to a small extent (3-4%) or gastrointestinal (GI) tract (2-10%).Methylmercury is easily absorbed into the GI tract (≥95%) and into the respiratory tract (≈80%).About 90% of methylmercury is excreted through the faeces via bile, and less than 10 % through urine.The absorbed mercury is distributed throughout all tissues within 30 hours.Its halflife ranges from 45 to 70 days. 37Higher concentrations (more than 100 ppm) of mercury have been known to cross the placenta and result in foetal defects. 149][40] Therefore, it would be worthwhile to study the leaching from the packaging material and reconsider the permissible limit if proven.
Ingestion of cadmium in higher concentration creates oxidative stress in the cells and increases the level of antioxidant uptake to protect against macromolecular cell damage, thus leading to prolonged exposure to cadmium, causing depletion of antioxidant levels in the body.Cadmium is generally a contaminant present as residues of the recycling and manufacturing processes, and hence determining their suitability in packaging materials coming in direct contact with foodstuffs is imperative.During the recycling process and cooling of cans, cadmium enters the food production system due to changes in temperature or as an additive used in the metal and glass industry.Cadmium can cause respiratory illnesses, lung fibrosis and cancer. 41Huff et al., found cadmium to be the major causative agent of lung cancer, and possibly prostate cancer.Studies in experimental animals have demonstrated that cadmium concentrations higher than those set by regulations (100 ppm) cause tumours at multiple tissue sites, by various routes of exposure, and in several species and strains. 42rther, these studies offer important context for our findings and support the urgent need for remedial action to mitigate heavy metal exposure through packaging materials.Studies by Sood et al., and Marcelo Enrique Conti, have reported similar trends in heavy metal presence, emphasizing the need for improved quality control measures and regulatory oversight in the packaging industry. 43,44Muhammad et al. conducted research on heavy metals in food-contact papers in 2019.This study looked at the levels of heavy metals in papers that come into touch with food, including paperboard and wrapping paper used in packaging. 45Concerns over consumer exposure to these contaminants were raised by the researchers' discovery that a sizable portion of the samples tested had increased levels of heavy metals, including cadmium and arsenic.A thorough investigation of heavy metal contamination in plastic materials used to package food and pharmaceutical goods was carried out by Khan et al. in 2015. 46Their research showed that heavy metals, such as lead and mercury, were widely present in a variety of plastic packaging types, raising the possibility of health risks related to these substances.The amounts of heavy metals in glass packaging materials that are often used for food and medicine goods were assessed by Turner and Andrew (2016). 47Their study brought attention to the prevalence of heavy metals in glass packaging, specifically lead and cadmium, and emphasized the necessity of regulation and oversight to protect consumers. 47A thorough assessment of research examining the effects of heavy metal pollution on food and medication packaging materials was carried out by Scutarasu and Trinca in 2023.Their findings emphasized that in order to reduce the dangers associated with heavy metal exposure through packing materials, more stringent quality control procedures and regulatory oversight are required. 48Together, these investigations offer insightful information about the frequency of heavy metal contamination in food and medicine packaging materials, emphasizing the necessity of regulatory action to safeguard the health and safety of consumers.
It is alarming to find high concentrations of toxic metals in several of the packaging materials studied.While cadmium seemed to be the least abundant contaminant, arsenic was the most prevalent heavy metal contaminant.Of all the types of packaging materials studied, sachets, which are manufactured using a plastic and aluminium amalgam, had the highest concentration of all the heavy metals.

Conclusions
In summary, a wide variety of food and drug packaging materials from an Indian market were tested for the presence of heavy metal, namely vanadium, arsenic, cadmium and mercury.Our study shows the presence of heavy metals in routinely used food packaging materials, measured quantitatively and qualitatively.All samples, irrespective of the results of qualitative analysis, were subjected to quantitative analysis, as ICP-OES is a sensitive technique for the detection of heavy metals.Of the thirteen samples analysed, arsenic, mercury and cadmium were found in all samples at concentrations above the regulated limits.Vanadium was present in lower concentration in tissue, cellophane and sachets.
Leaching of these heavy metals into the packaged food/drug may be potential health hazard that severely compromise the well-being of humans and the environment.This study calls for stringent regulatory guidelines and strict monitoring of packaging materials at all stages, starting from raw material selection, storage and production until it reaches the consumer.The post-packaging handling, transport, storage and conservation of the supply chain requirements further add to prospective leaching, which may be the scope of future studies.

Partly
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Atomic spectrometry, metals determination, ICP-OES I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.10) enclosed in the manuscript.
Query 5 -The authors state that the ICP-OES can detect heavy metals from 0.0002 ppm to 1000 ppm range.It depends on the metal.For mercury, a studied analyte in this research, the range of concentrations is much limited.The authors should provide the range of

Mahesh Ganesapillai
School of Chemical Engineering, Vellore Institute of Technology, Vellore, India The queries are addressed.The article can be accepted for indexing.

Is the work clearly and accurately presented and does it cite the current literature? Partly
Is the study design appropriate and is the work technically sound?

Xin-Yuan Huang
Nanjing Agricultural University, Nanjing, China In this manuscript, authors quantified four heavy metals in 13 types of food and drug packaging materials, including arsenic (As), vanadium (V), mercury (Hg) and cadmium (Cd).They found that 10 of 13 samples are above the permissible range for vanadium, all samples for arsenic, two samples for mercury and one sample for cadmium.This manuscript was well written but I have the following concerns.Concentrations of the 4 heavy metals shown in Table 3 are presented as parts per million (ppm), which represent the concentrations of these 4 heavy metals in the extract buffer (probably after dilution but not mentioned in the methods).The concentrations of heavy metals in the extract buffer depends on the amount of the food and drug packaging materials used for extraction.So the concentrations must be normalized to the weight of 1.
2. The word used in place of extraction is digestion, the same correction would be made in the updated version of the manuscript.Digestion of complete sample takes places in the process leaving the heavy metal in solution form and therefore the accuracy was 100%.
3. The detection for arsenic (As), vanadium (V), mercury (Hg) and cadmium (Cd) for our study was carried out in ppm (parts per million).The machine is highly sensitive and detects these heavy metals in ppm (parts per million), ppb (parts per billion ) and ppq (parts per quadrillion).For heavy metal samples ICP -OES can detect heavy metals from 0.0002ppm to 1000ppm range.This information would be added in the updated version of the manuscript.
4. The analysis was done with standards of heavy metals namely vanadium, cadmium, mercury and arsenic.For all these heavy metals, calibration curve was developed using standard solution and then the concentration was determined.Thus, no control was required as same solution was used for digestion.
Competing Interests: No competing interests were disclosed.
Reviewer Report 11 November 2022 https://doi.org/10.5256/f1000research.133336.r154512 The study conducted is well aligned with the scope of the journal and boasts considerable novelty in its approach.However, there are a few possible revisions which may alleviate the overall quality of the work: The objective of the study needs to be clearly outlined in the last paragraph of introduction.Please refer the following articles for a proper understanding of the expected phrasing 1,2,3 . 1.

Introduction:
Information on the various quantitative analyses conducted must be mentioned in this section.Such details provide readers with valuable context for the study and improves the logical flow of the manuscript.

2.
Extraction: "…a concentration of 80:20 used…" Why was this composition chosen?Please provide valid reference for the same.

Table 2:
The data represented lacks clarity.Consider incorporating a legend representing the symbols.There are several abbreviations in the manuscript -please include a nomenclature table for the convenience of readers.

Conclusions:
The section needs further improvement and inclusion of better inferences.The mere mention of the presence of heavy mental entities does not conclude the study adequately. 6.

Figure 4 .
Figure 4. Ochre colour indicates positive test for mercury.

Figure 2 .
Figure 2. Turquoise colour indicating positive test for vanadium.

Figure 7 .
Figure 7. Violet colour indicates positive test for cadmium.

Figure 10 .
Figure 10.Calibration curves for select heavy metals.

Figure 9 .
Figure 9. Yellow to ochre indicates positive test for arsenic.

Table 1 .
Digestion of heavy metals from the samples.Cardboard, medicinal blister packets and closures were positive for the presence for vanadium.Mercury was qualitatively identified in eight samples viz.aluminum can and bag, leak-proof bags, sachet, plastic bag, cellophane, medicinal blister packets and closure.Arsenic was present in samples: aluminum bag and box, sachet, cellophane and leak-proof bags.Cadmium was detected in plastic bag, aluminum bag, sachet, cardboard and leakproof bag (Table2).The qualitative analysis assay images are presented as Figures1-9below.

Table 2 .
Presence of heavy metals using qualitative analysis.

Table 3 .
Concentrations in parts per million (ppm) of heavy metals detected in study materials.Bold values indicate values of heavy metals in packaging materials below the or under acceptable amount that is within the given range and not higher than the prescribed limit.
The title is too general and should be rewritten to be more specific for the conducted research.Response 1 -The title is changed to -Assessment of Arsenic, Vanadium, Mercury, and Cadmium in Food and Drug Packaging Query 2 -The correct name of the ICP-OES technique is inductively coupled plasma-optical emission spectrometry (as it is written in the Abstract), not Inductively coupled plasma optical emission spectrophotometry (as it is written in the Introduction).It is not necessary to give the principle of functioning of this technique; it is well-known.Response 2 -The changes are made in the manuscript.

Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly Competing Interests:
No competing interests were disclosed.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Version 1
https://doi.org/10.5256/f1000research.133336.r178554© 2023 Huang X.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Not applicable Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Yes Competing Interests:
1. Ganesapillai M, Sinha A, Mehta R, Tiwari A, et al.: Design and Analysis of Artificial Neural Network (ANN) Models for Achieving Self-Sustainability in Sanitation.Applied Sciences.2022; 12 (7).Publisher Full Text 2. Ganesapillai M, Mondal B, Sarkar I, Sinha A, et al.: The face behind the Covid-19 mask -A comprehensive review.Environ Technol Innov.2022; 28: 102837 PubMed Abstract | Publisher Full Text 3. Biracyaza E, Habimana S, Rusengamihigo D, Evans H: Regular antenatal care visits were associated with low risk of low birth weight among newborns in Rwanda: Evidence from the 2014/2015 Rwanda Demographic Health Survey (RDHS) Data.F1000Research.2022; 10.Publisher Full Text No competing interests were disclosed.