|Year : 2018 | Volume
| Issue : 58 | Page : 587-596
Elemental profiling of smokeless tobacco samples using inductively coupled plasma-mass spectrometry, their chemometric analysis and assessment of health hazards
Hassan A Alhazmi1, Waquar Ahsan1, Ibraheem M Attafi2, Asaad Khalid3, Siddig I Abdelwahab3, Mohammad Al Bratty1, Shahnaz Sultana4
1 Department of Pharmaceutical Chemistry, College of Pharmacy; Smokeless Tobacco Research Group, Substance Abuse Research Centre, Jazan University, Jazan, Saudi Arabia
2 Poison Control and Medical Forensic Chemistry Center, General Directorate of Health Affairs, Jazan, Saudi Arabia
3 Smokeless Tobacco Research Group, Substance Abuse Research Centre, Jazan University, Jazan, Saudi Arabia
4 Smokeless Tobacco Research Group, Substance Abuse Research Centre; Department of Pharmacognosy, College of Pharmacy, Jazan University, Jazan, Saudi Arabia
|Date of Submission||17-May-2018|
|Date of Acceptance||29-Jun-2018|
|Date of Web Publication||21-Nov-2018|
Hassan A Alhazmi
Department of Pharmaceutical Chemistry, College of Pharmacy, Jazan University, P. O. Box: 114, Jazan
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Smokeless tobacco (ST), locally called shammah, is a form of tobacco that is widely used in Middle Eastern countries, including Saudi Arabia. Objective: A total of 21 ST samples were collected from the southern province of Jazan for elemental analysis. Materials and Methods: These samples were analyzed by inductively coupled plasma mass spectrometry to determine their element concentrations. Chemometric multivariate analysis such as hierarchical cluster analysis, Pearson correlation analysis, and principal component analysis were performed for better understanding and interpretation of the data. Concentrations obtained were used to determine the users' daily and weekly intake of elements and were compared with the acceptable daily intake and provisional tolerable weekly intake. Results: Metal ions present in maximum concentrations were strontium (11608.71 μg/kg) and manganese (3543.10 μg/kg), whereas those with minimum concentrations were silver (53.90 μg/kg) and chromium (62.33 μg/kg). Conclusion: Although the concentrations of all the elements fell under the safe limit, the concentrations of many toxic elements were significantly high and resulted in various health hazards on the intake of these elements with other sources.
Abbreviations used: ST: Smokeless tobacco; EDI: Estimated daily intake; ADI: Acceptable daily intake; PTWI: Provisional tolerable weekly intake; HQ: Hazard Quotient; RfD = Oral reference dose; ICH: International Council for Harmonisation; ICP-MS: Inductively coupled plasma-mass spectroscopy; AAS: Atomic absorption spectroscopy; CTA-OTL-1: Oriental Tobacco Leaves; % R. S. D.: % Relative standard deviation; ppb: Parts per billion; LOD: Limit of detection; SD: Standard Deviation; FAO: Food agriculture organization; WHO: World Health Organization; JECFA: Joint Expert Committee on Food Additives.
Keywords: Elements, heavy metals, inductively coupled plasma-mass spectroscopy, Jazan, Shammah, smokeless tobacco
|How to cite this article:|
Alhazmi HA, Ahsan W, Attafi IM, Khalid A, Abdelwahab SI, Al Bratty M, Sultana S. Elemental profiling of smokeless tobacco samples using inductively coupled plasma-mass spectrometry, their chemometric analysis and assessment of health hazards. Phcog Mag 2018;14:587-96
|How to cite this URL:|
Alhazmi HA, Ahsan W, Attafi IM, Khalid A, Abdelwahab SI, Al Bratty M, Sultana S. Elemental profiling of smokeless tobacco samples using inductively coupled plasma-mass spectrometry, their chemometric analysis and assessment of health hazards. Phcog Mag [serial online] 2018 [cited 2019 Apr 24];14:587-96. Available from: http://www.phcog.com/text.asp?2018/14/58/587/245848
- Twenty one different varieties of Smokeless Tobacco (Shammah) samples were collected from various regions of Jazan, Saudi Arabia
- Qualitative and quantitative determination of different types of elements was performed by ICP-MS
- Total intake of elements by means of tobacco consumption was calculated and compared with the reference doses provided by international agencies and their hazard quotient were calculated
- Various Chemometric statistical analyses were performed to analyze the correlation between elements present in samples.
| Introduction|| |
Smokeless tobacco (ST), also known as chewing, spitting, dipping, or snuffing tobacco, is a form of tobacco that is used other than smoking. ST is generally used by placing tobacco into the oral cavity, mostly between the lips or cheeks and gums, and ingesting the saliva produced thereafter. The general types of ST include the leaves of Nicotiana tabacum, Nicotiana rustica, Nicotiana glauca, or Nicrophorus nepalensis., Although, the trade of ST is illegal in some countries such as Saudi Arabia, Japan, Hong Kong, Singapore, Australia, and New Zealand, its use is continuously increasing with time. ST has over 300 million users worldwide, and most of these users are from South Asian and Middle Eastern countries. The use of ST is more prevalent in the southern part of Saudi Arabia (Jazan Province) due to its proximity to Yemen, where it is legal to use and trade.
ST is associated with a range of adverse health issues, including cancer and cardiovascular diseases. Some harmful substances are reported to be found in ST, including at least 28 chemicals as potential carcinogens, of which nitrosamines are the most harmful. There are some trace elements in the soil that are absorbed by tobacco plant which accumulates them in its leaves in large quantities. Some of the elements are too toxic to the human body even in minute quantities.,,, The concentrations of these trace elements in tobacco largely depend on the genotype; type and pH of water and soil; fertilizers and pesticides used; and various other environmental factors. Some of these elements are responsible for different types of ailments and diseases when consumed in quantities beyond the tolerable limits.
The regions of Jazan Province, where the use of ST (Shammah) was most prevalent, were selected for sample collection. These regions were Jazan, Sabya, Abu Areesh, Ahd Al Masaraha, and Samtah. Different types of tobacco samples collected were Green, Black Adani 1, Black Adani 2, Black Sudani, Suhail, Areeshi, and Special. A total of 21 different types of samples were collected and subsequently analyzed to investigate their metal ion concentrations using inductively coupled plasma mass spectrometry (ICP-MS). Different types of metal ions analyzed in this study were lithium (Li), beryllium (Be), vanadium (V), chromium (Cr), manganese (Mn), cobalt (Co), copper (Cu), zinc (Zn), gallium (Ga), arsenic (As), selenium (Se), rubidium (Ru), strontium (Sr), silver (Ag), cadmium (Cd), cesium (Cs), barium (Ba), thallium (Tl), lead (Pb), and uranium (U).
ICP-MS was used to determine the metal ion concentrations in tobacco samples because it is a robust, sensitive, and accurate method having high detection capability and can be used to accurately detect concentrations at parts per quadrillion level. Moreover, the advantage of ICP-MS over other elemental detection techniques, such as atomic absorption spectroscopy (AAS), is that the concentration of a single element can be measured in AAS at a specific time, whereas, all elements can be determined simultaneously in ICP-MS.
The present study was aimed to select various tobacco samples from different regions of Jazan Province and to determine the metal ion concentrations in their leaves by using ICP-MS and their comparison with the provisional tolerable weekly intake (PTWI) to assess the health hazards that they pose.
| Materials and Methods|| |
A total of 21 samples were collected from five different cities of Jazan Province: Khudra Jazan (Jazan Green; Sample no. T-1), Areeshi Jazan (Sample no. T-2), Adani Jazan (Jazan Black Adani type-1; Sample no. T-3), Adani Jeeban (Jazan Black Adani type-2; Sample no. T-4), Sudani Sabya (Sabya Black Sudani; Sample no. T-5), Suhail Sabya (Sample no. T-6), Khudra Sabya (Sabya Green; Sample no. T-7), Areeshi Sabya (Sample no. T-8), Khususi Masna Sabya (Sabya Special; Sample no. T-9), Adani Barid Abu Areesh (Abu Areesh Black Cool type; Sample no. T-10), Adani Haar Abu Areesh (Abu Areesh Black Hot type; Sample no. T-11), Khususi Abu Areesh (Abu Areesh Special; Sample no. T-12), Areeshi Abu Areesh (Sample no. T-13), Ahd Al Masaraha Areeshi (Sample no. T-14), Ahd Al Masaraha Khudra (Ahd Al Masaraha Green; Sample no. T-15), Ahd Al Masaraha Khususi (Ahd Al Masaraha Special; Sample no. T-16), Adani Baheel Ahd Al Masaraha (Ahd Al Masaraha Black Adani with Cardamom, Sample no. T-17), Adani Ahd Al Masaraha (Ahd Al Masaraha Black Adani; Sample no. T-18), Khudra Abu Areesh (Abu Areesh Green; Sample no. T-19), Areeshi Khudra Samtah (Samtah Green Areeshi; Sample no. T-20), and Khususi Samtah (Samtah Special; Sample no. T-21). The five cities of Jazan Province, from which the samples were collected, included Sabya, Abu Areesh, Ahd Al Masarha, and Samtah. The use of ST is most prevalent in these cities. The samples were collected and stored in dry condition away from direct sunlight until further analysis.
Chemicals and reagents
All the chemicals used were of AR grade and were purchased from Sigma Aldrich (Germany). Deionized water (18 MΩ) purchased from Sigma Aldrich (Germany) was used throughout the study. Microwave Digestion System Ethos I (Milestone, USA) was used for the digestion of tobacco samples. The elemental concentrations of the digested samples were analyzed using ICP-MS 7500 (Agilent, Germany).
To determine the total concentration of different metals, 0.1 g of dry and fine ST samples were taken and digested in a microwave digestion system ethos I in Teflon vessels with 4 mL of nitric acid. The digestion parameters were as follows: The programmable microwave power ranged from 800 W to 1400 W, temperatures ranged from 50°C to 200°C, ramp time was set to 10 min, and the total time of digestion was 70 min. On completion of the digestion process, Teflon vessels were allowed to cool at room temperature for 30 min. Thereafter, the Teflon vessels were opened under the hood and kept for 5 min until all fumes evaporated. The digested samples (0.5 mL) were then diluted to 10 mL with ultrapure deionized water and analyzed using the ICP-MS. Blanks were digested and prepared by following the same procedure.
Inductively coupled plasma-mass spectroscopy analysis
The metal concentrations of the ST were analyzed using ICP-MS 7500 (Agilent, Germany). All samples, blanks, and standards were analyzed in triplicate. Instrument quality control and tuning were performed using an instrument tuning solution at 1 ppb in 2% HNO3.
Preparation of calibration curve and validation
A six-point calibration curve of the multi-element standard was plotted at 1, 5, 10, 20, 50, and 100 ppb. Excellent linearity was obtained in case of all elements with a correlation coefficient (r) value > 0.99. The digestion and analytical methods were validated using standard reference tobacco leaves (CTA-OTL-1). Percentage relative standard deviation (% R. S. D.) was calculated by using the recovered value of each element from the selected method and certified value. The percentage recovery of each metal was calculated as:
% Recovery = 100 × (value of selected method/certified value)
To determine the precision and accuracy of the selected method five quality control samples of concentrations 5, 10, 25, 50, and 90 ppb were analyzed interday and intraday before doing the real-time analysis. % Accuracy was calculated by the following formula:
% Accuracy = 100 × (practically obtained concentration/theoretical concentration)
Limit of Detection (LOD) values were determined from the calibration curve according to the ICH guidelines of validation (Q2R1). The standard deviation of several blank samples (SDblank) was determined and was used in the following formula:
LOD = 3.3 × (SDblank/slope of calibration curve)
Human exposure assessment
Considering that a user/dipper holds the tobacco in his/her pinch and places it between his/her gums and lower lip, then the quantity of consumed tobacco at one time varies between 0.5 g and 1.0 g. In this study, the quantity of consumed tobacco for all calculations was set at 0.5 g. If on an average, a dipper consumes tobacco 10 times daily, then the total quantities of tobacco consumed daily and weekly are 5 and 35 g, respectively. From this quantity, the amounts of elements consumed through tobacco per week were calculated by using Equation 1:
Celement= concentrations of elements (μg/kg) in different samples,
Cw= amount of Tobacco consumed per week = 35 g.
Other assessment parameters, namely, estimated daily intake (EDI) and hazard quotient (HQ), were investigated. EDI refers to the concentration of elements consumed per kg body weight per day by a user. EDI (μg/kg bw/day) is calculated using Equation 2
Celement= concentrations of elements (μg/kg) in different samples,
Cd= amount of tobacco consumed per day = 5 g = 0.005 kg,
bw = average body weight (kg) of target population = 70 kg.
HQ is calculated by dividing EDI by the established RfD values. The significance of HQ is that if the value of HQ is <1, then the substance is not hazardous. HQ is calculated using Equation 3
EDI = estimated daily intake,
RfD = reference dose of the substance.
The analytical data were analyzed using SPSS statistical program 20.0 (IBM, SPSS, Armonk, NY). Three multivariate statistical analyses were employed for better interpretation of data. There were 20 rows expressing various elements and 21 columns indicated different ST samples. Hierarchical cluster analysis was performed to determine if there were any significant groups of samples with the same characteristics. Pearson correlation analysis was utilized to show the correlation between variables and is useful in determining divergence and coherence of data. The third multivariate technique performed was a principal component analysis on the whole data to check the variability on a scale by dividing variables into principal components.
| Results|| |
Validation of the method
All the method validation parameters were assessed according to the ICH guidelines (Q2R1), and the results are presented in [Table 1]. Multi-element calibration curve was constructed for the quantification of elements and excellent linearity (1> r > 0.99) was observed for all elements. % Recovery, % Accuracy, % R. S. D. and LOD values were calculated for each element, and the results are summarized in [Table 2]. % Recovery values were obtained to be in a range of 92%–105%, % Accuracy was obtained in a range of 96%–122% and % R. S. D. values were <2.0 in case of all elements. All the obtained values were within the prescribed limit as stated in the ICH guidelines.
|Table 2: Concentrations (μg/kg) of various elements in different tobacco samples as measured by inductively coupled plasma mass spectrometry|
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All 20 elements were analyzed simultaneously for each sample in triplicate, and the concentrations were determined in micrograms per kilograms (μg/kg). All the obtained results are summarized in [Table 2]. The highest concentration was found for Sr (average concentration = 11608.71 μg/kg), whereas the lowest concentration was for Cr (average concentration = 62.33 μg/kg) in all the samples. The concentration values of all the elements in different samples were almost similar with few exceptions. For example, Cr was present in sample T-1 in relatively high quantity (528.4 μg/kg), but it was below detection limit (BDL) in most samples. Li was present in the concentration range of 151.7–708.4 μg/kg with an average of 521.28 μg/kg. Similarly, Be (106.3–109.9; 108.2), V (48.38–227.8; 109.08), Cr (0–528.4; 62.33), Mn (1570–5886; 3543.1), Co (45.5–106.4; 73.58), Cu (280.6–587.5; 383.75), Zn (533.4–1263; 740.07), Ga (74.58–90.41; 83.12), As (99.6–132.6; 115.29), Se (292.5–309.4; 300.86), Rb (132–225.5; 172.94), Sr (6566–16350; 11608.71), Ag (53.15–59.13; 53.90), Cd (93.28–102.7; 96.7), Cs (68.9–73.71; 70.29), Ba (520.4–1854; 1202.54), Tl (109.3–109.8; 109.53), Pb (175.3–211.5; 187.36), and U (119.6–133.3; 125.45) were found to be in significant quantities in all the samples. [Figure 1] shows the comprehensive chart of various elements present in different tobacco samples for comparison.
|Figure 1: Comprehensive chart showing the concentrations of various elements in different tobacco samples (T1–T21)|
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The amounts of elements consumed per week [Table 3] by the user were calculated using Equation 1, and the resulting values were compared with the PTWI based on the guidelines set by the Food Agriculture Organization/World Health Organization and Joint Expert Committee on Food Additives (JECFA). The PTWI for some elements, such as Li, V, Cr, Mn, Co, Cu, Zn, As, Se, Cd, and Pb were set to be 600, 500, 200, 4000, 19, 1500, 15, 35, 7, and 25 μg/kg body weight, respectively.,, Therefore, for these elements, the tolerable limits per week for a 70 kg person would be 42, 35, 14, 280, 1.33, 245, 105, 1.05, 2.45, 0.49, and 1.75 mg for Li, V, Cr, Mn, Co, Cu, Zn, As, Se, Cd, and Pb from all sources, respectively.
|Table 3: Amounts of elements (μg) consumed per week for different samples|
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Human exposure assessment
These limits were compared with the consumption of elements per week using ST, and the results showed a significant share in the total dietary intake for tobacco users. The results for the estimated consumption of elements per week are summarized in [Table 3]. It shows the amounts of elements found in 35 g sample, which is the approximate weekly intake of tobacco by the user for each sample. Although all the results were under the PTWI, they are still alarming because they constitute a major part of the total dietary intake of these elements by all means. These elements are also present in other food sources, and together, they can cross the PTWI, which could be detrimental for the user.
The results for EDI and HQ are summarized in [Table 4]. The EDI for each element was calculated for different tobacco samples which gives the concentration of various elements consumed daily by the user per kg body weight. This result can be compared with acceptable daily intake (ADI) and PTWI, which were previously established along with RfD by JECFA and the United States Environmental Protection Agency (U. S. EPA). The HQ values of various elements were calculated by dividing the EDI with Oral RfD is the dose in μg that can be taken orally per kilogram body weight per day. If the HQ of an element is more than one, that is, if the EDI exceeds RfD, then the element is considered toxic. Similarly, if the HQ is <1, that is, the ADI is less than the RfD, then the element is not considered hazardous. All the HQ values were <1, indicating that the overall intake of elements from ST was within safe limits.
|Table 4: Estimated daily intake and hazard quotient values of various elements for different tobacco samples|
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Hierarchical cluster analysis
By performing the cluster analysis, for the elements, we could determine that there were four clusters of samples based the clustering method used. The cluster solution was seen as a sudden jump (gap) in the distance coefficient. The solution before the gap indicates the good solution. Samples T-2,-7,-12,-18,-19,-20, and -21 were distributed in the same cluster. Where sample T-3,-5,-10,-11, and -17 have the same characteristics to be include in the same cluster. Distribution of the samples based on their elemental concentrations in form of a dendrogram is shown in [Figure 2].
Pearson correlation analysis
For correlation analysis, values obtained that were higher than 0.50 were considered to correlate various data points and the results are summarized in [Table 5]. Values in bold indicated significant correlation between variables at higher confidence interval (**P < 0.01). If the values obtained were positive, it showed positive correlation among the variables, whereas, the negative values signified negative correlation. The correlation values near to “zero” indicated poor and nonsignificant positive or negative correlation and on the other hand, values closer to “one” indicated significant correlation among variables. Interestingly, no any pair of elements displayed significant negative correlation among themselves at both the confidence levels (P = 0.01 and P = 0.05). Rubidium (Rb) showed an interesting behavior by not showing any significant positive or negative correlation with any of the element. Another element, Zinc (Zn), showed positive significant correlation with only one element (Co). Therefore, these two elements were placed in two distinct groups. The remaining elements were placed in three different groups following the interpretation of the correlation matrix. Group I consisted of elements such as Li, As, Sr, Cd, U, Ba, Mn, Be, Se, Co, and Cu. Group II comprised Cs, V, Pb, and Ga whereas, elements such as Ag, Cr, and Tl were placed in group III. Group IV and Group V consisted of Zn and Pb, respectively. This grouping of elements was further analyzed through another multivariate analysis, i.e., principal component analysis.
Principal component analysis
Principal component analysis was performed on the whole data and the results obtained are summarized in [Table 6]. It shows the rotated component matrix of the principal component analysis. All the variables are grouped in five principal components (PC1 to PC5). Data in bold showed the absolute values >0.50. Elements of Group I were found to give major contribution to principal component 1. Similarly, Group II elements of the correlation matrix were the major contributor to principal component II. Three major principal components were observed as shown in [Figure 3] that depicts the component plot in rotated 3D-space. It shows the exact placement of elements in their principal components according to their absolute values.
|Table 6: Component matrix obtained for principal component analysis showing placements of elements in five principal components (PC1-PC5)|
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|Figure 3: Component plot obtained after Principal component analysis showing the placement of elements in three major principal components|
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| Discussion|| |
All the detected elements were classified into (a) trace, (b) toxic, (c) essential, and (d) nonessential elements based on their nature and abundance and their presence in tobacco samples were studied. Various trace elements present were Li, Zn, Cu, Cr, Co, V, As, Rb, Sr, Mn, and Se, whereas toxic elements such as As, Pb, Cd, Be, Cu, Li, Mn, Ag, Zn, Ba, and Tl were also present. These elements are considered to be toxic when consumed even in smaller quantities for a long period. Arsenic (As) is known to cause cancer and diabetes, whereas, Pb is responsible for anemia, plambism, and encephalopathy. Cadmium (Cd) causes high blood pressure, lung cancer, and osteomalacia, whereas, Be leads to cardiovascular disorders. Similarly, other elements have various harmful effects on the body when consumed regularly. Essential elements that were present in the tobacco samples were Co, Cu, Cr, Mn, Se, and Zn. These elements were present in trace quantities in all the samples. Furthermore, the presence of nonessential elements was also observed. These elements were V, As, Ba, Be, Cd, Ga, Ag, Pb, Li, Rb, Sr, Cs, Tl, and U which were present in low-to-high concentrations.
Metals, such as Co, Cu, Cr, Mn, Se, and Zn, are referred to as essential nutrients because these are required for various physiological and biochemical functions of the body. The insufficiency of the supply of these elements results in various diseases or syndromes. These elements have important roles as constituents of several enzymes and take part in several redox reactions in biological systems. For example, Cu is an essential cofactor for many oxidative stress-related enzymes, such as peroxidase, cytochrome c oxidases, catalase, monoamine oxidase, etc.,, Moreover, Cu has toxic properties too as it can exhibit transitions between Cu (II) and Cu (I), which generate reactive oxygen species (ROS), including superoxide and hydroxyl radicals.,,, Excessive Cu intake has been reported to cause cellular damage, which leads to Wilson disease in humans., Similar to Cu, other essential elements that are present in tobacco are required for biological functioning. However, excessive amounts of these metals produce damage to tissues and cells, resulting in various harmful effects and diseases in humans. Cu along with Cr has a very narrow range of concentrations between beneficial and toxic effects.,
Other elements, such as As, Ba, Be, Cd, Ga, Pb, Li, Ag, Sr, Tl, V, and Uranium (U), which are present in all tobacco samples in high to medium concentrations, do not have any established roles in biological functions and are thus considered non-essential elements. Strontium (Sr) was found to be present in highest concentrations in all the samples that is similar in properties and uses to calcium (Ca), but its use is restricted nowadays because of its harmful effects on the body including various cardiovascular disorders. After Sr, the other highly abundant metal found in tobacco samples was Mn, which comes under the essential metal ions category. Mn has an important role in maintaining human health and is essential for the development and metabolism in humans. However, its excessive intake causes a set of disorders called manganism, which is a neurodegenerative disorder that affects the dopaminergic nerves and leads to a condition similar to Parkinsonism.,
The presence of heavy metal ions in tobacco samples was also investigated, and their concentrations were compared with the prescribed limits. The heavy metals present in these tobacco samples were As, Cu, Cr, Co, Zn, Ag, Cd, Pb, Se, and Tl. Although, except Zn, the presence of other heavy metal ions was comparatively less but cannot be ignored. Heavy metals in biological systems are known to have pronounced effects on cellular components and various enzymes involved in metabolism and repair. They interact with the cellular components and cause conformational changes that damage these components and lead to cancer and apoptosis., Heavy metals, such as As,,, Cd, Cr,, and Pb,, are known to produce carcinogenic effects due to their capability to produce ROS in biological systems. These elements are among those that pose a significant hazard to human health. These elements are all systemically toxic and cause damage to many organs, even at low concentrations. Various experimental and epidemiological studies have reported that these elements are placed under the category “known” carcinogens by the U. S. EPA and the International Agency for Research on Cancer.
| Conclusion|| |
The concentrations of various elements in all 21 tobacco samples were measured successfully by ICP-MS. The tobacco samples were of different varieties, color, and origin, and most of the elements in these samples showed some variation in concentration. Elements were found in either significantly high or relatively low concentrations. Different types of heavy and toxic metals, which could lead to various health hazards like cancer and cardiovascular diseases, were found in all tobacco samples. Further studies should be conducted to discover other toxic substances that are present in these tobacco samples to justify their potential in causing diseases.
The authors are thankful to the Deanship of Scientific Research, Jazan University for providing financial assistance (Research Group no. RG-2-9) to carry out this research work.
Financial support and sponsorship
This work is supported by Deanship of Scientific Research, Jazan University, Jazan, Saudi Arabia as Group Research Project (no. RG-2-9).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]