Presence of the heavy metal lead in samples of tobacco (Nicotiana tabaccum) snuff in Nigeria.
Tobacco snuff is one of the varieties of tobacco products that are not smoked (smokeless tobacco). It consists of finely ground leaves of the tobacco plant (Nicotiana tabaccum), which is prepared into dry or moist forms, and is sometimes packaged in ready-to-use pouches. Dry snuff is usually sniffed or swallowed, whereas moist snuff is placed between the gum and the lips or cheek where it is slowly dissolved and absorbed. 
Tobacco snuff contains more than 19 known carcinogens and at least 30 metallic compounds comprising some heavy metals such as lead (Pb), chromium (Cr), nickel (Ni), cadmium (Cd), selenium (Se), and mercury (Hg).  Tobacco is a rich source of these heavy metals because they get preferentially absorbed by the leaves of tobacco during plant growth. 
Harmful effects on human health are associated with prolonged exposure to these heavy metals.  Lead poisoning results in the interference with a variety of body processes, and lead is toxic to many organs and tissues including the heart, bones, intestines, kidneys, and reproductive and nervous systems. [5,6] It is also unsafe in pregnancy. 
Symptoms of lead poisoning include abdominal pain, confusion, headache, anemia, irritability, kidney failure, and in severe cases seizures, coma, and death. [8-10] Children can inhale tobacco snuff indirectly from parent users or as it is being prepared leading to toxicity over a prolonged period. Lead is particularly toxic to children, causing potentially permanent learning and behavioral disorders. 
This study was specifically directed at evaluating the presence of the heavy metal lead in samples of tobacco (N. tabaccum) snuff in Nigeria.
Materials and Methods
Study location: The study was conducted at the Department of Pharmacy, Madonna University, Elele Campus, Rivers State, Nigeria, from January to July 2013. Materials/apparatus: Petri dish, digestion bottle, test tube, beaker, filter paper, separator funnel. Chemicals: De-ionized water, dilute 0.1-M nitric acid, distilled water, standard solution of lead. Equipment/instruments (atomic absorption spectrophotometer [AAS]): Electronic weighing balance (model 2610; Ohaus), centrifuge, atomic absorption spectrophotometer machine (SP 2900 model; Pye Unicam) [Figure 1].
Thirty samples of tobacco snuff were obtained from five geopolitical areas in Nigeria. The date of purchase, name of manufacturer, method of preparation, and place of purchase were all noted. Different brands were collected from each area. Samples of the same brand were mixed together to obtain a representative sample. Brand names were not disclosed because of legal requirement. The breakdown of sample, geographical area, and type are shown in Table 1.
Two grams of the dry snuff sample was weighed using an electrical weighing balance for specific analysis and pretreated as per standard procedure for different metals. This was done for each of the 30 samples.
Wet Acid Digestion Method
A known quantity of sample (2.0 g) was transferred into a digestion bottle. The sample was wet digested with 50 mL dilute nitric acid. This enhances the extraction of the metals in the tobacco snuff. The oven was set at a temperature of 110[degrees]C. The digested sample was kept in the oven for 2 h.
After removal from the oven, the digested sample was filtered using a filter paper to ensure that the residue obtained after digestion is free from organic matter, which otherwise acts as impurities during the metal analysis.
After filtration, the filtrate was further analyzed using AAS.
The filtrate obtained was further analyzed to detect the concentration of heavy metals using AAS. This is sensitive for the determination of heavy metals at low concentrations. It is based on the fact of absorption of radiation of definite wavelength characteristic of the element. Its high sensitivity is exemplified by the fact that most metals can be determined at the part per million and part per billion concentration level.
Atomic Absorption Spectroscopy
Atomic absorption spectroscopy is used principally in limit test for metals in drugs before their incorporation into formulation. The sample is generally dissolved in 0.1 M nitric acid to avoid formation of metal hydroxides from heavy metals, which are relatively volatile and suppress the AAS reading.
In atomic absorption spectroscopy, the metal atoms are volatilized in a flame and radiation is passed through the flame. The volatized atoms that are mainly in their ground state and not emitting energy will absorb radiation with an energy corresponding to the difference between their ground state and excited state.
Instrumentation of AAS
An AAS consists of the following components that enable effective analysis of heavy metals:
(a) Light source: This is made up of a hollow cathode lamp coated with the element being analyzed.
(b) Flame: This is usually air/acetylene providing a temperature of 250[degrees]C; nitrous oxides/acetylene may be used to produce temperatures up to 300[degrees]C, which are required to volatilize salts of elements such as aluminum or calcium.
(c) Monochromator. This is used to narrow down the width of the band of radiation being examined and is thus set to monitor the wavelength being emitted by the hollow cathode lamp. This cuts out interference by radiation emitted from the flame from the filter gas in the hollow cathode lamp and from other elements in the sample.
(d) Detector. This is a photosensitive cell.
The daily intake was calculated using the following equation of Dhaware et al. :
DI ([micro]g/day) = [C.sub.metal] * [W.sub.analyses] * [D.sub.intake]
where [C.sub.metal] is the metal concentration in tobacco snuff sample (TSS) taken for analysis (in [micro]g/g), [W.sub.analyses] are the weight of TSS taken for analysis (2.0 g in this study), and [D.sub.intake] is the daily intake (assuming 10 pouches per day, 5.0 g/pouch).
Target Hazard Quotient
For the assessment of health risks arising from the indirect intake of heavy metals through the consumption of TSSs, target hazard quotient (THQ) was calculated in accordance to the methodology described by the USEPA.  Target Hazard Quotient is one of the methods of estimating risks based on the noncarcinogenic effects of the toxicant and the reference dose.  THQ was determined based on the formula (modified) given by Chien et al. :
THQ = (EFr x [ED.sub.tot] TSIR x C/Rf[D.sub.o] x [BW.sub.a] x [AT.sub.n]) x [10.sup.-3]
where EFr is the exposure frequency = 312 days/year, equivalent to average lifetime; TSIR is the tobacco snuff ingestion rate = 50.0 g/day, equivalent to 10 pouches/day, 50.0 [micro]g/pouch of TSS; C is the concentration of metal in TSS in [micro]g/g; [RfD.sub.o] is the oral reference dose in mg/kg/day; [BW.sub.a] is the average body weight, adult = 60 kg; [AT.sub.n] is the average exposure time for noncarcinogens in days (EFr(312 days/ year) x [ED.sub.tot] (number of exposure years, assuming 70 years in this study); and [10.sup.-3] is the unit of conversion.
Table 2 shows the comparison of concentration of lead in each sample collected from the four geopolitical zones of purchase. The daily intakes based on the different geographical areas from the table show variation in lead concentration. Southwest region shows the highest mean lead concentration in snuff samples, followed by north central, southeast. Northwest shows the least concentration of lead. This difference in mean lead concentration in the four zones is statistically significant (p < 0.05).
Table 3 shows the levels of the metal intake through the daily consumption of various TSSs and the percentage Food and Agricultural Organization/World Health Organization (FAO/WHO) violation. The daily intakes were calculated based on the consumption of 10 pouches per day. The various daily concentrations of lead in each of the samples were compared to that of the FAO/WHO standard, which is 5 [micro]g/kg/day. The table shows that all the TSSs exceeded the permissible limit by the WHO except for TSSs 21, 24, 25, 26, and 27, which had concentrations of lead below the permissible limit stipulated by the FAO/WHO.
Table 4 gives the daily intake of the assayed metal, compared with the provisional tolerable weekly intake and the proposed maximum permissible level suggested by the FAO/WHO.
Table 5 shows the results of THQ calculations to assess the potential health risk in the consumption of the TSSs. These THQs were calculated using the oral reference doses (mg/kg/day) (Pb-1.5) of the metal as stipulated by the United State Environmental Protection Agency (USEPA). 
The mean concentration of lead from the chart shows that the foreign made products had higher concentration of lead when compared to the Nigerian or locally made products [Figure 2].
Permission was obtained from the ethics and research committee of Madonna University, Elele Campus, Rivers State, Nigeria, before the study was conducted.
The study showed that the levels of lead in 27 of the 30 samples exceeded the daily recommended limit of 5 [micro]g/day set by the FAO/WHO.  Thus, a continuous intake of snuff could lead to bioaccumulation of this heavy metal, with the resulting negative health implications.
With respect to the individual percentage violation limit, all the samples except samples 21 and 24-27 exceeded the FAO/ WHO violation limit. The concentration of lead in the TSSs in Nigeria ranged from 0.41 to 1.13 [micro]g/g for north central, 0.45 to 2.48 [micro]g/g for southwest, 0.01 to 1.48 [micro]g/g for southeast, and 0.02 to 1.16 for northwest. This showed that the highest concentration of the heavy metal lead was found in the samples obtained from southwest Nigeria (0.45-2.48 [micro]g/g).
Lagos, Ibadan, and Sango Otta, which are some of the major industrial hub of the country, lie in the southwest Nigeria. A lot of land pollution from industrial waste and effluents released by paint, cement, cosmetic, and other factories may have been the reason for this. Heavy metals in the polluted soils are easily absorbed and retained by the leaves of N. tabaccum.  Incidentally, the Nigeria Tobacco company, the major producer of tobacco products in Nigeria, is located in the southwest.
The THQ for each of the sample was less than 1 (<1.0), which makes them nonhazardous as per the USEPA.  However, the level of lead violated the permissible levels of the FAO/WHO. Cumulatively, the THQ might be exceeded considering the fact that tobacco snuff is used chronically, thus leading to adverse effects on health. 
The study also showed that all the foreign TSSs (1,2, 12, 13, 15, 16, and 18) exceeded violation limit for lead recommended by the FAO/WHO, in contrast to some of the locally produced TSSs (20, 22, and 23-27), which did not exceed the violation limit. This showed that foreign tobacco snuffs were more contaminated with lead and indeed other heavy metals than the locally made snuffs. Foreign countries are more industrialized than Nigeria, hence the tendency for their soils to be more heavily contaminated with chemical pollutants, which are then absorbed by the leaves of tobacco plant in the farms. 
Also, crop farmers in foreign countries have more access to fertilizers and pesticides than Nigeria farmers, hence the tendency for their tobacco plants to be exposed more easily to lead and other heavy metals that are thereafter retained in the leaves, which will undergo eventual processing to snuff and other tobacco product. [14,15]
The study showed varying levels of the heavy metal lead in different brands of tobacco snuff obtained from various geopolitical zones of Nigeria. This evaluation shows that the majority of the tobacco snuffs in Nigeria is highly contaminated with lead and therefore constitutes major health risk to the local population. It also showed that foreign tobacco snuffs have a relatively higher concentration of lead than the local ones. This means a more profound adverse health effects on our population as our people often seem to prefer foreign goods to the locally made ones, including snuff.
The Federal Ministry of Health (FMOH) should regulate further with a stricter measure the production and use of all tobacco products in Nigeria. Also, health-care workers should enlighten their patients and the general populace on the obvious dangers inherent in the use of tobacco snuffs.
We thank Miss Onyejiaka Chinaza Vivian for the contribution to this manuscript and pharmacist (Mrs.) Zelinjo Igweze for supervising the work.
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Source of Support: Nil, Conflict of Interest: None declared.
Prosper O Adogu (1), Joseph C Enye (2), Henry N Chineke (3), Irene A Merenu (3), Nonye B Egenti (4), Ifeoma A Modebe (1)
(1) Department of Community Medicine and PHC Nnamdi Azikiwe University Teaching Hospital/Nnamdi Azikiwe University, (NAU/NAUTH) Nnewi, Nigeria.
(2) Department of Pharmacy Madonna University Elele River State Nigeria.
(3) Department of Community Medicine Imo State University Owerri Nigeria.
(4) Department of Community Medicine, University of Abuja, Nigeria.
Correspondence to: Prosper O Adogu, E-mail: firstname.lastname@example.org
Received May 20, 2015. Accepted May 28, 2015
Table 1: Breakdown of sample geographical area Sample Geographical Town area 1 North central Babangida Market, Suleja 2 North central Babangida Market, Suleja 3 North central Kuje Market, Abuja 4 Southwest Iseyin Market, Oyo 5 Southwest Iseyin Market, Oyo 6 Southwest Iseyin Market, Oyo 7 Southwest Iseyin Market, Oyo 8 Southwest Agbeni Market, Ibadan 9 Southwest Agbeni Market, Ibadan 10 Southwest Illeshiro Market, Ibadan 11 Southwest Illeshiro Market, Ibadan 12 Southeast Main Market Onitsha 13 Southeast Main Market Onitsha 14 Southeast Main Market Onitsha 15 Southeast Main Market Onitsha 16 Southeast Main Market Onitsha 17 Southeast Main Market Onitsha 18 Southeast Main Market Onitsha 19 Southeast Main Market Onitsha 20 Northwest Aitiken Road, Sabon Gari Kano 21 Northwest Yoruba Road, Sabon Gari Kano 22 Northwest Yoruba road, Sabon Gari Kano 23 Southeast Ochanga Market, Onitsha 24 Southeast Ochanga Market, Onitsha 25 Southeast Ahia Ohuru Market, Aba 26 Southeast Ahia Ohuru Market, Aba 27 Southeast Umuahia Market 28 Southeast Umuahia Market 29 Southeast King's Palace, Aba 30 Southeast King's Palace, Aba Sample Type/name 1 Foreign snuff 2 Medicated 3 Local 4 Local 5 Local 6 Local 7 Local 8 Local 9 Local 10 Local 11 Local 12 UAC & Campbell foreign 13 Foreign 14 Local 15 Campbell, KTC, & Maxwell (foreign) 16 KTC (foreign) 17 Broken (local) 18 Medicated 19 Maxwell (foreign) 20 Local 21 Local 22 Medicated 23 Medicated 24 Local 25 Local 26 Medicated 27 Local 28 Broken 29 Local 30 Medicated Table 2: Concentrations of lead in each of the tobacco snuff samples by geopolitical zone Geopolitical Sample Lead Mean [+ or -] SD zone ([micro]g/g) North central TSS 1 1.4125 1.013 + 0.4 TSS 2 1.0612 TSS 3 0.4536 TSS 4 1.1252 Southwest TSS 5 0.6349 1.11 + 0.69 TSS 6 1.0882 TSS 7 0.4536 TSS 8 1.1720 TSS 9 0.5974 TSS 10 2.4770 TSS 11 1.3421 Southeast TSS 12 0.6942 0.58 + 0.52 TSS 13 0.4893 TSS 14 1.2571 TSS 15 0.6529 TSS 16 1.3694 TSS 17 0.7241 TSS 18 0.5059 TSS 19 1.0972 TSS 23 0.8053 TSS 24 0.0291 TSS 25 0.0072 TSS 26 0.0121 TSS 27 0.0049 TSS 28 0.1171 TSS 29 0.0692 TSS 30 1.4836 Northeast TSS 20 1.1610 0.43 + 0.4 TSS 21 0.0219 TSS 22 0.1067 Geopolitical Sample F-test/p-value zone North central TSS 1 F = 2.91 TSS 2 p < 0.05 TSS 3 TSS 4 Southwest TSS 5 TSS 6 TSS 7 TSS 8 TSS 9 TSS 10 TSS 11 Southeast TSS 12 TSS 13 TSS 14 TSS 15 TSS 16 TSS 17 TSS 18 TSS 19 TSS 23 TSS 24 TSS 25 TSS 26 TSS 27 TSS 28 TSS 29 TSS 30 Northeast TSS 20 TSS 21 TSS 22 Table 3: Daily intake and percentage violation of each TSS from the WHO/FAO standard Sample Pb ([micro]g/ Percentage kg/day) violation from the WHO/FAO standard (@ 5 [micro]g/ kg/day) TSS 1 141.0 2820 TSS 2 106.0 2120 TSS 3 45.0 900 TSS 4 113.0 2260 TSS 5 63.0 1260 TSS 6 109.0 2180 TSS 7 45.0 900 TSS 8 117.0 2340 TSS 9 60.0 1200 TSS 10 248.0 4960 TSS 11 134.0 2680 TSS 12 69.0 1380 TSS 13 49.0 980 TSS 14 126.0 2520 TSS 15 65.0 1300 TSS 16 137.0 2740 TSS 17 72.0 1440 TSS 18 51.0 1020 TSS 19 110.0 2200 TSS 20 116.0 2320 TSS 21 2.0 40 TSS 22 11.0 220 TSS 23 81.0 1620 TSS 24 3.0 60 TSS 25 1.0 20 TSS 26 1.0 20 TSS 27 0.5 10 TSS 28 12.0 240 TSS 29 7.0 140 TSS 30 148.0 2960 Table 4: Permissible intake levels as per the FAO/WHO recommendations Metal Provisional Per day For a 60-kg Reference tolerable intake individual weekly intake ([micro]g/ ([micro]g/day) ([micro]g/ kg/day) kg/week) Pb 25.0 5.0 300.0 FAO/WHO Table 5: Target hazard quotient for lead from consumption of TSSs TSS Frequency THQ 1, 16 2 0.0008 2, 4, 6, 19, 20 5 0.0006 3, 7, 9, 13, 18 5 0.0003 5, 12, 15, 17 4 0.0004 8, 11, 14 3 0.0007 10 1 0.0014 21,24-27, 29 6 0.0000 22, 28 2 0.0001 23 1 0.0005 30 1 0.2167 Figure 2: Daily mean concentration of lead ([mu]g/kg/day) in Nigerian and foreign TSSs. Tobacco snuff sample type Nigerian 74.3 Foeign 75.2 Note: Table made from bar graph.
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|Title Annotation:||Research Article|
|Author:||Adogu, Prosper O.; Enye, Joseph C.; Chineke, Henry N.; Merenu, Irene A.; Egenti, Nonye B.; Modebe, I|
|Publication:||International Journal of Medical Science and Public Health|
|Date:||Sep 1, 2015|
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