Introduction

Pakistan stands among the top four states and poses rapid increase in tobacco market. Tobacco is consumed in several ways, for example, cigarette smoking and chewing or sniffing. Tobacco that is used without burning is known as smokeless tobacco products (SLT). Usage of SLT is a common practice among the people of Asia including Pakistan (Middleton 2016). More than 19 carcinogens and around 30 metallic compounds including heavy metals are known to be present in tobacco. Several health disorders are associated with the exposure of heavy metals, especially mercury (Hg), which is one of these heavy metals (Dhaware et al. 2009). Plants have different tolerance mechanisms for higher levels of heavy metals (Schat et al. 2000). Tobacco plant efficiently absorbs Hg from soil (Hussein et al. 2007). It is introduced as a contaminant of lime, fertilizers, manures, poor quality urban waste composite and pesticides.

The SLT products are made of finely crushed tobacco leaves and are sold in either packed or loose form. Moist snuff is dipped between the gum and cheek, while dry snuff is sniffed through nasal or oral route to enlighten the head and used as stimulant and depressant (Bloomfield and Stephens 1996; Popova and Ling 2013; Wrangsjo et al. 2015). The snuff products are also called as dipped/spit tobacco products (Mirbod and Ahing 2000). These products comprises of several inorganic toxicants which release on continuous mastication and absorb into the soft tissues of oral cavity, while the residual portion may be absorbed in the digestive tract (Boffetta et al. 2008). Snuff is commonly used in all cultures across the globe; in Pakistan its common name is “naswar,” but we use the term snuff as its international name. The packets of snuff contain 5–10 g to be sold in markets (Kazi et al. 2013). These items are known to enhance the risk of oral cancer (Farrand et al. 2001; Gupta and Ray 2003; Mazahir et al. 2006; Subramanian et al. 2004). The habituated people may have oral submucous fibrosis, which become more severe to adolescents (Khawaja et al. 2005).

Mercury (Hg) is very poisonous and extremely bio-accumulative (Patrick 2002). It is thought to be most toxic element in the environment and one of the major global and environmental pollutants. It is released from the industrial wastewater, which may further polluted the agricultural soil (Wu and Cao 2010). It has been recently reported that anthropogenic activities are the main cause of Hg pollution. Maximum soil Hg originates from urban wastes, smelting and mining (Patra and Sharma 2000). The Hg enters in the body through food chain and causes severe health risk, especially to the nervous system (Gnamus et al. 2000). It has been reported that organic and aqueous extracts of Indian chewing tobacco, Swedish and American moist snuff originate chromosomal alterations in mammalian and microbial cell cultures (Emerging and Risks 2008). The literature-reported data indicate augmented development of micronuclei in oral epithelial cells as sign of chromosomal impairment in oral cancer patients due to the consumption of SLT products including snuff (Rodu et al. 2005; Rodu and Jansson 2004). The Hg could either be accidentally added in these products, as contaminant, or may be added during the process of manufacturing or through raw material.

The health risks associated with high exposure of Hg are well known (Bull 2011). However, risk associated with the long-term exposure of Hg at trace levels is still under debate for the researchers (Gnamus et al. 2000). However, less research is carried out on the oral exposure of toxic metals through the snuff products (Bolewska et al. 1990). The Hg is absorbed into the brain after passing from olfactory bulb, which was settle on mucous membrane in the upper nasal cavity as Hg vapors, during nose breathing. In addition, extraction rate for Hg from snuff products in saliva will differ according to each product. However, the frequent use of these products permits the accumulation of non-degradable and hazardous components in the consumer’s body (Pappas et al. 2008). Heavy metals have lethal effects, and even trace amounts of them can cause severe physiological disorders (García-Rico et al. 2007).

Recently, advance preconcentration methods for the analysis of trace levels of Hg have been observed in substantial depth (Carabias-Martınez et al. 2000). These preconcentration steps are crucial because the sensitivity or the selectivity of the methods could be affected by the presence of organic/inorganic contaminations (Coelho and Arruda 2005). Co-precipitation, liquid–liquid extraction, micellar, sorption and ion exchange system are the most commonly used techniques in various fields of analytical chemistry (Carabias-Martınez et al. 2000). The most common methodology is cloud point extraction for preconcentration and separation of metals in different matrices (Manzoori and Bavili-Tabrizi 2002a). In this method, solution becomes turbid above a specific temperature that is recognized as “cloud point temperature” (Coelho and Arruda 2005), above which the phase separation occurs as the bulk aqueous solution, comprising surfactant monomers and the surfactant-rich phase with very small volume (Manzoori and Bavili-Tabrizi 2002a). Micellar system offers several advantages, such as higher capacity to concentrate the inclusive range of analytes with elevated preconcentration factors, recoveries and low cost (Manzoori and Bavili-Tabrizi 2002b; Shemirani et al. 2005). Due to pure hydrophobic interactions between analyte complex and nonionic surfactant, metal chelate exists in surfactant phase.

After the advent of cloud point, micelle dispersion is imperative factor that has significant impact on analysis time and extraction efficacy (Guiñez et al. 2018). Dispersion of micelle in the solution could be carried out by different energy modes such as automatic agitation (mixer, colloid mill, stirrer, valve homogenizer) and ultrasound energy (Chiha et al. 2010). In recent times, ultrasound energy has attain huge acceptance to be used as a safe way for the dispersion of solution due to its greener nature and low cost (Khan et al. 2017; Miano et al. 2016). Sonication decreases the analysis time by increasing the analyte’s interaction with nonionic surfactant rapidly. Cavitation effect is produced by ultrasound energy, which improves the contact area between two phases and mass transfer (Mehrabi and Dil 2017). With the help of ultrasound energy, reduced concentration gradients and rapid mass transfer are the main advantages that eventually enhance the extraction proficiency of the method by reducing the internal/external resistance to analyte transportation (Ju et al. 2020; Khan et al. 2017).

Despite legal warning and proposal for prohibition of smoking and consuming SLT, the public still use these products, frequently. Lack of information on the levels of different toxic metals contamination of SLT products consumed in Pakistan/different parts of world should be seriously concerned and monitored by health administrative agencies. In the present study, the concentration of Hg will be determined in different brands of commercially available SLT (snuff) products and in their ASE. This study was undertaken to help well comprehend the level of Hg, to which the consumers of snuff products are exposed through risk assessment.

The purpose of present work was to develop dual-step ultrasonically modified cloud point extraction (DsUm-CPE) method to determine the total mercury (Hg) content in artificial saliva extract (ASE) of different types of snuff products (moist and dry) having different colors, green, brown and black. Ultrasound energy and vortex shaking were compared as dispersion modes for the extraction efficiencies. Several variables of developed methods including concentration of complexing agent, Triton X110, incubation time in ultrasonic bath, centrifugation time and concentration of back extraction reagent have been studied in order to achieve optimum recovery of Hg. The accuracy for the analysis of total Hg was checked by certified reference materials of tobacco, human hair and whole blood. In addition, the present study also aims to determine total Hg contents in the biological samples (scalp hair and blood) of the adult population (25–60 years) having sniffing habits. Healthy referent subjects of the same age group were also selected for comparison purpose; those don’t sniff or consume any tobacco product. Different risk factors associated with the Hg intake via consuming snuff products were also studied.

Material and method

Reagents and chemicals

For the preparation of samples/standard solutions, deionized water was utilized throughout the investigation, attained from ELGA lab water system (Bucks, UK). The complexing reagent and other chemicals such as ammonium O, O-diethyldithiophosphate (DDTP), concentrated HNO3 (65%), HCl (37%), H2O2 (30%) and α-amylase were of high purity/analytical grade. The octylphenoxypolyethoxyethanol (Triton X-114) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solution of mercury (1000 mg L−1) was attained from Fluka Kamica (Bushs, Switzerland) to prepare the working standard by successive dilution. The glassware was infused in 10% HNO3; then washed by means of distilled and deionized water; and kept in close containers to avoid any contamination. For accuracy of the analytical technique, to determine the total Hg concentrations in certified reference materials, Virginia tobacco leaves (ICHTJ-cta-VTL-2) Clincheck control-lyophilized human whole blood (Recipe, Munich Germany) and human hair (BCR 397, Brussels®, Belgium) were analyzed for total Hg concentrations.

Instrumentation

To determine the total Hg contents, the certified and real samples were acid digested in close PTFE tubes using a domestic microwave oven (Pel, Osaka, Japan). pH meter (Ecosan Ion 6, Malaysia) was used to measure the pH of samples. For systematic mixing, programmable ultrasonic water bath (0–80 °C temperature range and 35 kHz intensification frequency), model no: SC-121TH (Sonicor, Deep Park, NY, USA), and MX-E vortex mixer (Dragon Lab instrument, Beijing 101, 318 China) were utilized. WIROWKA Laboratory type WE-1, nr-6933 centrifuge machine (220/50 Hz, 0–6000 rpm speed range, 0–60 min timer, Mechanika Phecyzyjna, Poland) was used to separate both phases. An Analyst 700 atomic absorption spectrometer, PerkinElmer (Norwalk, CT, USA), MHS-15 chemical vapor generation system (PerkinElmer), joined with AA spectrometer system was used to produce cold vapor of Hg. A mercury hollow cathode lamp was used as the radiation source, functioning at 5 mA. Measurements were carried out in the integrated absorbance (peak area) mode at 253.7 nm, using a spectral band width of 2.6 nm. Argon with 99.99% purity was used as the carrier gas.

Sampling and treatment

Different types of 23 snuff samples (n = 10 of each) were purchased form markets of different cities of Pakistan during January to December 2018. The snuff samples were divided into two mains categories, on the basis of texture as moist and dry snuff samples. The snuff samples were further classified on the basis of their color as green moist snuff (GMS n = 7), brown moist snuff (BMS n = 7), dry brown snuff (DBS n = 5) and dry black snuff (DBKS n = 4). Brand identity of each sample has not been declared in the text due to authority rules. All snuff samples were prepared carefully in clean environment to avoid any contamination. The snuff samples were dried at 40 °C for 12 h, then ground into fine particles and passed through the nylon sieve with 125 μm of mesh size to remove the larger granules. Then, sieved samples were stored in clean polyethylene bags till further analysis.

A survey among male adults was carried out to collect information based on structured questionnaire. Biological samples (scalp hair and blood) were collected from male subjects (n = 100) age ranged from 25 to 60 years resided in two big cities of Pakistan, consuming different types of snuff. For comparative purposes, 60 age matched male subjects, not consuming any tobacco products were selected as referents. All contributors were aware about the purpose of the study; all of them gave consent to take part in the study and signed the form. Ethical committee of Sindh University Jamshoro approved this study. A 7-mm heparinized lithium Vacutainer® tube (Becton Dickinson) was used to collect 5-ml blood samples from the selected subjects. Blood samples were stored at − 20 °C till analysis. The scalp hair samples were taken from the nape of the neck of each study subjects and kept in separate labeled plastic bags. Hair samples were cut into small pieces, around 0.2 to 0.3 cm, and thoroughly washed with diluted Triton X-100, followed by acetone and ultrapure water. After drying at 80–85 °C, the samples were stored. The details about collection and pretreatment of scalp hair samples are given in our previous work (Akhtar et al. 2017).

Procedure for total mercury analysis

To determine the total Hg contents in replicate six (0.2 g) of each certified reference materials (scalp hair, blood and tobacco) and triplicate sets of each real samples of different types of snuff, scalp hair and blood samples (0.5 mL) of referents and snuff consumers were kept into Teflon PTFE flasks. The fresh mixture of concentrated H2O2 and HNO3 in the equal ratio was prepared and added 2 mL of it to each flask and reserved for 5 min at room temperature; then, enclosed vessels were positioned in microwave heating system for 2 to 5 min (Shah et al. 2009). Volume of the final digests was made up to 25 mL with 0.1 M HCl. Resulted solutions were analyzed by CVAAS to determine total Hg contents.

Extraction of Hg by Artificial saliva

Artificial saliva was prepared by following the procedure as reported in previous work (Arain et al. 2014). Replicate six (0.2 g) of each snuff sample (dray and moist) in PTFE flasks was weighted, and 25 mL of freshly prepared artificial saliva was added. Flasks were then kept for dissimilar time intervals (5–30 min) in an ultrasonic bath. After each interval, the contents of the flasks were centrifuged for 5 min and aqueous phase was separated by means of pasture pipette; 100 µL of HCl was added (to avoid fungal growth) and kept at 4 °C till further analysis.

Procedure

A DsUm-CPE was applied to the ASE of different types of snuff samples for preconcentration of Hg. For the optimization of different variables, two set of six replicate of Hg standards (2 µg L−1) and triplicates of each ASE (10 mL) of real samples were taken in PTFE flasks. Then, 0.1–0.5% (w/v) Triton X-114 (TX-114) and complexing agent (DDTP) were added in the concentration range of 0.2–1.0% (w/v). One set was placed for 1–5 min at 20–60 °C in an ultrasonic water bath for the dispersion/shaking, whereas other set was subjected to vortex mixer for 30–120 s. The content of the tubes was than centrifuged at 4000 rpm for 5 to 10 min to separate both phases. Flasks were then positioned in an ice bath for 5 min, to upsurge the viscidity of the sediment phase. The upper aqueous phase was discarded with the help of pipette and added 1.0 to 2.0 mol L−1 of HNO3 and HCl separately to the enriched phase and subjected to the second round of cloud point extraction (CPE) (sonication/vortex mixing, then cooling and centrifugation as given above). The aqueous phase is separated after centrifugation. Triplicate of each (500 µl) of sedimented samples was taken in PTFE flasks of the MHS-15 system and added 40 µl of the antifoam agent along with 1.0 M HCl (2.5 ml). After the addition of 3% (m/v) NaBH4 in PTFE flasks for 5 s, system was sealed. Hg vapors produced in the system were carried to the quartz cell using Argon stream, and the signals were noted.

Statistical Analysis

For statistical analysis and data processing different computer programs such as Minitab13.2 (Minitab Inc., State College, PA), XL State (Addin soft, NY, USA) and Excel 2003 (Microsoft Office ®) were used. Resulted data of all samples are given as means ± std. Significant difference was evaluated between certified and experimental values of CRM using Student’s t test.

Results and discussion

The DsUm-CPE method has not been commonly used for the Hg determination and limited to few environmental and biological samples (Yu 2005). Certified standard and real samples (ASE extracts of snuff) were treated with complexing agent; formed complex was entrapped in TX-114. It was manifested that the addition of complexing agent and Triton X-114 has numerous benefits, i.e., appreciable hydrophobicity of the DDTP-Hg complex, and quite low cloud point temperature of Triton X-114 (Borges et al. 2003). The parameters affecting the CPE performance were studied to attain an optimum recovery of Hg from ASE of different snuff samples. Different variables including volume of complexing agent, surfactant and back extracting agent, equilibration temperature, dispersion mode and time were selected.

The pH is an important factor to be studied to expand the preconcentration effectiveness of the methodology. However, in the present study, pH was not optimized because the Hg-DDTP complex is stable in the acidic media, so it is directly preconcentrated in previously acidified sample (ASE of snuff samples), without using any buffer (Dressler et al. 2002). For back extraction of analyte complex (Hg-DDTP) from surfactant enriched phase, 0.5 mL of 1 to 2 mol L−1 of HCl/HNO3 was added and subjected to ultrasonic energy only, for shaking and heating at 50 °C (second round of CPE), prior to analyzed by CVAAS.

The concentration of the DDTP was studied in the range of (0.2–1.0%) to attain the optimum recovery of Hg (Fig. 1). Maximum recovery of the analyte was observed at 0.4% DDTP (w/v). Significant decrease (> 20%) was observed in the extraction efficiency as the concentration of DDTP reached up to 1% (w/v). This action may be due to competition for the complexation in sample solution. DDTP was selected as complexing agent for Hg due to its high stability in acidic media and aqueous media. It can be added to the aqueous solutions of standards and samples.

Fig. 1
figure 1

Effect of complexing agent concentration on the %recovery of Hg using DsUm-CPE. Conditions: Hg: 2.0 µg L−1, 0.2% Triton X-114 (v/v).Other experimental conditions are described under procedure

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The TX-114 was chosen as the surfactant due to its less toxicity, accessibility in purified form, high density and low cost to enable the better phase separation. Moreover, it has low cloud point temperature (about 40 °C), which allows its application for the extraction of numerous hydrophobic complexes (Filik et al. 2006). The concentration of TX-114 was studied in the range of 0.05–0.3% as shown in Fig. 2. The extraction efficacy of the Hg was found to be highest at 0.2% (m/v). More upsurge concentration of surfactant leads to the lower recovery of Hg. For this purpose, back extracting agent was used to extract the analyte into acidic aqueous solution, and second round of CPE was performed.

Fig. 2
figure 2

Effect of the concentration of surfactant on the percent recovery of Hg using DsUm-CPE. Hg: 2.0 µg L−1, ligand (DDTP) 0.4% (w/v) and 0.2% Triton X-114 (v/v)

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Dispersion of the solution’s contents increases the interface between complexing agent and analyte. This actually enhances its entrapment by chelating agent in addition to mass transfer to the organic phase. It is anticipated that shaking the content of tubes after addition of chelating agent and surfactant has strong influence on the extraction efficacy of the developed method. This is because shaking enables additional protracted and close contact between extraction mixture and analyte in both phases. Two dispersion modes have been studied for the present methodology, which comprises vortex and ultrasound energy (Fig. 3). Among both dispersion modes, ultrasound was found to be best for the dispersion of extraction mixture as compared to vortex shaking, in relation to the analyte recovery, extraction efficacy, energy consumption, greener tactic, low cost and little chances of reagent loss. For the present study, sonication time was studied for the time duration of 1–5 min. The % recovery of Hg for the developed methodology increases with sonication time as shown in Fig. 4. The % recovery increases up to three min; further increase in time has no significant effect on the recovery. So, for the optimum recovery 3.0 min was selected and applied to rest of the experiments. Time for vortex mixing was studied in the range of (20–120) sec, and optimum recovery was observed at 90 s. However, in spite of lesser time period, the % recovery was around 10–20% lesser than those values obtained through sonication. Therefore, ultrasound energy was used for further experimental work, as it provides maximum extraction recoveries.

Fig. 3
figure 3

Comparison of sonication and vortex shaking on the extraction recovery of Hg

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Fig. 4
figure 4

Effect of sonication and vortex time on the % recovery of Hg using DsUm-CPE. Hg: 2.0 µg L−1, ligand (DDTP) 0.4% (w/v) and 0.2% Triton X-114 (v/v)

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The optimal incubation time and sonication temperature are required for the ease in phase separation and preconcentration step. It is obvious that the cloud point temperature of the surfactant is responsible for the precipitation and formation of the surfactant-rich phase. The efficiency of CPE has been strongly influenced by the incubation temperature of the nonionic surfactant (TX-114). Efficient extraction of analyte and better phase separation could be enhanced at the equilibration temperature above than cloud point temperature of TX-114. Volume of surfactant-rich phase may reduce due to an increase in cloud point temperature (40 °C). At higher temperature, it causes dehydration that results hydrogen bonds disruption. Incubation temperature is therefore required at shorter time for effectual phase parting and complete analyte extraction. In the current study, the incubation temperature was studied for ultrasonic assisted dispersion at 40 °C, at room temperature, whereas vortex shaking was also carried out at room temperature.

Back extraction of the Hg in second round of CPE was carried out by means of sonication for 5 min at 40 °C. The complete phase separation was achieved after centrifugation at rate of 4000 rpm at 5 min, whereas 10 min was required for those samples obtained after vortex mixing.

For the back extraction of the Hg from the enriched phase, both nitric and hydrochloric acid in the range of 1.0 to 2.0 mol L−1 were selected. The volume was studied in the range of 0.2–1.0 mL. The optimal extraction was attained at ≤ 0.4 mL of HCl (Fig. 5), whereas 10 to 15% less value of analyte was obtained in the case HNO3. The better recoveries were achieved at volume of 0.4 mL HCl at 1.5 mol L−1, which was selected for following experimentation.

Fig. 5
figure 5

Effect of the volume of back extracting agents (HNO3 and HCl) on the % recovery ofHg using DsUm-CPE. Hg: 2.0 µg L−1, ligand (DDTP) 0.4% (w/v) and 0.2% Triton X-114 (v/v)

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The consequence of interfering ions was studied for the recovery of Hg in ASE of snuff samples. For this purpose, various interfering analytes were added at diverse ratios to 10 mL solution of Hg (100 µg L−1), and developed methodology was applied to the solution. The results indicate that recovery of the target analyte was > 95% for all studied metals in the proposed DsUm-CPE method, as shown in Table 1.

Table 1 Effect of some foreign ions on the % recovery of Hg determined DsUm-CPE (μgL−1)
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Analytical figure of merit

The correlation coefficient of the calibration graph was found as 0.993 and 0.997 for vortex shaking and sonication, respectively. Preconcentration factor (PF) was calculated by the ratio among the volumes of the solution before and after the application of preconcentration method; however, the enhancement factor (EF) was intended as the ratio between the slope of calibration graph for with and without preconcentration step. The PF was found to be 78 and 63, with sonication and vortex shaking, respectively. The EF was found to be 97 and 84 with sonication and vortex shaking, respectively. The resulted limit of detection (LOD) was found as 0.004 µg L−1, which was adequate and low for the Hg determination at trace levels in ASE of snuff samples, using ultrasound energy as a dispersion mode. However, LOD for the developed method using vortex shaking was found as 0.25 µg L−1. Improved extraction efficiency 98 to 99.0% was observed in aqueous solutions by applying DsUm-CPE. The accuracy for the analysis of total Hg was checked by CRMs, and insignificant difference (p > 0.05) was found between certified and experimental values (Table 2). The analytical figure of merit for the proposed method is given in Table. 3. The developed method was found to be efficient when compared with the previously reported methods (Table 4) (Ali et al. 2017; Shah et al. 2009; Shirkhanloo et al. 2015; Stanisz et al. 2013; Yuan et al. 2012).

Table 2 Determination of mercury in certified sample of human hair, blood and Virginia tobacco leaf by microwave assisted digestion method (MAD) (n = 6)
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Table 3 Analytical performance and figure of merit of the proposed method for enrichment of Hg
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Table 4 Comparative data of analytical characteristics of DsUA-CPE for Hg with previously reported pre-concentration methods
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Application

The pH of all snuff samples was found to be very basic, in the range of 8.4–8.7. At this pH, tobacco-specific amines are formed making these products more toxic. The mean concentration of Hg with standard deviation for five composite samples of each type is shown in Table 5. The range of the Hg concentration in green and brown moist snuff was analyzed in the ranges of 0.13—0.62 μg g−1 and 0.24—0.71 μg g−1, respectively. The concentration ranges of Hg were found as 1.63–2.3 μg g−1 and 1.03–2.31 μg g−1 for dry black and brown snuff, respectively. The extractable Hg in ASE of green and brown moist snuff samples is found in the range of 0.053–0.243 μg g−1 and 0.146–0.277 μg g−1, respectively. The resulted data indicated that extractable Hg in ASE was found to be 30 to 48% of total Hg contents in all snuff products (Table 5), whereas extracted Hg in ASE of dry black and brown snuff samples was found as 0.424 to 0.598 μg g−1 and 0.304–0.682 μg g−1 corresponding to 23 to 34% of total Hg contents (Table 5).

Table 5 Concentration of total and artificial saliva extracted Hg in different types of snuff sample (µg g−1)
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Risk assessment

Estimated daily intake (EDI) is based on the concentration of Hg in chewing/sniffing products and the ingested amount of the selected SLT products. To calculate the EDI, the following equation was used. The calculations are based on the standard assumption recommended by United States Environmental Protection Agency (USEPA). Hg content in the snuff samples is expressed in µg Kg−1; average body weight is expressed in Kg; and amount of snuff was taken as 0.01 kg of snuff/person/day.

$${\text{EDI}} = \frac{{\left( {\text{Concentration of Hg x Amount of snuff}} \right)}}{{\text{Average body weight}}}$$

The EDI values intended for brown moist, green moist, dry black and dry brown snuff (10 g day−1) were calculated as 0.042–0.118, 0.022–0.103, 0.532–0.740, 0.512–0.823 µg kg−1bw, respectively. The resulted EDI values correspond to 8–15%, 2–13%, 21.5–48% and 33–48% of the proximal tolerable daily intake (PTDI) of Hg for brown moist snuff, green moist snuff, dry black and dry brown snuff, respectively. All calculated EDI values for Hg were found to be lower than the PTDI of Hg recommended by (Joint and Ng 2010). Highest intake of Hg is coming from consumption of dry black snuff, whereas lowest comes from green moist snuff sample. Maximum weekly intake of total Hg for adult persons is recommended as 5.6 µg kg−1bw (1.6 µg kg−1bw of methylmercury and 4 µg kg−1bw of inorganic mercury) by Joint FAO/WHO expert committee for provisional tolerable weekly intake (Joint and Ng 2010). Average daily dose (ADD) of Hg depends on both the amount of consumption of SLT and the metal concentration in SLT. The ADD of Hg was calculated by means of its average concentration in SLT by its consumed weight via an individual (body weight 60 kg for an adult in Pakistan). The equation used for the determination of ADD of Hg in SLT for adults is given below:

$$ADD = \frac{C*IngR*EF*ED*CF}{{AT*BW}}$$

where C signifies the concentration of Hg (mg g −1) in snuff products; exposure frequency (EF) and exposure duration (ED) signify exposure period of 365 days per year and 30 year for adults, respectively, for the present work. Ingestion rate (mg day−1) is represented by IngR; BW is adult’s body weight (60 kg); for non-carcinogenic risk, average time (AT) is calculated as ED*EF; however, conversion factor is signified as CF (10 −6 mg/kg). All these factors are available in the earlier intelligences of US EPA and in our formerly published work (Akhtar et al. 2016; Means 1989; Part 2011).

Non-carcinogenic risk associated with chronic exposure of Hg through SLT consumption for SLT consumers was evaluated based on the Hazard Quotient (HQ). HQ value < 1 signifies that population is improbable to have the risk. HQ was calculated by following equation (EPA 1992).

$$HQ = \frac{ADD}{{RfD}}$$

where ADD represents the average daily dose of Hg (mg kg−1 day−1) via snuff consumption and reference dose (RfD) for Hg is 0.0003 mg kg−1 day−1 which is recommended by the United States Environmental Protection Agency (EPA. 2013). The ADD values intended for brown moist, green moist, dry black and dry brown snuff samples based on consumption of 10 g day−1 are calculated as 0.07–0.123, 0.02–0.1, 0.17–0.39, 0.27–0.38 µg kg−1bw, respectively. FAO/WHO Joint Expert Committee on Food Additives (JECFA) and WHO, 2010, recommended Provisional Tolerable intake (PTI) of Hg, 5.6 µg Kg−1 body weight/week or 0.8 µg Kg−1 body weight/day (Joint 2003). The calculated ADD values for Hg were found to be lower than the tolerable daily intake level in brown and green moist snuff. However, consumption of higher weight and multiple time of snuff products might be created adverse impact on consumers. Highest intake of Hg is coming from consumption of dry black and dry brown snuff. The ADD values are shown in Table 6. The HQ values are found to be greater than 1 for dry black and dry brown snuff products, whereas for green and brown moist snuff HQ values were found to be < 1.0. It was indicated in the literature that the HQ values > 1 suggest adverse risk of human being (Akhtar et al. 2016; EPA 1992). Carcinogenic risk assessment in terms of cancer risk could not be performed due to unavailability of cancer slope factor for Hg.

Table 6 Risk assessment of mercury by ingesting 10 g of different types of snuff
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Exposure of Hg in population consumed snuff products

The Hg levels in hair samples of adult male subjects consuming different types of snuff were higher at 95% confidence interval (CI: 4.64–5.52 μg g−1) than referents male subjects not use any cigarette or/smokeless products have (CI: 1.68–2.09 μg g−1). The Hg levels in blood samples of adult male subjects consuming different snuff products were higher at 95% confidence interval (CI: 7.70–9.82 μg g−1), than referents male subjects not use any cigarette or/smokeless products have (CI: 4.23–6.17 μg g−1). The contents of Hg in hair samples of the population consuming different types of snuff have significantly higher levels than those who were not using any snuff product (p = 0.03–0.04). The resulted data indicate a correlation of sniffing habit with Hg content in biological samples of the adult population. The average values of Hg content in hair and blood samples of adult male subjects with standard deviation are given in Table 7.

Table 7 Mercury concentration in scalp hair and blood samples of referent and study subjects (n = 50), consuming different types of snuff products
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Conclusion

An innovative ultrasonically modified cloud point extraction method which eliminates the surfactant’s effects using acidic solutions and long ultrasonic-assisted shaking/heating on the extraction efficiency was developed for the preconcentration of Hg, whereas the acidic ASE of each snuff products was directly subjected for complexation with DDTP. In addition to this, two dispersion modes, i.e., ultrasound energy and vortex shaking, were compared. Dispersion of the micelle by the ultrasound energy for the proposed method improves the extraction efficiency immensely due to increased interaction of metal chelate with micelles formed after cloud point. Ultrasound energy improves the extraction efficiency by increasing the contact area between both phases (aqueous and surfactant rich phase) which ease the analytes mass transfer into the micelles enriched phase from the aqueous phase. This preconcentration method with higher extraction efficiency and valuable parameters facilitates the tough task of Hg determination in the ASE of smokeless tobacco products. Average Hg concentration in each SLT product indicates that the source of Hg may be related with preparation of SLT products or mixing unidentified constituents. Daily intake of Hg was greater than the tolerable daily intakes, which shows the significant risk related to the consumption of these products. The findings of the current study would be advantageous for health specialists and individuals consume SLT frequently. Moreover, extraction rate of the Hg from the snuff samples by the saliva is different for each type (dry and moist). It was observed that dry snuff, especially black, contained higher levels of Hg than moist green and brown snuff.